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Separation and purification method for Salvia Miltiorrhiza protoplast

A protoplast, separation and purification technology, applied in the field of biochemistry, can solve problems such as no one has studied

Inactive Publication Date: 2014-06-04
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, no one has studied the method of protoplast isolation from Salvia miltiorrhiza

Method used

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  • Separation and purification method for Salvia Miltiorrhiza protoplast
  • Separation and purification method for Salvia Miltiorrhiza protoplast
  • Separation and purification method for Salvia Miltiorrhiza protoplast

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Experimental program
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Embodiment Construction

[0034] Induction of callus and subculture

[0035] Aseptic seedlings were cultivated from the seeds of Salvia miltiorrhiza, and then the robust leaves on the aseptic seedlings were cut into explants with a size of 0.5 cm × 0.5 cm. Under sterile conditions, inoculate the explants on MS solid medium containing 1.0 mg / L NAA, 1.0 mg / L 6-BA, 1.0 mg / L 2,4-D, 5.5 g / L Agar and 30 g / L sucrose were used to induce callus formation under light conditions. The induced callus was subcultured every 20 days. The cultivation conditions were the same as above.

[0036] The callus that had been subcultured for 3 generations with stable traits was subcultured again for 12 days, and transferred to a 50 mL Erlenmeyer flask containing 25 mL of MS liquid medium (without growth regulator substances and agar), at a rotation speed of 125 r / min at a temperature of 25°C in a dark suspension culture. Cells cultured in suspension for 6 days can be used for the isolation of protoplasts.

[0037] Is...

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Abstract

The invention discloses a separation and purification method for a Salvia Miltiorrhiza protoplast. The method includes the steps of: induction of a callus and subculture, protoplast separation and protoplast purification. The extraction conditions for a Salvia Miltiorrhiza suspension culture cell's protoplast include that: an enzyme solution suitable for enzymolysis of the suspension culture cell is composed of 1.5% of cellulase, 0.3% of pectinase and 0.5% of macerozyme; a suitable mannitol concentration is 0.4M; the extraction time is 12h; centrifugation is carried out at 600r / min for 5min, then collection and purification are conducted so as to obtain the protoplast with a yield of 1.1*10<6> / gFW, FDA detection shows that the vitality of the protoplast is over 95%, and the calcium ion fluorescence probe fluo-3 / AM can be successfully loaded into protoplast. The protoplast extracted under the conditions of the method completely satisfies the follow-up series of study taking a single cell as the study object.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a method for separating and purifying salvia miltiorrhiza protoplasts. [0002] Background technique [0003] Plant protoplasts are living plant cells that have lost their cell walls and are surrounded only by a plasma membrane. The role of the plasma membrane in life activities is extremely important, such as information transmission, energy conversion, material transport and other life phenomena are closely related to the plasma membrane. The nature of protoplasts being naked and alive provides favorable conditions for the research related to the function of plant plasma membrane, and the essence of a series of life processes has been revealed at the cellular level. The plant's resistance to elicitors is carried out at the cellular level, and research at the overall level has been subject to various interferences, and the use of protoplasts as materials can e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04
Inventor 董娟娥朱楠姚雅琴刘文婷
Owner NORTHWEST A & F UNIV
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