PEG-Mediated Plastid Transformation in Higher Plants

Author(s): Waltraud Kofer, Christian Eibl, Klaus Steinmüller and Hans-Ulrich Koop
Source: In Vitro Cellular & Developmental Biology. Plant, Vol. 34, No. 4 (Oct. - Dec., 1998), pp.
303-309
Published by: Society for In Vitro Biology
Stable URL: http://www.jstor.org/stable/20065007
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 InVitro Cell. Dev. Biol?Plant 34:303-309, October-December, 1998
? 1998 Society for InVitro Biology
1054-5476/98 $05.00 + 0.00

REVIEW
PEG-MEDIATED PLASTID TRANSFORMATION INHIGHER PLANTS1

and HANS-ULRICHKOOP
WALTRAUDKOFER,2CHRISTIANEIBL,KLAUSSTEINM?LLER,

Botanical Institute, Ludwig-Maximilians-Universit?t, Menzinger Str. 67, 80638, M?nchen, Germany (W.K., C. E., H.-U. K.); and Institut
f?r Entwicklungs- und Molekularbiologie der Pflanzen, Heinrich-Heine-Universit?t, Universit?tsstrasse 1, D-40225 D?sseldorf (K. S.)

(Received 19 March 1998; accepted 23 May 1998; editor C. L. Armstrong)

Summary

Plastids are surrounded by an envelope consisting of a double membrane. This barrier has to be penetrated or overcome

by the DNA whentransforming the plastome. Both the biolistic and polyethylene glycol-mediated transformation techniques
accomplish this task, albeit by different mechanisms. We were the first laboratory to successfully use the polyethylene

glycol (PEG)-method for plastid transformation, yet we use the particle gun when appropriate. In this report we compare
the two methods and discuss their shortcomings and advantages. Plastid transformations with various constructs, mainly
using the aadk gene as a selective marker, were
performed. We point to potential problems likely to be encountered during
the transformation and selection processes and
offer possibilities for improvement. We give further examples of the suc
cessful application of plastome transformation and show its merits in addressing biological questions concerning the elu
cidation of plastid sequences of unknown function and the control of plastid gene expression.

Key words: plastome; PEG-transformation; tobacco; aadk gene; selection.

Introduction ported successful transformation of the plastome by the polyethylene

glycol (PEG)method (Golds et al., 1993), which is mainly used for
The chloroplast is a cellorganelle with two enclosing membranes cell fusion and the transformation of the nucleus. The
experiments
that form a barrier for nucleic acids. Although according to recent mechanism by which the DNA is transported from the cytoplasm into

(K?hler et al., 1997) plastids can fuse and thus potentially the chloroplast organelle by PEG treatment is unclear. In the PEG
findings
macromolecules, no mechanism has been described method, are with DNA
treated in the presence of PEG.
exchange by protoplasts
which such as large nucleic acids. The trans Both biolistics the and
PEG have
they take up molecules techniques, method, shortcomings.
fer of genetic information between the three (genetic) compartments
The biolistic method may be not affordable and the target tissue

of a higher plant cell seems to be a common in evolution as an intact leaf) must be to plants. The PEG
process (ordinarily regenerable
can be found in the nuclear and mitochondrial transformation system is inexpensive; however, it requires protoplast
plastid sequences
and Brennicke, culture experience and most the species must be regen
genomes (Schuster 1988, 1994). An example of the importantly,
erable from protoplasts.
transfer of nucleic acids from one cellular compartment to another
one is plastid Generally, with either transformation method, the target DNA is
t-RNA genes which have been moved during evolution
likely to be incorporated into only one plastid genome of a cell behind
into the chondriome; in the mitochondrial genome of wheat for ex
a background of 1000 to 10 000 untransformed
there exist six t-RNA copies. The ultimate
ample, genes of plastid origin (Joice and Gray,
there must be a mechanism acids can goal is to rid the cell of the 10 000 untransformed copies and to end
1989). Thus, by which nucleic
up with a higher plant that is homoplastomic. Except for a report by
pass the lipid bilayers enclosing the plastid organelle. Carrer et al. (1993) who used the nptll gene for kanamycin resis
Until recently the presence of this envelope seemed to be an im
tance, all plastome transformations reported in the literature have
penetrable barrier blocking attempts to transform the plastome. The
used the aadk-gene (aminoglycoside-3'-adenyltransferase), confer
came when the group of Boynton et al. (1988) used the
breakthrough to spectinomycin and streptomycin, as a selectable
ring resistance
biolistic method to directly introduce DNA into the plastid genome marker. Generally, the constructs used to transform the higher plant
of the green alga Chlamydomonas rheinhardtii. Only 2 yr later the are
plastome designed such that a plastid promoter region is fused
same was used Svab et al. to trans to the antibiotic
technique by (1990b) successfully resistance gene connected to a plastid-specific ter
form the plastome of Nicotiana tabacum. The biolistic method uses minator of either tobacco or This cassette is flanked
Chlamydomonas.
DNA-coated tungsten or which are shot the to promote recombination re
gold particles through by plastome sequences homologous
Thus, this technique does not need a in an insertion at a specific site of the higher
chloroplast envelope. biological sulting integration plant
mechanism to overcome the bilayers. In 1993, we re In this paper we report on our plastid transformation ex
lipid plastome.
periments and the biological questions they address. We will report
on a presentation on and will discuss to overcome
^ased in the symposium "Organelle Transformation" unexpected findings strategies prob
lems that should be anticipated when attempts are made to transform
during the 1997 SIVB Congress held inWashington, DC June 14-18, 1997.
2To whom correspondence should be addressed. the plastome.

303

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 304 KOFERET AL.

Results ml of calcium agar. Then 625 \i\ of the protoplast/alginate mixture is

added and allowed to solidify. After 30 to 60 min, the alginate has
We have previously described our protocol for PEG-mediated plas set and the mesh, now a thin layer of embedded
containing proto
tome transformation in detail (Koop and Kofer, 1995). Since then we
can be carefully removed from the calcium agar with a pair
plasts,
have refined our mainly the embedding, cul
technique, concerning of forceps. This procedure can be facilitated the addition of 0.5
by
ture, and selection systems. The main drawback of our system was
ml 20 mM calcium chloride solution (in 0.4 M mannitol, 550 mOsm)
that it took too much expertise to master the technique. The culture
around the periphery of the plastic netting. Now the nets can be quite
of protoplasts was labor intensive due to the frequent culture medium
easily separated from the calcium agar and transferred into a fresh
changes Thus, our goals were to simplify our method and
required.
petri dish (6 cm) containing 10 ml of culture medium (PCN). The
to shorten the time between transformation and the development of
nets are equilibrated for 1 h and transferred to 2 ml PCN medium.
transformed shoots.
We have omitted the stepwise reduction in osmolarity [as reported
in our earlier publications (Koop and Kofer, 1995; Koop et al., 1996)]
1. Transformation Principle the initial time of culture. we are now,
during Instead, only after 1
wk of liquid culture (550 mOsm), going directly to solid shoot re
are treated with a solution containing various ions,
Protoplasts
PEG and DNA. in the plasma membrane allow the DNA to generation medium, RMOP (Svab et al., 1990b), with 0.8% purified
Changes
agar. This method is less time-consuming and bears less risk of con
penetrate and move into the cytoplasm. Whether PEG is directly
tamination due to frequent our
involved in the delivery of DNA the chloroplast handling. We have improved plating
through envelope
efficiencies to about 95% and reduced the time span between trans
and the mechanism of this process are still unclear. The
integration
formation and shoot formation to less than 6 wk.
of the target DNA into the plastid chromosome is site-directed due
to the design of vectors with sequences homologous to a specific
3. Selection of Transformants
target area in the plastid genome. When a cell divides, the plastids
When by PEG treatment or
are randomly distributed to both daughter cells and then they mul transforming protoplasts mesophyll
cells the gun method, a few are
a transformed cell will give rise to daughter by particle only plastome copies
tiply by division. Thus,
To obtain cells or a selection scheme
cells with heterologous targeted. homoplastomic plants,
plastome populations until, by selective pres
has to be administered that aims at the selection and segregation of
sure, segregation is complete with cells having only transformed plas
cells with transformed plastome copies. Svab and Maliga (1993) de
tome copies.
a potent transformation cassette with the aadk gene that when
signed
integrated into the plastome, renders the plastid resistant to the an
2. Transformation and Culture Procedure
tibiotics spectinomycin and streptomycin (Goldschmidt-Clermont,
The culture of plants, the isolation of protoplasts and the trans 1991). An advantage of this antibiotic selection is that cells are not
were in Koop
formation described and Kofer (1995). The only dif killed when treated with the antibiotics, yet their
development is
ference was that the donor were grown from seedlings rather slowed and they fail to turn green. As soon as a green shoot has
plants
than shoot on selective medium 1 a, 1 6), that has formed
the
cuttings. developed (Fig.
culture. and first true leaves, rounds
of regeneration can be started to
Principle of alginate film Viability subsequent speed repeated
enhanced if obtain use a
of division and regeneration
of protoplasts is greatly homoplastomic plants. We the following procedure:
are in a thin layer of alginate
embedded (Golds et al., small piece of leaf (2X2 mm) is off the plantlet and placed
protoplasts picked
1992). It is important that the be thin, as the of on shoot regeneration medium containing either spectinomycin only
layer kept exchange
metabolites and the availability of nutrients from the outside culture or both spectinomycin and streptomycin (streptomycin slows the gen
medium decreases with increasing thickness which negatively affects eral development of plants and may thus be omitted after the initial
the development of the cells (Sch?ffler and Koop, 1990). To coun selection) (Fig. 1 a). Newly formed shoots are takenoff the leaf piece
teract this problem, we changed our embedding support from steel and transferred onto antibiotic-containing B 5 medium. The proce
mesh to polypropylene or nylon nets. There are three main advan dure should be repeated until all wild-type gene copies are elimi

tages to this new

system. First, technique the require does not nated.
out
the protoplast/alginate mixture on cellophane which
spreading
4. Verification of Transformants
wrinkles is difficult to handle, and consequently causes un
easily,
even thickness of the spread. Second, the alginate/protoplast Molecular As soon as the first green shoots or calli have
layer analysis.
is much thinner, and third, the plating of protoplasts and the analysis can We either Southern
efficiency regenerated, proceed. perform
for more sensitive is bet blots with total DNA the appropriate or use the PCR
protoplast regeneration (especially species) using probes
ter on the plastic mesh than on steel mesh even when the alginate method with primers that give unambiguous information concerning
are of equal thickness. We assume that the steel netting is the presence of the introduced DNA or
gene copies. If
layers wild-type
toxic to the cells, whereas or is biolog segregation is not complete, the probing for wild-type gene copies
slightly nylon polypropylene
ically inert. Generally, embedding of the protoplasts facilitates the has to be repeated after several rounds of regeneration.
of culture and selection medium. In Nicotiana, are maternally in
exchange Progeny analysis. chloroplasts
culture After the PEG transformation, 2.6 herited. A homoplastomic, transgenic plant which is resistant to the
Alginate film procedure.
ml of protoplast culture medium (PCN) (Koop and Kofer, 1995) are selective marker should yield the following progeny when germinated
added to the protoplast/transformation mix to make a final volume of on antibiotic-containing media: the progeny of self-pollinated plants
approximately 3 ml. To this, 3 ml of alginate is added and gently should all be resistant, the progeny of cross-pollinated plants should
mixed. An (autoclaved) square of plastic mesh (3.0 X 3.0 cm, mesh be all resistant if the maternal plant is the transformant, and all
size 10 X 10 mm) is placed onto a 10-cm petri dish containing 15 sensitive if the paternal plant is the transformant.

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 PEG PLASTIDTRANSFORMATION305

FlG. 1. Procedure for plastome transformants by the PEG method and the analysis of the transformed plants, a, Outline of events from
transformation to analysis; b, transformed calli embedded in alginate in a nylon mesh; c, genetic make-up of wild-type and transformant
plastomes and probes used to detect transformed plants as well as wild-type plastome copies. The top of c shows the integration site in
the wild-type plastome, followed by the integration of the transformation vector; c (bottom) shows the Southern blots with probes from rpl32
testing for wild-type gene copies, aadk testing for the presence of the selective marker gene, and uidk investigating the integration of the
reporter gene: plants 1-13 and 14-18 are putative plastid transformants. 435S' stands for a nuclear transformant and is not detectable
with the uidk probe due to its low copy number, wt stands for wild-type; d, a leaf with GUS expression is shown which was transformed
with the uidk gene and demonstrates segregation of the transformed plastome and wild-type gene copies; e, the reporter gene cassette
with editing site.

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 306 KOFERET AL.

TABLE 1
gion between rpl32 and trnL. A nucleotide
sequence (sprk) in this
has been to a role in RNA maturation
REGIONSOR GENE LOCIWHICH SERVEDAS
INTERGENIC region reported potentially play
and The same further characterized this
NEUTRALINTEGRATION
SITESFORPLASTIDTRANSFORMATION (Vera Sugiura, 1994). group
INTOBACCO site by deletion of sprk through plastid transformation. The resulting
transformed were and showed no obvious
plants healthy phenotype
1 2
Gene
Gene Reference
(Sugita et al., 1997).
16S-rDNA Svab et al. (1990b)
Golds et al. (1993) 7. Efficiency of Integration
?mA-16S-rDNA-/p5l2 Staub and Maliga (1992)
16S-rDNA O'Neill et al. (1993)
To assess the integration of a vector having a selectable and a
rbcL
accD Svab and Maliga (1993)
nonselectable gene within the same construct, we a vector
Carrer et al. (1993) designed
Staub and Maliga (1994a) with the aadk gene as a selectable marker and the uidk gene as a
Staub and Maliga ensure
(1995) reporter (Fig. 1 e). To expression of the uidk reporter in the
unpublished plastids only, we included the trnL 5'UTR editing signal, which leads
16S-rDNA
tmV Staub and Maliga (1993)
to the generation of the AUG start codon from AC G (as described in
trnY
rpsl2 Zoubenko et al. (1994)
Chaudhuri et al., 1995). The construct was further with the
McBride et al. (1994) equipped
McBride et al. (1995) 16S-rDNA promoter and the trailer of psbk.
Chaudhuri et al. (1995) Of 91resistant lines analyzed 68 (75%) con
by Southern blotting,
Bock and Maliga (1995)
tained the aadk selectable marker gene; 50 (55%) had both the
trnW
psbk Carrer and Maliga (1995)
aadk and the uidk cassettes inserted which demonstrates that a gene
trnk
trnl Staub and Maliga (1994b)
Bock et al. (1994) which is not selected for can be lost independently of the selective
psbj
petk
Bock et al. (1996) marker. Only 18 lines showed unexpected signals, meaning either
trnL
rpl32 Koop et al. (1996) the integration site was not correct or alternatively, part of the se
quence was lost. Another explanation, though unlikely, might be the
integration into the wrong genetic compartment, i.e., the mitochon
c shows the transformation cassettes
If there are any left in the transformed ma
drial genome. Fig. 1 integrated
wild-type gene copies
into the plastome and the probes used to detect the presence of either
ternal the progeny will not be uniformly resistant. In case the
plant,
cassette in the potential transformants. The probe named rp/32 which
construct has accidentally transformed the nucleus, the progeny
hybridizes to a DNA sequence to the rp/32 gene, was used to
close
should segregate according to Mendelian rules.
genetically
detect wild-type gene copies. the first shoot of a green
In the analysis,
line was before were
investigated repeated regeneration cycles
5. Construction of Vectors for Plastome Transformation
started. The blot shown at the top of Fig. 1 c demonstrates the pres

are ence of WT gene copies in all lines tested, albeit in different pro
Plastid genes transcribed by plastid-specific promoters and
relative to the transformant gene The second blot
use
plastid-specific termination signals; many plastid genes are tran portions copies.
as op?rons; two open frames can be inserted demonstrates the presence of the aadk gene in most green shoots
scribed thus, reading
into a vector in sequence without using an additional promoter. Al
that appeared. Resistant plants no. 10,12,15, and 17 do not contain

can be inserted in different both having the aadk gene and are thus false positives. Plants no. 3 and 9 which
ternatively, they orientations,
their own and promoters. The desired nucleo both contained the aadk gene show no positive integration signal
regulatory sequences
tide is placed between and terminator. Plastid with the uidk probe (blot no. 3). In these plants parts of the construct
sequence promoter
leaders untranslated and trailers untranslated were lost; only the selectable marker gene was retained.
specific (5' regions) (3'
can be placed upstream and downstream from promoter and
regions)
terminator sequences. The entire construct should be equipped with Pitfalls of the System

flanking sequences homologous to the plastome that are routinely

'False positives9. On average, of 100 green calli or shoots that form
about 1 kb long. There has not been a systematic analysis, however,
on selective medium, only 75 are transformants. Thus, 25
r?g?n?r
about the minimum required flank length.
we used ants are false positives. There are several explanations for the oc
In our constructs, the 16S-rDNA promoter, the aadk
as a selective currence of such First, the construct may have been inserted
gene marker, the Chlamydomonas rbcL terminator, and plants.
into the nuclear the transformation cassettes used
leaders and trailers of different plastid genes. Occasionally, uidk was genome. Although
as a reporter was to be quantitatively contain plastid regulatory sequences promoter and
elements, they
added gene when expression
can, when into the right environment, i.e., behind a strong
assessed. placed
nuclear promoter, render the cell resistant towards the antibiotic and

green shoots can develop. The aadk gene can be used as a selectable
6. Integration Sites
marker for nuclear transformations (Svab et al., 1990a; Schr?der et

most sitesapart from op?rons or open reading frames al., 1994).

Generally,
seem to be so-called silent or neutral sites, meaning that Another mechanism that can cause false positives are specific
integration
an into those sites does not interfere in the 16S-rDNA gene that confer resistance to spec
integration with plastome gene point mutations
or regulation. A seriesof plastome locations has been tinomycin or streptomycin, respectively (Harris et al., 1989). There
expression
used by different laboratories as target sites for DNA are about three different sites in the gene that can, upon mutation,
integration (see
Table insert our transformation cassette in the re cause resistance to spectinomycin. About six sites cause spontaneous
1). We commonly

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 PEG PLASTIDTRANSFORMATION307

resistance to streptomycin. Selection with both antibiotics (Svab et 55000

al., 1990b) may be advantageous as the likeliness for point mutations

occurring simultaneously at two different sites is slim.

50000 -I
The aadk-gene as a selective marker. For a number of species the

aadk-gene cannot be used for antibiotic selection. Our experiments

I
showed that, for example, Hordeum is immune to antibiotic
vulg?re
concentrations as as 2000 mg spectinomycin per 1. Similar prob
high
lems arise with Arabidopsis thaliana. Unlike other species that show
1 30000 -I
a higher tolerance level to spectinomycin than to streptomycin, in

Arabidopsis the contrary is true. There was no retardation in growth

even at concentrations of 1000 mg/1 and above. When spectinomycin
and streptomycin are both present at concentrations of 1000 mg/1 (5
calli and shoots grow vigorously. Potential transformants 10000 -
wild-type
are overgrown masses of shoots and cannot
by large yellowish-looking 1430 ^h 400
be detected.
A disadvantage of any antibiotic selection is that untransformed
B
parts of the plant or leaf may not be distinguishable from transformed
areas in early development. Without a visible marker, leaf areas may
FlG. 2. Effect of different regulatory elements on GUS expression in
be chosen for the repeated process that contain low
regeneration transgenic tobacco plants. Young leaves (about 100 mg) of transgenic and
levels of transformed the process of seg tobacco were harvested from greenhouse to
plastome copies, slowing wild-type plants and subjected
towards the fluorometric GUS assay. Column A shows the wild type with no significant
regation homoplasmy.
GUS activity. In column B the GUS expression of a nuclear transformant
of the transformation cassette. To obtain high expression
Design
elements and promoters having the 35 S promoter is demonstrated. Columns C and D show the GUS
levels of the aadk gene, plastid regulatory are controlled
activity of plastome transformants. Both plastid GUS constructs
are used. As these elements are taken from the wild-type plastome, by the 16 S rDNA promoter, but C had the leader and trailer of psbk, while
homologous recombination events may occur between these and na D had the minimal 5' UTR from rbcL with the trailer of rbcL.
tive plastome sites. In the most unfortunate cases, important regu
sequences may be disturbed or whole genes may be deleted.
latory
was used in our selection marker cassette. our recent find
or fail to turn green. Such Applying
The resulting plants are likely to be weak
elements in combination with the
never become as wild-type gene copies ings about plastid regulatory
plants may homoplastomic,
marker gene will help to boost gene expression of future transfor
need to be maintained to assure functioning of important processes
mation vectors. Interestingly, the level of GUS expression achieved
in plant metabolism.
with a 16S-rDNA promoter and both UTRs from the psbk gene is
To overcome these
problems, homologous regulatory sequences around 40 times higher than that of a nuclear transformant with the
can be replaced by elements of similar function in other organisms.
uidk gene under the control of the 35S promoter.
We use the rbcL terminator from Chlamydomonas that has only a We are currently of the
testing transplastomic plants with fusions
partial homology to a tobacco plastome nucleotide sequence. How uidk gene with other UTR sequences.
ever, a potential risk of this strategy might be that a foreign sequence Inactivation of ndh genes encoding the plastid NAD(P)H plasto
terminate less efficiently than a native one, poten in tobacco. The NAD(P)H-ubiquinone-oxi
may transcription quinone-oxidoreductase
with the expression of neighboring doreductase is the first complex of the respiratory chain of bacteria
tially interfering open reading
and mitochondria (Friedrich et al., 1995) and consists of more than
frames.
30 subunits in Neurospora crassa and more than 40 in bovine mito
chondria. The E. coli complex has merely 14 subunits which are
9. Applications of Plastome Transformations
coded for by a single operon. In the plastome of higher plants there
are 11 subunits are homologous to E. coli. There
(ndh A-K) which
Posttranscriptional regulatory mechanisms in plastid genes. Re
fore, plastids may possess a NADH-complex homologous to complex
porter gene cassettes were introduced into the plastome via PEG is not
I (Ohyama et al., 1988; Sugiura, 1992). So far the enzyme
treatment to address the influence of various plastid leader and trailer characterized as it was not possible to sufficiently
biochemically pu
sequences. The untranslated(UTRs) were fused with
regions the
rify it, neither has its biochemical function been elucidated. It is
a reporter put under a role in cyclic electron
coding region of the uidk gene?as gene?and assumed that the enzyme plays transport
the control of the 16S-rDNA promoter. The construct was further (Kubicki et al., 1996). Another function is thatNAD(P)H is involved
with the aadk as a selective marker. As the constructs in a process called chlororespiration that funnels off extra redox
equipped gene
elements which can evolve during the degradation of starch in
differ from each other only in their UTRs, with all other equivalents
darkness 1982). The NDH-complex would oxidize these
being the same or equal, the different levels of reporter gene ex (Bennoun,
redox equivalents and pass them into the plastoquinone pool.
measured can be considered a result of the influence of the
pression into the function oxido
To gain further insight of the NAD(P)H
UTR elements.
reductase in plastids we have mutagenized ndhC and ndh]
as complex
The combination of psbk sequences leader and trailer for the
by insertion of the aadk cassette into the coding sequence and ndhK
????A-coding region results in a very high level of GUS expression as well as ndhk and J by partial deletion of the coding sequence
(Fig. 2). By contrast, the combination of a 'minimal' 5'-UTR from followed by the insertion of the aadk cassette (Kofer et al., 1998).
rbcL with a 3'-trailer from the same gene reduces more After regeneration and repeated rounds of selection to rid the trans
expression
than 100-fold. This minimal 5'-UTR consists of a 26-bp fragment formants of all wild-type plastome copies, we characterized the
the ribosome site. The same short minimal UTR The redox state of the plastoquinone can be analyzed
including binding plants. pool

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 308 KOFERET AL.

through chlorophyll fluorescence measurements with a PAM fluorom laboratory experienced in one of the transformation techniques. This
eter (Krause and Weis, 1991). In the wild type, at the end of actinic is mainly due to the expense of setting up a particle gun transfor
illumination, the fluorescence rose for 2-4 min from F0 demonstrating mation system or due to the experience needed to establish a pro
a transient reduction of the plastoquinone The ndh mutants, toplast culture system with the PEG method. We tried to take these
pool.
show this rise or showed into account and simplified our method without loss in
however, did not only a very low rise in problems
fluorescence. of which subunit of the multi-enzyme com transformation efficiency. The embedding of the protoplasts in nylon
Regardless
mesh with alginate facilitates the transfer from one medium to the
plex we had mutagenized, all transformants showed this character
istic phenotype, that for the transient increase in darkness next. The omission of the stepwise reduction in osmolarity reduces
suggesting
a functioning NDH is required. It is feasible that this pro transfer time and handling of the protoplasts considerably and min
complex
imizes the risk of contamination. In our revised method, the trans
cess is involved in the oxidation of excess NAD(P)H which is pro
duced the previous with actinic
formed cells have to be transferred just once 1 wk after transforma
during illumination light. Another
tion and from then on at 3-wk intervals. As the transformation
characteristic feature of the transformants was the accumulation of
efficiency is high (approximately 50 transformants per million pro
starch after an 8-h dark treatment. starch grains were visible
Large
toplasts), it may suffice to pick green shoots or calli off the nylon
in electron of all transformants and missing in wild-type
micrographs
only twice and transfer them to shoot or root regeneration
mesh me
indicating that the transformants could not efficiently oxidize
plants,
dium. Therefore, after only three to four transfers and less than 2 mo.
glucose via glycolysis and the oxidative
pentose phosphate pathway.
time, transformed are ready to transfer to the
of the plas regeneration plants
This observation, together with the lack of the reduction
the chlororespiration greenhouse.
toquinone pool stated earlier, support hypoth
We have found the aadk gene to be a good plastome selectable
esis in which the NDH-complex is regarded as a valve
removing but it has a number of shortcomings. The of
marker, impossibility
excess reduction in the chloroplasts.
equivalents areas with a low percentage of transformed
visually distinguishing
Despite repeated antibiotic selection (8-10 rounds), we did not
makes
the selection time consuming and
plastome copies procedure
succeed in recovering any homoplasmic nd/i-transformants. All
labor intensive. Also, point mutations in the 16S-rDNA nucleotide
transformants contained from 1-5% wild-type gene copies by
sequence can lead to plants that are antibiotic-resistant but nontrans
Southern Immunoblot confirmed these data; even
analysis. analysis of the transformation vector into a
genic. Additionally, integration
in all transformed the concentration of NADH-H was can yield additional
though plants genetic compartment other than the plastome
drastically reduced compared to the wild type, there were faint sig 'false positives' to be dealt with. We have further experienced that a
nals visible in all of them. From this we deduce that the NDH com number of species are not sensitive to either spectinomycin or strep

plex is essential for the growth and metabolism of higher plants. and therefore, these antibiotics cannot be used for selection.
tomycin,
Finally, secondary plastome transformations will require additional

Discussion marker genes. Initial indications of the suitability of hygromycin

phosphotransferase for selection in plastome transformation have not

Both the PEG and the biolistic methods are tools for been confirmed so far. We are presently on the
powerful working development
transformation. However, the method has to be of new plastid selectable markers.
achieving plastid
as they may not work We have presented in this paper a summary of neutral
carefully chosen, equally well depending upon integration
the species to be transformed. For example, for Arabidopsis thaliana sites and pointed out sequences that, when included into the trans

the PEG would be our method of choice because the pro formation vector, will yield high expression levels of the introduced
technique
is very well established in our laboratory. In gene. We could show that with a potent construct for trans
toplast culture system foreign
contrast, the regeneration of bombarded leaves is difficult in Arabi formation (i.e., a strong promoter, leaders, and trailers) the expression
one wishes of genes inserted into the plastome can be greatly enhanced, many
to transform
dopsis and only few cells form shoots. When
to enrich times higher than when the same gene is inserted into the nucleus
specific target cells, it is possible the population of such
under the control of the 35S promoter. McBride et al. (1995) reported
trans
cellsby a specific procedure during protoplast isolation and
on plastome transformation to introduce the crylk gene which en
form only those cells. In contrast, the particle gun method does not
codes a Bacillus thuringiensis crystal toxin. The transformed plants
allow one select
specific target cells in a leaf or another organ. The
accumulated 3-5% of the total soluble as protoxin.
in leaves
is the technique of choice for species for which protein
particle gun method
Through extensive gene resynthesis, promoter optimization, and pro
no good system is established or for labora
protoplast regeneration tein targeting, it had been possible to raise the expression of nuclear
tories that do not have the expertise to work with protoplast culture,
cry genes to 0.8% of the total soluble protein as Bt toxin. However,
as in general, it is easier to regenerate a shoot from an intact leaf
due to position effects, only 1 of 10 nuclear transformants gave such
than to establish a culture system. When compared for
protoplast a between 0.001 and 0.6%
high expression level; the others produced
their efficiency, the two methods are equally potent. An experienced of their soluble protein in toxin.
person can easily transform 10 million protoplasts per d. From each In plastome transformations as opposed to nuclear transformations,
million tobacco we obtained about 50 transformed
protoplasts, the construct can be inserted into the plastid chromosome at a spe
As for the particle gun method, we routinely got 50 trans
plants. cific site by homologous recombination, thereby avoiding position
formants from six to eight bombarded tobacco leaves. While one to multiple events.
effects and effects due integration Site-specific
could bombard 50 leaves per d, it is time consuming to cut the leaves allow the study of gene function and regulation
integrations by direct
and transfer the resulting leaf pieces to petri dishes for the antibiotic of plastome nucleotide de
mutagenesis sequences through insertion,
selection and A manageable number would the plastid
regeneration procedure. letion, and replacement (Rochaix, 1997). As genome has
be the transformation of about 20 leaves per experiment. a prokaryotic gene structure in which several genes are transcribed

Many scientists in protoplast research shy away from either plas in one operon, it is possible to introduce several genes within the
tome transformation method and instead seek the collaboration of a same transformation vector. Nuclear transformations with the aim of

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 PEGPLASTIDTRANSFORMATION309

several genes several transformation Kofer, W; Koop, H. U.; Wanner, G, et al. Mutagenesis of the genes encoding
introducing frequently require
subunits A, C, H, I, J and K of the plastid NAD(P)H-plastoquinone
vectors. The problems connected with such an experiment are many.
oxidoreductase in tobacco by polyethylene glycol-mediated plastome
on the it may take several different resistance transformation. Mol. Gen. Genet, (in press).
Depending approach,
markers. Furthermore, constructs will end up in different chromo Koop, H. U.; Steinm?ller, K; Wagner, H., et al. Integration of foreign se
quences into the tobacco plastome via polyethylene glycol-mediated
somal locations and in different copy numbers which will lead to
protoplast transformation. Planta 199:193-201; 1996.
differences in expression levels, and finally, many plants will have
Koop, H. U.; Kofer, W. Plastid transformation by polyethylene glycol treat
to be screened to obtain transformants of the correct genetic com ment of protoplasts and regeneration of transplastomic tobacco plants.
and desired Genes from fre are Gene transfer to plants, Springer Lab Manual. Heidelberg: Springer
position phenotype. prokaryotes
to transform it necessary to Verlag, D.; 1995:75-82.
quently used eukaryotes which makes
Krause, G. H.; Weis, E. Chlorophyll fluorescence and photosynthesis: the
alter their eukaryotic codon usage. Functioning of these genes in the basics. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:313-349; 1991.

plastome does not require such alterations. Finally, in most Kubicki, A.; Funk, E.; Westhoff, P., et al. Differential transcription of plas
generally
tome-encoded ndh-genes in mesophyll and bundle-sheath chloro
are maternally inherited, they are
higher plants, plastids meaning
plasts of the C4-plant Sorghum bicolor indicates that the complex I
on the maternal parent. All progeny of the transformed oxidoreductase is involved in
passed by only homologous NAD(P)H-plastoquinone
gene as long as cyclic electron transport. Planta 199:276-281; 1996.
plant will therefore uniformly carry the introduced
the transformant itself was homoplastomic. The offspring is therefore McBride, K. E.; Schaaf, D. J.; Daley, M., et al. Controlled expression of plastid
transgenes in plants based on a nuclear DNA-encoded and plastid
defined. As genes cannot be on
genetically plastid passed through targeted T7 RNA polymerase. Proc. Nati. Acad. Sei. 91:7301-7305;
the pollen, the biological safety of plastome transformants is greater 1994.
than for nuclear transformants and may find more acceptance McBride, K. E.; Svab, Z.; Schaaf, D. J., et al. Amplification of a chimeric
by the
Bacillus gene in chloroplasts leads to an extraordinary level of an
public. insecticidal protein in tobacco. Bio/Technology 13:362-365; 1995.

Acknowledgments Ohyama, K; Kohchi, T.; Sano, Y., et al. Newly identified groups of genes in
chloroplasts. Trends Biochem. Sei. 13:19-22; 1988.
We thank Petra Essig and Stefan Kirchner for their excellent assistance
O'Neill, C; Horv?th, G. V; Horv?th, ?., et al. Chloroplast transformation in
the transformations and tissue culture. This work was supported is an al
concerning plants: polyethylene glycol (PEG) treatment of protoplasts
by various grants from Deutsche Forschungsgemeinschaft (DFG). ternative to biolistic delivery systems. Plant J. 3:729-738; 1993.
Rochaix, J. D. Chloroplast reverse genetics: new insights into the function of
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