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. However, transformation efficiencies vary greatly from laboratory to laboratory for the same genotype. 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 the bar gene as a selectable marker coupled with bialaphos as a selective agent. The protocol offers efficient transformation results 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 inbreds. Key words Agrobacterium tumefaciens, Hi-II, Maize, Simple binary vectors, bar
1
Introduction Genetic transformation has become an important tool for biological research and genetic improvement in maize. As a natural DNA delivery vehicle, Agrobacterium is a good choice for introducing foreign genes into host plants for successful expression. 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 recalcitrance of maize in vitro regeneration and Agrobacterium infection. Hiei et al. [1] developed a highly efficient Agrobacterium-mediated transformation method in rice using embryogenic callus and superbinary vectors containing an extra copy of virB, virC, and virG. Subsequently Ishida et al. [2] first reported stable transformation of maize inbred lines (A-188) at a frequency of 5.5 % employing
Robert J. Henry and Agnelo Furtado (eds.), Cereal Genomics: Methods and Protocols, Methods in Molecular Biology, vol. 1099, DOI 10.1007/978-1-62703-715-0_22, © Springer Science+Business Media New York 2014
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immature embryos and superbinary vectors. Zhao et al. [3–5] further improved maize transformation by deploying maize Hi-II immature embryos as a starting explant tissue in combination with improved infection conditions. These conditions included the use of the phenolic compound acetosyringone, a low pH medium, and superbinary vectors to enhance maize transformation. More recently, the use of the 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 a simple binary vector system [6]. To further improve maize transformation, we have previously optimized inoculation and cocultivation condition by employing the two antioxidants, L-cysteine and dithiothreitol (DTT), coupled with low-salt media [7]. Such improvements have allowed maize Hi-II transformation at over 12 % efficiency without the use of a superbinary vector. To extend Agrobacterium-mediated transformation methodology to maize inbred or elite lines, several attempts have been made using different target tissues, such as mature embryos [8], immature embryos [9, 10], and the seedling nodal area from dry seeds [11]. However, most public laboratories have adopted maize Hi-II transformation using immature embryos and simple binary vectors [6, 7]. 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 [12].
2
Materials 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 Agrobacterium tumefaciens and Media
All semisolid media for agrobacterial growth are solidified with 15 g/L Bacto agar (Fisher), contained in 100 × 15 petri dishes, and stored at −4 °C before use.
2.1
1. Use A. tumefaciens EHA101 [13] for Hi-II Agrobacteriummediated transformation. Strains LBA4404 [14] and AGL1 [15] can also be used. 2. AB salts 20×: 20 g NH4Cl, 6 g MgSO4·7H2O, 3 g KCl, 0.2 g CaCl2, and 0.05 g FeSO4·7H2O are dissolved in 1 L ddH2O. This stock solution is filter sterilized and stored at −4 °C. 3. AB buffers 20×: 60 g K2HPO4 and 20 g NaH2PO4 are dissolved in 1 L ddH2O. This stock solution is filter sterilized and stored at −4 °C. 4. Stock C: 250 g glucose is dissolved in 1 L ddH2O. This stock solution is filter sterilized and stored at −4 °C.
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5. 50 mg/mL Kanamycin sulfate stock in ddH2O: This stock solution is filter sterilized and stored at −20 °C. 6. 25 mg/mL Chloramphenicol stock in 50 % EtOH: This stock solution is filter sterilized and stored at −20 °C. 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. 7. ABC medium [16]: 880 mL ddH2O and 3.9 g MES with pH 5.6 are mixed well and autoclaved. After autoclaving, AB salts 20× (50 mL/L), AB buffers 20× (50 mL/L), stock C (20 mL/L), and appropriate antibiotics are added after filter sterilized. 8. YEP medium [17]: 10 g/L peptone, 5 g/L yeast extract, 5 g/L NaCl, pH 7.0. After autoclaving, the appropriate antibiotics are added. 9. #11 razor blade (Fisher). 2.3 Maize Transformation
All semisolid media are contained in 100 × 15 mm petri dishes and are stored at −4 °C before use. The pH of all media except for inoculation medium was adjusted to 5.8 before autoclaving. 1. 2,4-dichlotophenoxyacetic acid (2,4-D): Dissolve 100 mg of 2,4-D in 2 mL 1N NaOH and adjust the final volume of 100 mL with ddH2O. Store the stock solution at −4 °C (see Note 2). 2. Bialaphos: Dissolve 200 mg of bialaphos in 40 mL ddH2O. Filter sterilize the stock and store at −4 °C. 3. Silver nitrate: Dissolve 340 mg of silver nitrate in 40 mL ddH2O. Filter sterilize the stock and store at −4 °C (see Note 3). 4. Acetosyringone (AS): Dissolve 0.196 g of acetosyringone in 5 mL methanol first. Then add 5 mL ddH2O to make final volume to 10 mL. Filter sterilize the stock solution and store at −20 °C in 1 mL aliquots (see Note 4). 5. N6 vitamin (1,000×) [18]: Dissolve 0.1 g glycine, 0.05 g thiamine HCl, 0.025 g pyridoxine HCl, and 0.025 g nicotinic acid in 50 mL of ddH2O. Filter sterilize the stock solution and store at −4 °C. 6. MS vitamin (1,000×) [19]: Dissolve 5 g myoinositol, 0.025 g nicotinic acid, 0.005 g thiamin HCl, and 0.025 g pyridoxine HCl in 50 mL of ddH2O. Filter sterilize this stock solution and store at −4 °C. 7. Glycine: 100 mg glycine is dissolved in 50 mL ddH2O. Filter sterilize the stock solution and store at −4 °C. 8. PHI-A, inoculation medium: 2 g/L N6 salts, 68.5 g/L sucrose, 36 g/L glucose, 0.7 g/L L-proline, 0.5 g/L MES, 1.5 mL/L 2,4-D, 1 mL/L N6 vitamin, pH 5.2. Filter sterilize
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this medium and store at −20 °C for up to 2 months. AS is added right before use. 9. PHI-B, cocultivation medium: 2 g/L N6 salts, 30 g/L sucrose, 0.7 g/L L-proline, 0.5 g/L MES, 1.5 mL/L 2,4-D and 5 g/L agar (Sigma-Aldrich), pH 5.8. After autoclaving, add filter-sterilized 1 mL/L N6 vitamin, 0.1 mL/L silver nitrate, and 1 mL/L AS. Also add freshly dissolved 0.4 g L-cysteine and 0.154 g DTT in 10 mL ddH2O. 10. PHI-C, resting medium: 4 g/L N6 salts, 30 g/L sucrose, 0.7 g/L L-proline, 0.5 g/L MES, 1.5 mL/L 2,4-D, and 3 g/L Gelrite (Sigma-Aldrich), pH 5.8. After autoclaving, add filter-sterilized 1 mL/L N6 vitamin and 0.1 mL/L silver nitrate. Add freshly dissolved 250 mg/L cefotaxime in 10 mL ddH2O. 11. PHI-D1, selection I medium: 4 g/L N6 salts, 30 g/L sucrose, 0.7 g/L L-proline, 0.5 g/L MES, 1.5 mL/L 2,4-D and 3 g/L Gelrite (Sigma-Aldrich), pH 5.8. After autoclaving, add filtersterilized 1 mL/L N6 vitamin, 0.1 mL/L silver nitrate, and 0.3 mL bialaphos. Also add freshly dissolved 250 mg/L cefotaxime in 10 mL ddH2O. 12. PHI-D2, selection II medium: 4 g/L N6 salts, 30 g/L sucrose, 0.7 g/L L-proline, 0.5 g/L MES, 1.5 mL/L 2,4-D, and 3 g/L Gelrite (Sigma-Aldrich), pH 5.8. After autoclaving, add filter-sterilized 1 mL/L N6 vitamin, 0.1 mL/L silver nitrate, and 0.6 mL bialaphos. Also add freshly dissolved 250 mg/L cefotaxime in 10 mL ddH2O. 13. PHI-E, maturation medium: 4.3 g/L MS salts, 60 g/L sucrose, 3 g/L Gelrite (Sigma-Aldrich), pH 5.6. After autoclaving, add filter-sterilized 1 mL/L MS vitamin, 1 mL/L glycine, and 0.6 mL bialaphos. Also add freshly dissolved 250 mg/L cefotaxime in 10 mL ddH2O. 14. PHI-F, regeneration medium: 2.9 g/L MS salts, 30 g/L sucrose, 3 g/L Gelrite (Sigma-Aldrich), pH 5.6. After autoclaving, add filter-sterilized 1 mL/L MS vitamin and 1 mL/L glycine. 15. Parafilm tape.
3
Methods
3.1 Agrobacterium Culture Initiation
1. Streak EHA101 carrying simple binary vector from −80 °C stock on ABC medium with appropriate antibiotics. Make dilute series so that single colonies can develop. 2. Let culture grow for 3 days at 28 °C in darkness (see Note 5). 3. Pick up single colony and streak it on YEP medium containing appropriate antibiotics, and let culture grow at 20 °C for 3 days in darkness.
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Inoculation
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1. Add 5 mL of sterile PHI-A infection medium into 15 mL Falcon tube. 2. Take about two full loops of EHA101 from YEP plate and suspend in the tube. Let the pallet sit there for 2–3 min. 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. 550 and adjust it to OD550 of 0.35 (0.5 × 109 cfu/mL). 4. Shake the re-suspended culture in a shaker at 100 rpm for 4–5 h. 5. Aliquot 1 mL suspension into 1.5 mL sterile micro-centrifuge tube.
3.3 Embryo Isolation, Inoculation, and Cocultivation
1. Remove the husks and silk from ears which are harvested 10–13 days post pollination (with embryo size of 1.5 mm). Insert the forceps from the top end of the ear (see Note 6). 2. Soak fresh Hi-II ears in a sterile 1 L wide-mouth bottle containing about 0.5 L of 30 % commercial bleach with a few drops of Tween-20 for 20 min (see Note 7). 3. Wash ears three times with plenty of sterile water, and let the ear stand upright on a sterile 150 × 15 mm petri dish. 4. Remove top half of the kernels of each ear with a #11 razor blade (see Note 8). 5. Isolate 1.5 mm immature embryos from sterile ear with sterile spatula, and transfer 50–100 embryos to each tube. Wash the embryos with PHI-A solution three times to remove debris and starch. 6. Add the Agrobacterium suspension, allow the tube to stand for 5 min in the hood, and then pour the whole contents including all embryos onto the petri plate containing PHI-B, the cocultivation medium. 7. Draw off the Agrobacterium suspension using a pipette with a fine tip, spread out the embryos across 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 in the dark at 20 °C 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 the dark at 28 °C for 7 days.
3.5
Selection
1. Transfer the embryos to PHI-D1, selection I medium. Place 25 embryos in each plate, and seal the plate. Incubate the embryos in the dark at 28 °C during the first 2-week selection.
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2. Transfer the calli from PHI-D1, selection I medium, to PHI-D2, selection II medium. Subculture the calli every 2 weeks onto the same fresh medium for a total of 2 months. 3. Bulk up the herbicide-resistant calli by growing them 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, fastgrowing Hi-II embryogenic callus can be divided into smaller calli to select the best-quality Hi-II embryogenic calli (i.e., type II calli which are dry, friable, and fast growing) to enhance selection stringency and/or maintain callus cultures for a prolonged period of time. 3.6
Regeneration
1. Transfer opaque calli onto PHI-E, maturation medium, in the dark at 28 °C for 2–3 weeks to allow the somatic embryos to mature. 2. Transfer ivory white calli onto PHI-F, regeneration medium, at 28 °C under 16-h photoperiod until shoot and roots develop. 3. Transfer each small plantlet to a 25 × 150 mm tube containing PHI-F, regeneration medium, and grow under the same light conditions for 2–3 weeks. 4. Transfer the plants to pots with soil mixture in a greenhouse.
4
Notes 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 immature embryos of high vigor which are capable of tolerating low-salt medium conditions during the Agrobacterium infection stage. We routinely use immature embryos derived from Hi-II A × B: F2 ears. In fact, the Hi-II A × B: F1 ears offer higher embryo vigor than F2 ears, which leads to a much higher transformation frequency (unpublished) than we have reported previously [7]. Nonetheless, use of Hi-II A × B: F1 ears requires more extensive labor to make crosses and more greenhouse space. Consequently, we routinely use Hi-II A × 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 the dark. 4. For dissolving AS, it is recommended to use methanol instead of dimethyl sulfoxide (DMSO) and store at −20 °C. Use of methanol will avoid freeze–thaw steps as encountered with the use of DMSO. This will minimize the reduced potency of AS resulting from freezing and thawing.
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5. Streak EHA101 on ABC medium; the colonies on the agar plate can be used for up to 1 month when stored at 4 °C. 6. The size of immature embryos, i.e., between 1.5 and 1.8 mm, is crucial. We found that as the size of the embryo increases the transformation frequency decreases but the embryos are more tolerant of the low-salt medium condition (unpublished). Therefore, use of small embryos (less than 1.2 mm) requires a full-strength salt medium to avoid 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 method is to use 1.5 mm embryos combined with low-salt medium, achieving a high transformation frequency with ease in embryo isolation. 7. If the maize plants are grown in the field to obtain immature embryos, the use of 50 % commercial bleach is recommended for decontamination. Embryos from the field offer higher transformation frequency than the embryos from the greenhouse. 8. It is recommended that the kernels be removed from two rows at a time. This will help to maintain the desired high moisture and vigor of the embryos in the remaining rows. 9. When the immature embryos are cocultivated on the PHI-B medium, the embryo should be flat face down and be sure to check the orientation of the embryos under a dissecting microscope. References 1. Hiei Y, Ohta S, 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 et al (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745–750 3. Zhao ZY, Gu W, Cai T (1999) Methods for Agrobacterium-mediated transformation. United States Patent 5,981,840 4. Zhao ZY, Gu W, Gai T et al (2001) High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Mol Breed 8:323–333 5. Zhao ZY, Gu W, Cai T et al (2004) Methods for Agrobacterium-mediated transformation. United States Patent 963,096. Pioneer Hi-bred International, Inc., Des Moines, IA 6. Frame BR, Shou H, Chikwamba RK et al (2002) Agrobacterium tumefaciens-mediated transformation of maize embryos using a stan-
7.
8.
9.
10.
11.
dard binary vector system. Plant Physiol 129:13–22 Vega JM, Yu W, Kennon AR et al (2008) Improvement of Agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary vectors. Plant Cell Rep 27:297–305 Huang XQ, Wei ZM (2004) High-frequency plant regeneration through callus initiation from mature embryos of maize (Zea mays L.). Plant Cell Rep 22:793–800 Huang X, Wei Z (2005) Successful Agrobacterium-mediated genetic transformation of maize elite inbred lines. Plant Cell Tissue Organ Cult 83:187–200 Frame BR, McMurray JM, Fonger TM et al (2006) Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts. Plant Cell Rep 25:1024–1034 Sidorov V, Gilbertson L, Addae P et al (2006) Agrobacterium-mediated transformation of seedling-derived maize callus. Plant Cell Rep 25:320–328
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12. Lee BK, Kennon AR, Chen X et al (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 13. Hood EE, Helmer GL, Fralcy RT et al (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 PR, Hooykaas PJJ et al (1983) Binary vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid, Nature 303:179–180 15. Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology 9: 963–967
16. Zhang W, Subbarao S, Addae P et al (2003) Cre/lox mediated marker gene excision in transgenic maize (Zea may L.) plants. Theor Appl Genet 107:1157–1168 17. An G, Ebert PR, Mitra A et al (1988) Binary vectors. In: Gelvin SB, Schilperoort RA (eds) Plant molecular biology manual. Kluwer, Dordrecht, pp 1–19 18. Chu CC, Wang CC, Sun CS et al (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, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
1
Introduction Genetic transformation has become an important tool for biological research and genetic improvement in maize. As a natural DNA delivery vehicle, Agrobacterium is a good choice for introducing foreign genes into host plants for successful expression. 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 recalcitrance of maize in vitro regeneration and Agrobacterium infection. Hiei et al. [1] developed a highly efficient Agrobacterium-mediated transformation method in rice using embryogenic callus and superbinary vectors containing an extra copy of virB, virC, and virG. Subsequently Ishida et al. [2] first reported stable transformation of maize inbred lines (A-188) at a frequency of 5.5 % employing
Robert J. Henry and Agnelo Furtado (eds.), Cereal Genomics: Methods and Protocols, Methods in Molecular Biology, vol. 1099, DOI 10.1007/978-1-62703-715-0_22, © Springer Science+Business Media New York 2014
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immature embryos and superbinary vectors. Zhao et al. [3–5] further improved maize transformation by deploying maize Hi-II immature embryos as a starting explant tissue in combination with improved infection conditions. These conditions included the use of the phenolic compound acetosyringone, a low pH medium, and superbinary vectors to enhance maize transformation. More recently, the use of the 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 a simple binary vector system [6]. To further improve maize transformation, we have previously optimized inoculation and cocultivation condition by employing the two antioxidants, L-cysteine and dithiothreitol (DTT), coupled with low-salt media [7]. Such improvements have allowed maize Hi-II transformation at over 12 % efficiency without the use of a superbinary vector. To extend Agrobacterium-mediated transformation methodology to maize inbred or elite lines, several attempts have been made using different target tissues, such as mature embryos [8], immature embryos [9, 10], and the seedling nodal area from dry seeds [11]. However, most public laboratories have adopted maize Hi-II transformation using immature embryos and simple binary vectors [6, 7]. 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 [12].
2
Materials 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 Agrobacterium tumefaciens and Media
All semisolid media for agrobacterial growth are solidified with 15 g/L Bacto agar (Fisher), contained in 100 × 15 petri dishes, and stored at −4 °C before use.
2.1
1. Use A. tumefaciens EHA101 [13] for Hi-II Agrobacteriummediated transformation. Strains LBA4404 [14] and AGL1 [15] can also be used. 2. AB salts 20×: 20 g NH4Cl, 6 g MgSO4·7H2O, 3 g KCl, 0.2 g CaCl2, and 0.05 g FeSO4·7H2O are dissolved in 1 L ddH2O. This stock solution is filter sterilized and stored at −4 °C. 3. AB buffers 20×: 60 g K2HPO4 and 20 g NaH2PO4 are dissolved in 1 L ddH2O. This stock solution is filter sterilized and stored at −4 °C. 4. Stock C: 250 g glucose is dissolved in 1 L ddH2O. This stock solution is filter sterilized and stored at −4 °C.
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5. 50 mg/mL Kanamycin sulfate stock in ddH2O: This stock solution is filter sterilized and stored at −20 °C. 6. 25 mg/mL Chloramphenicol stock in 50 % EtOH: This stock solution is filter sterilized and stored at −20 °C. 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. 7. ABC medium [16]: 880 mL ddH2O and 3.9 g MES with pH 5.6 are mixed well and autoclaved. After autoclaving, AB salts 20× (50 mL/L), AB buffers 20× (50 mL/L), stock C (20 mL/L), and appropriate antibiotics are added after filter sterilized. 8. YEP medium [17]: 10 g/L peptone, 5 g/L yeast extract, 5 g/L NaCl, pH 7.0. After autoclaving, the appropriate antibiotics are added. 9. #11 razor blade (Fisher). 2.3 Maize Transformation
All semisolid media are contained in 100 × 15 mm petri dishes and are stored at −4 °C before use. The pH of all media except for inoculation medium was adjusted to 5.8 before autoclaving. 1. 2,4-dichlotophenoxyacetic acid (2,4-D): Dissolve 100 mg of 2,4-D in 2 mL 1N NaOH and adjust the final volume of 100 mL with ddH2O. Store the stock solution at −4 °C (see Note 2). 2. Bialaphos: Dissolve 200 mg of bialaphos in 40 mL ddH2O. Filter sterilize the stock and store at −4 °C. 3. Silver nitrate: Dissolve 340 mg of silver nitrate in 40 mL ddH2O. Filter sterilize the stock and store at −4 °C (see Note 3). 4. Acetosyringone (AS): Dissolve 0.196 g of acetosyringone in 5 mL methanol first. Then add 5 mL ddH2O to make final volume to 10 mL. Filter sterilize the stock solution and store at −20 °C in 1 mL aliquots (see Note 4). 5. N6 vitamin (1,000×) [18]: Dissolve 0.1 g glycine, 0.05 g thiamine HCl, 0.025 g pyridoxine HCl, and 0.025 g nicotinic acid in 50 mL of ddH2O. Filter sterilize the stock solution and store at −4 °C. 6. MS vitamin (1,000×) [19]: Dissolve 5 g myoinositol, 0.025 g nicotinic acid, 0.005 g thiamin HCl, and 0.025 g pyridoxine HCl in 50 mL of ddH2O. Filter sterilize this stock solution and store at −4 °C. 7. Glycine: 100 mg glycine is dissolved in 50 mL ddH2O. Filter sterilize the stock solution and store at −4 °C. 8. PHI-A, inoculation medium: 2 g/L N6 salts, 68.5 g/L sucrose, 36 g/L glucose, 0.7 g/L L-proline, 0.5 g/L MES, 1.5 mL/L 2,4-D, 1 mL/L N6 vitamin, pH 5.2. Filter sterilize
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this medium and store at −20 °C for up to 2 months. AS is added right before use. 9. PHI-B, cocultivation medium: 2 g/L N6 salts, 30 g/L sucrose, 0.7 g/L L-proline, 0.5 g/L MES, 1.5 mL/L 2,4-D and 5 g/L agar (Sigma-Aldrich), pH 5.8. After autoclaving, add filter-sterilized 1 mL/L N6 vitamin, 0.1 mL/L silver nitrate, and 1 mL/L AS. Also add freshly dissolved 0.4 g L-cysteine and 0.154 g DTT in 10 mL ddH2O. 10. PHI-C, resting medium: 4 g/L N6 salts, 30 g/L sucrose, 0.7 g/L L-proline, 0.5 g/L MES, 1.5 mL/L 2,4-D, and 3 g/L Gelrite (Sigma-Aldrich), pH 5.8. After autoclaving, add filter-sterilized 1 mL/L N6 vitamin and 0.1 mL/L silver nitrate. Add freshly dissolved 250 mg/L cefotaxime in 10 mL ddH2O. 11. PHI-D1, selection I medium: 4 g/L N6 salts, 30 g/L sucrose, 0.7 g/L L-proline, 0.5 g/L MES, 1.5 mL/L 2,4-D and 3 g/L Gelrite (Sigma-Aldrich), pH 5.8. After autoclaving, add filtersterilized 1 mL/L N6 vitamin, 0.1 mL/L silver nitrate, and 0.3 mL bialaphos. Also add freshly dissolved 250 mg/L cefotaxime in 10 mL ddH2O. 12. PHI-D2, selection II medium: 4 g/L N6 salts, 30 g/L sucrose, 0.7 g/L L-proline, 0.5 g/L MES, 1.5 mL/L 2,4-D, and 3 g/L Gelrite (Sigma-Aldrich), pH 5.8. After autoclaving, add filter-sterilized 1 mL/L N6 vitamin, 0.1 mL/L silver nitrate, and 0.6 mL bialaphos. Also add freshly dissolved 250 mg/L cefotaxime in 10 mL ddH2O. 13. PHI-E, maturation medium: 4.3 g/L MS salts, 60 g/L sucrose, 3 g/L Gelrite (Sigma-Aldrich), pH 5.6. After autoclaving, add filter-sterilized 1 mL/L MS vitamin, 1 mL/L glycine, and 0.6 mL bialaphos. Also add freshly dissolved 250 mg/L cefotaxime in 10 mL ddH2O. 14. PHI-F, regeneration medium: 2.9 g/L MS salts, 30 g/L sucrose, 3 g/L Gelrite (Sigma-Aldrich), pH 5.6. After autoclaving, add filter-sterilized 1 mL/L MS vitamin and 1 mL/L glycine. 15. Parafilm tape.
3
Methods
3.1 Agrobacterium Culture Initiation
1. Streak EHA101 carrying simple binary vector from −80 °C stock on ABC medium with appropriate antibiotics. Make dilute series so that single colonies can develop. 2. Let culture grow for 3 days at 28 °C in darkness (see Note 5). 3. Pick up single colony and streak it on YEP medium containing appropriate antibiotics, and let culture grow at 20 °C for 3 days in darkness.
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Inoculation
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1. Add 5 mL of sterile PHI-A infection medium into 15 mL Falcon tube. 2. Take about two full loops of EHA101 from YEP plate and suspend in the tube. Let the pallet sit there for 2–3 min. 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. 550 and adjust it to OD550 of 0.35 (0.5 × 109 cfu/mL). 4. Shake the re-suspended culture in a shaker at 100 rpm for 4–5 h. 5. Aliquot 1 mL suspension into 1.5 mL sterile micro-centrifuge tube.
3.3 Embryo Isolation, Inoculation, and Cocultivation
1. Remove the husks and silk from ears which are harvested 10–13 days post pollination (with embryo size of 1.5 mm). Insert the forceps from the top end of the ear (see Note 6). 2. Soak fresh Hi-II ears in a sterile 1 L wide-mouth bottle containing about 0.5 L of 30 % commercial bleach with a few drops of Tween-20 for 20 min (see Note 7). 3. Wash ears three times with plenty of sterile water, and let the ear stand upright on a sterile 150 × 15 mm petri dish. 4. Remove top half of the kernels of each ear with a #11 razor blade (see Note 8). 5. Isolate 1.5 mm immature embryos from sterile ear with sterile spatula, and transfer 50–100 embryos to each tube. Wash the embryos with PHI-A solution three times to remove debris and starch. 6. Add the Agrobacterium suspension, allow the tube to stand for 5 min in the hood, and then pour the whole contents including all embryos onto the petri plate containing PHI-B, the cocultivation medium. 7. Draw off the Agrobacterium suspension using a pipette with a fine tip, spread out the embryos across 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 in the dark at 20 °C 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 the dark at 28 °C for 7 days.
3.5
Selection
1. Transfer the embryos to PHI-D1, selection I medium. Place 25 embryos in each plate, and seal the plate. Incubate the embryos in the dark at 28 °C during the first 2-week selection.
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2. Transfer the calli from PHI-D1, selection I medium, to PHI-D2, selection II medium. Subculture the calli every 2 weeks onto the same fresh medium for a total of 2 months. 3. Bulk up the herbicide-resistant calli by growing them 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, fastgrowing Hi-II embryogenic callus can be divided into smaller calli to select the best-quality Hi-II embryogenic calli (i.e., type II calli which are dry, friable, and fast growing) to enhance selection stringency and/or maintain callus cultures for a prolonged period of time. 3.6
Regeneration
1. Transfer opaque calli onto PHI-E, maturation medium, in the dark at 28 °C for 2–3 weeks to allow the somatic embryos to mature. 2. Transfer ivory white calli onto PHI-F, regeneration medium, at 28 °C under 16-h photoperiod until shoot and roots develop. 3. Transfer each small plantlet to a 25 × 150 mm tube containing PHI-F, regeneration medium, and grow under the same light conditions for 2–3 weeks. 4. Transfer the plants to pots with soil mixture in a greenhouse.
4
Notes 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 immature embryos of high vigor which are capable of tolerating low-salt medium conditions during the Agrobacterium infection stage. We routinely use immature embryos derived from Hi-II A × B: F2 ears. In fact, the Hi-II A × B: F1 ears offer higher embryo vigor than F2 ears, which leads to a much higher transformation frequency (unpublished) than we have reported previously [7]. Nonetheless, use of Hi-II A × B: F1 ears requires more extensive labor to make crosses and more greenhouse space. Consequently, we routinely use Hi-II A × 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 the dark. 4. For dissolving AS, it is recommended to use methanol instead of dimethyl sulfoxide (DMSO) and store at −20 °C. Use of methanol will avoid freeze–thaw steps as encountered with the use of DMSO. This will minimize the reduced potency of AS resulting from freezing and thawing.
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5. Streak EHA101 on ABC medium; the colonies on the agar plate can be used for up to 1 month when stored at 4 °C. 6. The size of immature embryos, i.e., between 1.5 and 1.8 mm, is crucial. We found that as the size of the embryo increases the transformation frequency decreases but the embryos are more tolerant of the low-salt medium condition (unpublished). Therefore, use of small embryos (less than 1.2 mm) requires a full-strength salt medium to avoid 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 method is to use 1.5 mm embryos combined with low-salt medium, achieving a high transformation frequency with ease in embryo isolation. 7. If the maize plants are grown in the field to obtain immature embryos, the use of 50 % commercial bleach is recommended for decontamination. Embryos from the field offer higher transformation frequency than the embryos from the greenhouse. 8. It is recommended that the kernels be removed from two rows at a time. This will help to maintain the desired high moisture and vigor of the embryos in the remaining rows. 9. When the immature embryos are cocultivated on the PHI-B medium, the embryo should be flat face down and be sure to check the orientation of the embryos under a dissecting microscope. References 1. Hiei Y, Ohta S, 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 et al (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745–750 3. Zhao ZY, Gu W, Cai T (1999) Methods for Agrobacterium-mediated transformation. United States Patent 5,981,840 4. Zhao ZY, Gu W, Gai T et al (2001) High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Mol Breed 8:323–333 5. Zhao ZY, Gu W, Cai T et al (2004) Methods for Agrobacterium-mediated transformation. United States Patent 963,096. Pioneer Hi-bred International, Inc., Des Moines, IA 6. Frame BR, Shou H, Chikwamba RK et al (2002) Agrobacterium tumefaciens-mediated transformation of maize embryos using a stan-
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