Method for establishing woodbine high frequency regeneration system through somatic embryogenesis way

A high-frequency regeneration and systematic technology, applied in the field of plant cell engineering, can solve the problems of large seasonal restrictions, restrictions, and low regeneration rate of explants, and achieve the effects of normal plant development, uniform traits, and easy genetic engineering operations

Inactive Publication Date: 2008-04-16
INST OF FORESTRY CHINESE ACAD OF FORESTRY
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  • Application Information

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Problems solved by technology

[0008] Although the plant regeneration system of the genus Diana was established through the above methods, the regeneration rate is low, and the explants are limited by the seasons, so it is greatly limited in practical application.

Method used

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  • Method for establishing woodbine high frequency regeneration system through somatic embryogenesis way

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] In this experiment, the seeds of N. chinensis imported from Canada were used. Soak the seeds of N. chinensis in tap water at room temperature for 10 to 15 hours, then sterilize the surface with 70% alcohol for 2 minutes, rinse them twice with sterile water, and then wash them with 0.1% Mercury Chloride (HgCl 2 ) surface sterilization for 10 minutes, rinsed with sterile water 5 times, cut out the embryos with tweezers and a scalpel and inoculated them in the culture medium containing 6-BA 2.3mg / l, 2,4-D 3.8mg / l, 600mg / l hydrolyzed milk protein Improved B 5 culture medium (Table 1). The medium contains 30g / L sucrose and 6.5g / L agar. Use 1mol / L NaOH or HCl to adjust the pH value to 5.8-6.2, sterilize with high pressure steam for 20 minutes, culture temperature at 22-28°C, light-dark cycle 16h / 8h, light intensity 2000-25001x, relative humidity 60%-70%. 30 days to replace the fresh medium.

[0023] The callus began to appear in 10-12 days, and the callus grew rapidly aft...

Embodiment 2

[0030] The culture procedures were all carried out according to Example 1, except that in the first stage, 1.1mg / l 6-BA, 2.2mg / l 2,4-D and 400mg / l hydrolyzed milk protein LH were added to the culture medium, and in the second stage 0.9mg / l 2,4-D and 0.45mg / l 6-BA were added to the first subculture medium, and 0.5mg / l 2,4-D and 0.25mg / l 6-BA were added to the second subculture medium. l 6-BA, supplemented with 0.5mg / l 2,4-D and 0.25mg / l 6-BA in the third subculture medium, the embryogenic callus obtained by subculture in the third stage was transferred to Growth regulator, supplemented with 600mg / l hydrolyzed milk protein B 5 performed on culture medium.

Embodiment 3

[0032] The culture procedures were all carried out according to Example 1, except that in the first stage, 1.3mg / l 6-BA, 1.9mg / l 2,4-D and 500mg / l hydrolyzed milk protein LH were added to the medium, and in the second stage Add 1.25mg / l 2,4-D and 0.65mg / l 6-BA to the first subculture medium, add 0.65mg / l 2,4-D and 0.35mg / l to the second subculture medium 6-BA, 0.65mg / l 2, 4-D and 0.35mg / l 6-BA were added to the third subculture medium, and the embryogenic callus obtained by subculture was transferred to no growth medium in the third stage. Regulator, B with additional 580 mg / l hydrolyzed milk protein 5 performed on culture medium.

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Abstract

The invention relates to a method for establishing a regenerated system by using mature embryo of Parthenocissus quinque folia Planch as the material, which comprises the following three stages: (1) stripping mature embryo and culturing induced callus, (2) subculturing callus, (3) developing strains from somatic embryo in the callus. In the subculture, the density of 2,4-dichloro phenoxyacetic acid 2,4-D is lowered step by step. Thus the generation rate of the somatic embryo can reach 59%.

Description

technical field [0001] The invention relates to a method for establishing a regenerating system of broccoli in vitro. Specifically, it relates to a method for establishing a regeneration system through a somatic embryogenesis pathway by taking mature embryos of broccoli quinquefolium seeds as explants. It belongs to the field of plant cell engineering. Background technique [0002] Brocade is a deciduous vine belonging to the genus Parthenocissus of the family Vitaceae, which has strong adsorption and climbing capabilities. Five-leaf brocade is a garden, urban vertical greening and landscaping plant with good ornamental effect. The United Nations Biosphere and Environment Organization proposed: "The area of ​​urban green space reaches 60m 2 / person-hour is the best living environment”, and the visual feeling is the most comfortable when the green color accounts for more than 25% of the human visual field. However, the per capita green space area in my country’s cities is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04A01H4/00
Inventor 孙振元李正红江泽慧巨关升韩蕾杨学军李云
Owner INST OF FORESTRY CHINESE ACAD OF FORESTRY
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