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Method and special culture medium for efficiently separating, converting and regenerating potato protoplast

A technology of protoplast and culture medium, applied in the field of transgenic engineering

Inactive Publication Date: 2017-03-22
TIANJIN GENOVO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, studies on the transformation and regeneration of protoplasts using suspension cells have not been reported yet.

Method used

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  • Method and special culture medium for efficiently separating, converting and regenerating potato protoplast
  • Method and special culture medium for efficiently separating, converting and regenerating potato protoplast
  • Method and special culture medium for efficiently separating, converting and regenerating potato protoplast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] 1. Preparation of mother solution and medium for callus induction and subculture

[0118] 1) Induction medium:

[0119] Add the following components: MS medium with a final concentration of 4.4g / L, 2,4-dichlorophenoxyacetic acid with a final concentration of 0.5mg / L and sucrose with a final concentration of 30g / L, and make up the volume with water, Adjust the pH to 5.8, add plant gel with a final concentration of 3g / L, autoclave at 121°C for 20 minutes, and add naphthaleneacetic acid with a final concentration of 2.0mg / L and a final concentration of 2.0 when the temperature of the medium drops to about 60°C. mg / L of 6-benzylaminopurine to make a solid medium.

[0120] 2) Subculture medium:

[0121] Add the following components: MS medium with a final concentration of 4.4g / L, hydrolyzed casein with a final concentration of 0.25g / L, 2,4-dichlorophenoxyacetic acid with a final concentration of 2.0mg / L and Sucrose with a concentration of 30g / L, make up the volume with wa...

Embodiment 2

[0165] The original diploid potato cultivars "NDK47-33" and "NDK5-19" were cultivated according to the method in Example 1, and potato transgenic plants were obtained.

[0166] The invention discloses a purple potato (Solanum tuberosum L.) protoplast high-efficiency separation, transformation and regeneration system and a special culture medium. In the present invention, the stem section of potato plantlets is used as the starting material, and embryogenic callus with vigorous growth and loose structure is obtained through induction and three times of subculture for suspension culture, and cellulase, pectinase and isolated enzyme are used to obtain Potato suspension cells were digested and protoplasts were separated by density gradient sedimentation, and high-purity protoplasts were obtained. The target gene was introduced into the genome of potato protoplasts mediated by polyethylene glycol, and a large number of potato transgenic plants were bred through shallow liquid cultu...

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Abstract

The invention discloses a method and special culture medium for efficiently separating, converting and regenerating a potato protoplast. The method comprises the steps of carrying out induction and three-time subculture on a starting material, namely a stem segment of a potato test-tube seedling to obtain an embryogenic callus vigorous in growth and loosened in structure, and carrying out suspension culture; slaking potato suspension cells by using cellulases, pectases and macerases; and separating a protoplast by using a density gradient sedimentation method to obtain a high-purity protoplast. A target gene is introduced to a potato protoplast genome through polyethylene glycol mediation, and liquid shallow culture and subsequent differentiation regeneration are performed, so that a great number of transgenic plants of potatoes are cultured.

Description

technical field [0001] The invention relates to the field of transgenic engineering, in particular to a method for efficiently separating, transforming and regenerating potato protoplasts and a special culture medium thereof. Background technique [0002] Potatoes are the fourth staple food in the world and can make important contributions to the food security of China, India, the European Union and other countries. In 2015, at the same time that the U.S. approved GM potatoes, China (6 million hectares), the world’s largest potato producer, announced plans to double its potato acreage and designate potatoes as the fourth staple food after rice, corn and wheat . [0003] Plant protoplasts are good reactors for studying genetic theory and ideal recipients for genetic transformation. At the same time, the protoplast mutant plants also provide a large number of materials that can be selected and utilized for plant breeding. At present, genome editing technology has opened a n...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12N15/82A01H4/00A01H5/00
CPCC12N5/0025C12N5/04C12N15/8206C12N2500/30C12N2500/34C12N2500/35C12N2500/40C12N2500/76C12N2501/998C12N2509/00
Inventor 冉毅东高崑张康
Owner TIANJIN GENOVO BIOTECH CO LTD
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