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Method for culturing regeneration plants of Brassica oleracea microspores

A technology for culturing and regenerating plants of microspores, which is applied in the field of plant tissue culture, can solve the problems of affecting the quality of embryos, affecting the embryo production rate, inhibiting the breeding technology of microspore culture, etc., and achieves the effects of improving utilization efficiency and improving embryo rate.

Inactive Publication Date: 2011-11-16
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

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  • Method for culturing regeneration plants of Brassica oleracea microspores

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The cultivation method is carried out as follows.

[0036] (1) Medium preparation: the medium including the different culture stages of microspores, the components and the weight of each component in each liter of medium are:

[0037] Table 1 NLN and MS medium formula table

[0038]

[0039] 1) NLN-13 induction medium: NLN liquid medium 1L + sucrose 130g / L, pH 6.0, filter sterilization;

[0040] 2) Embryoid body differentiation medium: MS medium + sucrose 20g / L, agar 10g / L, pH 6.0, high temperature and high humidity sterilization;

[0041] 3) Rooting medium: MS medium + sucrose 30g / L, agar 6g / L, pH 5.8, sterilized at high temperature and high humidity;

[0042] (2) Cultivation of regenerated plants of broccoli microspores with high embryo rate:

[0043] 1) The selection of the flower buds of the donor plants: the healthy, disease-free inflorescences of broccoli and rapeseed are used as the donor plants for microspore culture; after 24 hours of induction at 4°C, the length of the pe...

Embodiment 2

[0053] Except for step (1) in 1) NLN-13 induction medium: NLN-13 liquid medium 1L + sucrose 130g / L, pH 6.1, 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, pH 5.9, high temperature and high humidity sterilization;

[0054] (2) Cultivation of regenerated plants of broccoli microspores with high embryo rate:

[0055] 1) Selection of flower buds of donor plants: Take broccoli and rapeseed healthy, disease-free inflorescences as donor plants for microspore culture; after induction at 3°C ​​for 48 hours, the length of petals and anthers on the inflorescences is compared 1.2. Flower buds from late single nucleus to early dual nucleus;

[0056] 2) Sterilization of flower buds: use 5.6% (mass percentage concentration) sodium hypochlorite aqueous solution 56ml / L + absolute ethanol 100ml / L + detergent 8 drops + sterile water to...

Embodiment 3

[0065] Except for step (1) in 1) NLN-13 induction medium: NLN-13 liquid medium 1L + sucrose 130g / L, pH 6.2, filter sterilization; 2) embryoid body differentiation medium: MS medium + sucrose 20g / L, agar 12g / L, pH 6.0, high temperature and high humidity sterilization; 3) Rooting medium: MS medium + sucrose 25g / L, agar 6.5g / L, pH 5.6, high temperature and high humidity sterilization;

[0066] (2) Cultivation of regenerated plants of broccoli microspores with high embryo rate:

[0067] 1) Selection of flower buds of donor plants: take broccoli and rapeseed healthy, disease-free inflorescences as donor plants for microspore culture; take flower buds with a ratio of petals and anthers on the inflorescences of 0.9 in the late mononuclear to early dinuclear;

[0068] 2) Sterilization of flower buds: use 5.6% (mass percentage concentration) sodium hypochlorite aqueous solution 56ml / L + absolute ethanol 100ml / L + detergent 9 drops + sterile water to prepare sterile solution; mix rape and bro...

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Abstract

The invention discloses a method for culturing regeneration plants of Brassica oleracea microspores, which comprises the following steps of: taking flower buds from inflorescences of Brassica oleracea and Brassica napus directly or taking the flower buds from the inflorescences of the Brassica oleracea and the Brassica napus after inducing; sterilizing, adding an induced culture medium NLN-13 to prepare a suspension, filtering to obtain filter liquor, and centrifuging to obtain precipitates; adding the induced culture medium NLN-13 and mixed solution of active carbon sequentially to obtain a microspore suspension; performing heat activation, and culturing to obtain embryoid in the cotyledon stage; inoculating to an embryoid differential medium until the embryoid is differentiated to germinate; cutting regenerated sprouts, inoculating to a rooting culture medium to perform rooting culture, and hardening seedlings and transplanting to obtain the regeneration plants; and taking tender leaves of the regeneration plants to detect the ploidy of the regeneration plants, and identifying regenerated seedlings of the Brassica napus and regenerated seedlings of the Brassica oleracea from a regeneration plant colony. In the method, the Brassica napus of which the embryos are easy to germinate and the Brassica oleracea of which the embryos are difficult to germinate are mixed to be cultured, and materials of which the embryos are easy to germinate drives materials of which the embryos are difficult to germinate to germinate the embryos, so that the embryo growth rate of the Brassica oleracea is improved.

Description

Technical field [0001] The invention relates to the technical field of plant tissue culture, in particular to a method for culturing broccoli microspores to regenerate plants. Background technique [0002] Broccoli (B rassica oleracea L var.italica), also known as broccoli, green cauliflower, broccoli, etc., is one or two herbaceous plants in the Brassica spp. The green curd formed at the top of the main stem and lateral branches is the product. It is rich in nutrients, has good color, fragrance and taste. It is a famous and special vegetable that is very popular in the international market. At present, most of the main varieties of broccoli in my country are from abroad, and the varieties with our independent intellectual property rights are scarce. The reason is that our own broccoli breeding resources are few and we can't prepare good materials, so we urgently need to pass breeding The means of self-crossing and separation of varieties used in the market for many years. The t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00
Inventor 张振超周伟军庄义庆许玲耿鑫鑫
Owner ZHEJIANG UNIV
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