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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

Inactive Publication Date: 2017-05-31
WUHAN INST OF BIOENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on osmotic regulators in medium components

Method used

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Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a rape microspore culture method utilizing mannitol as an osmotic regulator, belonging to the technical field of plant tissue culture. The method comprises the following steps: fetching cabbage type rape as a donor plant for microspore culture; fetching the flower bud on the donor plant; sterilizing and adding an NLN-13 liquid medium to obtain a suspension; filtering to obtain filtrate, and centrifuging to obtain precipitate; adding a mannitol-containing NLN liquid medium, a colchicine solution and active carbon mixed liquid to obtain a microspore mixed suspension; performing heat shock treatment or direct culture to obtain a cotyledon-stage embryoid; inoculating into a regeneration medium till bud regeneration; cutting the regenerated bud and inoculating to a rooting medium for rooting culture; performing seedling hardening and transplanting to obtain a regenerated plant; and fetching the tender leaves of the regenerated plant and detecting the ploidy of each regenerated plant. Through the method disclosed by the invention, the microspore quality, regeneration rate and doubling rate are remarkably improved.

Description

technical field [0001] The invention relates to the technical field of plant tissue culture, in particular to a rape microspore culture method using mannitol as an osmotic regulator. Background technique [0002] As the third largest oil crop in the world, rapeseed meets at least 13% of the world's edible oil demand. However, my country's output ranks first in the world and has high economic value. The rapeseed cultivated in my country mainly includes Chinese cabbage-type rapeseed ( brassica rapa , n=10), Brassica napus ( Brassica juncea , n=18) and Brassica napus ( Brassica napus , n=19) three types. Among them, because of its high yield, strong resistance, excellent quality, and good taste, Brassica napus has been vigorously promoted since it was introduced into my country, and the planting area has expanded rapidly. It is also the main research object of domestic rapeseed breeders. In the breeding process of conventional rape varieties, it takes at least 5 years ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00
CPCA01H4/008A01H4/001
Inventor 耿鑫鑫徐飞赵华燕
Owner WUHAN INST OF BIOENG
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