Establishment Of Transgenic Acceptor System And Transformation Of Leaf Senescence-inhibition Gene P_SAG12-IPT In Wheat (Triticum Aestivum L.)

In the agricultural production, the application of fine variety to increase crop yield is an economic and feasible way. At present, leaf presenility is a relatively serious problem for some wheat variety (pedigree) and breeding material in China. It may limit yield potential and new variety culture. Cytokinin has the characteristic of delaying plant cell and tissue senescence. As the limits of traditional method, using modern, advanced biotechnology-plant transgenic technology to is new way to culture wheat senescence-inhibition variety. With leaf senescence -inhibition gene P_SAG12-IPT that was designed by Gan and Amasino transformed tobacco and rice, the transgenic plants showed significant to delay leaf senescence and to increase yield obviously. In this study, transgenic wheat plants of 9848 with P_SAG12-IPT gene were obtained by micro-projectile bombardment and pollen tube pathway. Through establishment of transgenic acceptor system in wheat (Triticum aestivum L.) and two-transformation system, a feasible technology was established. The main result showed as follows: 1. Establishment of transgenic acceptor system in wheat (Triticum aestivum L.) Effect of few factors on wheat immature embryo callus re-differentiation ability was studied. The resulted showed that the re-differentiation frequency of wheat immature embryo callus inducted in dark culture was higher than that of wheat immature embryo callus inducted in nature light and 2 000 lx light. The callus re-differentiation frequency of immature embryos obtained from field was higher than that of immature embryos obtained from greenhouse. The callus re-differentiation frequency of immature embryos obtained from main stem was higher than that of immature embryos obtained from tiller. Different genotype varieties and different development stage embryos were different in the re-differentiation characteristic of immature embryo callus. In the optimal age of wheat immature embryos, genotype had little effects on callus induction frequency of wheat immature embryos while had remarkable effect on the frequency of embryogenic callus induction frequency of wheat immature embryos. KT in medium significantly promoted the formation and regeneration of embryogenic calli derived from wheat immature embryos. The induction frequency of embryogenic callus is highest when KT concentration is 0.5mg/L, while the regeneration frequency of embryogenic callus is highest when KT concentration is 0.3mg/L. The optimum medium for the regeneration of embryogenic callus of wheat immature embryos was MS medium with KT 1.5mg/L, NAA 0.5mg/L. 2. Effect of factors on particle bombardment method Psag12-ipt gene was delivered into wheat immature embryo cells by using particle bombardment method. The effects of gold particle concentration and DNA amount per bombardment on transformation efficiency of wheat immature embryo cells were studied using GUS transient expression. The result showed that the gold particle concentration has little affect on transient expression of immature embryos and callus, while it has a remarkable role in the cell transient expression at a relatively high gold particle amount per bombardment. It was found that the transformation frequency of wheat immature embryo cells was the superior when coated by 2.0ug DNA and bombarded for 2 times per dish. The expression efficiency of GUS gene was reducing with increasing of callus culture time and it was stability after 28d.The examination of GUS gene transient expression should be at 1d and the examination of GUS gene stable expression should be after 28d. 3. Wheat genetic transformation by particle bombardment method Immature embryos and calli of a cultivars 9848 of wheat((Triticum aestivum L.)were bombarded using PDS 1000/He particle delivery system in the presence of plasmid pCMLA35-1 which contained a PASAG12-IPT and selectable marker NPT gene. Resistant calli were selected on kanamycin (40mg/ L) containing medium. 65 and 18 green plants were regenerated from resistant calli derived from 1870 and 682 bombarded immature embryos and callus respectively. By PCR analysis 2 transgenic plants with PASAG12-IPT gene were obtained from transformed immature embryos .The transformation frequency was about 0.107% averagely. No transgenic plant was obtained from transformed calli. 4. Wheat genetic transformation by pollen tube pathway The plasmid pCMLA35-1 with NPT and PASAG12-IPT gene was transferred into wheat(Triticum aestivum L.) 9848 with the plasmid 100,300,500 and 700ng/uL by pollen tube pathway .The result showed the transformation frequency of wheat was 0.19%, 0.41%, 0.73%, and 0.71% respectively; it has a remarkable role in the seed setting and seeds germination of T0 progeny at a relatively high plasmid concentration. It was found that the transformation frequency of wheat was the superior when plasmid concentration was 500ng/uL.2098 transformed seeds were selected with kanamycin,in which 2128 germinated and 96 expressedresistance to kanamycin .By PCR analysis 9 transgenic plants with PASAG12-IPT gene were obtained .The transformation frequency was about 0.29% averagely.