Preparing and applying method of cutting propagation growth promoting agent of tea tree endogenous herbaspirillum seropedicae

Preparing and applying method of cutting propagation growth promoting agent of tea tree endogenous herbaspirillum seropedicae

  • CN 104,585,241 A
  • Filed: 01/13/2015
  • Published: 05/06/2015
  • Est. Priority Date: 01/13/2015
  • Status: Active Application
First Claim
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1. the preparation of sward spirillum cottage propagation growth-promoting agent in tea tree, is characterized in that the steps include:

  • 1), the screening of sward spirillum in tea treeRinse disease-free camellia tree sample well with clear water, then use 75% alcohol disinfecting 0.5 hour, sterilize 3 minutes with the mercury solution of percent concentration 1% subsequently;

    Finally use aseptic water washing 3-5 time, draw the sterile water 100 μ

    l of last flushing material, on coating beef-protein medium flat board, 37 DEG C cultivate 3 days aseptic drop out existing after, again the tea leaf of disinfecting and tissue are cut marginal portion, interior tissue being cut fritter is positioned in beef extract-peptone culture plate, cultivates after 48 hours for 28-37 DEG C and obtains endophyte bacterium colony;

    Carry out bacterium colony purifying subsequently and systematic bacteriology is identified, finally obtain sward spirillum also-80 DEG C of preservations in tea tree;

    Described beef-protein medium is 3g beef extract, 10g peptone, 5g NaCl, 1.5% agar, adds water to 1000ml;

    2), the cultivation of sward spirillum and the preparation of growth-promoting agent in tea treeGet sward spirillum bacterial classification in tea tree that-80 °

    of C preserve, inoculation 5mL LB liquid nutrient medium, 150 turns of shakes per minute, 37 °

    of C overnight incubation;

    Accessed in fresh LB liquid nutrient medium or KB liquid nutrient medium to amplify with 1;

    100 ratio by bacterium liquid after activation and cultivate, condition of culture is 200 turns per minute of shaking speed, and temperature is 37 °

    of C;

    Treat bacterial culture 8-10 hour, OD 600value is for collecting whole inoculums during 1.2-1.5, after adding 1% glycerine, deposit in 4 °

    of C for subsequent use or directly use, above-mentioned inoculum is sward spirillum in tea tree of the present invention skewerinsert breeding growth-promoting agent;

    Described LB liquid nutrient medium is 10g tryptone, 5g yeast extract, and 10g NaCl, adds water to 1000ml, 10 μ

    g/mL spectinomycins;

    Described KB liquid nutrient medium is 20 g tryptone 1.5 g K 2hPO 4, 15 ml glycerine, 1M MgSO 4, add water to 1000 ml, 10 μ

    g/mL spectinomycins.

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