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A kind of eucalyptus genetic transformation method

A genetic transformation method, eucalyptus technology, applied in biochemical equipment and methods, horticultural methods, botanical equipment and methods, etc., to achieve the effects of increasing germination rate, promoting callus differentiation, and avoiding vitrification

Active Publication Date: 2022-06-14
CENTRAL SOUTH UNIVERSITY OF FORESTRY AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, if the eucalyptus seed germination medium in patent 2018113475844 is adopted, the seed germination rate and germination speed, the callus growth rate of the callus induction medium are relatively slow, and the rooting rate of the rooting medium is far lower than that of the present invention. Patents 201410267835.3; 201410463255.1 are adopted; 201410607050.6; 201510124023.8; 201510231119.4; 201610697486.8; 201711019129.7 The effects of adventitious bud differentiation, proliferation medium, rooting medium and other formulas are not ideal
In short, there is currently no complete set of rapid tissue culture methods for eucalyptus transgenics with outstanding effects, which can not only successfully construct a eucalyptus transgenic system, but also be able to culture from seed germination, explant stem induction, callus differentiation to adventitious buds, and rooting. Efficiency and other aspects to achieve very good results

Method used

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  • A kind of eucalyptus genetic transformation method
  • A kind of eucalyptus genetic transformation method
  • A kind of eucalyptus genetic transformation method

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Experimental program
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Embodiment 1

[0067] The preparation of embodiment 1 eucalyptus seed aseptic seedling

[0068] Use 5, 6, and 10 mesh sieves to screen eucalyptus seeds, soak the seeds larger than the pore size of the mesh sieve in 70% concentrated sulfuric acid for 3-4 minutes, wash the seeds repeatedly with 50g / L sodium bicarbonate solution to completely neutralize the concentrated sulfuric acid, and then use 50g Soak the seeds in 1 / L sodium bicarbonate aqueous solution for 10-12min, disinfect: 70% ethanol for 45s, 0.1% mercuric chloride for 2-10min, rinse with sterile water for more than three times to obtain aseptic seeds, and carry out germination-promoting culture after disinfection, among which eucalyptus Seed germination promotion solid medium DKW basic medium, add according to the following table 1: 6BA, 1.5mg / L, 2.0mg / L, NAA, 0.2mg / L, 0.4mg / L, GA 3 , 0.4mg / L, 0.6mg / L, agar powder 4g / L and gellan gum 1.2g / L were fixedly added. Placed in the environment of 25±2℃, 16 / 8h photoperiod environment, the l...

Embodiment 2

[0072] Example 2 Explant Preparation and Induction of Callus

[0073] (1) Select the stem section of the dormant bud, soak it for more than 12 hours, wash it with running water for more than 3 hours, cut it into a stem section with 1-2 buds, and disinfect it: 70% ethanol for 45 seconds, 0.1% mercury chloride for 8 minutes, sterile water Rinse more than three times to obtain sterile explants, transfer to YD medium and cultivate until new shoots grow (sterile seedlings). The sterile seedlings were transferred to the subculture medium (also YD medium) and allowed to grow to 5-8 cm. YD medium is MS basal medium, supplemented with 6-BA 2mg / L, IBA 0.1mg / L, TDZ 0.5mg / L. Placed in the environment of 25±2℃, 16 / 8h photoperiod environment, the light intensity is 60μmol m -2 the s -1 .

[0074] (2) choose the aseptic seedling of 5-8cm or the aseptic seedling in embodiment 1, cut it into the stem section of about 0.5cm with a sterilized blade, transfer to the rapid induction medium of ...

Embodiment 3

[0087] Embodiment 3 Agrobacterium bacterium liquid preparation

[0088] (1) Stored at -80°C with the 35S promoter, hyper-enriched Southeast Jingtian HMA3 (AJF37113.1) Cd transporter gene, GFP gene (for transformation detection) and resistance gene (for transformation detection) The Agrobacterium LBA4404 strain of the plasmid pBI1302 for screening) was melted on ice (the plasmid pBI1302 was purchased from Shanghai Baige Biological Co., Ltd., and transformed into Agrobacterium by chemical transformation method), and 200 μL was drawn and spread on an LB plate (without antibiotics), 28 °C, 200rpm for 24-48h, until a single colony grows.

[0089] (2) Pick a single colony and inoculate it into 2ml of LB liquid medium (containing 50mg / L Kana, 25mg / L Rif) containing appropriate amount of antibiotics, culture at 28°C with shaking at 200rpm overnight to OD 600 0.6-0.8.

[0090] (3) Inoculate the bacterial liquid into 40ml LB liquid medium (containing 50mg / L Kana, 25mg / L Rif), shake an...

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Abstract

The invention discloses a genetic transformation method of eucalyptus, comprising: (1) preparation of explants of eucalyptus; (2) rapid induction of callus of eucalyptus; (3) infection and co-cultivation of Agrobacterium bacterium liquid; Bacteria removal and screening culture of injured tissue; (5) differentiation culture of adventitious buds; (6) rooting culture. In this eucalyptus genetic transformation method, aseptic seedlings are germinated from eucalyptus seeds, the callus induced by various tissue parts is used as the receptor, and the GFP gene is used as the reporter gene to carry out genetic transformation through the method mediated by Pseudomonas, using hygromycin resistance The sex site gradient screened the positive transformation recipient callus, and the callus was differentiated, cultivated and developed into a complete plant to realize plant regeneration, and an efficient and rapid genetic transformation system of eucalyptus was established.

Description

technical field [0001] The present invention relates to the technical field of eucalyptus genetic transformation and tissue culture, in particular to a method for genetic transformation using eucalyptus callus as a receptor for Agrobacterium-mediated, hygromycin (Cef) gradient selection of positively transformed callus, wherein It also includes a series of complete eucalyptus genetic transformation methods such as rapid acquisition of eucalyptus sterile seedlings and high-efficiency medium ratios required for callus induction, differentiation, and rooting. Background technique [0002] Eucalyptus is a plant of the genus Euania in the Sapindaceae family. It grows in the calcium-based soil produced by weathering of limestone. The plant is resistant to salinity, drought and short-term floods, has a deep root system, and has strong adaptability to the environment. These characteristics make eucalyptus an important pioneer tree species for phytoremediation in heavy metal polluted...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00A01H6/00
CPCC12N15/8205C12N15/8212A01H4/008A01H4/001
Inventor 王平周韬白磊何其浩孙吉康
Owner CENTRAL SOUTH UNIVERSITY OF FORESTRY AND TECHNOLOGY
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