Method for generating grape somatic embryo

A technology for somatic embryos and grapes, applied in horticultural methods, botanical equipment and methods, plant regeneration, etc., can solve the problems of complex operation procedures, high technical difficulties, only suitable for individual varieties, etc., and achieve uniform traits and easy genetic engineering. The effect of simple operation and operation steps

Active Publication Date: 2012-06-20
宁夏林业研究院股份有限公司
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Although there have been a large number of studies on somatic embryogenesis in grapevine, there are still the following problems: ①Due to the differences in grape genotypes, most of the established grape regeneration systems are only suitable for individual varieties; ②The embryogenic callus of some regeneration systems The induction rate is low, only below 20%, which is not suitable for the later operation of transgenic technology; ③Although the induction rate of embryogenic callus in some regeneration systems is relatively high, the operation procedures are more complicated and the technology is more difficult

Method used

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  • Method for generating grape somatic embryo
  • Method for generating grape somatic embryo
  • Method for generating grape somatic embryo

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] (1) During the seedless flowering period of the grape variety Moritani, collect flower buds from healthy plants 5 days before flowering, store the collected flower buds at 4°C for 2 days, and use detergent (such as washing powder, detergent, soapy water, laundry detergent, etc.) for 5 minutes, rinsed with tap water for 20 minutes, sterilized with 75% volume percent ethanol for 30 seconds, 2% volume percent sodium hypochlorite solution for 6 minutes, washed 4 times with sterile water, and then blotted dry with sterile filter paper Reserve the excess water on the buds;

[0020] (2) Place the flower buds obtained in step (1) under a dissecting microscope, puncture the petals with a sterile dissecting needle, take out a single stamen and inoculate it on the medium for cultivation, the cultivation condition is dark cultivation, the temperature is 28±1°C ;

[0021] The medium formula is NN basic medium and add 1.0mg·L -1 2,4-Dichlorophenoxyacetic acid, 2.0mg·L -1 6-Benzyla...

Embodiment 2

[0027] (1) During the full flowering period of the grape variety Cabernet Sauvignon, collect flower buds from healthy plants 7 days before flowering, store the collected flower buds at 4°C for 3 days, and wash them with detergent (such as washing powder, detergent, soap, etc.) water, laundry detergent, etc.) for 10 minutes, rinsed with tap water for 30 minutes, sterilized with 75% volume percent ethanol for 30 seconds, 2% volume percent sodium hypochlorite solution for 6 minutes, washed 4 times with sterile water, and then dried the buds with sterile filter paper Add excess water for later use;

[0028] (2) Place the flower buds obtained in step (1) under a dissecting microscope, puncture the petals with a sterile dissecting needle, take out a single stamen and inoculate it on the medium for cultivation, the cultivation condition is dark cultivation, the temperature is 28±1°C ;

[0029] The medium formula is NN basic medium and add 1.2mg·L -1 2,4-Dichlorophenoxyacetic acid, ...

Embodiment 3

[0035] (1) is consistent with the step (1) in embodiment 2, and different varieties are replaced by snake dragon ball;

[0036] (2) Place the flower buds obtained in step (1) under a dissecting microscope, puncture the petals with a sterile dissecting needle, take out a single stamen and inoculate it on the medium for cultivation, the cultivation condition is dark cultivation, the temperature is 28±1°C ;

[0037] The medium formula is NN basic medium and add 1.0mg·L -1 2,4-Dichlorophenoxyacetic acid, 1.5mg·L -1 6-Benzylaminoadenine, 5.0mg·L -1 AgNO 3 , 60g·L -1 Sucrose and 6g·L -1 Agar; if the pH value of the prepared medium is not 5.8-6.0, it is also necessary to adjust the pH value of the medium to 5.8-6.0 with 1N hydrochloric acid or sodium hydroxide;

[0038] (3) consistent with step (3) in embodiment 2;

[0039] (4) consistent with step (4) in embodiment 2;

[0040] (5) is consistent with step (4) in embodiment 2.

[0041] Schedule 1:

[0042] NN Basic Medium ...

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Abstract

The invention relates to a method for building grape high-efficiency regeneration system by a somatic embryo generation way, in particular to a method for generating grape somatic embryo. The method comprises the following steps of: (1) collecting flower buds 5-7 days before blooming, cleaning and disinfecting, and sucking water for later use; (2) taking out single pistil, inoculating to a culture medium and culturing; (3) selecting light yellow or white, granular and compact embryonic callus, inoculating to a culture medium and culturing; (4) carrying out sub-culture on the somatic embryo formed by inducing in the same culture medium; and (5) carrying out sub-culture on the obtained somatic embryo in the culture medium. By adopting the method provided by the invention, grape pistil is cultured in vitro to build the regeneration system; the obtained regenerated plants are uniform and stable in the characters; and the operation steps are simple and are easy to operate in genetic engineering.

Description

technical field [0001] The invention relates to a method for establishing a high-efficiency regeneration system of grapes through somatic embryogenesis, in particular to a method for somatic embryogenesis of grapes. Background technique [0002] Grape regeneration is difficult, the growth cycle is long, and the number of genes is mostly in a heterozygous state, which seriously restricts the development of molecular breeding. At present, grape grafting technology is mainly used to improve cultivars in grape production. With the deepening of molecular biology research With the maturation of transgenic technology, although grape transgenic breeding has also been successful, the rapid development of grape transgenic breeding has been affected due to the imperfection of the transgenic receptor system. [0003] Currently, there are mainly organogenesis pathways and somatic embryogenesis pathways in the grape regeneration system, but compared with organogenesis, the embryogenic cel...

Claims

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Application Information

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IPC IPC(8): A01H4/00
Inventor 徐美隆柳金凤李健秦彬彬陈春伶
Owner 宁夏林业研究院股份有限公司
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