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玉米幼胚 诱导遇上

2010-08-08  bengua1985   |  转藏
   

Maize Somatic Embryogenesis




  1. Initiation:
    • Explant:
      • Immature zygotic embryos (lines: A188, Hi-II or B73) should be harvested when they are 1-2 mm in lenght (8-14 days post pollination).
      • Ears can be used immediately or held in a refrigerator for 1-2 days at 4°C while still in the husk.
      • Surface sterilize dehusked ears 20 minutes in 50% commercial bleach + some wetting agent (tween 20, dish soap, etc...).
      • Rinse 3x in sterile water to remove bleach.
      • Slice off top half of kernals, and separate the endosperm from the embryo.
      • Place embryo on the medium with the FLAT (embryo axis) side down in contact with the medium.
      • Wrap plates with Micropore tape and culture in the dark at 28°C.

       

    • Initiation Medium ("N6-1-25-100-Ag"):

      • N6 salts
      • N6 vitamins
      • 2% sucrose
      • 1 mg/L 2,4-D
      • 25 mM Proline
      • 100 mg/L Casien Hydrolysate
      • 10 mg/L Silver Nitrate
      • 0.4 % gellan gum as a solidifying agent
      • pH 5.8

  2. Maintenance:
    • Type II selection:

      • Callus will start to become visible after about 7 days post initiation.
      • Friable type II callus should separated from more organized callus and/or watery unorganized callus 2-3 weeks after iniation.
      • Friable type II callus should be visually selected under a stereoscope at each subsequent transfer to maintain an optimal phenotype.
      • Callus should be transferred to fresh medium at 2-4 week intervals depending on growth rate.
      • Wrap plates with Parafilm. Callus can be maintained at 25-28°C in the dark.

       

    • Maintenance Medium ("N6-1-25-100"):

      • N6 salts
      • N6 vitamins
      • 2% sucrose
      • 1 mg/L 2,4-D
      • 25 mM Proline
      • 100 mg/L Casien Hydrolysate
      • 0.4 % gellan gum as a solidifying agent
      • pH 5.8

  3. Regeneration:
  4. (After Register, J.C., et al, 1994.)

    • Step 1 - Somatic embryo maturation:
      • Transfer friable type II callus to N6-1N-6S regeneration medium 1.
      • Wrap plates with Parafilm and culture at 25-28°C in the dark for 2-3 weeks.

        Regeneration Medium 1 ("N6-1N-6S"):
        • N6 salts
        • N6 vitamins
        • 6% sucrose
        • 1 mg/L NAA
        • 0.4 % gellan gum as a solidifying agent
        • pH 5.8

         

    • Step 2 - Plantlet promotion:
      • Callus should have become more hard/white in appearence after 2-3 weeks on medium 1.
      • Transfer callus to MS0 regeneration medium 2.
      • Wrap plates with Micropore tape and culture at 25-28°C in the light for 2-3 weeks (the higher the light intensity the better).

        Regeneration Medium 2 ("MS0"):
        • MS salts
        • MS vitamins
        • 2% sucrose
        • 2 g/L myo-inositol
        • 0.3 % gellan gum as a solidifying agent
        • pH 5.8

         

    • Step 3 - Plantlet maturation:
      • Green shoots should have developed from the hard/white callus after 2-3 weeks in the light.
      • Transfer shoots to magenta boxes containing regeneration medium 3 (1/2 MS0).
      • Culture at 25-28°C in the light until plants are well rooted and shoots have elongated to the top of the box (1-2 weeks).

        Regeneration Medium 3 ("1/2 MS0"):
        • 1/2 MS salts
        • 1/2 MS vitamins
        • 2% sucrose
        • 1 g/L myo-inositol
        • 0.3 % gellan gum as a solidifying agent
        • pH 5.8

         

      • Step 4 - Transplant to soil:
        • Remove plantlets from magenta box.
        • Carefully wash gelrite off of roots with room temperature water.
        • Place transplants into peat pots.
        • Move plants to growth chamber or greenhouse.
        • Water well and cover plants in peat pots with 1-2 layers of cheese cloth to shade and reduce water loss.
        • Remove cheese cloth after 2-3 days.
        • Transplant plants in peat pots into large pots or directly into the field as soon as roots are seen growing through the peat pot.

          Soil mix:
          • 1 volume of peat-lite
          • 1 volume of soil

           

References
Register, J.C., et al, 1994. Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment. Plant Molecular Biology 25: 951-961.

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