Method for increasing incidence rate of isolated microspore embryos of Brassica campestris var. peruviridis

A technology of microspores and occurrence rate, applied in the biological field, can solve the problems of low culture efficiency and achieve the effect of improving the embryo formation rate

Inactive Publication Date: 2013-05-29
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The object of the present invention is to provide a kind of method that improves the free microspore embryogenesis rate of little Chinese cabbage for the low problem of the culture efficiency that the free microspore of Chinese cabbage exists in the above-mentioned prior art

Method used

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  • Method for increasing incidence rate of isolated microspore embryos of Brassica campestris var. peruviridis
  • Method for increasing incidence rate of isolated microspore embryos of Brassica campestris var. peruviridis
  • Method for increasing incidence rate of isolated microspore embryos of Brassica campestris var. peruviridis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The method for improving the induction rate of embryos cultured with free microspores of Chinese cabbage includes:

[0021] 1 Genotype: NHCC Autumn-631; NHCC Autumn-632; NHCC Autumn-634; NHCC Autumn-635; NHCC Autumn-636.

[0022] 2 Configuration and sterilization of medium

[0023] The basic medium is NLN medium with halved macroelements, that is, 1 / 2NLN medium;

[0024] ① High-sugar medium (1 / 2NLN-17): 1 / 2NLN+17% sucrose+0.8mg / L colchicine, pH 6.0;

[0025] ②Low-sugar medium (1 / 2NLN-13): 1 / 2NLN+13% sucrose+0.8g / L activated carbon with a pH of 6.0;

[0026] The above medium was sterilized by suction filtration through a 0.22 μm microporous membrane.

[0027] ③ microspore extract (B 5 -13 medium): B 5 Basic medium + 13% sucrose, pH 5.8;

[0028] ④Differentiation medium: B 5 Basic medium+13% sucrose+0.2mg / L 6-BA+0.02mg / L NAA+0.8% agar+0.5g / L activated carbon, pH 5.8;

[0029] The microspore extract and differentiation culture above are sterilized at 121°C and 1.1M...

Embodiment 2

[0042] The method for improving the induction rate of embryos cultured with free microspores of Chinese cabbage includes:

[0043] 1 Genotype: NHCC Autumn-633; NHCC Autumn-607; Elysium.

[0044] 2 Materials collection: Flower buds with a length of 2-3mm were selected in January 2011, and the flower buds were kept moist and stored in a refrigerator at 4°C for 24 hours.

[0045] 3 Disinfection: The selected flower buds are disinfected with 75% alcohol for 30s, and then sterilized with 0.1% HgCl 2 Disinfect for 6 minutes, and finally rinse with sterile water 3 times for use;

[0046] 4 Separation and purification of microspores: put the sterilized flower buds in a mortar, add microspore extract (B 5-13 culture medium), the microspores were dissociated by the extrusion method, then filtered with a 400-mesh filter, the filtrate was collected in a centrifuge tube, centrifuged at 800rpm for 5min, the supernatant was discarded, and repeated 3 times;

[0047] 5 Suspend the purified ...

Embodiment 3

[0062] The method for improving the induction rate of embryos cultured with free microspores of Chinese cabbage includes:

[0063] 1 Genotype: NHCC Autumn-633.

[0064] 2 Materials collection: Flower buds with a length of 2-3mm were selected in January 2011, and the flower buds were kept moist and stored in a refrigerator at 4°C for 24 hours.

[0065] 3 Disinfection: The selected flower buds are disinfected with 75% alcohol for 30s, and then sterilized with 0.1% HgCl 2 Disinfect for 6 minutes, and finally rinse with sterile water 3 times for use;

[0066] 4 Separation and purification of microspores: put the sterilized flower buds in a mortar, add microspore extract (B 5 -13 culture medium), the microspores were dissociated by the extrusion method, then filtered with a 400-mesh filter, the filtrate was collected in a centrifuge tube, centrifuged at 800rpm for 5min, the supernatant was discarded, and repeated 3 times;

[0067] 5. High sugar and colchicine treatment: suspend ...

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Abstract

The invention discloses a method for increasing the incidence rate of isolated microspore embryos of Brassica campestris var. peruviridis. According to the method, buds of Brassica campestris var. peruviridis in a late uninucleate stage are selected and disinfected; microspores are separated from the buds and purified; purified microspores are suspended in a culture medium with high glucose; the density of the microspores is adjusted to 1*10<5> to 1*10<6> / ml; the culture medium is treated in darkness at a temperature of 33 to 35 DEG C for 12 to 24 h; a supernatant obtained after centrifugation is discarded, and then a rest product of centrifugation is added into a culture medium with low glucose so as to obtain a suspension of the microspores; macroscopic embryoids appear when the suspension of the microspores is treated in darkness for 10 to 14 d, and the suspension of the microspores is cultured in a shaking incubator; when cotyledon stage embryos appear, the suspension of the microspores is immediately placed in the sunlight so as to enable the cotyledon stage embryos to turn green, and then the cotyledon stage embryos are inoculated on a differential medium for subculture so as to enable a regenerated plant to form. An improved culture method for microspores provided in the invention enables the variety of Brassica campestris var. peruviridis which has no response to routine culture to generate embryoids, and a success rate of a variety with an induced genotype reaches 60%.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for increasing the embryonic rate of free microspores of sage cabbage, which is specially used for the cultivation of free microspores of twig cabbage. technical background [0002] Small cabbage (Brassica campestris var. peruviridis) is a biennial herb of the family Cruciferae, and is a variety of non-heading Chinese cabbage. Originated in Asia, it has been systematically improved in Japan to form a variety with unique flavor. It is eaten from young plants, tender in quality, delicious in taste, emerald green in color after cooking, high in nutritional value, surpassing spinach, which is the first green leafy vegetable, and has the health care function of preventing cancer. It is supplied on the Japanese market every year and has become one of the most sold vegetable varieties. my country's Ningbo City began large-scale planting in 2002, and it has become one of the main fo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01H4/00
Inventor 侯喜林于文佳
Owner NANJING AGRICULTURAL UNIVERSITY
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