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Method for establishing hevea brasiliensis embryogenic cell pure line with high somatic embryogenic capacity and maintaining embryogenic form of hevea brasiliensis embryogenic cell pure line

An embryogenic cell and rubber tree technology, applied in the field of plant tissue culture and cell engineering, can solve the problems of low frequency of embryogenic callus, loss of embryogenicity, restriction of genetic transformation of rubber tree, etc., and achieve somatic embryo induction rate and induction coefficient. The effect of improving, high somatic embryogenesis, good source of material

Pending Publication Date: 2022-07-08
RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the prior art, the frequency of obtaining embryogenic callus by the above two methods is not high and takes a long time, and the degree of heterogeneity of embryogenic callus in the subculture process is high, and the ability of somatic embryogenesis increases with the number of subcultures. Usually, the embryonicity will be gradually lost after 2-3 years of subculture, which greatly limits the development and follow-up research of rubber tree genetic transformation and protoplast culture research.

Method used

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  • Method for establishing hevea brasiliensis embryogenic cell pure line with high somatic embryogenic capacity and maintaining embryogenic form of hevea brasiliensis embryogenic cell pure line
  • Method for establishing hevea brasiliensis embryogenic cell pure line with high somatic embryogenic capacity and maintaining embryogenic form of hevea brasiliensis embryogenic cell pure line
  • Method for establishing hevea brasiliensis embryogenic cell pure line with high somatic embryogenic capacity and maintaining embryogenic form of hevea brasiliensis embryogenic cell pure line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] A method for establishing a pure line of rubber tree embryogenic cells with high somatic embryogenic capacity:

[0058] (1) Induction of primary callus: Select male flower buds of Reken 525 that are yellow-green immature, rinse with running water for 20 minutes, place them on an ultra-clean workbench, soak and disinfect with 75% (v / v) alcohol for 60s, and use After rinsing once with sterile water, disinfect with 0.1% mercuric chloride aqueous solution by mass for 10 minutes, and rinse with sterile water for 5 times, 3 minutes each time.

[0059] Peel off the intact stamens from the sterile male flower buds with dissecting needles and tweezers, inoculate them on the callus induction medium, and culture them in a dark room at 27°C for 30 days to obtain primary callus. figure 1 -A shown.

[0060] The callus induction medium is based on improved MS, and the following content components are added: KT 1 mg / L, NAA 1 mg / L, 2,4-D 1.5 mg / L, CaCl 2 0.5g / L, coconut water 5%, suc...

Embodiment 2

[0073] Methods for maintaining the embryogenic morphology of a clonal line of rubber tree embryogenic cells with high somatic embryogenic capacity:

[0074] The pure line of embryogenic cell mass established in Example 1 was transferred into the subculture medium, and subcultured once every 20 d. Each time, 0.4 cm of fresh embryogenic tissue was picked with tweezers and inoculated into the subculture medium. Placed in a dark room at 27°C, the embryogenic cell mass was stably proliferated and the embryogenic shape remained consistent, such as Figure 4 -A shown.

[0075] The suspension cell mass kept in the liquid medium according to the subculture method of step (3) of Example 1 was used as the control group.

[0076] In the subculture medium and liquid medium, the cell mass after continuous subculture for 2 years was transferred to the somatic embryo induction medium, and 8 explants were inoculated in each dish, and the cells maintained in different subculture mediums were m...

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Abstract

The invention provides a method for establishing a rubber tree embryogenic cell pure line with high somatic embryogenic capacity and maintaining the embryogenic form of the rubber tree embryogenic cell pure line, and relates to the technical field of plant tissue culture and cell engineering. The method specifically comprises the following steps: inducing a primary callus, obtaining an embryogenic callus, and carrying out low-density suspension culture on the embryogenic callus, so as to establish the embryogenic cell pure line with high somatic embryogenesis capability. The invention also provides a subculture preservation method and a subculture medium for maintaining the embryogenic cell pure line. By utilizing the method provided by the invention, the obtaining time of the fragile embryogenic callus of the rubber tree can be shortened to be within 6 months, and an embryogenic cell pure line with the somatic embryo induction rate as high as 100% is obtained; when the subculture medium provided by the invention is used for subculture, an embryogenic cell pure line can still maintain about 60% of somatic embryogenesis capability after being subcultured for 2 years, and a good material source with a relatively consistent state is provided for rubber tree gene transformation, gene editing and protoplast culture and fusion.

Description

technical field [0001] The invention belongs to the technical field of plant tissue culture and cell engineering, in particular to a method for establishing a pure line of rubber tree embryogenic cells with high somatic embryogenesis ability. Background technique [0002] Rubber tree (Hevea brasiliensis Muell.Arg.) belongs to the genus Euphorbiaceae and is a perennial cross-pollinating tree. The natural rubber produced by it is an important industrial raw material and strategic material, and occupies a pivotal position in the national economy. Obtaining regenerated plants through somatic embryogenesis is an important method for factory production and genetic transformation of plant tissue culture seedlings. [0003] The fragile embryogenic callus of rubber tree is the best receptor and material source for genetic transformation and protoplast culture and fusion of rubber tree. At present, the fragile embryogenic callus of rubber tree is mainly obtained in the following two...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04A01H4/00
CPCC12N5/04A01H4/005A01H4/002C12N2500/38C12N2500/16C12N2500/14C12N2500/12C12N2500/20C12N2500/30C12N2500/05C12N2500/34C12N2501/998Y02P60/40
Inventor 戴雪梅彭素娜顾晓川黄天带成镜李季周权男
Owner RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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