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Preparation method of physcomitrella patens protoplast

A technology of Physcomitrella patens and protoplasts, which is applied in the field of preparation of Physcomitrella patens protoplasts, can solve the problems of low protoplast preparation efficiency and regeneration efficiency, and achieve the effect of improving transgenic efficiency and increasing the regeneration of protoplasts

Inactive Publication Date: 2016-11-16
KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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AI Technical Summary

Problems solved by technology

However, as the main source of transgenic materials, the quantity and quality of protoplasts are the main factors that limit the transgenic efficiency. The preparation efficiency and regeneration efficiency of protoplasts in the prior art are low, and improvement is urgently needed to improve the transgenic efficiency.

Method used

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  • Preparation method of physcomitrella patens protoplast
  • Preparation method of physcomitrella patens protoplast
  • Preparation method of physcomitrella patens protoplast

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Embodiment 1

[0022] 1. Materials and methods:

[0023] 1.1 Research materials:

[0024] The materials used in the present invention are Physcomitrella patens GD2004 species, which are stored in an incubator in a laboratory after multiple generations of rapid propagation of plant tissue.

[0025] 1.2 Research methods:

[0026] 1.2.1 Experimental design: Mix 3 g of Physcomitrella patens protonema with 15 ml of sterile ultrapure water, grind and pulverize it with a mechanical homogenizer, transfer it to 10 dishes of BCD medium, and control the pH value of 6.5 and 4.5 Two parameters, respectively at 23 °C, 80 μmolphotons m -2 the s -1 Light intensity, under 16 hours of long daylight, repeat the process once after culturing for 5 days, check the growth status of the cells under a microscope after 5 days, and collect 20 dishes of protoceles. The obtained protoplasts were purified by enzymolysis of the cell wall with disintegrating enzyme for 15 to 30 minutes, counted microscopically, transfe...

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Abstract

The invention discloses a preparation method of moss-physcomitrella patens protoplast, comprising the following steps: mechanically polishing and grinding the raw material-protonema of the protoplast and aseptic ultra-pure water, transferring to a 10-dish BCD culture medium, controlling the pH value at 4.5, the temperature at 23 DEG C and the light intensity at 80mu mol photons m<-2>s<-1>, culturing for 5 days under long irradiation of 16 hours, then repeating the processes once, 5 days later, examining the cell growth condition by a microscope, and collecting 20-dish protonema; freshly preparing 20ml of driselase with the concentration of 0.7% and dissolved in 8% mannitol, dissolving the collected protonema for 20 minutes at room temperature under a dark condition, centrifuging for 5 minutes at1000rpm, collecting cells, washing with 8% mannitol three times, dissolving in 5ml of mannitol, and microscopically counting to obtain about 9.6*10<6>; and transferring to a regeneration culture medium for culturing for 3 days, and then microscopically observing so as to discover that the regeneration efficiency is about 99%. Compared with the prior art, the obtained regeneration protoplast is doubled.

Description

[0001] Field: [0002] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of Physcomitrella patens protoplasts. Background technique [0003] Moss is a group of higher plants transitioning from aquatic to terrestrial. It has a unique evolutionary status. There are about 21,200 species in the world, which are found in every corner of the earth except the ocean, and even grow on deserts, tundra and rocks. It is drought-resistant, cold-resistant, and barren-resistant, and has strong adaptability. It is a pioneer and pioneer of nature (Kidron, 2014; Cao Tong et al., 2014). [0004] Physcomitrella patens is a moss of the genus Physcomitrium in the family Funariaceae of the order Funariales. Genomics and molecular biology research conducted on Physcomitrella patens as an object found that in order to adapt to land life, Physcomitrella patens acquired terrestrial stress response genes such as drought resistance genes, high temperat...

Claims

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Application Information

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IPC IPC(8): C12N5/04
CPCC12N5/04
Inventor 杨红李萍刘莉鲁元学章成君关艳龙
Owner KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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