玉米幼胚 诱导遇上

Maize Somatic Embryogenesis

---

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

---

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

---

Regeneration:

(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.