Method for inducing embryogenic cells by using Atropa belladonna L explant

A technology of embryogenic cells and explants, applied in the biological field

Inactive Publication Date: 2012-04-04
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Through belladonna embryogenic regeneration plants, belladonna virus-free seedlings can be rapidly multiplied in large quantities, and embryogenic cells can also create and screen excellent clones to obtain plants with relatively high and stable TAs content, which can also be used as recipients of transgenes for cultivation High-yielding transgenic belladonna provides high-quality new drug sources for the production of TAs. At present, there is no related report except the research results of our laboratory

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Obtaining sterile explants of belladonna

[0021] Method 1: Using leaves to establish sterile explants of belladonna

[0022] Take the mature leaves of belladonna, or the stems with joints, rinse with running water for 1 hour; treat with 75% ethanol for 30 seconds, and then use 2% (M / V) NaClO solution or 0.1% (M / V) mercuric chloride (HgCl 2 ) soaked for 10 minutes, rinsed 6 times with sterile water; then inoculated in the medium for inducing cluster buds (cultivation based on 121 0 C sterilized for 20 minutes and distributed in culture bottles or plates), the formula of the medium is: MS basic medium, adding plant growth regulator 2.0 mg / L BAP (benzyl adenine), 0.3 mg / L NAA ( Naphthalene acetic acid), 30g / L sucrose, pH value is 5.8, then add 5% agar powder. Cultivate the young shoots of belladonna in the light incubator, the culture condition is: 25 0 C, 12 hours of light, the light intensity is 55μmol.m -2 .s -1 . After 40 days, aseptic belladonna aseptic explant...

Embodiment 2

[0028] Establishment of Embryogenic Cell Suspension Culture System

[0029] Method 1: Obtaining embryogenic cells by inducing shoot tips or lateral buds of belladonna aseptic seedlings

[0030] Under the Nikon SMZ800 stereomicroscope, magnify and observe, and use a syringe needle to strip the belladonna shoot tip or lateral bud meristem with a length of about 0.5mm, and inoculate it in MS solid medium supplemented with 2,4-D 3.0mg / L (MSA ) on induction medium, cultured at 25°C under light, and embryogenic cells can be induced after 4-6 weeks. The induced embryogenic cells were screened under the condition of 600-800 times magnification under a stereo microscope, and the induced somatic embryos could be subcultured into a new MS solid medium (MSB) supplemented with 2,4-D 2.0mg / L Preserved from generation to generation.

[0031] Method 1: Using belladonna seeds to induce embryogenic cells

[0032] The mature and plump seeds were screened by water selection, rinsed with runnin...

Embodiment 3

[0034] Screening, multiplication and subculture of embryogenic cells

[0035] Induced embryogenic cells can be subcultured to a new embryogenic cell induction medium, subcultured once a month, and non-embryogenic callus is removed to purify somatic embryos, somatic embryos are purified and multiplied for preservation and provide material for suspension cultures.

[0036] After 6-8 weeks of proliferation of embryogenic callus, break the embryogenic callus into small cell clusters through a 0.5mm sieve, and transfer them to a triangular flask or plastic bottle with 30-50ml of MS suspension medium Suspension culture in medium, subculture once every 3-10 days, and need to be crushed with a 0.5mm sieve during subculture. Suspension culture conditions are: 30° C., shaker rotation speed 120 rpm.

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Abstract

The invention provides a method for inducing embryogenic cells by using an Atropa belladonna L explant. The method relates to establishment of an in-vitro culture system, induction of the embryogenic cells, suspension culture of the embryogenic cells, and regeneration of plants obtained by using the embryogenic cells. The method comprises the following steps: inducing the embryogenic cells by using Atropa belladonna L aseptic seedling, propagating and subculturing the embryogenic cells, and inducing plants to regenerate by the embryogenic cells. The embryogenic cells obtained by the method can be used for producing virus-free seedling, and can produce new induction strain by using induction and mutation of a genetic material, and can be used as a transformation receptor to obtain a transformant capable of producing tropane alkaloids.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method utilizing belladonna ( Atropa belladonna L.) A method for sterile seedlings to induce embryogenic cells and embryogenic cells to regenerate plants. Background technique [0002] Scopolamine (scopolamine, C 17 h 21 o 4 N) and hyoscyamine (hyoscyamine, C 17 h 23 o 3 N) (the racemate is atropine) are all Tropane alkaloids (Tropane alkaloids, TAs are), which are two important basic drugs widely used in clinic. It is mainly used for analgesia, anesthesia, anti-motion sickness drugs, treatment of Parkinson's disease, improvement of microcirculation, detoxification, treatment of pesticide poisoning, etc. The market demand is huge. With the continuous development of new functions of TAs, its demand is still growing rapidly. TAs are mainly obtained from the Solanaceae belladonna ( Atropa belladonna ), Tianxianzi ( Hyoscyamus niger ), Mandala ( Datura stramonium ), the o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00
Inventor 廖志华杨春贤陈敏唐克轩张磊
Owner SOUTHWEST UNIVERSITY
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