Method for quickly and efficiently obtaining free microspores of Brassica vegetables and cultivating regenerated plants

A technology for culturing microspores and regenerating plants is applied in the field of plant tissue culture, which can solve the problems of low seedling efficiency and save time.

Active Publication Date: 2016-05-18
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a method for quickly and efficiently obtaining free microspores of Brassica vegetables to cultivate and regenerate plants for the problem of low seedling efficiency in the prior art obtained from free microspores for regenerated plants

Method used

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  • Method for quickly and efficiently obtaining free microspores of Brassica vegetables and cultivating regenerated plants
  • Method for quickly and efficiently obtaining free microspores of Brassica vegetables and cultivating regenerated plants
  • Method for quickly and efficiently obtaining free microspores of Brassica vegetables and cultivating regenerated plants

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The preparation of embodiment 1 culture medium

[0043] 1. Preparation of mother liquor

[0044] Before the experiment, the macroelements, microelements, organic elements and iron salts of the various culture media that need to be used in the method of the present invention are formulated into mother liquor and stored in 4 ℃ of refrigerators, and the preparation concentration is respectively: 20 times (hereinafter used 20X), 200 times (hereinafter expressed as 200X), 100 times (hereinafter expressed as 100X) and 100 times (hereinafter expressed as 100X), the specific components are shown in Table 1.

[0045] Various mother solution formulas of free microspore culture in table 1

[0046]

[0047]

[0048] 2. Preparation and sterilization of medium

[0049] ① Microspore medium (1 / 2NLN-13): 1 / 2NLN+13% sucrose, pH 6.0-6.2;

[0050] The culture medium was sterilized by suction filtration with a 0.22 μm microporous membrane;

[0051] ②Microspore extract (B 5 -13): ...

Embodiment 2

[0059] Example 2 Culture of free microspores of Brassica vegetables and induction of embryos

[0060] 1. Genotype: non-heading Chinese cabbage NHCC-549; broccoli A36-5, A47-4, A48-3.

[0061] Non-heading Chinese cabbage NHCC-549 - a hybrid of "Maertou" and "Komatsucai" (from "Maertou" as the female parent and "Komatsucai" as the male parent by conventional methods); Broccoli A36- 5—the product name is "Excellent No. 1"; broccoli A47-4—the product name is "Jiulu"; broccoli A48-3—the product name is "Fudao". All were purchased from Mingda Seed Management Department of Jiangsu Academy of Agricultural Sciences.

[0062] 2. Selection of flower buds: at the initial flowering stage and the full flowering stage, determine the developmental stage of the microspores and the corresponding relationship with the length of the flower buds through the microscopic examination of the acetic acid magenta tablet method, and accurately find the flower buds whose developmental stage is the mononu...

Embodiment 3

[0074] Example 3: Inducing embryoid bodies to obtain regenerated shoots

[0075] 1. Shaking culture: transfer the embryoid body obtained in Example 2 to 2000lx, 25°C, 60r / min shaker for shaking culture; 2. Induce the embryoid body to obtain regenerated buds: transfer the cotyledon-type embryos that have been turned green for the same number of days Transfer to two B5 solid media containing 2.5% sucrose, 1% agar powder and 2.5% sucrose, 0.8% agar powder (ck) to continue culturing for 15-20 days. The culture conditions are 90uE / m2 / s, 12hr / d. A culture room at 25°C. Observe its growth and make statistical records.

[0076] The implementation results are as follows:

[0077] The results in Table 3 show that: ① NHCC-549 and A48-3 were almost completely browned on the B5 solid medium containing 0.8% agar, and the embryogenesis rate was extremely low, but on the B5 solid medium containing 1% agar, they all induced The germination rates of regenerated buds were 0.50 and 0.33 respe...

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Abstract

The invention discloses a method for rapidly and efficiently obtaining regeneration plants by culturing the free microspores of brassica vegetables. The method comprises the following steps: transferring microspore embryoids into a shaking table at 2000 lx (Lux), 25 DEG C and 60 r / min (Revolutions Per Minute) for shaking culturing at the time of the generation of the microspore embryoids; after 3 to 7 days, transferring cytoledon-stage embryos turning green into a B5 solid culture medium containing 2.5% of saccharose and 1% of agar powder for continuous culturing so as to obtain regeneration buds; after 15 to 20 days, transferring the regeneration buds into a MS (Murashige Skoog) solid culture medium containing 10% of coconut juice, 3% of saccharose and 0.78% of agar powder; after 20 to 30 days, obtaining results that the majority of plants grow strongly and the rooted seedlings can be domesticated and transplanted, wherein all the culturing conditions are carried out at 90uE / m<2> / s, 12hr / d and 25 DEG C. The average planting percent of the regeneration plants can reach 83.5%. As the method is used, the problem that the microspore embryoids are easy to brown and vitrify is effectively solved; the time of obtaining the DH (Double Haploid) plants by culturing the free microspores is effectively shortened; the DH plants are safer because the addition of any hormones in the DH plants is not required.

Description

technical field [0001] The invention relates to a method for quickly and efficiently obtaining free microspores of Brassica vegetables to cultivate and regenerate plants, and belongs to the technical field of plant tissue culture. Background technique [0002] Brassica vegetables are cross-pollinated plants with obvious heterosis. It takes 5 to 7 years to obtain a pure line in breeding, but free microspore culture can obtain double haploid plants in 1 to 2 years, which greatly saves purification. time and workload. At the same time, the existence of haploid in the regenerated population is also conducive to the expression of recessive traits, which enriches the breeding resources. In addition, free microspores are single cells, which have special advantages in mutagenesis breeding, mutant screening, and gene transformation. DH populations obtained by spontaneous or artificial doubling are ideal materials for molecular markers and gene mapping. [0003] Free microspore cult...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01H4/00
Inventor 李英刘环环侯喜林刘同坤
Owner NANJING AGRICULTURAL UNIVERSITY
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