Identification Of Transgenic Rice Plants Harboring HVA1 And Establishment Of A System For Propagating It

The regenerated plantlets harboring a HVA1 gene and a bar gene were used as experiment materials, this studies are carred out from two aspects,one includes herbicide resistance anlysis .molecular analysis ,field investigation and the salt tolerance of transgenic materials; the other is the establishment of the propagating system of transgenic materials to reserve and propagate the transgenic materials which failed to produce the seeds. 1.The results of identification of transgenic plants are as follows:1.1 Test of tolerance to Basta of transgenic plants indicated that there was a significant difference between the transgenic plants and the control ones in the response to Basta. A week after spayed with 4mg/l Basta, transgenic plants grew nomally and their leaves remained green, while at the same time the growth of control plants was inhibited and leaves of these plants became yellow. Sometimes the control plants even withered to death.1.2 Southern blot indicated that most of the transgenic rice plants contained the expected 1.2kb hybridization band, and several copy had been integrated into the genome of transgenic rice plant.1.3 Investigation of transgenic plants in the field showed that: there were various phenotypic alterations in the transgenic rice. Compared with the control plants, transgenic plant tended to be shorter, its growth duration became longer, tiller increased and the pollen fertility was almost lost. It can't seed by either inbreeding or outcrossing.1.4 The identification of resistance to salt stress of transgenic materials: In vitro, tillering seedlings of the TO transgenic rice and tissue culture seedlings were used to test the resistance to salt stress, and the result indicated that transgenic plants were more resistant to salt stress; two weeks after treated with 0.75% NaCl ,the transgenic plants still grew well, but the growth of control plants were inhibited; In vivo (callus), callus induced from transgenic young inflorescene were tested in the subculture medium, regeneration medium and rooting medium respectively, the results also showed that the transgenic materials were more resistant to salt stress than the control ones.These results confirmed that HVA1 gene had been integrated into the genome of transgenic rice plant,and it conferred increased tolerance to salt stress, thus it hold considerable potential for use as molecular tools for genetic crop improvement toward stress tolerance.2. The results of establishment of the propagating system of transgenic materials are as follows:2.1 The regenerating system of young inflorescene was established. Different levels of cytokinins (6-BA, KT, ZT) were used, and it was found that 2mg/L 6-BA was the best effective concentration for regeneration.2.2 The suspension culture lines had been established. The effect of La3+ on the suspension culture lines was observed, the results showed that low concentration of La3+(lmg/L) benefited the development of suspension culture lines in some aspects, such as stimulating the growth, increasing the activity of SOD, decreasing the content of MDA, and enhancing the regeneration of suspension culture cell, while high concentration was harmful for the suspension culture lines.2.3 The rapid propagating system of the stem nodes was established. Investigating effects of different concentrations of 6-BA on rapid propagation of transgenic seedlings induced from stem nodes, we found 20mg/L is the most effective concentration to induce the adventitious buds and axillary buds during the stage of inducement. In the subculture stage of rapid propagation, 10mg/L is the optimum concentration for the propagation of axillary buds, and 4mg/L for the propagation of adventitious buds. In the stage of rooting, the regeneration seedlings were transferred to the 1/2MS medium with 1mg/1 IAA. At last a large number of transgenic seedlings were abstained.