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A method for electrofusion of sugarcane protoplasts

A protoplast and electrofusion technology, applied in biochemical equipment and methods, botany equipment and methods, microorganisms, etc., can solve the problems of poor cold resistance and achieve the effect of improving the fusion rate

Inactive Publication Date: 2019-01-15
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Guangxi has not yet been able to cultivate sugarcane varieties with high sugar, high yield and strong stress resistance
Xintaitang No. 22 is the leading sugarcane variety in Guangxi. It has planting traits such as high germination rate, high tillering rate, high yield, high sugar, and high drought tolerance. However, this variety has poor cold resistance, while Guitang No. 28 has high sugar and cold resistance. , strong stress resistance, wide adaptability and other characteristics, it is necessary to develop a new variety that combines the excellent genes of the above two sugarcane varieties. At present, there are few experimental cases of successful somatic cell fusion of sugarcane

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The method of sugarcane protoplast electrofusion provided by the invention is as follows:

[0022] A method for sugarcane protoplast electrofusion, comprising the following steps:

[0023] (1) Preparation of protoplasts: Weigh 0.5 g each of the tender leaves of ROC22 sugarcane seedlings and GT28 sugarcane seedlings, and cut the young leaves of the seedlings into tissue pieces of the same size; then place the above two parental tissue pieces respectively in 4.5 L of 13% CPW solution containing 13% mannitol was housed and sealed for 27 minutes, and then the 13% CPW solution in the two containers was removed respectively, and the two containers were cleaned with 9% CPW solution containing 9% mannitol. Tissue slices in each container twice; then add 4.5mL enzyme solution to the two containers respectively according to weight, seal the parafilm respectively, put them in a shaker, and enzymolyze them for 3 hours at a speed of 35r / min under dark conditions. , to obtain the en...

Embodiment 2

[0028] The method of sugarcane protoplast electrofusion provided by the invention is as follows:

[0029] A method for sugarcane protoplast electrofusion, comprising the following steps:

[0030] (1) Preparation of protoplasts: Weigh 0.7g each of the tender leaves of ROC22 sugarcane seedlings and GT28 sugarcane seedlings, and cut the tender leaves of the seedlings into tissue pieces of the same size; then place the above two parental tissue pieces in The container containing 5L of 13% CPW solution containing 13% mannitol was sealed for 30 minutes, and then the 13% CPW solution in the two containers was removed respectively, and then the two containers were cleaned with 9% CPW solution containing 9% mannitol. Tissue slices in the container twice; then add 5mL of enzyme solution to the two containers by weight, respectively seal the parafilm and put them in a shaker, and enzymolyze at a speed of 40r / min for 3.5h in the dark. Obtain the enzymolysis products of the two parents, s...

Embodiment 3

[0035] The method of sugarcane protoplast electrofusion provided by the invention is as follows:

[0036] A method for sugarcane protoplast electrofusion, comprising the following steps:

[0037] (1) Preparation of protoplasts: Weigh 0.8g each of the tender leaves of ROC22 sugarcane seedlings and GT28 sugarcane seedlings, and cut the tender leaves of the seedlings into tissue pieces of the same size; then place the above two parental tissue pieces in 5.5 L of 13% CPW solution containing 13% mannitol was housed and sealed for 33 minutes, and then the 13% CPW solution in the two containers was removed, and the two containers were cleaned with 9% CPW solution containing 9% mannitol. Tissue slices in each container for 3 times; then add 5.5mL enzyme solution to the two containers respectively according to the weight part, seal the sealing film respectively, put them in a shaker, and enzymatically hydrolyze at a speed of 45r / min for 4h under dark conditions. , to obtain the enzyma...

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Abstract

The invention belongs to the technical field of plant somatic cell hybridization, in particular to a method for electrofusion of sugarcane protoplasts, comprising the following steps: (1) preparationof parent protoplasts; (2) protoplast inactivation; (3) the addition of protoplast fusion inducer: agglutinating and binding plants to the surface of one parent protoplast and binding biotin to the surface of another parent protoplast; (4) protoplast fusion. The invention utilizes the principle that plant lectin can combine with biotin, plant agglutination and biotin binding to the surface of thetwo parent protoplasts. The two parental protoplasts could be fused by lectin and biotin to improve one-to-one fusion rate. The hybrid progeny possessed the genes of ROC22 sugarcane with high yield, high sugar content and high drought tolerance, and also possessed the genes of GT28 sugarcane with high sugar content and cold tolerance.

Description

【Technical field】 [0001] The invention belongs to the technical field of plant somatic cell hybridization, and in particular relates to a method for electrofusion of sugarcane protoplasts. 【Background technique】 [0002] Plant protoplasts include nucleus, cytoplasm and cell membrane, and refer to the viable part obtained after removal of the plant cell wall. Under suitable culture conditions, isolated plant protoplasts can reproduce, differentiate and regenerate into complete plants. Because the protection of the cell wall is removed, the fluidity of the cell membrane can be fully reflected. The plant somatic cell hybrid breeding technology through the fusion of plant protoplasts depends on the fluidity of the cell membrane. Moreover, this breeding method can shorten the breeding period, and can also overcome the obstacle of distant hybridization incompatibility. Therefore, plant somatic hybrid breeding technology has been a research hotspot in recent years. It has broad a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/05C12N13/00
CPCC12N13/00C12N15/02
Inventor 李素丽宋亚妮刘芳君龙安四
Owner GUANGXI UNIV
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