MAIZE PLASTID TRANSFORMATION METHOD

Abstract

The present invention relates to processes for the transformation of plant tissues with a genetic construct which comprises a transgene and a selection gene. The selection gene preferably encodes an auxin biosynthetic polypeptide, thus allowing for selection of transformed plants on media lacking plant auxins. The invention particularly relates to processes wherein the selection step is carried out under a light/dark cycle.

Claims

1. A process for producing a transformed plant tissue, the process comprising the steps: (i) transforming plant tissue with a genetic construct, wherein the genetic construct comprises a transgene and a selection gene, wherein the selection gene encodes an auxin biosynthetic polypeptide; and (ii) selecting for transformed plant tissue using a light/dark cycle on media which is lacking plant auxin.

2. A process as claimed in claim 1, wherein the process comprises: initiating cell differentiation from a plant tissue; and/or pre-culturing the plant tissue on osmotic medium, prior to the transforming step.

3. A process as claimed in claim 1, wherein the process comprises: a post-transformation recovery interval prior to the selection step.

4. A process as claimed in claim 1, wherein the process comprises: regenerating mature somatic embryos to produce shoots/roots, preferably on media which is lacking plant auxin using a light/dark cycle.

5. A process as claimed in claim 1, wherein the transforming step is carried out using a biolistic transformation method.

6. A process for producing somatic plant embryos, the process comprising the steps: (i) initiating cell differentiation from immature plant embryos on a callusing medium comprising auxin; (ii) pre-culturing the immature plant embryos on an osmotic medium in the dark; (iii) transforming the immature plant embryos with a genetic construct using a biolistic transformation method, wherein the genetic construct comprises a transgene and a gene encoding one or more auxin biosynthetic polypeptides; (iv) optionally culturing the immature plant embryos on a callusing medium; (v) selecting for transformed immature plant embryos on media which is lacking plant auxin on a medium lacking 2,4-D in the dark; and (vi) selecting mature somatic embryos on media which is lacking plant auxin using an optional continuous light cycle and then using a light/dark cycle, wherein the optional continuous light cycle is for about 2-4 days, and the light/dark cycle is approx. 16 hour light/8 hour dark cycle for 2-8 days.

7. A process for producing a transformed plant, the process comprising the steps: (i) initiating cell differentiation from immature plant embryos to produce plant calli on a callusing medium comprising auxin; (ii) pre-culturing the plant calli on an osmotic medium in the dark; (iii) transforming the plant calli with a genetic construct using a biolistic transformation method, wherein the genetic construct comprises a transgene and a gene encoding one or more auxin biosynthetic polypeptides; (iv) optionally culturing the bombarded plant calli on an osmotic medium; (v) selecting for transformed plant calli on media which is lacking plant auxin on a medium lacking 2,4-D in the dark; (vi) selecting plant calli on media which is lacking plant auxin using a light/dark cycle, wherein the light/dark cycle is approx. 16 hour light/8 hour dark cycle for 2-8 days; and (vii) optionally regenerating a transformed plant from the calli.

8. A process as claimed in claim 1, wherein the plant is a monocot or a dicot.

9. A process as claimed in claim 1, wherein the plant is selected from the group consisting of cereals, legumes, oil crops, cash crops, vegetable crops, fruit trees, nut trees, beverages, timber trees, mosses and duckweed.

10. A process as claimed in claim 1, wherein the plant is maize.

11. A process as claimed in claim 9, wherein the plant tissue is a plant embryo or plant callus.

12. A process as claimed in claim 1, wherein the genetic construct is targeted to plastids within the plant tissue.

13. A process as claimed in claim 1, wherein the transgene encodes one or more antibodies, antibiotics, herbicides, vaccine antigens, enzymes, enzyme inhibitors or design peptides.

14. A process as claimed in claim 1, wherein the auxin biosynthetic polypeptide is selected from the group consisting of iaaH/iaaM, AMI1, TAA1, TAR1, TIR2, YUC, AAO1, CYP79B2 and TDC.

15. A process as claimed in claim 1, wherein the process comprises: regenerating mature somatic embryos or plants on selection media using a light/dark cycle.

16. A process for making a transgene product, the process comprising a process for producing a transformed plant tissue as claimed in claim 1, and additionally comprising purifying the transgene product from the regenerated plant.

17. A transgene product obtained or obtainable by a process as claimed in claim 1.

18. A process as claimed in claim 2, wherein the process comprises: a post-transformation recovery interval prior to the selection step.

19. A process as claimed in claim 2, wherein the process comprises: regenerating mature somatic embryos to produce shoots/roots, preferably on media which is lacking plant auxin using a light/dark cycle.

20. A process as claimed in claim 3, wherein the process comprises: regenerating mature somatic embryos to produce shoots/roots, preferably on media which is lacking plant auxin using a light/dark cycle.

21. A process as claimed in claim 18, wherein the process comprises: regenerating mature somatic embryos to produce shoots/roots, preferably on media which is lacking plant auxin using a light/dark cycle.

22. A process as claimed in claim 2, wherein the transforming step is carried out using a biolistic transformation method.

23. A process as claimed in claim 3, wherein the transforming step is carried out using a biolistic transformation method.

24. A process as claimed in claim 18, wherein the transforming step is carried out using a biolistic transformation method.

25. A process as claimed in claim 4, wherein the transforming step is carried out using a biolistic transformation method.

26. A process as claimed in claim 19, wherein the transforming step is carried out using a biolistic transformation method.

27. A process as claimed in claim 21, wherein the transforming step is carried out using a biolistic transformation method.

28. A process as claimed in claim 6, wherein the plant is a monocot or a dicot.

29. A process as claimed in claim 7, wherein the plant is a monocot or a dicot.

30. A process as claimed in claim 6, wherein the plant is selected from the group consisting of cereals, legumes, oil crops, cash crops, vegetable crops, fruit trees, nut trees, beverages, timber trees, mosses and duckweed.

31. A process as claimed in claim 7, wherein the plant is selected from the group consisting of cereals, legumes, oil crops, cash crops, vegetable crops, fruit trees, nut trees, beverages, timber trees, mosses and duckweed.

32. A process as claimed in claim 30, wherein the plant tissue is a plant embryo or plant callus.

33. A process as claimed in claim 31, wherein the plant tissue is a plant embryo or plant callus.

34. A process as claimed in claim 10, wherein the plant tissue is a plant embryo or plant callus.

35. A process as claimed in claim 6, wherein the genetic construct is targeted to plastids within the plant tissue.

36. A process as claimed in claim 7, wherein the genetic construct is targeted to plastids within the plant tissue.

37. A process as claimed in claim 6, wherein the transgene encodes one or more antibodies, antibiotics, herbicides, vaccine antigens, enzymes, enzyme inhibitors or design peptides.

38. A process as claimed in claim 7, wherein the transgene encodes one or more antibodies, antibiotics, herbicides, vaccine antigens, enzymes, enzyme inhibitors or design peptides.

39. A process as claimed in claim 6, wherein the auxin biosynthetic polypeptide is selected from the group consisting of iaaH/iaaM, AMI1, TAA1, TAR1, TIR2, YUC, AAO1, CYP79B2 and TDC.

40. A process as claimed in claim 7, wherein the auxin biosynthetic polypeptide is selected from the group consisting of iaaH/iaaM, AMI1, TAA1, TAR1, TIR2, YUC, AAO1, CYP79B2 and TDC.

41. A process as claimed in claim 6, wherein the process comprises: regenerating mature somatic embryos or plants on selection media using a light/dark cycle.

42. A process as claimed in claim 7, wherein the process comprises: regenerating mature somatic embryos or plants on selection media using a light/dark cycle.

43. A process for making a transgene product, the process comprising a process for producing a transformed plant tissue as claimed in claim 6, and additionally comprising purifying the transgene product from the regenerated plant.

44. A process for making a transgene product, the process comprising a process for producing a transformed plant tissue as claimed in claim 7, and additionally comprising purifying the transgene product from the regenerated plant.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0255] FIG. 1 shows schematic diagrams of the targeting region in the maize plastid genome and the resulting transplastome following integration of the pAD001 transgene cassette. The transgenes are targeted to the region between the 5rps12 and clpP genes. Construction of the plastid transformation vector pAD001: 5rps12 homologous recombination sequence (nt 68231-69454, accession NC_001666.2 Zea mays chloroplast genome DNA); PrrnT7g10L: Plastidic ribosomal RNA (rrn) operon promoter (nt 139983-14065, accession Z00044 Nicotiana tabacum chloroplast genome DNA) fused to the leader sequence of bacteriophage T7 gene 10; iaaM gene from Agrobacterium tumefaciens; IEE, putative processing element (Zhou, F., Karcher. D. and Bock., R. Identification of a plastid intercistronic expression element (IEE) facilitating the expression of stable translatable monocistronic mRNAs from operons. The Plant Journal (2007) 52, 961-972); iaaH gene from Agrobacterium tumefaciens; TpsbA: psbA polyA addition sequence (nt 141-536, accession Z00044 Nicotiana tabacum chloroplast genome DNA); clpP; homologous recombination sequence (nt 69455-71184, accession NC_001666.2 Zea mays chloroplast genome DNA).

[0256] FIG. 2 shows images of regeneration in maize following bombardment of immature maize embryos. (A) Immature maize embryos 1 day post bombardment with pAD001 construct. (B) Immature maize embryos 7 days post bombardment on selection medium () 2,4-D in the dark. A number of immature maize embryos remain white and continue to proliferate and grow as callus. Browning of other immature maize embryos signifies death. (C) Green calli (red circles) visible 2 to 3 weeks post bombardment on selection medium following the introduction of light. (D) Immature maize embryos on the control plate 2 weeks post bombardment on selection medium.

[0257] FIG. 3 shows images of regeneration in maize following bombardment of immature maize embryos. (A) Following the introduction of light, green calli is visible 2 weeks post bombardment of immature maize embryos with pAD001 construct. (B) Immature maize embryos bombarded with an empty vector (control) turn brown and die 2 weeks post bombardment.

[0258] FIG. 4 shows images of regeneration in maize following bombardment of callus derived from immature maize embryos. (A) Calli derived from immature maize embryos 1 day post bombardment with pAD001 construct on selection media () 2,4-D. (B) Green calli is visible 4 weeks post bombardment following 4 weeks incubation in the dark and 3 days under a 16/8 hr light dark cycle. (C) Green calli approximately 4 weeks following bombardment with pAD001 construct.

[0259] FIG. 5 shows PCR analysis confirming the presence of the iaaM-iaaH transgenes. (A) Schematic diagram showing the approximate annealing position of the iaaM-F and iaaH-R primers used to confirm the presence of the iaaM-iaaH transgenes. Amplification of a PCR product confirmed the presence of the iaaM-iaaH transgenes in putative transformed calli derived from immature maize embryos (B) and in putative transformed immature maize embryos (C).

[0260] FIG. 6 shows PCR analysis confirming the correct integration of the pAD001 transformation vector into the left homologous recombination border region of the maize plastome. (A) Schematic diagram showing the approximate annealing position of the Ext-F and iaaM-R primers used to confirm the correct integration of the pAD001 transformation vector into the left homologous recombination border region of the maize plastome. (B) Amplification of a PCR product confirmed the correct integration of the pAD001 transformation vector in putative transformed calli derived from immature maize embryos.

EXAMPLES

[0261] The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

[0262] Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0263] The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.

Example 1

[0264] The plastid transformation vector pAD001 was constructed as detailed in FIG. 1 using a 1223 bp homologous recombination sequence (nt 68231-69454) and a 1729 bp homologous recombination sequence (nt 69455-71184) from the chloroplast genome from Zea mays (Maier, R. M., Neckermann, K., Igloi, G. L. and Kossel, H. (1995). Complete sequence of the maize chloroplast genome: gene content, hotspots of divergence and fine tuning of genetic information by transcript editing. J. Mol. Biol. 251 (5), 614-628 (1995)) on either side of the gene cassette.

[0265] For proof-of-principle purposes, the iaaM-iaaH transgene cassette was cloned in between the homologous recombination sequences to generate pAD001 (FIG. 1). This vector was then bombarded into both immature maize embryos and maize callus as detailed in Appendix 1. For the transformation of immature maize embryos, ears were collected 10-13 days after pollination from greenhouse grown Hi II plants (A188B73 origin, Armstrong, C. L., Green, C. E., and Phillips, R. L. (1991) Development and availability of germplasm with high Type II culture formation response. Maize Genetics Coop Newsletter 65: 92-93.). Ears were sterilized in 30-50% commercial bleach containing 3 drops of Tween 20 for 30 minutes and washed in sterilized water three times. Immature zygotic embryos were excised from the ears and placed embryo axis side down on N6E callus initiation medium (containing 2,4D) for 2 days in the dark as scutellum-derived callus is most likely the producer of transgenic events. For the transformation of Hi II Type II callus, ears were sterilized as described previously. Immature maize embryos were excised and placed on N6E callus initiation medium (containing 2,4D) for six to eight weeks in the dark.

[0266] The pAD001 construct was transformed into plastids using the protocol shown in Appendix 1 followed by auxin mediated selection and regeneration (FIGS. 2A, 2B, 3A, 3B, 4A and 4B). PCR verification of iaaM-iaaH presence in maize cells was carried out on genomic DNA prepared from regenerated maize calli (FIG. 2C, 4C) and the primers iaaM-F and iaaH-R, which span the iaaM-iaaH junction in the transgene cassette (FIG. 5A). FIG. 5B and FIG. 5C show the presence of the iaaM-iaaH transgene in putative transgenic callus derived from immature maize embryos and immature maize embryos respectively. To determine whether plastid integration had occurred, PCR analysis was carried out using the primers Ext-F (which anneals to sequence external to the homologous recombination sequence on the transformation sequence) and iaaM-R, which anneals internal to the iaaM transgene (FIG. 6A). FIG. 6B shows the correct integration of the transformation vector into the plastid genome on the left homologous recombination side in callus derived from immature maize embryos.

Example 2: Expression of Proteins Conferring Abiotic Stress Resistance

[0267] Abiotic stresses such as drought, salinity and temperature can be very detrimental to plants because of their sessile existence and can result in severe reduction in crop yields worldwide. The described system allows for the introduction and selection of transgenes, which can confer tolerance to abiotic stresses, in the maize plastid genome.

[0268] Transgenes e.g. the betaine aldehyde dehydrogenase gene, which confers tolerance to salinity and trehalose phosphate synthase, which confers drought tolerance, can be inserted into the pAD001 vector (FIG. 1) between the PrrnT7g10L and the TpsbA sequence. The construct is transformed into plastids using the protocol shown in Appendix 1 followed by auxin mediated selection and regeneration. The use of this system allows for a high level of transgene containment as plastids are predominantly maternally inherited in most crops. Maternal inheritance stops the escape of plastid genes and transgenes by pollen transmission, which is a significant advantage over nuclear transformation. In addition, environmental as well as health concerns in relation to the integration of antibiotic resistance genes in transformed plants is eliminated as this selection system does not contain an antibiotic selectable marker resulting in improved safety.

Example 3: Expression of Proteins Conferring Biotic Stress Resistance

[0269] Biotic stresses such as bacterial, viral and fungal pathogens in addition to weeds and pests affect crop yields yearly and can result in significant financial losses to both farmers and industry alike.

[0270] As described in Example 2, biotic stress resistant transgenes e.g. B. thuringiensis (Bt) cry1A(c), may be inserted into the pAD001 vector followed by transformation, selection and regeneration as described previously.

Example 4: Expression of Proteins Conferring Both Abiotic and Biotic Stress Resistance

[0271] A major limitation that both farmers and scientists face in crop production worldwide is the loss of up to 30-60% crop yield each year due to a combination of both biotic and abiotic stresses (Dhlamini. Z., Spillane. C., Moss. J P., Ruane. J., Urquia. N., Sonnino. A., (2005). Status of research and applications of crop biotechnologies in developing countries: Preliminary assessment, Roma, Food and Agriculture Organization of the United Nations [ISBN 92-5-105290-5]). As described in Examples 2 and 3, a combination of transgenes conferring both abiotic and biotic stress resistance may be inserted into the pAD001 vector, followed by transformation, selection and regeneration as previously described.

Example 5: Removal of the Auxin Biosynthetic Genes Post Selection

[0272] An increase in auxin due to the integration of the iaaM-iaaH transgene cassette may alter the growth characteristics of transformed plant species. To avoid this issue, the system described in Example 1 can be combined with a system for eviction such as a RIRS system from Zygosaccharomyces rouxii, Flp/frt from Saccharomyces cerevisiae, and Gin/gix from bacteriophage Mu removing the iaaM-iaaH transgene cassette and thus eliminating the problem.

Example 6: Generation of Whole Transformed Plants

[0273] The generation of whole transformed plants may be achieved by the following protocol. Transformation vectors containing the iaaM-iaaH gene cassette as a selectable marker are constructed and then bombarding into maize tissue as described above. Following the selection and confirmation of putative transformed calli, as described above, shoot regeneration could then be achieved using a cocktail of plant growth regulators (e.g. cytokinins etc.) to promote organogenesis. Alternatively, an antibiotic resistant gene may be incorporated in addition to the auxin genes (iaaM-iaaH) and the use of a two step selection system, first utilizing the iaaM-iaaH gene cassette for initial selection of transformants in the dark and secondly utilizing the antibiotic resistance gene once the calli are moved into the light.

APPENDIX 1

Protocol for Chloroplast Transformation

Time Course

[0274] The standard procedures produce transformed plants in 3-5 months.

Equipment Set Up

Helium Gun Bio-Rad PDS 1000

[0275] Rupture disk PSI: 900 [0276] Gap between rupture disk retaining cap and macrocarrier over cover lid: [0277] Spacer rings below stopping screen support: 2 [0278] Level of macrocarrier launch assembly: 1 (from top) [0279] Level of Petri dish holder: 3 (from top) [0280] Vacuum inflow rate: Maximum [0281] Vacuum release rate: attenuate the release so it approximates the speed of vacuum inflow.

Stock Solutions

[0282] 2.5 M CaCl.sub.2 filter sterilized [0283] 0.1 M Spermidine Free Base in sterilized H.sub.2O [0284] dH.sub.2O [0285] DNA at 1 g/l in dH.sub.2O or 1 TE [0286] 100% Ethanol [0287] 70% Isopropanol

Consumables

[0288] 900 PSI rupture disks [0289] Stopping screens [0290] Macrocarriers [0291] Gold particles

Preparation of the DNA-Gold Particle Mix

[0292] 50 mg of gold particles are suspended in 1 ml of 100% ethanol as stock [0293] Take 0.25 ml of Gold stock suspension and centrifuge for 5 seconds. Remove ethanol and wash three times with sterile distilled H.sub.2O, centrifuging 3 minutes between washings. [0294] Resuspend Gold in 0.25 ml dH.sub.2O. [0295] Aliquot 50 l of Gold-H.sub.2O suspension into Eppendorf tubes. [0296] Into each Eppendorf tube add the following in succession: [0297] 10 l DNA at 1 g/l [0298] 50 l of 2.5 M CaCl.sub.2 [0299] 20 l of 0.1 M Spermidine free base [0300] Vortex for 5 minutes at highest speed. [0301] Add 200 l of 100% ethanol to each tube. [0302] Centrifuge at 3000 rpm for 10 seconds. [0303] Remove as much supernatant as possible and rinse pellet in 100% ethanol once, centrifuging at 3000 rpm for 10 seconds. [0304] Resuspend pellet in 30 l 100% ethanol (makes 4-5 shots). Store mixture on ice.

Preparing the Biolistic Gun and Consumables

[0305] Sterilize the gun vacuum chamber and surfaces with 70% ethanol. [0306] Sterilize the stopping screens and macrocarrier holders by autoclaving. [0307] Sterilize the rupture disks in 70% isopropanol. [0308] Sterilize the macrocarriers in 100% ethanol. Air dry in hood. [0309] Open helium tank. Set the helium tank regulator to 1100 psi (or 200 psi above the rating of the rupture disk.

Bombardment

[0310] Particle bombardment was carried out using a biolistic PDS-1000/He gun (Bio-Rad). [0311] Place sterile macrocarriers into the macrocarrier holders. [0312] Pipet 5 l of vortexed gold/DNA mixture onto the center of each sterile macrocarrier and leave at room temperature for 10 minutes. [0313] Insert a sterile rupture disc into the recess of the retaining cap and tightly screw onto the gas acceleration tube. [0314] Place a sterile stopping screen on the support and install the macrocarrier holder on the rim of the fixed nest. [0315] Screw the macrocarrier lid onto the assembly and place the macrocarrier launch assembly in the top slot inside the bombardment chamber. [0316] Place the target shelf at the desired distance, 6 cm from the macro-projectile stopping screen (three from top) and place the Petri dish containing the target tissue on it. [0317] Open the helium tank to 1100 psi (200 psi greater than the capacity of the rupture disc). [0318] Close the door of the gene gun; evacuate the chamber to 28 Hg (inches of mercury) and hold at this vacuum. [0319] Press the fire button and release once the rupture disc has burst. [0320] Vent the chamber, remove the Petri dish and repeat the procedure for subsequent shots. [0321] At the end of the experiment, turn off the helium tank. Pull a vacuum in the gun to release the remaining helium through the gun and then turn off the helium regulator.
The key to successful bombardment is usually in the spread of particles on the macrocarrier. The gold-DNA mixture should be spread evenly over the center of the macrocarrier. The resulting spread should be void of any clumps, which can result in an increased frequency of cell death. Each 30 l gold-DNA mix usually gives 4-5 bombardments.

Maize Preparation and Regeneration Media

N6E (Callus Initiation):

[0322] 4 g/L N6 salts (Chu et al., 1975)
1 ml/L (1000) N6 vitamin stock
2 mg/L 2,4-D
100 mg/L myo-inositol
2.76 g/L proline
30 g/L sucrose
100 mg/L casein hydrolysate
2.5 g/L agar,
20 pH 5.8 and autoclave
Silver nitrate (25 M) added after autoclaving.

N6OSM (Osmotic Medium):

[0323] 4 g/L N6 salts
1 ml/L N6 vitamin stock
2 mg/L 2,4-D
100 mg/L myo-inositol
0.69 g/L proline
30 g/L sucrose
100 mg/L casein hydrolysate
36.4 g/L sorbitol
36.4 g/L mannitol (Vain et al, 1993)
2.5 g/L agar
pH 5.8 and autoclave.
Silver nitrate (25 M) added after autoclaving.

N6S (Selection Minus Auxin (2,4-D)):

[0324] 4 g/L N6 salts
1 ml/L N6 vitamin stock
100 mg/L myo-inositol
30 g/L sucrose
2.5 g/L agar
pH 5.8 and autoclave.
Silver nitrate (25 M) added after autoclaving.

Tissue Culture

Pre-Bombardment

[0325] Dehusk ear. Cut off and discard top 1 cm of ear. Place ear into a clean beaker. [0326] Add 700 ml of 70% ethanol (or enough to cover ear), swirl for 1-2 minutes and discard the ethanol. Wash the ear three times with sterile distilled H.sub.2O. [0327] Add 700 ml sterilizing solution (30-50% commercial bleach in water and 3 drops of Tween 20) to cover ear. Throughout the 30 minute disinfection, swirl the ears to dislodge air bubbles for thorough surface sterilization of ear. [0328] Pour off the bleach solution, rinse the ears three times in sterile distilled H.sub.2O and the ears are ready for embryo dissection. [0329] In a large (15015 mm) sterile petri-plate, cut off the kernel crowns (the top 1-2 mm) with a sharp scalpel blade. Re-sterilize the forceps and scalpels intermittently throughout this protocol to avoid contamination. [0330] Excise the embryos by inserting the narrow end of a narrow pointed forceps between the endosperm and pericarp at the basipetal side of the kernel. The embryo is gently coaxed onto the tip of the forceps and plated with the embryo-axis side down (scutellum side up) onto the N6E media (approximately 30 embryos/plate). [0331] Wrap the plate with vent tape and incubate for 2 or 3 days (if transforming immature maize embryos) or 6-8 weeks (if transforming callus derived from immature maize embryos) at 28 C. in the dark. [0332] Four hours prior to bombardment, use sterile forceps to transfer the embryos or calli onto the osmotic medium (N6OSM), (Vain et al., 1993). Center the embryos or calli in the center of the plate. Embryos should be facing scutellum side up at bombardment since it is from this surface that subsequent callus initiation begins and from which transformed cells are then selected.

Post Bombardment

[0333] The bombarded embryos or calli (still on N6OSM) are gently wrapped with vent tape and incubated in 28 C. in the dark.

Selection for Stable Transformed Events (Immature Maize Embryos)

[0334] The next day (16-20 hours after bombardment), embryos are transferred off the N6OSM and onto N6E media to continue callus initiation. Embryos are again oriented scutellum side up and plates are wrapped with vent tape. [0335] After 7 days, embryos are transferred to selection medium (lacking 2,4D) and placed in the dark at 28 degrees for a further 7 days. [0336] Embryo selection plates are then placed under continuous light for 3 days, followed by 16/8 hour light/dark cycle for a further 6 days. [0337] Green calli are visible 3 days after being placed in light (2 weeks post bombardment).
Selection for Stable Transformed Events (Callus Derived from Immature Maize Embryos) [0338] 24 hours post bombardment; Calli are transferred to selection medium (without 2,4-D) and placed in the dark at 28 C. for 7 days. [0339] Four weeks post bombardment; plates removed from the dark and placed under a 16/8 hour light/dark cycle at 28 C. Green calli begin to appear 3 days later (approximately 4.5 weeks post bombardment).

Regeneration Protocol

[0340] A standard protocol is used as for nuclear transformation.