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Preparation and conversion method of orchid protoplast and application thereof

A protoplast and plant technology, applied in the field of preparation and transformation of orchid plant protoplasts, can solve the problems of poor protoplast transformation effect, no Phalaenopsis protoplast transformation, and limited application fields

Pending Publication Date: 2020-05-26
BGI SHENZHEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many researches about Phalaenopsis tissue culture at present, adopt tissue culture to propagate seedling more, carry out factory production, and the report about the separation of protoplast is still few, and existing method can only realize the preparation of Phalaenopsis protoplast, but It is a method for preparing protoplasts from suspension cells obtained from embryogenic callus culture, which limits its application field; and there is currently no method for transforming Phalaenopsis protoplasts
[0004] "Optimization of Phalaenopsis Protoplast Extraction Method" "Plant Physiology Bulletin" Volume 44, No. 6, December 2008, it disclosed that under the same conditions, 1g of Phalaenopsis leaves were added to 10mL enzymatic hydrolysis solution, and the enzymatic hydrolysis solution contained 1.0% cellulase R-10, 1.0% pectinase, 0.5mol / L mannitol, enzymatic hydrolysis for 3 hours, the yield of viable protoplasts was the highest, and the yield was 1.3×10 5 Individual / g, the activity reaches 81.90%, but the transformation effect of the protoplasts prepared by the above method is not good
CN 106167787A discloses a method for preparing and instantaneous transformation of birch xylem protoplasts, which extracts the protoplasts of birch glabra and realizes instantaneous transformation; CN107488675A discloses a method for separating and transforming rapeseed protoplasts, which extracts Rapeseed protoplasts were obtained and transformed; however, Phalaenopsis is not the same as smooth birch and rapeseed, and the method of directly preparing and transforming protoplasts from smooth birch or rapeseed is not significant for Phalaenopsis.

Method used

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  • Preparation and conversion method of orchid protoplast and application thereof
  • Preparation and conversion method of orchid protoplast and application thereof
  • Preparation and conversion method of orchid protoplast and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0073] The preparation of embodiment 1 protoplast (I)

[0074] The preparation of Phalaenopsis protoplast, concrete steps are as follows:

[0075] (1) Choose about 1g of Phalaenopsis leaves with a leaf age of 2 months, and cut them into thin strips with a width of 0.4mm and a length of 1.0cm;

[0076] (2) Move the cut leaves in step (1) to a petri dish containing 5 mL of PIS enzyme solution, seal with a parafilm, and culture in dark for 5 h at 25° C. and a rotation speed of 50 rpm;

[0077] (3) Filter the protoplast suspension after enzymolysis twice with a 40 μm cell sieve into a sterile round-bottom centrifuge tube, add 5 mL of W5 solution to the filtered enzymolysis mixture for washing and counting, and centrifuge at 100 g for 5 min for a total of centrifuged twice to obtain the protoplast cells;

[0078] Wherein, the PIS enzyme solution includes cellulase with a mass volume fraction of 0.8%, pectinase with a mass volume fraction of 0.8%, 8mM CaCl 2 , 18mM MES at pH 5-6, 0...

Embodiment 2

[0079] The preparation of embodiment 2 protoplasts (II)

[0080] The preparation of Chunlan protoplast, concrete steps are as follows:

[0081] (1) Choose about 1g of Chunlan leaves with a leaf age of 2 months, and cut them into thin strips with a width of 0.6mm and a length of 1.0cm;

[0082] (2) Move the cut leaves in step (1) to a petri dish containing 5 mL of PIS enzyme solution, seal with a parafilm, and culture in dark at 25° C. and a rotation speed of 40 rpm for 6 h;

[0083] (3) Filter the protoplast suspension after enzymolysis twice with a 40 μm cell sieve into a sterile round-bottom centrifuge tube, add 5 mL of W5 solution to the filtered enzymolysis mixture for washing and counting, and centrifuge at 100 g for 5 min for a total of centrifuged twice to obtain the protoplast cells;

[0084] Wherein, the PIS enzyme solution includes cellulase with a mass volume fraction of 0.8%, pectinase with a mass volume fraction of 0.8%, 8mM CaCl 2 , 18mM MES at pH 5-6, 0.2M D-...

Embodiment 3

[0085] The preparation of embodiment 3 protoplasts (III)

[0086] The preparation of Cymbidium protoplasts, the specific steps are as follows:

[0087] (1) Choose about 1g of Cymbidium leaves with a leaf age of 3 months, and cut them into thin strips with a width of 0.3mm and a length of 1.0cm;

[0088] (2) Move the cut leaves in step (1) to a petri dish containing 5 mL of PIS enzyme solution, seal with a parafilm, and culture in dark at 25°C and 60 rpm for 8 hours;

[0089] (3) Filter the protoplast suspension after enzymolysis twice with a 40 μm cell sieve into a sterile round-bottom centrifuge tube, add 5 mL of W5 solution to the filtered enzymolysis mixture for washing and counting, and centrifuge at 150 g for 5 min for a total of centrifuged twice to obtain the protoplast cells;

[0090] Wherein, the PIS enzyme solution includes cellulase with a mass volume fraction of 1.2%, pectinase with a mass volume fraction of 1.2%, 12mM CaCl 2 , 23mM MES pH 5-6, 0.6M D-mannitol, ...

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Abstract

The invention relates to a protoplast preparation and conversion method, in particular to a preparation and conversion method of an orchid protoplast and an application thereof, and the method comprises the following steps: selecting orchid tissues, carrying out enzymolysis by adopting a PIS enzyme solution, and separating protoplast; wherein the PIS enzyme solution is prepared from cellulase, pectinase, CaCl2, D-mannitol, KCl, BSA and MES. The optimized PIS enzyme solution is adopted in the process of preparing the orchid protoplast; the components of the PIS enzyme solution are adjusted, the interaction of the components in enzymolysis is enhanced, the PIS enzyme solution is more suitable for enzymolysis of orchids, specific rotating speed and dark culture time are matched in the enzymolysis process, complete enzymolysis can be achieved to the maximum extent, the protoplast preparation efficiency is improved, and a large amount of high-quality protoplasts are efficiently obtained.

Description

technical field [0001] The invention relates to the field of biotechnology, and relates to a method for preparing and transforming protoplasts, in particular to a method for preparing and transforming protoplasts of Orchidaceae plants, and more specifically, to a method for preparing and transforming protoplasts for Orchidaceae plants and apply. Background technique [0002] Plant protoplast separation and transformation technology has been realized in various plants (Arabidopsis thaliana, rice, wheat, lettuce, etc.), which plays an important role in promoting the scientific research of this species. Species vary by material type, so isolation and transformation methods will need to be optimized and redeveloped for specific species. [0003] Phalaenopsis is a plant of the genus Phalaenopsis in the family Orchidaceae, and it is the most widely cultivated and one of the most popular species in the Orchidaceae. Because of its elegant flower posture, beautiful flower color and...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12N15/82
CPCC12N5/04C12N15/82
Inventor 夏科科章登位郝璋颖顾颖沈玥
Owner BGI SHENZHEN CO LTD
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