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.)**
# of spots
( + S.E.)
|a C R b pl (2mg/ml)|
|yg c sh wx b pl (10mg/ml)|
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