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Agrobacterium Protocols
Agrobacterium Protocols
Agrobacterium
Protocols
Volume 2
Third Edition
METHODS
IN
M O L E C U L A R B I O LO G Y
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
Agrobacterium Protocols
Volume 2
Third Edition
Edited by
Kan Wang
Center for Plant Transformation, Plant Sciences Institute, and Department of Agronomy,
Iowa State University, Ames, IA, USA
Editor
Kan Wang
Center for Plant Transformation
Plant Sciences Institute
Department of Agronomy
Iowa State University
Ames, IA, USA
ISSN 1064-3745
ISSN 1940-6029 (electronic)
ISBN 978-1-4939-1657-3
ISBN 978-1-4939-1658-0 (eBook)
DOI 10.1007/978-1-4939-1658-0
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2014950243
Springer Science+Business Media New York 2015
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Dedication
To Marc Van Montagu and Jeff Schell (19352003), my Ph.D. mentors, for their inspiration and encouragement.
Preface
Agrobacterium tumefaciens is a soil bacterium that for more than a century has been known as
a pathogen causing the plant crown gall disease. Unlike many other pathogens, Agrobacterium
is able to deliver DNA to plant cells and permanently alter the plant genome. The discovery of
this unique feature has provided plant scientists with a powerful tool to genetically transform
plants for both basic research purposes and for agricultural advancement.
The first transgenic plants were reported a little over 30 years ago in 1983 by three
independent research groups. Using disarmed Agrobacterium vectors, these groups produced antibiotic-resistant transgenic tobacco Nicotiana tobaccum (Herrera-Estralla et al.,
1983, Nature 303: 209), Nicotiana plumbaginifolia (Bevan et al., 1983, Nature 304: 184),
and petunia (Petunia hybrid, Fraley et al., 1983, Proceedings of the National Academy of
Sciences 80: 4803). The three scientists who led the landmark work, Marc Van Montagu,
Mary-Dell Chilton, and Robert Fraley, were the laureates for the 2013 World Food Prize
(http://www.worldfoodprize.org/en/laureates/2013_laureates/#StatementAchievem
ent). As the statement of achievement of the World Food Prize says, each conducted
groundbreaking molecular research on how a plant bacterium could be adapted as a tool to
insert genes from another organism into plant cells, which could produce new genetic lines
with highly favorable traits.
While other methods such as biolistic gun, electroporation or polyethylene glycol can
also be used for introducing DNA molecules into plant cells, the Agrobacterium-mediated
transformation method remains the method of choices for most plant species in many laboratories due to its efficiency and its propensity to generate single or a low copy number of
integrated transgenes with defined ends.
When the first edition of Agrobacterium Protocols was published in 1995, exactly 20
years ago, only a handful of plants could be routinely transformed using Agrobacterium.
The second edition, which was published in 2006, collected transformation protocols for
59 plant species. In this third edition, we have updated protocols for 32 plant species from
the second edition and added protocols for 17 new species. Together with the first and
second editions, these two new volumes offer Agrobacterium-mediated genetic transformation protocols for a total of 76 plant species.
The third edition of Agrobacterium Protocols contains 57 chapters (two volumes)
divided into 9 parts. This edition emphasizes on agricultural crops or plant species with
economic values. For a number of important plants such as rice, barley, wheat, and citrus,
multiple protocols using different starting plant materials for transformation are included.
Like the second edition, plants are grouped according to their practical uses rather than
their botanical classifications.
Agrobacterium Protocols provides a benchtop manual for tested protocols involving
Agrobacterium-mediated transformation. All chapters are written in the same format as that
used in the Methods in Molecular Biology series. Each chapter is contributed by authors
who are leaders or veterans in their respective areas. The Abstract and Introduction
sections provide outlines of protocols, the rationale for selection of particular target tissues,
and information regarding overall transformation efficiency. The Materials section lists
the host materials, Agrobacterium strains and vectors, stock solutions, media, and other
vii
viii
Preface
supplies necessary for carrying out these transformation experiments. The Methods section
is the core of each chapter. It provides a step-by-step description of the entire transformation procedure from the preparation of starting materials to the harvest of transgenic plants.
To ensure the reproducibility of each protocol, the Notes section lists possible pitfalls in
the protocol and suggests alternative materials or methods for generating transgenic plants.
Typically, most laboratories only work on one or a few plant species. Each laboratory or
individual researcher has his or her own favorite variation or modification of any given plant
transformation protocol. The protocols presented in this edition represent the most efficient methods used in the laboratories of the contributors. They are by no means the only
methods for successful transformation of your plant of interest.
The broad range of target tissue selection and in vitro culture procedures indicate the
complexity in plant transformation. It is the intention of this book to facilitate the transfer
of this rapidly developing technology to all researchers for use in both fundamental and
applied biology. I take this opportunity to thank all my colleagues whose time and effort
made this edition possible. Special thanks go to my family for their unconditional love and
support during the process of editing this book.
Ames, IA, USA
Kan Wang
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PART I
INDUSTRIAL PLANTS
1 Brassica rapa. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tom Lawrenson, Cassandra Goldsack, Lars Ostergaard,
and Penny A.C. Hundleby ne Sparrow
2 Cotton (Gossypium hirsutum L.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Keerti S. Rathore, LeAnne M. Campbell, Shanna Sherwood,
and Eugenia Nunes
3 Jatropha (Jatropha curcas L.). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Devendra Kumar Maravi, Purabi Mazumdar, Shamsher Alam,
Vaibhav V. Goud, and Lingaraj Sahoo
4 Sesame (Sesamum indicum L.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sonia Kapoor, Sanjay S. Parmar, Manju Yadav, Darshna Chaudhary,
Manish Sainger, Ranjana Jaiwal, and Pawan K. Jaiwal
5 Sunflower (Helianthus annuus L.). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Laura M. Radonic, Dalia M. Lewi, Nilda E. Lpez, H. Esteban Hopp,
Alejandro S. Escandn, and Marisa Lpez Bilbao
PART II
11
25
37
47
ROOT PLANTS
PART III
vii
xiii
59
67
85
97
ix
111
121
Contents
12 Cherry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Guo-Qing Song
13 Chestnut, American (Castanea dentata (Marsh.) Borkh.) . . . . . . . . . . . . . . . .
Charles A. Maynard, Linda D. McGuigan, Allison D. Oakes,
Bo Zhang, Andrew E. Newhouse, Lilibeth C. Northern,
Allison M. Chartrand, Logan R. Will, Kathleen M. Baier,
and William A. Powell
14 Chestnut, European (Castanea sativa) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Elena Corredoira, Silvia Valladares, Ana M. Vieitez,
and Antonio Ballester
15 Grapevine (Vitis vinifera L.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Laurent Torregrosa, Sandrine Vialet, Anglique Adivze,
Pat Iocco-Corena, and Mark R. Thomas
16 Melon (Cucumis melo) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Satoko Nonaka and Hiroshi Ezura
17 Peach (Prunus persica L.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Silvia Sabbadini, Tiziana Pandolfini, Luca Girolomini,
Barbara Molesini, and Oriano Navacchi
18 Strawberry (Fragaria ananassa) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Roberto Cappelletti, Silvia Sabbadini, and Bruno Mezzetti
19 Walnut (Juglans) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Charles A. Leslie, Sriema L. Walawage, Sandra L. Uratsu,
Gale McGranahan, and Abhaya M. Dandekar
PART IV
143
163
177
195
205
217
229
TROPIC PLANTS
PART V
133
245
259
275
293
307
319
331
Contents
xi
347
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
373
357
365
Contributors
ANGLIQUE ADIVZE Montpellier SupAgro-INRA, UMR AGAPGenetic Improvement
and Adaptation of Mediterranean and Tropical Plants, Montpellier Cedex, France
SHAMSHER ALAM Department of Biotechnology, Indian Institute of Technology Guwahati,
Guwahati, India
NURIA ALBURQUERQUE Grupo de Biotecnologa de Frutales, Departamento de Mejora,
CEBAS-CSIC, Murcia, Spain
FREDY ALTPETER Plant Molecular and Cellular Biology Program, Agronomy Department,
Genetics Institute, University of Florida, Gainesville, FL, USA
KATHLEEN M. BAIER Department of Environmental and Forest Biology, SUNY College
of Environmental Science and Forestry, Syracuse, NY, USA
ANTONIO BALLESTER Instituto de Investigaciones Agrobiolgicas de Galicia, IIAG, CSIC,
Santiago de Compostela, Spain
JEAN-CHRISTOPHE BREITLER Centre de Coopration Internationale en Recherche
Agronomique pour le Dveloppement, UMR RPB, Montpellier, France
SIMON E. BULL Plant Biotechnology Group, ETH Zurich, Universitaetstrasse,
Zurich, Switzerland
LORENZO BURGOS Grupo de Biotecnologa de Frutales, Departamento de Mejora,
CEBAS-CSIC, Murcia, Spain
LEANNE M. CAMPBELL Institute for Plant Genomics and Biotechnology, Texas A&M
University, College Station, TX, USA
ROBERTO CAPPELLETTI Department of Agriculture, Food and Environmental Sciences,
Universit Politecnica delle Marche, Ancona, Italy
MING-TSAIR CHAN Academia Sinica Biotechnology Center, Tannan, Taiwan; Academia
Sinica Agricultural Biotechnology Research Center, Taipei, Taiwan
ALLISON M. CHARTRAND Horn Performance and Environmental Science, Northwestern
University, Evanston, IL, USA
DARSHNA CHAUDHARY Centre for Biotechnology, M. D. University, Rohtak, India
ZENN-ZONG CHEN Division of Silviculture, Taiwan Forestry Research Institute,
Taipei, Taiwan
VENKATESWARI J. CHETTY Plant Transformation Research Center, University of California
Riverside, Riverside, CA, USA
VINCENT L. CHIANG Forest Biotechnology Group, Department of Forestry, North Carolina
State University, Raleigh, NC, USA
JIHONG LIU CLARKE Bioforsk- Norwegian Institute for Agricultural and Environmental
Research, s, Norway
ELENA CORREDOIRA Instituto de Investigaciones Agrobiolgicas de Galicia, IIAG,
CSIC, Santiago de Compostela, Spain
ABHAYA M. DANDEKAR Plant Science Department, University of California, Davis,
CA, USA
EVELINE DCHAMP Centre de Coopration Internationale en Recherche Agronomique
pour le Dveloppement, UMR RPB, Montpellier, France
xiii
xiv
Contributors
Contributors
xv
xvi
Contributors
KEERTI S. RATHORE Institute for Plant Genomics and Biotechnology, Texas A&M University,
College Station, TX, USA; Department of Soil and Crop Sciences, Texas A&M University,
College Station, TX, USA
SILVIA SABBADINI Scienze Agrarie, Alimentari ed Ambientali D3A, Universit Politecnica
delle Marche, Ancona, Italy
LINGARAJ SAHOO Center for Energy and Department of Biotechnology, Indian Institute
of Technology Guwahati, Guwahati, India
MANISH SAINGER Centre for Biotechnology, M. D. University, Rohtak, India
RONALD R. SEDEROFF Forest Biotechnology Group, Department of Forestry, North Carolina
State University, Raleigh, NC, USA
ALKA SHANKAR Citrus Research and Education Center, University of Florida/IFAS,
Lake Alfred, FL, USA
SHANNA SHERWOOD Institute for Plant Genomics and Biotechnology, Texas A&M
University, College Station, TX, USA
GUO-QING SONG Department of Horticulture, Plant Biotechnology Resource
and Outreach Center, Michigan State University, East Lansing, MI, USA
JINGYUAN SONG Institute of Medicinal Plant Development, Chinese Academy of Medical
Sciences and Peking Union Medical College, Beijing, China
MARK R. THOMAS Horticulture Unit, CSIRO Plant Industry, Glen Osmond, SA, Australia
TAGE THORSTENSEN Bioforsk- Norwegian Institute for Agricultural and Environmental
Research, s, Norway
LAURENT TORREGROSA Montpellier SupAgro-INRA, UMR AGAPGenetic Improvement
and Adaptation of Mediterranean and Tropical Plants, Montpellier Cedex, France
SANDRA L. URATSU Plant Science Department, University of California, Davis, CA, USA
SILVIA VALLADARES Instituto de Investigaciones Agrobiolgicas de Galicia, IIAG, CSIC,
Santiago de Compostela, Spain
SANDRINE VIALET Unit Exprimentale de Pech-Rouge, INRA, UEPR, Gruissan, France
ANA M. VIEITEZ Instituto de Investigaciones Agrobiolgicas de Galicia, IIAG, CSIC,
Santiago de Compostela, Spain
SRIEMA L. WALAWAGE Plant Science Department, University of California, Davis,
CA, USA
OWEN S.D. WALLY Department of Plant Sciences, University of Manitoba, Winnipeg,
MB, Canada
ZENG-YU WANG Forage Improvement Division, The Samuel Roberts Noble Foundation,
Ardmore, OK, USA
LOGAN R. WILL The American Chestnut Research and Restoration Project,
SUNY College of Environmental Science and Forestry, Syracuse, NY, USA
HAO WU Agronomy Department, Plant Molecular and Cellular Biology Program,
Genetics Institute, University of Florida, Gainesville, FL, USA
MANJU YADAV Centre for Biotechnology, M. D. University, Rohtak, India
CHENMIN YANG Forest Biotechnology Group, Department of Forestry, North Carolina
State University, Raleigh, NC, USA
TING-FENG YEH School of Forestry and Resource Conservation, National Taiwan
University, Taipei, Taiwan
JANICE ZALE Citrus Research and Education Center, University of Florida/IFAS,
Lake Alfred, FL, USA
BO ZHANG Therapeutic Proteins International, LLC, Chicago, IL, USA
YUN J. ZHU Hawaii Agriculture Research Center, Kunia, HI, USA
Part I
Industrial Plants
Chapter 1
Brassica rapa
Tom Lawrenson, Cassandra Goldsack, Lars Ostergaard,
and Penny A.C. Hundleby ne Sparrow
Abstract
Within this chapter we outline an A. tumefaciens-mediated transformation method for B. rapa using
4-day-old cotyledonary explants and the genotype R-o-18. Transformation efficiencies are typically
achieved in the region of 1 % (based on 2 PCR-positive independent shoots from 200 inoculated explants).
This system has been developed to work with gentamicin selection.
Key words Agrobacterium tumefaciens, Brassica rapa, Diploid, Gentamicin selection, Oilseed,
Transformation
Introduction
Brassica rapa has historically been the most recalcitrant of Brassica
species to transform, with published work focusing on Chiffu, the
genotype of the sequencing program, and other B. rapa pekinensis
genotypes (Chinese cabbage). However, one significant downside
to these genotypes is that they are vegetable types and also highly
self-incompatible. As a transformation resource, the economic cost
of generating enough seed for routine transformation, as well as
the time to hand-pollinate transgenic lines to obtain nextgeneration material, makes these undesirable candidates for routine transformation studies. The B. rapa variety R-o-18 was chosen
as the target genotype for studies in our laboratories as its plant
architecture is similar to B. napus oilseed rape. R-o-18 is derived
from a B. rapa oilseed crop grown in Pakistan and India and is
therefore already a crop in its own right. Moreover, the genotype
is rapid cycling and self-compatible, enabling the production of
large seed stocks to use in transformation studies, as well as generating next-generation transgenic material cost-effectively without
the need for laborious hand pollination. This genotype is also used
as a model B. rapa genotype by a number of research labs, in particular complementing the R-o-18 TILLING resource available via
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_1, Springer Science+Business Media New York 2015
Materials
2.1 Plant
Culture Media
and Components
Brassica rapa
1. LB medium: 5 g/L yeast extract, 10 g/L NaCl, 10 g/L peptone 15 g/L Bacto agar (for solid/liquid medium); autoclave at 120 C for 20 min.
2. 20 AB salts: To prepare 1 L, add 20 g NH4Cl, 6 g
MgSO4 7H2O, 3 g KCl, 0.26 g CaCl2 H2O, and 0.05 g
FeSO4 7H2O in SDW. Store at 20 oC in 50 mL aliquots.
3. Induction media: To prepare 200 mL, add 10 mL 20 AB
salts, 4 g glucose, 1.18 g MES, 0.06 g NaH2PO4, and 0.06 g
Na2HPO4 in SDW. Adjust pH to 5.6 with 1 M NaOH. Filter
sterilize and store at 4 C for up to 1 month.
4. Acetosyringone (20 mg/mL in dimethyl sulfoxide). Aliquot
into 20 L volumes and store at 20 C.
2.3
Seed Source
Methods
The protocol described below is applicable to B. rapa genotype
R-o-18. Transformation is based on a previously reported method
for B. napus [5].
3.1 Seed
Sterilization
and Germination
3.2 Agrobacterium
Preparation
Brassica rapa
3.4
Selection
1. After 72 h explants are moved to CIM-S plates with the exception of the two control plates (one inoculated and the other
not exposed to Agrobacterium) which are moved to fresh
CIM-C plates.
2. Plates are returned to the same growth conditions for a further
48 h, before being transferred to shoot induction medium
(with the two controls moved to shoot induction with no
selection). At this point the shallow lid of the tissue culture
dish is replaced with a deeper base of a fresh dish, and the two
are joined with Micropore tape (see Note 4).
3. On day 11 (after explant isolation), explants are moved to shoot
elongation selection media, retaining the base-lid format.
Explants are moved to fresh shoot elongation selection media
Fig. 1 Stages in Agrobacterium-mediated Brassica rapa transformation. Four-day-old R-o-18 seedlings ready
for explant isolation, showing (a) excision line, (b) scalpel about to make cut, and (c) explant with correct petiole length. Explant cultures 14 days after isolation/inoculation seen from the basal side of plate showing (d)
control explants; no Agrobacterium inoculation and no gentamicin selection, (e) control explants; inoculated
but maintained in the absence of selection and (f) inoculated explants maintained on gentamicin selection.
Explant cultures 3 weeks after isolation/inoculation seen from the top side of plate showing (g) proliferation of
shoots in control explants where no inoculation or gentamicin selection was used; (h) proliferation of shoots in
control explants inoculated but where no gentamicin selection has been applied; and (i) inoculated explants on
gentamicin selection with one putative transgenic shoot (circled); (j) an enlarged view of the marked shoot in
(i). Shoots isolated and moved to rooting media at approximately 4 weeks postinoculation (k) and root development after a further 2 weeks (l)
Brassica rapa
Shoot Isolation
1. When using R-o-18, the emergence of green shoots on control plates (no selection) occurs between 14 and 21 days after
explant isolation (Fig. 1g, h), with 90100 % of explants producing shoots. Transgenic (green) shoots can be seen from 3
weeks onward on selection plates (Fig. 1i, j). From 3 weeks
transgenic shoots can be isolated to rooting media (Fig. 1k).
The period when most transgenic shoots are likely to appear is
between 21 days and 1 month after inoculation.
2. Putative transgenic (green) shoots are excised and transferred
to 100 mL jars containing 25 mL of rooting medium using the
base of 50 mm petri dishes (Sterilin 124) as lids and maintained
at 23 C under 16-h day length of 40 mol/m2/s (see Note 5).
3. After root elongation (Fig. 1l) (to approximately 20 mm in
length), plantlets are transferred to sterile peat pots to allow
further root growth (approx. 2 weeks) before being transferred to the glasshouse (see Note 6).
3.6 Transfer of
Plants to Greenhouse
10
Notes
1. Typically, 1.2 g of seed is enough for a 300 explant transformation. Seed should be placed onto the surface of the medium
and not embedded.
2. AGL1 is the Agrobacterium tumefaciens strain routinely used
in our lab.
3. We used a plant selection cassette consisting of a double 35S
promoter driving the aacC1 coding region with a CaMV terminator to provide gentamicin resistance.
4. Using large petri dishes (20 90 mm) and the deeper base as a
lid gives a larger vessel volume which helps prevent the accumulation of ethylene and water vapor. Sealing with Micropore
tape also helps with better gas exchange. We have found that
these changes result in a less stressful environment, leading to
healthier shoots.
5. It may be possible to omit the inclusion of gentamicin at the
root induction stage, as the number of escapes making it
through to this stage will be few; however, we recommend
that you confirm transgene presence by PCR.
6. R-o-18 plants are often on the verge of flowering during the
rooting period in peat pots. To reduce the risk of flowering,
the time spent in vitro once roots are established and moving
to the greenhouse should be kept as short as possible. Once
established in greenhouse pots, early flowers may fail to set,
but plants should gather vigor to produce fruit.
Acknowledgments
The authors acknowledge the support of the Biotechnology and
Biological Science Research Council (BBSRC) Strategic Tools and
Resources Grant BB/I023763/1 Development of an efficient B.
rapa transformation system to facilitate studies on fruit development in a diploid Brassica oilseed crop and further support by
BBSRC Strategic Programme Grant B/J004588/1 (GRO) and
the John Innes Foundation.
References
1. Murashige T, Skoog F (1962) A revised medium
for rapid growth and bioassays and tobacco tissue
culture. Physiol Plant 15:437497
2. Gamborg OL, Miller RB, Ojima K (1968)
Nutrient requirements of suspension cultures of
soybean root cells. Exp Cell Res 50:151158
3. Rusholme RL, Higgins EE, Walsh JA, Lydiate
DJ (2007) Genetic control of broad-spectrum
resistance to turnip mosaic virus in Brassica rapa
(Chinese cabbage). J Gen Virol 88:31773186
Chapter 2
Cotton (Gossypium hirsutum L.)
Keerti S. Rathore, LeAnne M. Campbell, Shanna Sherwood,
and Eugenia Nunes
Abstract
Cotton continues to be a crop of great economic importance in many developing and some developed
countries. Cotton plants expressing the Bt gene to deter some of the major pests have been enthusiastically
and widely accepted by the farmers in three of the major producing countries, i.e., China, India, and the
USA. Considering the constraints related to its production and the wide variety of products derived from
the cotton plant, it offers several target traits that can be improved through genetic engineering. Thus,
there is a great need to accelerate the application of biotechnological tools for cotton improvement. This
requires a simple, yet robust gene delivery/transformant recovery system. Recently, a protocol, involving
large-scale, mechanical isolation of embryonic axes from germinating cottonseeds followed by direct transformation of the meristematic cells has been developed by an industrial laboratory. However, complexity
of the mechanical device and the patent restrictions are likely to keep this method out of reach of most
academic laboratories. In this chapter, we describe the method developed in our laboratory that has undergone further refinements and involves Agrobacterium-mediated transformation of cotton cells, selection of
stable transgenic callus lines, and recovery of plants via somatic embryogenesis.
Key words Agrobacterium, Regeneration, Somatic embryogenesis, Transformation, Transgenic
cotton
Introduction
Cotton, the most important source of natural fiber worldwide, is
grown in more than 80 countries across five continents. Compared
to the synthetic fibers, cotton provides some major environmental/societal benefits. Firstly, unlike the petroleum-based synthetics,
it is a renewable resource. Secondly, its cultivation, processing, and
use in textile manufacturing provide for the livelihoods of a much
higher number of people compared to the synthetic fibers.
Of course, the superiority of cotton clothing in terms of its comfort level cannot be matched by the synthetic versions. With an
unrelenting growth in global population and the resulting demand
for food and feed, cottonseed is also becoming an increasingly
precious commodity. A major by-product, it is used as cattle feed,
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_2, Springer Science+Business Media New York 2015
11
12
13
culture passages and reducing the length of time for the recovery
of transformants and, therefore, somaclonal variations. Another
major advantage of this system is that regeneration from shoot apices is genotype independent.
Various systems for obtaining transgenic cotton were thoroughly evaluated in our laboratory [7, 19] using a reporter gene
encoding the green fluorescent protein (GFP) [20]. GFP expression provided an excellent tool to measure the efficiency of T-DNA
transfer to cotton cells and was also useful in revealing the timing
and localization of transient transgene expression [7]. Cells at the
cut edge of the cotyledon segments proved to be the most susceptible to Agrobacterium-mediated transformation as indicated by
the transient GFP activity. This was followed by conversion of
some of these transiently transformed cells to stable transformation
events. Fewer cells at the cut surface of the hypocotyl showed transient GFP activity as compared to the cotyledonary tissue. The cells
that showed GFP expression were seen as a ring of fluorescent cells
in the middle of the cut surface that appeared to be part of the
vascular tissue; however, their true identity was masked by the
hypersensitive response displayed by cells around them. Despite
the lower rate of transient transformation, hypocotyl segments
gave rise to several stable transgenic events that grew as small fluorescent clusters at the cut surface of hypocotyls during the selection on kanamycin-supplemented medium over 34 weeks. Thus,
although hypocotyl segments showed a low level of transient activity as compared to the cotyledons, these explants were still capable
of producing several stable transgenic events [7, 19]. Cotyledonary
petiole segments are also as efficient as the hypocotyl segments in
terms of producing stable transformation events. Thus, the use of
GFP as the reporter gene in combination with neomycin phosphotransferase II (npt II) gene as a selectable marker showed that
cotyledons, hypocotyls, and cotyledonary petioles are highly
competent explants for Agrobacterium-mediated transformation
[7, 19]. A single experiment involving 50 donor seedlings can yield
several hundred independent transgenic events in the form of
kanamycin-resistant calli. Although most of the experiments with
GFP were conducted with cv. Coker 312, we have shown previously that cotyledon segments of several Texas cultivars are also
competent for Agrobacterium-mediated T-DNA transfer and are
capable of yielding stable transgenic callus [7].
As mentioned earlier, there are a few studies that have reported
transformation of cells within the shoot apical meristem via the
Agrobacterium method [1518]. However, these investigations
relied heavily on the ability of explants to survive selection pressure
as a measure of their transgenic status and/or did not provide convincing molecular and genetics data to support their claims. In our
laboratory, the competence of shoot apices for Agrobacteriummediated transformation was evaluated by cocultivating these with
14
Table 1
Screening of five different genotypes of G. hirsutum for their embryogenic response using the tissue
culture protocol described in this chapter (# of lines undergoing somatic embryogenesis 8 months
following culture initiation/# of cultured lines)
Coker 312
TM-1
Tamcot 22
Tamcot 73
DP50
Hypocotyl
67/86
1/16
0/7
0/7
0/5
Petiole
45/52
0/14
0/3
0/6
0/8
Cotyledon
5/6
0/9
0/6
0/11
0/14
Root
37/45
0/8
0/6
0/2
0/4
Combined total
154/189
1/47
0/22
0/26
0/31
15
Materials
All media are autoclaved at 121 C for 20 min after adjusting the
pH and after the addition of the gelling agent. Autoclaved media
(with the gelling agent) should be cooled to <60 C before adding
filter-sterilized antibiotics or acetosyringone.
2.1 Agrobacterium
Media
16
g
g
to
to
17
Methods
3.1 Preparation
of Agrobacterium
Inoculant
3.3
Transformation
1. Cut 3- to 4-mm-long segments from either hypocotyl or cotyledonary petiole. Place 1012 segments horizontally on sterile
filter paper over P1-AS medium in a Petri dish (100 15 mm).
2. Apply 5 L of acetosyringone-induced Agrobacterium suspension to each cut surface. Keep the plates under light (70 mol/
m2/s, 16-h photoperiod) at 25 C for 3 days for cocultivation
(see Note 4).
3. Transfer hypocotyl/petiole pieces to the P1-c4k50 medium.
Keep the plates under light (70 mol/m2/s, 16-h photoperiod) at 28 C for 34 weeks without subculture (see Note 5).
18
3.4 Selection/
Proliferation
3.5 Regeneration
(Somatic
Embryogenesis),
Embryo Germination,
and Plantlet
Development
3.6
Transfer to Soil
19
Notes
1. Agrobacterium tumefaciens strain LBA4404 provides higher
transformation efficiencies in cotton as compared to the strain
EHA105 [7].
2. Alternatively, it is possible to simply grow Agrobacterium on
YEP plates under appropriate antibiotic selection, harvest the
cells with a loop, and resuspend in PIM + AS before cocultivation. This simpler procedure eliminates the need for a shaker.
3. We have also used acid-delinted seeds following a modified
sterilization protocol. Seeds, soaked for 3 h under running tap
water, are treated with 70 % ethanol for 1 min and washed
twice with sterile DW. This is followed by sterilization with
20 % commercial bleach (+two drops of Tween 20) under
vacuum for 5 min and then 15 min without vacuum. After
rinsing three times, the seeds are germinated on MSO medium
in jars at 25 C, under fluorescent light (70 mol/m2/s, 16-h
photoperiod) for 10 days. For uniform germination, the seeds
should be soaked overnight, and the seed coat should be
removed before placing the kernel in the jar.
4. We use Parafilm to wrap the Petri dishes. Nescofilm and
Millipore tape were also tested as substitutes but offered no
advantage over Parafilm.
5. Although the transformation/regeneration protocol described
is for hypocotyl and cotyledonary petiole segments, it can be
used also for cotyledon segments. Individual transgenic events
arising at the edges of the cotyledon segments can be used to
obtain transgenic plants by following the method described in
this chapter.
6. Often, more than one transgenic events are observed growing
at the cut surface of the hypocotyl or the petiole segments.
The longer these are permitted to grow on the explant, the
greater the likelihood that two or more transgenic events will
converge with each other. In this situation, the possibility
remains that the two plants obtained from the same excised
callus tissue may have arisen from separate transgenic events.
As is the case with the transformation efficiency of any species,
experiment-to-experiment variability is also observed with
cotton. This necessitates performing several small-scale experiments for a given construct rather than a few large-scale ones.
7. If the excised calli are smaller than 3 mm, culture these for 7
days on P1-c4k50 plates at 28 C under a light intensity of
~10 mol/m2/s (16-h photoperiod) prior to transferring
them to P7-c4k50 plates. The additional week of culture on
P1-c4k50 medium improves the survival of small-sized transgenic events; however, this may delay embryogenesis.
20
Table 2
Effect of two types of lighting conditions on the efficiency of conversion of
somatic embryos to healthy plantlets (# of somatic embryos transferred
from MSEm medium to EG3 medium/# of healthy plantlets obtained)
Experiment no.
Fluorescent lighting
Fluorescent +
incandescent lighting
5/124 (4.0 %)
24/148 (16.2 %)
5/124 (4.0 %)
8/124 (6.4 %)
3/250 (1.2 %)
22/250 (8.8 %)
6/113 (5.3 %)
22/250 (8.8 %)
21
22
Acknowledgments
The authors wish to thank Ms. Lauren Tollack for excellent technical assistance. Experimental work in our laboratory has been supported by Cotton Inc., Texas Cotton Biotechnology Initiative
(TxCOT), and Texas AgriLife Research.
References
1. Perlak FJ, Deaton RW, Armstrong TA et al
(1990) Insect resistant cotton plants.
BioTechnology 8:939943
2. Perlak FJ, Oppenhuizen M, Gustafson K et al
(2001) Development and commercial use of
Bollgard cotton in the USA early promises
versus todays reality. Plant J 27:489501
3. Johnson SR, Strom S, Grillo K (2007)
Quantification of the impacts on US agriculture of biotechnology-derived crops planted in
2006. National Center for Food and
Agricultural Policy. www.ncfap.org. http://
www.ncfap.org/documents/2007biotech_
report/Quantification_of_the_Impacts_on_
US_Agriculture_of_Biotechnology.pdf .
Accessed 15 Sept 2013
4. Brookes G, Barfoot P (2012) GM crops: global
socio-economic and environmental impacts
19962010. PG Economics Ltd., UK. https://
www.landesbioscience.com/journals/gmcrops/
article/20061/. Accessed 15 Sept 2013
5. Umbeck P, Johnson G, Barton K et al (1987)
Genetically transformed cotton (Gossypium hirsutum L.) plants. BioTechnology 5:263266
6. Firoozabady E, DeBoer DL, Merlo DJ et al
(1987) Transformation of cotton (Gossypium
hirsutum L.) by Agrobacterium tumefaciens
and regeneration of transgenic plants. Plant
Mol Biol 10:105116
7. Sunilkumar G, Rathore KS (2001) Transgenic
cotton: factors influencing Agrobacteriummediated transformation and regeneration.
Mol Breeding 8:3752
8. Finer JJ, McMullen MD (1990) Transformation
of cotton (Gossypium hirsutum L.) via particle
bombardment. Plant Cell Rep 8:586589
9. Rajasekaran K, Hudspeth RL, Cary JW et al
(2000) High-frequency stable transformation
of cotton (Gossypium hirsutum L.) by particle
bombardment of embryogenic cell suspension
cultures. Plant Cell Rep 19:539545
10. Cousins YL, Lyon BR, Llewellyn DJ (1991)
Transformation of an Australian cotton cultivar: prospects for cotton improvement through
genetic engineering. Aust J Plant Physiol 18:
481494
22.
23.
24.
25.
26.
27.
28.
29.
23
30. Firoozabady E, DeBoer DL (1993) Plant regeneration via somatic embryogenesis in many cultivars of cotton (Gossypium hirsutum L.). In Vitro
Cell Dev Biol 29P:166173
31. Rajasekaran K, Grula JW, Hudspeth RL et al
(1996) Herbicide-resistant Acala and Coker cottons transformed with a native gene encoding
mutant forms of acetohydroxyacid synthase. Mol
Breeding 2:307319
32. Trolinder NL, Xhixian C (1989) Genotype
specificity of the somatic embryogenesis
response in cotton. Plant Cell Rep 8:133136
33. Sakhanokho H, Zipf A, Rajasekaran K et al
(2001) Induction of highly embryogenic calli
and plant regeneration in upland (Gossypium
hirsutum L.) and Pima (Gossypium barbadense
L.) cottons. Crop Sci 41:12351240
34. Sakhanokho H, Zipf A, Rajasekaran K et al
(2004) Somatic embryo initiation and germination in diploid cotton (Gossypium arboreum
L.). In Vitro Cell Dev Biol 40P:177181
35. Han GY, Wang XF, Zhang GY et al (2009)
Somatic embryogenesis and plant regeneration
of recalcitrant cottons (Gossypium hirsutum).
Afr J Biotechnol 8:432437
36. Murashige T, Skoog F (1962) A revised medium
for rapid growth and bioassays with tobacco
tissue cultures. Physiol Plant 15:473497
37. Chen Y, Rivlin A, Lange A et al (2014) High
throughput Agrobacterium tumefaciens-mediated
germline transformation of mechanically isolated
meristem explants of cotton (Gossypium hirsutum
L.). Plant Cell Rep 33:153164
Chapter 3
Jatropha (Jatropha curcas L.)
Devendra Kumar Maravi, Purabi Mazumdar, Shamsher Alam,
Vaibhav V. Goud, and Lingaraj Sahoo
Abstract
The seed oil of Jatropha (Jatropha curcas L.) as a source of biodiesel fuel is gaining worldwide importance.
Commercial-scale exploration of Jatropha has not succeeded due to low and unstable seed yield in semiarid
lands unsuitable for the food production and infestation to diseases. Genetic engineering is promising to
improve various agronomic traits in Jatropha and to understand the molecular functions of key Jatropha
genes for molecular breeding. We describe a protocol routinely followed in our laboratory for stable and
efficient Agrobacterium tumefaciens-mediated transformation of Jatropha using cotyledonary leaf as
explants. The 4-day-old explants are infected with Agrobacterium tumefaciens strain EHA105 harboring
pBI121 plant binary vector, which contains nptII as plant selectable marker and gus as reporter. The putative transformed plants are selected on kanamycin, and stable integration of transgene(s) is confirmed by
histochemical GUS assay, polymerase chain reaction, and Southern hybridization.
Key words Agrobacterium tumefaciens, Cotyledonary leaf, Jatropha curcas, Kanamycin resistance,
Plant binary vector, Stable transformation
Introduction
Jatropha (Jatropha curcas L.) is an important nonedible oilseed
crop receiving worldwide attention as a biodiesel feedstock. The
seeds contain 4050 % of oil, which can be blended directly with
petro-diesel or transesterified for use as biodiesel. The short gestation period, drought endurance, low cost of seeds, high oil content, and easy adaptation on degraded soil unsuitable for food
crops make Jatropha as the most sought oil seed crop among the
nonedible oil-yielding crops for biodiesel production [1, 2]. The
huge demand of Jatropha seeds is expected to be met through its
large-scale cultivation and increasing its productivity. However,
several undesirable traits of Jatropha such as low and unstable
seed yield, dependent on the growth conditions and environment,
limit its commercial use [3]. Furthermore, large-scale cultivation of Jatropha through monocrop plantation especially under
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_3, Springer Science+Business Media New York 2015
25
26
Materials
Plant Materials
2.2 Agrobacterium
tumefaciens Strain
and Vector
2.1
27
Day
Day
Day
Day
Day
Day 6
Day
Day
2.3
Stock Solutions
28
2.3.2 Antibiotics
1. Kanamycin sulfate: Prepare the stock of 100 mg/ml by dissolving 500 mg of kanamycin powder in 5 ml of sterile dH2O,
filter sterilize, and store at 20 C (see Note 4).
2. Rifampicin: Prepare the stock of 10 mg/ml by dissolving
10 mg of rifampicin in few drops of dimethyl sulfoxide
(DMSO) and then make up the volume by adding sterile
dH2O, filter sterilize, and store at 20 C.
3. Meropenem: Prepare the stock of 50 mg/ml by dissolving 1 g
of meropenem salt in 20-ml sterile distilled water, filter sterilize, and store at 4 C.
2.3.4 Culture
Medium Stock
29
30
Methods
3.1 Preparation
of Explant for
Inoculation
1. Remove the seed coat and soak in distilled water for overnight
at room temperature.
2. Surface-sterilize the de-coated seeds with 0.1 % sodium hypochlorite solution supplemented with few drops of Tween 20
for 15 min and rinse with distilled water several times.
3. Surface-sterilize the seeds with 70 % ethanol for 5 min and
rinse three times with sterilized distilled water.
4. Finally, sterilize with 0.2 % mercuric chloride for 2 min and
rinse with sterile distilled water 34 times under the laminar
air flow cabinet.
5. Blot dry the seeds using sterile filter paper.
6. Carefully dissect the endosperm to expose out embryos with
papery cotyledonary leaves using forceps and scalpel.
7. Separate out the papery cotyledonary leaves and germinate on
MS basal medium.
8. Cut the papery cotyledonary leaves into four segments (8 mm)
with their edges removed and use as explant for Agrobacteriummediated transformation.
3.2 Agrobacterium
Culture for Infection
3.3 Infection,
Cocultivation,
and Regeneration
31
32
3.5 GUS
Histochemical Assay
1. Take 100-mg leaf tissue, wash with sterile distilled water, and
blot dry with tissue paper to remove water.
2. Place the tissue in a prechilled mortar and pestle and homogenate to powder with liquid nitrogen; transfer the powder to
sterile microcentrifuge tube.
3. Add 700 l of extraction buffer (preheated at 60 C for
15 min) to homogenate, mix gently to avoid shearing of DNA,
and incubate in water bath at 65 C for 45 min.
4. Bring down the sample temperature to room temperature,
add 700-l chloroform-isoamyl alcohol (24:1), and mix gently
by inverting for a period of 5 min.
5. Spin at 7,155 g for 10 min at 25 C; transfer the supernatant
to a fresh microcentrifuge tube.
6. Add 150 l of 5 M NaCl and mix properly.
7. Add 0.6 volume of cold isopropanol. Mix gently and allow the
mixture to stand at 4 C for 45 min (see Note 19).
8. After 60 min, spin the samples at 11,180 g for 10 min at
25 C, discard the supernatant, and wash the pellet with 80 %
ethanol.
9. Dry the pellet in vacuum for 15 min and dissolve in 200 l of
high-salt TE buffer.
10. Add 5 l of RNase and incubate at 37 C for 45 min.
11. Extract with equal volume of chloroform-isoamyl alcohol
(24:1), mix gently, and spin at 10,000 rpm for 10 min.
12. Transfer the aqueous layer to a fresh 1.5-ml centrifuge tube,
and add 2 volume of cold ethanol; keep in 20 C for 1 h.
13. Spin at 10,000 rpm at 4 C for 10 min, perform ethanol (80 %)
wash, and spin for 10 min at 10,000 rpm.
14. Dry the pellet and suspend in sterile double distilled water
(see Note 20).
15. Measure the DNA concentration by running the sample on
0.8 % agarose gel or taking the absorbance at 260 nm
(see Note 21).
16. PCR reaction mixture: in an Eppendorf tube, add 2.5-l 10
Taq buffer, 0.5-l 10-mM dNTPs, 1-l forward primer
33
(50 pmol/l), 1-l reverse primer (50 pmol/l) for nptII and
gus gene, respectively (see Note 22), 0.5-l Taq polymerase
(5 U/l), ~100-ng DNA template, then make up the final
volume up to 25 l.
17. PCR condition: 95 C for 5 min (1 cycle), 95 C for 1 min
(denaturation), 58 C for 1 min (annealing), 72 C for 1 min
(extension) for 35 cycles, followed by the final extension at
72 C for 5 min (1 cycle).
18. Resolve the PCR-amplified product by electrophoresis on 1 %
agarose gel and visualize with ethidium bromide staining
under UV transilluminator and document in gel documentation system (Bio-Rad Laboratories).
Notes
1. The seeds are collected from the shade house-grown Jatropha
curcas of Indian Institute of Technology Guwahati.
2. Selectable marker neomycin phosphotransferase encoding
gene II (nptII) confers kanamycin resistance to transformed
plant cells.
3. Antibiotic stocks are aliquots in 1.5-ml sterile Eppendorf tubes
and stored at 20 C.
4. All filter sterilization is carried out with 0.22-mm syringe
filter.
5. Mercuric chloride (HgCl2) is a highly effective surface sterilant
and extremely toxic and must be disposed off according to safety
regulations in the laboratory. It may be necessary to use a specially designated sink for toxic chemicals for the washing step.
6. Prepare KH2PO4 and NaH2PO4 2H2O separately and then
mix and bring the final volume up to 100 ml with distilled
water.
7. AB salt solution may show yellow precipitates after autoclaving
which is dissolved by shaking vigorously just before use.
8. Increased concentration of acetosyringone up to 100 M
enhances the transient transformation efficiency and decreases
with further increases in concentration.
9. The 15-mg/l kanamycin is the optimal concentration to select
transformed explants, and 25-mg/l meropenem is used to
eliminate the growth of Agrobacterium.
10. Agrobacterium culture lose their viability when kept in
LB + Rif + Kan plate at 4 C for more than 3 weeks; therefore,
subculture the plate in regular intervals.
11. A 3-day cocultivation period is optimum for transient transformation experiment. Cocultivation period longer than 3-day
34
13.
14.
15.
16.
17.
18.
35
Chapter 4
Sesame (Sesamum indicum L.)
Sonia Kapoor, Sanjay S. Parmar, Manju Yadav, Darshna Chaudhary,
Manish Sainger, Ranjana Jaiwal, and Pawan K. Jaiwal
Abstract
Sesame (Sesamum indicum L.) is an important oilseed crop grown in India, China, Korea, Russia, Turkey,
Mexico, South America, and several countries of Africa. Sesame seeds are rich in oil, proteins, unsaturated
fatty acids, vitamins, minerals, and folic acid. Nearly 70 % of the worlds sesame is processed into oil and
meal, while the remainder is channeled to food and confectionery industries. Production of sesame is limited by several fungal diseases, water logging, salinity, and shattering of seed capsules during harvest.
Introgression of useful genes from wild species into cultigens by conventional breeding has not been successful due to postfertilization barriers. The only alternative for the improvement of S. indicum is to transfer genes from other sources through genetic transformation techniques. Here, we describe a simple, fast,
and reproducible method for the Agrobacterium-mediated genetic transformation of S. indicum which
may be employed for the transfer of desirable traits into this economically important oilseed crop.
Key words Agrobacterium tumefaciens, Genetic transformation, nptII, Sesame, Sesamum indicum, uidA
Introduction
Sesame (Sesamum indicum L.), an oilseed crop of the family
Pedaliaceae, is one of the oldest cultivated crops of the world with
total production of 3.3 million tons (FAOSTAT data 2008). Sesame
seeds are rich in oil (5060 %), protein (25 %), unsaturated fatty
acids, vitamins, minerals, and folic acid. They are used in baking,
candy making, health-care products, and biomedicine. Nearly 70 %
of sesame seeds are processed into oil [1]. Sesame oil has numerous
health benefits as it contains potent natural antioxidants such as sesamolin, sesamin, and sesamol [2] and is also a source of linoleic acid.
It has low levels of saturated fatty acids (15 %) and recently found to
be beneficial in lowering cholesterol levels and hypertension [3, 4]
and in reducing incidence of certain cancers [5, 6]. Production of
sesame is severely affected by biotic as well as abiotic constraints
which mainly include fungal diseases, photosensitivity, and early
senescence resulting in losses ranging from 10 to 90 % of the yield.
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_4, Springer Science+Business Media New York 2015
37
38
2
2.1
Materials
Tissue Culture
39
40
Methods
3.1 Explant
Preparation and
Culture Conditions
3.2 Agrobacterium
Inoculum Preparation
and Cocultivation
41
Fig. 1 In vitro regeneration of multiple shoots and Agrobacterium-mediated transformation of Sesamum indicum cv. HT-1. (a) Cotyledon explants excised from 2-day-old seedlings. (b) Direct shoot regeneration from
cotyledon explants on MS medium supplemented with 25.0 M BA after 4 weeks of culture. (c) Induction of
roots from in vitro regenerated shoot cultured on MS basal medium supplemented with 2.0 M IBA. (d) In vitro
regeneration of shoots from non-transformed control explants cultured on MS + 25.0 M BA medium without
kanamycin (i), non-transformed control explants cultured on MS + 25.0 M BA medium containing 25.0 mg/L
kanamycin showing regeneration of completely bleached shoots (ii), and explants inoculated with Agrobacterium
tumefaciens strain EHA105 (pCAMBIA2301) and cultured on MS + 25.0 M BA medium containing 25.0 mg/L
kanamycin showing regeneration of green shoots (iii). (e) Fertile transgenic plant growing in pot. Scale bars:
1 mm (a), 2.5 mm (b), 7.5 mm (c), 5 mm (d), 2 mm (e) (Reproduced from Yadav et al. 2010 [16], with permission from Springer)
4. Blot the inoculated explants on sterile filter paper and coculture in Petri dish lined with sterile filter paper moistened with
liquid cocultivation medium (CM) for 3 days under a 16-h
photoperiod with light intensity of 80 mol/m2/s at 25 2 C
(see Notes 1 and 2).
3.3 Shoot
Regeneration
and Selection
42
Rooting
3.5 Hardening
and Acclimatization
of the Plants
3.6 Screening
of Transgenics
Independent transgenic lines transferred to the greenhouse are subjected to molecular analysis to check the presence, integration, and
expression of transgenes (see Notes 6 and 7). Transient and stable
histochemical GUS analysis can be performed on various tissues.
1. Transient GUS activity is determined immediately after cocultivation. Explants thoroughly washed with sterile distilled
water are analyzed.
2. Stable expression of the uidA gene is determined by performing GUS assay for all plants in all generations. Various tissues
like leaves, stem, roots, and pollen of putative plants established in soil can be used for assay.
3. Immerse the tissues in freshly prepared GUS assay solution
and incubate overnight at 37 C (see Note 5).
43
Fig. 2 Transient and stable GUS activities in various tissues of transgenic plants. Transient GUS assay in
(a) non-transformed (control) explants not showing GUS activity and (b) cotyledon explants showing GUS activity after 2 days of cocultivation with Agrobacterium tumefaciens EHA105 (pCAMBIA2301). Stable GUS assay in
roots from non-transformed (c) and transformed (d) plants; shoots from non-transformed (e) and transformed
(f) plant; anthers from non-transformed (g) and transformed (h) plant; pollen grains from non-transformed
(i) and transformed (j) plant; and germinating seeds from non-transformed (k) and transformed (l) plant.
Transgenic plants were recovered and rooted on kanamycin-containing medium and germinated T1 seeds
(Reproduced from Yadav et al. 2010 [16], with permission from Springer)
44
Notes
1. Inoculation and cocultivation steps are crucial for the success
of any transformation experiment. Bacterial concentration at
106 cells/mL, bacterial inoculation time for 20 min, and
cocultivation for 2 days in the presence of BAP 25.0 M and
acetosyringone 20.0 M at pH 5.5 under light conditions
were effective in improving transformation efficiency.
2. Inclusion of a combination of thiol compounds, L-cysteine
(400 mg/L), and DTT (1.0 mM) in the cocultivation medium
completely inhibited the browning and necrosis of explants. It
increased the percentage of explants showing intense transient
GUS activity and also improved the survival of explants on the
selection medium.
3. Kanamycin at a concentration of 5 mg/L completely inhibited
rooting in non-transformed control plants indicating that root
induction is more sensitive to kanamycin than shoot organogenesis which is inhibited at 25 mg/L.
4. At the onset of pod maturation, the pods should be covered
with polythene bags to prevent seed loss due to pod
shattering.
5. GUS assay solution should be stored at 20 C in small aliquots of 1 mL to avoid repeated freezing and thawing. Before
use, thaw at room temperature since the components degrade
in heat immediately.
6. To check presence and integration of transgenes, extract total
genomic DNA from young leaves of putative transgenic and
non-transformed (control) plants by GenElute Plant Genomic
DNA Miniprep Kit (Sigma). Perform PCR for presence of
nptII and uidA genes using specific primers. To analyze the
putative transformants by Southern blotting, digest the
genomic DNA with restriction enzyme that has a single recognition site in the plasmid, resolve on 0.8 % agarose gel, transfer
onto nylon membrane (Hybond N+, Amersham) using standard protocol, and probe the blot with labeled (radioactive or
nonradioactive) PCR-amplified fragment of the nptII gene.
7. Analyze the progeny of self-pollinated transformants for the
presence of uidA gene by PCR and RT-PCR. For RT-PCR,
isolate total RNA from leaves of T1 transformed (positive for
uidA gene) and non-transformed control plants using RNeasy
Plant Mini Kit (Qiagen, USA). Use 100 ng of total RNA and
perform RT-PCR as per the instructions of the manufacturer
using the Single-Step RT-PCR Kit (Qiagen, USA). Separate
the amplified products on 1 % agarose gel and stain with ethidium bromide to visualize the bands.
45
References
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2. Brar GS, Ahuja L (1997) Sesame: its culture,
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3. Sankar D, Sambandam G, Rao MR, Pugalendi
KV (2004) Impact of sesame oil on nifedipine
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4. Frank J (2005) Beyond vitamin E supplementation: an alternative strategy to improve vitamin status. J Plant Physiol 162:834843
5. Hibasami H, Fujikawa T, Takeda H, Nishibe
S, Sato T, Fujisawa T, Nakashima K (2000)
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6. Miyahara Y, Hibasami H, Katsuzaki H, Imai
K, Komiya T (2001) Sesamolin from sesame
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cells. Int J Mol Med 7:369371
7. Rao KR, Kishore PBK, Vaidyanath K (2002)
Biotechnology of sesamean oilseed crop.
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8. Saravanan S, Nadarajan N (2005) Effects of
media supplements on in vitro response of sesame (Sesamum indicum L.) genotypes. Res J
Agric Biol Sci 1:98100
9. Ram R, Catlin D, Romero J, Cowley C (1990)
Sesame: new approaches for crop improvement. In: Janick J, Simon JE (eds) Advances in
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10. Baskaran P, Jayabalan N (2006) In vitro mass
propagation and diverse callus orientation on
(Sesamum indicum L.) an important oil plant.
J Agric Technol 2:259269
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callus induction and root-shoot bud formation
depend on seed coat of sesame genotypes.
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13.
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Chapter 5
Sunflower (Helianthus annuus L.)
Laura M. Radonic, Dalia M. Lewi, Nilda E. Lpez,
H. Esteban Hopp, Alejandro S. Escandn,
and Marisa Lpez Bilbao
Abstract
Sunflower (Helianthus annuus L.) is still considered as a recalcitrant species to in vitro culture and
transformation in spite of the publication of different protocols. Here we describe a routine transformation
system of this crop which requires mature HA89 genotype seeds and Agrobacterium tumefaciens EHA105
strain for gene delivery, being both easily available. Selection of transformed shoots depends on root development in kanamycin-selective media, instead of shoot color, avoiding selection of escapes. The establishment of this protocol proved successful for the incorporation of both reporter and agronomic important
genes and also for the evaluation of the specific expression patterns of different promoters in transgenic
sunflower plants. Stable expression of the incorporated transgenes was confirmed by RT-PCR and GUS
reporter gene visualization. Stable inheritance of transgenes was successfully followed until T2 generation
in several independent lines.
Key words Agrobacterium tumefaciens EHA105 strain, HA89 mature seeds, Helianthus annuus L,
Kanamycin selection, Sunflower transformation, Transgenic plants
Introduction
Sunflower (Helianthus annuus L.) is one of the most important
sources of edible oil and is becoming important for biofuel production. Total world sunflower seed production is approximately
25.8 million tons which goes almost exclusively to oil extraction,
providing 8.2 % of total world volume. Sunflower oil is considered
a good-quality oil for its light taste and appearance but especially
because it supplies more vitamin E than any other vegetable oil.
Sunflower and peanut are the only major vegetable oil-yielding
crops that have no GM varieties authorized for commercial use.
Sunflower biotechnological improvement is limited to molecular
markers as a result of the lack of an available routine and efficient
transformation protocol, which is one of the main reasons why it is
still described as a recalcitrant species [1].
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_5, Springer Science+Business Media New York 2015
47
48
2
2.1
Materials
Genotypes
2.2 Agrobacterium
tumefaciens Strain
and Plasmid
2.3 A. tumefaciens
Culture Media
49
50
Methods
3.1 Seed
Disinfection
3.2 Explant
Preparation
1. Place the disinfected seeds in a glass Petri dish under the flow
hood.
2. Dehull seeds with the help of a scalpel and place ten seeds per
Petri dish containing 20-mL MS1/2.
3. Culture in the dark for 24 h. Seeds that do not develop a growing radicle are discarded.
3.3 A. tumefaciens
Culture
3.4
Cocultivation
51
52
3.5 Selection
and Regeneration
of Transgenic Plants
3.6 Transfer
to the Greenhouse
3.6.2 Grafting
53
Fig. 2 Transfer to greenhouse by grafting. Each shoot (a) is cut obliquely at the base (b) and placed on the
rootstock (c). Rootstock tip was previously removed (d) and prepared by performing a longitudinal cut of the
same size as the shoot oblique cut (e, f). Shoot is placed on the rootstock making sure that conduction tissues
of both parts are in close contact, and grafting area is tied up with a cotton ribbon (g), fixed with a clothespin
(h). The whole plant is covered with a nylon bag to prevent dehydration
54
Notes
1. Complete healthy seeds, with unbroken and undamaged hull,
from field-grown plants.
2. Plates should not be older than 5 days.
3. Culture time depends on the initial inoculum and shaker speed.
4. Smaller pot usage is not recommended, as it may impair root
development and prevent full-length aerial growth.
5. We strongly suggest using CTAB genomic DNA extraction
method [18] including a posterior phenol extraction step.
Acknowledgments
This study was developed at the Instituto de Biotecnologa,
Instituto Nacional de Tecnologa Agropecuaria (INTA),
Argentina, and was supported by grants of ANPCyT (PICTO
ASAGIR N08-13164, PICTO INTA N08-12925) and INTA
(PE AEGR3425, PE AEGR3426).
The authors would like to thank Valeria Peralta and Agustn
Montenegro for greenhouse technical support.
References
1. Mayor ML, Nestares G, Vega T, Zorzoli R,
Picardi LA (2010) Sunflower propagation. In:
Jain SM, Ochatt SJ (eds.) Protocols for in vitro
propagation of ornamental plants, vol. 589.
Methods in molecular biology. Humana,
New York. pp 271280
2. Hahne G (2001) Sunflower. In: Hui Y,
Khatchtourians G, McHughen A, Nip W,
Scorza R (eds) Handbook of transgenic plants.
Marcel Dekker, New York, pp 813883
3. Lewi D, Esteban Hopp H, Escandn A (2006)
Sunflower (Helianthus annuus L.). In: Wang K
(ed.) Agrobacterium protocols, vol. 343.
Methods in molecular biology. Humana Press,
New York. pp 291298
14.
15.
16.
17.
18.
55
Part II
Root Plants
Chapter 6
Carrot (Daucus carota L.)
Owen S.D. Wally and Zamir K. Punja
Abstract
Plants are susceptible to infection by a broad range of fungal pathogens. A range of proteins have been
evaluated that can enhance tolerance to these pathogens by heterologous expression in transgenic carrot
tissues. The protocols for carrot transformation with Arabidopsis NPR1 (Non-Expressor of PathogenesisRelated Proteins 1) are described in this chapter, using the herbicide resistance gene bar, which encodes
phosphinothricin acetyltransferase, as a selectable marker. In this protocol, petiole segments (0.51.0 cm
long) from aseptically grown carrot seedlings are exposed to Agrobacterium tumefaciens strain LBA4404
for 1030 min and cocultivated for 23 days. Herbicide selection is then imposed for 812 weeks on a
series of different tissue culture media until embryogenic calli are produced. The transfer of the embryogenic calli to hormone-free medium results in embryo development which eventually gives rise to transgenic plantlets. Embryogenic calli can also be propagated in suspension cultures. This protocol has yielded
transgenic carrot plants with defined T-DNA inserts at the rate of between 1 and 3 Southern-positive
independent events out of 100.
Key words Carrot, Disease resistance, Genetic engineering, Pathogenesis-related proteins, Herbicide
resistance
Introduction
Carrot (Daucus carota L. subsp. sativus), a member of the family
Apiaceae, is grown worldwide for its edible taproot, which provides a source of vitamin A and fiber in the diet. Carrot is readily
amenable to tissue culture and transformation, with the production of transgenic plants possible within 6 months. Numerous
transformation methods are available for carrot; however,
Agrobacterium tumefaciens-based methods are by far the most
common, and numerous strains of A. tumefaciens have been shown
to be successful [1]. Additionally, the ability of carrot tissue cultures to rapidly produce somatic embryos makes it a logical target
for molecular pharming of transgenic health products, such as antigens [2, 3], interferons [4], and vaccines [5].
One of the greatest challenges to carrot crop production is the
management of fungal diseases. There are a number of widespread
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_6, Springer Science+Business Media New York 2015
59
60
Materials
Plant Materials
2.2 Agrobacterium
Strains and Binary
Vector
2.1
61
DL-Phosphinothricin
62
Methods
3.1 Sterile
Carrot Tissue
3.2 Agrobacterium
Cultures
63
Transformation
64
65
Notes
1. Agrobacterium tumefaciens LBA4404 was used in our experiments; however, other more aggressive strains have also proven
successful by other groups [1]. Typically, less callus development will be observed with more aggressive strains of
Agrobacterium and can result in the direct formation of somatic
embryos from the necrotic explants.
2. DL-PPT was used for selection in our experiments; however,
glufosinate ammonia can be substituted at identical concentrations, and Basta can be used at 0.75 the PPT concentration.
Alternatively, for cost saving, Liberty herbicide can be used
directly in the medium following filter sterilization to provide
the appropriate glufosinate ammonia concentration; however,
there are some surfactants within the solution that could lead
to enzymatic inhibition of sensitive assays performed from tissue culture-grown plants.
3. When LBA4404 cultures are resuspended in 1/10 MS solution, they will often form clumps; this is completely normal
and does not affect the transformation.
4. Carrot petioles are dissected on top of sterile filter paper and
cut into appropriate sizes. This alleviated some of the difficulties associated with the petioles dehydrating and sticking to
smooth surfaces, such as a Petri plate. Following dissection of
each petiole (1025 explants), they are immersed in the
Agrobacterium solution. This caused some explants to have
longer exposure to the inoculum; however, it was necessary to
avoid the suberization of wound sites.
5. Some of the PPT-resistant plants still show some susceptibility
to the 0.4 % Liberty application; however, it was much less
than the control plants. The minimum lethal concentration of
Liberty to each plant needs to be ascertained by a preliminary
experiment. The concentrations range between 0.1 and 0.4 %.
Cotton swabs are used to gently paint the herbicide solution
over the leaf surface pre-marked with a waterproof ink or marker
pen. Typical phytotoxicity symptoms develop after 7 days.
6. All of the somatic embryos that arise from an individual callus
are typically a unique transformation event; however, careful
tracking and Southern blot confirmation are required to identify events. Typically, we reinitiate the callus from individual
sterile plant lines in order to propagate clonal populations.
7. There are significant differences in the transformation frequency depending on the cultivar selected. Danvers Half Long
and Nantes Coreless have efficiencies greater than 3 %; Nanco
and HCM cultivars have efficiencies of less than 1 % [4]. These
efficiencies are the number of independent Southern-positive
events from 100 explants.
66
Acknowledgment
Funding for this research was provided by the Natural Sciences and
Engineering Research Council of Canada (NSERC), Discovery
Grants Program.
References
1. Baranski R (2008) Genetic transformation of
carrot (Daucus carota) and other apiaceae species. Trans Plant J 2:1838
2. Kalbina I, Wallin A, Lindh I, Engstrom P,
Andersson S, Strid A (2011) A novel chimeric
MOMP antigen expressed in Escherichia coli,
Arabidopsis thaliana, and Daucus carota as a
potential Chlamydia trachomatis vaccine candidate. Prot Express Purif 80:194202
3. Rosales-Mendoza S, Soria-Guerra RE,
Moreno-Fierros L, Han YP, Alpuche-Solis AG,
Korban SS (2011) Transgenic carrot tap roots
expressing an immunogenic F1-V fusion protein from Yersinia pestis are immunogenic in
mice. J Plant Physiol 168:174180
4. Luchakivskaya Y, Kishchenko O, Gerasymenko
I, Olevinskaya Z, Simonenko Y, Spivak M,
Kuchuk M (2011) High-level expression of
human interferon alpha-2b in transgenic carrot (Daucus carota L.) plants. Plant Cell Rep
30:407415
5. Zhang HX, Liu M, Li YJ, Zhao YH, He H,
Yang GD, Zheng CC (2010) Oral immunogenicity and protective efficacy in mice of a
carrot-derived vaccine candidate expressing
6.
7.
8.
9.
10.
11.
Chapter 7
Cassava (Manihot esculenta Crantz)
Simon E. Bull
Abstract
Genetic transformation of plants is an indispensable technique used for fundamental research and crop
improvement. Recent advances in cassava (Manihot esculenta Crantz) transformation have facilitated the
effective generation of stably transformed cassava plants with favorable traits. Agrobacterium-mediated
transformation of friable, embryogenic callus has evolved to become the most widely used approach and
has been adopted by research laboratories in Africa. This procedure utilizes axillary meristem tissue (buds)
to produce primary and secondary somatic embryos and subsequently friable, embryogenic callus.
Agrobacterium harboring a binary expression cassette is used to transform this tissue, which is regenerated
via cotyledons and shoot organogenesis to produce rooted in vitro plantlets. This chapter details each step
of the procedure using the model cultivar 60444 and provides supplementary notes to successfully produce transgenic cassava.
Key words Agrobacterium tumefaciens, Axillary meristems, Buds, Cassava, Cultivar 60444, Friable
embryogenic callus, Manihot esculenta Crantz, Somatic embryogenesis, Tissue culture, Transformation
Introduction
Cassava is an ancient plant that is gaining prominence in crop
biotechnology programs. The domesticated species (Manihot esculenta Crantz) is grown for its starch-rich storage roots, providing
food and energy security in tropical and subtropical countries
[16]. Traditional breeding has contributed significantly towards
improving the cassava germplasm [711]. However, this approach
is hampered by poor flowering and seed development, heterozygosity, and allopolyploidy, meaning that introgression of desirable
trait(s) into farmer-preferred cultivars is difficult and considerably
time-consuming [1214]. Advances in cassava biotechnology offer
an additional strategy to expedite research [15] and has led to the
generation of lines with improved traits, for example, virus resistance [16, 17], reduced cyanide content [18, 19], delayed postharvest physiological deterioration [20, 21], and waxy starch [22].
During the 1990s, electroporation, particle bombardment,
and Agrobacterium-mediated transformation techniques were
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_7, Springer Science+Business Media New York 2015
67
68
Simon E. Bull
69
2
2.1
Materials
Stock Solutions
70
Simon E. Bull
2.2
Plant Material
71
72
Simon E. Bull
73
Agrobacterium strain LBA4404 [60] is used predominantly in cassava transformation and carries resistance to rifampicin and streptomycin. To visualize transformation success, it is advisable to use
a CaMV35S:uidA containing plasmid in parallel to the experimental expression construct(s) that can be used in GUS assays [58].
1. Agrobacterium medium (YEBA and YEB): Dissolve 1 g Bacto
yeast extract, 5 g Bacto beef extract, 5 g Bacto peptone,
and 5 g sucrose in approximately 700 mL of water and then
adjust to 1 L (final volume), adjust pH to 7.2, add 15 g Bacto
agar, and autoclave. Allow the media to cool before adding
2 mL rifampicin (25 mg/mL) and 1 mL streptomycin
(100 mg/mL) required for bacterial selection. Also add appropriate antibiotic for selection of expression cassette. Pipette
25 mL into 90 mm Petri dishes. For liquid medium, omit the
addition of Bacto agar and add 2 mL of MgSO4 (1 M) with
the antibiotics after sterilization. Store at 4 C.
2. Agrobacterium solution for transformation (GDS): Dissolve
2.7 g of GD medium (including vitamins) and 20 g sucrose in
approximately 700 mL water. Add 1 mL picloram (12 mg/mL);
adjust to a final volume of 1 L and pH to 5.8. Autoclave and
store at 4 C.
Methods
To avoid/minimize contamination, it is imperative that all steps
are performed using sterile tools and media in a biological safety
cabinet. Where otherwise indicated, light intensity in growth cabinets was 4050 mol/m2/s. Handle all genetically modified material according to local regulations and good laboratory practice.
3.1 Enlargement
of Axillary Buds
from Stem Cuttings
74
Simon E. Bull
Fig. 2 Procedure for producing transgenic cassava plants. (a) Swollen axillary bud on CAM. (b) Immature
primary somatic embryos (indicated by arrows) developing on a cushion of NEFC on CIM. (c) Maturing somatic
embryos on CIM. White line indicates approximate suggested division for further propagation. (d) Cluster of
FEC on GD appropriate for Agrobacterium inoculation. (e) FEC following cocultivation spread onto mesh on
GD + C250. (f) Developing embryo/cotyledon on MSN + C250 + H15 (indicated by arrow). Putative transformed
FEC seen as swollen, yellowish structures. Non-transformed are smaller, white clusters. (g) Developing
embryo/cotyledon transferred to CEM + C100. (h) Appearance of immature shoots following several weeks on
CEM + C100. (i) In vitro transgenic cassava plantlet. (j) Developing embryos/cotyledons from MSN + C250 + H15
used for GUS assay. Blue precipitate clearly visible throughout all tissue. (k) GUS-stained leaves. (l) Rooting
assay of transgenic plantlets (left and center ) and wild-type 60444 (right ) on CBM + C50 + H10. Examples
showing regeneration of cassava harboring the hptII antibiotic selection gene. Figure reproduced with kind
permission from [34]
75
1. Divide the embryo clusters, gently removing NEFC, and transfer to GD. Seal the plates with Parafilm, wrap in aluminum foil,
and incubate at 28 C. After 2 weeks, use a binocular light
microscope to check for FEC growth. FEC are clusters of offwhite/yellowish spherical units 1 mm in diameter (Fig. 2d).
Incubation for a further 2 weeks should aid development and
thus identification of FEC.
2. Using sterile syringe needles, gently transfer the FEC to GD
(see Note 10). Aim to have approximately ten clusters per Petri
dish. Seal the plates with Parafilm, wrap in aluminum foil, and
incubate at 28 C for 2 weeks. After this incubation, the clusters should have approximately doubled in size and any that do
not should be discarded.
3. Every 2 weeks, divide the clusters and culture (as described
above) on fresh GD medium (Fig. 2d). The aim of this process
is to multiply and maintain healthy FEC material (see Notes 11
and 12). A sample of FEC should be plated on MSN + C250
and incubated (approximately 4 weeks at 28 C, 16 h light and
8 h dark) to determine regeneration efficiency.
3.3 Preparation
of Agrobacterium
tumefaciens Inoculum
76
Simon E. Bull
3.5 Recovery
and Maturation
of Transformed FEC
3.6 Regeneration
of Putative Transgenic
FEC
77
2. Small green cotyledons will appear after a few weeks (Fig. 2f).
Transfer them to CEM + C100 using sterile syringe needles,
gently removing FEC or NEFC stuck to their surface (see Note
20; Fig. 2g). Seal the plates with Parafilm and incubate at
28 C (16 h light and 8 h dark).
3. Every 1014 days, transfer the developing tissue to fresh
CEM + C100 (see Note 21). Callus may develop and should be
removed using a scalpel during transfer (see Note 22). Continue
to propagate material until juvenile shoots and leaves appear
(usually 23 weeks onwards; Fig. 2h).
4. Remove and culture in CBM + C50 juvenile shoots (usually
approximately 23 cm in length), seal the pot with Parafilm or
Micropore tape, and incubate at 28 C (16 h light and 8 h
dark). Roots should appear within 12 weeks (see Note 23;
Fig. 2i).
3.7 Rooting
Experiment to Screen
for Transgenic
Plantlets
3.8 Transfer
of In Vitro Plantlets
to Soil and Glasshouse
Environment
The approach to transfer in vitro plantlets to soil and the maintenance of mature cassava plants will vary between different facilities/research centers. Ergo, the following steps should be regarded
as suggested guidelines.
78
Simon E. Bull
Notes
1. Carbenicillin disodium forms a viscous solution in water at this
concentration. Therefore, add the powder gradually and stir
continuously to dissolve. A gelatinous lump will form if the
powder is added too quickly.
2. Experiments have been conducted to assess the impact of
Noble agar (high-grade agar from red algae) and Gelrite
(a gellan gum derived from Pseudomonas elodea) on cassava
tissue development [61]. Hyperhydricity of somatic cassava tissue appeared to occur on Gelrite-containing medium,
whereas high-quality tissue was generated on medium containing Noble agar. Gelrite (which is usually cheaper than Noble
agar) is still used for in vitro plantlet stock propagation where
it had no discernible negative impact on growth.
3. Following sterilization via autoclaving, tissue culture medium
should be plated; do not reheat because this may affect chemical/
nutrient content and caramelize the sucrose.
4. To prevent heat-induced degradation of antibiotics, the media
should be allowed to cool to approximately 50 C before
carbenicillin is added.
5. Wrapping the Petri dishes in aluminum foil creates a dark environment that aids bud enlargement and slows development.
Additionally, it will be easier to handle the stem cuttings in this
and subsequent steps if they are approximately 510 mm in
length on either side of the node. To minimize tissue multiplication in subsequent steps, it is advisable to use stem cuttings
from approximately 75 in vitro plantlets.
6. Leaves larger than approximately 1 cm in diameter should be
removed from the propagated apical shoot to reduce risk of
leaf senescence before the plantlet is established. Additionally,
to encourage root growth, ensure the cutting has a node in the
medium.
7. The incubator used for cassava tissue culture can affect tissue
quality [61]. Most prominent is the accumulation of moisture
on Petri dish lids that may be detrimental to light penetration
79
and media properties. Ergo, it is advisable to use precise climatecontrolled chambers (e.g., Sanyo/Panasonic MLR Plant
Growth Chamber). Culture plates can be stacked 23 high with
no discernible compromise to tissue growth or quality.
8. Clusters of embryogenic tissue should be no smaller than
approximately 5 mm in diameter; otherwise their growth may
be attenuated.
9. At this stage, you should aim to have more than 20 CIM plates
of tissue to ensure enough material is available for transfer and
FEC production.
10. It is important not to disrupt or spread the clusters of FEC as
this will attenuate their growth.
11. The extent to which FEC material can be subcultured repeatedly is somewhat dependent on experience and growth conditions. However, it is advisable that a batch of FEC is maintained
for no more than six months to minimize the risk of somaclonal variation [37].
12. It is imperative that the FEC clusters are not completely disassociated as this will negatively affect development. Using
syringe needles, gently scoop clumps of FEC avoiding where
possible NEFC and other unwanted tissue.
13. The rate of bacterial growth can be variable. Therefore, it is
recommended that two or three cultures are inoculated at various times during the day to improve the probability that one
of them will be at the optimal OD when required by the
researcher.
14. Try to avoid flooding the plate with the Agrobacterium suspension as this will likely result in overaccumulation of bacterial growth.
15. Use material from approximately six culture plates per 25 mL
of GDS + C500 for washing. Too much material makes resuspension more difficult and the washing is less effective.
16. It is important that the FEC are spread thinly on the mesh;
regeneration is compromised if too thickly layered and may
also exacerbate Agrobacterium growth. As a general guide,
1218 pieces of mesh would be prepared for FEC from six
cocultivation plates.
17. This step provides the FEC with a period of recovery and
the carbenicillin in the medium suppresses Agrobacterium
growth.
18. The stepwise increase in antibiotic concentration and weekly
transfer to fresh medium allow transformed material to
acclimatize while minimizing changes in culturing/media
conditions. A sample of material transformed with a
CaMV35S:uidA construct may be used at this stage for a
80
Simon E. Bull
Acknowledgments
This protocol was developed by the author at the University of
Bath (UK) and ETH Zrich (Switzerland) and partially funded by
the Bill & Melinda Gates Foundation (BioCassava Plus Program
Phase I). The author thanks Herv Vanderschuren (ETH Zrich,
Switzerland) for helpful comments and discussion.
81
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83
Chapter 8
Potato (Solanum tuberosum L.)
Venkateswari J. Chetty, Javier Narvez-Vsquez,
and Martha L. Orozco-Crdenas
Abstract
Agrobacterium-mediated transformation is the most common method for the incorporation of foreign
genes into the genome of potato as well as many other species in the Solanaceae family. This chapter
describes protocols for the genetic transformation of three species of potato: Solanum tuberosum subsp.
tuberosum (Desir), S. tuberosum subsp. andigenum (Blue potato), and S. tuberosum subsp. andigena
using internodal segments as explants.
Key words Agrobacterium tumefaciens, Andigena, -Glucuronidase (uidA), Blue potato, Desir,
Neomycin phosphotransferase II (npt II), Plant transformation, Solanum tuberosum
Introduction
Potato (Solanum tuberosum L.) is the worlds third most important
food crop next to rice and wheat in terms of human consumption
with its production exceeding 300 million metric tons as reported
by International Potato Center [1]. Potato is a critical crop in
terms of food security. More than one billion population around
the globe consume potato. Potato is vegetatively propagated,
meaning that a whole plant can be grown from a potato tuber or a
piece of it. The new plant can produce 520 new tubers, which will
be genetic clones of the mother plant. Potato enjoys a long history
of improvement through traditional breeding. Breeders target
multiple traits, including resistance to biotic and abiotic stresses,
and tuber quality [2].
Recently, the full sequence of the potato genome has been
completed [3], opening a broad spectrum of possibilities to understand gene function and the genetic manipulation through plant
transformation for the improvement of this important crop.
Agrobacterium-mediated transformation is the most common
technique used for functional genomic studies in potato, and for
the introduction of novel traits into commercial potato varieties,
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_8, Springer Science+Business Media New York 2015
85
86
Fig. 1 Steps in the transformation of potato with Agrobacterium tumefaciens strain AGL1 harboring plasmid
pBI121. (a) Four-week-old in vitro plants used as source of inter nodal explants. (b) Pre-culture of internodal
explants on CIM for 2 days. (c) Callus formation on CIM with antibiotic selection in 23 weeks. (d) Shoot regeneration on SIM with antibiotic selection (4 weeks). (e) Shoot elongation on SIM with antibiotic selection
(2 weeks). (f) Rooting on RIM with antibiotic selection (3 weeks). (g) Histochemical detection of GUS expression
in transgenic shoots
2
2.1
Materials
Plant Material
2.3
87
Stock Solutions
In vitro micropropagated plants are used as source of stem internodal explants for transformation. Solanum tuberosum subsp.
tuberosum cv Desir and S. tuberosum subsp. andigenum (blue
potato) plants were originally established in vitro from tuber
sprouts. The plants are grown at 25 C under 16 h light/8 h dark
cycle with fluorescent light (irradiance of 60 mol/m2/s). Fourweek-old plantlets are used for the transformation studies.
1. Agrobacterium tumefaciens strain AGL1.
2. Binary vector: The binary vector pBI121 (Clontech, Palo
Alto, CA, USA) contains -glucuronidase (uidA) and neomycin phosphotransferase II (npt II) genes under the regulation of
the CaMV 35S promoter [17, 18]. The binary vector pBI121
is transformed into AGL1 using electroporation. Transformed
cells are selected on YEP plates with kanamycin (100 mg/L).
Kanamycin-resistant colonies are screened by colony PCR
using npt II primers. A PCR-positive colony is grown in 5 mL
of YEP with kanamycin, and Agrobacterium glycerol stocks
are prepared by mixing equal volumes of glycerol and the
Agrobacterium culture (1:1 ratio v/v). Glycerol stocks are
stored at 80 C.
All chemicals for stock solutions and culture media can be
obtained from different vendors including Sigma-Aldrich (www.
sigmaaldrich.com), Plantmedia (www.plantmedia.com), PhytoTechnology Laboratories (www.phytotechlab.com), and Caisson
Laboratories (www.caissonlabs.com). Unless otherwise specified,
all stock solutions are filter sterilized and stored at 20 C:
1. Gamborgs B5 vitamin solution (1,000): Dissolve 200 mg
nicotinic acid, 200 mg pyridoxine hydrochloride, and
2,000 mg of thiamine hydrochloride in 180 mL of ddH2O
[19]. Bring the volume up to 200 mL with ddH2O.
2. MS vitamin solution (1,000): Dissolve 100 mg thiamine
HCl, 50 mg pyridoxine HCl, 50 mg nicotinic acid, and
200 mg glycine in 100 mL of ddH2O [20].
3. Indole-3 butyric acid (IBA) solution (1 mg/mL): Dissolve
20 mg of IBA in 2 mL of 95 % ethanol. Bring the volume up
to 20 mL with ddH2O.
4. Naphthalene acetic acid (NAA) solution (1 mg/mL): Dissolve
20 mg of NAA in 1 mL of 1 M NaOH. Bring the volume up
to 20 mL with ddH2O.
5. Zeatin solution (1 mg/mL): Dissolve 50 mg zeatin in 1 mL of
1 M NaOH. Bring up the volume to 50 mL with ddH2O. Filter
sterilize, dispense into1 mL aliquots, and store at 20 C.
88
Culture Media
89
Table 1
Composition of medium for the induction of callus, shoots, and roots from stem internode
segments in three different genotypes of potato
Desir
Media components (L)
CIM
SIM
RIM
Blue potato
S. andigena
CIM/SIM RIM
CIM
SIM
RIM
MS salts (g)
4.33
4.33
4.33
4.33
4.33
4.33
4.33
4.33
Sucrose (g)
20
20
20
30
20
Glucose (g)
16
16
16
100
100
100
100
100
100
100
100
IAA (mg)
0.5
0.05
BAP (mg)
0.1
NAA (mg)
0.2
0.02
0.02
Zeatin (mg)
2.5
2.2
GA3 (mg)
0.02
0.02
0.15
pH
5.8
5.8
5.8
5.8
5.8
5.8
5.8
5.8
Gelrite (g)
Agar (g)
Myoinositol (mg)
CIM callus induction medium, SIM shoot induction medium, RIM root induction medium
90
91
Methods
3.1 Growth
of In Vitro Plants
from Tuber Sprouts
92
3.4 Preparation
of Explants
for Agrobacterium
Inoculation
3.5 Agrobacterium
Infection and
Cocultivation
93
3.8 Transplanting
and Acclimation
of Rooted Shoots
94
3.11 Histochemical
Analyses for GUS
Assay
95
Notes
1. The EDTA disodium salt will not go into solution until the
pH of the solution is adjusted to ~8.0 by the addition of
NaOH.
2. Ethidium bromide is a strong mutagen and a possible carcinogen or teratogen. Its hazardous properties require the use
gloves for handling and safety disposal.
3. Rifampicin (25 mg/L) and kanamycin (50 mg/L) are added
to grow Agrobacterium strain AGL1 transformed with pBI121.
4. To calculate the final dilution volume of Agrobacterium (V1),
use the equation C1V1 = C2V2, where C1 and C2 are respectively
the initial and final OD and V2 the final volume. Use AIM to
dilute the Agrobacterium cells. Diluted Agrobacterium culture
has to be used immediately to avoid aggregation.
5. Shoots regenerated from the two cut ends of the explants can
be considered independent events. It is important to cut off
the calli at the base from the shoot.
6. Transgenic shoots start rooting after 35 days.
References
1. Potato (2013) Retrieved from http://cipotato.
org/potato
2. Facts and figures about potato (2013)
Retrieved from http://cipotato.org/potato/
publications/pdf/005449.pdf
3. Xu X, Pan S, Cheng S, Zhang B, Mu D, Ni P,
Visser RG (2011) Genome sequence and analysis of the tuber crop potato. Nature
475:189195
4. Bradeen JM, Carputo D, Douches D (2009)
Part 7 Transgenic sugar, tuber and fiber crops.
doi:10.1002/9781405181099. k0704 in
compendium of transgenic crop plants
5. De Block M (1998) Genotype-independent
leaf disc transformation of potato (Solanum
tuberosum) using Agrobacterium tumefaciens.
Theor Appl Genet 76:767774
6. Stiekema WJ, Heidekamp F, Louwerse JD,
Verhoeven
HA,
Dijkhuis
P
(1998)
Introduction of foreign genes into potato
cultivars Bintje and Desiree using an
Agrobacterium tumefaciens binary vector.
Plant Cell Rep 7:4750
7. Sheerman S, Bevan MW (1988) A rapid transformation method for Solanum tuberosum
using binary Agrobacterium tumefaciens vectors. Plant Cell Rep 7:1315
8. Tavazza R, Tavazza M, Ordas RJ, Ancora G,
Benvenuto E (1988) Genetic transformation
9.
10.
11.
12.
13.
14.
96
15.
16.
17.
18.
19.
20.
21.
22.
Chapter 9
Taro (Colocasia esculenta (L.) Schott)
Xiaoling He, Susan C. Miyasaka, Maureen M.M. Fitch, and Yun J. Zhu
Abstract
Genetic engineering of taro is an effective method to improve taro quality and the resistance to various
diseases of taro. Agrobacterium tumefaciens-mediated transformation of taro is more efficient than the
particle bombardment transformation method based on current research. The development of a regeneration system starting from taro shoot tip explants could produce dasheen mosaic virus (DsMV)-free plantlets.
Highly regenerative calluses could be developed from DsMV-free, in vitro plantlets on the Murashige and
Skoog (MS) medium with 2 mg/L BA and 1 mg/L NAA (M5 medium). The Agrobacterium tumefaciensmediated transformation method is reported in this chapter. The highly regenerative calluses were selected
and cocultivated with the Agrobacterium strain EHA105 harboring the binary vector PBI121 with either
a rice chitinase gene chi11 or a wheat oxalate oxidase gene gf2.8. After cocultivation for 34 days, these
calluses were transferred to selection medium (M5 medium) containing 50 mg/L Geneticin G418 and
grown for 3 months in the dark. Transgenic shoot lines could be induced and selected on the MS medium
containing 4 mg/L BA (M15 medium) and 50 mg/L Geneticin G418 for 3 months further in the light.
Molecular analyses are used to confirm the stable transformation and expression of the disease resistance
gene chi11 or gf2.8. Pathologic bioassays could be used to demonstrate whether the transgenic plants had
increased disease resistance to taro pathogens Sclerotium rolfsii or Phytophthora colocasiae.
Key words Agrobacterium tumefaciens, Colocasia esculenta, Dasheen mosaic-free plantlets, Genetic
engineering, Shoot tip explants, Taro, Transformation
Introduction
Taro (Colocasia esculenta (L.) Schott) is an important tropical
root crop which is cultivated worldwide especially in Southeast
Asia and the Pacific Islands [1, 2]. Traditionally, taro is propagated vegetatively using underground stems from sucker plants
(i.e., cormels) [3]. Approximately 10 % of the previous crops
cormels need to be used for propagation [3]. Several tissue culture protocols for various taro cultivars have been developed. The
development of an efficient taro tissue culture system could
reduce the usage of the cormels for propagating materials and
increase the speed and production of the taro propagation. For
example, Chand et al. in 1999 [4], Fukino et al. in 2000 [5],
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_9, Springer Science+Business Media New York 2015
97
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Xiaoling He et al.
99
2
2.1
Materials
Plant Material
2.2 Growth
Regulator Stock
Solutions
Taro cv. Bun Long cormels were obtained from the University of
Hawaiis Waiakea Experiment Station. In vitro plantlets in culture
were established from primary shoot apices and axillary buds on
taro cormels. Highly regenerative calluses were induced from the
DsMV-free in vitro plantlets. These calluses were the targets of
Agrobacterium tumefaciens-mediated transformation.
1. -Naphthalene acetic acid (NAA): 100 ml 0.5 mg/mL stock is
prepared by dissolving 50 mg of NAA in 2 mL 1 N NaOH and
making up to volume with 98 mL of sterile distilled water.
Stock solution can be stored at 4 C for 6 months.
2. 6-Benzylaminopurine (BA): 100 ml 0.5 mg/mL stock is prepared by dissolving 50 mg of BA in 2 mL 1 N NaOH and making up to volume with 98 mL of sterile distilled water. Stock
solution can be stored at 4 C for 6 months.
2.3 Antibiotics
Stock Solutions
100
Xiaoling He et al.
2.5
Culture Media
2.5.1 Plant
Culture Medium
101
2.5.2 Agrobacterium
Culture Medium
1. Disarmed Agrobacterium tumefaciens strain EHA105 [15] containing the binary vector pBI121/ricchi11 or pBI121/gf2.8.
All EHA105 culture stocks are maintained in 30 % sterile
glycerol (v/v), 1 mL aliquots in 2 mL Eppendorf tubes stored
at -80 C.
2. Both vectors pBI121/ricchi11 [7] and pBI121/gf2.8 [9]
contain the selectable marker gene neomycin phosphotransferase II (nptII) for kanamycin and G418 resistance under the
NOS promoter and the reporter gene -glucuronidase (gus)
driven by the cauliflower mosaic virus (CaMV) 35S promoter.
In addition to these two genes, pBI121/ricchi11 contains the
rice chitinase gene chi11 driven by the CaMV 35S promoter.
In a separate construct, pBI121/gf2.8 contains the wheat oxalate oxidase gene gf2.8 driven by the gf2.8 promoter.
Methods
3.1 Establishment
of In Vitro Plantlets
In vitro shoot or plantlet cultures are maintained under environmental conditions of 25 2 C, 16 h photoperiod at light intensity
of 15 mol/m2/s. In vitro shoot or plantlet cultures in liquid
medium are placed on a shaker with shaking at 95 rpm under the
same environmental conditions. In vitro callus cultures are maintained in the dark at 25 2 C:
1. Wash the cormels in running tap water for 5 min.
2. Excise approximately 1 cm3 cormel sections containing one
primary shoot apex or axillary bud each section.
3. Immerse cormel cubes in 70 % ethanol with 1 drop of
Tween-80 for 5 min.
4. Peel and remove cormel section outer layer tissues and pick
and excise the 0.51.5 mm shoot tips from the center of shoot
apices or axillary buds using a sterile needle. Microscope can be
used to help find the shoot tips (see Note 1).
102
Xiaoling He et al.
5. Place shoot tips into a sterile petri dish containing surfacesterilizing solution (1.25 % sodium hypochlorite plus 1 drop of
Tween-80) for 16 s (see Note 1).
6. Transfer shoot tips to another sterile petri dish containing sterile distilled H2O; lightly shake the petri dish by hand for 2 min
to rinse the shoot tips.
7. Transfer the shoot tips to the test tubes containing 3 mL M15
liquid media, with one shoot tip per test tube. Shake culture
for 2 months. Multiple shoots could be induced after 2 months
(see Fig. 1a). ELISA assay for DsMV can be conducted at this
stage (see Note 1).
8. Excise multiple DsMV-free shoots (approximately 10 mm in
length) and transfer them to M5 petri dish plate for inducing
calluses. Place these culture plates in the dark for 34 months.
Subculture every month to remove brown and dead tissues.
Fig. 1 (a) The multiple shoots induced from one shoot tip explant in the M15 liquid media. (b) The calluses
induced after transferring the DsMV-free shoots onto the M5 plate for 34 months. (c) The multiple shoots and
plantlets induced after transferring the callus onto the M15 plate for 34 months. (d) The cocultivated calluses
selected on the callus selection medium CSM. (e) The PCR-positive multiple shoots of the independent line C6
with the rice chitinase gene chi11 further selected on the stage two shoot selection medium SSM2. (f) The
plantlets of the independent line g5 with the wheat oxalate oxidase gene gf2.8 further selected on the stage
three shoot selection medium SSM3
103
3.3 Infection
and Cocultivation
104
Xiaoling He et al.
3.4 Selection
and Regeneration
for Independent
Transgenic Lines
3.5
Transplanting
105
Notes
1. Unlike other protocols of in vitro shoot tip culture, this protocol describes directly surface-sterilizing the 0.51.5 mm shoot
tips for a very short time of 16 s. Using this protocol, contamination can be easily controlled and most explants remain
106
Xiaoling He et al.
107
108
8.
9.
10.
11.
Xiaoling He et al.
transformation of taro (Colocasia esculenta (L.)
Schott) with a rice chitinase gene for improved
tolerance to a fungal pathogen Sclerotium rolfsii. Plant Cell Rep 27:903909
He X, Miyasaka SC, Fitch MM, Zhu YJ (2010)
Regeneration and transformation of taro
(Colocasia esculenta) with a rice chitinase gene
enhances resistance to Sclerotium rolfsii.
HortScience 45:10141020
He X, Miyasaka SC, Fitch MM, Khuri S, Zhu YJ
(2013) Taro (Colocasia esculenta) transformed
with a wheat oxalate oxidase gene for improved
resistance to taro pathogen Phytophthora colocasiae. HortScience 48(1):2227
Murakami K, Kimura M, Matsubara S (1995)
Plant regeneration from protoplasts isolated
from callus of taro. J Jpn Soc Hortic Sci
63(4):773778
Deo PC, Harding RM, Taylor M, Tyagi AP,
Becker DK (2009) Somatic embryogenesis,
organogenesis and plant regeneration in taro
12.
13.
14.
15.
16.
Part III
Nuts and Fruits
Chapter 10
Apricot (Prunus armeniaca L.)
Csar Petri, Nuria Alburquerque, and Lorenzo Burgos
Abstract
A protocol for Agrobacterium-mediated stable transformation of whole leaf explants of the apricot (Prunus
armeniaca) cultivars Helena and Canino is described. Regenerated buds were selected using a two-step
selection strategy with paromomycin sulfate and transferred to bud multiplication medium 1 week after
they were detected for optimal survival. After buds were transferred to bud multiplication medium, antibiotic was changed to kanamycin and concentration increased gradually at each transfer to fresh medium
in order to eliminate possible escapes and chimeras. Transformation efficiency, based on PCR analysis of
individual putative transformed shoots from independent lines, was 5.6 %. Green and healthy buds,
surviving high kanamycin concentration, were transferred to shoot multiplication medium where they
elongated in shoots and proliferated. Elongated transgenic shoots were rooted in a medium containing
70 M kanamycin. Rooted plants were acclimatized following standard procedures. This constitutes the
only transformation protocol described for apricot clonal tissues and one of the few of Prunus.
Key words Agrobacterium tumefaciens, Apricot, Fruit trees, Prunus armeniaca, Rosaceae
Introduction
Fruit trees are among the most recalcitrant of plants to regenerate
adventitious shoots from cultured explants. In most woody fruit
species, transformation and regeneration are generally difficult and
often limited to a few genotypes or to seedlings [1]. This feature is
the major limiting factor preventing the development of gene
transfer technologies for fruit trees. Apricot (Prunus armeniaca
L.) is not an exception. For many years our research group at
CEBAS-CSIC in Murcia (Spain) has been working in the development of a reproducible and efficient procedure of apricot transgenic shoots regeneration by using clonal tissues as the source of
the explants.
Back in the late 1990s, some commercial cultivars were established in vitro and successfully micropropagated, as the first
requirement for regeneration and transformation protocols [2, 3].
At this point, an adventitious shoot regeneration protocol from
clonal mature tissues (leaves) was developed and later optimized
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_10, Springer Science+Business Media New York 2015
111
112
Materials
2.1 Agrobacterium
tumefaciens Strain
and Vector
2.2
Plant Material
2.3
Stock Solutions
113
2.3.3 Antibiotics
2.3.4 Others
2.4
Culture Media
114
Methods
115
Fig. 1 Apricot leaf transformation. The four younger, expanded leaves from apricot micro shoots (a) are
detached and cut two or three times perpendicular to the midrib but without fully separating the segments (b).
After Agrobacterium infection, coculture and transfer to regeneration medium adventitious buds regenerated
from the reactive leaves (c). Regenerated buds have to be rescued as soon as possible for optimal survival and
transferred to bud multiplication medium where selection is increased gradually until 100140 M kanamycin
is reached. Only surviving, green, and healthy buds (d) are then transferred to selective shoot multiplication
medium where they elongate in shoots and proliferate (e). Finally elongated shoots can be rooted in selective
medium (f) and acclimatized (g). Bar represents 1 cm
3.2 Explants
Preparation
3.3 Agrobacterium
Culture and
Preparation
116
Coculture
3.5 Washing
(See Note 7)
1. After coculture, rinse the explants briefly in liquid SRM supplemented with 1.26 mM CEF and 0.26 mM VAN.
2. Dry the explants on sterile filter paper.
3.6 Transgenic
Shoot Regeneration
3.7 Transgenic
Shoot Elongation
and Multiplication
3.8
Rooting
3.9
Acclimatization
117
Notes
1. As an example in this protocol, sgfp or uidA reporter genes
have been used for monitoring the transgenic shoot regeneration. These genes may be substituted for the gene of interest in
each case.
2. Dissolving all salts in the same recipient may lead to some salts
precipitation.
3. We recommend making fresh stock solution after 4 weeks for
all growth regulator stock solutions.
4. Autoclave all media at 121 C for 20 min.
5. Shoot multiplication medium composition is a key factor for
the subsequent successful adventitious bud regeneration [5].
Different apricot cultivars may need a different multiplication
medium.
6. Proceed to this step with a few explants at a time to avoid
explant dehydration.
7. This step is only necessary if overgrowth of Agrobacterium is
observed after coculture; otherwise go to Subheading 3.6.
8. Culture a maximum of seven leaves per petri dish.
9. Paromomycin was the only aminoglycoside antibiotic tested
that allowed the production of transgenic buds at the early
regeneration stages [10]. However, this antibiotic did not
effectively kill the escaped buds once they regenerated and did
not produce bleaching. We have found that kanamycin is much
more aggressive, producing bleaching and killing nontransgenic buds (Fig. 2).
118
Fig. 2 Elimination of escapes and chimeras by culturing buds in multiplication medium with increasing kanamycin concentrations. The differential effect of the antibiotic eliminating escapes and chimeras can be
observed in a petri dish (a) but also within a developed bud, where green and healthy meristems are appearing
in a bud with bleached leaves and necrotic areas (b)
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
119
Chapter 11
Blueberry (Vaccinium corymbosum L.)
Guo-Qing Song
Abstract
Vaccinium consists of approximately 450 species, of which highbush blueberry (Vaccinium corymbosum) is
one of the three major Vaccinium fruit crops (i.e., blueberry, cranberry, and lingonberry) domesticated in
the twentieth century. In blueberry the adventitious shoot regeneration using leaf explants has been the
most desirable regeneration system to date; Agrobacterium tumefaciens-mediated transformation is
the major gene delivery method and effective selection has been reported using either the neomycin
phosphotransferase II gene (nptII) or the bialaphos resistance (bar) gene as selectable markers. The
A. tumefaciens-mediated transformation protocol described in this chapter is based on combining
the optimal conditions for efficient plant regeneration, reliable gene delivery, and effective selection. The
protocol has led to successful regeneration of transgenic plants from leaf explants of four commercially
important highbush blueberry cultivars for multiple purposes, providing a powerful approach to
supplement conventional breeding methods for blueberry by introducing genes of interest.
Key words Agrobacterium tumefaciens, Genetic transformation, Leaf explant, plant regeneration,
Transgenic plant
Introduction
Vaccinium is a genus of terrestrial shrubs in the family Ericaceae
(Syn. Heath) [1]. It consists of approximately 450 species, of which
three Vaccinium fruit crops (blueberry, cranberry, and lingonberry) have been domesticated since the twentieth century [2].
Vaccinium crops are economically important fruit crops due in
part to their exceptional nutritional value and high amounts of
antioxidants and anti-inflammatory capacities that benefit human
health [3].
Highbush blueberry (Vaccinium corymbosum L.) is by far the
most important commercial blueberry (Vaccinium sp.), a highly
heterozygous, polyploid crop [4]. To date, all blueberry cultivars
have been generated exclusively through the traditional breeding
approaches (i.e., controlled hybridization and deliberate selection).
In general, it takes over 10 years to produce a finished cultivar
through these approaches. A major limitation is that not every
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_11, Springer Science+Business Media New York 2015
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Guo-Qing Song
123
was confirmed by Southern hybridization [6]. The A. tumefaciensmediated transformation protocol described in this chapter is routinely used in our laboratory.
2
2.1
Materials
Plant Material
1. Starting plant materials: 1-year-old softwood branches of highbush blueberry cvs. Aurora (Michigan State University cv.),
Bluecrop, Brigitta, and Legacy, preferably taken from
greenhouse-grown plants.
2. Leaf explants are taken from in vitro cultured stock shoots.
124
Guo-Qing Song
125
Method
3.1 Establishment
of Stock Cultures
3.2 Preparation
of Leaf Explants
3.3 Infection
and Cocultivation
126
Guo-Qing Song
Fig. 1 Agrobacterium tumefaciens (strain EHA105)-mediated transformation of blueberry cultivar Legacy using
a pCAMBIA-derived vector that contains a blueberry C-repeat binding factor (CBF) gene. (a) Leaf explants
inoculated with A. tumefaciens strain EHA105 containing the CBF. (b) Leaf explants after 6-day cocultivation.
(c) Kanamycin-resistant shoots produced from inoculated leaf disks after 10-week selection. (d) Proliferation
of kanamycin-resistant shoots. (e) Transgenic shoots rooting in planting medium. Bars: 1 cm
3.4 Selection
and Regeneration
127
3.5
Regrowth
3.6
Rooting
128
Guo-Qing Song
Greenhouse Care
Notes
1. Although EHA105 seems more efficient than the other two
strains in transient transformation studies [6, 21], leaf explants
of blueberry cultivars are also found susceptible to octopine
strain LBA4404 and nopaline strain GV3101, and both
A. tumefaciens strains may be used for stable transformation.
2. For both selectable marker genes, either the cauliflower mosaic
virus 35S (CaMV 35S) promoter or the nopaline synthase
(nos) promoter is able to drive an effective selection [911].
3. The change of pH caused by the addition of Km, cefotaxime,
and acetosyringone to media is usually not considered.
However, addition of zeatin or zeatin riboside to the stock
culture medium after autoclaving will increase the pH of the
medium due to the solvent (1 N NaOH) in the zeatin stock
solution. Thus, to get a final pH = 5.2 for stock culture medium,
preliminary experiments should be performed to work out
how much (X) the pH will increase after the addition of a certain amount of zeatin or zeatin riboside; then adjust the pH for
stock culture medium to 5.2-X prior to autoclaving. X is variable to different stock solutions of zeatin or zeatin riboside.
4. Either zeatin or zeatin riboside works for blueberry proliferation. The difference is that we include the zeatin riboside to
our medium prior to autoclaving.
5. WPM2Z is used when you see your blueberry cultures (e.g.,
cvs. Aurora and Brigitta) give a high shoot-proliferation rate
but the tiny leaves on the shoots. The reduced zeatin (or zeatin
riboside) amount from 4 to 2 mg/L promotes shoot elongation and leaf expanding.
129
Table 1
Regeneration medium for different blueberry cultivars
Regeneration medium
Cultivar
Duke
Biloxi, Emerald
130
Guo-Qing Song
131
Chapter 12
Cherry
Guo-Qing Song
Abstract
Agrobacterium tumefaciens-mediated transformation of sour cherry (Prunus cerasus L.) Montmorency
and sweet cherry rootstocks Gisela 6 and Gisela 7 (P. cerasus P. canescens) is described. Briefly, leaf
explants from in vitro shoots are cocultivated with A. tumefaciens either directly (for Gisela 6 and
Gisela 7) or after pretreatment (for Montmorency) on cocultivation medium; selection and regeneration of transformed shoots are carried out on selection medium containing 50 mg/L kanamycin (Km) and
250 mg/L timentin (or cefotaxime) for 35 months. In this protocol, the optimal media for shoot proliferation and shoot regeneration from leaf explants are genotype dependent.
Key words Agrobacterium tumefaciens, Cherry, genetic transformation, Leaf explant, plant regeneration, Transgenic plant
Introduction
Stone fruits (i.e., almonds, apricots, cherries, nectarines, peaches,
and plums) are a group of fruits in the genus Prunus. The true
cherries are derived from the genus Prunus, subgenus Cerasus,
family Rosaceae [1]. Sweet cherry (Prunus avium) and sour cherry
(Prunus cerasus) are the major eating cherries that have become
recognized for potential benefits to human health and are favorite
fruits of many people.
Traditional approaches for cherry breeding, such as hybridization, clone selection, and mutagenesis, are generally difficult and
long-term processes due to heterozygosity, polyploidy, length of
field trials, and the interval between generations [2]. Thus, transformation of cherries offers an attractive approach to complement
these breeding methods by efficiently introducing single or multiple desired traits such as improved fruit quality and resistance to
insects and diseases.
Prunus spp. are not amenable for plant tissue culture and
genetic transformation. To date, genetic transformation has been
reported only for a few commercially important cherry species,
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_12, Springer Science+Business Media New York 2015
133
134
Guo-Qing Song
including sour cherry (P. cerasus L.), chokecherry (P. virginiana L.),
black cherry (Prunus serotina Ehrh.), and the cherry rootstocks
Rosa (P. subhirtella autumno), Gisela 6 (P. cerasus P. canescens),
Colt (P. avium P. pseudocerasus), Inmil (P. incisa P. serrula),
and Damil (P. dawyckensis) [311].
In general, a reliable transformation system depends on an efficient plant regeneration system, which is usually genotype dependent. The Agrobacterium tumefaciens-mediated transformation
protocol described here is based on our optimized conditions for
shoot regeneration from leaf explants, gene delivery using A. tumefaciens, and selection of transgenic shoots using the neomycin
phosphotransferase II (nptII) as the selectable marker gene [810].
This protocol has resulted in successful transformation of a major
sour cherry Montmorency and two sweet cherry rootstocks
Gisela 6 and Gisela 7 [9, 12].
2
2.1
Materials
Plant Material
Cherry
135
136
Guo-Qing Song
Method
All cultures are maintained at 25 C, 3040 mol/m2/s of 16 h/day
from cool white fluorescent tubes unless as otherwise mentioned.
3.1 Establishment
of Stock Cultures
preferably
from
2. Cut off the leaves, wash the branches under running tap water
for 20 min, and then trim the branches to 510 cm in length.
3. Soak the branch segments in 70 % ethanol for 1 min in a 50 mL
Corning tube (Corning Inc.) and then pour off the ethanol.
4. Surface sterilize the segments in sterilizing solution for 20 min.
5. Rinse the branches five times (2 min per time) with sterile distilled water.
6. Cut the branches into 12 cm pieces each with a single bud
using sterile dissecting scissors.
7. Place branch pieces horizontally, three for each dish, onto
10 mL SCM in 60 mm 15 mm Petri dishes.
8. Check the dishes every other day. If any visible contamination
buds are observed, transfer the sterile ones to fresh medium in
Petri dishes.
9. After incubating explants for 2 weeks, excise sterile shoots and
insert them into fresh SCM in Magenta GA7 boxes.
10. Subculture the shoots and stem nodes, ten per glass jars, every
46 weeks.
Cherry
137
138
Guo-Qing Song
Cherry
3.4 Selection
and Regeneration
139
3.5
Regrowth
3.6
Rooting
140
Guo-Qing Song
Fig. 1 Agrobacterium tumefaciens (strain EHA105)-mediated transformation of sweet cherry rootstock Gisela
6 using a pART27-derived RNAi vector for silencing Prunus necrotic ring spot virus. (a) Kanamycin-resistant
shoots produced from inoculated leaf disks after 10-week selection. (b) Proliferation of kanamycin-resistant
shoots. (c) Transgenic shoots rooting in planting medium. (d) Transgenic plants growing in the greenhouse
3.7
Greenhouse Care
Notes
1. In our transient transformation studies, three opine-type
strains EHA105, GV3101, and LBA4404 showed no significant difference in gene delivery [8].
Cherry
141
142
7.
8.
9.
10.
11.
Guo-Qing Song
iron on the cyclic secondary somatic embryogenesis and plant regeneration of transgenic
cherry rootstock Colt (Prunus avium x P.
pseudocerasus). Plant Cell Tiss Org Cult 79:
223232
Dai W, Magnusson V, Johnson C (2007)
Agrobacterium-mediated transformation of
chokecherry (Prunus virginiana L.). HortScience
41:140142
Song G-Q, Sink KC (2005) Optimizing shoot
regeneration and transient expression factors
for Agrobacterium tumefaciens transformation
of sour cherry (Prunus cerasus L.) cultivar
Montmorency. Sci Hortic 106:6069
Song G-Q, Sink KC (2006) Transformation of
Montmorency sour cherry (Prunus cerasus L.)
and Gisela 6 (P. cerasus P. canescens) cherry
rootstock mediated by Agrobacterium tumefaciens. Plant Cell Rep 25:117123
Song G-Q, Sink KC (2007) Transformation of
cherry: Prunus cerasus L. Montmorency and
Prunus cerasus x P. canescen Gisela 6 mediated
by Agrobacterium tumefaciens and a two-step
selection system. Acta Hortic 738:683689
Liu X, Pijut PM (2008) Plant regeneration
from in vitro leaves of mature black cherry
(Prunus serotina). Plant Cell Tiss Org Cult 94:
113123
Chapter 13
Chestnut, American (Castanea dentata (Marsh.) Borkh.)
Charles A. Maynard, Linda D. McGuigan, Allison D. Oakes, Bo Zhang,
Andrew E. Newhouse, Lilibeth C. Northern, Allison M. Chartrand,
Logan R. Will, Kathleen M. Baier, and William A. Powell
Abstract
The key to successful transformation of American chestnut is having the correct combination of explant
tissue, selectable markers, a very robust DNA delivery system, and a reliable regeneration system. The most
important components of this transformation protocol for American chestnut are the following: starting
out with rapidly dividing somatic embryos, treating the embryos gently throughout the Agrobacterium
inoculation and cocultivation steps, doing the cocultivation step in desiccation plates, and finally transferring the embryos into temporary-immersion bioreactors for selection. None of these departures from
standard Agrobacterium transformation protocols is sufficient by itself to achieve transgenic American
chestnut, but each component makes a difference, resulting in a highly robust protocol.
The average transformation efficiency that can be expected using the described protocol is approximately 170 stable embryogenic transformation events per gram of somatic embryo tissue, a considerable
improvement over the 20 transformation events per gram we reported in 2006 (Maynard et al. American
chestnut (Castanea dentata (Marsh.) Borkh.) Agrobacterium protocols, 2nd ed., 2006). We have regenerated nearly 100 of these events, containing 23 different gene constructs, into whole plants. As of the fall
of 2013, we had a total of 1,275 transgenic chestnut trees planted at eight locations in New York State and
one in Virginia. Based on a combination of field-trial inoculations, greenhouse small-stem inoculations,
and detached-leaf assays, we have identified three transgenes that produce stronger resistance to chestnut
blight than non-transgenic American chestnut. Depending on the transgene and the event, this resistance
can be either intermediate between American chestnut and Chinese chestnut, approximately equal to or
even higher than the resistance naturally found in Chinese chestnut.
Key words bar, Bioreactor, Cocultivation, Desiccation, nptII, Oxalate oxidase, Paromomycin,
Periodic immersion, Somatic embryogenesis
Introduction
The American chestnut was one of the most important deciduous
tree species in the eastern United States. Towering over most other
trees, it often made up 25 % or more of the overstory [1]. In 1904,
the disease that later became known as chestnut blight was discovered
in the Bronx Zoological Park [2] by the zoos chief forester,
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_13, Springer Science+Business Media New York 2015
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Fig. 1 The first of two transgenic American chestnut trees were planted in the
field on June 7, 2006, by Charles Maynard (left) and Linda McGuigan (right)
146
Fig. 2 (a) Periodic immersion bioreactors (RITA) used to select transformed somatic embryos of American
chestnut; (b) a closer view of American chestnut somatic embryos being selected for the bar marker gene
147
Fig. 3 (a) A GFP-positive root emerging from a transgenic American chestnut shoot; (b) the same shoot under
white light; (c) a GFP-positive radical emerging from a nut from a F1 cross between a transgenic a wild-type
American chestnut; (d) the same radical under white light. The scale bars are 1.0 mm
148
Fig. 4 American chestnut plantlets transplanted to potting mix and topped with
small plastic bags to maintain high relative humidity (a). The pots are maintained
in a specially modified high-humidity growth chamber (b)
Materials
2.1 Specialized
Equipment
and Supplies
2.2 Solutions
and Media
All solutions should be made with sterile distilled water unless otherwise noted. Media containing Phytagel are dispensed into three
149
150
Plant Material
2.4 Agrobacterium
Strains and Vectors
151
Methods
152
153
154
3.5 Rooting
and Acclimatization
of Chestnut
Microshoots
155
156
Notes
1. Nephelo sidearm flasks can be inserted directly into a spectrophotometer equipped with a suitable adapter, allowing for a
quick nondestructive determination of optical density. The
attached flask sticks up too far to close the spectrophotometer
lid but a thick, dark-colored cloth can be draped over the flask
to prevent ambient light from interfering with spectrophotometer readings.
2. As an option, 2 g/L of activated charcoal can be added to the
rooting medium instead of the 4-day dark treatment. If charcoal is to be used, it should be autoclaved separately with 1/2
the final volume of distilled water. The autoclaved and partially
cooled (~55 C) rooting medium made up at 2 the final volume should then be added to the containers in a laminar flow
hood, swirled briefly to mix, and allowed to cool. Alternatively,
when we have several hundred shoots to root, we use sterile
disposable plastic clamshell fast-food containers. Purchased
by the case (500/case), these cost ~7 cents each. They will hold
~200 mL of rooting medium with ~20 to 30 shoots per case.
3. Collect only from chestnut trees in flowering groups. Castanea
is almost completely self-sterile so ovules from isolated trees
will abort. Expect to see large tree-to-tree differences in contamination rates and ease of establishment of cell lines; therefore, it is better to collect a few burs (perhaps 1015) from
many different trees rather than a lot of burs from a few trees.
Burs can be kept at 4 C in sealed plastic bags for a week or
longer, but if the spines turn brown, the ovules inside are probably brown too, meaning they are dying.
4. The original pVspB-OxO vector carries three genes: a selectable
marker (bar), a scorable marker (gfp), and a putative blight resistance gene (OxO). The bar gene codes for a phosphinothricin
acetyltransferase [39] and is controlled by a potato ubiquitin
(Ubi3) promoter and terminator [40]. This gene conveys resistance to glufosinate-ammonium. The gfp gene codes for a modified green fluorescent protein (mgfp5-ER). It is driven by a
CaMV 35S promoter and terminator [41]. The OxO gene codes
for a wheat germin-like oxalate oxidase gene [42]. It is driven by
a soybean vegetative storage protein (VspB4) promoter [43]
and has an actin 2 (Act2) terminator. Details of the construction
of pVspB-OxO are in [26]. When we used this vector alone, we
included 30.27 M glufosinate-ammonium in the selection
medium. When we did co-transformations with two plasmids,
one plasmid had the gfp and bar genes while the second had an
nptII gene and the gene of interest. We included both glufosinate-ammonium and Paromomycin in the original three
selection media (Solid, Liquid and Embryo Initiation + Par).
157
In 2013, when we stopped doing co-transformations, we eliminated the plasmid containing the bar gene and with it the need
to include glufosinate-ammonium.
5. Be careful not to cut into the nut or else the bleach solution
will kill the ovules. Treat only as many nuts as can be processed
in 1 day. With practice, it is possible to extract and plate out
ovules from approximately 15 to 20 nuts in 1 h, but it is tiring
because of the tough seed coats. We found that one person
could work for approximately 3 h at a sitting.
6. In immature chestnuts, all the ovules are in a little cluster at the
pointy end of the nut.
7. Castanea nuts are polyembryonic with 12 or more ovules in
each nut. Soon after fertilization, all the ovules begin to grow,
but within 6 weeks, one will have expanded to fill the nut while
the rest will have aborted. The optimum stage of development
for establishing somatic embryo cell lines is when the cluster of
ovules has begun to develop, but the dominant ovule is still
less than three times the size of the other ovules in the group.
If the largest ovule in the cluster is too big and the others are
brown or black, discard the nut.
8. Expect a high contamination rate. Also expect some trees to
have higher contamination than others.
9. There is usually a burst of initial contamination from fastgrowing fungi and bacteria, but slow-growing bacteria can
show up after a month or more. If apparently clean ovules
are repeatedly rescued, these slow-growing contaminants
may not be discovered for many weeks.
10. A broad range of tissue types will emerge from the ovules. The
key is being able to distinguish between callus and somatic
embryos. Somatic embryos look like clusters of balloons or
grapes. They are smooth and regular in shape. Callus is more
rough-surfaced and irregular in shape than the somatic embryos.
11. It requires a large number of nuts, preferably collected from a
number of different trees, to eventually end up with a small number of vigorous somatic cell lines. In 2004, our lab received burs
from 18 trees, explanted more than 3,000 ovules, and 6 months
later had five cell lines suitable for transformation studies.
12. If cell cultures are stressed due to infrequent transfers to fresh
medium, transformation efficiency will plummet.
13. The purpose of this step is to induce the VIR genes, not to
grow more bacteria.
14. Screening transformation events for correct DNA integration
is an important follow-up for the transformation process, but
requires equipment and expertise well beyond that available in
most tissue culture laboratories. The simplest screen is to use
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159
20. Transfer all shoots, even those not rooted, to the PR low
BA. More shoots will produce roots during this step. Shoots
that are still not rooted at the end of this step can also be potted; they may produce ex vitro roots. This will also allow time
for shoots with tip dieback to begin elongating an axillary bud.
During this stage, it is normal to witness shoot-tip dieback. In
some cases, it can be as severe as 50 % of the total shoot height.
21. Even though the soil mix is not sterile, it is important to maintain a high degree of cleanliness in this step. Estimate 500 mL
of potting mixture per 7 tube. Many potting mixes have fertilizer incorporated in the mix; however, Fafard Super-Fine
Germinating Mix does not. We add Scotts Micromax Granular
Micronutrients at the rate of 1.0 g/L of dry potting mix. After
thoroughly incorporating the dry components, we add ~1 L of
tap water per liter of potting soil and then mix again until uniformly moistened.
22. The roots will be very fragile. It is helpful to break up the solid
medium with forceps before removing the plantlets. This stage
is the beginning of non-sterile conditions, which is why all of
the tissue culture medium must be removed from the roots.
Any medium left on the roots will promote fungal growth. As
an additional precaution, plantlets can be dipped in a fungicide
solution.
23. The plastic bag functions much like the sweat tent used in
horticulture to propagate cuttings. It is important to begin
increasing the light level at this stage.
24. The Bt treatments start 2 weeks after the bags are clipped and
alternate with the fertilization of Hoagland solution.
25. Many commercial potting mixes that contain peat moss also
harbor viable fungus gnat eggs. These hatch into fungus gnat
larvae, which have insatiable appetites for plant roots. We routinely treat all acclimatizing plantlets every 2 weeks with a Bt
soil drench.
26. For the combined rooting and acclimatization process, expect
to see an overall survival rate of 50 %. In the greenhouse and
subsequent field planting, we have found that micropropagated plantlets lag behind seedlings of the same age for the first
growing season.
Acknowledgments
Financial support was provided by the Forest Health Initiative, the
Monsanto Fund, the American Chestnut Foundation (New York
chapter and National), USDA-Biotechnology Risk Assessment
Grant program (BRAG), the Consortium for Plant Biotechnology
Research (CPBR), and ArborGen LLC.
160
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
161
springer.com/article/10.1007%2Fs11248013-9708-5#
Newhouse AE, Zhang B, Northern L, Maynard
CA, Powell WA (2010) Analysis of transgenic
American chestnut. Phytopathology 100:S89
Komari T, Hiei Y, Saito Y, Murai N, Kumashiro
T (1996) Vectors carrying two separate
T-DNAs for co-transformation of higher plants
mediated by Agrobacterium tumefaciens and
segregation of transformants free from selection markers. Plant J 10:165174
McGuigan L, Northern LC, Stewart KE,
Powell WA, Maynard CA (2012) Transforming
American chestnut somatic embryos using a
temporary-immersion
bioreactor
system.
Poster presented at the Fifth International
Chestnut Symposium, 48 Sep 2012, National
Conservation Training Center, Shepherdstown,
WV
Cheng M, Hu T, Layton J, Liu C, Fry JE
(2003) Desiccation of plant tissues postAgrobacterium infection enhances T-DNA
delivery and increases stable transformation
efficiency in wheat. In Vitro Cell Dev Biol
Plant 39:595604
Hoagland DR, Arnon DI (1950) The waterculture method for growing plants without
soil, vol Circ. 347. Univ. of Calif. Agric. Exp.
Station, Berkley
Hood EE, Gelvin SB, Melchers LS, Hoekema
A (1993) New Agrobacterium helper plasmids
for gene transfer to plants. Transgenic Res 2:
208218
White J, Chang SY, Bibb MJ (1990) A cassette
containing the bar gene of Streptomyces hygroscopicus: a selectable marker for plant transformation. Nucleic Acids Res 18:1062
Garbino JE, Belknap WR (1994) Isolation of a
ubiquitin-ribosomal protein gene (ubi3) from
potato and expression of its promoter in transgenic plants. Plant Mol Biol 24:119127
Haseloff J, Siemering KR (1998) The uses of
GFP in plants. In: Chalfie M, Kain S (eds)
Green fluorescent protein: properties, applications, and protocols. Wiley-Liss, New York,
pp 191220
Dratewka-Kos E, Rahman S, Grzekzak ZF,
Kennedy TD, Murray RK, Lane G (1989) The
polypeptide structure of germin as deduced
from cDNA sequencing. J Biol Chem 264:
48964900
Mason HS, DeWald DB, Mullet JE (1993)
Identification of a methyl jasmonate-responsive
domain in the soybean vspB promoter. Plant
Cell 5:241251
Chapter 14
Chestnut, European (Castanea sativa)
Elena Corredoira, Silvia Valladares, Ana M. Vieitez, and Antonio Ballester
Abstract
Development of a system for direct transfer of antifungal candidate genes into European chestnut (Castanea
sativa) would provide an alternative approach to conventional breeding for production of chestnut trees
that are tolerant to ink disease caused by Phytophthora spp. Overexpression of genes encoding PR proteins
(such as thaumatin-like proteins), which display antifungal activity, may represent an important advance in
control of the disease. We have used a chestnut thaumatin-like protein gene (CsTL1) isolated from
European chestnut cotyledons and have achieved overexpression of the gene in chestnut somatic embryogenic lines used as target material. We have also acclimatized the transgenic plants and grown them on in
the greenhouse. Here, we describe the various steps of the process, from the induction of somatic embryogenesis to the production of transgenic plants.
Key words Agrobacterium tumefaciens, Castanea sativa, Forest biotechnology, Genetic transformation, gfp, Somatic embryogenesis, Thaumatin-like protein
Introduction
European chestnut (Castanea sativa Mill.) is a tree of great historical,
ecological, and economic significance, and it is distributed within
25 European countries, covering an area of over two million hectares [1]. Chestnuts, which have been cultivated for centuries, were
used as a staple food [2], and chestnut wood was used to make
house frames and furniture, for tannin production and as source of
renewable energy.
Ink disease, mainly caused by Phytophthora cinnamomi Rand
and P. cambivora (Petri) Buis, is one of the most destructive diseases
affecting European chestnut [3]. The species is also threatened by
the ascomycete fungus Cryphonectria parasitica (Murr.), which
causes blight or canker disease. However, the trees partially recover
from this disease as a result of the natural occurrence of hypovirulence [4], which causes nonlethal, superficial (healing) cankers that
are restricted to the outer parts of the bark.
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_14, Springer Science+Business Media New York 2015
163
164
165
2
2.1
Materials
Plant Material
2.2 Agrobacterium
tumefaciens Strain
and Vector
166
LB
T-nos
npt II
Pro-nos
T-S
CsTL1
Pro-S Pro-rolD
egfpER
T-S
Fig. 1 Structure of T-DNA region of the binary vector pK7WG2D-TAU [10]. T-nos, Pro-nos, terminator, and promoter of nopaline synthase gene, respectively; nptII, neomycin phosphotransferase marker gene; T-35S,
Pro-35S, terminator, and promoter of CaMV 35S RNA gene, respectively; CsTL1 gene encoding a thaumatinlike protein, Pro-rolD the rol root loci D (rolD) promoter, egfpER enhanced green fluorescence protein gene with
endoplasmic reticulum-targeting signal, RB, LB T-DNA right and left border, respectively
driven by the rol root loci D (rolD) promoter (see Note 3) and
a neomycin phosphotransferase (nptII) selectable marker gene
driven by the nopaline synthase (nos) promoter.
2.3
Stock Solutions
2.4
Culture Media
2.4.1 For
Agrobacterium
1. Agrobacterium growth medium (Luria Bertani (LB) [18] + antibiotics): 10 g/L Bacto-tryptone, 5 g/L Bacto yeast extract,
10 g/L NaCl, kanamycin (50 mg/L), nalidixic acid (50 mg/L),
and pH 7. Add Bacto Agar (1.5 %) to prepare solid LB medium.
Antibiotics are added after autoclaving when medium is cooled.
1. Axillary shoot proliferation medium: Gresshoff and Doy (GD)
medium [19] (Duchefa, Netherlands), BA (0.44 M), sucrose
(3 %) and Bacto Agar (0.7 %) (Table 1).
2. Somatic embryo induction medium for zygotic embryos (M1Z): Murashige and Skoog (MS) medium [20], sucrose (3 %),
agar (0.6 %), casein hydrolysate (500 mg/L), 2,4-D (4.52 M),
and BA (0.88 M) (Table 1).
MS
500
2.22 or 4.44
5.37 or 21.48
30
Basal medium
Glutamine (mg/L)
BA (M)
NAA (M)
IBA (M)
2,4-D (M)
Sucrose (g/L)
Maltose (g/L)
30
4.52
0.88
500
MS
SE induction
in ZE (M1-Z)
30
0.54
0.44
438
MS
SE proliferation
30
MS
SE maturation
30
0.49
0.44
200
MS
SE germination
30
0.44
GD
Axillary shoot
proliferation
6.5
30
122.5
1/3GD
Shoot
rootinga
BA 6-benzyladenine, 2,4-D 2,4-dichlorophenoxyacetic acid, GD Gresshoff and Doy, 1/3 GD GD with 1/3 macronutrients, IBA indole-3-butyric acid, MS Murashige and
Skoog, MS MS half-strength macronutrients, NAA naphthaleneacetic acid, SE somatic embryos, ZE zygotic embryos
a
After 2448 h in rooting medium, shoots were transferred to the same medium without AIB for 4 weeks
SE induction in
leaves/apex (M1-L)
Components
Table 1
Culture media used in the different steps of somatic embryogenesis in European chestnut
168
169
Methods
3.1 Induction
of Somatic
Embryogenesis
from Zygotic Embryos
170
3.2 Induction
of Somatic
Embryogenesis
from Leaf and Shoot
Apex Explants
Fig. 2 Different steps in the production of transgenic plants of European chestnut. (a) Chestnut shoot multiplication cultures from which apex and leaf explants (b) are isolated for induction of somatic embryogenesis.
(c) Somatic embryos initiated from leaf explant. (d) Cluster of putatively transformed somatic embryos isolated
after 8 weeks in selection medium. (e) Transformed cluster of somatic embryos observed under white light.
(f) The same cluster of somatic embryos observed under blue light showing green fluorescence. (g) The gfp
expression on shoot apex of transgenic plant visualized with an epi-fluorescence stereomicroscope.
(h) Transgenic plants after 6 weeks acclimatization in the growth chamber. Scale bars in ce, 1 mm
171
3.4 Preparation
of Agrobacterium
Inoculum for Infection
3.5 Infection
and Cocultivation
of Somatic Embryos
172
3.6 Plantlet
Regeneration
173
1. Isolate cotyledonary somatic embryos (46 mm) from transgenic cultures and transfer them to Petri dishes containing
25 mL maturation medium for 4 weeks.
2. At the end of this culture period, transfer the somatic embryos
to Petri dishes containing 25 mL pregermination medium and
store the Petri dishes at 4 C for 2 months.
3. The transgenic somatic embryos must then be cultured for
8 weeks on Petri dishes containing 25 mL germination medium
(Table 1).
4. At the end of this period, determine both the number of germinating embryos showing signs of plant conversion (with
development of both root and shoot) as well as those embryos
exhibiting only shoot development (see Note 12).
5. Excise the shoots from germinating embryos showing only
shoot development and subculture them on shoot proliferation
medium, according to the previously defined procedure [13].
6. Elongated shoots (1520 mm) were cultured in glass jars containing 30 mL shoot rooting medium for 2448 h and subsequently transfer to an auxin-free medium (Table 1) [13].
7. After 4 weeks under standard growth conditions, record the
percentage of rooting obtained in both transgenic and control
(wild-type) shoots.
8. Confirm the transgenic nature of the regenerated plantlets by
molecular analyses. Fluorescence in different tissues (shoots,
leaves, roots) should also be observed (Fig. 2g).
9. Isolate the rooted shoots from the glass jars, avoiding damage
to the roots, which should be washed with tap water. Transplant
the rooted plantlets in pots (with drainage holes) containing
sterilized peat to perlite (3:1) and acclimatize them in a phytotron or growth chamber at 25 1 C and 90 % relative humidity under a 16-h photoperiod (100 mol/m2/s) for 68 weeks
or until resumption of shoot growth is evident (Fig. 2h).
10. Transfer the plants to a greenhouse for further growth. Irrigate
the plants once a week with Hoaglands solution and then with
tap water when necessary.
Notes
1. Currently European chestnut can be micropropagated from
both juvenile and mature material through axillary shoot
proliferation method. Efforts have focused on regeneration
systems that enable clonal propagation of selected mature
chestnut trees. Large-scale propagation is often challenging,
as the protocols require optimization for a specific cultivar.
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175
7. In chestnut, the multiplication and maintenance of embryogenic capacity can be carried out by either of two methods:
(1) secondary or repetitive embryogenesis from isolated
somatic embryos at torpedo-cotyledonary stages which develop
secondary embryos from the root-hypocotyl zone and (2) subculture of nodular embryogenic masses or proembryogenic
masses (PEMs) [21]. PEMs are produced from the surface of
somatic embryos. Despite the low induction rates from original explants, chestnut embryogenic cultures show a high
capacity for secondary embryogenesis, which ensures the
maintenance of embryogenic competence.
8. As the induction of somatic embryogenesis, the transformation
efficiency is clearly genotype dependent [9, 10], and the use of
different genotypes is highly recommended for these purposes.
9. The coculture period is clearly genotype dependent and should
be evaluated with the specific target material used, although in
our experience with European chestnut, 45 days of coculture
is sufficient for all the genotypes tested [810].
10. The concentration of kanamycin was selected on the basis of
previous results, but should be reevaluated for a specific chestnut material. The original creamy-yellowish color of the
embryo clumps became brownish/blackish after 23 weeks of
culture in selection medium. However, the addition of antioxidants such as cysteine, ascorbic acid, or acetosyringone in
either cocultivation or selective media is not necessary in
European chestnut and even may be detrimental for the transformation efficiency [8, 10].
11. Each transformation event should be taken as the start of a
putative transgenic line. Each transgenic embryogenic line
should be derived from one somatic embryo to confirm that
the line is the consequence of a unique transformation event.
The line should be routinely maintained by secondary embryogenesis separated from the other transgenic lines produced.
12. Regardless of the genotype, the rate of embryo conversion
(shoot + root development) is very low for both European and
American chestnut. The limited number of plantlets produced
makes subsequent analyses that should be carried out with
these plants difficult. However, some of the embryos cultured
on germination medium develop only a shoot as a partial
germination response, and this provides an opportunity to
multiply and root these shoots to obtain an unlimited number
of transgenic European chestnut plants by proliferation of axillary shoot cultures. Efforts should obviously be made to
increase plantlet conversion from transgenic somatic embryos;
however, at present this method (axillary shoot proliferation) is
the only realistic alternative. At this stage, the plants may be
used for both molecular analyses and/or for testing the resistance to fungal attacks.
176
References
1. Conedera M, Manetti MC, Giudici F et al
(2004) Distribution and economic potential of
the sweet chestnut (Castanea sativa Mill.) in
Europe. Ecol Mediter 30:4761
2. Bellini E (2005) The chestnut and its resources:
images and considerations. Acta Hort 693:
8596
3. Vannini A, Natili G, Anselmi NA et al (2010)
Distribution and gradient analysis of ink disease in chestnut forests. For Pathol 40:7386
4. Heiniger U, Rigling D (1994) Biological control of chestnut blight in Europe. Annu Rev
Phytopathol 32:581599
5. Vieitez E, Vieitez ML, Vieitez FJ (1996) El
castao. Edilesa, Len
6. Rodrguez L, Cuenca B, Lpez CA et al
(2005) Selection of Castanea sativa Mill. genotypes resistant to ink disease in Galicia
(Spain). Acta Hort 693:645651
7. Maynard CA, Powell WA, Polin-McGuigan
LD et al (2008) Chestnut. In: Kole C, Hall TC
(eds) Compendium of transgenic crop plants:
transgenic forest tree species. Blackwell,
Chichester, pp 169192
8. Corredoira E, Montenegro D, San-Jos MC
et al (2004) Agrobacterium-mediated transformation of European chestnut embryogenic
cultures. Plant Cell Rep 23:311318
9. Corredoira E, San-Jos MC, Vieitez AM et al
(2007) Improving genetic transformation of
European chestnut and cryopreservation of
transgenic lines. Plant Cell Tiss Org Cult 91:
281288
10. Corredoira E, Valladares S, Allona I et al (2012)
Genetic transformation of European chestnut
somatic embryos with a native thaumatin-like
protein (CsTL1) gene isolated from Castanea
sativa seeds. Tree Physiol 32:13891402
11. Vanblaere T, Szankowski I, Schaart J (2011)
The development of a cisgenic apple plant.
J Biotechnol 154:304311
Chapter 15
Grapevine (Vitis vinifera L.)
Laurent Torregrosa, Sandrine Vialet, Anglique Adivze,
Pat Iocco-Corena, and Mark R. Thomas
Abstract
Grapevine (Vitis) is considered to be one of the major fruit crops in the world based on hectares cultivated
and economic value. Grapes are used not only for wine but also for fresh fruit, dried fruit, and juice production. Wine is by far the major product of grapes, and the focus of this chapter is on wine grape cultivars.
Grapevine cultivars of Vitis vinifera L. have a reputation for producing premium quality wines. These
premium quality wines are produced from a small number of cultivars that enjoy a high level of consumer
acceptance and are firmly entrenched in the market place because of varietal name branding and the association of certain wine styles and regions with specific cultivars. In light of this situation, grapevine improvement by a transgenic approach is attractive when compared to a classical breeding approach. The transfer
of individual traits as single genes with a minimum disruption to the original genome would leave the
traditional characteristics of the cultivar intact. However, a reliable transformation system is required for a
successful transgenic approach to grapevine improvement. There are three criteria for achieving an efficient
Agrobacterium-mediated transformation system: (1) the production of highly regenerative transformable
tissue, (2) optimal cocultivation conditions for both grapevine tissue and Agrobacterium, and (3) an efficient
selection regime for transgenic plant regeneration. In this chapter, we describe a grapevine transformation
system that meets these criteria. We also describe a protocol for the production of transformed roots
suitable for functional gene studies and for the production of semi-transgenic grafted plants.
Key words Agrobacterium, Antibiotic sensitivity, Embryogenic callus, Grapevine, Hairy roots,
Reporter genes, Plant regeneration, Semi-transgenic grafted plants, Selectable markers, Transformation
efficiency, Transgenic, Vitis
Introduction
Most of the known grapevine wine varieties have been vegetatively
propagated for several centuries. The reasons for the persistence of
traditional European grapevine (Vitis vinifera L.) cultivars for wine
production are many with both plant and human factors involved
[1]. All V. vinifera cultivars are highly heterozygous and do not
breed true from seed. The combination of genes in a heterozygous
genome responsible for wine quality is conserved by vegetative
propagation. Thus, classical breeding programs, particularly those
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_15, Springer Science+Business Media New York 2015
177
178
179
Fig. 1 Successive stages of the plant transformation procedure in grapevine. (a) Inflorescence with immature
flower buds produced on cutting and ready for anther culture (bar = 5 mm). (b) Embryogenic calli grown on
GS1CA prior to cocultivation (bar = 1 mm). (c) Bright clusters of gfp-expressing cells observed 5 weeks after
cocultivation (bar = 50 m). (d) Clusters of kanamycin-resistant embryogenic cells visually selected among
dead tissue 2 months after cocultivation (bar = 1 mm). (e) Germination of a kanamycin-resistant embryo on
MG1 medium (bar = 5 mm). (f) Well-developed embryo before trimming of cotyledons and roots (bar = 5 mm).
(g) Growth and axillary branching of the embryo apical meristem on BFe2 medium with 50 g/mL kanamycin
(bar = 5 mm). (h) Rooted transformed plantlets on micropropagation medium. Both plants from the left are
transformed by VlmybA1-2 gene that activates anthocyanidin pigmentation in all vegetative organs, both
plants from the right being untransformed controls
180
181
2
2.1
Materials
Plant Material
2.2 Agrobacterium
Strains
182
183
12. Amino acid mix (1,000, [17]): 100 g/L of glutamine, 10 g/L
of phenylalanine, and 2 g/L of glycine.
13. 2,4-Dichlorophenoxyacetic acid (2,4-D, 1 mM): Dissolve
44.2 mg in 1 mL 10 N NaOH. Add nanopure water and heat
until completely dissolved. Make up the volume to 200 mL in
nanopure water.
14. 6-Benzylaminopurine (BAP, 1 mM): Dissolve 45.04 mg in
1 mL of 10 N NaOH. Add nanopure water to make a volume
of 200 mL.
15. Thidiazuron (TDZ, 500 M): Dissolve 22 mg in 1 mL
Dimethyl sulfoxide (DMSO). Add nanopure water to make a
volume of 200 mL.
16. 3-Naphthoxyacetic acid (NOA, 1 mM): Dissolve 40.44 mg in
1 mL 10 N NaOH. Add nanopure water to make a volume of
200 mL.
17. Indole-3-acetic acid (IAA, 1 mM): Dissolve 35.0 mg in 1 mL
10 N NaOH. Add nanopure water to make a volume of
200 mL.
18. -Naphthaleneacetic acid (NAA, 1 mM): Dissolve 37.2 mg in
1 mL 10 N NaOH. Add nanopure water to make a volume of
200 mL.
19. Timentin (Smith-Kline Beecham, Boronia, Australia).
20. Augmentin: Dissolve in nanopure water at a concentration of
250 mg/mL. Store as aliquots at 20 C.
21. Claforan or cefotaxime stock solution: Dissolve in nanopure
water at a concentration of 250 mg/mL. Store as aliquots at
20 C.
22. Kanamycin monosulfate: Dissolve in nanopure water at a concentration of 100 mg/mL. Store as aliquots at 20 C.
23. Hygromycin B: Dissolve in nanopure water at a concentration
of 25 mg/mL.
24. Rifampicin: Dissolve in dimethyl sulfoxide (DMSO) at a concentration of 25 mg/mL. Store as aliquots at 20 C.
25. Acetosyringone: Dissolve in absolute ethanol at a concentration
of 100 mM. Store as aliquots at 20 C.
2.5 Plant Material
and Tissue Culture
184
185
1. Flower and stem surface disinfectant: 7 % (w/v) calcium hypochlorite filtered solution, corresponding to 5 % active chlorine, and containing 0.1 % (v/v) Tween 20 as wetting agent
(see Note 2).
2. 70 % (v/v) ethanol.
3. Soil mixture for the growth of transgenic plants: Peat moss,
compost, and sand with a ratio of 1:1:1 (v/v/v).
4. Millipore 100 m nylon net filter (St. Quentin-Yveline,
France).
5. Corning 50 mL centrifuge tube (Corning Incorporated, NY).
6. Whatman No. 1 filter paper (Whatman, Springfield Milland, UK).
7. Magenta GA7-3 vessels (Life Technologies).
8. Parafilm M.
9. Tween 20.
10. 55 and 90 mm Petri dishes.
11. Stone wool substrate (4 cm diameter) (http://www.cultilene.nl).
Methods
3.1 Plant
Transformation
3.1.1 Initiation
and Maintenance
of Embryogenic Cultures
186
3.1.3 Cocultivation
of Embryogenic Callus
with Agrobacterium
1. Add 20 mL of the bacterial suspension to each gram of embryogenic calli in a 50 mL Corning centrifuge tube and then shake
vigorously for 12 s.
2. After a 10-min incubation at 25 C, separate the calli with
gentle shaking from the liquid phase with a 100 m 3 M nylon
net filter.
3. Briefly blot on sterile Whatman filter paper and transfer onto a
90 mm Petri dish containing CM medium (see Note 7). Seal
the Petri dishes with Parafilm to prevent the culture from
drying out and incubate in the dark at 22 C for 48 h.
4. After cocultivation, wash the embryogenic calli in a 50 mL
Corning tube with 20 mL LCM medium plus 1,000 g/mL
Timentin.
5. Retrieve the calli with a 100 m nylon net and blot briefly on
filter paper. Gently fragment the calli with forceps and distribute evenly onto 55 mm Petri dishes containing GS1CA
medium with 1,000 g/mL Timentin. Incubate cultures in
the dark at 28 C for 2 weeks (see Note 8).
3.1.4 Selection
of Transgenic Embryos
and Regeneration
of Transgenic Plants
187
1. Transfer the resulting healthy well-rooted plantlets into individual, water-saturated 4 cm diameter stone wool blocks. Place
the blocks in a plastic box containing few mm of water at the
bottom. Cover the boxes with plastic film and maintain under
in vitro culture conditions.
2. During this period, maintain water saturation of the blocks
irrigating with MS/2 liquid medium without sugar until and
progressively open plastic film.
3. After 24 weeks, when the roots arise from the blocks, transfer
the block and the plant in 10 cm diameter plastic containing
compost and incubate in same culture conditions for 2 or 3
188
1. Young stem sections (1050 cm from the tip) are taken from
actively growing plants from the greenhouse or the field.
Leaves and tendrils are sectioned 1 cm from their base and
maintained in a cold room (24 C) until use.
2. Stem explants are sectioned into short fragments composed of
a node with 1 cm of internode above and 5 cm below and
immediately sterilized by immersion for 15 min into a solution
of filtered 70 g/L calcium hypochlorite with a few drops of
Tween 20 (see Note 12).
3. Explants are rinsed three times with sterile distilled water and
trimmed with a scalpel (5 mm) to remove injured tissues
(see Note 13). The cuttings are then inserted in 200 mm long
culture tubes containing 20 mL of MS/2 solid medium and
transferred under lower light (approximately 45 E/m2/s).
4. After 58 weeks, shoots arising from axillary buds are extracted
and cut into single-node micro cuttings and propagated on the
same medium.
5. When plantlets are 810 cm high (approx. 3 months time),
the stem is sectioned at 1 cm above a node and the two last
leaves removed by sectioning 23 mm from blade in order to
let 0.51 cm of petiole attached to the stem (see Note 14).
A typical experiment being is based on 12 vitro plantlets (36
inoculation ends).
3.2.2 Agrobacterium
Culture Preparation
and Inoculation
189
Notes
1. The medium used to induce and maintain embryogenic calli
cultures initiated from anthers can be successfully applied to
induce and maintain embryogenic calli from leaf explants [31].
2. Sodium hypochlorite solution with the same percentage of active
chlorine can be used in place of calcium hypochlorite. Solutions
can be stored overnight but for a maximum of 2 days.
3. This technique can be applied throughout the year with greenhouse material. Alternatively, inflorescences can be collected in
the vineyard at about 1214 days before anthesis.
190
Fig. 2 Successive stages of the root transformation procedure in grapevine. (a) Formation of calli from stem
and petiole stub 2 weeks after inoculation, ready to be explanted (bar = 5 mm). (b) Induction of non-transformed
adventitious roots below the point of inoculation (right). Plant on left has most roots only from callus at site of
inoculation (bar = 5 mm). (c) Browning of the callus 3 weeks after inoculation (bar = 2 mm). (d) Hairy roots (big
roots) arising from the callus 1 week after callus extraction (red hairy roots expressing the transgene
VlmybA1-2, white hairy roots only transformed by pRi oncogenes) (bar = 5 mm). (e) Hairy roots growing on LG0
medium with 200 g/mL Claforan and Augmentin, 2 weeks after extraction (bar = 10 mm). (f) Well-developed
hairy roots culture ready for a second round of propagation (bar = 10 mm). (g) Chimeric grapevine plant associating a non-transformed scion grafted on a root system deriving from a hairy root (bar = 10 mm)
191
192
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
193
194
Chapter 16
Melon (Cucumis melo)
Satoko Nonaka and Hiroshi Ezura
Abstract
Genetic transformation is an important technique used in plant breeding and to functionally characterize
genes of interest. The earliest reports of Agrobacterium-mediated transformation in the melon (Cucumis
melo) were from the early 1990s (Fang and Grumet, Plant Cell Rep, 9: 160164, 1990; Dong et al., Nat
Biotechnol 9: 858863, 1991; Valles and Lasa, Plant Cell Rep 13: 145148, 1994). These early studies
described three problems that decreased the efficiency of transformation: tetraploidy, chimeras, and escape.
Using a liquid culture system for somatic embryogenesis, Akasaka-Kenedy et al. (Plant Sci 166: 763769,
2004) overcame these problems and established an efficient transformation system; the protocol introduced in this chapter is based on this method.
Key words Agrobacterium-mediated transformation, Chimera, Escape, Liquid culture, Somatic
embryo, Tetraploidy
Introduction
In addition to being an important horticultural crop, the melon
(Cucumis melo L.) is also a useful experimental organism for understanding fruit ripening, as physiological and biochemical changes
in flavor development and texture occur during the ripening of the
fruit [1]. An understanding of the molecular mechanism underlying melon fruit ripening will require the isolation and functional
verification of genes contributing to this trait.
Transformation is a technique used to characterize the functions of genes of interest. In the last two decades, several types of
genetic transformation techniques have been developed in the
melon. Agrobacterium-mediated techniques reported in the early
1990s [24] identified three major problems affecting transformation in the melon: the induction of tetraploidy, chimeras, and
escape. Embryogenesis via cotyledon explants was later found to
reduce the occurrence of tetraploidy [5]. Embryogenesis is a useful
regeneration system because the embryos originate from a single
cell and the number of chimeric plants regenerated is therefore
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_16, Springer Science+Business Media New York 2015
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196
reduced. Problems with escape appear to be caused by the inefficient selection of transformed cells. Because the whole explant is
exposed to antibiotics when suspended in liquid media but not
when cultured on solid media, the liquid culture system achieves a
more effective selection of transformed cells. Akasaka-Kenedy et al.
[6] demonstrated that the use of a liquid culture system for
Agrobacterium-mediated melon transformation reduced the occurrence of tetraploidy, chimeras, and escape. The protocol introduced in this chapter is based on the method of Akasaka-Kenedy
et al. [6]. One mature melon seed was chopped into 1220 segments for the preparation of explants for genetic transformation.
These explants were cultured in liquid embryo induction (EI)
medium containing MS salts, MS vitamins, 3 % sucrose, 2 mg/l
2,4-D, and 0.1 mg/l BA for 2 days. After this 2-day pre-culture
period, the segments were inoculated with Agrobacterium
tumefaciens containing the desired transgene and cocultured on
solid EI medium with 0.8 % agar for 4 days in the dark. To select
transformed calli and embryos, the inoculated segments were
transferred to liquid EI medium containing 25 mg/l kanamycin
and 375 mg/l Augmentin and subcultured every 2 weeks.
Antibiotic-resistant embryos appeared on the surface of the
explants 34 months after A. tumefaciens transfection. For regeneration and plant development, the embryos were cultured on
solid MS medium containing 50 mg/l kanamycin and 375 mg/l
Augmentin without plant growth regulators (PGRs) for 1 month.
The transgenic melon plants were acclimated and grown in a
greenhouse. Three transgenic lines were obtained from 130
explants that, in theory, could have been derived from 6.5 seeds.
Thus, the transformation efficiency of this method was 2.3 % per
explant and 46.1 % per seed.
Materials
2.1 Agrobacterium
tumefaciens Strain
and Vector
2.2
Plant Material
197
2.3
Stock Solutions
2.3.1 MS salt
2.3.2 Vitamins
and Phytohormones
2.3.3 Antibiotics
and Selective Agents
198
2.4
Culture Media
Methods
The various steps of the protocol are summarized in Fig. 2 and
described in further detail below.
1. Seeds were wrapped with moisten paper towel for 1 week for
germination.
2. Germinated seedlings were in soil in 10 cm pot with molding.
The seedlings were grown in the greenhouse. The photoperiod and light intensity are natural condition in Tsukuba, Japan
(36.0 N, 140.0 E). Lowest temperature is 15 C, and highest
temperature is 25 C (see Note 2).
3. After the three leaves appearance, transplant each plant into a
40 cm pot with molding (one plant per pot).
199
200
3.2
Plant Preparation
Fig. 3 Plant regeneration and transformation in the melon (C. melo L. var. cantalupensis cv. Vedrantais).
(a) Sterilized melon seeds without seed coats. (b) Sterilized seeds are cut into 14 mm2 pieces. (c) Calli formed
at the cut surfaces of explants after 3 weeks of culture (bar = 2 mm). (d) Embryos developed in liquid EI
medium after 4 weeks of culture (bar = 4 mm). (e) Regenerated plants exhibited severe vitrification in liquid MS
medium after 8 weeks of culture. (f) Healthy plantlets growing in 1.0 % agar-solidified MS medium after
8 weeks of culture (bar = 6 mm). (g) A regenerated plant obtained from a somatic embryo after 12 weeks of
culture. (h) Blue spots were observed at the cut surfaces of explants 4 days after infection. (i) Strong GUS
expression was observed on proliferated calli on the explants grown in liquid EI selection medium 6 weeks
after infection. (j) Stable GUS expression was observed throughout the embryo 9 weeks after infection (color
figure online)
3.3 Preparation
of Agrobacterium
Culture for Infection
201
3.4 AgrobacteriumMediated
Transformation
3.5 Selection
for Putative
Transgenic Embryos
and Calli
1. After cocultivation, the explants were cultured in 10 ml of liquid EI medium containing 25 mg/l kanamycin and 375 mg/l
Augmentin with 100 ml volume of Erlenmeyer flask on a
rotary incubator shaker (100 rpm) at 25 C under a 16 h photoperiod with light intensity of 50 mol/m2/s (see Note 3).
2. The explants were subcultured and washed in water every
2 weeks (see Note 4).
3. After 34 months of culture, somatic embryos and calli were
observed at the cut surfaces of the explants (Fig. 3c, d, i, and j).
3.6 Regeneration
of Transgenic Plants
202
Notes
1. Melon cultivars: The protocol described is applicable to other
melon varieties such as C. melo L. var. reticulatus cv. Earls
Favourite.
2. The suitable temperature for melon cultivation is between 15
and 25 C. And melon grows well in low humidity condition.
3. Escape and chimeras: In this protocol, a liquid culture system
was used to select transgenic embryos derived from explants.
Because somatic embryos derive from a single cell and because
whole embryos are exposed to kanamycin, the occurrence of
escape and chimeras was reduced, even though a lower concentration of kanamycin was used than in the standard melon protocol. This result indicates that a liquid culture system is
effective for the selection of transformed embryos in the melon.
4. Overgrowth of Agrobacterium: When using a liquid culture
selection system, it is difficult to eliminate A. tumefaciens from
liquid medium containing sugar during the selection of transgenic embryos. Particular care must therefore be taken to
change the liquid EI medium when using this protocol. During
the subculture, the explants must be washed in sterilized water
and new culture vessels must be used.
5. Vitrification: Melon tissues are sensitive to vitrification. Somatic
embryos derived from liquid culture have often been found to
exhibit typical vitrification and fail to develop into plantlets
[5]. To avoid vitrification, 1.0 % agar-solidified G medium was
used for germination in this protocol. Embryos did not grow
in 0.4 % Gelrite-solidified medium. Using surgical tape to seal,
the culture vessels were also important for keeping the humidity low during plant regeneration.
203
References
1. Ezura H, Owino WO (2008) Melon, an alternative model plant for elucidating fruit ripening.
Plant Sci 175:121129
2. Fang G, Grumet R (1990) Agrobacterium tumefaciens mediated transformation and regeneration of muskmelon plants. Plant Cell Rep
9:160164
3. Dong JZ, Yang MZ et al (1991) Transformation
of melon (Cucumis melo L.) and expression from
the cauliflower mosaic virus 35S promoter in
transgenic melon plants. Nat Biotechnol
9:858863
4. Valles MP, Lasa JM (1994) Agrobacteriummediated transformation of commercial melon
(Cucumis melo L., cv. Amarillo Oro). Plant Cell
Rep 13:145148
5. Guis M, Amor MB et al (2000) A reliable system
for the transformation of cantaloupe charentais
melon (Cucumis melo L. va. Cantalupensis) leading to a majority of diploid regenerants. Sci
Hortic 84:9199
6. Akasaka-Kenedy Y, Tomita K et al (2004)
Efficient plant regeneration and Agrobacteriummediated transformation via somatic embryogenesis in melon (Cucumis melo L.). Plant Sci
166:763769
7. Deblaere R, Bytebier B, De Greve H, Deboeck
F, Schell J, van Montagu M, Leemans J (1985)
Efficient octopine Ti plasmid-derived vectors for
Agrobacterium-mediated gene transfer to plants.
Nucleic Acids Res 13:47774788
8. Hiei Y, Ohta S, Komari T, Kumashiro T (1994)
Efficient transformation of rice (Oryza sativa L.)
mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J
6:271282
Chapter 17
Peach (Prunus persica L.)
Silvia Sabbadini, Tiziana Pandolfini, Luca Girolomini,
Barbara Molesini, and Oriano Navacchi
Abstract
Until now, the application of genetic transformation techniques in peach has been limited by the difficulties
in developing efficient regeneration and transformation protocols. Here we describe an efficient regeneration protocol for the commercial micropropagation of GF677 rootstock (Prunus persica Prunus
amygdalus). The method is based on the production, via organogenesis, of meristematic bulk tissues characterized by a high competence for shoot regeneration.
This protocol has also been used to obtain GF677 plants genetically engineered with an empty hairpin
cassette (hereafter indicated as hp-pBin19), through Agrobacterium tumefaciens-mediated transformation.
After 78 months of selection on media containing kanamycin, we obtained two genetically modified
GF677 lines. PCR and Southern blot analyses were performed to confirm the genetic status.
Key words Agrobacterium tumefaciens, Fruit trees transformation, GF677 rootstock, Peach,
Regeneration via organogenesis
Introduction
Stone fruits, especially peach (Prunus persica), are among the most
important tree species of the Mediterranean basin. Viral diseases
represent one of the major problems affecting stone fruit trees and
causing significant agronomic and economic losses. Many viruses
are difficult to eradicate because antiviral treatments are either not
available or not effective; thus, preventing diseases from entering
and spreading in the fields is the most efficient and cost-effective
method for controlling infections. However, prevention and control tactics are often not effective and associated to environmental
sustainability issues and excessive costs for farmers. The genetic
transformation of these fruit species provides huge potentiality for
the obtainment of cultivars with increased resistance to pathogens
and also for the improvement of fruit production and quality. Until
now, the main problem in the genetic transformation of peach
has been the lack of an efficient in vitro regeneration protocol.
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_17, Springer Science+Business Media New York 2015
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207
2
2.1
Materials
Plant Material
1. 6-Benzylaminopurine (BAP, 1 mg/mL): Prepare stock by adding 50 mg of BAP to a 50-mL tube and add a few drops of 1 M
KOH until dissolved by stirring. Bring to volume with ultrapure water (Milli-Q system water). Stirring the solution while
adding water may be required to keep the material in solution.
Store at 20 C for up to 6 months.
LB
Pro-S
InLAX
HindIII
KpnI
BamHI
The hairpin cassette (hp-pBin19) (Fig. 1) used to genetically transform the rootstock GF677 plants contains the 543-base-long constitutive promoter 35S of the CaMV (Cauliflower mosaic virus),
followed by the 115-base-long intron of the LAX1 (InLAX) gene of
Medicago truncatula and the 253-base-long terminator sequence of
the nopaline synthase (NOS) gene of A. tumefaciens. The hairpin
cassette was subcloned in the T-DNA region of a derivative of pBin19
binary vector [12], containing the 984-base-long sequence of the
neomycin phosphotransferase II gene (npt II) and its regulatory
regions (i.e., the 307-base-long promoter sequence and the 256-baselong terminator sequence of NOS gene) (see Note 1). The recombinant plasmid was inserted into A. tumefaciens strain GV2260.
EcoRI
NOS-ter
NOS-Ter/npt II/Pro-NOS RB
Fig. 1 Schematic drawing of the T-DNA region of the hairpin cassette (hp-pBin19). Pro-35S, CaMV 35S promoter; InLax, the intron of the LAX1 (InLAX) gene of Medicago truncatula; NOS-ter, the terminator sequence of
the nopaline synthase (NOS) gene of A. tumefaciens; NOS ter/npt II/Pro-NOS, the plant selectable marker gene
cassette, neomycin phosphotransferase II gene (npt II ) controlled by the NOS promoter and terminator
208
Media
After pH adjustment, all media should be autoclaved at the temperature of 121 C and 1 bar for 20 min (see Note 2).
1. YEB medium for A. tumefaciens culture: 5 g/L of yeast extract,
1 g/L of peptone, 5 g/L sucrose, 480 mg MgSO4, 50 mg/L
kanamycin, 100 mg/L streptomycin, 100 mg/L rifampicin,
and 7.2 g/L agar (for solid medium) adjusted to pH 7.2 with
NaOH.
2. Meristematic bulk initiation medium (IM): 1,050 mg/L
KNO3, 400 mg/L NH4NO3, 200 mg/L KH2PO4, 400 mg/L
MgSO47H2O, 750 mg/L CaNO3, 200 mg/L NaH2PO4, MS
209
microelements and vitamins [13], 3 % sucrose, 0.7 % commercial agar, and 0.01 mg/L NAA. IM medium was supplemented
with increasing concentration of BAP during subsequent subcultures (from 1 mg/L up to 3 mg/L). The medium is adjusted
to pH 5.65.7 with KOH.
3. Meristematic bulk selection medium (SM): IM medium
enriched with 3 mg/L of BAP, increasing concentration of
kanamycin monosulfate (from 25 mg/L up to 70 mg/L) and
200 mg/L cefotaxime (see Note 3).
4. Meristematic bulk rooting medium: IM medium supplemented
with 1.5 mg/L IBA adjusted to pH 5.65.7 with KOH.
5. MS20 medium meristematic bulk infection solution: 4.4 g of
MS salts including vitamins, 20 g/L sucrose, adjusted to pH
5.2 with KOH.
6. MSH0 Agrobacterium-plant co-culturing medium: 4.4 g of
MS salts including vitamins, 30 g/L sucrose, 7 g/L plant agar,
adjusted to pH 5.2 with KOH, 1 L/mL of proline (200 mg/
mL stock solution), and 1 L/mL of acetosyringone (20 mg/L
stock solution). These last two components are added after
sterilization, as they are heat sensitive. The infection solution
has to be prepared fresh as required.
2.5 Transgenic Plant
Analysis
Methods
3.1 Initiation
and Regeneration
of Meristematic Bulks
Meristematic bulks (MBs) of the peach rootstock GF677 (P. persica P. amygdalus) are initiated from shoot tips that are mechanically and chemically treated in order to induce the formation of
MBs, which consist of organogenic calli able to efficiently differentiate and regenerate new adventitious shoots.
210
Fig. 2 Main steps of the rootstock GF677 regeneration and in vitro propagation: (a) Meristematic bulk obtained
after the four subcultures, after the elimination of the apical domes; (b) slice from a meristematic bulk able to
regenerate many adventitious shoots; (c) slices obtained from MB (approx. 1 cm2, 2 mm thick) and used for
in vitro micropropagation or genetic transformation
1. The production of MBs starts with the elimination of the apical dome from in vitro proliferating shoots, placed on a standard proliferation medium used for GF677 commercial
propagation (Fig. 2a).
2. The remaining basal cluster is subcultured on an initiation
medium (IM) supplemented with increasing concentrations of
BAP (from 1 up to 3 mg/L) for a total of four subcultures
(every 4 weeks for the first and second phases, and every 60
and 90 days, for the third and fourth phases). At the beginning
of each subculture, the apical domes of the initial proliferating
shoots originated from the MBs (Fig. 2b) are eliminated, and
the remaining meristematic tissues are cut in small slices
(1 cm2, 2 mm thick) that are used to repropagate MBs and also
used as starting plant material for the transformation experiments (Fig. 2c).
3. During the first two subcultures, the regenerated MBs are
transferred to new media every 4 weeks. The concentration of
BAP is increased from 1 mg/L (first subculture) to 2 mg/L
(second subculture).
4. During the last two subcultures, the regenerated MBs are
transferred to new media, after 60 days for the first one, and
after 90 days for the second one. The concentration of BAP is
raised from 2 to 3 mg/L.
5. MBs, when initiated, can be maintained on IM medium added
with 3 mg/L of BAP for a long period by keeping them proliferating and transplanted, at 24 C under a photoperiod of
16-h light (70 mol/m2/s) provided by white fluorescent
tubes.
3.2 A. tumefaciens
Preparation and Plant
Tissue Infection
211
3.3 Regeneration/
Selection
and Acclimatization
of Putative
Transformed Lines
212
Fig. 3 Main steps of the selective process: (a) regeneration and selection of GF677 at kanamycin concentration
of 25 mg/L; (b) rooting of GF677 selected lines; (c) acclimatization of putative transgenic lines
Table 1
Regeneration/selection scheme
Kanamycin concentration
2 subcultures-1 month
25 mg/L (pot)
2 subcultures-1 month
50 mg/L (pot)
4 subcultures-2 month
70 mg/L (pot)
4 subcultures-2 month
2 subcultures-1 month
Total period of 7 months
213
Notes
1. The neomycin phosphotransferase II gene (npt II), which confers resistance against kanamycin, has been used as selectable
marker gene, which is the most commonly used, and it is recommended for the highest efficiency in cleaning transgenic
regenerants at the chimeric state and finally in selecting stable
transgenic clones.
2. The antibiotics are added to the culture media after sterilization, as they are heat sensitive.
3. Cefotaxime is used to avoid A. tumefaciens contamination in
the regeneration/selection medium.
4. The electroporation protocol exploits the use of an electroporator (Bio-Rad) set up on 2.5 kW/cm. Immediately after
electroporation, 1 mL of liquid YEB medium is added to the
bacterial cells, and then the bacterial suspension is incubated at
28 C for 1 h and subsequently plated on solid YEB medium
containing the selective antibiotics rifampicin (100 mg/L),
kanamycin (50 mg/L), and streptomycin (100 mg/L), for
2 days at 28 C. Finally, the transformation reactions are verified by PCR analysis.
5. At the beginning of each subculture, the regenerating shoots
originated from the slices are eliminated, and then the proliferated MBs are fragmented again to expose new regenerating
cells to the selective agent.
6. The first two subcultures, at 25 mg/L of kanamycin, which
follow the transformation protocol, are made in Petri dishes,
while the regenerants are transferred on solid medium contained in glass pots for the all subsequent steps.
7. The regenerants are kept in a plant growth chamber at 24 C
under a 16-h photoperiod for all the selective process and following proliferating and rooting steps.
8. The transformation trials have been performed on a total of
600 meristematic sections of the rootstock GF677 that showed
a percentage of bulk regeneration of about 94 % during the
first phases of the selective process, which decreased to 86 %
during the last steps, with a maximum number of ten adventitious shoots regenerated per slice. After 78 months, we
obtained the first stable transgenic lines, which correspond to
a transformation efficiency of 0.33 %. The regeneration efficiency was very high even on media with increased kanamycin
concentration, but the fact that at the end of the selection procedure most of such regenerants were not transformed can be
explained by the fact that the cell regeneration events are rarely
originated by a single cell but mostly by a larger number of
214
initials cells, as generally happens in the organogenesis processes [14], creating as a consequence a high number of chimeric adventitious shoots, then difficult to bring at stable and
homogenous transgenic line. As a consequence, they perform
as escapes or unstable chimeric lines. Therefore, the selection
procedure remains the key factor for the achievement of new
stable transgenic lines.
9. For the analysis of putative transgenic lines, the genomic DNA
isolation is performed using the commercial kit Nucleon
Phytopure (GE Healthcare) starting from 1 g of leaves. PCR
analysis is performed using a couple of primers specifically
designed onto 35S promoter of the genetic construct, placed
next to the left border of the T-DNA. For Southern blot analysis, 15 g of genomic DNA was digested with Hind III, electrophoresed on a 0.7 % agarose gel in 1 TBE buffer, and
transferred for capillarity to a nylon membrane (Hybond-N+,
GE Healthcare). The DNA probe, corresponding to 540-bp
sequence of the NPTII gene, was labelled with [32P] dCTP
using Ready to go DNA labelling beads (-dCTP) (GE
Healthcare). Unincorporated nucleotides were removed with
ProbeQuant G-50 micro columns (GE Healthcare). The
membrane was hybridized overnight at 42 C in ULTRAhyb
buffer (Ambion). Labelled probe (106 cpm/mL) was added to
the hybridization buffer. The membrane was washed twice in
2 SSC containing 0.1 % SDS for 5 min and twice in 0.1 SSC
containing 0.1 % SDS for 15 min at 42 C. Autoradiography
was then performed using Kodak X-AR5 film (Fig. 4).
215
References
1. Meng X, Zhou W (1981) Induction of embryoid ad production of plantlets in vitro from
endosperm of peach. Acta Agric Univ Peking
7:9598
2. Hammerschlag FA, Bauchan G, Scorza R
(1985) Regeneration of peach plants from callus derived from immature embryos. Theor
Appl Genet 70:248251
3. Mante S, Scorza R, Cordts JM (1989) Plant
regeneration from cotyledons of Prunus persica. Prunus domestica and Prunus cerasus.
Plant Cell Tiss Org Cult 19:111
4. Scorza R, Morgens PH, Cordts JM, Mante S,
Callahan AM (1990) Agrobacterium-mediated
transformation of peach Prunus persica
L. Batsch leaf segments, immature embryos
and long term embryogenic callus. In Vitro
Cell Dev Biol 26:829834
5. Bhansali RR, Driver JA, Durzan DJ (1990)
Rapid multiplication of adventitious somatic
embryos in peach and nectarine by secondary
embryogenesis. Plant Cell Rep 9:280284
6. Pooler MR, Scorza R (1995) Regeneration of
peach Prunus persica L. Batsch rootstock cultivars from cotyledons of mature stored seed.
HortSci 30:355356
7. Zhou HC, Li M, Zhao X, Fan XC, Guo AG
(2010) Plant regeneration from in vitro leaves
of the peach rootstock Nemaguard (Prunus
persica x P. davidiana). Plant Cell Tiss Org
Cult 101:7987
Chapter 18
Strawberry (Fragaria ananassa)
Roberto Cappelletti, Silvia Sabbadini, and Bruno Mezzetti
Abstract
Genetic transformation in strawberry (Fragaria spp.) can be achieved by using the Agrobacterium-mediated
procedure on leaves from in vitro proliferated shoots. Regardless of the sufficient regeneration levels
achieved from leaf explants of some commercial strawberry genotypes, the regeneration of transformed
strawberry plants remains difficult and seems to be strongly genotype dependent. In fact, the main factors
that play an important role in the success of strawberry genetic transformation are the availability of an
efficient regeneration protocol and of an appropriate selection procedure of the putative transgenic shoots.
The strawberry genetic transformation protocol herein described relates to three genotypes resulted
from our experience with the highest regeneration and transformation efficiency. The study includes two
octoploid Fragaria ananassa cultivars, Sveva and Calypso, and a diploid F. vesca cultivar (Alpina W.O.).
All the different steps related to the leaf tissue Agrobacterium infection, coculture, and selection of regenerating adventitious shoots, as well as the following identification of selected lines able to proliferate and
root on the selective agent (kanamycin), will be described.
Key words Agrobacterium infection, Kanamycin selection, Leaf tissue regeneration, Strawberry
Introduction
The Agrobacterium tumefaciens-mediated transformation represents
one of the most common techniques of recombinant DNA, and it
is largely employed to obtain genetically modified plants. Efficient
transformation and regeneration methods are a priority for successful application of recombinant DNA technology to vegetative propagated plants such as strawberry.
To date, the most resourceful plant differentiation process for
recombinant DNA technology in strawberry remains adventitious
shoot organogenesis directly from somatic tissue or a previous callus
formation. However, detailed developmental and physiological characterization of the whole sequence of the organogenic processes in
strawberry somatic tissues is still mainly lacking [1]. The genotype and
type of explants represent important factors affecting the regeneration process and consequently the genetic transformation efficiency.
For several Fragaria ananassa genotypes, efficient regeneration
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_18, Springer Science+Business Media New York 2015
217
218
219
(about 5 %) was observed from the genotypes with the highest leaf
tissue regeneration efficiency (100 % of leaf tissue regeneration),
such as Sveva, a newly released octoploid cultivar (F. ananassa),
and the diploid F. vesca cv. Alpina W.O. Genotypes such as cv.
Onda and cv. Paros, performing with an intermediate leaf tissue
regeneration efficiency (about 80 % of leaves with adventitious
shoots), showed a reduced Agrobacterium transformation efficiency (13 %). Strawberry genotypes showing much lower percentages of leaf tissue regeneration (lower then 40 %) can be quite
more difficult to be transformed [23].
The achievement of stable genetically transformed plants is also
strictly related to the antibiotic (kanamycin) selection protocol,
starting from the early stage of leaf tissue regeneration, immediately after the Agrobacterium infection and coculture, and including
the in vitro proliferation and rooting of newly selected shoots.
Depending on the efficiency of the regeneration and transformation protocol, the first stable newly produced transgenic line can be
available after 56 months from the first transformation experiment. Selection of trasgenic plants using antibiotics must be optimize depending of genotype used through toxicity threshold tests
[24] and evaluating the possibility to use iterative or non iterative
method of selection [25]. GFP fluorescence technique nowadays
seems to be an alternative approach in order to help and in some
case replace antibiotics in strawberry selection protocols [26].
Materials
2.1 Agrobacterium
tumefaciens Strains,
Vector, and Plant
Selectable Markers
Explant Material
2.2
220
Media
After pH adjustment, all media should be autoclaved at the temperature of 121 C and 1 bar pressure for 20 min.
1. Shoot-proliferating medium: 4.3 g/L of MS [17] micro- and
macroelements, 5 mg/L of pyridoxine, 5 mg/L of nicotinic
221
222
Methods
The protocol includes three main steps: (1) the supply of plant
material (in vitro proliferating shoots), (2) the Agrobacterium
infection, and (3) the regeneration and selection of stable transformed plantlets.
3.1 Preparation
of Strawberry Leaf
Tissue
1. For each genotype start in vitro proliferating shoots by sterilizing lateral buds collected from runners (vegetative parts of
the mother plant) and treating them with 2 % (v/v) chlorideactive solution for 20 min.
2. After rinsing 45 times with sterile distilled water, transfer the
meristematic tissues to sterile glass tubes (length 11 cm) containing the shoot-proliferating medium, to let meristematic tissues develop and to generate shoots.
3. Incubate the proliferating shoots in growth chambers under
16 h at 70 mol/m2/s and 8 h dark, at 24 2 C, and subculture regularly at 4-week intervals.
4. The transformation and regeneration experiments are carried
out by using young expanded leaves detached from 4-weekold in vitro proliferating shoots, after a minimum of 45 subcultures from the initial explants. When detached, incise the
leaf laminar on transversal rib and keep in distilled sterile water
until starting the transformation experiment (see Notes 13).
3.2 Agrobacterium
tumefaciens
Preparation and Plant
Tissue Infection
223
1. Infection and cocultivation is the main step of leaf tissue transformation. When the detached and cut leaves are ready and
maintained in sterile H2O to prevent drying, gently wash the
leaves in 30 mL infection solution containing the Agrobacterium
tumefaciens strain for 15 min at 100150 rpm.
2. After infection the leaves are blotted with sterile filter paper
and transferred to the Agrobacterium-plant coculturing
medium for 48 h at 25 C in dark condition. It is necessary to
put the leaves with their inferior-abaxial side in contact with
the medium.
3. At the end of the coculturing period, stop the infection by
transferring the leaves to the washing solution for 5 h at 25 C
with shaking at 100150 rpm.
1. The washed leaves are then blotted with sterile filter paper and
placed on leaf explant regeneration and selection medium,
always by keeping in contact with the medium the abaxial side
of the leaf.
2. For strawberry, the highest leaf tissue regeneration response is
promoted by a first period of incubation (2 weeks) at continuous dark and then under 16 h at 70 mol/m2/s and 8 h dark,
at 24 2 C. Leaf tissue has to be subcultured regularly at
2-week intervals on freshly prepared media.
3. At each subculture, the type of morphogenic activity that
occurs for each explant should be monitored. Generally, yellow
pale callus will form at the cut leaf edge around the end of the
first subculture. After moved to light (16/8 photoperiod),
224
some callus cultures start to form green dot nodules, while the
remaining parts display progressive necrosis.
4. The leaves bearing callus and green dot nodules are subcultured every 2 weeks on a freshly prepared regeneration and
selection medium. Keep subculturing the leaf and callus tissues
as long as it remains a proliferation and differentiation activity
(up to 6 months).
5. When transplanting, detach the selectable green and elongated
regenerated shoots (about 24 cm in size) from the callus tissues and transfer in glass tube (12-mm diameter) containing
the shoot-proliferating medium supplemented with the selective agent (kanamycin 25 mg/L) and the decontaminant
(cefotaxime 200 mg/L) antibiotic used in the regeneration
medium. A larger number of isolatable regenerants generally
occur at the second and third subculture (see Note 4).
6. The proliferation stage of the isolated shoots is another critical
stage for the identification of stable homogenous transgenic
lines. Again, 23 subcultures on the same proliferation medium
supplemented with antibiotics are generally useful to identify
the regenerated lines showing the more homogenous green
tissues (see Note 5). The type of agar (plant agar or Gelrite)
and their concentration inside the medium (from 7.5 to 7 g/L)
could help to expose regenerating plant to the antibiotics in
order to make the selection more efficient.
3.5 Rooting
and Transplanting
to Soil
225
Notes
1. Experiment planning: Each experiment has to be carried out
using young trifoliate strawberry leaves.
2. Experiment repetition: Plan at least three subsequent transformation experiments, with at least 50 or 100 leaf explants each,
depending on the efficiency of the regeneration protocol.
3. It is of extreme importance to ensure sterile conditions for all
experiment processes.
4. Each newly selected regenerant has to be identified with the
corresponding leaf tissue origin. This leaf tissue can be maintained for other subsequent subcultures, but if other regenerating shoots will occur later, they can be isolated, but it is
better to identify them as a subclone of the first regenerated
shoot already isolated from the same explant. If both will grow
stably on proliferation/selection medium, only the molecular
characterization (Southern blot) can confirm their origin from
different transformation events. If the regeneration and transformation protocols are really efficient, as soon as a regenerated shoot is isolated, the leaf explant can be discarded.
5. The identification and selection of greener stably proliferating
and rooting shoots are fundamental aspects of the in vitro procedure to avoid the risk of selecting chimeric clones.
6. Blend of white and frozen through black sphagnum peat.
Structure medium grade, optimum air to water ratio. pH 6
226
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
227
Chapter 19
Walnut (Juglans)
Charles A. Leslie, Sriema L. Walawage, Sandra L. Uratsu,
Gale McGranahan, and Abhaya M. Dandekar
Abstract
Walnut species are important nut and timber producers in temperate regions of Europe, Asia, South
America, and North America. Trees can be impacted by Phytophthora, crown gall, nematodes, Armillaria,
and cherry leaf roll virus; nuts can be severely damaged by codling moth, husk fly, and Xanthomonas
blight. The long generation time of walnuts and an absence of identified natural resistance for most of
these problems suggest biotechnological approaches to crop improvement. Described here is a somatic
embryo-based transformation protocol that has been used to successfully insert horticulturally useful traits
into walnut. Selection is based on the combined use of the selectable neomycin phosphotransferase (nptII)
gene and the scorable uidA gene. Transformed embryos can be germinated or micropropagated and
rooted for plant production. The method described has been used to establish field trials of mature trees.
Key words Agrobacterium tumefaciens, California black walnut, Eastern black walnut, Gene transfer,
Juglans hindsii, Juglans nigra, Juglans regia, Paradox, Persian walnut, Somatic embryo
Introduction
There are approx 20 species of walnut worldwide, many of which
are valued for their nut production and timber quality. The principal species used for commercial walnut production, the Persian
walnut (Juglans regia L.), is native to the mountain ranges of central Asia. It is now cultivated in many temperate regions including
much of the Mediterranean, Central Asia, northern India, China,
South Africa, Argentina, Chile, and Australia. In the United States,
this crop is produced almost entirely in California, where orchard
trees are grafted onto rootstock of either the native California black
walnut (Juglans hindsii) or J. hindsii J. regia hybrids known as
Paradox. The eastern black walnut (Juglans nigra), native to the
eastern United States, is highly valued for its timber quality and has
been introduced into Europe and China for this purpose.
Walnuts are susceptible to a number of insect, disease, and
nematode problems. The key insect pest is codling moth (Cydia
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_19, Springer Science+Business Media New York 2015
229
230
Walnut (Juglans)
231
2
2.1
Materials
Plant Material
2.2 Transformation
Vectors
and A. tumefaciens
Strains
2.3
Stock Solutions
1. Kanamycin sulfate (50 mg/mL): Dissolve 5 g kanamycin sulfate powder in 100 mL of water, filter-sterilize, and freeze in
1015 mL aliquots in 25 mL screw-cap vials.
2. Gentamicin sulfate (25 mg/mL): Dissolve 250 mg gentamicin
sulfate powder in 10 mL of water, filter-sterilize, and store
frozen.
3. Timentin (100 mg/mL): Dissolve 6.2 g (two 3.1 g bottles) of
timentin powder in 62 mL of water, filter-sterilize, and freeze
in 1015 mL aliquots in 25 mL screw-cap vials.
4. Hygromycin B (12.5 mg/mL): Dissolve 125 mg hygromycin
B powder in 10 mL of water, filter-sterilize, and store frozen
(see Note 3).
232
Media
Walnut (Juglans)
233
234
Methods
3.2 Agrobacterium
Preparation
235
Walnut (Juglans)
( Amount
( A600
( 25 mL ) ( 0.5)
= 3.36 mL.
( 0.371) 10
6. If performing co-transformation, mix the two bacterial cultures
in a 1:1 ratio before preparing the final volume (see Note 7).
7. Using a sterile pipette, place the calculated volume of culture
solution into a sterile plastic-capped 50 mL centrifuge tube
and centrifuge sufficiently to lightly pellet the bacteria (e.g.,
10 min at 4,000 g).
8. Pour or pipette the supernatant into an autoclavable waste container and resuspend the pellet in the cocultivation medium.
The pellet is easier to resuspend in a small volume (0.5 mL first)
using a pipette tip. Then add the full volume. If needed, set up
a tube for a no-bacteria control using only cocultivation medium.
9. Return the tubes to the shaker until ready to use.
3.3
Cocultivation
236
Walnut (Juglans)
3.6 PCR
Confirmation
of Gene Insertion
237
3.7 Germination
and Plant Production
After sufficient additional somatic embryos have developed, desiccate some to initiate germination. Choose well-formed somatic
embryos and place them in 35 10 mm sterile Petri plates with no
medium. Cover the plates but leave unsealed (do not wrap with
Parafilm) and place them in the dark at room temperature on the
rack of a well-sealed desiccator containing 1015 mL of saturated
ZnSO4 or NH4NO3 in the bottom.
1. After 27 days, when the embryos become opaque white with
the consistency of popcorn but before they brown, remove the
embryos from the desiccator and place them on DKW shoot
medium in Magenta GA-7 vessels or glass jars with similar
headspace. Culture at room temperature under cool white fluorescent lights (16 h/day photoperiod, approx 100 mol/
m2/s) for 28 weeks.
2. Most embryos will produce roots, but typically fewer than
10 % of embryos develop shoots. Roots will usually emerge
from embryos in a week to 10 days. A few shoot buds may also
begin to push quickly. In this case, the plants should be
removed from the medium as soon as possible and planted in
potting soil. If the embryos are left on the shoot medium, the
roots begin to deteriorate but more shoot buds will push.
These can be excised and micropropagated. Alternatively
embryos can be placed on shoot medium for 1 week to initiate
238
Notes
1. Walnut is susceptible to wide variety of Agrobacterium strains.
This was discussed in an earlier publication [23] where we
showed the susceptibility of walnut vegetative tissues to a variety of strains. Among the strains that are particularly infective
are the derivatives of A281, A6, and C58. We have used mainly
the disarmed versions of A281 and C58 in all of our transformation experiments. With the exception of apical meristems,
we found most vegetative tissues quite susceptible to the infection with Agrobacterium including somatic embryos; the latter
was noted in our earlier publication [6]. The nptII gene works
when higher concentrations of kanamycin are used, typically
100 g and above. Lower concentrations do not work particularly well. Because of the weak selection with kanamycin and
the variability in the efficiency of transformation this may produce, we used GUS to confirm the transformants. Among the
GUS constructs, the gene that contains an intron has the least
background, but the others work as well too.
2. We use electroporation to introduce DNA into Agrobacterium.
Briefly, 1 L of plasmid DNA (10100 ng) is added to
Walnut (Juglans)
239
240
idea of how many you will have to work with before you get
too far into the rest of the work. If you need more, wait another
wk or lower your selection standards. To achieve the best transformation efficiency, it is important to use rapidly multiplying
embryos. Embryos multiply continuously and a culture will
have a mixture of all sizes, ages, and qualities of embryos.
If you want to measure transformation rates and timing or otherwise need uniform initial material for experimental purpose,
choose small, white (25 mm) embryos of good somatic
embryo form (two visible cotyledons) and semitranslucent
rather than ivory white opaque appearance. This gives starting
material that is distinct and can be counted easily, and these
embryos will continue to multiply well. Opaque white embryos
often move toward germination and produce fewer new
embryos. Once you have selected the embryos, distribute them
equally across treatments. Mark one or more plates for each
treatment and then select similar sets of embryos to be used for
each treatment. Continue this process until all the embryos are
assigned to a treatment. Be very careful with your sterile technique because you can contaminate everything during this
process. If the goal is only to obtain some transformants, use
any rapidly multiplying embryo culture material except browning older embryos.
9. Do this soon after placing embryos in the wells so they dont
dry out. Use enough medium to cover the embryos (approx
8 mL/well) if you have enough; otherwise, distribute the
medium over the embryos so they all get wet. Label carefully
and be sure to avoid cross contamination. You may want to use
a different plate for each vector.
10. Physical wounding is not necessary and is, in fact, detrimental
to successful transformation. Wound sites develop callus rather
than new somatic embryos.
11. We tested the efficiency of applying kanamycin immediately
after cocultivation vs applying at a later stage. Early application
gave a better yield of transformants. Appropriate kanamycin
and hygromycin concentrations were determined by performing kill curves on untransformed embryos (unpublished data).
12. Transfer embryos in a consistent pattern on each plate so that
if resistant bacteria begin to multiply, they are not moved to all
the embryos on the plate.
13. The X-Gluc is not always toxic. If you use a filter-sterilized
X-Gluc solution, dispense it into sterile wells, and return the
tissue to selection medium as soon as the blue color becomes
apparent, then tissue pieces that turn blue can sometimes
themselves develop embryogenic cultures in addition to using
the embryo from which it was excised.
Walnut (Juglans)
241
References
1. McGranahan GH, Tulecke W, Arulsekar S,
Hansen JJ (1986) Intergeneric hybridization in
the Juglandaceae: Pterocarya sp. x Juglans
regia. J Am Soc HortSci 111:627630
2. Driver JA, Kuniyuki AH (1984) In vitro propagation of Paradox walnut (Juglans hindsii x
Juglans regia) rootstock. HortSci 19:507509
3. McGranahan GH, Leslie CA, Driver JA (1988)
In vitro propagation of mature persian walnut
cultivars. HortSci 23:220
4. Tulecke W, McGranahan GH (1985) Somatic
embryogenesis and plant regeneration from
cotyledons of walnut, Juglans regia L. Plant Sci
(Limerick) 40:5764
5. Tulecke W, McGranahan GH, Ahmadi H
(1988) Regeneration by somatic embryogenesis of triploid plants from endosperm of walnut,
Juglans regia L. cultivar Manregian.. Plant
Cell Rep 7:301304
6. McGranahan GH, Leslie CA, Uratsu SL,
Martin
LA,
Dandekar
AM
(1988)
Agrobacterium mediated transformation of
walnut somatic embryos and regeneration of
transgenic plants. Nat Biotechnol 6:800804
7. McGranahan GH, Leslie CA, Uratsu SL,
Dandekar AM (1990) Improved efficiency of
the walnut somatic embryo gene transfer system. Plant Cell Rep 8:512516
8. Dandekar AM, McGranahan GH, Vail PV,
Uratsu SL, Leslie C, Tebbets JS (1994) Low
levels of expression of wild type Bacillus
thuringiensis var. Kurstaki cryIA(c) sequences
in transgenic walnut somatic embryos. Plant
Sci 96:151162
9. Dandekar AM, McGranahan GH, Vail PV,
Uratsu SL, Leslie CA, Tebbets JS (1998)
High levels of expression of full-length
cryIA(c) gene from Bacillus thuringiensis in
transgenic somatic walnut embryos. Plant Sci
131:181193
10. Vahdati K, McKenna JR, Dandekar AM et al
(2002) Rooting and other characteristics of a
transgenic walnut hybrid (Juglans hindsii x J.
regia) rootstock expressing rolABC. J Am Soc
HortSci 127:724728
11. Escobar MA, Leslie CA, McGranahan GH,
Dandekar AM (2002) Silencing crown gall disease in walnut (Juglans regia L.). Plant Sci
163:591597
12. Walawage SL, Britton MT, Leslie CA, Uratsu
SL, Li Y (2013) Stacking resistance to crown
gall and nematodes in walnut rootstocks. BMC
Genomics 14:668
13. Polito VS, McGranahan GH, Pinney K,
Leslie C (1989) Origin of somatic embryos
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Part IV
Tropic Plants
Chapter 20
Citrus Transformation Using Juvenile Tissue Explants
Vladimir Orbovi and Jude W. Grosser
Abstract
The most frequently used method for production of citrus transgenic plants is via Agrobacterium-mediated
transformation of tissues found on explants obtained from juvenile seedlings. Within the last decade and
especially within the last 56 years, this robust method was employed to produce thousands of transgenic
plants. With the newly applied screening methods that allow easier and faster detection of transgenic
shoots, estimates of transformation rate for some cultivars have gone up making this approach even more
attractive. Although adjustments have to be made regarding the (varietal) source of the starting material
and Agrobacterium strain used in each experiment preformed, the major steps of this procedure have not
changed significantly if at all. Transgenic citrus plants produced this way belong to cultivars of rootstocks,
sweet oranges, grapefruits, mandarins, limes, and lemons.
Key words Agrobacterium tumefaciens, Citrus, Genetic transformation, Juvenile tissue
Introduction
Researchers have found many applications for Agrobacteriummediated transformation of citrus. Out of two methods of transformation using stem explants as a starting material, the one based
on juvenile tissue is far more prevalent and efficient than the
method based on mature tissue. The latter will be discussed in
details in Chapter 21. As a reflection of the present state of citrus
industry worldwide, most of the efforts invested in production of
genetically modified citrus are directed toward improvements of
tolerance to citrus diseases [18]. However, a plethora of genes
were introduced into citrus plants for the purpose of improvement
of tolerance to environmental stress [911], manipulation of hormonal balance [12, 13], alteration in development [14, 15], and
even changes in fruit flavor [16, 17].
In the previous edition of this chapter [18], the availability of
starting material was stated as a major advantage for this method
over the use of mature tissue explants. That statement still stands
firm. Extraction of seeds from fruit, seed processing, and their
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_20, Springer Science+Business Media New York 2015
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2
2.1
Materials
Plant Material
247
248
Methods
3.1 Preparation
of Plant Material
249
Fig. 1 Photographs of materials used in different phases of citrus transformation. (a) Processed seeds 4 days
after planting; (b) 37-day-old seedlings ready for cutting; (c) explants after incubation with Agrobacterium;
(d) explants with sprouted shoots 35 days after cocultivation with Agrobacterium; (e) plate with harvested
shoots; (f) transgenic shoot 14 days after grafting in vitro
250
1. Two days before the experiment (see Note 6), start liquid cultures of Agrobacterium by inoculating 50 mL of YEP + Rif + Kan
medium with one colony in 125 mL Erlenmeyer flask. These
cultures are placed in an incubator that maintains the temperature at 28 C and shaking speed at 220 rpm.
2. One day before the experiment, examine the culture, and if it
appears dense and opaque (optical density OD600 higher than
1), replenish the medium and antibiotics in the bacterial culture. Take 2 mL of growing culture using a sterile pipette and
discard the rest. Use the culture from the pipette as an inoculum for the new 50 mL culture with fresh YEP and antibiotics.
If the culture is growing slowly and is translucent, leave it in
the incubator without changing the medium or incubation
conditions.
3. On the day of the transformation experiment, examine
Agrobacterium culture. It should be growing vigorously at this
time. Replenish the medium in the same flask by using 3 mL
from growing culture and add 50 mL of fresh YEP (as described
in step 2) supplemented with antibiotics to obtain actively
growing bacteria for maximum effect at the desired time.
Allow this culture to grow for additional 45 h.
4. Harvest 35 mL of Agrobacterium culture by centrifuging it at
3,000 g for 10 min then resuspend the pelleted bacteria in
35 mL of liquid CCM medium.
5. The optical density (OD600) of resuspended culture is measured and adjusted to the desired level with additional amounts
of CCM. Choice of citrus cultivar affects the optical density of
bacterial suspension used in the experiment (see Note 7). For
the series of experiments presented herein, we have adjusted
OD600 to 0.7.
3.3 Co-incubation
of Explants
with Bacteria
251
1. Take a group of explants (about 50) out of the plate with CCM
and place them on sterilized paper towel to remove most of
CCM liquid. This step (takes about 1 min) is necessary to
avoid further dilution of bacterial suspension by each consecutive group of explants carrying some CCM.
2. Transfer the explants to the Petri dish (20 mm 100 mm) with
30 mL of Agrobacterium suspension where they should be
soaked for 12 min.
3. Following co-incubation with bacteria, place the explants on
sterilized paper towels again to remove the excess of bacterial
suspension. Since in the next step explants are placed on solid
CCM medium that does not contain any antibiotics, getting
rid of excess of bacteria is needed to prevent their overgrowth
that may cause loss of the explants.
4. Place 16 infected explants on plate with solid CCM. Seal plates
containing the explants with Nescofilm and place them in the
incubator for 2 days. Temperature in the incubator is maintained at 26.1 C for 16 h photoperiod (35 mol/m2/s) and
at 24.5 C during 8 h of dark period.
5. Transfer explants from plates with CCM to RM medium, seal
plates, and leave them in the incubator for about 5 weeks.
Incubation conditions same as in step 4. Shoots should be easy
to count and harvest at this time as they are 47 mm in length
(see Fig 1d).
3.4 Detection
of Reporter Gene
in Transgenic Shoots
(Green Fluorescent
Protein, GFP)
1. For detection of GFP [20] as a reporter gene for transformation, observe shoots under the fluorescent microscope.
This is a binocular microscope that has a source of blue light
attached to it. When blue light hits molecules of GFP, it
excites them to emit green light. As a result of the presence
of GFP in transgenic tissue, shoots appear completely green,
light pink, or red with green patches of different sizes. Under
the same conditions, wild-type shoots appear red because
chlorophyll emits red light after excitation with blue light.
The pink color of shoots indicates presence of low levels of
GFP in the tissue and the concomitant emission of light by
both GFP and chlorophyll.
2. Following selection, excise transgenic shoots from the explants
and place them on GM medium (see Note 8).
252
Fig. 2 Photograph of the shoot with the corked-out circular piece of tissue that
will be used for the PCR-based primary screen for transgenic shoots. Diameter
of the cork borer is 0.5 mm
2. Carefully cork out (see Fig. 2) a piece of leaf and plunge it into
already prepared PCR mix. This test is based on the activity of
Phire hot start II DNA polymerase (see Fig. 3 and Note 9).
Cork borers used for removal of small explants from leaves of
shoots have a diameter of the cutting tube of 0.5 mm (Harris
Uni-Core 0.5 mm multipurpose sampling tool, see Note 10).
3.6 Micrografting
of Transgenic Shoots
In Vitro
1. Plants that are used as a rootstock are grown the same way as
the plants that are used to obtain explants (see Subheading 3.1),
meaning they should be fully etiolated on the day of grafting.
For rootstock, select only plants which have at least 5 cm
of straight shoot and root when measured from cotyledons
(see Note 11).
2. Pull the plant out of the tube with sterile forceps, make a transverse cut about 2 cm above cotyledons with a sterile surgical
blade mounted on scalpel handle, and discard the stem.
3. Cut the root about 45 cm below cotyledons and remove root
hairs if there are any. At this point, everything should be done
as quickly as possible to prevent the drying of cut surfaces of
plant tissue.
4. Take the putative transgenic shoot and carefully cut it at the
bottom so that lowest portion of the stem appears as a letter V.
5. Make a 23 mm deep longitudinal cut along the center of the
cut surface of rootstock with a surgical blade. While the blade
is in the rootstock, wiggle it to the left and right so that the slit
widens a little and is ready to accept the graft.
6. Insert the wedged part of the shoot into the slit of rootstock,
and transfer this plant into the tube with grafting medium.
253
Fig. 3 Photographs of two agarose gels with products of PCR reactions performed as a part of primary screen
for transgenic shoots. All seven shoots that provided corked-out explants for positive PCR reactions shown
on the photo were tested in additional PCR for the native Agrobacterium gene. Especially suspicious were PCR
products in lanes 7 and 25, and indeed it turned out that the shoot number 7 was not transgenic (see Note 9).
Numbers associated with lanes designate the number of shoots tested. Lane labeled H2O was loaded with the
products of PCR reaction where water was added instead of DNA sample. Lane labeled (+) was loaded with
the products of PCR reaction where the DNA of binary vector that was mobilized into Agrobacterium was used
as a template
254
Notes
1. If you are extracting larger numbers of seeds from freshly harvested fruit on a regular basis, buy a commercial grade juicer as
those marketed for household use have motors that burn out
easily.
2. Binary vectors were mobilized into Agrobacterium by a freezeand-thaw method [25]. Once the binary vector of interest has
been mobilized into an appropriate strain, two stocks of newly
created strains are made. Glycerol stock is kept at 80 C and
working stock is kept at 4 C on plates of YEP medium supplemented with appropriate antibiotics.
3. Liquid CCM is used for temporary incubation of cut explants
before they get infected in Agrobacterium suspension. Solid
CCM is used for incubation of explants following incubation
with Agrobacterium.
4. Phire hot start DNA polymerase is an enzyme with very high
activity due to the presence of special DNA-binding domain.
Because of high potency, this enzyme can multiply DNA molecules from a small number of templates and is therefore useful
for our application where small piece of leaf tissue and not
isolated DNA serves as a source of template. The use and storage of both an enzyme and a buffer are done according to
manufacturers suggestions.
5. There is a report stating that transformation efficiency increases
if explants are cut longitudinally as a result of higher surface of
cambial tissue being exposed to action of Agrobacterium [26].
We confirmed these results (data not shown) but also noticed
that stems of many citrus cultivars are not sturdy enough to
sustain longitudinal cuts. For that reason, as well as for labor
efficiency, we chose not to cut explants longitudinally but
made slanted instead of transversal cuts on ends of explants,
thereby increasing the surface area of internal tissues that came
in contact with Agrobacterium.
6. Cultures of Agrobacterium start to lose their viability when
kept on YEP + Rif + Kan plates at 4 C for more than 3 weeks.
Because of that, it is prudent to start the cultures for experiments 2 days earlier to allow bacteria to attain vigorous growth;
a 24-h period may not be enough.
7. Although we never made exact calculations, we have noticed
that different cultivars of citrus go through co-incubation with
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9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
257
taste-modifying
protein,
in
transgenic
Miyagawa Wase Satsuma mandarin (Citrus
unshiu Marc.). J Korean Soc Appl Biol Chem
54:2429
Orbovi V, Grosser JW (2006) Citrus: sweet
orange (Citrus sinensis L. Osbeck Valencia)
and Carrizo citrange [Citrus sinensis (L.)
Osbeck x Poncirus trifoliata (L.) Raf.]. In:
Wang K (ed) Agrobacterium protocol, Methods
in molecular biology. Humana Press Inc,
Totowa, NJ, pp 177189
Jefferson RA (1987) Assaying chimeric genes
in plants: the GUS gene fusion system. Plant
Mol Biol Rep 5:387405
Mankin SL, Thompson WL (2001) New green
fluorescent protein genes for plant transformation: intron-containing, ER-localized, and
soluble-modified. Plant Mol Biol Rep 19:
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Hood EE, Gelvin SB, Melchers LS, Hoekema
A (1993) New Agrobacterium helper plasmids
for gene transfer to plants. Transgenic Res
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Murashige T, Skoog F (1962) A revised medium
for rapid growth and bioassays and tobacco
tissue culture. Physiol Plant 15:473497
Murashige T, Tucker DPH (1969) Growth
factor requirements of Citrus tissue culture.
Proc 1st Int Citrus Simp 3:11551161
Sambrook J, Russell DW (2001) Molecular cloning - a laboratory manual. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY
Walkerpeach
CR,
Velten
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(1994)
Agrobacterium-mediated gene transfer to plant
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Yu C, Shu H, Chen C, Deng Z, Ling P, Gmitter
FG (2002) Factors affecting the efficiency of
Agrobacterium-mediated transformation in
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Hood EE, Helmer GL, Fraley RT, Chilton
M-D (1986) The hypervirulence of
Agrobacterium tumefaciens A281 is encoded in
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Bacteriol 168:12831290
Lazo GR, Stein PA, Ludwig RA (1991) A
DNA transformation-competent Arabidopsis
library in Agrobacterium. Nat Biotechnol
9:963967
Chapter 21
Citrus Transformation Using Mature Tissue Explants
Vladimir Orbovic, Alka Shankar, Michael E. Peeples,
Calvin Hubbard, and Janice Zale
Abstract
Mature tissue protocol for production of transgenic Citrus plants via Agrobacterium-mediated transformation
uses explants derived from branches of mature, fruit-bearing trees. Through the multiple cleaning steps consisting of grafting of apical tip meristems on rootstock plants grown under sanitary conditions, mother
plants are produced that will serve as a source of budding material. These buds are grafted onto rootstock
plants grown under the same, highly sanitary conditions. Newly obtained, one meter tall, young grafted plants
serve as a source of explants for co-incubation experiments with Agrobacterium. Following successful
transformation with Agrobacterium, selected transgenic shoots are micrografted onto rootstock plants in vitro
where they are allowed to grow for a couple of months. Grafted transgenic plantlet together with the
associated rootstock plant is taken out of culture tubes, severed from the root, and regrafted in terra on a
1-year-old rootstock plant. With the application of proper horticultural techniques, such a plant will yield first
fruit about 1215 months later.
Key words Agrobacterium tumefaciens, Citrus, Genetic transformation, Mature tissue
Introduction
The method of using mature tissue explants for production of
Citrus transgenic plants via Agrobacterium-mediated transformation was described almost 15 years ago [1]. Despite the fact that it
cannot be considered as new, only a few laboratories have
attempted to employ this protocol with varying levels of success
[2, 3]. The most important advantage of this approach to producing genetically modified Citrus plants is that they will be fruiting
within short period of time of about 18 months (see Note 1).
Compared to the method of using juvenile tissue explants as a
starting material ([4], see associated chapter) in transformation
experiments, mature tissue transformation yields fruiting plants
34 times faster. Having transgenic plants that are true to type and
capable of producing fruit allows for rapid evaluation of commercially important traits of transgenic plants associated with fruit and
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_21, Springer Science+Business Media New York 2015
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juice quality together with the trait that was being improved via
the introduction of transgene.
Production of starting material for co-incubation with
Agrobacterium in this protocol requires highly organized plantgrowing facility manned with skilled personnel. Uninterrupted
production of rootstock plants is a major part of this operation.
Rootstocks need to be available for grafting in batches that can
number up to 100 plants, which upon grafting will yield high
number of scion plants that will be cut into explants two times over
seven to eight months. Also, additional rootstock plants need to be
ready whenever there is a positively identified transgenic plant that
should be grafted in terra. Figure 1 depicts the phases of production and the growth of plants that will be cut into explants for coincubation experiments.
Having sufficient amounts of starting material for co-incubation
experiments will lead to success in transformation only if the quality of explants is at the most satisfactory level. The absence of any
microorganisms in the tissue of the plants to be cut into explants is
the crucial parameter of quality. As much as following good horticultural practices in growing these plants to be vigorous and of
proper size is important, it fades in comparison to making sure that
these plants, both rootstocks and scions, do not contract any infection before they get used in transformation protocol. From the
time of seed planting and germination of rootstock, and all phases
of growth, extreme care has to be exercised in keeping these plants
away from outside pests which could carry bacterial and/or fungal
infection. In our facility, plants are grown in walk-in chambers that
are contained within another building. Entrances to the building
and to all the rooms where different activities associated with the
maintenance of plants are performed are equipped with an air
curtain to prevent insects from entering. Growth rooms themselves do not have air curtains installed. All personnel working
within the facility wear protective/disposable coats and footwear
that is used only on those premises. A special system is in place to
purify water which is used for irrigation and fertigation and to feed
humidifiers that are operational within the growth chamber.
Because many pieces of equipment used for proper maintenance of
plants are technical in nature, support for this type of the facility
has to include a crew of maintenance personnel as well.
Although this methodology holds the promise of yielding fruiting Citrus plants within 1824 months, the relatively large initial
financial investment and low output have made its adoption rare.
Table 1 contains the data from two experiments that differ in choice
of binary vector. The vector named pTLAB21 carries a gene for green
fluorescent protein (GFP) between T-DNA borders [5], and pCAMBIA2301 binary vector is available commercially. High percentage
transformation rate recorded in the experiment where pTLAB21 vector was used is highly unusual and is most probably due to the small
number of shoots that were regenerated from explants.
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Table 1
Transformation rate recorded in two experiments with Hamlin sweet orange cultivar
Bin. vector
Number
of explants
Number
of sprouted
Number
of (+) shoots
Transformation
efficiency (%)
pCAMBIA2301
490
236
0.8
pTLAB21
530
16
12.8
2
2.1
Materials
Plant Material
1. Seeds for the rootstock plants used for bud grafting to produce
plants that will be cut into explants are obtained from commercial nursery. For this purpose, we use Swingle citrumelo
[Citrus paradisi Poncirus trifoliata] and Citrus macrophylla
and obtain the seeds from Willits and Newcomb company
(Bakersfield, CA).
2. Seeds of Carrizo [Citrus sinensis (L.) Osbeck Poncirus trifoliata (L.) Raf.] rootstock used for micrografting of transgenic
shoots are obtained either from the nursery or by extraction
from harvested fruit. Plants propagated in the growth room by
bud grafting from clean mother plants came from the following clone of sweet orange (Citrus sinensis. L.) cultivars:
Hamlin 1-4-1. Although we presented here the data from
experiments with Hamlin, we also use different clones of
sweet orange cultivar Valencia and grapefruit (Citrus paradisi) cultivar Ray Ruby.
2.2 Agrobacterium
Strain and Plasmid
263
264
265
Methods
3.1 Preparation
and Growth of Plant
Material
3.1.1 Germination
of Seeds for Production
of Rootstock Plants
for Bud Grafting
3.1.2 Transplanting
Rootstock Seedlings
to Be Used as Plants
for Bud Grafting
266
3.1.4 Pesticide
Application
267
1. Both seed coats are peeled from Carrizo citrange seeds that are
surface-sterilized by shaking for 10 min in 5 % solution of commercial bleach. These seeds are rinsed 56 times with sterile water.
2. Sow individual seeds into 25 150 mm glass tubes filled with
25 mL of SGM and incubate at 26 C in the dark for 2 weeks.
1. Decapitate Carrizo seedlings leaving 11.5 cm of the epicotyls.
Shorten the roots to 46 cm and remove the cotyledons and
their axillary buds. Place the regenerated shoot onto the apical
end of the cut surface of the decapitated epicotyl, in such a way
to establish a contact between the vascular tissues of both scion
and the rootstock (see Fig. 2a).
2. Culture grafted plants in grafting medium and maintain under
conditions of 25 C, 16 h light/8 h dark photoperiod, and
45 E/m2s of illumination. Scions develop 24 expanded
leaves within about 34 weeks after grafting.
268
Fig. 2 Photographs depicting: (a) primary micrografting, (b) secondary grafting, (c) initial stages of growth of
transgenic plant, and (d) 19-month-old plant (the inset shows developing flowers)
269
3.2.2 Preparation
of Explants
and Cocultivation
with Agrobacterium
1. Cut the bud stick (see Note 6) into pieces (about 4050 cm
long; see Fig. 1g) from first flushes of propagated plants leaving
two nodes for regeneration of the second flush. Remove the
leaves and thorns, and collect them in plastic bag. Move this
material to the laboratory. Next steps are all done in the
laboratory.
2. Done outside of laminar flow hood: place the bud sticks in a
tub containing tap water and soap. Scrub them carefully with a
soft brush. Rinse the bud sticks with distilled water. Collect the
clean bud sticks in graduated cylinder that will accommodate
length of bud sticks. Add 20 % bleach solution with 56 drops
of Tween-20 and wash bud sticks for 10 min while inverting
the cylinder. The cylinder should be sealed with Parafilm.
Transfer to laminar flow hood.
3. Done inside laminar flow hood: rinse all bud sticks five times
with sterile deionized water. Cut bud stick internodes transversely into 15 mm long explants with the help of forceps and
sterile clippers on the autoclaved Whatman filter paper. Collect
270
3.3.2 Harvest
of Putatively
Transgenic Shoots
1. Take a 96-well ELISA plate and aliquot 100 L of GUS substrate solution into each well.
2. Cut out the shoots from the explants with the help of forceps
and sterile surgical scalpel. Cut a thin cross section at the base
of the stem or small piece of leaf and place it in one of the wells
of ELISA plate.
3. Seal the plate with Parafilm and keep it at 37 C overnight.
4. Tissue from the transgenic shoots will turn blue in color. Stop
the reaction by washing the tissue three times with 100 mM
Tris, pH 7.0.
5. Bleach out the green color from the tissue (originating from
the presence of chlorophyll) to better visualize the blue staining. For these washes use increasing series of ethanol: 30, 50,
70, and 100 % until the green color is lost.
271
Fig. 3 Photograph of two explants with transgenic shoots carrying functional GFP
in their tissue. Explants were exposed to both blue light and dim white light. Blue
light was used to induce GFP emission and white light to allow photographing of
explants. Scale line 12 mm
Notes
1. Preparation of cleaned mother plants used as source of budding material is not included into time necessary to produce
fruiting plants as it requires considerable amount of time itself.
Obtaining clean budwood from the certified nursery will be
of great help in this endeavor. In our experience, even some of
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273
References
1. Cervera M, Juarez J, Navarro A, Pina JA,
Duran-Vila N, Navarro L, Pea L (1998)
Genetic transformation and regeneration of
mature tissues of woody fruit plants bypassing
the juvenile stage. Transgenic Res 7:5159
2. Almeida WAB, Mourao Filho FAA, Pino LE,
Boscariol RL, Rodriguez APM, Mendes BMJ
(2003) Genetic transformation and plant
recovery from mature tissue of Citrus sinensis
L. Osbeck. Plant Sci 164:203211
3. He YR, Chen SC, Peng AH, Zou XP, Xu LZ,
Lei TG, Liu XF, Yao LX (2011) Production and
evaluation of transgenic sweet orange (Citrus
sinensis Osbeck) containing bivalent antibacterial peptide genes (Shiva A and Cecropin B) via
a novel Agrobacterium-mediated transformation
of mature axillary buds. Sci Hort 128:99107
4. Orbovi V, Grosser JW (2006) Citrus: sweet
orange (Citrus sinensis L. Osbeck Valencia)
and Carrizo citrange [Citrus sinensis (L.)
Osbeck Poncirus trifoliata (L.) Raf.]. In:
Wang K (ed) Agrobacterium protocol methods in molecular biology. Humana, Totowa,
NJ, pp 177189
5. Orbovi V, Pasquali G, Grosser JW (2007) A
GFP-containing binary vector for Agrobacterium
6.
7.
8.
9.
10.
11.
Chapter 22
Coffee (Coffea arabica L.)
Eveline Dchamp, Jean-Christophe Breitler,
Thierry Leroy, and Herv Etienne
Abstract
Coffee (Coffea sp.) is a perennial plant widely cultivated in many tropical countries. It is a cash crop for
millions of small farmers in these areas. As for other tree species, coffee has long breeding cycles, which
makes conventional breeding programs time-consuming. For that matter, genetic transformation can be
an effective way to introduce a desired trait in elite varieties or for functional genomics. In this chapter, we
describe two highly efficient and reliable Agrobacterium-mediated transformation techniques developed
for the C. arabica cultivated species: (1) A. tumefaciens to study and introduce genes conferring resistance/
tolerance to biotic (coffee leaf rust, insects) and abiotic stress (drought, heat, seed desiccation) in fully
transformed plants and (2) A. rhizogenes to study candidate gene expression for nematode resistance in
transformed roots.
Key words Abiotic stress, Agrobacterium rhizogenes, Agrobacterium tumefaciens, Coffee leaf rust
resistance, Embryogenic cultures, Functional genomics, Hairy roots, Meloidogyne sp., Nematode
resistance
Introduction
With more than seven million tons of green coffee beans produced
every year on about 11 million hectares all over the intertropical
countries, coffee is an extremely important agricultural crop.
Growing regions typically offer moderate sunshine and rain, steady
temperatures around 26 C, and rich, porous soil. In terms of economic importance on the international markets, coffee is the second
natural commodity in value after petroleum. The total coffee production for year 2009/2010 was of 120.6 million bags (www.ico.
org). Brazil is responsible for about a third of all coffee production
(39.4 million bags of 60 kg), making it by far the most important
coffee-producing country, followed by Vietnam, Indonesia, and
Colombia. About 125 million people depend on coffee for their
livelihood in Latin America, Africa, and Asia.
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_22, Springer Science+Business Media New York 2015
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for other species [44, 45]. Our procedure enables rapid and
routine generation of hairy roots, as well as their maintenance.
Two Coffea arabica genotypes were used for these experiments.
The Caturra variety, which is widely grown in Latin America, is
susceptible to the root-knot nematode Meloidogyne exigua. The
second variety used, Iapar 59 (a Catimor-type interspecific hybrid),
is resistant to numerous diseases and pests. Its resistance to
Meloidogyne exigua has been demonstrated [46].
Materials
2.1 Regeneration
of Whole Transgenic
Plants Using
A. tumefaciens
280
281
The Agrobacterium rhizogenes strain A4RS was used for transformation (see Note 4). This strain derived from the wild strain A4
modified for the resistance to rifampicin and spectinomycin antibiotics [51]. Construct has been integrated in the pBin19 plasmid
[47]. The uidA bacterial gene isolated from Escherichia coli coding
for -glucuronidase (GUS) was introduced [52], with an additional intron for specific expression in plants [53]. The gene was
controlled by the cauliflower mosaic virus (CaMV) 35S promoter
and terminator. The A4RS including the pBIN19 plasmid with the
above construct was called armed A4RS.
1. MYA medium: 5 g/L yeast extract, 0.5 g/L casein hydrolysate, 8 g/L mannitol, 2 g/L MgSO4, 5 g/L NaCl, and 15 g/L
agar, pH 6.6.
2. Kanamycin sulfate: 50 mg/mL stock solution in water. Sterilize
by filtration through a 0.2 m membrane. Dispense 1 mL aliquots into Eppendorf tubes and store at 20 C.
3. Rifampicin: 25 mg/mL stock solution in water; use a few
drops of 100 % methanol for correct dilution. Sterilize by filtration and store at 20 C.
4. Spectinomycin: 20 mg/mL stock solution in water. Sterilize by
filtration through a 0.2 m membrane and store at 20 C.
5. MYA semisolid medium containing 50 mg/L rifampicin (both
for wild and armed A4RS), 500 mg/L spectinomycin (both
for wild and armed A4RS), 50 mg/L kanamycin (only for
armed A4RS), pH 6.6.
282
Methods
3.1 Protocol
for Producing Whole
Transgenic Plants
Using A. tumefaciens
3.1.1 Leaf Explant
Sterilization
3.1.3 Establishment
of Maintained Embryogenic
Callus Cultures
3.1.5 Agrobacterium
tumefaciens Culture
Preparation
283
3.1.7 Selection
and Regeneration
284
Fig. 1 Regeneration of transformed coffee plants from maintained embryogenic cultures [43]. (a) Calli in selective media containing 100 mg/L hygromycin and 125 mg/L cefotaxime, 4 months after cocultivation with
A. tumefaciens LBA1119 without plasmid, used as a control. (b) Regeneration of resistant calli in selective media
containing 100 mg/L hygromycin and 125 mg/L cefotaxime, 4 months after cocultivation with A. tumefaciens
LBA1119 carrying pMDC32; the yellow calli are resistant to hygromycin. (c) Regeneration of torpedo-shaped
somatic embryos 6 months after cocultivation. (d) In vitro plantlet development 8 months after cocultivation.
(e) Acclimatization of transgenic plants in the greenhouse 12 months after cocultivation
285
3.2.2 Transformation
and Hairy Root Induction
286
Fig. 2 Regeneration of Coffea arabica transgenic roots (hairy roots) using Agrobacterium rhizogenes [33, 34].
(a) Regeneration of a transgenic C. arabica root at the wounded and infected site (hypocotyl) 6 weeks after
transformation. Bar = 0.4 cm. (b) Several 4 to 6 cm long roots develop from the inoculation site. (c) Composite
plants ready for transfer to greenhouse. (d) C. arabica composite plants in soil substrate ready for nematode
inoculation in resistance tests. (e) Histochemical localization of -glucuronidase (GUS) gene expression in
transgenic roots of C. arabica transformed with the p35S-gusA-int gene construct. The blue staining allows the
localization of tissues that actively express the chimeric GUS gene. The staining is strong in all the meristematic regions and central cylinders. Bar = 3 mm. (f) Axenic culture of hairy roots of C. arabica cv. Caturra;
long-term maintenance in the presence of IBA in the dark
287
288
Notes
1. Tests showed that this methodology also worked with somatic
embryos and could therefore be used on heterozygous
materials.
2. Screenable marker: We used the reporter gene GFP5 isolated
from Aequorea victoria jellyfish coding for green fluorescent
protein. The gene was controlled by the constitutive cauliflower mosaic virus (CaMV) 35S promoter.
3. Selectable marker: We used the hygromycin phosphotransferase gene (HPTII) isolated from E. coli conferring resistance to
hygromycin for selection of transformed cells and regeneration
of transgenic plants.
4. Five Agrobacterium rhizogenes wild strains were compared for
transformation efficacy: 1583 also called A4 (agropin mannopin strain), ARqua1 (agropin mannopin strain), 1724 (mikimopin strain), 2659 (cucumopin strain), and 8196 (mannopine
strain). The A4 strain was the most efficient (80 % transformation efficacy), then ARqua1 (30 %), 1724 (10 %), and 2659
(5 %). Hairy roots were not obtained with the 8196 strain.
5. The transformation frequency (number of transformed calli/
total number of calli subjected to the transformation treatment
100) is variable from one genotype to another. For C. arabica, the process is efficient and reliable; the transformation
efficiency can vary between 50 and 90 % depending on the
genotype and the quality of the embryogenic tissues.
6. Both YEP and LB (10 g/L bacto-tryptone, 10 g/L yeast
extract) media can be used for bacteria growing. However,
Agrobacterium grows slightly slower in Luria-Bertani (LB)
medium than in YEP medium.
7. Several binary plasmids have been successfully used for coffee
transformation (pBIN19, pCAMBIAA1, PMDC32).
8. Hygromycin is the most efficient antibiotics for coffee transformation [43]. If kanamycin is used, it should be used at high
concentrations (more than 400 mg/L).
9. The temperature during the coculturing stage had a marked
effect on transformation efficiency with A. rhizogenes. For
example, the results obtained were 7080 % for embryos transformed at 18 C and 2030 % at 27 C [33].
289
11.
12.
13.
14.
15.
16.
17.
18.
290
43.
44.
45.
46.
47.
291
Chapter 23
Pineapple [Ananas comosus (L.) Merr.]
Gaurab Gangopadhyay and Kalyan K. Mukherjee
Abstract
The efficacy of Agrobacterium-mediated pineapple transformation technique has been improved (mean
percentage of transgenic micro-shoots regenerated from initial callus explants up to 20.6 %) using a novel
encapsulation-based, antibiotic selection procedure. The detailed protocol using a standard plant transformation vector (pCAMBIA1304) as reported in an elite Indian variety (Queen) of pineapple [Ananas
comosus (L.) Merr] can be applied to other varieties of pineapple for introgression of target genes.
Key words Agrobacterium tumefaciens, Antibiotic selection, Encapsulation, Pineapple, Transformation
Introduction
Pineapple [Ananas comosus (L.) Merr, family Bromeliaceae] is
considered as one of the most economically important tropical
fruits. It is the number one agricultural commodity in certain parts
of the world. However, its production often becomes severely limited due to a number of soilborne pathogens (http://www.nhb.
gov.in/fruits/pineapple/pin002.pdf) since the normal practice of
its propagation is through crowns (tops) and slips (side shoots
arising in older leaf axils) (http://www.uga.edu/fruit/pinapple.
htm). Classical pineapple plant breeding is based on crosses, backcrosses, and selection [1]. Because of the long generation cycle of
pineapple, the conventional breeding programs are extremely
time-consuming and can hardly keep pace with the rapid evolution
of pathogenic fungi [2].
Genetic engineering is an attractive tool for improving
elite pineapple clones. This has been attempted with varying
degrees of success either through micro-projectile-mediated gene
delivery [3, 4], selection by micropropagation in temporary
immersion bioreactors (TIBs) [5], or Agrobacterium-mediated
transformation [6] techniques. The target explants for cocultivation
vis--vis transformation range from leaf bases of micropropagated
shoots [3] to embryogenic cell clusters [6]/callus pieces [4, 5].
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_23, Springer Science+Business Media New York 2015
293
294
Each gene delivery system and choice of target tissue has its own
inherent advantage as well as limitations. Of the three gene delivery systems, the biolistic and use of temporary immersion bioreactors seem to be more technically demanding in comparison to
the Agrobacterium-mediated transformation, which has delivered
success in most of the plant system targeted for transgenic production till date. Regarding the target explant, which would
show high regeneration frequency after cocultivation either
through organogenesis or somatic embryogenesis, direct organogenesis from explants or an intermediate callus stage is a preferred one over regeneration from long-term maintained
embryogenic calli or cell clusters due to relative higher frequency
of the former than the latter. Finally, the efficacy of any transformation program for large-scale production depends on stringent
selection as far as practicable so that true targeted transgenics
can be picked up rightly from the large crowd of false-positive
antibiotic/GUS. This is evident in the entire successful pineapple transformation programs where relatively few transgenic lines
have been confirmed by the workers in spite of obtaining a large
number of GUS-positive regenerants/transformants initially. The
biolistic approach provided an acceptable efficiency of 0.21
1.5 % in case of Smooth Cayenne cultivar of pineapple [4],
while an efficiency of 6.6 % of transgenic plant recovery was
reported [5]. This cultivar while worked out with Agrobacteriummediated transformation technique again encountered a large
number of escape plants, which was gradually eliminated through
a very rigorous and time-consuming stepwise selection procedure [6]. Furthermore, regeneration from embryogenic callus
pieces with occasional somaclonal variation was another bottleneck with this approach [6].
Considering all these factors, the present protocol of an
Agrobacterium-mediated transformation for pineapple using a standard plant transformation vector (pCAMBIA1304) with subsequent encapsulation-based novel antibiotic selection technique was
designed to minimize the occurrence of false-positive plants, which
in consequence would elevate the transformation frequency.
Precisely, in this two-step selection protocol, micro-shoots regenerating from the organogenic calli after cocultivation and initial
screening in agar-gelled media for antibiotic resistance were encapsulated in alginate beads containing hygromycin, the selection antibiotic. The hypothesis was the alginate beads containing sublethal
concentration of antibiotic would provide a uniform environment
for selection to the entrapped micro-shoot that has overcome the
first step of screening in antibiotic containing agar-gelled media.
This endeavor of stringent selection resulted in higher transformation frequency (mean percentage of transgenic micro-shoots regenerated from initial organogenic callus explants up to 20.6 %). The
work was reported by our group in elite Indian variety (Queen) of
pineapple [7]. The scheme of this protocol is presented in Fig. 1.
295
Action
Subculture
of
the
responding explants in
RM2
Day of
Action/observation
1
30
45-55
Plantlets
separated
out
aseptically and subcultured
individually in maintenance
media in MM
Events
Regeneration
plantlets
from
bases
of
leaf
60
Culture
of
excised
meristematic
leaf
base
regions of the aseptically
maintained plantlets in RM1
30-40
10 mm
Development
of
organogenic calli
296
Action
Infection of organogenic
callus pieces (explants)
with
Agrobacterium
followed by co cultivation
in induction media (RM1
+ acetosyringone)
Transfer of explants to
agar gelled selection
media (containing 20 mg
/L hygromycin)
Day of
Action/observation
1
Efficiency cascade
11
18
25
32
~65 70 explants
Encapsulation
of
micro
shoots (excised aseptically
from
regenerated
calli,
individual micro shoot ~1.5
2.0 mm long, rate of
regeneration
6-10
per
explant)
in
Ca-alginate
beads containing 60 mg /L
hygromycin; beads kept in
sterile petri dishes under
culture room conditions
40
Fig. 1 (continued)
10 mm
Step
Action
Day of
Action/observation
70
297
Efficiency cascade
(per experimental set)
10
11
Histochemical
GUS
assay of portions of the
selected micro shoots
Multiplication
of
the
selected trasformants in
RM2
for
three
successive sub cultures
of three weeks each (~60
days)
Fig. 1 (continued)
70
130
135
onwards
The
uidA
fragment
(1051 bp) amplified in
DNA samples from
~85%
plantlets
(84/99);
30.54%
considering the total
number
of
viable
micro
shoots
(84/275);
20.58%
considering the initial
infected
callus
explants (84/408). All
randomly
selected
plantlets showed low
to
moderate
copy
number
of
gene
integration.
298
2
2.1
Materials
Plant Material
2.2 Agrobacterium
tumefaciens Vectors
and Strains
2.3 Plant
Culture Media
2.4 Bacterial
Culture Media
1. YEB medium: 0.1 % yeast extract, 0.5 % beef extract, 0.5 % peptone, 0.5 % sucrose, 0.049 % MgSO4, pH 7.2, containing kanamycin (100 mg/l, for pCAMBIA1304 selection) and rifampicin
(50 mg/l, for chromosomal background selection) (see Note 1).
2. The autoclaving condition of bacterial culture medium is
similar to that of plant culture medium.
299
10 ml with double-distilled water (DDW). To be filtersterilized and stored in aliquots at 4 C. Put in water bath
(~3540 C prior use).
3. Stock solution of auxins (IAA/NAA): Weigh 10 mg IAA/
NAA powder and dissolve with 23 drops of ethanol, volume
to be made up to 10 ml with DDW. To be filter-sterilized and
stored in aliquots at 4 C. Put in water bath (~3540 C prior
use).
4. Presterilized (-irradiated; of TARSONS/AXYGEN/others)
plastic Petri plates (90 mm and/or 35 mm diameter) are to
be used for callus induction/coculture/bacterial culture
maintenance.
5. For increased T-DNA transfer, 400 M acetosyringone
(3,5-dimethoxy-4-hydroxyacetophenone) is to be used.
6. Filter papers for coculture step: Whatman, qualitative grade 1.
7. CaCl22H2O and sodium alginate are to be procured from
SIGMA/FLUKA.
8. X-Gluc (5-bromo-4-chloro-3-indolyl
SIGMA preferably is to be used.
glucuronide)
from
300
Methods
3.1 Establishment
and Aseptic
Maintenance of Plant
Cultures
3.2 Establishment
of Plant Regeneration
System Suitable
for Transformation
3.3 Maintenance
of Bacterial Culture
3.4 Inoculation
and Cocultivation
301
4. Explants (ca. 10 mm callus pieces with initiation of shoot budlike structures) have to be simultaneously excised and aseptically pooled in MS liquid medium without growth regulator.
From that pool about 100 explants per experimental set are to
be placed in 10 ml of preinduction medium in which 10 ml of
preinduced liquid bacterial culture is to be finally added to
obtain a 20 ml coculture.
5. Explants are then to be subjected to vacuum infiltration for
5 min in vacuum desiccators followed by shaking at 100 rpm for
2.5 h. Excess of bacterial suspension has to be removed by filtration with sterile filter papers in Bchner funnel (see Note 4), and
Agrobacterium-infected explants are placed on semisolid MS
induction medium (same as RM1, supplemented with 400 M
acetosyringone, poured in 35 mm Petri plates).
6. The plates are to be left for cocultivation under a photoperiod
of 16 h (white fluorescent light, 4080 mol/m2/s) at
25 2 C and 78 % relative humidity.
3.5 Selection
of Transformants Prior
to Encapsulation
3.6 Selection
of Transformants After
Encapsulation in Ca
Alginate Beads
302
1. Randomly selected hygromycin-resistant micro-shoots emerging from the beads are subjected to histochemical GUS (betaglucuronidase) assays according to the protocol of Jefferson
et al. [10].
2. The whitish to green basal zones of the micro-shoots are
immersed in an X-Gluc (5-bromo-4-chloro-3-indolyl glucuronide) solution (see Note 8).
3. Tissues are incubated overnight in the dark at 37 C for staining and subsequently cleared using an ethanol series (70, 85,
95, and 100 %, 1 h at each concentration).
4. GUS-positive shoots/tissues turn blue, while the white ones
can be discarded as GUS-negative.
3.8 Multiplication
of Selected
Transformants
3.9 Molecular
Analysis
of Transgenic Plants
3.9.1 PCR Analysis
303
3.9.2 Southern
Hybridization Analysis
304
Notes
1. Antibiotics are not to be autoclaved along with culture media;
rather they are to be added in media from stock solutions after
filter sterilization through a 0.2 hydrophobic filter in laminar
flow hood chamber. Stock solutions of antibiotics can be stored
at 20 C or can be prepared freshly as per need.
2. Concentration of mercuric chloride and the duration of the
treatment can be increased considering the rate of contamination vis--vis infection-free established explants in culture. Since mercuric chloride is a hazardous chemical,
caution is needed to handle the chemical and also to dispose
the solution.
3. Acetosyringone dissolves in DMSO (dimethyl sulfoxide).
Required amount to be added from prefilter-sterilized stock
solution.
4. Porcelain components of Bchner funnel have to be presterilized by autoclaving.
5. The concentration of CaCl22H2O has to be pre-standardized
(usually it is 0.17 M).
6. The concentration of sodium alginate for optimum gelling has
to be pre-standardized. Usually, it is 2.5 % (w/v). Extreme care
has to be taken during autoclaving media supplemented with
sodium alginate since this chemical has a tendency of losing
gelling capacity if over autoclaved.
7. Petri plates for culture/coculture/selection experiments are to
be sealed properly with PARAFILM (American National Can)
to avoid contamination and losing desirable transformants.
8. X-Gluc solution consists of 2 mM X-Gluc, 100 mM TrisHCl,
pH 7.0, 50 mM NaCl, 2 mM potassium ferricyanide, and
0.1 % v/v Triton X-100.
9. The composition of 50 TAE buffer (100 ml): Tris 24.2 g,
glacial acetic acid 5.71 ml, 0.5 M EDTA 10 ml, pH adjusted to
8.0 with HCl.
10. Ethidium bromide is an extremely hazardous chemical; proper
care should be taken during weighing and dispensing it. Stock
solution: 10 mg dissolved in 1 ml of DDW; for 100 ml of agarose gel, 11.5 l from stock.
11. Unique EcoRI and BamHI restriction sites occur within
pCAMBIA1304 T-DNA, 3,6503,700 bp upstream from the
right T-border.
305
Acknowledgment
A critical reading of the manuscript by Ms. Ranjana Prasad is duly
acknowledged. Authors are thankful to the director of Bose
Institute.
References
1. Botella JR, Cavallaro AS, Cazzonelli CI (2000)
Towards the production of transgenic pineapple to control flowering and ripening. Proc 3rd
Int Pineapple Symp. Acta Hortic 529:115122,
Subhadrabandhu S, Chairidchai P (eds.)
2. Cabot C, Lacoevilhe JC (1990) A genetic
hybridization programme for improving pineapple quality. Acta Hortic 275:395400
3. Sripaoraya S, Marchant R, Power JB, Davey
MR (2001) Herbicide tolerant transgenic
Pineapple (Ananas comosus) produced by micro
projectile bombardment. Annal Bot (Lond)
88:597603
4. Ko HL, Campbell PR, Jobin-Dcor MP,
Eccleston KL, Graham MW, Smith MK (2006)
The introduction of transgenes to control
Blackheart in Pineapple (Ananas comosus L.)
cv. Smooth Cayenne by micro projectile bombardment. Euphytica 150:387395
5. Espinosa P, Lorenzo JC, Iglesias A, Yabor L,
Menendez E, Borroto J, Hernandez L,
Arencibia AD (2002) Production of pineapple
transgenic plants assisted by temporary immersion bioreactors. Plant Cell Rep 21:136140
6. Firoozabady E, Hecker M, Gutterson N
(2006) Transformation and regeneration of
pineapple. Plant Cell Tiss Org Cult 84:116
Chapter 24
Sugarcane (Saccharum Spp. Hybrids)
Hao Wu and Fredy Altpeter
Abstract
Genetic transformation of sugarcane has a tremendous potential to complement traditional breeding in
crop improvement and will likely transform sugarcane into a bio-factory for value-added products. We
describe here Agrobacterium tumefaciens-mediated transformation of sugarcane. Embryogenic callus
induced from immature leaf whorls was used as target for transformation with the hypervirulent
Agrobacterium strain AGL1 carrying a constitutive nptII expression cassette in vector pPZP200. Selection
with 30 mg/L geneticin during the callus phase and 30 mg/L paromomycin during regeneration of
shoots and roots effectively suppressed the development of non-transgenic plants. This protocol was successful with a commercially important sugarcane cultivar, CP-88-1762, at a transformation efficiency of
two independent transgenic plants per g of callus.
Key words Agrobacterium tumefaciens-mediated transformation, Embryogenesis, Genetic transformation, Saccharum spp. hybrids, Sugarcane
Introduction
The current sugarcane cultivars are typically interspecific hybrids
between Saccharum officinarum and Saccharum spontaneum.
Some of the interspecific hybridizations also involve S. robustum,
S. sinense, S. barberi, Miscanthus, or Erianthus [1].
Sugarcane is an economically important crop in the tropical
and subtropical regions of the world. Over 75 % of the worlds
sugar is derived from sugarcane. Sugarcane is also the most efficient feedstock for the production of the biofuel ethanol. The cost
of sugarcane ethanol production in Brazil is lower than that of corn
ethanol in the USA, and its production emits less greenhouse gases
[2]. Genetic transformation allows targeted improvement of key
traits in crop and biofuel production.
The first transgenic sugarcane plants were described in 1992
following biolistic gene transfer [3]. Agrobacterium-mediated
gene transfer for the production of transgenic sugarcane plants was
first reported in 1998 [4]. In contrast to Agrobacterium-mediated
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_24, Springer Science+Business Media New York 2015
307
308
2
2.1
Materials
Plant Material
2.2 Agrobacterium
Strain and Vector
2.3
Stock Solutions
309
310
Media
2.4.1 Media
for Agrobacterium
311
3
3.1
Methods
Timeline
312
Callus Induction
1. Disease- and insect-free tops are cut below the top visible node
when 58 aboveground nodes are visible. Expanded leaves are
cut from tops, and the immature leaf whorl is surface sterilized
by wiping with 70 % ethanol.
2. After removing the top layer of the leaf whorl, the tops are
placed into a laminar hood and wiped again with 70 % ethanol.
One to two layers of the leaf whorl are aseptically removed
until the apical meristem becomes visible.
3. Ten to twenty leaf whorl cross sections of 12 mm thickness
are aseptically cut from the leaf whorl section above the apical
meristem using sharp razor blades and placed onto solidified
CI3 medium (see Note 5) [22].
4. Seven days after culture initiation, leaf whorl cross sections are
separated into half circle segments and then subcultured to
fresh CI3 medium every 7 days at 28 C and in darkness (see
Note 6). Callus growth was visible 2 weeks after culture initiation. Calli are used for inoculation with Agrobacterium 68
weeks after culture initiation and 35 days after the last subculture (see Note 4).
3.3 Preparation
of Agrobacterium
Culture
313
1. Pellet the bacteria by centrifugation for 30 s in a microcentrifuge. Resuspend in 2 volume of VGIM medium. Shake gently
at 20 C with 50 rpm (Innova 42 shaker) for 1424 h. Pellet
the bacteria by centrifugation 30 s in a microcentrifuge [23].
Resuspend in equal volume of liquid cocultivation medium.
2. Transfer calli to a sterile 150 mL flask.
3. Prepare the inoculum by diluting the resuspended bacteria in
liquid cocultivation medium in a 1:49 ratio (see Note 7).
4. Pour the inoculation solution into the flask containing the
calli. Ensure all the calli are submerged in the solution. Incubate
the calli in the inoculation solution at room temperature for
20 min, with occasional and gentle shaking.
5. Transfer the calli onto sterile filter papers (Whatman #1004090)
to remove excessive liquid.
6. Prepare the cocultivation plates by placing three layers of filter
papers into a sterile 9 cm petri dish and pipette 45 mL cocultivation medium onto the filter papers.
7. With a pipette, remove the excess cocultivation medium from
the petri dish and transfer the calli to the filter paper with
cocultivation medium. Seal the petri dish with Parafilm, and
incubate in the dark at 19 C for 3 days.
8. Spread the cocultivated calli on three layers of sterile dry filter
paper to remove excessive moisture. Repeat this process with
fresh filter papers until the filter papers with calli remain dry.
Expose the calli on dry filter paper to the air for 1020 min in
a laminar hood for further drying (see Note 8).
3.5 Recovery,
Selection,
and Regeneration
314
3.6 Transplanting
and Greenhouse Care
Notes
1. When alternative genotypes are considered for this protocol,
preselect suitable genotypes on the basis of their ability to generate highly embryogenic callus and their plant regeneration
potential from embryogenic callus.
2. Do not freeze this stock solution; instead make it fresh before
use to preserve its potency.
3. When media are supplemented with geneticin or paromomycin media, use agarose instead of Phytagel or agar to prevent
the reagents from precipitating.
4. Callus growth rates vary with the developmental stage of the
explant and the genotype. Use fast-growing, highly embryogenic callus as soon as it easily detaches from the explant (68
weeks after culture initiation). Longer callus induction periods
increase both the transformation efficiency and the frequency
of undesirable somaclonal variation.
5. The orientation of the leaf whorl cross sections on the medium
influences the induction of embryogenic callus. The removed
surface that is distal to the apical meristem should be in contact
with the medium.
315
9.
10.
11.
12.
13.
14.
316
15.
16.
17.
18.
19.
Part V
Other Important Plants
Chapter 25
Hemp (Cannabis sativa L.)
Mistianne Feeney and Zamir K. Punja
Abstract
Hemp (Cannabis sativa L.) suspension culture cells were transformed with Agrobacterium tumefaciens
strain EHA101 carrying the binary plasmid pNOV3635. The plasmid contains a phosphomannose isomerase (PMI) selectable marker gene. Cells transformed with PMI are capable of metabolizing the selective
agent mannose, whereas cells not expressing the gene are incapable of using the carbon source and will
stop growing. Callus masses proliferating on selection medium were screened for PMI expression using a
chlorophenol red assay. Genomic DNA was extracted from putatively transformed callus lines, and the
presence of the PMI gene was confirmed using PCR and Southern hybridization. Using this method, an
average transformation frequency of 31.23 % 0.14 was obtained for all transformation experiments, with
a range of 15.155.3 %.
Key words Agrobacterium-mediated transformation, Callus, Cannabis sativa, Chlorophenol red
assay, Hemp, Mannose selection, Phosphomannose isomerase, Plant tissue culture, Suspension
culture, Transformation protocol
Introduction
Hemp (Cannabis sativa L.) is regaining importance as a cultivated
crop after decades of legal prohibitions. Hemp cultivation is now
permitted in Canada under strict governmental control. The species,
Cannabis sativa, has been selected for very different qualities; while
hemp varieties are cultivated for seed, oil, and fiber and may contain
only trace amounts of the psychoactive drug, 9-tetrahydrocannabinol
(THC), related marijuana varieties are selected for high THC content [13]. Hemp and marijuana are very difficult to distinguish
morphologically but are biochemically distinct.
There is interest in developing improved varieties of hemp that
are resistant to disease and pest pressures and possess enhanced
qualities [1, 4]. Plant tissue culture and genetic transformation are
biotechnological approaches that can be used to compliment conventional breeding toward hemp improvement [5]. Clonal multiplication of hemp plants can be achieved through micropropagation by
shoot tip culture [6]. A method was also identified to encapsulate
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_25, Springer Science+Business Media New York 2015
319
320
axillary bud explants using synthetic seed technology [7]. Hemp has
long been considered recalcitrant to both regeneration and transformation; explants readily form callus and develop roots but have had
a very poor ability for shoot formation [4, 810]. However, recent
progress is now shifting this notion. Indirect shoot organogenesis
was demonstrated in a variety of explant sources and cultivars, but
the efficiency of plantlet regeneration was low [11]. A much higher
frequency plant regeneration and acclimatization were achieved by
direct shoot organogenesis from nodal segments containing axillary
buds [12]. At present, there is no established protocol for regeneration of hemp by somatic embryogenesis.
Hemp has been shown to be amenable to Agrobacteriummediated transformation [10]. Recently, Wahby et al. [13] successfully demonstrated the ability to establish stable transformed
hemp tumor and hairy root lines using a wide variety of
Agrobacterium tumefaciens and A. rhizogenes strains, respectively.
However, to our knowledge, Agrobacterium-mediated transformation has only rarely been applied toward the improvement of
hemp with desirable traits. In the only account that we are aware,
MacKinnon et al. [4] reported to have developed Botrytis-resistant
hemp plants using an Agrobacterium tumefaciens-mediated transformation procedure; however, details of their work were not
described. Our objective was to demonstrate that gene transfer can
occur in callus cultures, with the anticipation that a regeneration
protocol will be established involving an intervening callus phase.
With the recent advancements in hemp regeneration [11, 12], this
goal is now becoming more attainable.
This chapter describes the Agrobacterium-mediated transformation of hemp callus with the selectable marker gene phosphomannose isomerase (PMI). The PMI gene confers a metabolic
advantage to the plant cell, allowing growth on a selective medium
containing a sugar, mannose, as the selective agent [14]. Methods
are outlined for the initiation and establishment of hemp callus and
suspension cultures. Callus growing on selection is screened for
PMI expression using a biochemical assay. DNA is extracted from
putatively transformed callus lines and analyzed by PCR and
Southern hybridization techniques to detect the gene of interest.
An average transformation frequency of 31.23 % 0.14 was obtained
for all transformation experiments, with a range of 15.155.3 %.
This value represents an average of 31 mannose-metabolizing independent events in 100 explants targeted for transformation.
Materials
1. Hemp seeds cv. Anka are monoecious and cultivated for seed.
2. Potting mix soil: Sunshine Mix No. 1 (Sun Gro Horticulture,
Bellevue, WA).
321
1. Agrobacterium tumefaciens strain EHA101 [17] contains plasmid pNOV3635 as a binary vector [10]. The plasmid
pNOV3635 carries a PMI gene under control of the Arabidopsis
thaliana ubiquitin promoter (Ubq3) and the nopaline synthase
terminator (NOS). Spectinomycin and kanamycin selectable
markers are present on the pNOV3635 plasmid and the Ti
plasmid carrying the virulence genes, respectively.
2. LB (Luria-Bertani medium): 10 g/L bacto-tryptone, 5 g/L
bacto-yeast extract, 10 g/L NaCl, and 15 g/L bacteriological
agar (for solid medium).
3. Spectinomycin dihydrochloride: dissolve in ddH2O at
0.25 M. Filter sterilize using a 0.2 m filter and store in aliquots at 20 C.
4. Kanamycin monosulfate: dissolve in ddH2O at 0.17 M. Filter
sterilize using a 0.2 m filter and store in aliquots at 20 C.
5. Agrobacterium cultures are centrifuged using a Beckman
GS-6R centrifuge (Beckman Coulter Inc., Fullerton, CA).
322
6. Acetosyringone (3,5-dimethoxy-4-hydroxyacetophenone):
dissolve in a small quantity of 100 % methanol. Dilute with
ddH2O at 100 mM. Filter sterilize using a 0.2 m filter and
store in aliquots at 20 C.
2.3
Transformation
2.3.1 Inoculation
and Cocultivation
2.3.2 Selection
2.4
PMI Assay
2. Polyvinylpolypyrrolidone (PVPP).
323
Methods
324
3.3
Transformation
3.3.1 Inoculation
and Cocultivation
3.3.2 Selection
325
Fig. 1 Selection of Anka callus transformed with pNOV3635 on MB2.5D with 300 mg/L Timentin and 1 % mannose after 4 weeks. (a) Non-transformed cell masses are arrested in growth. (b) Transformed cell masses are
distinguished by their enlarged size compared to untransformed callus. (c) Transformed callus on mannose
medium, forming large, pale yellow callus protruding from small, dark-yellow parental callus. Photos are of
9 cm diameter dishes. Scale bar: 5 mm for (c). (d, e) Chlorophenol red PMI assay after 34 days. Medium is
composed of MB2.5D, 8 g/L agar, and 0.1 g/L chlorophenol red with either 3 % sucrose or 2 % mannose.
Control wells do not contain callus. (T ) Transformed callus harboring the PMI gene grew on both sugar sources,
turning the medium pale yellow. (AS ) Non-transformed callus metabolized sucrose and acidified the medium,
turning it a pale yellow color. (AM ) Callus incubated with Agrobacterium lacking the PMI plasmid did not acidify
the medium. Well diameter in each dish is 1.5 cm (Reproduced with permission from ref. 10)
PMI Assay
326
3.5.2 PCR
327
Notes
1. Assaying other hemp varieties or tissue types may require
adjusting the concentration of chlorophenol red within the
assay medium. Higher concentrations of chlorophenol red (up
to 2 mg/mL) incorporated into the PMI assay medium caused
transgenic callus to absorb the dye and produced no color
change within wells. The callus did not resume growth when
transferred back to mannose- or sucrose-containing medium.
An excess uptake of chlorophenol red from the assay medium
may arrest tissue metabolism and cause cell death.
2. Hemp seedlings attract thrip insect pests. Thrip eggs are difficult to see and can survive the sterilization process and ruin
experiments. A thrip predatory mite (Amblyseius cucumeris)
can be successfully used as a biological control agent to significantly lower or eliminate the thrip population before a new
batch of seeds are planted for experiments. The predatory
mites have not interfered with experiments and can be purchased from local garden stores.
3. After sterilization, it is important to place seedling material
onto sterile moistened filter paper while cutting explants.
Seedling tissues quickly wilt under flow hood conditions,
which may affect callus development. The moistened filters
keep tissues turgid during processing.
4. Agrobacterium cultures should be frozen at 80 C for longterm storage. To do this, transfer 750 L bacteria and 250 L
50 % (v/v) sterile glycerol to a labeled, sterile cryotube (Nunc,
Thermo Fisher Scientific). Gently invert tube to mix, quickly
freeze in liquid nitrogen, and store at 80 C. To start a culture
for experiments, scrape cells from the frozen culture with a
sterile loop and streak an LB plate containing the appropriate
selection.
328
Acknowledgments
We thank S. Clemens for providing technical assistance with
Southern hybridizations. This research was funded by the Natural
Sciences and Engineering Research Council of Canada to ZKP and
the John Yorston Scholarship in Pest Management to MF. In
accordance with Health Canada regulations, all hemp cultures
used in this research were subjected to regular analyses for THC
content over the duration of the study to ensure they did not
exceed the legal allowable limit of 0.3 % THC. The cultures were
grown under permit No. 00-F0041-R-01 and disposed of according to the requirements.
329
References
1. Clarke RC (1999) Botany of the genus
Cannabis. In: Ranalli P (ed) Advances in hemp
research. Haworth, Binghamton, NY, pp 119
2. Lata H, Chandra S, Techen N, Khan IA, ElSohly
MA (2011) Molecular analysis of genetic fidelity
in Cannabis sativa L. plants grown from synthetic (encapsulated) seeds following in vitro
storage. Biotechnol Lett 33:25032508
3. Stott CG, Guy GW (2004) Cannabinoids for the
pharmaceutical industry. Euphytica 140:8393
4. MacKinnon L, McDougall G, Aziz N, Millam
S (2001) Progress towards transformation of
fibre hemp. In: Macfarlane Smith WH,
Heilbronn TD (eds) Annual Report of the
Scottish Crop Research Institute, 2000/2001,
SCRI, Invergowrie, Dundee, pp 8486
5. Ranalli P (2004) Current status and future
scenarios of hemp breeding. Euphytica 140:
121131
6. Wang R, He L-S, Xia B, Tong J-F, Li N, Peng
F (2009) A micropropagation system for cloning of hemp (Cannabis sativa L.) by shoot tip
culture. Pak J Bot 41:603608
7. Lata H, Chandra S, Khan IA, ElSohly MA
(2009) Propagation through alginate encapsulation of axillary buds of Cannabis sativa L.: an
important medicinal plant. Physiol Mol Biol
Plants 15:7986
8. Fisse J, Braut F, Cosson L, Paris M (1981)
tude in vitro des capacits organogntiques
de tissus de Cannabis sativa L.; effet de diffrentes substances de croissance. Pl Md
Phytopath 15:217223
9. Mandolino G, Ranalli P (1999) Advances in
biotechnological approaches for hemp breeding and industry. In: Ranalli P (ed) Advances in
hemp research. Haworth Press, Binghamton,
NY, pp 185212
10. Feeney M, Punja ZK (2003) Tissue culture and
Agrobacterium-mediated transformation of
hemp (Cannabis sativa L.). In Vitro Cell Dev
Biol Plant 39:578585
11. Slusarkiewicz-Jarzina A, Ponitka A, Kaczmarek
Z (2005) Influence of cultivar, explant source
and plant growth regulator on callus induction
and plant regeneration of Cannabis sativa
L. Acta Biol Crac Ser Bot 47:145151
12. Lata H, Chandra S, Khan I, ElSohly MA
(2009) Thidiazuron-induced high-frequency
direct shoot organogenesis of Cannabis sativa
L. In Vitro Cell Dev Biol Plant 45:1219
13. Wahby I, Caba JM, Ligero F (2013)
Agrobacterium infection of hemp (Cannabis
sativa L.): establishment of hairy root cultures.
J. Plant Interact 8:312320
Chapter 26
Orchids (Oncidium and Phalaenopsis)
Chia-Wen Li, Chia-Hui Liao, Xia Huang, and Ming-Tsair Chan
Abstract
This chapter describes an efficient and reproducible method for large-scale propagation of Oncidium and
Phalaenopsis protocorm-like bodies (PLBs) using floral stalk sections and seeds, respectively. The propagated PLBs can be used for Agrobacterium-mediated transformation. An advanced transformation system
for Oncidium and Phalaenopsis orchids has been established. This protocol demonstrates that the time
during which the PLBs are cocultivated with Agrobacterium is the key to promoting transformation
efficiency. Modified DNA and RNA extraction methods are also provided to diminish polysaccharide
contamination and to improve the quality for further molecular analysis.
Key words Agrobacterium tumefaciens, Oncidium orchid, Phalaenopsis, Protocorm-like body (PLB)
Introduction
The elegant shapes of orchids make them very popular flowers.
The orchid Phalaenopsis is an important floriculture product in
Taiwan. The export value of Phalaenopsis reached USD
114,175,000 in 2012 (Agricultural Trade Statistics Query System,
Council of Agriculture, Executive Yuan, Taiwan). Oncidium spp.
are also commercially important in Taiwan as cut flowers and as
flowering potted orchid plants. During the 1980s, a commercial
Oncidium Gower Ramsey was introduced into Taiwan and has
become a most important cut orchid flower variety. By 2012, the
planted area of Oncidium had increased to more than 200 ha, and
cut flower exports had reached 1,618 t (valued at USD 18,462,000)
to the Japan floral cutting market, which accounts for 94.3 % of all
cut and potted Oncidium flower exports from Taiwan (Agricultural
Trade Statistics Query System, Council of Agriculture, Executive
Yuan, Taiwan).
Oncidium Gower Ramsey has brought economic benefits, but
the unitary flower colors and patterns of this variety limit market
expansion. Oncidium orchids are self-incompatible, so it is difficult
to obtain new traits by traditional breeding. Since the 1990s, gene
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_26, Springer Science+Business Media New York 2015
331
332
Chia-Wen Li et al.
Materials
1. Floral stalk of Oncidium Gower Ramsey orchid (see Fig. 1a, b).
333
Fig. 1 Development of Oncidium explants during transformation procedures. (a) Floral stalk of Oncidium Gower
Ramsey. Arrows show the nodes. (b) Stalk sections for PLB initiation. (c) PLB formation on NDM medium.
(d) PLB propagation on G10 medium. (e) Cultured Agrobacterium broth and PLBs. (f) PLBs cocultivated with
Agrobacterium in NDM liquid medium. (g, h) PLBs selected on G10 medium with antibiotics. (i) GUS staining of
PLBs after hygromycin selection. (j) Regeneration of transgenic Oncidium seedlings on G10 medium.
(k) Transgenic plants in jars (G10 medium). (l) Transgenic plant in pot (Sphagnum moss medium). (m) Flowering
Oncidium plant
334
Chia-Wen Li et al.
Fig. 2 Development of Phalaenopsis explants during transformation procedures. (a) Phalaenopsis equestris
silique. (b) PLB propagation on NDM solid medium. (c) PLBs cocultivated with Agrobacterium in NDM liquid
medium. (d, e) PLBs selected on T2 medium with antibiotics. (f) Regeneration of transgenic Oncidium seedlings on T2 medium. (g) Transgenic plants in jar (T2 medium). (h) Transgenic plant in pot (Sphagnum moss
medium). (i) Flowering Phalaenopsis equestris plant. (j) Molecular analysis of transgenic plants. Upper panel:
RT-PCR. Lower panel: Southern blot. GH, plants grown in greenhouse; CR, plants grown in culture room;
W, wild-type plant, pflp, sweet pepper ferredoxin-like gene; HPT, hygromycin phosphotransferase gene; 18S,
18S ribosomal RNA
335
336
Chia-Wen Li et al.
337
Methods
Different transformation stages of Oncidium and Phalaenopsis are
illustrated in Figs. 1 and 2, respectively. The transformation flow
charts of Oncidium and Phalaenopsis are presented in Figs. 3 and 4,
respectively.
3.1 Initiation
of Protocorm-Like
Bodies (PLBs)
3.1.1 Initiation
of Oncidium PLBs
from Stalk Buds (4 Months)
3.1.2 Initiation
of Phalaenopsis PLBs
from Seeds (5 Months)
338
Chia-Wen Li et al.
4M
Propagate PLBs on G10 medium
21 D
Pretreatment of PLBs in NDM/AS liquid medium
3D
Co-cultivate the PLBs with Agrobacterium in NDM liquid
medium (A600<0.6)
3-7 D
Wash and blot dry the infected PLBs
M
1
2
3
4
5
6
7
8
9
10
11
5M
Subculture selected explants to G10/Myron medium with
selection agent and subculture at 4 weeks interval
2M
Genomic PCR and GUS histochemical confirmation
12
13
14
15
16
17
18
19
4M
Molecular analysis
20
21
22
23
24
12 M
Flowering
25
26
27
28
339
5M
Propagate PLBs in NDM solid medium, and subculture at
3 weeks interval
21 D
Pretreatment of PLBs in NDM/AS liquid medium
3D
Co-cultivate the PLBs with Agrobacterium in NDM liquid
medium (A600<0.6)
3-7 D
Wash the infected PLBs
5M
Propagate selected explants to T2/Myron medium with
selection agent and subculture at 4 weeks interval
5M
Genomic PCR and GUS histochemical confirmation
Plant 0.5-1 cm height seedlings on T2 medium
6M
Molecular analysis
M
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
3.1.3 Propagation
of PLBs (21 Days)
340
Chia-Wen Li et al.
3.3 Agro-infiltration
and Cocultivation
(57 Days)
1. Newly differentiated Oncidium/Phalaenopsis PLBs (21-dayold culture) were selected from PLB propagation medium
(G10/NDM solid medium) (see Figs. 1d and 2b).
2. Healthy PLBs (about 3 g) were transferred into a 125 ml flask,
30 ml NDM/AS liquid medium was added, and the flask was
incubated in the dark at 26 C for 3 days (optional, see Note 4).
3. AS-pretreated PLBs were transferred into a new 125 ml flask,
and 30 ml NDM liquid medium and 1 ml overnight-cultured
Agrobacterium (A600nm = 0.30.5 in MGL medium) were added
(see Figs. 1e and 2c). The PLBs were cocultivated with
Agrobacterium with shaking (70 rpm) in the dark for 17 days
at 26 C (see Note 9). During the cocultivation period, cocultivated broth was removed and NDM medium added to keep
the bacterial concentration below 0.6 A600nm (see Note 10).
4. The infected PLBs were washed five times in sterilized ddH2O
supplemented with 40 mg/l meropenem (see Note 11).
3.4 Selection
of Transformed
PLBs (5 Months)
3.5 Plant
Regeneration
and Conversion
(6 Months)
341
342
Chia-Wen Li et al.
13. The pellet was washed with cold 70 % EtOH, rinsed with
100 % EtOH, and then dried.
14. The DNA was dissolved in 50100 l TE and quantified by
spectrophotometry (NanoDrop).
3.6.2 PCR Amplification
343
3.6.6 Reverse
Transcription (RT)-PCR
1. For 30 l RT reaction, 13 g total RNA was mixed thoroughly with 2 l 10 M dT(1518) and H2O to a final volume of
11 l, then incubated at 72 C for 10 min.
2. RT mixture (19 l comprising 7.4 l H2O, 6 l 5 RT buffer,
5 l 2.5 mM dNTP, 0.5 l MMLV-reverse transcriptase
(Promega), 0.1 l RNase inhibitor (Takara)) was added and
then incubated at 42 C for 70 min.
344
Chia-Wen Li et al.
1. For GUS staining, antibiotic-selected PLBs or leaves of regenerated plants were soaked in X-Gluc staining solution (pH 8.0)
overnight at 37 C.
2. The chlorophyll was removed by soaking in 95 % EtOH.
Notes
1. The concentration of phytohormones for PLB initiation can
differ among cultivars.
2. If the selection antibiotic is kanamycin, the gelatinous substance
Phytagel should be replaced with agar. Note that the kanamycin
selection system has a higher false-positive ratio than the hygromycin selection system in orchid transformation.
3. Charcoal can improve the growth and development of
Phalaenopsis orchid seedlings by absorbing harmful secondary
metabolites secreted by the orchid. However, it is not a necessary ingredient for Oncidium PLB propagation and regeneration or for Phalaenopsis orchid seed germination.
4. Additional acetosyringone (AS) is optional for Oncidium
transformation. Application of AS has little effect on the transformation efficiency of Oncidium Gower Ramsey and
Phalaenopsis orchids.
5. Other strains of Agrobacterium, such as LBA4404 and
GV3101, can also be employed, while EHA105 demonstrated
a better transformation efficiency as tested in both Oncidium
Gower Ramsey and Phalaenopsis orchids.
6. The antibiotic used for transformation depends on the selection marker of the binary vector; the antibiotic concentration
can differ among cultivars.
7. The age and condition of the PLBs can influence the regeneration efficiency. Differentiated small shoots should be removed
before propagating transformation materials.
8. The antibiotics used for Agrobacterium preculture depend on
the Agrobacterium strain and the cloning selection marker
in the binary vector. MGL broth is better than YEP broth for
Agrobacterium preculture. Agrobacterium cultured in YEP
broth can aggregate, reducing the transformation efficiency.
345
9. The length of the cocultivation period can influence transformation efficiency and can differ among test orchid cultivars
and Agrobacterium strains. Over-cocultivation will lead to
hyperhydricity of the PLBs and make it difficult to eliminate
Agrobacterium on the selection medium.
10. Overgrowth of Agrobacterium needs to be prevented to avoid
the death/desiccation of infected PLBs during the cocultivation period.
11. Timentin (200 mg/l) or other antibiotics can be employed to
replace meropenem (40 mg/l) to eliminate Agrobacterium,
but a higher antibiotic concentration entails more cost.
12. Starting materials (protocorms or PLBs) are small, and the
transformation efficiency is difficult to calculate. In average,
1030 transgenic lines can be obtained from 20 g fresh weight
of activated grown PLBs. In addition, the newly propagated
PLBs both from seeds and floral stalks have higher transformation efficiency than those that have been subcultured for a long
time.
13. RNAmate (BioChain Catalog No.: L1011100, Hayward, CA)
can efficiently reduce polysaccharide and proteoglycan contamination in orchid DNA. RNAmate can also be used for
RNA extraction by mixing with an RNA phenol extraction
reagent such as TRIzol. However, the conventional extraction
method followed by LiCl precipitation can also be used to
eliminate the coprecipitated polysaccharides.
Acknowledgments
This work was supported by grants from Academia Sinica and the
National Science Council of the Republic of China.
References
1. Hieber AD, Mudalige-Jayawickrama RG,
Kuehnle AR (2006) Color genes in the orchid
Oncidium Gower Ramsey: identification, expression, and potential genetic instability in an interspecific cross. Planta 223:521531
2. Liau CH, Lu JC, Prasad V, Hsiao HH, You SJ,
Lee JT, Yang NS, Huang HE, Feng TY, Chen
WH, Chan MT (2003) The sweet pepper
ferredoxin-like protein (pflp) conferred resistance against soft rot disease in Oncidium orchid.
Transgenic Res 12:329336
3. Chan YL, Lin KH, Sanjaya, Liao LJ, Chen WH,
Chan MT (2005) Gene stacking in Phalaenopsis
346
Chia-Wen Li et al.
Chapter 27
Poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch)
M. Ashraful Islam, Tage Thorstensen, and Jihong Liu Clarke
Abstract
Genetic engineering is an important tool for introducing desired genes into poinsettia (Euphorbia pulcherrima
Willd. ex Klotzsch). We describe in this chapter an Agrobacterium tumefaciens-mediated transformation
protocol for poinsettia. A detailed description of genetic transformation, antibiotic selection, subsequent
regeneration via somatic embryogenesis, and rooting as well as molecular and morphological analyses is
included. The methodology described here could facilitate the future engineering of poinsettia for research
purpose as well as commercial production of poinsettia plants with improved resistance or novel traits.
Key words Agrobacterium tumefaciens-mediated transformation, Poinsettia, Agrobacterium
tumefaciens, Somatic embryogenesis, Binary vector, Transgenic plants
Introduction
Poinsettia, a nonfood, non-feed ornamental plant, is a contemporary
symbol of Christmas in most parts of the world. Since it was introduced to the USA in 1825 from Mexico, poinsettia has become the
primary potted flower produced and sold in North America,
Europe, Asia, and Australia [1, 2]. The annual production in the
USA and the EU is 50 million and 100 million plants, respectively,
and the yearly production in 2008 was estimated to comprise a
value of around 155 million US dollars [3]. Hence, the innovation
potential of poinsettia is considerable and has become the driving
force for the development of genetic engineering protocols for
poinsettia.
Genetic engineering is an effective tool for breeding ornamental plants with the addition of desirable traits such as engineering
of gibberellin biosynthetic pathway to induce dwarfism in poinsettia reducing the chemical spray (Clarke et al. unpublished results),
novel colors, and resistance to pathogens and insects [46]. This
technology has been successfully utilized in the production or
improvement of a number of important ornamental crops, e.g.,
blue roses [7], novel carnations (www.florigene.com), transgenic
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_27, Springer Science+Business Media New York 2015
347
348
2
2.1
Materials
Plant Material
2.2 Agrobacterium
tumefaciens Strain
and Vector
349
Fig. 1 Hairpin RNA (hpRNA) gene construct containing coat protein-encoding gene (Partially reproduced from
Clarke et al. [13], an OA publication with permission from Springer)
Stock Solutions
350
Media
351
Methods
3.1 Transformation
of Agrobacterium
tumefaciens
by Electroporation
1. Isolate plasmid DNA from E. coli cultures by using a commercial kit or by following a standard laboratory mini-prep protocol. The concentration of DNA should be about 100 ng/L.
2. Thaw commercially purchased A. tumefaciens LBA4404 cells
(Invitrogen, California, USA) on wet ice (see Notes 4 and 5).
3. Take 20 L competent cells in two new precooled microcentrifuge tubes and add 2 L undiluted and 1 L 10 diluted plasmid DNA respectively in each tube.
4. Mix gently; pipette the cell and DNA mixtures (22 L) immediately into the 0.1 cm cuvette and electroporate using an ECM
630 Electro Cell Manipulator (BTX Harvard Apparatus) with
conditions: 2.0 kV, 200 resistance, and 25 F capacitance.
5. Add 1 mL LB medium to the cuvettes, mix gently by pipetting, and transfer the solutions to a 15 mL tube.
6. Shake for 23 h at 225 rpm and 30 C in the incubator.
7. In the meantime plates with LBA and appropriate antibiotic
(50 g/mL) should be prepared.
8. Transfer the cells to the plates, spread them evenly, and wrap
with plastic film.
9. Keep the plates upside down at 28 C for 2 days or until bacterial colonies appear.
10. Select one colony to proceed with for the glycerol stock (for
long-term storage at 80 C) (see Note 6).
3.2 Preparation
of Agrobacterium
Culture
352
3.4 Agrobacterium
Infection,
Cocultivation,
and Plant
Regeneration
353
Fig. 2 Agrobacterium tumefaciens-mediated transformation of poinsettia. (a) Stem segments placed on the
callus induction medium (CIM) after Agrobacterium inoculation. (b) Somatic embryos at different development
stages emerged from embryogenic calli on the somatic embryo induction medium (SEIM). (c) An embryogenic
callus with early-stage somatic embryos. (d) Cotyledonary stage of somatic embryos. (e) Plantlets with welldeveloped roots growing on the root induction medium (RIM). (f) Regenerated plants with roots are transferred
into soil in the greenhouse (Partially reproduced from Clarke et al. [13], an OA publication with permission from
Springer)
354
Notes
1. Agrobacterium strains must be propagated at optimal conditions (shaker speed: 180 rpm, incubator temperature: 28 C)
to reach the desired optical density (OD600) 0.60.8.
2. Acetosyringone application can increase the transformation
frequencies in many species [17]. It can be used to induce virulence genes in Agrobacterium [18, 19].
3. Antibiotics (50 g/mL) can be added to LB or LB Agar
(LBA), after the medium is cooled down to approximately
50 C. Too high temperature (>50 C) is likely to destroyed or
inactivated antibiotics. For the LBA plates, petri dishes (9 cm
in diameter) are used, and the LBA plates are stored at 4 C.
4. All the bacterial culture preparations are done in a laminar air
flow hood under sterile conditions.
5. A. tumefaciens LBA4404 competent cells can also be prepared
in the laboratory according to the standard molecular protocols. However, the transformation efficiency may be affected
due to the quality of homemade A. tumefaciens cells.
6. Bacterial stocks containing 15 % glycerol are important for
long-term storage of plasmids. Glycerol stabilizes the frozen
bacteria and prevents damage to the cell membranes as well as
keeping the cells alive. A bacterial glycerol stock can be stored
stably at 80 C for many years.
7. Individual stem explants excised from greenhouse-growing
poinsettia plants are kept in water before approximately 2530
stem explants are collected for a transformation experiment.
After sterilization, stem segments (11.5 mm thickness) are
generated and kept under moist conditions on plates (sterile
filter papers are soaked with MS-2 on a plate) prior to transformation avoiding dehydration of stem segments which can lead
to low Agrobacterium tumefaciens-mediated transformation
efficiency.
8. Experiment size can be adjusted based on the explants available. In a standard poinsettia transformation experiment at our
355
laboratory, we use about 300 stem segments and the corresponding volume of Agrobacterium cell culture.
9. The dark culture condition can be achieved by either placing
all petri dishes in a box and cover with aluminum foil to avoid
any light or keep the petri dishes in a dark room.
10. Subculture every third week is necessary to promote the root
development.
11. It is important to cover plantlets with a clear plastic covering
before putting into a tray in the greenhouse. Also put up very
thin cloths (for a few weeks) to prevent direct sunlight as well as
to prevent wilting. Water carefully to regulate soil humidity.
After the plants are established, water should be given every day.
12. Four- to six-week-old putative transgenic poinsettia plants are
at the ideal stage for collecting plant materials for molecular
and various other analyses.
Acknowledgments
Thanks are due to Sissel Haugslien, Dag-Ragnar Blystad, Erling
Flistad, and Carl Spetz for their practical support; CSIRO Plant
Industry for providing pHANNIBAL and pART27 vectors; and
J. Kristiansen nursery for the poinsettia plants. We are grateful to
Dr. Nicholas Clarke for the linguistic correction. This research was
supported by the Research Council of Norway grants 147147/140
and 199398 to Dr. Jihong Liu Clarke.
References
1. Ecke P III, Faust JE, Higgins A, Williams J
(2004) The Ecke poinsettia manual. Ball,
Batavia, IL
2. Williams J (2005) Poinsettia production.
FlowerTech 8:69
3. USDA (2009) [NASS] Floriculture crops:
2008 summary. Sp Cr 61. (http://usda.
mannlib.cornell.edu/usda/current/
FlorCrop/FlorCrop-04-23-2009.pdf)
4. Deroles SC, Boase MR, Lee CE, Peters TA
(2002) Gene transfer to plants. In: Vainstein A
(ed) Breeding for ornamentals: classical and
molecular approaches. Kluwer, Dordrecht,
pp 156196
5. Hammond J, Hsu HT, Huang Q, Jordan R,
Kamo K, Pooler M (2006) Transgenic
approaches to disease resistance in ornamental
crops. J Crop Improv 17:155210
6. Ltken H, Clarke JL, Mller R (2012) Genetic
engineering and sustainable production of
7.
8.
9.
10.
11.
12.
ornamentals: current status and future directions. Plant Cell Rep 31:114111573
Yoshikazu T (2004) Visual biotechnology.
Blue rose realized by biotechnology. Biosci Ind
62:789790
Kamo K, Hammond J, Roh M (1997)
Transformation of Gladiolus for disease resistance. J Kor Soc Hort Sci 38:188193
Teixeira da Silva JA (2004) Ornamental chrysanthemum: improvement by biotechnology.
Plant Cell Tiss Org Cult 79:118
Smith F, Chou TS, Eisenreich R, Sanford J,
Blowers A, Van Eck J (1997) Production of
transgenic poinsettia. US Patent 7,119,262,
pp 136
Vik NI, Hvoslef-Eide AK, Gjerde H, Bakke K
(2001) Stable transformation of poinsettia via
electrophoresis. Acta Hort 560:101103
Clarke JL, Klemsdal SS, Flistad E, HvoslefEide AK, Haugslien S, Moe R, Blystad DR
356
Chapter 28
Populus trichocarpa
Quanzi Li, Ting-Feng Yeh, Chenmin Yang, Jingyuan Song,
Zenn-Zong Chen, Ronald R. Sederoff, and Vincent L. Chiang
Abstract
Populus trichocarpa Nisqually-1 is a clone of black cottonwood that is widely used as a model woody plant.
It was the first woody plant to have a full genome sequence and remains today as the model for growth,
metabolism, development, and adaptation for all woody dicotyledonous plants. It is one of the bestannotated plant genomes available. It is also currently studied to improve bioenergy feedstocks and to
learn about responses to environmental variation that may result from climate change. It is the best characterized woody plant for lignin biosynthesis. In spite of its role as a model woody plant, many important
genetic applications have been limited because it was particularly difficult for DNA transformation. The
ability to transform P. trichocarpa is a central component of a systems biology approach to the study of
metabolic and developmental processes, where in combination with genome and transcriptome sequencing, all the expressed genes for specific pathways can be defined, cloned, and characterized for biological
function.
We previously reported on a method for Agrobacterium-mediated genetic transformation in P. trichocarpa (Song et al. Plant Cell Physiol 47: 15821589, 2006). Since then, we have optimized the protocol
based on many experiments that varied in tissue manipulation, media, DNA constructs and Agrobacterium
strains. A modified step-by-step protocol for Agrobacterium-mediated transformation of stem explants is
described here. The health of the tissue explants and the time of cocultivation are among the critical steps
in the protocol for successful transformation. This updated protocol should be helpful to many laboratories that are currently carrying out P. trichocarpa transformation. It should also encourage many labs that
have not yet had success with P. trichocarpa to try again.
Key words Agrobacterium tumefaciens, Genetic transformation, Populus trichocarpa, Transgenic trees
Introduction
Agrobacterium-mediated transformation is a widely used and
powerful tool for molecular genetics and plant biotechnology.
Transgenic plants are unique materials to study gene function.
Near-whole-genome sequence is now available for many species
and is being applied to many problems through new techniques,
such as next-generation sequencing and ChIP-seq, to gain
unprecedented insights into biological structure and function.
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Quanzi Li et al.
Overexpression and knockdown/knockout transgenics are increasing our understanding of gene function and the roles of specific
genes in genetic regulatory networks. Forest trees, which produce
large amounts of lignocellulosic biomass, are major resources for a
diverse array of wood products and as raw materials for construction and pulp/paper and biofuel production [2]. In the genus
Populus, the only species with a complete genome sequence is
Populus trichocarpa [3]. However, this species is difficult to transform compared to other widely studied poplar species, such as P.
tremula or P. tremuloides or several poplar hybrids. Previously we
established a simple and efficient Agrobacterium-mediated transformation protocol for P. trichocarpa (Nisqually-1) using stems as
explants [1]. Since then we have carried out extensive transformation experiments testing different tissue manipulations, types of
constructs, Agrobacterium strains, and media. Overexpression
constructs using either the CaMV35S or a P. trichocarpa 4CL3
promoter always have a two- to threefold higher transformation
efficiency than RNAi constructs using the same 35S or 4CL promoter. After the P. trichocarpa genome was sequenced, many labs
have generated transgenics to study gene function because it is
possible to readily determine the location of most transformation
events. Some labs are still struggling to carry out P. trichocarpa
transformation, because they are not aware of some critical steps in
the transformation process.
In this chapter, we describe an updated stepwise protocol for
Agrobacterium-mediated P. trichocarpa transformation. In this
protocol, stems of fifth to eighth internodes of 5- to 6-month-old
trees are used as explants for infection with A. tumefaciens strain
C58 or GV3101 harboring a binary vector pBI121 or pBI121derived vector. Transgenic shoots can be obtained within 58
months under kanamycin selection. The average transformation
rate is 10 %, and a frequency as high as 20 % can be achieved with
careful adherence to this protocol. This updated protocol should
help more researchers to successfully obtain transgenic P. trichocarpa plants.
2
2.1
Materials
Plant Materials
2.2 Agrobacterium
Strains and Vectors
Populus trichocarpa
359
2.3
Stock Solutions
1. Kanamycin (100 mg/mL): Dissolve 5 g kanamycin monosulfate in distilled H2O and sterilize by passing through a 0.2 m
filter, and store at 20 C.
2. Cefotaxime (200 mg/mL): Add 10 mL sterile distilled H2O to
the bottle to dissolve the 2 g Claforan (cefotaxime) inside.
Additional sterilization of the solution with a filter is not
required. Store at 20 C.
3. Timentin (200 mg/mL): Add 10 mL sterile distilled H2O into
a bottle containing 2 g Timentin. Additional sterilization with
a filter is not required. Store at 20 C.
4. Murashige and Skoog (MS) basal medium with vitamins (10
stock): Put one bag of MS basal medium with vitamins in 1 L
distilled H2O, stir at room temperature for at least 8 h, and
store at 4 C. Shake well before using.
5. Acetosyringone (AS, 200 mM): Dissolve 0.196 g AS in 3 mL
dimethyl sulfoxide (DMSO) and bring volume to 5 mL with
distilled H2O. Pass through a 0.2 m filter into 1.5 mL
Eppendorf tubes and store at 20 C.
6. Kinetin (1 mg/mL): Dissolve 100 mg kinetin in ~2.5 mL
0.5 N HCl and bring volume to 100 mL with distilled
H2O. Sterilization is not needed. Store at 4 C.
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1. Callus Induction Medium 1 (CIM1): 1 MS salts and vitamins, 30 g/L sucrose, 100 mg/L inositol, 0.5 mg/L kinetin,
0.05 mg/L 2.4-D, 6.5 g agar. To prepare, dissolve sucrose and
inositol in ~750 mL water, add 100 mL of 10 MS salts with
vitamins, 500 L of 1 mg/mL kinetin, 50 L of 1 mg/mL
2,4-D, and then adjust pH to 5.7, bring volume to 900 mL,
add 6.5 g agar, autoclave, cool, add 100 mL coconut water.
2. Callus Induction Medium 2 (CIM2): 1 MS salts and vitamins, 30 g/L sucrose, 100 mg/L inositol, 100 mg/L inositol,
0.5 mg/L kinetin, 0.05 mg/L 2.4-D, 50 mg/L kanamycin,
500 mg/L cefotaxime, and 6.5 g agar. Prepare the medium as
CIM1, bring volume to 1 L. After the medium is autoclaved
and cooled, add 500 L of 100 mg/mL kanamycin and 2.5 mL
of 200 mg/mL cefotaxime.
3. Shoot Induction Medium 1 (SIM1): MS salts and vitamins, 30 g/L sucrose, 100 mg/L inositol, 132 g/mL TDZ,
50 mg/L kanamycin, 500 mg/L cefotaxime, and 6.5 g agar.
To prepare, dissolve 30 g sucrose and 100 mg inositol in
~900 mL water, add 50 mL of 10 MS salt with vitamin, adjust
pH to 5.7. Add 6.5 g agar, autoclave, and cool. Add 264 L of
0.5 mg/mL TDZ, 500 L of 100 mg/mL kanamycin and
2.5 mL of 200 mg/mL cefotaxime.
4. Shoot Induction Medium 2 (SIM2): 1 WPM with vitamins,
30 g/L sucrose, 100 mg/L inositol, 2 mg/L zeatin, 50 mg/L
kanamycin, 500 mg/L cefotaxime, and 6.5 g agar. To prepare,
dissolve one bag of WPM with vitamins, 30 g sucrose and
100 mg inositol in 1 L water, adjust pH to 5.7, add 6.5 g agar.
After autoclaving and cooling, add 1 mL of 2 mg/mL zeatin,
50 L of 100 mg/mL kanamycin and 2.5 mL of 200 mg/mL
cefotaxime.
Populus trichocarpa
361
3
3.1
Methods
Plant Growth
3.2 Agrobacterium
Culture Preparation
P. trichocarpa plants were grown in pots containing half MiracleGro soil (Scotts Miracle-Gro, Marysville, OH) and half Metro-Mix
200 (Sun Gro, Bellevue, WA) in a greenhouse at 1726 C, 16 h
light/8 h dark cycle (with supplemental light of ~300 E/m2/s).
Miracle-Gro Plant Food (Scotts Miracle-Gro) was applied once a
week (see Note 2).
1. Pick a bacterial colony 2 days before infection from a LuriaBertani (LB) plate (up to 2 weeks old) into 10 mL LB liquid
media containing appropriate antibiotics (50 g/mL kanamycin and 50 g/mL gentamicin) and shake at 220 rpm at 28 C
for 1 day.
2. Inoculate 5200 L of the culture into 25 mL liquid LB in a
125 mL Erlenmeyer flask. Add acetosyringone to a concentration
of 200 M following inoculation. Culture Agrobacterium overnight with shaking at 220 rpm at 28 C. The Agrobacterium culture is ready when the OD600 reaches ~0.4 (see Notes 3 and 4).
3.3 Inoculation
with Agrobacterium
and Cocultivation
1. Sterilize double-edged razor blades by immersion in 70 % ethanol in a petri dish for 20 min (see Note 5).
2. Collect stems that contain the fifth to eighth internodes
(Fig. 1b) from a 56-month-old tree grown in the greenhouse,
cut into two fragments (each ~15 cm long), and immediately
put them in a bottle containing enough 10 % Clorox (0.83 %
sodium hypochlorite) to submerge the segments.
3. Hold stems in Clorox for 20 min with gentle shaking to sterilize the stem fragments.
4. Rinse with 5 L sterile distilled water for the stems from two
trees. After rinsing, put the stem fragments into sterile water in
a sterile glass beaker, leaving about 2 cm out of the water.
5. Cut the stem sections into 0.4 cm long segments in a petri dish
(size 150 15 mm) using a double-edged razor blade held by
a serrated forceps (Fig. 1c), and immediately put the segments
into the Agrobacterium solution. Swirl gently for 35 min by
hand or on a shaker.
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Quanzi Li et al.
3.5 Rooting
of Shoots
and Propagation
in Magenta Boxes
Notes
1. TDZ may precipitate. Mix well before adding TDZ into the
warm SIM1 medium.
2. The health of plants is very important for successful transformation, because healthy explants can survive under a high
concentration of acetosyringone (200 M) and for a longer
period of Agrobacterium infection (>46 h).
Populus trichocarpa
363
Chapter 29
Tall Fescue (Festuca arundinacea Schreb.)
Yaxin Ge and Zeng-Yu Wang
Abstract
Tall fescue (Festuca arundinacea Schreb.) is the predominant cool-season perennial grass in the United
States. It is widely used for both forage and turf purposes. This chapter describes a protocol that allows for
the generation of a large number of transgenic tall fescue plants by Agrobacterium tumefaciens-mediated
transformation. Embryogenic calli induced from caryopsis are used as explants for inoculation with
A. tumefaciens. The Agrobacterium strain used is EHA105. Hygromycin phosphotransferase gene (hph) is
used as the selectable marker, and hygromycin is used as the selection agent. Calli resistant to hygromycin
are obtained after 46 weeks of selection. Soil-grown tall fescue plants can be regenerated 45 months
after Agrobacterium tumefaciens-mediated transformation.
Key words Agrobacterium, Festuca arundinacea, Forage and turf grass, Tall fescue, Genetic transformation, Transgenic plant
Introduction
Tall fescue (Festuca arundinacea Schreb.) is the most important
forage species worldwide of the Festuca genus. It forms the basis
for beef cow-calf production in the east-central and southeast
United States, supporting more than 8.5 million beef cows, and is
used for sheep and horse production [1]. It is also widely used for
general purpose turf and low-maintenance grass cover and plays an
important role in environmental protection [2]. The widespread
use of tall fescue is due to its adaptation to a wide range of soil
conditions, tolerance of continuous grazing, high yields of forage
and seed, persistence, long grazing season, compatibility with varied management practices, and low incidence of pest problems [1].
Tall fescue is a polyploid (2n = 6 = 42), wind-pollinated monocot species with a high degree of self-incompatibility. This makes
breeding management difficult and selection schemes complex,
resulting in slow breeding progress, especially for traits with low
heritability [3]. There has been considerable interest in manipulating tall fescue by genetic transformation in the past decade with
Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224,
DOI 10.1007/978-1-4939-1658-0_29, Springer Science+Business Media New York 2015
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366
the aim of improving its agronomic traits [4, 5]. Transgenic tall
fescue plants have been obtained by direct gene transfer to protoplasts, microprojectile bombardment, and Agrobacterium
tumefaciens-mediated transformation [4, 6, 7].
The protocol outlined in this chapter is based on our work [6]
in tall fescue transformation. We used embryogenic calli as explant
and hygromycin as selection agent. Transformation frequency is
about 8.7 % based on the number of transgenic plants recovered
and the number of original intact calli used. The use of highly
embryogenic calli is one of the key factors affecting transformation
frequency.
2
2.1
Materials
Plant Material
2.2 Agrobacterium
tumefaciens Strain
and Selectable Marker
2.4
Tissue Culture
1. Calcium hypochlorite: Prepare fresh 3 % (w/v) calcium hypochlorite solution in a glass bottle; add a few drops of Tween-80.
2. 2,4-Dichlorophenoxy-acetic acid (2,4-D) solution: 1 mg/mL
(PhytoTechnology Laboratories, Shawnee Mission, KS).
3. M5 medium: Dissolve 4.43 g Murashige & Skoog basal
medium with vitamins (PhytoTechnology Laboratories) in
800 mL distilled water, add 30 g sucrose and 5 mL 2,4-D
(1 mg/mL), make up the volume to 1,000 mL with distilled
water. Adjust pH to 5.8 and add 7.5 g agar. Autoclave at
121 C for 15 min. Cool to 50 C and pour 25 mL aliquots
into Petri dishes (100 15 mm). Store at 24 C.
4. M1 liquid medium: Dissolve 4.43 g Murashige & Skoog basal
medium with vitamins (PhytoTechnology Laboratories) in
800 mL distilled water, add 30 g sucrose and 1.5 mL 2,4-D
367
368
Methods
3.1 Sterilization
and Callus Induction
3.2 Agrobacterium
Preparation
3.3 Inoculation
of Explants
and Cocultivation
369
Fig. 1 Transgenic tall fescue (Festuca arundinacea) plants obtained after Agrobacterium tumefaciens-mediated
transformation of embryogenic calli. (a) Embryogenic callus induced from caryopses. (b, c) Detailed view of
the embryogenic calli. (d) Hygromycin-resistant calli obtained 5 weeks after Agrobacterium tumefaciensmediated transformation and selection of infected callus pieces on M1 medium. (e) Shoot differentiation of
hygromycin-resistant calli 4 weeks after transfer onto regeneration medium MSK. (f) Rooted transgenic plants
4 weeks after transfer of the differentiated shoots to rooting medium. (g) Greenhouse-grown transgenic plants
5 months after Agrobacterium tumefaciens-mediated transformation. (h) Fertile transgenic tall fescue after
vernalization
3. Transfer tall fescue calli into 6.6 cm culture vessels and break
up the calli into small pieces.
4. Add Agrobacterium suspensions to the culture vessels and
immerse the callus pieces.
5. Place the culture vessels in a polycarbonate desiccator and draw
vacuum (62 cm Hg) for 510 min.
6. Release vacuum, incubate the callus pieces and Agrobacteria
for 20 min with gentle shaking on a rotary shaker.
370
3.5 Greenhouse
Care, Vernalization,
and Seed Harvest
371
length to 12 h and grow the plants for 3 days; and (6) transfer
the vernalized plants back to the greenhouse (390 mol/m2/s,
16 h day/8 h night at 24/20 C).
5. After vernalization, plants normally flower in about 2 months
(Fig. 1h). Because tall fescue is an outcrossing species, crosses
need to be made between independent plants (e.g., transgenic
and non-transgenic plants) in order to obtain seeds. For crosspollination, emasculate recipient inflorescence and then bag
them together with two panicles from the pollen donor plant;
supply water to the donor panicles from a 50 mL conical tube
fixed to a bamboo stake [10]. Seeds can be harvested 1 month
after cross-pollination.
Notes
1. Transformation efficiency varies with the cultivar used. By
using this protocol, we have been able to produce transgenic
plants from another widely used cultivar, Kentucky-31.
Endophyte-free seeds should be used.
2. We have been able to produce transgenic tall fescue with either
EHA105 or LBA4404 strain.
3. If the seeds are dirty, the first sterilization time can be extended
to 2.5 h or even 3 h. Make sure all seeds have good contact
with the solution.
4. The majority of seed bracts (lemma and palea) surrounding
caryopsis become detached after the second sterilization. The
surface sterilization procedure will not remove the endophyte
in the seeds. If the seeds contain an endophyte, then the endophyte should be removed by treatment with high humidity and
relatively high temperature. Older seeds contain less viable
endophyte.
5. The amount of callus pieces on each filter paper was equivalent
to approximately 20 original intact calli.
6. Before transfer to soil, rinse the roots with water or remove
excessive medium with a damp paper towel.
References
1. Sleper DA, West CP (1996) Tall fescue. In: Moser
LE, Buxton DR, Casler MD (eds) Cool-season
forage grasses. Madison, WI: American Society
of Agronomy; Crop Science Society of America;
Soil Science Society of America, pp 471502
2. Sleper DA, Buckner RC (1995) The fescues.
In: Barnes RF, Miller DA, Nelson CJ, Heath
372
INDEX
overview......................................................................34
seed source ......................................................................5
seed sterilization and germination ..................................6
selection ......................................................................79
shoot isolation ................................................................9
A
Ananas comosus (L.) Merr. See Pineapple
Apricot
Agrobacterium
preparation............................................................ 115
strains and vectors ................................................. 112
coculture and washing ................................................116
donor plants ........................................................114115
efficiencies ..................................................................112
kanamycin........................................................... 113, 116
materials .............................................................112114
overview..............................................................111112
pBIN19 ...................................................................... 112
transgenic plants
acclimatization ......................................................117
elongation and multiplication ...............................116
regeneration .......................................................... 116
rooting ..................................................................116
B
Blueberry
bacterial strains and binary vector...............................123
bialaphos .....................................................................123
culture media and stock solutions .......................123124
greenhouse care...........................................................128
infection and cocultivation.................................. 125126
kanamycin................................................... 122, 124, 127
leaf explant preparation ..............................................125
overview..............................................................121123
regrowth .....................................................................127
rooting ................................................................127128
selection and regeneration ..........................................127
stock culture establishment .........................................125
Brassica rapa
Agrobacterium
culture ...................................................................5, 6
preparation............................................................67
culture media ..............................................................4, 5
efficiency.........................................................................4
explant isolation, inoculation,
and cocultivation ...........................................7
gentamicin selection ...................................................79
greenhouse care...............................................................9
materials .....................................................................46
C
Cannabis sativa L. See Hemp
Carrot
Agrobacterium
cultures ....................................................... 60, 6263
strains and binary vectors ........................................60
herbicide resistant .........................................................60
materials .................................................................6062
overview..................................................................5960
pCAMBIA1300 ..................................................... 60, 63
seed sterilization and tissue preparation........................62
transformation procedure .......................................6364
transformation rate ........................................... 59, 63, 65
Cassava
Agrobacterium
inoculum preparation .............................................. 75
strains and culture media ........................................73
antibiotics selection......................................69, 72, 76-77
axillary buds enlargement and somatic embryos
development..........................................7375
cassava tissue culture media ..........................................71
efficiency....................................................................... 69
friable, embryogenic callus (FEC) ................... 68, 69, 71,
72, 74, 76, 77, 79
generation of ...........................................................75
materials .................................................................6973
overview..................................................................6769
transformation procedure
cocultivation............................................................76
recovery and maturation .........................................76
regeneration ......................................................7677
rooting and screening..............................................77
transfer to soil and glasshouse care ...................7778
Castanea dentata (Marsh) Borkh. See Chestnut, American
Castanea sativa. See Chestnut, European
Cherry
bacterial strains and binary vector...............................134
culture medium and stock solutions....................134136
greenhouse care...........................................................140
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373
AGROBACTERIUM PROTOCOLS
374 Index
Cherry (cont.)
infection and co-cultivation ........................................138
kanamycin selection .................................... 134, 139, 140
leaf explant preparation ......................................137138
overview..............................................................133134
plant materials ............................................................134
regrowth .....................................................................139
rooting ........................................................................ 139
selection and regeneration ..........................................139
stock culture establishment
using bud woods with dormant leaf
buds ..................................................136137
using newly formed softwood branches ................136
Chestnut, American
Agrobacterium
preparation....................................................152153
strains and vectors .................................................151
bar gene selection........................................................156
bioreactor ............................................ 146, 148, 150, 153
desiccation .................................................. 146, 148, 153
efficiency............................................................. 143, 157
materials .............................................................148151
overview..............................................................143148
paromomycin selection ....................... 150, 153154, 156
rooting and acclimatization ................................154155
shoot culture ...............................................................154
somatic embryos
establishment ................................................151152
transformation ..............................................153154
Chestnut, European
Agrobacterium-mediated transformation
efficiency ............................................... 165, 172, 175
infection and cocultivation............................171172
kanamycin selection ...................... 166, 168, 172, 175
materials .......................................................165169
overview ........................................................163165
plantlet regeneration .............................................173
preparation for infection .......................................171
somatic embryogenesis induction .................169171
somatic embryos proliferation
and maintenance .......................................171
strain and vector ...........................................165166
forest biotechnology ........................................... 163165
thaumatin-like protein ................................ 164166, 174
Citrus
juvenile tissue transformation
Agrobacterium culture preparation .........................250
Agrobacterium strain and plasmid ..........................247
co-incubation ........................................................ 251
efficiency ...............................................................254
green fluorescent protein detection ....................... 251
materials .......................................................246248
micro-grafting ..............................................252254
overview ........................................................245246
pBI121 ..................................................................247
plant materials preparation ...........................248250
transgene detection by PCR .........................251252
mature tissue transformation
Agrobacterium culture preparation .........................269
Agrobacterium strain and plasmid ..........................262
co-cultivation ........................................ 261, 269270
efficiency ...............................................................262
explant materials preparation ........................269270
fertilizer application ..............................................266
Green fluorescent protein (GFP)
detection ..................................................271
histochemical GUS assay ..........................................270
kanamycin resistance gene ....................................262
materials .......................................................262265
overview ........................................................259262
pesticide application .............................................266
primary micro-grafting ................................. 267, 268
rootstock production for bud grafting...................265
secondary grafting.........................................267269
selection of transgenic shoots........................270271
transplanting .................................................265266
transgenic shoots harvest ......................................270
Coffea arabica L. See Coffee
Coffee
Agrobacterium rhizogenes-mediated transformation
A. rhizogenes strains and transformation
vectors .......................................................281
culture media ........................................................281
molecular analysis ......................................... 287288
overview ........................................................ 275279
plant materials ......................................................281
seed sterilization and zygotic embryo
germination...............................................285
transformation and hairy root
induction ...........................................285287
Agrobacterium tumefaciens-mediated transformation
A. tumefaciens culture preparation .................282283
callus culture preparation ......................................282
culture media and stock solutions .................279280
infection and cocultivation....................................283
leaf explant sterilization ........................................282
materials .......................................................279281
molecular analysis .................................................285
overview ........................................................277278
selection and regeneration ............................283284
strains and transformation vectors ........................ 279
breeding ..............................................................276278
economic importance.......................................... 275, 276
genertic engineering applications ....................... 276, 278
pBIN19 and pMDC32............................... 279, 283, 288
species .................................................................276279
AGROBACTERIUM PROTOCOLS
375
Index
Colocasia esculenta (L.) Schott. See Taro
Cotton
Agrobacterium-mediated transformation
aseptic seed germination .........................................17
cotton tissue culture ................................................16
bacterial inoculant preparation................................ 17
efficiency ..................................................... 14, 19, 21
explant inoculation and cocultivation .....................17
kanamycin resistance test ........................................ 18
materials ...........................................................1516
media ................................................................1516
overview ............................................................1115
regeneration, embryo germination, and plantlet
development................................................18
selection and proliferation.......................................18
soil transfer .............................................................18
transformation ........................................................17
genetic engineering application ....................................12
green fluorescent protein reporter .................................13
Cucumis melo L. See Melon
D
Daucus carota L. See Carrot
E
Euphorbia pulcherrima Willd. ex Klotzsch. See Poinsettia
F
Festuca arundinacea Schreb. See Tall fescue
Fragaria x ananassa. See Strawberry
G
Gossypium hirsutum L. See Cotton
Grapevine
Agrobacterium rhizogenes-mediated hairy root transformation
Agrobacterium preparation and
inoculation ........................................188189
calli and hairy root recovery ..................................189
in vitro plantlets production and
inoculation ................................................188
Agrobacterium tumefaciens-mediated plant transformation
Agrobacterium culture preparation .........................186
cocultivation.......................................................... 186
embryogenic culture initiation and
maintenance ......................................185186
kanamycin selection ...................................... 186-187
selection and regeneration ............................186187
transplanting and greenhouse
care....................................................187188
materials .............................................................181185
overview..............................................................177181
H
Helianthus annuus. See Sunflower
Hemp
Agrobacterium culture conditions ................ 321322, 324
Agrobacterium strains and binary vectors. .................... 321
inoculation and cocultivation .............................. 322, 324
mannose selection ............................... 320, 322, 326, 328
materials .............................................................320323
overview..............................................................319320
phosphomannose isomerase assay ....................... 325326
phosphomannose isomerase
selection .................................... 320, 324325
pNOV3635 ......................................... 321, 324, 325, 328
polymerase chain reaction ................................... 326327
Southern hybridization ....................................... 323, 327
tissue culture ....................................... 320321, 323324
transformation frequency ............................................320
J
Jatropha
Agrobacterium culture for infection ...............................30
culture media and stock solutions
for Agrobacterium ..................................................... 30
for plant ..................................................................29
efficiency........................................................... 26, 33, 34
infection, cocultivation and regeneration ......................31
kanamycin resistance ..............................................31, 33
materials .................................................................2630
overview..................................................................2526
plant materials ..............................................................26
root induction and acclimatization ...............................31
seed sterilization and explant preparation ..................... 30
transgenic plants
genomic DNA isolation and PCR analysis ...........3233
GUS histochemical assay ........................................32
Jatropha curcas L. See Jatropha
Juglans. See Walnut
M
Manihot esculenta Crantz. See Cassava
Melon
Agrobacterium
preparation............................................................201
strain and vector ...................................................196
donor plants preparation .....................................198199
efficiency............................................................. 196, 201
explant preparation ..................................................... 200
kanamycin selection ............................................ 196, 201
materials .............................................................196198
overview..............................................................195196
pIG121-Hm ....................................................... 196, 201
AGROBACTERIUM PROTOCOLS
376 Index
Melon (cont.)
transformation procedures
Agrobacterium inoculation ..................................... 201
regeneration .................................................. 201202
selection ........................................................ 201202
transplanting and acclimation ...............................202
O
Oncidium. See Orchid
Orchid
Agrobacterium preparation ...........................................340
bacterial strain and DNA construct ............................335
culture media ......................................................332335
hygromycin selection .......................................... 333, 344
materials .............................................................332337
overview..............................................................331332
pCAMBIA1304 ................................................. 335, 340
plant materials ............................................................ 332
protocorm-like bodies (PLBs)
initiation for Oncidium ........................................337
initiation for Phalaenopsis ............................337338
propagation ...........................................................339
transformation process
Agro-infiltration and co-cultivation .....................340
regeneration and conversion .........................340341
selection ................................................................340
transgenic plant analysis
genomic DNA isolation ................................341342
histochemical GUS staining .................................344
PCR amplification ................................................342
reverse transcription-PCR ............................343344
RNA extraction ....................................................343
Southern blotting..................................................342
P
Peach
Agrobacterium
preparation............................................................211
strain and binary vector ........................................207
efficiency............................................................. 206, 213
GF677 rootstock ................................................ 206, 207
kanamycin selection ............................ 206, 207, 211212
materials .............................................................207209
meristematic bulks initiation and
regeneration ......................................209210
overview..............................................................205207
pBIN19 .............................................................. 207, 211
plant acclimatization...........................................211212
plant tissue infection ...................................................211
regeneration and selection .................................. 211212
Phalaenopsis. See Orchid
Pineapple
Agrobacterium
culture maintenance .............................................. 300
vectors and strains .................................................298
explants
establishment and maintenance ............................300
regeneration ..........................................................300
frequency ....................................................................294
histochemical GUS assay............................................302
hygromycin selection .................................. 298, 301302
inoculation and co-cultivation ....................................300
materials .............................................................298299
molecular analysis
by PCR .................................................................303
by Southern hybridization ....................................303
overview..............................................................293297
pCAMBIA1304 ......................................... 294, 298, 303
selected transformants multiplication .........................302
transformants selection
after encapsulation in Ca-alginate beads .........301302
prior to encapsulation ...........................................301
Poinsettia
Agrobacterium
culture preparation ........................................351352
DNA introduction by electroporation ..................351
strain and vector ...........................................348349
explant preparation .....................................................352
frequency .................................................................... 348
infection and co-cultivation ................................352353
materials .............................................................348351
neomycin phosphotransferase (npt II) ........................ 349
overview..............................................................347348
transgenic plants
propagation/greenhouse cultivation ..............353354
regeneration ..................................................352353
Populus trichocarpa
Agrobacterium
culture preparation ................................................361
strains and vectors .........................................358359
callus induction and shoot regeneration .....................362
frequency ....................................................................358
inoculation and co-cultivation ............................361362
kanamycin selection ............................................ 358, 360
materials ............................................................. 358361
overview..............................................................357358
pBI121........................................................................358
plant materials ............................................................358
rooting and propagation ............................................. 362
Potato
Agrobacterium
preparation..............................................................92
strain and binary vector ..........................................87
culture media and stock solutions ...........................8789
explant preparation, inoculation and cocultivation.......9293
frequency ......................................................................86
in vitro plant propagation .......................................9192
materials .................................................................8791
overview..................................................................8586
pBI121.............................................................. 86, 87, 95
plant material ................................................................87
AGROBACTERIUM PROTOCOLS
377
Index
transgenic plants
callus selection and shoot induction........................93
DNA extraction and PCR analysis .........................94
greenhouse care.................................................9394
GUS histochemical analysis....................................94
shoot elongation and root induction .......................93
transplanting and acclimation .................................93
Prunus armeniaca L. See Apricot
Prunus persica L. See Peach
Prunus spp. See Cherry
S
Saccharum spp. Hybrids. See Sugarcane
Sesame
Agrobacterium
inoculum preparation ........................................4041
strains and selectable markers .................................39
cocultivation ...........................................................40, 41
culture media ..........................................................3940
efficiency.................................................................38, 44
explant preparation .......................................................40
materials .................................................................3840
overview..................................................................3738
pCAMBIA2301 ...........................................................39
transgenic plants
hardening and acclimatization ................................42
rooting ....................................................................42
screening using histochemical GUS
analysis ..................................................4243
shoot regeneration and selection .......................4142
Sesamum indicum L. See Sesame
Solanum tuberosum L. See Potato
Strawberry
Agrobacterium preparation ...................................222223
efficiency............................................................. 219, 225
infection.............................................................. 222223
kanamycin selection ............................................ 219, 224
leaf tissue preparation .................................................222
materials .............................................................219222
overview..............................................................217219
pBI121........................................................................ 219
regeneration and selection ..................................223224
rooting and soil transplantation ..........................224225
Sugarcane
Agrobacterium
culture preparation ........................................312313
strain and vector ...................................................308
callus induction ...........................................................312
efficiency............................................................. 308, 314
geneticin/paromomycin selection ...................... 309310,
313, 314
inoculation and co-cultivation ....................................313
materials .............................................................308311
neomycin phosphotransferase (npt II) ........................308
overview..............................................................307308
T
Tall fescue
Agrobacterium
culture media ........................................................366
preparation............................................................368
strain and selectable marker ..................................366
greenhouse care, vernalization and seed
harvest...............................................370371
hygromycin selection .......................................... 366, 370
inoculation and cocultivation ..............................368370
materials .............................................................366368
overview..............................................................365366
pCAMBIA 1305.1 .....................................................366
selection and regeneration ..........................................370
sterilization and callus induction ................................368
tissue culture .......................................................366368
transformation frequency ............................................366
Taro
Agrobacterium
culture preparation ................................................103
strains and vector ..................................................101
culture media and stock solutions ......................... 99100
efficiency................................................................. 98, 99
infection and cocultivation..........................................103
in vitro plantlets establishment ...........................101103
neomycin phosphotransferase II (npt II) .................... 101
overview..................................................................9799
pBI121........................................................................ 101
plant material ................................................................99
transgenic plants
selection and regeneration ....................................104
transplanting .........................................................105
AGROBACTERIUM PROTOCOLS
378 Index
V
Vaccinium corymbosum L. See Blueberry
Vigna unguiculata (L.) Walp. See Cowpea
Vitis vinifera L. See Grapevine
W
Walnut
Agrobacterium
preparation....................................................234235
transformation vectors and strains ........................231
cocultivation .......................................................235236
efficiency..................................................................... 231
germination and plant production ......................237238
histochemical GUS analysis .......................................236
kanamycin/hygromycin selection ................................236
materials .............................................................231233
media and stock solutions ...................................231233
overview..............................................................229231
PCR confirmation ......................................................237
selection ......................................................................236
somatic embryo culture ....................................... 230, 234