Genetic Transformation Of Leymus Chinensis With Laccase Gene

Leymus chinensis is an important grass of the Gramineae family.It widely grows at the northern part grassland of our country,its value of nutrition is very high and it can grow very well in the bad surroundings.There is important economical value.Leymus chinensis can stand up cold,drought,wet, trample,poor soil and some Basta stress,its adapt is very wide.Hoever,because of the grassland has been excess exploited,it makes the grassland obsolete.Next,because of the exorbitant lignin in Leymus chinensis,it is too difficult to digest by animal.So in order to promote the development of graziery,it is our need to find a new variety.Laccase is a kind of protein which link with some copper ion.The essence of it is covellite oxidase,it exists in fungus,bacterium and plants.lt can survive in atmosphere.The output of its rrespondence is only water,so its essence is an enzyme which has no pollution.we can use laccase to transfer Leymus chinensis with the screening of bar,the aim is to lower the lignin.We have got someresults as below.(l)By improving the condition of regeration,we have established anew system..In order to improve the embryogenic calli induction rate from mature embryos,different media with different hormones are used in the embryo genic calli induction.The experiment results indicated that selection suitable medium is basis to improve the induction rate and the quantity of embryogenic calli from mature embryos.The hormone treatments have certain effects on the calli induction,with the effects having significant difference for different.The mature calli induction of different media is quite different.N6 gain very well induction efficiency.During the different of hormones,concentration 2,4-D 2.0mg/L can obviously enhance efficiency of induction and embryos induction.(2) Leymus chinensis calli subcultureIn experiment,MB and N6 media can improve the growth of mature calli and can obtain embryogenic calli.Adding 1.5-2.0mg/L 2,4-D is the best concentration in Leymus chinensis calli subculture. Concentration of sugar and proline should be 40g/L and 2500mg/L in subculture media.(3) Differentiation and regeneration of Leymus chinensis calliRegeneration system is essential basis of genetic transformation.Regeneration system of Leymus chinensis calli is belonging to embryo occurrence type.The ability of regeneration becomes low with the subculture time.When it is cultured more than 6 months,Leymus chinensis calli do not regenerate.So it do not admit too long time when the calli subculture.Differentiation rate can be 25%when there is 0.5mg/L 6-BA in the tissue culture media.It is the best concentration to add 2.0mg/L NAA into root-regeneration media.Pre-regeneration 10—30 days before differentiation could increase plant regeneration.It also can improve the state of calli.2.The sensitive characteristic on Basta of Leymus chinensis calli According to the state and weight index of calli,20mg/L Basta is the limit concentration condition of calli selection.The resist Basta plant can be selected in high concentration of Basta.Increasing the concentration of Basta,we get the 6 mutation seedling.It is different from the natural plant that there are light-green leaves,and the mutation plant dwarfism.3.Parameters of Gene-gun(PDS-1000) transformation of mature embryo calli were studied by expression.Bar and laccase gene is transferred into mature embryo calli by Gene-gun.Bar and laccase gene had been inserted into Leymus chinensis genome demonstrated by PCR.When the fire target distance is 6cm, 9cm,12cm,and the transient expression is different.The best distance is 9cm.Dosage of DNA is 6ul in every fire;1100psix9cm is the best effect.While the target distance is fixed of 9cm,fire for 1,2,3 times, the transient expression rate is 88.13%,41.12 and 18.21.The best effect is 1 time.4.The transient expression of Leymus chinensis:by using Gus staining,we get positive result.