LONDON, ONTARIO, CANADA

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)
*Helium gas at 900 psi; 1 mm gold particles were used.  1st shelf = most distant (11cm) from delivery source.
**(N) = number of meristems; S = scutellum portion; P = plumule (leaves); R = radical portions of a growing plant at the time of transient assay; S.E. = standard error of the mean.


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