Rape microspore culture method utilizing mannitol as osmotic regulator
A technology of microspore culture and osmotic regulator, which is applied in the field of plant tissue culture to achieve high doubling rate, low pollution and good culture effect
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Embodiment 1
[0035] (1) Culture medium preparation
[0036] ① NLN liquid medium, calculated in 1L, consists of: KNO 3 125mg, Ca(NO 3 ) 2 4H 2 O 500mg, MgSO 4 ·7H 2 O125mg, KH 2 PO 4 125 mg, H 3 BO 3 6.2 mg, MnSO 4 ·H 2 O 18.95mg, ZnSO 4 ·7H 2 O 10mg, Na 2 MoO 4 2H 2 O0.25mg, CuSO 4 ·5H 2 O 0.025mg, CoCl 2 ·6H 2 O 0.025mg, vitamin B1 0.5mg, vitamin B6 0.5mg, biotin 0.05mg, folic acid 0.5mg, Na 2 EDTA 37.3mg, FeSO 4 ·7H 2 O 27.8mg, inositol 100mg, glycine 2mg, niacin 5mg, L-glutamine 800mg, glutathione 30mg, serine 100mg and the rest of sterile water, filter sterilized.
[0037] ② NLN-13 liquid medium, calculated by 1L, consists of: 1L NLN liquid medium and 130g sucrose, pH 6.0, filter sterilized.
[0038] ③NLN liquid medium containing 36.5g / L mannitol, calculated by 1L, consists of: 1L NLN liquid medium, 36.5g mannitol, 65g sucrose, pH 6.0, filter sterilized.
[0039] ④ MS liquid medium, calculated in 1L, consists of: NaH 2 PO 4 ·H 2 O 150mg, CaCl 2 113.24mg, KI...
Embodiment 2
[0052] (1) Culture medium preparation
[0053]① NLN liquid culture medium, with embodiment 1.
[0054] ② NLN-13 liquid medium, the same as in Example 1.
[0055] ③NLN liquid medium containing 54.8g / L mannitol, calculated by 1L, consists of: 1L NLN liquid medium, 54.8g mannitol, 32.5g sucrose, pH 6.0, filter sterilized.
[0056] 4. MS liquid culture medium, with embodiment 1.
[0057] 5. regeneration medium, with embodiment 1.
[0058] 6. rooting medium, with embodiment 1.
[0059] (2) Microspore culture method using mannitol as an osmotic regulator
[0060] 1) Selection of flower buds of donor plants: take the inflorescences of Brassica napus in the early flowering stage that grows healthy and free from diseases and insect pests as the donor plants for microspore culture; take the inflorescences with a petal to anther length ratio of 0.5, late uninucleate to early binucleate 40 flower buds.
[0061] 2) Sterilization of flower buds: use sodium hypochlorite 56mL / L + absolu...
Embodiment 3
[0069] (1) Culture medium preparation
[0070] 1. NLN liquid culture medium, with embodiment 1.
[0071] ②NLN-13 liquid medium, same as Example 1.
[0072] ③NLN liquid medium containing 73g / L mannitol, calculated by 1L, consists of: 1L NLN liquid medium, 73g mannitol, 1g sucrose, pH 6.0, filter sterilized.
[0073] 4. MS liquid culture medium, with embodiment 1.
[0074] 5. regeneration medium, with embodiment 1.
[0075] 6. rooting medium, with embodiment 1.
[0076] (2) Microspore culture method using mannitol as an osmotic regulator
[0077] 1) Selection of flower buds of donor plants: take the inflorescences of Brassica napus in the early flowering stage that grows healthy and free from diseases and insect pests as the donor plants for microspore culture; take the inflorescences with a petal to anther length ratio of 0.5, late uninucleate to early binucleate 40 flower buds.
[0078] 2) Sterilization of flower buds: use sodium hypochlorite 56mL / L + absolute ethanol 10...
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