Transformation: ovary injection
--Ding, Q; Xie, Y; Dai, J; Mi, J; Li, T; Qiao, L; Tian, Y and Mang, K
About twelve hours after artificial pollination, plasmid (containing the Bt
gene) was injected into young ovaries. These injected ovaries were kept
growing until seeds matured, then harvested and sowed in soil. The
chromosomal DNA was isolated from leaves of plantlets and tested with dot
blotting, PCR and Southern blotting analysis. A total of 5 out of 363 plantlets
demonstrated positive tests in Southern blotting analysis. One of the five was
comprehensively identified with a Bt gene fragment probe (EcoRI digested): the
undigested sample of its chromosomal DNA showed a hybridization signal at a
size of about 30kb, EcoRI digested sample showed an expected 1.2kb band, and
AccI(which has a cut site in the region of probe) digested sample showed one
10kb signal band and another weak band at about 0.8kb size while the BglII
digested sample showed a 9kb signal band and light smear (Ding Qunxing et al.,
Science in China (Series B) 37(5):563-572). This transgenic plant was normally
fertile and its offspring were tested with Southern blotting analysis also.
Interestingly, the BglII digested samples of the offspring's chromosomal DNA
showed a different hybridization pattern: one had four bands siting from 30kb
to 8kb with almost the same intensity while another had two close bands near
23kb. It seems that the integrating position on chromosome or the copy number
of the Bt gene might change. Recently, the R4 and R5 generations have been
obtained and genetic analysis results will be reported soon. Meanwhile the
mechanism of injection transformation has been studied also. Carbon powder
and tungsten particles (1nm, with plasmid DNA precipitated onto the particles)
were used as tracks respectively in light microscope and electron microscope.
The results proved that carbon powder and tungsten particles could enter the
embryo sac and spread following its development. This transformation
technique is genotype independent, evading tissue culture and regeneration. It's
considered that several factors are important: the first is the injection time,
which must be after sperms enter the sac and before the fertilized egg divides,
which also depends on the varieties, length of silks and temperature etc.; the
second is avoiding heavy injury on the ovaries, for this we have designed a
micro-glass-tubed (diam. 2-5µm) injector; the third is keeping the injected
ovaries vital enough to mature; the fourth, is that a special solution for plasmid
DNA (100µg/ml final) was used: 20mmol/L MgCl2, 1.5mmol/L HBO3, 10mmol/L glycine,
5mmol/L spermidine, 5% PEG6000(w/v).
Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors
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