Culture method for brassica oleracea L. var. acephala microspore regeneration plant

A technology for kale and plant regeneration is applied in the field of plant tissue culture, which can solve the problems of affecting the quality of embryos, affecting the embryo production rate, inhibiting microspore culture and breeding technology, etc., so as to achieve the effects of improving utilization efficiency and improving embryo rate.

Inactive Publication Date: 2012-11-07
ZHEJIANG UNIV +1
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Problems solved by technology

A large number of studies on the culture of free microspores of Brassica have shown that the genotype of the donor plant is extremely important for the microspore embryogenesis, which not only affects the embryo production rate, but also affects the quality of the embryo (Chuong et al., 1988) , inhibiting the application of microspore culture breeding technology

Method used

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  • Culture method for brassica oleracea L. var. acephala microspore regeneration plant
  • Culture method for brassica oleracea L. var. acephala microspore regeneration plant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The cultivation method is carried out as follows.

[0038] (1) Culture medium preparation: comprise the culture medium of microspore different culture stages, its component and the weight that each component contains in every liter of medium are:

[0039] Table 1 NLN and MS medium composition table

[0040]

[0041]

[0042] 1) NLN-13 induction medium: NLN liquid medium 1L + sucrose 130g / L, pH6.0, filter sterilized;

[0043] 2) Embryoid body differentiation medium: MS medium + sucrose 20g / L, agar 10g / L, pH 6.0, high temperature and high humidity sterilization;

[0044] 3) Rooting medium: MS medium + sucrose 30g / L, agar 6g / L, pH5.8, high temperature and high humidity sterilization;

[0045] (2) Cultivation of the regenerated plants of microspores of kale with high germination rate:

[0046] 1) Selection of flower buds of donor plants: take healthy and pest-free inflorescences of kale and rapeseed as donor plants for microspore culture; after induction at a low t...

Embodiment 2

[0056] In addition to step (1) in 1) NLN-13 induction medium: NLN-13 liquid medium 1L + sucrose 130g / L, pH6.1, filter sterilization; 2) embryoid differentiation medium: MS medium + sucrose 20g / L, agar 11g / L, pH6.1, high temperature and high humidity sterilization; 3) Rooting medium: MS medium + white sugar 20g / L, agar 7g / L, pH5.9, high temperature and high humidity sterilization;

[0057] (2) Cultivation of the regenerated plants of microspores of kale with high germination rate:

[0058] 1) Selection of flower buds of donor plants: take healthy and pest-free inflorescences of kale and rapeseed as donor plants for microspore culture; after induction for 48 hours at a low temperature of 3°C, the length of petals and anthers on the inflorescences are taken at a ratio of 1.1. Flower buds from late mononucleate to early binucleate;

[0059] 2) Sterilization of flower buds: use 5.6% (mass percentage concentration) sodium hypochlorite aqueous solution 56ml / L+ absolute ethanol 100m...

Embodiment 3

[0068] In addition to step (1) in 1) NLN-13 induction medium: NLN-13 liquid medium 1L + sucrose 130g / L, pH6.2, filter sterilization; 2) embryoid differentiation medium: MS medium + sucrose 20g / L, agar 12g / L, pH6.0, high temperature and high humidity sterilization; 3) rooting medium: MS medium + sucrose 25g / L, agar 6.5g / L, pH6.0, high temperature and high humidity sterilization;

[0069] (2) Cultivation of the regenerated plants of microspores of kale with high germination rate:

[0070] 1) Selection of flower buds of donor plants: take the inflorescences of kale and rapeseed that grow healthily and are free of diseases and insect pests as donor plants for microspore culture; take flower buds with a petal to anther length ratio of 0.8 on the inflorescence, late mononucleate to early binucleate;

[0071] 2) Sterilization of flower buds: use 5.6% (mass percentage concentration) sodium hypochlorite aqueous solution 56ml / L+dehydrated alcohol 100ml / L+clean liquid 9 drops+sterile wa...

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Abstract

The invention discloses a culture method for a brassica oleracea L. var. acephala microspore regeneration plant. The method comprises the following steps: taking inflorescences of brassica oleracea L. var. acephala and rape, directly taking or taking after induction alabastrum of the inflorescences, adding the alabastrum into NLN-13 induced medium after disinfection to form fluid suspension, carrying out filtering, and centrifuging the filtrate to obtain a precipitate; adding NLN-13 induced medium and active carbon mixed liquor in order so as to obtain a microspore fluid suspension; carrying out a heat shock treatment, followed by culturing so as to obtain embryoids in cotyledon stage; inoculating the embryoids to embryoid differential medium until the embryoids are differentiated and regenerates into buds; cutting the regenerated buds to a rooting medium for rooting culture, and hardening and transplanting seedlings to obtain regenerated plants; taking young leaves of the regeneratedplants for the detection of ploidy of corresponding regenerated plants and determining regenerated seedlings of rape and brassica oleracea L. var. acephala in the regenerated plants. According to themethod provided in the invention, rape which is easy to generate embryos and brassica oleracea L. var. acephala which is difficult to generate embryos are mixedly cultured; the material which is easyto generate embryos is used to spur the material which is difficult to generate embryos; therefore the ratio of embryos of brassica oleracea L. var. acephala is improved.

Description

technical field [0001] The invention relates to the technical field of plant tissue culture, in particular to a method for cultivating kale microspore regeneration plants. Background technique [0002] Collard kale (Brassica oleracea L.var.acephala), also known as leaf peony, is a variety of Brassica oleracea, a 2-year-old herb with beautiful leaves and changeable colors. The whole plant is shaped like a peony flower , High ornamental value. In recent years, my country has imported kale from the United States, Japan, the Netherlands and other places, because kale contains a lot of vitamins A, C, and B. 2 And a variety of minerals, especially high in calcium, iron, and potassium, its nutritional value is much higher than that of ordinary cabbage, and it is mostly cultivated as a special vegetable. Because some kale varieties have bright and beautiful leaves, red, yellow, and green, with special shrinkage and different shapes, they can be used to decorate banquet tables, arr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01H4/00
Inventor 张振超周伟军潘跃平戴忠良刘旦
Owner ZHEJIANG UNIV
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