Extraction and fusion method for strawberry protoplast

A technology of protoplasts and fusion methods, applied in biochemical equipment and methods, botany equipment and methods, plant cells, etc., can solve the problems of mixed varieties, poor resistance, and quality decline, and achieve the effect of mild action and easy operation

Inactive Publication Date: 2012-06-20
TONGHUA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Strawberry production has developed rapidly since the 1980s. Most varieties of strawberry production in my country are imported from abroad, and the varieties are mixed, and there are generally problems such as poor resistance and quality decline. There are not many varieties that are truly suitable for cultivation in different climatic conditions in my country. , there is an urgent need to select and breed fine varieties suitable for cultivation in our country
my country is at the international advanced level in some aspects of strawberry modern biotechnology breeding research (such as the acquisition of haploid plants, the regeneration of transformed plants, etc.), but there is a big gap compared with foreign counterparts in many aspects. Screening of mutants, culture of protoplasts and genetic transformation of strawberry are still blank

Method used

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  • Extraction and fusion method for strawberry protoplast
  • Extraction and fusion method for strawberry protoplast
  • Extraction and fusion method for strawberry protoplast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The extraction and fusion method of strawberry protoplasts, the steps are as follows:

[0026] 1) Get green-leaf oriental strawberry and rupee strawberry test-tube plants respectively and move into the MS medium that has reduced the sucrose concentration and cultivate for 10 days. The MS medium contains 6-benzyl adenine (6-BA) 1.0mg / L, sucrose 1.5% and agar 0.7%;

[0027] 2) Take the pretreated leaves in step 1) and cut them into 2.0mm×2.0mm sized strips with scissors and remove the veins;

[0028] 3) Mix the material and the enzyme solution according to the volume ratio of 1:10, seal with Parafilm, seal with tinfoil paper, place on a shaker at a constant temperature of 26°C, and perform dark enzymatic hydrolysis at a speed of 60r / min, every hour after 7 hours Observing the enzymatic hydrolysis under a microscope, the composition of the enzyme solution is 1.0% Cellulase Onozuka R-10+0.5% Pectolyase Y-23+0.6mol / L Mannitol+0.1-0.2% 2-(N-morphine Phyloline)ethanesulfonic...

Embodiment 2

[0035] The extraction and fusion method of strawberry protoplasts, the steps are as follows:

[0036] 1) Get green-leaf oriental strawberry and rupee strawberry test-tube plantlets respectively and move into the MS medium that has reduced the sucrose concentration and cultivate for 10 days. The MS medium contains 6-benzyl adenine (6-BA) 1.5mg / L, sucrose 1.5% and agar 0.7%;

[0037] 2) Take the pretreated leaves in step 1) and cut them into 2.0mm×2.0mm sized strips with scissors and remove the veins;

[0038] 3) Mix the material with the enzyme solution at a ratio of 1:10, seal it with Parafilm, seal it with tinfoil paper, place it on a shaker, and carry out dark enzymolysis at a constant temperature and low speed (60r / min) at 26°C, every hour after 7 hours Observing the enzymatic hydrolysis under a microscope, the composition of the enzyme solution is 2.0% Cellulase Onozuka R-10+0.5% Pectolyase Y-23+0.6mol / L Mannitol+0.1-0.2% 2-(N-morphine Phyloline)ethanesulfonic acid MES+0...

Embodiment 3

[0045] The extraction and fusion method of strawberry protoplasts, the steps are as follows:

[0046] 1) Take the test-tube plantlets of green-leaf oriental strawberry and rupee strawberry respectively and culture them in the dark at 26±1°C for one week;

[0047] 2) Take the pretreated leaves in step 1) and cut them into 2.0mm×2.0mm sized strips with scissors and remove the veins;

[0048] 3) Mix the material with the enzyme solution at a ratio of 1:10, seal it with Parafilm, seal it with tinfoil paper, place it on a shaker, and carry out dark enzymolysis at a constant temperature and low speed (60r / min) at 26°C, every hour after 7 hours Observing the enzymatic hydrolysis under a microscope, the composition of the enzyme solution is 1.0% Cellulase Onozuka R-10+1.0% Pectolyase Y-23+0.6mol / L mannitol+0.1-0.2% 2-(N-morphine Phyloline)ethanesulfonic acid MES+0.05mol / L CaCl 2 combination, the enzyme digestion time is 13 hours;

[0049] 4) Filter the two kinds of strawberry mesop...

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PUM

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Abstract

The invention provides an optimal isolation and fusion method for strawberry protoplasts, which utilizes strawberry leaves as research objects. According to the method, the protoplasts with high activity are obtained with both the pre-treatment method and the enzymolysis method, and 30-45% polyethylene glycol (PEG) 6000 is combined with high Ca<2+> method and high pH method so as to induce one-to-one protoplast fusion with improved probability. The method disclosed by the invention has the advantages of mild action, more than 85% of the activity of the isolated protoplast, one-to-one fusion rate up to 11.7%, simple operation, and possibility of forming hybrid cell. The method is suitable for inducing and culturing the new breed by overcoming distant hybridization barrier, and has certain significance in the biological breeding aspect.

Description

technical field [0001] The invention relates to the technical field of plant variety improvement, in particular to a method for extracting and fusing strawberry protoplasts. Background technique [0002] Strawberry belongs to Rosaceae (Rosaceae) Fragaria (Fragaria) plants, perennial herbaceous fruit trees, its economic value and nutritional value are very high, and its planting range is wide. Strawberry production has developed rapidly since the 1980s. Most varieties of strawberry production in my country are imported from abroad, and the varieties are mixed, and there are generally problems such as poor resistance and quality decline. There are not many varieties that are truly suitable for cultivation in different climatic conditions in my country. , which is an urgent need to select and breed good varieties suitable for cultivation in my country. [0003] At present, strawberry breeding at home and abroad mainly focuses on strawberry stem tip culture, anther culture, scre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12N15/05
Inventor 冯颖顾地周朱俊义杨丽娟
Owner TONGHUA NORMAL UNIV
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