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Agrobacterium tumefaciens-mediated dioscorea zingiberensis transgene method

An Agrobacterium-mediated technology of Dioscorea scutellaria, applied in the biological field, can solve problems such as unstable content of saponin in high-yielding plants

Inactive Publication Date: 2010-08-04
ZHEJIANG NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These technical means currently on the market have not realized the improvement of the genetic varieties of Dioscorea scutellaria, and the high-yielding plants of saponin obtained through these screenings have problems such as unstable content.

Method used

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  • Agrobacterium tumefaciens-mediated dioscorea zingiberensis transgene method
  • Agrobacterium tumefaciens-mediated dioscorea zingiberensis transgene method
  • Agrobacterium tumefaciens-mediated dioscorea zingiberensis transgene method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. Induction of callus of Dioscorea scutellaria

[0031] 1. Take the surface-sterilized stem section of Dioscorea scutellaris, cut it into a single-segment stem containing a single bud, and inoculate it on solid medium MS+BA1.0~2.0mg / L+NAA 0.5mg / L for axillary bud induction, pH 5.8, 25 ± 1 ℃, light to cultivate sterile seedlings;

[0032] 2. Take 120 leaves and stems of sterile seedlings, cut the leaves into about 0.5cm 2 Size, stem segments were cut into about 1 cm in size and inoculated in solid callus induction medium MS+BA 3.0mg / L, 25±1°C, cultured in the dark, and 139 calli were obtained after one month.

[0033] 2. Agrobacterium infection and transformation

[0034] 1. Prepare engineering bacteria with GUS gene, cultivate and activate them, pick a single colony of Agrobacterium, and add them to YEP liquid medium for shaking culture to OD 600 Value 0.4~0.6;

[0035] 2. Add the embryogenic callus to the Agrobacterium bacterial liquid, infect for 20-30 minutes, t...

Embodiment 2

[0042] 1. Induction of callus of Dioscorea scutellaria

[0043] 1. Take the sterilized surface-sterilized stem section of Dioscorea peltifolius, cut it into a single-segment stem containing a single bud, and inoculate it on a solid medium for axillary bud induction MS+BA2.0mg / L+NAA 0.5mg / L, pH 5.8, 25±1 Cultivate sterile seedlings under light;

[0044] 2. Take the stems of sterile seedlings and inoculate them in solid callus induction medium MS+BA 2.0~3.0mg / L+NAA 0~0.5mg / L, 25±1℃, culture in the dark, inoculate 360, one 294 calluses were obtained one month later;

[0045] 2. Agrobacterium infection and transformation

[0046]1. Prepare the engineering bacteria of Agrobacterium tumefaciens strain EHA105, the expression vector is D2-8, cultivate and activate, pick a single colony of Agrobacterium tumefaciens, and add it to YEP liquid medium for shaking culture until the OD value is 0.3-0.7;

[0047] 2. Add the embryogenic callus to the Agrobacterium bacterial liquid, infect f...

Embodiment 3

[0053] 1. Induction of callus of Dioscorea scutellaria

[0054] 1. Take the sterilized surface-sterilized stem section of Dioscorea peltifolius, cut it into a single-segment stem containing a single bud, and inoculate it on a solid medium for axillary bud induction MS+BA2.0mg / L+NAA 0.5mg / L, pH 5.8, 25±1 Cultivate sterile seedlings under light;

[0055] 2. Take the stems of sterile seedlings and inoculate them in the solid callus induction medium MS+BA 2.0~3.0mg / L+NAA 0~0.5mg / L, 25±1℃, cultivate in the dark, and it can be obtained after one month callus.

[0056] 2. Agrobacterium infection and transformation

[0057] 1. Prepare the engineering bacteria of Agrobacterium tumefaciens strain EHA105, the expression vector is D1-6, cultivate and activate, pick a single colony of Agrobacterium tumefaciens, add it into YEP liquid medium and shake it to cultivate to OD value of 0.3-0.7;

[0058] 2. Add the embryogenic callus to the Agrobacterium bacterial liquid, infect for 15-45 min...

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Abstract

The invention discloses an agrobacterium tumefaciens-mediated dioscorea zingiberensis transgene method, which belongs to the technical field of biotechnology. The method lays a foundation for deeply researching the medicinal value of dioscorea zingiberensis by transgene technology. The agrobacterium tumefaciens-mediated dioscorea zingiberensis transgene method comprises the following steps: (1) obtaining an embryonic callus acceptor material through a high-frequency regeneration system; (2) picking individual agrobacterium tumefaciens colonies, and adding the colonies into a YEP culture medium to perform culture; (3) adding the induced callus into the agrobacterium tumefaciens liquid, infecting the callus, taking an explant out and transferring the explant to a co-culture medium to perform culture and transition culture, and then transferring the cultured explant to a screening culture medium to perform screening; and (4) picking resistant callus to perform differentiation and rooting.

Description

technical field [0001] The invention relates to the technical field of biotechnology, in particular to a transgenic breeding technology of Dioscorea scutellaria. Background technique [0002] Dioscorea Zingiberensis C.H Wright (Dioscorea Zingiberensis C.H Wright) is an important medicinal plant of the genus Dioscorea in the family Dioscoreaceae. An important raw material of hormone drugs, with the highest content in the genus Dioscorea. Thousands of steroid hormone drugs and water-soluble saponin preparations can be obtained after the structure of diosgenin is modified, which is called "medicinal gold" by the medical field. Therefore, as an important medicinal source plant, Dioscorea scutellaria has high economic value. In recent years, with the increasing demand of Dioscorea scutellaria and the lack of scientific guidance in the process of excavation, the wild resources of Dioscorea scutellaria scutellaria are increasingly depleted, and the quality is seriously degraded. ...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 马伯军刘帅顾志敏
Owner ZHEJIANG NORMAL UNIVERSITY
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