Introduction Of Senescence-Inhabiting Gene Into Wheat (Triticum Aestivum L) Mediated By Agrobacterium Tumefaciens

The senescence of wheat, accompanying with the development and maturity of seed, is a main limitation of yield. By inhabiting senescence, the yield of wheat can be significantly increased. Exogenous phytohormone and other chemical agents, which inhibit leaf senescence, are generally associated with the problems of cost and environment. By linking a cytokinin synthase gene to a senescence-induced promoter SAG 12, Gan and coworkers constructed an automatically regulatory element for inhabiting senescence (SAGi2-ipt), which has been proved to postpone leaf senescence in several crops.Great progress in wheat transformation has been made since the birth of the first transgenic wheat in 1992. More than ninety percent of transgenic wheat were produced through Biolistics, and there were less reports about transformation of wheat mediated by Agrobacterium. To produce a senescence-inhabiting wheat, a plasmid pCBSI, harboring SAGn-ipt gene and bar gene which encoded resistance to Basta, was transformed into wheat via Agrobacteriwn, using a optimized method. The main results are as followed.1. Establishment of regeneration systemIt was demonstrated that, culture with scutellia facing up and addition of 0.5 mg/L ABA could obviously increase the differentiation frequency of immature embryos from all the five wheat cultivars investigated. An efficient regeneration system had been established: callus induction medium, MS+2mg/L 2,4-D+500mg/L LH+0.5mg/L ABA+3% sucrose~K).7%ager; regeneration medium, MS-K).5mg/L NAA+0.5mg/L KT+500mg/L LH; shoot induction medium, l/2MS+5mg/L IBA; pH was 5.8 in all the medium.2. Sensitivity tests of callus to selection agentshi order to test the effects of defferent selection agents and their concentration on callus inhibition, calli were transferred to selection medium containing Km, G418 and Basta of different concentration. The best inhabiting concentration of Km, G418 and Basta were determined as 50mg/L, 20mg/L, and 5mg/L, respeactively.3. Optimization of the transformation systemThe effect of infection time, AS, Silwet on transformation were tested, and the transformationsystem was optimized to 30min infection with A. twnefaciens re-suspended in MS+lOOumol/L AS+0.05% Silwet. It was also found that vacuum and empty-microprojectile bombardment increase the frequency of resistant callus.Another method is to co-culture callus and A. tumefaciens differentiation medium without selection of Cef and Basta.4. Obtainment of resistant plant and PCR assaysAfter selection with Basta, five resistant plants were obtained from the variety Bobwhite and three from variety 95319. PCR assay showed that the 5 out of the 8 resistant plants had integrated bar gene(s).Without addition of Basta and Cef, 31 Plants were regenerated from the co-culture of Agrobacterium and the calli from variety Bobwhite, among which 7 showed phenotypic variation and two were showed positive in PCR assays.5. Phenotypes of transgenic wheatTransgenic plants showed three types of phenotypic variations: ㏕he number of spikes increased. Multiple spikes formed in one stem, all of which could flower and set seeds. ㏕he number of florets increased. Compared to control on which generally three florets in a spikelet could set seeds, in some transformants 5-7 florets per spikelet could set seeds. ㏕he senescence was inhibited. While spikes changed into yellow and the seeds became full and hard, the leaf and stem of tansformants remained green.