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Method for screening MSAP sites relevant to regeneration in vitro of lycium ruthenicum

A technology of black fruit Lycium barbarum and in vitro regeneration, applied in the field of plant epigenetics, can solve the problems of limited establishment and application, unclear molecular mechanism of callus, etc.

Inactive Publication Date: 2018-09-28
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the molecular mechanism of dedifferentiation of woody plant explants to form callus is still unclear, which limits the establishment and application of woody plant regeneration systems in vitro

Method used

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  • Method for screening MSAP sites relevant to regeneration in vitro of lycium ruthenicum
  • Method for screening MSAP sites relevant to regeneration in vitro of lycium ruthenicum
  • Method for screening MSAP sites relevant to regeneration in vitro of lycium ruthenicum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 A method for screening MSAP sites related to in vitro regeneration of Lycium barbarum

[0062] 1. Obtain sterile seedlings

[0063] Put the mature seeds of wolfberry black fruit in warm water at 37 ℃ for 24 hours; soak the seeds in 70-75% alcohol in a sterile ultra-clean workbench for 30 s-1min, and quickly soak the seeds in 0.2% mercuric chloride aqueous solution 2 min; after rinsing with sterile water for 4 times, blot the water on the surface of the seeds and inoculate them horizontally on 1 / 2MS solid medium without any plant growth regulator (add 0.45% agar and 2% sucrose, pH The value was adjusted to 5.8 with 1M KOH, autoclaved at 121°C for 16 minutes), and the seeds and medium must be in close contact. The inoculated materials were cultured at 25 °C under the photoperiod conditions of "light 12 h / dark 12 h", and the light was 3000 lx.

[0064] 2. Inoculation of leaf explants to obtain callus and regenerated plants

[0065] When the seeds inoculated in...

Embodiment 2

[0132] Example 2 A method for screening MSAP sites related to in vitro regeneration of Lycium barbarum

[0133] 1. Obtain sterile seedlings

[0134] Put the mature seeds of wolfberry black fruit in warm water at 37 ℃ for 24 hours; soak the seeds in 70-75% alcohol for 30 s-1 min in a sterile ultra-clean workbench, and quickly place the seeds in 0.1% mercuric chloride aqueous solution Soak for 5 min; after rinsing with sterile water for 5 times, blot the water on the surface of the seeds and inoculate them horizontally in 1 / 2MS solid medium without any plant growth regulator (add 0.45% agar and 2% sucrose, Adjust the pH value to 5.8 with 1M KOH, autoclave at 121°C for 16 minutes), pay attention to the close contact between the seeds and the medium; place the inoculated material at 25°C, under the photoperiod conditions of "light 12 h / dark 12 h" For cultivation, the light is 3000 lx.

[0135] 2. Inoculation of leaf explants to obtain callus and regenerated plants

[0136] When...

Embodiment 3

[0201] Example 3 A method for screening MSAP sites related to in vitro regeneration of Lycium barbarum

[0202] 1. Plant sterile seeds to obtain sterile seedlings

[0203] Put the mature seeds of wolfberry black fruit in warm water at 37 ℃ for 24 hours; soak the seeds in 70-75% alcohol for 30 s-1 min in a sterile ultra-clean workbench, and quickly place the seeds in 0.2% mercuric chloride aqueous solution Soak for 2 min; after rinsing with sterile water for 4 times, blot the water on the surface of the seeds and inoculate them horizontally in 1 / 2MS solid medium without any plant growth regulator (add 0.45% agar and 2% sucrose, Adjust the pH value to 5.8 with 1M KOH, autoclave at 121°C for 16 minutes), pay attention to the close contact between the seeds and the medium; place the inoculated material at 25°C, under the photoperiod conditions of "light 12 h / dark 12 h" For cultivation, the light is 3000 lx.

[0204] 2. Inoculation of leaf explants to obtain callus and regenerate...

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Abstract

The invention discloses a method for screening MSAP sites relevant to regeneration in vitro of lycium ruthenicum. The method comprises the steps that (1) sterile seedlings are cultured; (2) leaf explants are inoculated, and thus calluses and regenerated plants are obtained; (3) donor plants and leaf-regenerated plants are acclimated and transplanted; (4) DNA of the calluses, DNA of transplanted and non-transplanted donor plant leaves and DNA of transplanted and non-transplanted regenerated plant leaves are extracted; (5) all the types of extracted DNA are subjected to double enzyme digestion through combination of EcoR I and Msp I or combination of EcoR I and Hpa II, and double-chain connectors are connected; (6) a PCR pre-amplification reaction is conducted; (7) a selective PCR amplification reaction is conducted; (8) capillary electrophoresis is conducted; (9) statistic analysis is conducted, and the MSAP sites relevant to regeneration in vitro of lycium ruthenicum leaves are screened; and (10) specific bands are recovered, sequenced and analyzed. According to the method for screening the MSAP sites relevant to regeneration in vitro of the lycium ruthenicum, the MSAP sites relevant to leaf regeneration can be screened rapidly and are unrelated to transplanting and acclimatizing; and excavation of the type of the sites has important significance to finally disclose dedifferentiation and redifferentiation mechanisms of woody plant leaves, and the content of plant epigenetics is enriched.

Description

technical field [0001] The invention belongs to the field of plant epigenetics, and in particular relates to a method for screening MSAP sites related to in vitro regeneration of Lycium barbarum. Background technique [0002] Black Goji Berry ( Lycium ruthenicum Murr.) is a perennial thorny shrub of Lycium barbarum of the Solanaceae family. It is a halophytic medicinal plant and an important ecological and economic tree species. In my country, black fruit wolfberry is mainly distributed in Ningxia, Shanxi, Gansu, Qinghai, Xinjiang, Tibet and other provinces. Black fruit wolfberry contains a variety of essential amino acids and trace elements, which can improve human immunity; black fruit wolfberry is known as the king of proanthocyanidins, and its proanthocyanidins can scavenge free radicals, enhance immunity, prolong the lifespan of fruit flies, and reduce the risk of mice Serum total cholesterol and triglycerides, increase serum, liver tissue and body's total antioxidan...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/6855C12Q1/6895
CPCC12Q1/6858C12Q1/6855C12Q1/6895C12Q2600/13C12Q2600/156C12Q2531/113C12Q2565/125C12Q2521/313C12Q2525/191
Inventor 王钦美王皓崔进崔建国李娜林梅
Owner SHENYANG AGRI UNIV
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