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Culture method of high diplont rate sporule regeneration plant of broccoli

A technology for regenerating plants and culturing methods, applied in the field of plant tissue culture, can solve the problem of low diploid rate of regenerated plants, and achieve the effects of increasing the diploid rate and the embryo rate.

Inactive Publication Date: 2011-09-07
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The object of the present invention is, aiming at the low defect of the diploid rate of regenerated plants existing in the existing cauliflower microspore culture technology system, to provide a kind of medium and culture method that can improve the diploid rate of cauliflower microspore regenerated plants, and Use the regenerated plants with high diploid rate as breeding parents to improve the process and efficiency of cauliflower breeding

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  • Culture method of high diplont rate sporule regeneration plant of broccoli

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Embodiment 1

[0027] Embodiment 1: (cultivation method 1 of cauliflower high diploid rate microspore regeneration plant)

[0028] The method proceeds as follows:

[0029] (1) The preparation of culture medium: comprise the culture medium of each stage of microspore culture, their component and each component contain weight in every liter of medium:

[0030] 1) Flower bud pretreatment medium: NLN-13 liquid medium + 130 g / L sucrose + 0.1% colchicine + 2% DMSO, pH 5.6, filter sterilized. The formula of NLN-13 medium is shown in Table 1;

[0031] 2) Embryoid body induction medium: NLN-13 liquid medium + sucrose 130g / L, pH5.8, filter sterilized;

[0032] 3) Embryoid body differentiation medium: B 5 Liquid medium+trans-ZT 1.0mg / L+IAA 0.1mg / L+BAP 1.0mg / L+xylose 250mg / L+white sugar 30g / L+agarose 3g / L, pH 5.6, high temperature sterilization; where B 5 The medium formula is shown in Table 1;

[0033] 4) germination, rooting medium: B 5 Liquid medium + sugar 30g / L + agar 8g / L, pH6.0, high temper...

Embodiment 2

[0046] Embodiment 2: (cultivation method 2 of cauliflower high diploid rate microspore regeneration plant)

[0047] In this embodiment, the bud pretreatment medium is NLN-13 liquid medium + sugar 130g / L + 0.05% colchicine + 3% DMSO, pH is 6.0; select 15 single-nucleus mid-term flower buds, petals and anthers The length ratio is 0.6; the flower buds are cultured in a petri dish containing flower bud pretreatment medium at 4°C in the refrigerator for 5 days; mL of activated carbon mixture prepared by NLN-13 liquid medium + agarose 5g / L + 1g / L activated carbon and sterilized at high temperature, mixed into microspore suspension; The dishes were divided into sterile plastic petri dishes with a diameter of 6 mm, a total of 10 dishes, and sealed with parafilm film after being covered; culture dishes were placed at 33°C for 1 day; the obtained embryoid bodies were placed in embryoid body differentiation Medium B 5 +trans-ZT 0.5mg / L+IAA 0.05mg / L+BAP 0.5mg / L+xylose 500mg / L+sucrose 20...

Embodiment 3

[0048] Embodiment 3: (cultivation method 3 of cauliflower high diploid rate microspore regeneration plant)

[0049] The bud pretreatment medium of this embodiment is NLN-13 liquid medium + sucrose 130g / L + 0.2% colchicine + 1% DMSO, pH is 5.8; select 15 uninucleate late flower buds, the length ratio of petals and anthers is 1.0 ; Flower buds were cultured in a petri dish containing flower bud pretreatment medium at 4°C in a refrigerator for 1 day; after the microspores were isolated, 40 mL of embryoid body induction medium was added with a pH of 6.0, and 0.5 mL of NLN- 13 liquid culture medium + agarose 2g / L + 1g / L activated carbon, the activated carbon mixture prepared by high temperature sterilization, mixed into microspore suspension; the microspore suspension was divided into 4mL / dish In a sterile glass culture dish with a diameter of 9mm, 10 dishes in total, cover with a parafilm film; culture the culture solution at 31°C for 3 days; place the obtained embryoid bodies in ...

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Abstract

The invention discloses a culture method of high diplont rate sporule regeneration plant of broccoli, which belongs to the technical field of plant tissue culture and comprises the following steps: (1), preparing a culture medium; (2), culturing the high diplont rate sporule regeneration plant of broccoli: 1), selecting a donor plant and a bud, 2), sterilizing the bud, 3), pre-treating and culturing the bud, 4), separating, mixing and subpackaging of bud sporule, 5), culturing sporule embryoid, 6), differentiating and germinating and culturing a regeneration plant, 7), rooting and transplanting the regeneration plant and 8), detecting ploidy of the regeneration plant. The invention obviously increases the germ extraction rate and the rate of emergence of the sporule culture of the broccoli and the diplont rate of the regeneration plant respectively to 80 embryos / bud, 70 percent and more than 70 percent from common 60 embryos / bud, 50 percent and about 50 percent, thereby improving the breeding efficiency of the broccoli. The method can be popularized and applied to a vegetable breeding department or company.

Description

technical field [0001] The invention relates to the technical field of plant tissue culture, in particular to a method for cultivating cauliflower microspore regeneration plants with high diploid rate. Background technique [0002] Cauliflower (Brassica oleracea L.var.botrytis) is widely cultivated all over the world because of its delicious taste and rich nutrition. The breeding cycle of traditional breeding of new cauliflower hybrid varieties generally takes 6 to 8 years, of which 5 to 6 years are required for the breeding of parent materials. The parent material of diploid regenerated plants can be quickly created by using the microspore culture method, which can significantly improve the breeding efficiency and shorten the breeding cycle to 3-5 years. [0003] In the past, there have been reports on the culture technology of cauliflower microspores at home and abroad. In 1992, the cultivation of cauliflower microspores was first successful (Duijs JC, et al. Euphytica, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01G31/00A01H4/00
Inventor 顾宏辉朱丹华虞慧芳赵振卿盛小光王建升张晓辉
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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