The University of Western Ontario
Transient expression of B-peru gene in bombarded (PDS-2000) immature shoot apical meristems
--V. R. Bommineni, D. B. Walden and J.C. Sanford*
*New York State Agricultural Research Experiment Station, Cornell University, Geneva, New York
A helium gas driven device (PDS-2000, 'wand' design) was used to transform immature shoot apical meristems (MNL 63: 87-88; Plant Cell Tissue Organ Culture 19:225-234, 1989) with a dominant anthocyanin B-peru gene color marker (gene construct was provided by Dr. Vicki L. Chandler, University of Oregon).
Shoot apical meristems were dissected from 12-14 days old pollinated ears of a C R b pl, and yg2 c sh wx b pl genotypes and arranged in a circle on the agar plate in such a way that the apical dome of the meristem was facing towards the microprojectile source. The procedure for preparation of DNA was described by Klein et al. (Bio/Technology 6:339-563, 1988) with the following modifications. Mix and vortex all the components (gold particles, DNA, CaCl2, and spermidine) and leave the solution in the microtubes for 5 min. Decant the supernatant and rinse the particle mixture with 140 µl of 70% EtOH and pulse vortex, decant the supernatant and rinse with 140 µl of 100% EtOH. Pulse vortex again, decant the supernatant and resuspend the particles with 48 µl of 100% EtOH. Sonicate the mixture three times at one second intervals. Place 5 µl of suspended microprojectile solution on flying disc (kapton membrane) in a ring, and set them in a small dessicator in the culture hood until use. The design and operation of the gun was described by Johnston (Nature 346: 776-777, 1990).
A summary of the transient expression (96 h) of B-peru gene in bombarded shoot apical meristems is provided in Table 1. Many of the bombarded explants expressed the B-peru gene. A larger number of spots was observed in the meristems bombarded once (1x) on the lowest (1st) shelf than on the 3rd shelf; however, bombardment three times produced a fewer number of spots in the meristems located on the 1st shelf than on the 3rd shelf. The pattern of B-peru gene expression was consistent among the meristematic portions (scutellum, plumule or leaf, and radicle). A similar pattern was observed between the two genotypes, but approximately four times more expression was observed with 10 µg/µl DNA (yg2 c sh wx b pl) than 2 µg/µl DNA (a C R b pl) in the microprojectile solution.
Table 1. Transient expression (96h) of B-peru gene in bombarded* immature apical meristems.
No. DNA Mean number of brown spots ( + S.E.)**
| Genotype and
treatment** |
(N) |
# of
spots |
Number of
explants bombarded |
Number of
explants expressed B-peru gene |
S |
P |
R |
Mean total
# of spots ( + S.E.) |
| a C R b pl (2mg/ml) | ||||||||
| 1x | ||||||||
| 1st shelf | 10 | 0 | 32 | 30 | 16.3(2.5) | 7.6(1.1) | 5.4(0.7) | 29.3(3.5) |
| 2nd shelf | 10 | 0 | 41 | 31 | 3.0(0.8) | 1.8(0.4) | 1.6(0.4) | 6.4(1.0) |
| 3rd shelf | 11 | 0 | 19 | 11 | 4.5(1.9) | 2.2(0.6) | 2.5(1.5) | 9.2(3.0) |
| Total | 31 | 0 | 92 | 72 | 8.8(1.4) | 4.3(0.6) | 3.3(0.5) | 16.4(2.1) |
| 3x | ||||||||
| 1st shelf | 9 | 0 | 18 | 17 | 6.9(1.6) | 3.5(0.3) | 4.4(0.8) | 14.8(2.2) |
| 2nd shelf | 12 | 0 | 13 | 12 | 8.8(2.3) | 6.6(1.2) | 6.8(1.7) | 22.2(4.3) |
| 3rd shelf | 9 | 0 | 22 | 19 | 11.6(2.3) | 7.1(2.3) | 8.1(1.4) | 26.8(4.2) |
| Total | 30 | 0 | 53 | 48 | 9.3(1.3) | 5.7(1.0) | 6.4(0.8) | 21.4(2.3) |
| yg c sh wx b pl (10mg/ml) | ||||||||
| 1x | ||||||||
| 1st shelf | 15 | 0 | 18 | 17 | 22.2(4.5) | 38.4(8.1) | 43.4(6.7) | 104.0(14.3) |
| 2nd shelf | 14 | 0 | 21 | 19 | 12.9(2.4) | 6.3(2.0) | 23.4(5.7) | 44.6(9.1) |
| 3rd shelf | 15 | 0 | 18 | 15 | 4.8(2.9) | 3.4(1.5) | 13.5(9.0) | 21.7(13.1) |
| Total | 44 | 0 | 57 | 51 | 13.6(2.2) | 16.1(3.6) | 27.3(4.5) | 57.0(8.5) |
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