Genetic Transformation Of Cotton With Isopentenyl Transferase Gene And Regeneration Of Transgenic Plants

The cotton of Yangtze River area is easy to decrepitude on the condition of high temperature in summer. A number of experiments show that delayed leaf senescence and increasing its photosynthesis functional period can improve plant yield and quality. Plant genetic engineering provides a modern and effective method for delaying senescence breeding .Isopentenyl transferase gene might promote the biosynthesis of cytokinin,,which was proved having the function of delaying leaf senescenceIn this study ,the systen of cotton rgeneration has been establishe,we also investigated factors affecting transformation efficiency. Ttansgenic. plants has been obtained via A.Tumefaciens mediated and ovary - injecting. The main results are as follows:1 The high effective system of regeneration has established.The results showed that IBA can induce a number of embryos within 2 months.The best hormone combinations for callus induction and subculture were MSB medium with 0.5~lmg/L IBA and 0.2mg/L KT.Thus it can greatly shorten the time of plant regeneration compared with other hormone combinations such as IAA+KT and 2,4-D+KT.2 In order to establish an A.grobacterium-mediated transfoumation systen on cotton.we investigated factors affecting transformation efficiency. The hypocotyls segments of 5-day old seedling(3 dark +2 light days) and the shoot apex of 2~3 -day old seedle and bright yellow andrelatively loose granular callus were the best physiological condition as receptor.Ashrt preculture , vacuum treatment and AS can raise the efficiency of transformation.Different tissue receptors have different efficiency of transformation,the transformated efficiency of hypocotyl segments is 42.3~80.3%,and efficiency of embyos is 17.7-44.7%,and efficiency of shoot apex is 3.7-11.8%.3 PCR and Southern blot analysis showed that most of transgenic cullus were positive .Sixty-five transgenic plants from embros were obtained by Agrobacterium-meadiated embryos.4 At present,54 transgenic plants were obtained by Agrobacterium -mediated shoot apex transformation. Some of the transgenic plants have fruited and the seed were collected and planted this year.5 Direct gene transfer to cotton via ovary-injection of DMA.We selected the overies one or two days after pollination as accepter for injecting plasmid DMA.The concentration of DNA is 2~4ug.PCR and Southern blot analysis showed tha three plants have been transformed.5 Transformation of cotton pollen via Biolistic Bombardment has no obtained transgenic plants.