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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 problems of non-segregation of offspring, inhibiting utilization efficiency, etc., and achieve the effect of increasing the diploid rate

Active Publication Date: 2010-01-06
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the regenerated plants of broccoli obtained through microspore culture have several types such as haploid, diploid, triploid, tetraploid and chimera. The occurrence frequency of diploid regenerated plants required for non-segregation and breeding is only about 50%, which seriously inhibits the utilization efficiency of breeding by microspores

Method used

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

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Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1: (cultivation method 1 of broccoli high diploid rate microspore regeneration plant)

[0027] The cultivation method is carried out as follows:

[0028] (1) preparation of medium, including the medium of each stage of microspore culture, their components and the weight contained in each component in every liter of medium are:

[0029] 1) Flower bud pretreatment medium: B 5 Liquid medium + 0.1% colchicine + 2% DMSO, sugar 130g / L, pH5.8, filter sterilized;

[0030] Among them, B 5 The formula of liquid medium is shown in Table 1;

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

[0032] Wherein, the formula of NLN-13 liquid medium is shown in Table 1;

[0033] 3) Embryoid differentiation medium: MS medium + NAA 0.05mg / L + BAP 1.0mg / L, sugar 25g / L, agar 8g / L, pH 5.8, high temperature sterilization;

[0034] Among them, the formula of MS medium is shown in Table 1;

[0035] Table 1 NLN-13, B 5 an...

Embodiment 2

[0048] Embodiment 2: (cultivation method 2 of broccoli high diploid rate microspore regeneration plant)

[0049] In this embodiment, the flower bud pretreatment medium is B 5 Liquid medium + sucrose 130g / L + 0.05% colchicine + 3% DMSO, the pH is 6.0; select 10 mononuclear mid-term flower buds, and the length ratio of petals to anthers is 0.6; the flower buds are placed in the petri dish of flower bud pretreatment medium Cultivate for 4 days in a refrigerator at 4°C; separate the pretreated flower buds from microspores, centrifuge at 850 rpm for 5 minutes, discard the supernatant, add 40 mL of embryoid body induction medium with a pH of 5.6, and add 0.5 mL of NLN-13 liquid medium + agarose 5g / L+1g / L activated carbon preparation, the activated carbon mixture obtained by high temperature sterilization, mixed into microspore suspension; the microspore suspension was divided into 4mL / per dish Put them in sterile plastic culture dishes with a diameter of 6 mm, 10 dishes in total, c...

Embodiment 3

[0050] Embodiment 3: (cultivation method 3 of broccoli high diploid rate microspore regeneration plant)

[0051] In this embodiment, the flower bud pretreatment medium is B 5 Liquid medium + sucrose 130g / L + 0.2% colchicine + 1% DMSO, the pH is 5.6; select 10 uninucleated middle and late flower buds, the ratio of the length of petals to anthers is 1.0; Cultivate in a petri dish at 4°C in a refrigerator for 2 days; separate the microspores from the pretreated flower buds, centrifuge at 900 rpm for 4 minutes, discard the supernatant, add 40 mL of embryoid body induction medium, pH 6.0, and then add 0.5mL of activated carbon mixture prepared by NLN-13 liquid medium + agarose 2g / L + 1g / L activated carbon, sterilized at high temperature, mixed into microspore suspension; press 4mL / Each dish was divided into sterile glass petri dishes with a diameter of 6mm, a total of 10 dishes, sealed with parafilm film after being covered; the culture solution was placed at 32.5°C for 2 days; t...

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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 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 120 embryos / bud, 70 percent and more than 70 percent from common 80 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 broccoli microspore regeneration plants with high diploid rate. Background technique [0002] Broccoli (Brassica oleracea L.var.italica), also known as broccoli and green cauliflower, is rich in nutrients and ranks in the forefront of the top ten anti-cancer vegetables, and is favored by consumers. At present, most of the seeds used for the production of broccoli in my country come from abroad, and the seeds are expensive. Because the period of routine breeding of new broccoli hybrid varieties and self-bred parents is long, generally F 1 It takes 5 to 6 generations to breed the homozygous parents of the first generation of hybrids, and it takes 6 to 8 years to obtain new hybrid varieties by retesting and matching combinations. However, the parents of diploid regenerated plants can be quickly obtained by using microspore culture technology, t...

Claims

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Application Information

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IPC IPC(8): A01H4/00A01G31/00
Inventor 顾宏辉虞慧芳赵振卿王建升盛小光张晓辉
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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