Method for improving embryogenesis efficiency and plant regeneration efficiency of stem nodule mustard microspore embryo

A technology for embryogenesis and regeneration efficiency, applied in plant regeneration, botany equipment and methods, horticultural methods, etc., can solve problems such as vacancy of microspore culture technology

Active Publication Date: 2013-10-02
NINGBO ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the differences in regional cultivation of stem tuber mustard, the microspore culture technology of stem tuber mustard is still vacant in China

Method used

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  • Method for improving embryogenesis efficiency and plant regeneration efficiency of stem nodule mustard microspore embryo
  • Method for improving embryogenesis efficiency and plant regeneration efficiency of stem nodule mustard microspore embryo
  • Method for improving embryogenesis efficiency and plant regeneration efficiency of stem nodule mustard microspore embryo

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Experimental program
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Effect test

Embodiment 1

[0031] The preparation of embodiment 1 culture medium

[0032] 1. Preparation of mother liquor

[0033] Before the experiment, the culture medium required in the method of the present invention is prepared into a mother liquor and stored in a 4°C freezer. It is divided into a large amount, four kinds of iron salt, trace and organic, and its preparation concentration is respectively 20 times, 20 times, 200 times and 200 times, the specific composition is shown in Table 1.

[0034] Various mother liquor formulas of table 1 microspore culture

[0035]

[0036]

[0037] 2. Preparation of B5-13 medium

[0038] In this operation, it is a 1L medium formula, and the operation is as follows: add 50ml, 50ml, 5ml and Mix 5ml into 600ml distilled water and mix well. Then add 0.1g inositol and 130g sucrose in sequence, stir with a stirrer (domestic GL3250A) until completely dissolved, set the volume to 1000ml, and adjust the pH value to 5.75-5.80 with a pH meter (Mettler FE20K). D...

Embodiment 2

[0045] The selection of embodiment 2 microspore development period

[0046] In this embodiment, take the stem mustard material Yongzai No. 2 (Y04), Qingdao mustard mustard (Y06), Zhongdu mustard (Y08), and Yongzai No. 2╳Qingdao mustard (Y09) (Yongzai used in the present invention Both No. 2 and Qingdao mustard mustard can be bought in the market. Yongzae No. 2╳Qingdao mustard is a variety available in the market for hybridization (F1 generation) of the main inflorescence flower buds, and the anthers of 2mm, 3mm, and 4mm flower buds were respectively taken for 1% Acetate magenta (mix 1 g of magenta, 45 ml of glacial acetic acid and 55 ml of distilled water, boil for 2 hours, filter with gauze after cooling, add 1 drop of 4% iron alum) and observe under a microscope (Olympus, Japan). Within one week of the flowering period, the proportion of flower buds with a length of 2-3mm in the single-core side stage and the double-core stage reached 80%-90%.

Embodiment 3

[0047] The separation of embodiment 3 free microspores and the induced embryo

[0048] (1) Material collection period: The initial flowers appeared as 2-3mm flower buds within one week of flowering on the first day of the first flowering stage (refer to Example 2) for isolation of free microspores.

[0049] (2) Material sterilization method: Since the flowering period of the stem mustard material is more likely to be in the rainy weather, the humidity of the flower buds affects the efficiency of sterilization. In this experiment, 0.1% HgCl 2 Comparing the sterilization time of 8 and 12 minutes, it is found that the contamination rate of materials sterilized for 8 minutes is 85%, and when the time increases to 12 minutes, the contamination rate is 10%. Therefore, the sterilization time in rainy weather can be increased to 12 minutes as the best.

[0050] (3) Cleaning: Place the flower buds of the material in step (2) into sterile water and wash three times, 3 minutes each time...

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Abstract

The invention discloses a method for improving the embryogenesis efficiency and the plant regeneration efficiency of a stem nodule mustard microspore embryo. The method comprises the following steps of: (1) selecting materials, namely observing flower buds by a microscope, and selecting the flower buds with the diameters of 2-3 mm; (2) performing sterilization, namely performing sterilization by adopting 0.1 percent of mercury bichloride for 12 min, and washing the flower buds by germfree water for three times; (3) extracting pollens, namely adding a B5-13 liquid culture medium for grinding and filtration, and performing centrifugation; (4) performing heat-activation treatment, namely transferring sedimented pollens into an NLN-16 liquid culture medium containing a colchicine solution, culturing the pollens in the dark at the temperature of 32 DEG C for 48-52 hours; (5) performing induction to obtain the embryo, namely performing centrifugation, adding the NLN-13 liquid culture medium, packaging the pollens into a culture dish according to a unit of 1.5 flower buds per culture dish, and culturing the flower buds in the dark at the temperature of 25 DEG C; and (6) performing induction to obtain seedlings, namely after the embryo is formed, transferring the embryo into a B5-3 solid culture medium for culture. The invention creates a novel method of a stem nodule mustard free microspore culture technology and has a wide application prospect in breeding of stem nodule mustards.

Description

technical field [0001] The invention relates to the field of plant breeding, in particular to a method for improving the microspore embryogenesis and plant regeneration efficiency of tuber mustard ( mustard mustard ) . Background technique [0002] Rapidly obtaining homozygous double haploid plants through microspore culture can effectively improve breeding efficiency and speed up breeding, and has always been an important means in breeding. Since Japan’s Sato et al first used free microspore culture technology to obtain double haploid plants of Chinese cabbage in 1989, Lichter, Takahata et al. have successively achieved success in the microspore culture of kale and head cabbage. In 1991, duijs succeeded in culturing microspores of broccoli. The microspore culture of Brassica in China has also been carried out since the 1990s. The free microspore technology of Brassica cabbage and Brassica napus has been continuously developed. progress. [0003] Although microspore techn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00
Inventor 杨明贵王毓洪任锡亮孟秋峰
Owner NINGBO ACAD OF AGRI SCI
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