Research On Transformation Of DREB1A Gene From Arabidopsis Thaliana Into Alfalfa (Medicago Sativa L.)

The growth of plant often has been severely affected by the drought,high salt and low temperature stress.So the research on improvement of plant stress resistance is of practical significance.Alfalfa is a very important leguminous pasture in the world,and also an excellent plant for soil and water conservation.Accordingly, study on enhancement of drought,salt and cold resistance using genetic engineering technique is of great significance.In this study the DREB1A gene from Arabidopsis thaliana was transfered into alfalfa via Agrobacterium-mediated method and pollen-tube pathway to lay a foundation for high stress resistance of alfalfa.The main results were as following:1.The tissue culture regeneration system had been studied and optimized systemically.By selecting and optimizing diverse factors effected on plant regeneration such as different cultivar of alfalfa(Algonguin,West blend and WL323),different explant(hypocotyl and cotyledon),different basic medium(MS and SH) as well as different combination and concentration of exogenous hormone, finally the results were concluded that the optimum material,explant and basic medium was Algonguin,hypocotyl and MS respectively.Among different combination and concentration of exogenous hormone,2 mg/L 2,4-D+ 0.1 mg/L 6-BA is the best for cllus induction and 2mg/L 6-BA+0.1 mg/L NAA had highest differentiation rate.The regenerated plant was obtained in the rooting medium of 1/2 MS+1.0 mg/L IBA.2.The transformation system mediated by Agrobacterium tumefaciens had been studied and optimized systemically based on the tissue culture regeneration system which had been established previously.By optimizing diverse factors effected on plant transformation,the results were concluded that the optimum pro-cultivated time and co-cultivated time was 7 days and 3 days respectively.The optimum selection pressure of gentamicin for callus induction and rooting was 20 mg/L and 10 mg/L, respectively.After PCR detection of transgenic regenerated plant,the result indicated DREB1A gene was integrated into genome of alfalfa.3.The transformation system via pollen-tube pathway had been studied and established systemically.Four plant expression vectors such as pPZP211(35S-DREB1A-NOS),pPZP221(prd29A-DREB1A-NOS),pPZP211(prd 29A-GUS-NOS),pPZP221(35S-GUS-NOS) were transfered into alfalfa at eight different transformation time through two different transformation methods.The results of PCR detection showed that foreign DNA was inserted into the genome of receptor plant and the transformation rate of T₀ seedling from 20 to 48h after pollination was relatively high.