Stability And Genetic Expression Of Exogenous Gene In Transgenic Apple

It is critical for the transgenic plants to become commercialization that the exogenous gene can persist stably after the gene transformation, which needs a series inspection of transgenic plants for their performance in either current or the following generations. In this experiment, the transgenic plants carring exogenous CpTI gene either subcultured in vitro for twelve years or planted in field for four to six years were used to study the stability of exogenous gene in transgenic plants, the characteristics of the pollen grains and the fruits, and the rule of exogenous gene separation in hybrid F₁ generation by using embryo culture.The main results were as follows:1. The kanamycin resistance screen and PCR analysis to the transgenic lines of Gala, Orin, Jonagold, and Fuji cultivars subcultured in vitro for twelve years showed that all of the 59 tested lines had CpTI gene positive bands. All transgenic lines showed the kanamycin resistance, except 2 of them from Orin cultivar showed sensitive to the 50 mg/L kananmycin applied in the culture medium. It demonstrates that the exogenous nptⅡand CpTI gene persists in the transgenic lines subcultured in vitro for a long time.2. The PCR analysis to the 4 to 6 years old transgenic lines in field showed that the exogenous CpTI gene positive bands could be found in leaves and roots of Gala, Orin and Fuji cultivars, and in pollen grains and fruits of Gala cultivar, with an exception in a few Gala lines in the roots, the pollen grains, and the fruits that showed negative. The exogenous CpTI gene could be found in the sexual tissue and possessed the ability to pass onto the offsprings.3. The transformation of exogenous gene had some effect on the amount and viability of pollen grains. The number of pollen grains per anther, the germination rate, and the viability of pollen grains varied from the transgenic lines, and had a tendency to be lower compared to the non-transformated control.4. The seeds collected from the hybrid fruits of transgenic lines of Gala with non-trangenic plants of Fuji were used to study the appropriate condition for the embryo culture. That was as treating seeds at about 8℃for four weeks prior to facilitating seed germination at 25℃. The seeds after germination were sterilized with 0.1 % HgCl2 for 8-10 min, and then were inoculated on the medium. The hybrid offsprings in vitro were obtained.5. The hybrid offsprings in vitro from either transgenic lines as female or male were used for the kanamycin resistance screen and PCR analysis. The segregation of the kanamycin characteristics in F₁ offsprings followed the ratio of 1:1, and that indicated the nptⅡgene was a dominant heterozygous gene and could pass onto the offsprings through sexual hybridization. The PCR analysis to the offsprings showed that the nptⅡgene and CpTI gene were tightly linked to each other and did not segregation.