Research On Transformation Of With Marker-free Expression Vector CMO And BADH Bivalent Gene On Alfalfa

The salting of soil is the serious problem that affects the agricultural production and the ecological environment.The alfalfa can improve soil,maintains the water and it's a kind of important grass crops.The transgenic alfalfa research carries on widely and successful.It is extremely slowly to use traditional breeding method to selective salt tolerance variety.(Along with the development of molecular biology,) In order to improve the salt tolerance of alfalfa,we transfer the salt tolerance gene into alfalfa by agrobacterium-mediated transformation.This research mainly takes explant from sterile cotyledon of alfalfa to induce somatic embryogenesis.Throught optimization of culture medium main influencing factors,completed the entire regenerative process which contains embryogenic callus induction,differentiation, seedling emergence and radiate.It is proven initially the resistance to drought and salt genes had transfer to alfalfa plant,and has carried on the physiological laboratory value to it.The primary coverage is as follows:First:This experiment separately optimized the culture medium ingredient of alfalfa at each time:(MS+2,4-D 2.0mg/L+KT 0.25 mg/L,sugarcane 3%,pH5.8) medium can induce somatic embryogenesis;(MS+NAA 0.05mg/L+ 6BA 0.5 mg/L,sugarcane 3%,pH5.8)和MS,1/2MS(sugarcane 1.5%,pH5.8) medium can nomal development and germination.Second:This experiment transfer BADH and CMO gene to alfalfa by Agrobacterium-mediated.In this research,we discuss the transformation effect on different invades dyes time,different density of bacterium.The result indicated that the density(the OD value) about 0.3-0.5,infest 25 minute time to injury the organization the effect to be best.Using culture medium contains 40mmol/L salt-bearing to carry on screening。Third:Through the PCR and the PCR-Southern blotting,The results showed that,PCR testing positive for the two plants have positive control the size and entirely consistent with a hybrid,and fully consistent PCR test results,but the non-transgene adult plant has not hybridized the signal to appear,so it explained that the PCR product is truly BADHgene,but is not genome team's homologous sequence in the alfalfa.It proved that BADH and CMO bivalent genes had transfer to genome of alfalfa.Forth:The physiological index contains:contrast to betaine content,membrane permeability, dissociation praline content,MDA content,SOD activity and POD activity.The experimental result indicated that the content of betaine,dissociation praline and MDA are increasing along with the salty coercion.Under 1.0%salt water irrigation,contrast with non-transformation plant, betaine content has increased,but membrane permeability and MDA content has obvious reduction.However the SOD,POD activeness activity are on the contrary.Proline has no remarkable changes.