Chapter 22

Agrobacterium-mediated Transformation of Maize (Zea mays) Immature Embryos

Hyeyoung Lee and Zhanyuan J. Zhang

Abstract

Agrobacterium tumefaciens-mediated transformation is one of the most efficient and simple gene
delivery systems for genetic improvement and biology studies in maize. This system has become
more widely used by both public and private laboratories. Meanwhile, given the same maize
genotype, the transformation efficiencies vary greatly from laboratory to laboratory. Here, we
illustrate our advanced Agrobacterium-mediated transformation method in Hi-II maize using
simple binary vectors. The protocol utilizes immature embryos as starting explants and bar gene
as selectable marker coupled with bialaphos as a selective agent. The protocol offers efficient
transformation result with high reproducibility, provided that some experimental conditions are
well controlled. This transformation method, with minor modifications, can be also employed to
transform certain maize inbred.

Keywords: Agrobacterium tumefaciens, Hi-II, Maize, simple binary vectors, bar

1. Introduction

Genetic transformation has become an important tool for biology study and genetic
improvement in maize. As a natural DNA delivery vehicle, Agrobacterium is a good choice for
introducing foreign genes into the host plants to express the gene products. This is because this
delivery tool usually inserts a high percentage of single or low copy numbers of T-DNA into the
host genome with relatively rare rearrangement.
Routine maize regeneration and Agrobacterium-mediated transformation were difficult
previously because of the maize recalcitrance to the in vitro regeneration and Agrobacterium
infection. Hiei et al. (1) developed the highly efficient Agrobacterium-mediated transformation
method in rice using embryogenic callus and superbinary vector containing an extra copy of
virB, virC, and virG. Afterwards Ishida et al. (2) first reported stable transformation of maize
inbred lines (A-188) at a frequency of 5.5% employing immature embryos and superbinary
vector. Zhao et al (3-5) further improved maize transformation by deploying maize Hi-II
immature embryos as starting explant tissues in combined use with improved infection
conditions. These conditions included the use of phenolic compound acetosyringone, low pH
medium, and superbinary vector to enhance maize transformation. More recently, the use of
antioxidant L-cysteine has made it possible not only to improve maize Hi-II transformation (5)
but also to transform three inbred lines (B104, B114, and ky21) more efficiently using simple
binary vector system (6). To further improve maize transformation, we have previously
optimized inoculation and co-cultivation condition by employing both antioxidants L-cysteine
and DTT (dithiothreitol) coupled with low salt media (7). Such improvements have made maize
Hi-II transformation achieve over 12% transformation efficiency without a need of using the
superbinary vector.
To extend transformation methodology to maize inbred or elite lines, several attempts
have been made in using different target tissues, such as mature embryos (9), immature embryos
(10-11), and seedling nodal area from dry seeds (12) by Agrobacterium tumefaciens. However,
most of the public laboratories have adopted maize Hi-II transformation using immature embryos
and simple binary vectors (6-7). Therefore, here we describe our advanced maize Hi-II
transformation protocol (7) with tips for high frequency and reproducible transformation results.
This protocol, with minor modifications, can be also used in transformation of certain maize
inbred (8).

2. Materials

2.1. Plant materials

Maize Hi-II immature embryos derived from the self-pollinated ears (F2) of the F1 cross
between Hi-II A and Hi-II B are used as starting material. (see, Note 1)

2.2. Stock solution and culture media for Agrobacterium tumefaciens

The A. tumefaciens strain used for Hi-II Agrobacterium-mediated transformation is EHA101

(13). Strains LBA4404 (14) and AGL1 (15) can be also used.

2.2.1. Stock solution

1. AB salts 20X: 20g NH4Cl, 6g MgSO4.7H2O, 3g KCl, 0.2g CaCl2, and 0.05g FeSO4.7H2O are
dissolved in 1L ddH2O. This stock solution is filter-sterilized and stored at -4℃.

2. AB buffers 20X: 60g K2HPO4, and 20g NaH2PO4 are dissolved in 1L ddH2O. This stock
solution is filter-sterilized and stored at -4℃.

3. Stock C: 250g Glucose is dissolved in 1L ddH2O. This stock solution is filter-sterilized and
stored at -4℃.

4. Kanamycin sulfate (Sigma-Aldrich, St. Louis, Mo, USA): 50mg/ml stock in ddH2O. This
stock solution is filter-sterilized and stored at -20℃.

5. Chloramphenicol (Sigma-Aldrich): 25mg/ml stock in 50% EtOH. This stock solution is filter-
sterilized and stored at -20 ℃. To dissolve chloramphenicol in 50% EtOH, let the
chloramphenicol dissolve in 100% EtOH (half of final stock volume) and then make 1:1 dilution
with ddH2O before it is filter-sterilized.

2.2.2. Agrobacterium culture media

All semi-solid media for agrobacterial growth are solidified with 15g/l Bacto agar (Fisher),
contained in 100x15 petri dishes, and stored at -4℃ before use.

1. ABC medium (16): 880 mL ddH20 and 3.9 g MES with pH 5.6 are mixed well and autoclaved.
After autoclaving, AB salts 20X (50ml/l), AB buffers 20X (50ml/l), Stock C (20ml/l) and
appropriate antibiotics are added after filter-sterilized.

2. YEP medium (17): 10g/l peptone, 5g/l yeast extract, 5g/l NaCl, pH7.0. After autoclaving, the
appropriate antibiotics are added.

2.3. Stock solutions and culture media for maize transformation

2.3.1. Stock solutions

1. 2,4-D: 100 mg 2,4-dichlotophenoxyacetic acid (2,4-D) (Sigma-Aldrich) is dissolved in 2ml 1N

NaOH and adjusts the final volume of 100 ml with ddH2O. The stock solution is stored at -4℃.
(see, Note 2)

2. Bialaphos: 200mg bialaphos (Gold Biotechnology, St. Louis, USA) is dissolved in 40ml
ddH2O. The stock solution is filter-sterilized and stored at -4℃.

3. Silver nitrate: 340mg Silver nitrate is dissolved in 40ml ddH2O. The stock solution is filter-
sterilized and stored at -4℃. (see, Note 3)

4. Acetosyringone (AS): 0.196g acetosyringone is dissolved in 5 ml methanol first. Then add 5

ml ddH2O to make final volume 10 ml. This stock solution is filter-sterilized and stored at -20℃
in 1 ml aliquots. (see, Note 4)

5. N6 vitamin (1000X) (18): 0.1g glycine, 0.05g thiamine HCl, 0.025g pyridoxine HCl, and
0.025g nicotinic acid are dissolved in 50ml ddH2O. This stock solution is filter-sterilized and
stored at -4℃.

6. MS vitamin (1000X) (19): 5g myo-inositol, 0.025g nicotinic acid, 0.005g thiamin HCl, and
0.025g pyridoxine-HCl are dissolved in 50ml ddH2O. This stock solution is filter-sterilized and
stored at -4℃.

7. Glycine: 100mg glycine is dissolved in 50ml ddH2O. The stock solution is filter-sterilized and
stored at -4℃.

2.3.2. Maize culture media

All semi-solid media are contained in 100x15 mm Petri dishes and are stored at -4 ℃ before use.
The pH of all media except for inoculation medium was adjusted to 5.8 before autoclaving.
1. PHI-A, Inoculation medium: 2g/l N6 salts, 68.5g/l sucrose, 36g/l glucose, 0.7g/l L-proline,
0.5g/l MES, 1.5ml/l 2,4-D, 1 ml/l N6 vitamin, pH5.2. This medium is filter-sterilized and stored
at -20℃ for up to 2 months. AS is added right before use.

2. PHI-B, Co-cultivation medium: 2g/l N6 salts, 30g/l sucrose, 0.7g/l L-proline, 0.5g/l MES,
1.5ml/l 2,4-D and 5g/l Agar (Sigma-Aldrich), pH5.8. After autoclaving, filter-sterilized 1ml/l N6
vitamin, 0.1ml/l silver nitrate, and 1ml/l AS are added. And freshly dissolved 0.4g L-cysteine
and 0.154g DTT in 10 ml ddH2O are added.

3. PHI-C, Resting medium: 4g/l N6 salts, 30g/l sucrose, 0.7g/l L-proline, 0.5g/l MES, 1.5ml/l
2,4-D and 3g/l Gelrite (Sigma-Aldrich), pH5.8. After autoclaving, filter-sterilized 1 ml/l N6
vitamin and 0.1ml/l silver nitrate are added. And freshly dissolved 250mg/l cefotaxime in 10ml
ddH2O is added.

4. PHI-D1, Selection I medium: 4g/l N6 salts, 30g/l sucrose, 0.7g/l L-proline, 0.5g/l MES,
1.5ml/l 2,4-D and 3g/l Gelrite (Sigma-Aldrich), pH5.8. After autoclaving, filter-sterilized 1 ml/l
N6 vitamin, 0.1ml/l silver nitrate and 0.3ml bialaphos are added. And freshly dissolved 250mg/l
cefotaxime in 10ml ddH2O is added.

5. PHI-D2, Selection II medium: 4g/l N6 salts, 30g/l sucrose, 0.7g/l L-proline, 0.5g/l MES, 1.5
ml/l 2,4-D and 3g/l Gelrite (Sigma-Aldrich), pH5.8. After autoclaving, filter-sterilized1 ml/l N6
vitamin, 0.1 ml/l silver nitrate and 0.6 ml bialaphos are added. And freshly dissolved 250 mg/l
cefotaxime in 10 ml ddH2O is added.

6. PHI-E, Maturation medium: 4.3g/l MS salts, 60g/l sucrose, 3g/l Gelrite (Sigma-Aldrich), pH
5.6. After autoclaving, filter-sterilized 1ml/l MS vitamin, 1ml/l glycine and 0.6ml bialaphos are
added. And freshly dissolved 250mg/l cefotaxime in 10ml ddH2O is added.

7. PHI-F, Regeneration medium: 2.9g/l MS salts, 30g/l sucrose, 3g/l Gelrite (Sigma-Aldrich),
pH5.6. After autoclaving, filter-sterilized 1ml/l MS vitamin and 1ml/l glycine are added.

3. Methods

3.1. Agrobacterium culture initiation

1. Streak EHA101 carrying simple binary vector from -80℃ stock on ABC medium with
appropriate antibiotics. Make dilute series so that single colonies can develop. Let culture growth
for 3 days at 28℃ in darkness. (see, Note 5)

2. Pick up single colony and streak it on YEP medium containing appropriate antibiotics and let
culture growth at 20℃ for 3 days in darkness.

3.2. Inoculation

1. Add 5ml of sterile PHI-A infection medium into 15ml Falcone tube (Fisher).
2. Take about two full loops EHA101 from YEP plate and suspend in the tube. Let the pallet sit
there for 2 to 3 minutes. Shake the tube to suspend bacterial cells well.

3. Transfer 1 ml of such suspension to spectrophotometer cuvette and check the density of the
suspension culture at O.D. at 550 and adjust it to OD550 of 0.35 (0.5 X 109 cfu/ml).

4. Shake the re-suspended culture in a shaker at 100 rpm for 4 to 5 hours.

5. Aliquot 1 ml suspension into 1.5ml sterile microcentrifuge tube.

3.3 Embryo isolation, inoculation, and co-cultivation

1. Remove the husks and silk from ears which are harvested 10-13 days post-pollination (with
embryo size of 1.5mm). Insert the forceps from the top end of ear. (see, Note 6)

2. Soak fresh Hi-II ears in a sterile 1L wide-mouth bottle containing about 0.5L of 30 %
commercial bleach with a few drops of tween-20 for 20 min. (see, Note 7)

3. Wash ears 3 times with a plenty of sterile water and let the ear stand up right on a sterile
150X15 mm petri dish.

4. Remove top half of the kernels of each ear with a #11 razor blade (Fisher). (see, Note 8)

5. Isolate 1.5 mm immature embryos from sterile ear with sterile spatula and transfer 50 to 100
embryos to each tube. Then, wash the embryos with PHI-A solution three times to remove debris
and starch.

6. Add to the tube the Agrobacterium suspension and allow tube to stand 5 min in the hood and
then pour the whole content including all embryos onto petri plate containing PHI-B, the co-
cultivation medium.

7. Draw off Agrobacterium suspension using a pipette with fine tips, spread out the embryos
cross the plate (so that they are quite evenly spaced) and place embryos flat face down on the
medium. (see, Note 9)

8. Seal the plate with parafilm tape and incubate at dark at 20℃ for 3 days.

3.4. Resting

1. Transfer the embryos to PHI-C, resting medium. Avoid damaging the embryos.

2. Seal the plate with parafilm and incubate in dark at 28℃ for 7 days.

3.5. Selection
1. Transfer embryos to PHI-D1, selection I medium. Place 25 embryos per plate and seal the
plate. The embryos are incubated in the dark at 28℃ during the first 2-week selection.

2. Transfer calli from PHI-D1, selection I medium, to PHI-D2, selection II medium. The calli are
then subcultured every 2 weeks onto the same fresh medium for a total of 2 months.

3. Bulk up the herbicide-resistant calli by growing then on the same fresh medium for another 2
weeks until the diameter of the calli is about 1.0 cm. During this stage, each large, fast growing
Hi-II embryogenic callus can be divided into smaller calli to select the best quality of Hi-II
embryogenic calli (i.e., type II calli which are dry, friable, and fast growing), enhance selection
stringency and/or maintain callus cultures for prolonged period of time.

3.6. Regeneration

1. Transfer opaque calli onto PHI-E, maturation medium in the dark at 28℃ for two 2 to 3 weeks
to allow somatic embryos to mature.

2. Transfer ivory white calli on PHI-F, regeneration medium at 28℃ under 16 hours photoperiod
until shoot and roots developed.

3. Transfer each small plantlet to a 25 X 150 mm tube containing PHI-F, regeneration medium,
and grow under the same light conditions for 2 to 3 weeks.

4. Transfer the plants to pots with soil mixture in a greenhouse.

4. Note

1. The quality of ears is the most important factor in Hi-II maize transformations, given other
conditions are well under the control. High quality ears contain the immature embryos of high
vigor which are capable of tolerating low salt medium condition during the Agrobacterium
infection stage. We routinely use immature embryos derived the Hi-II A x B: F2 ears. In fact, the
Hi-II A x B: F1 ears offer higher embryo vigor than F2 ears, which leads to a much higher
transformation frequency (unpublished) than we reported previously (7). Nonetheless, use of Hi-
II A x B: F1 ears requires more extensive labor to make crosses and more greenhouse space.
Consequently, we routinely use Hi-II A x B: F2 instead of F1 ears.

2. Do not heat the solution while dissolving 2,4-D in NaOH.

3. Silver nitrate is sensitive to light and therefore the chemical should be kept in dark.

4. For dissolving AS, it is recommended to use methanol instead of DMSO (dimethyl sulfoxide)
and store at -20 ℃. Use of methanol will avoid freeze-thaw steps as encountered by the use of
DMSO. This will minimize the reduced potency of AS due to the freeze-thaw.

5. After streak EHA101 on ABC medium, the colonies on the agar plate can be used for up to
one month when stored at -4℃.
6. The size of immature embryos, i.e., between 1.5 mm to 1.8 mm, is crucial. We found that as
the size of embryo increases the transformation frequency decreases but the embryos are more
tolerant to the low salt medium condition (unpublished). Therefore, use of small size of embryos
(less than 1.2 mm) requires a full-strength salt medium without suffering from a high mortality
of the embryos. However, it is much more difficult to isolate small embryos than large ones. As a
result, our most practical way is to use 1.5mm embryos in combined use with low salt medium,
achieving a high transformation frequency with ease in embryos isolation.

7. If the maize plants are grown in the field to obtain immature embryos, it is recommended to
use 50 % commercial bleach for decontamination. Embryos from field offer higher
transformation frequency than the embryos from the greenhouse.

8. It is recommended to remove top half of kernels from two rows at a time. This will help to
keep a desired high moisture and vigor of the embryos in the remaining rows.

9. When the immature embryos are co-cultivated on the PHI-B medium, the embryo should flat
face down and be sure to check the orientation of the embryos under dissecting microscope.

Reference

1. Hiei, Y., Ohta, S., and Komari, T. (1994) Efficient transformation of rice (Oryza sativa L.)
mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J.
6:271-282

2. Ishida, Y., Saito, H., Ohta, S., Hiel, Y., Komari, T., and Kumashiro, T. (1996) High efficiency
transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat.
Biotechnology. 14:745-750

3. Zhao, Z.Y., Gu, W., and Cai, T. (1999) Methods for Agrobacterium-mediated transformation.
United States Patent No 5981840

4. Zhao, Z.Y., Gu, W., Gai, T., Tagliani, L., Hondred, D., Bond, D., Schroeder, S., Rudert, M.,
and Pierce, D. (2001) High throughput genetic transformation mediated by Agrobacterium
tumefaciens in maize. Mol. Breed. 8:323-333

5. Zhao, Z.Y., Gu, W., Cai, T., and Pierce, D.A. (2004) Methods for Agrobacterium-mediated
transformation. United States Patent No. 963096. Pioneer Hi-bred International, Inc. (Des
Moines, IA)

6. Frame, B.R., Shou, H., Chikwamba, R.K., Zhang, Z., Xiang, C., Fonger, T.M., Pegg, S.E.K.,
Li, B., Nettleton, D.S., Pei, D., and Wang, K. (2002) Agrobacterium tumefaciens-mediated
transformation of maize embryos using a standard binary vector system. Plant Physiol. 129:13-
22
7. Vega, J.M., Yu, W., Kennon, A.R., Chen, X., and Zhang, Z. (2008) Improvement of
Agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary
vectors. Plant Cell Rep. 27:297-305

8. Lee, B.K., Kennon, A.R., Chen, X., Jung, T.W., Ahn, B.O., Lee, Z.Y., and Zhang, Z. (2007)
Recovery of transgenic events from two highly recalcitrant maize (Zea mays L.) genotypes using
Agrobacterium-mediated standard–binary-vector transformation. Maydica, 52:457-469

9. Huang, X.Q., and Wei, Z.M. (2004) High-frequency plant regeneration through callus
initiation from mature embryos of maize (Zea mays L.). Plant Cell Rep. 22:793-800

10. Huang, X., and Wei, Z. (2005) Successful Agrobacterium-mediated genetic transformation of
maize elite inbred lines. Plant Cell Tissue Organ Cult. 83:187-200

11. Frame, B.R., McMurray, J.M., Fonger, T.M., Main, M.L., Taylor, K.W., Torney, F.J., Paz,
M.M., and Wang, K. (2006) Improved Agrobacterium-mediated transformation of three maize
inbred lines using MS salts. Plant Cell Rep. 25:1024-1034

12. Sidorov, V., Gilbertson, L., Addae, P., and Duncan, D. (2006) Agrobacterium-mediated
transformation of seedling-derived maize callus. Plant Cell Rep. 25:320-328

13. Hood, E.E., Helmer, G.L., Fralcy, R.T., and Chilton, M.D. (1986) The hypervirulence of
Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J.
Bacteriol. 168:1291-1301

14. Hoekma, A., Hirsch, P.R., Hooykaas, P.J.J. and Schilperoort, R.A. (1983). Nature 303, 179-
180

15. Lazo, G.R., Stein, P.A., and Ludwig, R.A. (1991) A DNA transformation-competent
Arabidopsis genomic library in Agrobacterium. Bio. Technol. 9:963-967

16. Zhang, W., Subbarao, S., Addae, P., Shen, A., Armstrong, C., Peschke, V., Gilbertson, L.
(2003) Cre/lox mediated marker gene excision in transgenic maize (Zea may L.) plants. Theor.
Appl. Genet.107:1157-1168

17. An, G., Ebert, P.R., Mitra, A., and Ha, S.B. (1988) Binary vectors. In: Gelvin SB,
Schilperoort RA (eds) Plant molecular biology manual. Kluwer Academic. Dordrecht. pp 1-19

18. Chu, C.C., Wang, C.C., Sun, C.S., Hsu, C., Yin, K.C., Chu, C.Y., and Bi, F.Y. (1975)
Establishment of an efficient medium for anther culture of rice through comparative experiments
on the nitrogen source. Sci Sin. 18:659-668

19. Murashige, T, and Skoog, F. (1962) A revised medium for rapid growth and bioassays with
tobacco tissue cultures. Physiol. Plant 15:473–497(1962)