Studies On The Establishment Of Salix Regeneration System And The Sophora Japonica Transformation With Rd29A Gene By Agrohczcterium Tumefaciens

Drought,salt and the low temperatures are the factors that the nature is widespread to repress plants growth existently, these conditions could cause many physiology bio-chemical variety, the plants must adapt these conditions and then they can exist enough, therefore, how to develop and make use of the salt soils, the poor and unproductive lands of the big area , not only is the demand of increment agriculture in our country that can make use of the resources, also is the demand of improving the ecosystem environment, the increment green plants, overlay the rate of exaltation forest, The modern technology development provided the new method and paths for the farm crop species that grows the anti-to go against, having already become the economic and valid means of improvement the salt soils.The salix and the Sophora japonica have many utility, our country has the cultivation everywhere, having the important ecosystem and economies value to use, If tolerance can be increased, which will having the very important meaning towards backing the farmland to return the wood, developing livestock husbandry and improvement the ecosystem environment.This research mainly establishs the regeneration system of the salix firstly, which provides the dependable basis of turn the genetic engineering. at the same time, The gene rd29A has the anti-resisting function of dry, salt and cold, therefore, making using of the Agrobacterium-mediated transformation of rd29A gene in the Sophora japonica, in older to acquire the new species with the anti-drought, anti-salt, bearing the low temperature, thus enlargement expansion area, improving the ecosystem environment. The main research result is as follows:1. The establishment of salix regeneration system(1) Using one axillary bud stem as explants, the two mediums used alternately was the best, the axillary bud have obtained better multiplication on medium supplement with 0.5mg/L6-BA,0.05mg/L NAA and medium supplement with 0.3mg/L6-BA,0.2mg/LKT, 0.1mg/LNAA, 30g/L cane sugar, It could made the bud lengthen and the leaf open, also the leaf colour became deeply green;(2) The explant was leaf with Petiole, Two steps in the method of differentiation induction:Callus induction was initiated on MS basic medium supplemented with 1.0 mg/L 6-BA and 0.3 mg/L NAA, After three weeks of growth, callus induction rate achieved 100%, in the process, darkness time was 15 days, then cultured on MS medium supplemented with 1.0mg/L 6-BA,1.5mg/L KT and 0.1mg/L IBA for shoot induction, the average differentiation rate was 83%, average differentiation number was 5.3; (3)Root induction was on 1/2MS medium supplemented with 20g/L cane sugar, the root rate could amount to 100%, Selecting the strong and young plants through taming to 15～20 days, the living rate could amount to above 90% in big farmland.2. the Sophora japonica transformation with rd29A gene by Agrohczcterium tumefaciens Making using of the Agrobacterium-mediated transformation, turning the rd29A gene into the Sophora japonica . The results enunciation: (1)The big leaves of absorbing the more nourishment were the most optimum explants; the leaves didin't pre-culture;(2)The GV3103 surpassed the LBA4404 in different strains of Agrohczcterium tumefaciens; the D600 of Agrohczcterium tumefaciens was 0.3; (3)Using liquid differentiation medium was the best , infection time for 15 min; (4)The best co-culture time was 3 days , the co-culture medium pH was 5.5; (5) The 400mg/L Cefotaxime sodium had good effects in inhibiting Agrohczcterium tumefaciens effectively; (6)growed in 27℃darkness conditions in 15 days, the delay choice of 15 days, the Phosphinothricin and Kanamycin were the most Antibiotics, Firstly, selective time was 30days with using 30mg/L Kanamycin , then continued selective with using 3mg/L Phosphinothricin, at last, selective in the root medium with using 50mg/L Kanamycin.3.The transgenic plants of Sophora japonica were obtained:(1) The transgenic plants which growed well and rooted in the kanaycin medium by distinctive PCR, and 2100bp positive signals were obtained, the first step indication that rd29A gene may be already integrated the genome of Sophora japonica; (2) The transgenic plant could growed regularly in 0.6% to 0.8% NaCI medium but the un-transgenic plant couldn't growed regularly, The transgenic plant was improved obviously on salt-tolerance...