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Tissue culture method for inducing immature zygotic embryos of paris polyphylla to generate somatic embryos

A somatic embryo and tissue culture technology, which is applied in horticultural methods, botany equipment and methods, plant regeneration, etc., can solve the problem of lower induction rate of adventitious buds, insignificant tissue culture effect, and low reproduction coefficient of callus of P. problems, to achieve the effect of avoiding endophyte infection, good plasticity, and low infection rate

Inactive Publication Date: 2017-02-22
SOUTHWEAT UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, there are many endophytic bacteria in the rhizome of Pachyphylla polyphylla, and the tissue culture is difficult. The current tissue culture effect is not significant, and its reproduction coefficient is low.
In addition, with the increase of the number of subcultures, the induction rate of adventitious buds in P.
Therefore, there is currently no feasible large-scale reproduction technology of P. polyphylla seedlings, and it is imperative to explore the tissue culture and rapid propagation method of expanding P. polyphylla breeding in the short term to alleviate the pressure of P. polyphylla resource demand.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] 1) Sampling and disinfection: The suitable sampling time for the explants used is August. The best when the fruit is about to mature, but the skin has not cracked. Cut the whole fruit of C. Chonglou and open it, remove the attached fibrous tissue of C. Chonglou seeds with fleshy seed coats, wash in running water, and soak in tap water for 24 hours, and pass 0.1% HgCl under sterile conditions. 2 After 5 minutes of disinfection, wash with sterile water 10 times;

[0015] 2) Peeling of immature zygotic embryos: Use tweezers to peel off the fleshy testa of the sterilized seeds in 1) under aseptic conditions, use ultra-fine tissue culture forceps to fix the peeled endosperm under a stereo microscope, and use surgery The blade cuts the endosperm longitudinally. Because the color of the embryo is the same as that of the endosperm, but the tissue density is different, the vertical light intensity of the microscope can be adjusted to find the shadow-like immature zygotic embryo in ...

Embodiment 2

[0019] 1) Sampling and disinfection: In September each year, remove the attached fibrous tissues of the seeds with fleshy seed coats, wash them in running water, and soak them in tap water for 24 hours, and pass them to 0.1% HgCl under aseptic conditions. 2 After disinfection for 5 minutes, wash with sterile water 10 times;

[0020] 2) Stripping of immature zygotic embryos: peel off the fleshy testa from the sterilized seeds under aseptic conditions, fix the peeled endosperm with tissue culture forceps, and cut the endosperm longitudinally with a scalpel;

[0021] 3) Induction of somatic embryos: inoculate the stripped immature embryos in a pH 5.8 medium (medium is 1 / 2MS, agar powder concentration is 7.5g / L, sucrose concentration is 30g / L), at 19℃ Continuous dark light training;

[0022] 4) Establishment of embryogenic tissue culture system: After 6 months of culture, after 6 months of culture, the somatic embryos obtained by induction are stripped and placed on a new induction mediu...

Embodiment 3

[0024] 1) Sampling and disinfection: In August each year, remove the attached fibrous tissues of the seeds with fleshy seed coats, wash them in running water, and soak them in tap water for 24 hours, and pass them to 0.1% HgCl under aseptic conditions. 2 After disinfection for 5 minutes, wash with sterile water 10 times;

[0025] 2) Stripping of immature zygotic embryos: peel off the fleshy testa from the sterilized seeds under aseptic conditions, fix the peeled endosperm with tissue culture forceps, and cut the endosperm longitudinally with a scalpel;

[0026] 3) Induction of somatic embryos: inoculate the stripped immature embryos in a pH 5.8 medium (medium is 1 / 2MS, agar powder concentration is 7.5g / L, sucrose concentration is 30g / L) at 21℃ Continuous dark light training;

[0027] 4) Establishment of embryogenic tissue culture system: After 6 months of culture, the somatic embryos obtained by induction are stripped and placed on a new induction medium to continue culturing to make...

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PUM

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Abstract

The invention discloses a tissue culture method for inducing immature zygotic embryos of paris polyphylla to generate somatic embryos. The tissue culture method includes removing fiber tissues adsorbed to paris polyphylla seeds with succulent testa, washing the paris polyphylla seeds with running water, soaking the paris polyphylla seeds in tap water, and sterilizing and washing the paris polyphylla seeds; stripping the succulent testa from the sterilized seeds under an aseptic condition, and slitting endosperms longitudinally; inoculating a culture medium with the stripped immature embryos to continue dark culture; after six-month culture, allowing the successfully-induced somatic embryos to grow into spindle shapes, stripping the somatic embryos, and putting the somatic embryos in a new inducing culture medium to continue culture to obtain the paris polyphyllato somatic embryos with the induction success rate of 90%. The tissue culture method has the advantages that the induced somatic embryos can be directly induced into complete plants with roots and buds, and better plasticity is achieved as compared with calluses; the growth and differentiation speed of the obtained somatic embryos is much higher than that of calluses induced by nutritive organs, and the somatic embryos have higher genetic stability; the difference between the paris polyphylla plants generated through induction of the somatic embryos and original plants in paris polyphylla saponin component and content is small.

Description

Technical field [0001] The invention relates to the technical field of Chinese medicine tissue culture, in particular to a tissue culture method for inducing immature zygotic embryos to produce somatic embryos. Background technique [0002] Chonglou is an authentic rare and precious medicinal material in Yunnan and Sichuan of my country. It is the main raw material of Chinese patent medicines such as "Yunnan Baiyao", "Gongxue Ning" and "Jidesheng Snake Pill". It can also be directly used as medicine for hemostasis, analgesia and treatment of snakes. Bite, fracture, mumps, tumor and immune disorders. Because of the extremely high medicinal value, the demand for heavy buildings increases by nearly 20% every year. In addition, Chonglou seeds have a long dormancy period, low germination rate, and a long resource regeneration cycle. At the same time, due to the huge demand, the phenomenon of uncontrolled and plundered collection of Chonglou wild resources is extremely serious, which h...

Claims

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Application Information

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IPC IPC(8): A01H4/00
CPCA01H4/001A01H4/008
Inventor 侯大斌余马舒晓燕段莎莎何静徐冬梅刘丹刘宏伟
Owner SOUTHWEAT UNIV OF SCI & TECH