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).
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