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A tissue culture method for inducing double building immature zygotic embryos to produce somatic embryos

A technology of somatic embryo and tissue culture, applied in horticultural methods, botanical equipment and methods, plant regeneration, etc., can solve the problem of reduced induction rate of callus adventitious buds, insignificant tissue culture effect, low reproduction coefficient, etc. problem, to avoid the problem of endophyte infection, good plasticity, and low infection rate

Inactive Publication Date: 2018-04-20
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 best sampling time for the explants used is August, when the fruit of Chonglou is close to maturity, but the pericarp has not yet cracked. Cut off the whole fruit of Chonglou, remove the attached fibrous tissue from the seeds of Chonglou with fleshy seed coat, wash them in running water, soak in tap water for 24 hours, and pass through 0.1% concentration of HgCl under sterile conditions 2 After 5 minutes of disinfection, wash with sterile water 10 times;

[0015] 2) Stripping of immature zygotic embryos: peel off the fleshy seed coat of the sterilized seeds in 1) with tweezers under aseptic conditions, fix the endosperm with the peeled off seed coat under a stereomicroscope with ultra-fine tissue culture tweezers, and use surgical The blade slices the endosperm longitudinally. Because the color of the embryo is the same as that of the endosperm, but the tissue density is different, the shadow-shaped immature zygotic embryo located in t...

Embodiment 2

[0019] 1) Sampling and disinfection: In September every year, remove the attached fibrous tissue from the seeds of P. chinensis with fleshy seed coat, wash them in running water, soak them in tap water for 24 hours, and pass them through 0.1% concentration of HgCl under sterile conditions. 2 After 5 minutes of disinfection, wash with sterile water 10 times;

[0020] 2) Stripping of immature zygote embryos: peeling off the fleshy seed coat of the sterilized seeds under aseptic conditions, fixing the peeled endosperm with tissue culture tweezers, and longitudinally cutting the endosperm with a scalpel;

[0021] 3) Somatic embryo induction: Inoculate the stripped immature embryos on a medium with a pH of 5.8 (medium is 1 / 2MS, agar powder concentration is 7.5g / L, sucrose concentration is 30g / L), at 19°C Continuous dark light cultivation;

[0022] 4) Establishment of the embryogenic tissue culture system: after 6 months of culture, the somatic embryos obtained by the induction wer...

Embodiment 3

[0024] 1) Sampling and disinfection: In August every year, remove the attached fibrous tissue from the seeds of P. edulis with fleshy seed coat, wash them in running water, soak them in tap water for 24 hours, and pass them through 0.1% concentration of HgCl under sterile conditions. 2 After 5 minutes of disinfection, wash with sterile water 10 times;

[0025] 2) Stripping of immature zygote embryos: peeling off the fleshy seed coat of the sterilized seeds under aseptic conditions, fixing the peeled endosperm with tissue culture tweezers, and longitudinally cutting the endosperm with a scalpel;

[0026] 3) Somatic embryo induction: Inoculate the stripped immature embryos on a medium with a pH of 5.8 (medium is 1 / 2MS, agar powder concentration is 7.5g / L, sucrose concentration is 30g / L), at 21°C Continuous dark light cultivation;

[0027] 4) Establishment of embryogenic tissue culture system: After 6 months of culture, the induced somatic embryos were stripped and placed on a n...

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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 tissue culture of traditional Chinese medicines, in particular to a tissue culture method for inducing immature zygotic embryos of Polyphylla polyphylla to produce somatic embryos. Background technique [0002] Zhonglou is an authentic rare and precious medicinal material in Yunnan and Sichuan regions of my country. It is the main raw material of Chinese patent medicines such as "Yunnan Baiyao", "Gongxuening" and "Jidesheng Snake Tablets". It can also be directly used in medicine to stop bleeding, relieve pain and treat snakes Bites, fractures, mumps, tumors, and immune disorders. Because of its high medicinal value, the demand for heavy building is increasing by nearly 20% every year. Moreover, the seeds of chrysanthemum have a long dormancy period, low germination rate, and long resource regeneration cycle. At the same time, due to the huge demand, the phenomenon of unrestrained predatory collection of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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