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High-frequency Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee in-vitro tissue culture method

A technology of tissue culture and Chinese cabbage, applied in horticultural methods, botany equipment and methods, horticulture, etc., can solve the problems affecting the application of genetic engineering, Agrobacterium is not easily infected, and the regeneration of adventitious buds is difficult, so as to achieve simple operation and easy formation , The effect of high regeneration rate

Active Publication Date: 2020-10-02
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The cabbage crops carrying the AA genotype belong to the Brassica species with a relatively low regeneration rate. It is difficult to regenerate adventitious buds. Agrobacterium is not easy to infect and the cells are not easy to differentiate after infection, which affects the application of genetic engineering in the improvement of cabbage varieties.

Method used

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  • High-frequency Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee in-vitro tissue culture method
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  • High-frequency Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee in-vitro tissue culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The influence of different seedling ages of embodiment 1 on the differentiation of adventitious buds

[0036] 1. Materials

[0037] The variety of Chinese cabbage tested was "Youlu 501", which was bred by the Guangzhou Academy of Agricultural Sciences, and the seeds were self-bred for multiple generations in our laboratory.

[0038] 2. Test method

[0039] 2.1 Obtaining sterile vaccines

[0040] Select the full-grained cabbage seeds and put them into a centrifuge tube, rinse them twice with sterile water, soak them in 75% ethanol solution for 2 minutes, wash them twice with sterile water, soak them in 7.5% NaClO for 10 minutes, and finally Rinse with sterile water three times and place on filter paper, blot the excess water, inoculate the seeds evenly on the seeding medium (1 / 2MS medium+10g / L sucrose+6g / L agar, pH=5.9), every 50-80 capsules in a bottle. The environmental conditions are 25°C, 16h light / 8h dark, light use 110μmol / m 2 the s 1 white LED lights.

[00...

Embodiment 2

[0048] The influence of embodiment 2 different explant types on adventitious bud differentiation

[0049] 1. Material: with embodiment 1;

[0050] 2. Test method

[0051] 2.1 The acquisition of sterile vaccine: same as Example 1;

[0052] 2.2 The effect of different explant types on the differentiation of adventitious buds

[0053] Select aseptic seedlings of Chinese cabbage with a seedling age of 3 days, take cotyledon, cotyledon with handle (the length of the petiole of the cotyledon with handle is 0.5 cm), and stem segment (length 1-1.5 cm) as explants, and inoculate them in the differentiation stage. Medium (MS+0.3mg / L NAA+5mg / L 6-BA+30g / L sucrose+8g / L agar+2mg / L AgNO 3, pH=5.8). The cotyledon and cotyledon with handle are inserted vertically, and both ends of the stem are in contact with the culture medium. They are placed in a tissue culture room and cultured for 40 days. 2 the s 1 The white LED light, record the growth every 10d. Adventitious bud regeneration rat...

Embodiment 3

[0059] The influence of embodiment 3 different differentiation medium formulations on adventitious bud differentiation

[0060] 1. Material: with embodiment 1;

[0061] 2. Test method

[0062] 2.1 The acquisition of sterile vaccine: same as Example 1;

[0063] 2.2 Effects of Different Differentiation Medium Formulas on Adventitious Bud Differentiation

[0064] Different medium formulations from different 6-BA / NAA ratios, different osmotic pressures, different agar concentrations and different AgNO 3 Concentration is dealt with in four areas. Select the cotyledon with handle (the length of the cotyledon petiole of the handle cotyledon is 0.5cm) of seedling age 3d sterile seedlings as explants, inoculate in (1) containing 6-BA / NAA different concentration ratio (5:0.1, 5 :0.3, 5:0.5, 3:0.3, 7:0.3) in the differentiation medium, (2) in the differentiation medium adding 20, 30, 40, 50g / L sucrose, (3) adding 0, 1, 2 , 4mg / LAgNO 3 (4) Add 6, 8, 12, 14g / L agar in the differentia...

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Abstract

The invention relates to the application field of cell engineering, in particular to a high-frequency Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee in-vitro tissue culture method. According to the method, seeds of Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee are subjected to surface disinfection and then placed on a sowing culture medium for culture to obtain sterile seedlings with a seedling age of 3-5 d, then cotyledons with petiole are stably cut above growth points as explants, and the explants are vertically inserted into a differentiation culture medium for differentiation culture to obtain adventitious buds; the adventitious buds are transferred to a rooting culture medium for rooting culture to obtain tissue culture seedlings; and the tissue cultureseedlings are subjected to bottle opening and seedling hardening, and the follow-up routine management is performed. The Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee high-frequency regeneration system established by the invention has high adventitious bud regeneration rate, easy formation of callus, low pollution rate, simple and convenient operation, and high comprehensive performance, and a foundation for the establishment of a Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee genetic transformation system is laid.

Description

technical field [0001] The invention relates to the application field of cell engineering, in particular to a method for culturing high-frequency cabbage heart isolated tissue. Background technique [0002] Caixin (Brassica campestris L.ssp.chinensis var.utilis Tsen et Lee) is a variety of Brassicaceae Brassica subspecies. It is the vegetable with the largest cultivation area and yield in South China. Caixin takes cabbage as the main edible organ. It is tender in quality, unique in flavor and rich in nutrition, and is loved by consumers. It has wide adaptability, short growth cycle, and can be used for annual production. It occupies an important position in vegetable supply. [0003] It is urgent to establish a stable and efficient regeneration system to obtain excellent germplasm resources for cultivating new varieties of Chinese cabbage that are resistant to diseases and insect pests and have other excellent traits. This just needs to select suitable explant and the mediu...

Claims

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Application Information

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IPC IPC(8): A01H4/00
CPCA01H4/001A01H4/008
Inventor 宋世威梁雯雯苏蔚郝彦伟陈日远刘厚诚孙光闻
Owner SOUTH CHINA AGRI UNIV
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