Method for culturing isolated microspore of common head cabbage to obtain regeneration plant
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A technology of microspore culture and head cabbage, applied in the biological field, can solve the problems of low efficiency and slow progress, and achieve the effect of improving the embryo rate
Inactive Publication Date: 2010-07-14
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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Problems solved by technology
The main problem is that the efficiency is low, the progress is slow, and there is still a certain distance from the application in actual breeding
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Embodiment 1
[0034] 1 microspore culture
[0035] 1.1 The experimental materials of head cabbage (Brassica oleracea var.capitata) were planted in the solar greenhouse of the Vegetable Research Institute of Jiangsu Academy of Agricultural Sciences; clean and non-polluted flower buds with a length of 2.1-6.0mm were taken at the early stage of flowering, and the flower buds were kept moist and placed in a refrigerator at 4°C. Pretreatment 1 ~ 3d.
[0036] 1.2 Disinfection Disinfect with 75% alcohol by volume for 60 seconds, then disinfect the surface with 8% sodium hypochlorite solution by mass for 15 minutes, rinse with sterile water 3 times and set aside.
[0037] 1.3 Separation of microspores Place the sterilized flower buds in a sterilized mortar, add a small amount of microspore free culture B5-13 liquid medium with a pH of 5.8 as the washing liquid, use the extrusion method to free the microspores, and then use 450 Mesh nylon mesh filter, the filtrate is collected in a centrifuge tube,...
Embodiment 2
[0047] 1 microspore culture
[0048] 1.1 The experimental materials of head cabbage (Brassica oleracea var. Refrigerator low temperature pretreatment 1d.
[0049] 1.2 Disinfection Disinfect with 75% alcohol by volume for 60 seconds, then disinfect the surface with 8% sodium hypochlorite solution by mass for 15 minutes, rinse with sterile water 3 times and set aside.
[0050] 1.3 Separation of microspores Place the sterilized flower buds in a sterilized mortar, add a small amount of microspore free culture B5-13 liquid medium with a pH of 5.8 as the washing liquid, use the extrusion method to free the microspores, and then use 450 Mesh nylon mesh filter, the filtrate is collected in a centrifuge tube, 1000r min -1 Centrifuge for 5 minutes, discard the supernatant, add microspore-free medium and centrifuge, repeat 3 times.
[0051] 1.4 Culture of microspores Suspend the washed microspores in liquid medium 1 (NLN-13 medium with halved macroelements + 0.05 mg·L -1 6-BA+20mg·L...
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Abstract
The invention discloses a method for culturing isolated microspore of a common head cabbage to obtain a regeneration plant, which belongs to the field of biotechnology. The method comprises the following steps: choosing a flower bud of appropriate length, pre-treating the flower bud under the low temperature of 4 DEG C for 24 hours, using the squeezing and pressing method to make the microspore isolated after sterilization, filtering and centrifuging the microspore, suspending the purified microspore in a liquid culture medium, and thermally pre-treating the microspore under the high temperature of 32.5 DEG C for 24 hours and transferring to dark cultivation under the temperature of 25 DEG C for 15-20 days; when a macroscopic embryoid emerges, placing in the sunshine for cultivation for 5-7 days until the emergence of a leaf type embryoid, after the greening of the embryoid, switching to a secondary culture medium for germination and transferring to a root culture medium to root, and obtaining the integrated regeneration plant; and then opening the bottle for hardening off, and hardening and transplanting into a culture pan with sterilization matrix. In case of using the method for cultivation, the embryoid with high frequency can be obtained, the obtained haploid regeneration plant is not only a good breeding material, but also an excellent material for research on molecular markers, genetic mapping, gene clone, trans gene and the like.
Description
1. Technical field [0001] The invention discloses a method for culturing free microspores of cabbage to obtain regenerated plants, which belongs to the field of biotechnology and is specially used for tissue culture to obtain regenerated plants. 2. Technical background [0002] Heading cabbage (Brassica oleracea var.capitata) is a biennial plant of the genus Brassica in the family Brassicaceae. It has a wide cultivation area in my country, strong adaptability, high nutritional value, and storage and transportation resistance. one of the main vegetables. At present, the hybrids used in production are mainly obtained through the isolation and purification of multi-generation self-crossing of materials imported from abroad, and the breeding of self-incompatible lines. Generally, 6 to 8 generations of separation are required. As cabbage is a green body vernalization crop, it is not easy to reproduce, so the breeding cycle is long, and there is a phenomenon of plant decline after...
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