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Method for inducing wild cabbage type cole in vitro microspores and screening mutant

A technology of brassica napus and microspores, which is applied in the field of plant cell engineering, can solve the problem of the theory and method research of in vitro mutagenesis of microspores without ultraviolet radiation, the correlation between irradiation time and dose rate and variation, and the distance of in vitro microspores. And the radiation time is different, to achieve the effect of convenient use of radiation source, saving time and cost, safety and security of operators

Inactive Publication Date: 2008-08-27
HUAZHONG AGRI UNIV
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  • Description
  • Claims
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Problems solved by technology

However, the UV lamp power and irradiation dose rate used by various researchers are different, and the distance and irradiation time of the isolated microspores from the UV lamp are different. The measured microspore half-lethal dose (LD) 50 ) exposure times vary greatly
Moreover, the correlation between irradiation time and dose rate and variation has not been studied, and it is not clear how much dose rate and how long irradiation time can be used to obtain more variation. So far, there has been no research on the theory and method of ultraviolet mutagenesis of isolated microspores. Systematic research, the technology is not yet mature, let alone programmed

Method used

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  • Method for inducing wild cabbage type cole in vitro microspores and screening mutant
  • Method for inducing wild cabbage type cole in vitro microspores and screening mutant
  • Method for inducing wild cabbage type cole in vitro microspores and screening mutant

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Isolation and In Vitro Mutagenesis of Microspores of Brassica napus

[0035] During the flowering stage of rapeseed, the main plants of Brassica napus varieties Zhongyou 821, Huashuang 5, Huashuang 2, Huahuang 1, and Huayou 8 (the above-mentioned varieties are publicly promoted in China) were taken from the test field. Take the stem or branch inflorescence to the laboratory, select the flower buds whose microspores have developed to the uninucleate late stage, and then soak the flower buds in 70% ethanol aqueous solution for 0.5-1 minute, and then soak them in 0.1% mercury liter aqueous solution for 8-10 minutes. Minutes (the following operations are all performed on a clean bench), and then rinsed with sterile water 3 times. Put the sterilized flower buds on the surface into a sterile test tube (the following utensils are all sterilized), crush them with a glass rod, and then add the microspore extract containing 13% sucrose to the test tube (according to the number of...

Embodiment 2

[0041] Chromosome doubling and embryoid body induction of microspores of Brassica napus

[0042] The rapeseed microspores suspended in colchicine NLN medium and irradiated with ultraviolet rays were cultured in the dark at 32°C for 2 days to perform chromosome doubling. Afterwards, the colchicine medium containing the microspores in the petri dish was transferred into a centrifuge tube with a pipette and centrifuged (500 rpm, 3 minutes) on the ultra-clean workbench to precipitate the microspores. Then use a straw to suck off the colchicine medium, add 6ml of colchicine-free modified NLN medium into a centrifuge tube, suspend the microspores, then pour them into a 7.5ml diameter petri dish, seal the lid with paraffin film, and put Culture in a culture room at about 25°C in the dark to induce embryoid bodies.

Embodiment 3

[0044] Induction of Regenerated Plants of Brassica napus

[0045] When the microspores cultured in the dark at about 25° C. produce small embryoid bodies visible to the naked eye (usually 10 days after the microspores are isolated), put the petri dish into a shaker for shaking culture (50 rpm). When the embryoid develops from the late torpedo stage to the early stage of the cotyledon, stop the shaking culture, transfer it to the B5 solid medium supplemented with 2% sucrose, and cultivate it in a culture room at about 25°C (12 hours of light per day, light intensity 3000LUX) , to induce embryoid body germination. When the embryoid bodies produced plantlets with leaves and stems or deformed plantlets, the roots of these plantlets were excised, and then transferred to B5 strong seedling medium supplemented with 0.12 mg / L paclobutrazol to cultivate robust regeneration plants.

[0046]The preparation of the above-mentioned B5 strong seedling medium: adopt the original B5 medium co...

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Abstract

The invention belongs to the technical field of plant cell engineering and in particular relates to a method for screening brassica napus in vitro microspores ultraviolet mutation and mutants, which is characterized in that the method comprises: using aqueous sucrose solution to separate brassica napus microspores, suspending the microspores on NLN culture medium which is modified through adding colchicine to be irradiated with ultraviolet and to be doubled with chromosome, transferring doubled microspores on the NLN culture medium which is modified to induce embryoid, then, transferring the embryoid on B5 culture medium to be induced into double haploid plants, transplanting the plants into filed to indentify, and screening out the mutants. The morphological character mutant rate is 10.53-21.57%, and the variant rates of sulfuric glucoside, the oil content and the erucic acid content are respectively 25.53-78.7%, 22.22-59.09% and 38.89-46.97%. The method of the invention has the advantages that the operation procedure is convenient, the time is saved, the cost is low, and the like. The method is suitable for screening the microspores ultraviolet mutation and mutants of cruciferae crops.

Description

technical field [0001] The invention belongs to the technical field of plant cell engineering, and in particular relates to a screening method for in vitro microspores and mutants of Brassica napus induced by ultraviolet rays Background technique [0002] Using physical radiation or chemical mutagens to mutate rape seeds or tissues, organs and single cells is one of the important methods to create new germplasm and improved varieties and to establish mutant libraries. However, the mutants produced by mutagenizing seeds, tissues and organs and multicellular callus are chimeras of mutant and non-mutated genes, and recessive gene mutations do not appear in contemporary times and cannot be identified. Therefore, the discovery and selection of stable mutants must be done through selfing, especially for traits controlled by recessive genes and polygenes, multiple generations of selfing are required to obtain homozygous, genetically stable mutants. The period of obtaining mutants ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/01C12N13/00C12N3/00A01H4/00
Inventor 石淑稳吴江生
Owner HUAZHONG AGRI UNIV
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