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Low-temperature preservation culture medium of rubber tree embryonic callus and low-temperature preservation method of culture medium

A technology of embryogenic callus and cryopreservation, which is applied in the direction of plant preservation, horticultural methods, botanical equipment and methods, etc., and can solve the problems of reducing operation and saving labor, losing the ability to recover growth and regeneration, and difficult operation , to achieve a good low temperature storage state, strengthen the antioxidant capacity, and maintain the effect of dynamic balance

Active Publication Date: 2017-12-29
HAINAN UNIVERSITY
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  • Application Information

AI Technical Summary

Problems solved by technology

During the long-term preservation of plant tissues and cells, if subculture is adopted at room temperature, the tissue materials generally need to be subcultured once every 4 to 6 weeks, which consumes a lot of manpower and material resources.
Theoretically, cryopreservation has the advantages of long-term and stability, but due to the complexity of cryopreservation procedures, improper freezing and thawing procedures will damage cells and lose the ability to resume growth and regeneration, and the regeneration rate of plants is also restricted, including rubber trees. The bottleneck of cryopreservation of most plants including the plant; in addition, the preservation process requires the use of high-end equipment such as expensive program-controlled cooling devices, ultra-low temperature refrigerators, etc., which makes the use of cryopreservation methods in the preservation of rubber tree embryogenic callus not popular enough and difficult to operate. Technology needs to be further explored and perfected
Slow-growth preservation refers to a preservation method that limits the growth of preservation materials by adjusting and changing one or several culture conditions, minimizes cell growth but does not die, thereby prolonging the interval between subcultures, reducing operations and saving labor. The biggest advantage of this preservation method is that it can maintain the slow and continuous growth of medium and short-term preservation materials, and it is easy to resume growth in the later stage. However, most tropical and subtropical plants lack the ability to adapt to low temperature, and the cells will be damaged when the ambient temperature is lower than 10°C. , seriously affecting the normal growth, development and even death of plant tissue cells

Method used

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  • Low-temperature preservation culture medium of rubber tree embryonic callus and low-temperature preservation method of culture medium
  • Low-temperature preservation culture medium of rubber tree embryonic callus and low-temperature preservation method of culture medium
  • Low-temperature preservation culture medium of rubber tree embryonic callus and low-temperature preservation method of culture medium

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Experimental program
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Effect test

Embodiment 1

[0064] The material is Hevea brasiliensis Müll.Arg. variety Reyan 7-33-97, and the rubber inflorescences were collected from the experimental farm of the Chinese Academy of Tropical Agricultural Sciences. Submerge the inflorescences with 0.1% light soapy water ( figure 1 -A) 10min, rinse with running water. Use sterilized small scissors to cut out unopened, healthy rubber tree male flowers ( figure 1 -B), most of the pollen grain development period is the uninucleate marginal stage ( figure 1 -D). Wrap it with sterile gauze, disinfect the surface with 70-75% alcohol for 40 seconds, and use 0.1% HgCl 2 Soak and disinfect for 12 minutes, then rinse with sterile water for 6 times, and then peel off a single anther under a dissecting microscope and inoculate it on the anther callus induction medium, with 30 anthers per dish.

[0065] Table 1 Correspondence between the pollen development period and the external shape of flower buds of rubber tree Reyan 7-33-97 variety

[0066]...

Embodiment 2

[0075] The embryogenic callus ( image 3-A), inoculated into the cryopreservation medium, the cryopreservation medium is modified MS as the basic medium, including the following components: L-glutamine 250-350mg / L, vitamin C 0.3-0.8mg / L, salicylic acid 18-23mg / L, hydrolyzed casein 280-320mg / L, coconut milk volume concentration 8%-12%, sucrose 65-75g / L, mass concentration 0.08%-0.12% activated carbon and plant gel 2.0~2.5g / L; the improved MS medium uses water as a solvent, including ammonium nitrate 1100mg / L, potassium nitrate 1267mg / L, calcium chloride dihydrate 800mg / L, magnesium sulfate heptahydrate 247mg / L, diphosphate Potassium hydrogen 226mg / L, potassium iodide 0.83mg / L, boric acid 9mg / L, manganese sulfate tetrahydrate 22.3mg / L, zinc sulfate heptahydrate 8.6mg / L, sodium molybdate dihydrate 0.25mg / L, copper sulfate pentahydrate 0.025 mg / L, cobalt chloride hexahydrate 0.025mg / L, disodium edetate 37.3mg / L, ferrous sulfate heptahydrate 27.8mg / L, inositol 100mg / L, glycine 1m...

Embodiment 3

[0078] By visual inspection, callus survival rate (%)=(light yellow fresh and alive callus number / inoculated callus total number)×100%, measure the embryogenic callus preserved at low temperature (4° C.) for 25 days in Example 2 For the survival rate of the tissue, the embryogenic callus cultured for 25 days under the condition of 25°C inoculated on the cryopreservation medium was used as the control group 1, which was preserved for 25 days at low temperature (4°C) on the proliferation medium described in Example 1. The embryogenic callus was the control group 2. The result is as Figure 4 shown.

[0079] Depend on Figure 4 It can be seen that there was no significant difference in the survival rate between the embryogenic callus preserved at low temperature and the embryogenic callus cultured normally at 25°C (control group 1), but it was higher than that cultured in conventional callus medium and cultured at the same low temperature. The survival rate of the embryogenic ...

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Abstract

The invention provides a low-temperature preservation culture medium of rubber tree embryonic callus and a low-temperature method of the culture medium, belonging to the technical field of tissue culture. Exogenous antioxidant L-glutamine, oxidation-resistant vitamin VC and exogenous hormone salicylic acid are added in the low-temperature preservation culture medium; endogenous antioxidant enzyme glutathione reductase GR action is motivated to maintain intracellular high-proportion GSH / GSSG and SG so as to participate in ascorbic acid glutathione circulation AsA-GSH of a key approach for eliminating ROS in a plant, other antioxidant enzyme is supplemented to synergistically and effectively remove residual ROS in the embryonic callus preserved at low temperature so as to improve the cold stress resistance of the embryonic callus. The callus survival rate of the embryonic callus preserved by a low-temperature preservation culture medium is not significantly different from that of the embryonic callus cultured in a dark condition at 25 plus / minus 2 DEG C, and the FDA fluorescence detection indicates that the cell viability of the embryonic callus subjected to low-temperature preservation is maintained stronger.

Description

technical field [0001] The invention belongs to the technical field of tissue culture, and in particular relates to a low-temperature storage medium for rubber tree embryogenic callus, a low-temperature storage method, and a recovery growth method. Background technique [0002] Hevea brasiliensis Müll. Arg. belongs to Euphorbiaceae (Euphorbiaceae) rubber tree genus (Hevea), is a perennial cross-pollinated tree. Rubber tree is the main source of natural rubber, while the self-sufficiency rate of natural rubber in my country is only 18%. my country's rubber tree researchers should work together to provide a technical system for the research on gene functions related to rubber tree resistance and high yield, in order to obtain a batch of genes with important application value and independent intellectual property rights as soon as possible. At the same time, through genetic engineering technology to cultivate new varieties of major transgenic rubber trees with high yield, high...

Claims

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Application Information

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IPC IPC(8): A01H4/00A01N3/00
CPCA01H4/001A01H4/005A01N3/00
Inventor 陈健妙何朝族栾林莉宋玉凤
Owner HAINAN UNIVERSITY
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