Method of promoting generation of lateral buds of Heveabrasiliensis by trans-AtWUS (Arabidopsisthaliana WUSCHEL) gene

Method of promoting generation of lateral buds of Heveabrasiliensis by trans-AtWUS (Arabidopsisthaliana WUSCHEL) gene

  • CN 104,031,936 A
  • Filed: 06/16/2014
  • Published: 09/10/2014
  • Est. Priority Date: 06/16/2014
  • Status: Active Application
First Claim
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1. turn the method that AtWUS gene promotes that rubber tree lateral bud occurs, it is characterized in that, this turns AtWUS gene and promotes the method for rubber tree lateral bud generation to comprise:

  • Step 1, Arabidopis thaliana AtWUS gene clone and plant expression vector construction, clone'"'"'s Arabidopis thaliana AtWUS gene, builds pCAMBIA2301-35s-AtWUS plant expression vector;

    Step 2, according to beta-Glucuronidase (β

    -glucuronidase, GUS) number of gene (uidA) transient expression rate and the height of cell survival rate, in conjunction with the correlative factor that affects agrobacterium tumefaciens genetic transformation, correlative factor comprises preculture time, bacterial concentration, Syringylethanone AS concentration, time of infection, is total to incubation time and common culture temperature, contrived experiment, determines the optimal conditions of the continuous subculture friable embryogenic of rubber tree callus genetic transformation;

    Step 3, grind the growth velocity of 88-13 friable embryogenic callus in the shoot proliferation substratum that contains Kan concentration gradient as index taking rubber tree kind heat, it is 100~

    125mg/L to Kan sensitive concentration that mensuration heat is ground 88-13 friable embryogenic callus, for the screening of resistant calli;

    Step 4, resistant calli, the acquisition of embryoid and regeneration plant, after cultivation finishes altogether, heat is ground to 88-13 friable embryogenic callus to be proceeded to and in micro-organisms base, carries out antibacterial processing, after 18 days, transfer in the screening culture medium that kantlex is 100~

    125mg/L, through the screening of 4 months, grow the resistant calli of aureus, dye through GUS callus propagation positive inducing embryoid body and plant regeneration after 1~

    2 month, result is ground 88-13 from heat and has been obtained 5 resistance friable embryogenic callus systems, 843 embryoids are gone out from resistant calli coinduction, transformed plant is intended in 21 strains,Step 5, histological chemistry is detected and Molecular Detection, cuts 14 strain heat and grinds 88-13 resistance and intend the cotyledon of transformed plant and carry out GUS dyeing, and result has 6 strains to be blue positive, and positive plant has certain phenotypic characteristic, choose 7 strain heat and grind 88-13 resistant plant carry out Molecular Detection together with 3 strains contrasts.

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