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Manish N. Raizada and Virginia Walbot Dept. of Biological Sciences, Stanford University, Stanford, CA 94305-5020 This is a detailed procedure of the biolistic transformation of maize embryogenic callus as first described by Fromm et al. (1990) and Gordon-Kamm et al., (1990) and adapted by us. pAHC20 – The ubiquitin promoter- bar herbicide resistance plasmid, pAHC20, is kindly provided by Peter Quail (PGEC, Albany, CA) (Christensen and Quail, 1996). The bar gene encodes resistance to the herbicide Basta® or bialaphos (De Block et al., 1987). Establishment of Embryogenic Callus Cultures A188 X B73 (HiII) seed (Armstrong and Green, 1985) is obtained from Monsanto (Chesterfield, MO) and sib- or self-pollinated. Nine to thirteen days after pollination, 1-2 mm embryos are dissected in the dark, and callus is induced on N6 1-100-25 media (Armstrong, 1994) containing N6 salts+ vitamins, 1 mg/L 2,4-D, 100 mg/L casamino acids, 25 mM proline, 20 g/L sucrose, 2 g/L Phytagel (Sigma) and 10 µM AgNO3 (Armstrong and Green, 1985; Songstad et al., 1991). Calli are maintained in complete darkness at 27ºC, 75% RH in 100 x 25 mm Petri dishes during the entire callus induction, bombardment and herbicide selection procedures. Following 4 weeks on induction media with one subculture, calli are transferred to media lacking AgNO3, and maintained for up to 3 months, with a 2 week subculture regime, selecting for white, embryogenic, friable tissues at each subculture. Every 3 months, new cultures are initiated. Bombardment Plasmid and double-stranded M13 DNA are isolated as supercoiled DNA using a Wizard Maxiprep Kit (Promega), then extracted once with phenol:chloroform and ethanol precipitated. DNA is precipitated onto 1µM spherical gold particles (Alameda Scientific Instruments, ASI) and accelerated onto immature somatic embryoid tissue using a Helium PDS 1000HE device (BioRad) (see Sanford et al., 1993) following the procedure of Wan et al., 1994. Equimolar quantities of all plasmids are coprecipitated. All DNA precipitation and bombardment steps are performed under sterile conditions at room temperature. DNA precipitation for three bombardments uses 2 mg of gold resuspended in ethanol; gold is centrifuged in a Beckman TJ-6 swinging bucket rotor at 2000 rpm in an Eppendorf tube. The pellet is rinsed once in distilled water, recentrifuged, and resuspended in 25 µL of 1 µg/µL total DNA. In between the addition of each of the following reagents, the tube is briefly vortexed: 220 µL H20, 250 µL 2.5M CaCl2, 50µL 0.1M spermidine, free base (Sigma or Aldrich). Fresh spermidine is found to be the most critical reagent in this procedure. The precipitating DNA mix is then placed on ice for 5 min. Each tube is then vortexed for 1-2 min at room temperature, and centrifuged at 500 rpm for 5 min in a Beckman TJ-6 centrifuge. The supernatant is removed and the pellet is resuspended in 600 µL ethanol, and centrifuged for 1 min on a table-top microfuge at 14,000 rpm. The final pellet is resuspended in 36 µL of ethanol and used immediately or stored on ice for up to 4 hr prior to bombardment. All steps involving aqueous solutions are performed efficiently to prevent agglomerization of the DNA (Sanford et al., 1993). For bombardment, tissues are used 5-9 days following subculture. Four to six hours prior to bombardment, the best embryogenic, friable calli clumps growing rapidly on the surface of callus cultures are gently removed as ~5 mm diameter clumps and crowded together (embryoids facing up) in a ~3x3 cm area on the surface of a Baxter S/P Filter, Grade 363 (5.5 cm) filter, and placed on osmotic induction media (N6 1-100-25, 0.2 M mannitol, 0.2 M sorbitol [Armstrong, 1994; Kemper et al., 1996; Vain et al., 1993]) at 27ºC in darkness. Ten plates are prepared for each transgenic line, along with an additional plate that is bombarded with plasmids encoding the anthocyanin regulators pR and pC1 (Ludwig et al., 1990) as a positive control for the DNA precipitation and bombardment procedures. Red spots are scored 16-40 hr after bombardment. A few minutes prior to shooting, filters are removed from the medium and placed onto sterile opened Petri dishes to allow the calli surface to partially dry. Ten µL of the 36 µL gold-DNA-ethanol solution are spread onto the surface of a macrocarrier (ASI), briefly dried, and accelerated in a vacuum of 27 psi against a wire mesh screen (ASI). Each plate is typically shot once with a 650 psi disc and a second time with a 1100 psi disc that has been briefly soaked in isopropanol to promote a good seal to the rupture disk holder. The distance from the rupture disc to the macrocarrier is 1.0 cm and from the mesh screen to the target, 5.9 cm. If gold residue is not seen on the surface of the calli, they are bombarded a third time. Following bombardment, the filters holding the calli are transferred back onto osmotic media and incubated for 16 hr in darkness at 27ºC. The filters are then transferred onto callus maintenance media N6 1-100-25 (no herbicide) for 1-2 days to promote tissue recovery prior to herbicide selection. Herbicide Selection Each callus is removed from the bombardment filters and placed onto herbicide selection media: N6 2-0-0 (N6 salts + vitamins, 2 mg/mL 2,4-D, no proline or casamino acids) containing 3 mg/mL bialaphos (Meiji Seika Kaisha Ltd, Japan) (Spencer et al., 1990; Denney et al., 1994). All tissues are nonselectively subcultured without breaking every 12 days for 10-12 weeks. After the first subculture, the tissue is partially flattened on the media to promote direct contact with the herbicide. After 6-8 weeks on herbicide, white, fast-growing sectors can be detected growing out of the nonproliferating and partially necrotic mother calli. These resistant sectors are permitted to grow to a diameter of 1 cm, and then they are selected and subcultured onto fresh herbicide media. Resistant calli are permitted to proliferate to occupy an entire plate, and are subcultured every two weeks, during which embryogenic sectors are aggressively chosen. If necessary, 0.4 M sorbitol are added to the media for 1-5 days to promote the induction of embryogenic sectors. Individual resistant callus lines (designated cA1, cA2, cA3, etc.) are checked by RNA gel blot hybridization for the presence of transgene expression and selectively regenerated. Regeneration Regeneration is performed following the procedure of Armstrong (1994). Embryogenic calli are placed in the dark onto Regeneration 1 media for two weeks. This media contains MS salts + vitamins (Sigma), 2% sucrose, 0.1 mg/mL 2,4-D and 0.1 µM ABA to promote the formation of somatic embryos, and 1 mg/mL bialaphos. Embryogenic calli are then transferred to Regeneration 2 media and maintained in the dark for 2 weeks. Regeneration 2 media contains N6 salts + vitamins, 1 mg/mL bialaphos and 6% sucrose to promote embryo enlargement and maturation. Differentiated, white somatic embryoids are then placed onto Regeneration 3 (MS salts + vitamins, 2% sucrose, 1 mg/mL bialaphos, no hormones) media <2 weeks in 100 mm x 25 mm Petri dishes to promote seedling germination; conditions are16 hr day (70 µMol m-2s-1) and 8 hr night at 25-27ºC. Immature seedlings are transferred onto the same media in baby jars, and transferred to soil after substantial root development. Five or more plants from each regenerated line are transferred to the greenhouse. Greenhouse Hardening Off Greenhouse conditions are 16 hr day, 8 hr night, 28ºC day and 22ºC night, with a mixture of 1000W sodium vapor and metal halide lamps. Seedlings are transferred into 15 cm round pots into loose soil containing peat moss and perlite (Sunshine Mix or Metromix 350) with Nutricote 13-13-13 slow release fertilizer, and maintained in a high humidity environment under individual 1 pint supermarket plastic cups for 3 days at 225-500 µMol m-2 s-1, or summer partial shade sunlight. After hardening off, the seedlings are maintained in full summer sunlight or ~1600 µMol m-2s-1 greenhouse light and transferred to potting soil. Plants are outcrossed to promote vigorous kernel development. The initial regenerated plants are called T0 while the first seed belongs to the T1 generation. Leaf Herbicide Test To test for bialaphos resistance, a 5 cm diameter marked leaf surface is painted with 0.75% glufosinate ammonium (Ignite 600, 50% solution, Hoescht, Canada) with 0.1% Tween 20 using a Q-tip. The area is visually scored for the presence or absence of necrosis 5-7 days later.
References Armstrong, C.L., and Green, C.E. (1985). Establishment and maintenance of friable, embryogenic maize callus and the involvement of L-proline. Planta 164, 207-214 Armstrong, C.L. (1994). Regeneration of plants from somatic cell cultures: Applications for in vitro genetic manipulation. In The Maize Handbook, M. Freeling and V. Walbot, eds (New York: Springer-Verlag), pp. 663-671. Christensen, A.H., and Quail, P.H. (1996). Ubiquitin promoter-based vectors for high level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res. 5, 213-218. De Block, M., Botterman, J., Vandewiele, M., Dockx, J., Thoen, C., Gossele, V., Rao Movva, N., Thompson, C., Van Montagu, M., and Leemans, J. (1987). Engineering herbicide resistance in plants by expression of a detoxifying enzyme. EMBO J. 6, 2513-2518. Denney, B.K., Petersen, W.L., Ford-Santino, C., Pajeau, M., and Armstrong, C.L. (1994). Comparison of selective agents for use with the selectable marker gene bar in maize transformation. Plant Cell, Tissue Organ Culture 36, 1-7. Fromm, M.E., Morrish, F., Armstrong, C., Williams, R., Thomas, J., and Klein, T.M. (1990). Inheritance and expression of chimeric genes in the progeny of transgenic maize plants. Bio/Technology 8, 833-839. Gordon-Kamm, W.J., Spencer, T.M, Mangano, M.L., Adams, T.R., Daines, R.J., Start, W.G., O’Brien, J.V., Chambers, S.A., Adams, W.R., Jr., Willetts, N.G., Rice, T.B., Mackey, C.J., Krueger, R.W., Kausch, A.P., and Lemaux, P.G. (1990). Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2, 603-618. Kemper, E.L., da Silva, M.J., and Arruda, P. (1996). Effect of microprojectile bombardment parameters and osmotic treatment on particle penetration and tissue damage in transiently transformed cultured immature maize (Zea mays L.) embryos. Plant Science 121, 85-93. Ludwig, S.E., Bowen, B., Beach, L., and Wessler, S.R. (1990). A regulatory gene as a novel visible marker for maize transformation. Science 247, 449-450. Sanford, J.C., Smith, F.D., and Russell, J.A. (1993). Optimizing the biolistic process for different biological applications. Methods in Enzymology 217, 483-509. Songstad, D.D., Armstrong, C.L., and Petersen, W.L. (1991). AgNO3 increases Type II callus production from immature embryos of maize inbred B73 and its derivatives. Plant Cell Rep. 9, 699-702. Spencer, T.M, Gordon-Kamm, W.J., Daines, R.J., Start, W.G., and Lemaux, P.G. (1990). Bialaphos selection of stable transformants from maize cell culture. Theor. Appl. Genet. 79, 625-631. Vain, P., McMullen, M.D., and Finer, J.J. (1993). Osmotic teatment enhances particle bombardment-mediated transient and stable transformation of maize. Plant Cell Rep. 12, 84-88. Wan, Y.C., Widholm, J.M., and Lemaux, P.G. (1994). Type I callus as a bombardment target for generating fertile, transgenic maize (Zea mays L.). Planta 196, 7-14.
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