Agrobacterium mediated method for obtaining transgenic plant of hevea brasiliensis

Agrobacterium mediated method for obtaining transgenic plant of hevea brasiliensis

  • CN 102,676,574 A
  • Filed: 04/19/2012
  • Published: 09/19/2012
  • Est. Priority Date: 04/19/2012
  • Status: Active Application
First Claim
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1. the method for an agriculture bacillus mediated acquisition rubber tree transfer-gen plant is characterized in that, comprises the steps:

  • 1) engineering bacteria infects suspension cell;

    soak 1~

    5min with putting into 20~

    30ml engineering bacteria liquid behind the rubber tree embryonal suspension cell removal substratum, let engineering bacteria infect the rubber tree embryonal suspension cell;

    2) suspension cell and engineering bacteria are cultivated altogether;

    after the rubber tree embryonal suspension cell after will infecting removes bacteria-removing liquid and blots with aseptic filter paper, be seeded on the common substratum and cultivate altogether, 20~

    25 ℃

    of culture temperature are cultivated 2~

    8d;

    The effective constituent of described substratum altogether is MS substratum (MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 300~

    550mg/L, potassium primary phosphate 300~

    500mg/L, the Calcium Chloride Powder Anhydrous 150~

    550mg/L of improvement;

    Manganese sulfate monohydrate 15~

    45mg/L, cupric sulfate pentahydrate 0.1~

    0.35mg/L) adds 2;

    4-dichlorphenoxyacetic acid 0.1~

    3.0mg/L, NAA 0.1~

    2.0mg/L, kinetin 0.1~

    3.0mg/L;

    Inositol 0.1~

    0.2g/L, Sucus Cocois 30~

    50ml/L, sucrose 30~

    70g/L;

    Syringylethanone 50~

    100 μ

    M, plant gel 2~

    3g/L;

    3) bacterium that takes off of suspension cell is cultivated;

    through the rubber tree embryonal suspension cell of cultivation altogether with sterilized water after the abundant rinsing 3~

    5 times;

    Be inoculated into and take off on the Agrobacterium substratum;

    Dark 15~

    the 30d that cultivates removes Agrobacterium and induces the generation embryo callus under 25~

    28 ℃

    of conditions;

    The described MS substratum that takes off the effective constituent of Agrobacterium substratum for improvement adds 2,4 dichlorophenoxyacetic acid 0.1~

    3.0mg/L;

    NAA 0.1~

    2.0mg/L, kinetin 0.1~

    3.0mg/L, inositol 0.1~

    0.2g/L;

    Sucus Cocois 30~

    50ml/L;

    Sucrose 30~

    70g/L, ticarcillin 200~

    500mg/L, plant gel 2~

    3g/L;

    4) screening of resistance embryo callus;

    the inductive embryo callus is received on the screening culture medium;

    25~

    28 ℃

    of dark cultivations;

    Per 15~

    30d is transferred on the fresh screening culture medium, and screening and culturing 2~

    4 times is picked out resistant calli and continued succeeding transfer culture on same medium separately;

    Described screening culture medium effective constituent is the MS substratum of improvement, adds 2,4 dichlorophenoxyacetic acid 0.1~

    3.0mg/L;

    NAA 0.1~

    2.0mg/L, kinetin 0.1~

    3.0mg/L, inositol 0.1~

    0.2g/L;

    Sucus Cocois 30~

    50ml/L, sucrose 30~

    70g/L, ticarcillin 200~

    500mg/L;

    Kantlex 50~

    200mg/L, plant gel 2~

    3g/L;

    5) resistant calli inducing embryoid body;

    the resistant calli behind the succeeding transfer culture is received on the somatic embryo inducement substratum, secretly cultivated under 25~

    28 ℃

    of conditions, per 25~

    30d is transferred on the fresh substratum ripe until somatic embryo development;

    The effective constituent of described somatic embryo inducement substratum is the MS substratum of improvement, adds kinetin 1.0~

    3.0mg/L, NAA 0.1~

    0.5mg/L;

    6-benzyl aminoadenine 0.1~

    3.0mg/L, Plant hormones regulators,gibberellins 0.1~

    3.0mg/L, gac 1~

    2g/L;

    Inositol 0.1~

    0.2g/L;

    Sucus Cocois 30~

    50ml/L, sucrose 30~

    70g/L, plant gel 2~

    3g/L;

    6) plant regeneration;

    ripe embryoid is inoculated into the regeneration plant substratum, illumination cultivation under 25~

    28 ℃

    of conditions, the photoperiod is 16h/d, until growing complete regenerated plant;

    The effective constituent of described regeneration plant substratum is the MS substratum of improvement, adds kinetin 0.5~

    3.0mg/L, NAA 0.01~

    1.0mg/L;

    Plant hormones regulators,gibberellins 0.1~

    3.0mg/L, sucrose 50~

    90g/L, activated carbon 1~

    2g/L;

    Sucus Cocois 30~

    50ml/L, plant gel 2~

    3g/L.

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