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Method for propagating Tortula muralis protonema by cell engineering cultivation

A cell engineering and protonema technology, applied to plant cells and other directions, can solve the problems of difficulty in collection, small size of bryophytes, difficult to distinguish and separate, etc., and achieve the effects of simple preparation method, consistent development degree and low cost.

Inactive Publication Date: 2009-07-22
YUNNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the bryophytes are small in shape, mixed and clustered, and it is difficult to distinguish and separate the species. There is no mature system for the artificial cultivation and propagation of a single moss, and it is difficult to collect sufficient samples for some species, which has led to the research on related products of bryophytes. Development faces raw material dilemma

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] 1. Sterilization of mature spores:

[0039] Select the mature and full capsules of Physcomitrella pannatum, cut off the middle part of the stalk 1cm below the capsule, wash the surface of the capsule with clean water, then soak the capsule with 75% alcohol, and pre-sterilize the surface of the tissue for 0.5 minutes; then use Mercury chloride with a concentration of 1.0g / L and a solution containing 1.0% meal detergent are used to disinfect the surface of the material spore capsule for 3 minutes; then soak and clean the spore capsule 4 times with sterilized water repeatedly to clean the material spore capsule. Mercury chloride and detergent residue on the capsule surface; then fix the capsule handle with sterile tweezers, cut the top of the capsule horizontally with sterile ophthalmic scissors to expose the internal spores, squeeze the spores into a centrifuge filled with sterile water with tweezers in the tube to disperse and suspend the spores; seal the centrifuge tube...

Embodiment 2

[0050] Basically the same as embodiment 1, the difference is:

[0051] 1. When sterilizing mature spores, soak the capsules in 75% alcohol, pre-disinfect the surface of the tissue for 1 minute, sterilize with mercury chloride for 5 minutes, and stimulate the water bath for 50 seconds.

[0052] 2. Germination on solid medium at a spore density of 10 6 / ml, the concentration of 2,4-D is 5mg / L.

[0053] 3. Protofilament mass propagation and collection:

[0054] Use tweezers to gently pick up a volume of 200mm 3 Protoceles, inoculated into 2,4-D2.5mg / L, sodium silicate 2.0g / L, sucrose 2.0%, pH5.8 MS liquid medium volume is 500ml Erlenmeyer flask, each Erlenmeyer flask The culture solution added in the medium was 1 / 3 of the volume of the Erlenmeyer flask, cultured on a shaker at 120rpm for 7 days, the ambient temperature was controlled at 24°C, and the light source was diffuse light; then the same culture medium and control conditions were used for rotary flask culture, and obta...

Embodiment 3

[0057] Basically the same as embodiment 1, the difference is:

[0058] 1. When sterilizing mature spores, soak the capsules in 75% alcohol, pre-disinfect the surface of the tissue for 1.5 minutes, sterilize with mercury chloride for 3 minutes, and stimulate the water bath for 45 seconds.

[0059] 2. Germination on solid medium at a spore density of 10 5 / ml, the concentration of 2,4-D is 5mg / L.

[0060] 3. Protofilament mass propagation and collection:

[0061] Use tweezers to gently pick up a volume of 1000mm 3 Protoceles, inoculated into 2,4-D3mg / L, sodium silicate 2.0g / L, sucrose 2.0%, pH5.8 MS liquid medium volume is 1000ml Erlenmeyer flask, add in each Erlenmeyer flask The culture medium is 1 / 3 of the volume of the Erlenmeyer flask, cultivated on a shaker at 120rpm for 7 days, the ambient temperature is controlled at 24°C, and the light source is diffuse light; then the same medium and control conditions are used for rotary flask culture, and a large amount of raw mate...

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PUM

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Abstract

The invention relates to a method for cultivating and breeding pangenesis tortula protonema by using cell engineering, which belongs to the technical field of biology and comprises following concrete steps: a. mature spores are sterilized, and capsulea surface is sterilized for 0.5 to 1 minutes by a solution that contains 1.0% of mercury bichloride and 1.0% of a washing agent; b. germination is implemented on a solid culture medium, the spores are inoculated to an MS culture medium that has the pH value of 5.8 and is added with 2 to 5 mg / L of 2,4-D, 2.0g / L of sodium silicate and 2.0% of sucrose, and cultivated at the temperature of 25 plus or minus 3 DEG C and illumination intensity of 4000lx for 10 to 20 days; c. mass propagation of the protonema is implemented in the MS fluid culture medium for spore germination that has pH value of 5.8 and is added with 2 to 5 mg / L of 2,4-D, 2.0g / L of sodium silicate and 2.0% of sucrose, shaking cultivation is implemented at the temperature of 25 plus or minus 3 DEG C and the speed of 120rpm for 7 to 10 days and the power source is diffused lights; or augmentation cultivation is implemented in a fermentation flask charged with sterile air at the speed of 200L per hour; a small amount of the protonema is collected by 800rpm centrifugation, and a large amount of the protonema is collected through the filtration by 4 to 8 gauze layers. The invention has the advantages of simple and convenient preparation method, low cost and consistent maturity of the obtained protonema.

Description

Technical field: [0001] The invention relates to a method for cultivating and propagating protocelium of Phyllostachys panphyta by cell engineering, which belongs to the field of biotechnology. Background technique: [0002] In the plant kingdom, bryophytes are a group of plants that transition from aquatic life to terrestrial life, and are one of the oldest groups of terrestrial plants on earth. At present, there are footprints of bryophytes in different ecosystems on the earth, including Antarctica, except for mountains and oceans above 5,000 meters. Bryophytes belong to the spore plants in higher plants. There are about 23,000 species in the world, and the number of species is second only to angiosperms. It is an important category in the plant kingdom. [0003] The life history of bryophytes is dominated by gametophytes, and the common green bryophytes belong to the gametophyte generation. The sporophyte of bryophytes is attached to the gametophyte, sometimes the sporo...

Claims

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Application Information

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IPC IPC(8): C12N5/04
Inventor 陈穗云陈正贵沈霏汪家乐李育中张鸽范静黎兴江
Owner YUNNAN UNIV
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