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Honeysuckle protoplast separation and culture method and special culture medium

A technology of protoplasts and culture methods, applied in the direction of plant cells, etc., to achieve the effects of increasing yield and vitality, increasing strength, and improving enzymatic hydrolysis effect

Pending Publication Date: 2020-07-31
林瑞娥
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, the research on the isolation, purification and cultivation of honeysuckle protoplasts is still blank.

Method used

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  • Honeysuckle protoplast separation and culture method and special culture medium
  • Honeysuckle protoplast separation and culture method and special culture medium
  • Honeysuckle protoplast separation and culture method and special culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] A method for separating and cultivating honeysuckle protoplasts, comprising the following steps:

[0032] (1) Disinfection of explants

[0033] Select the young leaves of robust honeysuckle test-tube plantlets as explants, wash them with detergent and rinse them in running water for 10 minutes, then disinfect them with 75% alcohol for 30 seconds, and then use 0.1% HgCl 2 The solution was sterilized for 10 minutes, and finally washed 3 times with sterile water.

[0034] (2) Pretreatment of explants

[0035] Place the sterilized leaves in a petri dish, add plasmolysis solution for pretreatment for 60 minutes; the composition of plasmolysis solution is as follows: KNO 3 190mg / L, CaCl 2 2H 2 O 75mg / L, MgSO 4 ·7H 2 O 125mg / L, KH 2 PO 4 17.5mg / L, MnSO 4 4H 2 O 2.23mg / L, H 3 BO 3 0.62mg / L, KI 0.083mg / L, Na 2 MoO 4 2H 2 O 0.025mg / L, CoCl 2 ·6H 2 O0.0025mg / L, ZnSO 4 ·7H 2 O 0.86mg / L, FeSO 4 ·7H 2 O 2.79mg / L, Na 2 -EDTA 3.73mg / L, vitamin B10.05mg / L, vitam...

Embodiment 2

[0045] The influence of embodiment 2 pretreatment time on honeysuckle protoplast separation

[0046] For the pretreatment of the explants in Example 1, five gradient pretreatment times were set in this experiment, namely: 0, 30 min, 60 min, 90 min, and 120 min. Protoplasts were isolated and purified from the explants pretreated for different times, and the yield and viability of the protoplasts were measured (see Table 1).

[0047]

[0048] The results in Table 1 show that with the prolongation of the pretreatment time, the yield and vigor of honeysuckle protoplasts first increased and then decreased. When the pretreatment time was 60 min, the yield and vigor of honeysuckle protoplasts reached the maximum. 5.73×10 6 individuals / g and 85.4%; with the further extension of pretreatment time, its yield and vigor decreased sharply, therefore, the most suitable pretreatment time for honeysuckle leaves was 60 minutes.

Embodiment 3

[0049] The influence of embodiment 3 different enzymolysis methods on honeysuckle protoplast separation

[0050]For the separation of protoplasts in Example 1, three enzymatic hydrolysis methods were set up in this experiment, which are: standing enzymolysis, that is, mixing the leaves with the enzymatic hydrolysis solution and then standing enzymatic hydrolysis at 28°C for 8 hours in the dark; shaking Bed shaking enzymolysis, that is, mixing the leaves with the enzymatic solution and placing them on a shaking table (28°C, 40rpm) for 8 hours of oscillating enzymolysis; first standing still and then oscillating enzymolysis, that is, mixing the leaves with the enzymatic solution and then standing for 4 hours , and then placed on a shaker (28°C, 40rpm) for oscillating enzymatic hydrolysis for 4 hours. The protoplasts obtained through different enzymatic hydrolysis methods were purified, and then the yield and activity of the protoplasts were measured (see Table 2).

[0051]

...

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Abstract

The invention discloses a honeysuckle protoplast separation and culture method and a special culture medium, and belongs to the technical field of plant tissue and cell culture. The method comprises the following steps: (1) disinfection of explants; (2) pre-treatment of the explants: pre-treating leaves in a plasmolysis solution for 60 minutes; (3) protoplast separation: mixing the leaves with anenzymatic hydrolysate, standing and carrying out enzymolysis for 4 hours, and then carrying out low-speed oscillation enzymolysis for 4 hours; (4) purification of a protoplast: purifying the separatedprotoplast by adopting a precipitation method; and (5) protoplast culture: carrying out liquid shallow culture by adopting a protoplast culture medium. Honeysuckle test-tube plantlet leaves are usedas explants, the honeysuckle protoplast with high yield and high activity is obtained through separation and purification, a protoplast culture system is preliminarily established, and the method hasgreat significance in development of honeysuckle crossbreeding, gene engineering and other related biotechnology researches.

Description

technical field [0001] The invention relates to a method for separating and cultivating honeysuckle protoplasts and a special culture medium, belonging to the technical field of plant tissue and cell culture. Background technique [0002] honeysuckle( Lonicera japonica Thunb), also known as silver flower, double flower, honeysuckle, and Erbao flower, is a perennial semi-evergreen woody vine of the genus Lonicera in the family Lonicera. Honeysuckle has single leaves opposite, the leaves are ovate or long ovate, both sides are pilose, and there are two flowers in the axil of each leaf to form a small cyme, the flowers are symmetrical on both sides, the corolla is lip-shaped, it is white when it first blooms, and then turns into a cyme. Golden yellow, hence the name honeysuckle. Traditional medicine believes that honeysuckle stems, leaves, vines, and flower buds can all be used as medicine, especially the flower buds. The dried flower buds or the first bloomed flowers are us...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04
CPCC12N5/04
Inventor 林瑞娥
Owner 林瑞娥
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