Separation and culturing method of saussurea involucrate protoplast

A technology of protoplasts and culture methods, applied in the field of isolation and cultivation of Xinjiang Saussurea sauraceae protoplasts, to achieve the effects of expanding the genetic material base, overcoming incompatibility barriers, and strong regeneration ability

Inactive Publication Date: 2010-03-17
XINJIANG TECHN INST OF PHYSICS & CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is a feasible and effective way to use the protoplast culture

Method used

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  • Separation and culturing method of saussurea involucrate protoplast
  • Separation and culturing method of saussurea involucrate protoplast
  • Separation and culturing method of saussurea involucrate protoplast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] a. Screen out the full-bodied snow lotus seeds, rinse them with tap water for 2 hours to clean the surface, then soak them in 75% alcohol for 10 seconds on the ultra-clean workbench, sterilize them with 0.1% mercuric chloride for 1 minute, and treat them with 15% hydrogen peroxide. 20min, rinse 3 times with sterile water, and finally soak in sterile water for 10min;

[0031] b. Inoculate the sterile snow lotus seeds treated in step a on the MS medium, the temperature is 23 ± 1°C, the light is 2500lx, the time is 16h / d, and germination begins on 10d;

[0032] c. Cut the cotyledons and true leaves of the aseptic seedlings germinated in step b into 1cm fragments, insert medium MS+NAA 1.0mg / L+6-BA 0.1mg / L+2,4-D 0.1mg / L, placed in a temperature of 23±1°C, light 30001x, time 16h / d, to induce and differentiate embryogenic callus;

[0033] d. Subculture the embryogenic callus that grows rapidly in step c, is bright green in color, has a compact structure and is loosely combin...

Embodiment 2

[0039] a. Screen out the full-bodied snow lotus seeds, rinse them with tap water for 2 hours to clean the surface, then soak them in 75% alcohol for 20 seconds on the ultra-clean workbench, sterilize them with 0.1% mercuric chloride for 3 minutes, and treat them with 15% hydrogen peroxide. 25min, rinse 4 times with sterile water, and finally soak in sterile water for 10min;

[0040] b. Inoculate the sterile snow lotus seeds treated in step a on the MS medium, the temperature is 23 ± 1°C, the light is 28001x, the time is 16h / d, and germination begins on 12d;

[0041] c. Cut the cotyledons and true leaves of the aseptic seedlings germinated in step b into 1.5cm fragments, insert medium MS+NAA 1.2mg / L+6-BA 0.3mg / L+2,4-D 0.2mg / L, placed at a temperature of 23±1°C, illuminated at 3200lx, for 16h / d, to induce and differentiate embryogenic callus;

[0042] d. Subculture the embryogenic callus that grows rapidly in step c, is bright green in color, has a compact structure, and is lo...

Embodiment 3

[0048] a. Screen out the full-bodied snow lotus seeds, rinse them with tap water for 3 hours to clean the surface, then soak them in 75% alcohol for 60 seconds on the ultra-clean workbench, sterilize them with 0.1% mercuric chloride for 10 minutes, and treat them with 15% hydrogen peroxide for 30 minutes , washed 4 times with sterile water, and finally soaked in sterile water for 10 minutes;

[0049] b. Inoculate the aseptic snow lotus seeds treated in step a into hormone-free MS medium, at a temperature of 23 ± 1° C., with a light of 2700 lx, time of 16 h / d, and germinate on 15 d;

[0050] c. Cut the cotyledons and true leaves of the aseptic seedlings germinated in step b into 2cm fragments, insert medium MS+NAA 1.5mg / L+6-BA 1.0mg / L+2,4-D 0.5mg / L L, placed in a temperature of 23±1°C, light of 3800 lx, and time of 16 h / d, to induce and differentiate embryogenic callus;

[0051] d. Subculture the embryogenic callus that grows rapidly in step c, bright green in color, compact i...

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Abstract

The invention relates to a separation and culturing method of Sinkiang saussurea protoplast, comprising four steps of culturing of sterile saussurea involucrate seedlings, inducement and differentiation of embryonic callus, separation and purification of plasmogen and culturing of the protoplast which are all applicable to the separation and culturing of the Sinkiang saussurea involucrate protoplast. The invention has simple and easily-operated method and lower requirements on the equipment and culturing conditions; in addition, the enzymolysis method is practical, the conditions for cell wallremoval are mild, and the separated protoplast has high yield and strong activity, and is easy for the cultuing of the saussurea involucrate protoplast and a plant regeneration, thus laying the foundations for the culturing and fusing of the protoplast as well as the germplasm resource innovation of the Sinkiang saussurea involucrate and the research of transgenic saussurea involucrate.

Description

technical field [0001] The invention relates to the establishment of a suspension cell line of Saussurea sauraceae, in particular to a method for isolating and culturing protoplasts of Saussurea sauvignon. Background technique [0002] Snow lotus, (Saussurea involucrata Kar.et Kir.et Maxim) belongs to Compositae and is a national second-class endangered plant. It is a perennial herb with a height of 15-25 (-35) cm; the rhizome is thick, dark brown, and the base is brown. Brown; stems are solitary, erect, hollow, 2-4 cm in diameter, glabrous, with dense leaves, subleathery, green, oblong or egg-shaped oblong leaves, about 14 cm long, 2-4 cm wide, Apex obtuse or slightly pointed, base decurrent, margin sparsely serrated, with papillary glandular hairs, uppermost bracts 13-17, membranous transparent, pale yellow, margin with neat sparse teeth, slightly glandular hairy, apex obtuse Acute, base contracted, often 2 times beyond inflorescence. Capitulum 10-30, gathered at the end...

Claims

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Application Information

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IPC IPC(8): C12N5/04
Inventor 王晓军袁永娴郝秀英赵民安刘敏徐琴康喜亮
Owner XINJIANG TECHN INST OF PHYSICS & CHEM CHINESE ACAD OF SCI
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