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Genetic transformation method of Chinese fir

A genetic transformation method, fir technology, applied in biochemical equipment and methods, horticultural methods, botanical equipment and methods, etc., can solve problems such as limitations, lack of transformation methods and regeneration systems, and achieve strong stress resistance and low cost Effect

Active Publication Date: 2013-05-08
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But in conifers it is severely limited by the lack of good transformation methods and regeneration systems

Method used

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  • Genetic transformation method of Chinese fir
  • Genetic transformation method of Chinese fir
  • Genetic transformation method of Chinese fir

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Establishment of transforming receptors.

[0031] (1) Take about 5 mL of embryogenic callus cells of Chinese fir subcultured for 3 weeks, inoculate them into a 250 mL sterile Erlenmeyer flask, and gently tap the wall of the flask to disperse the cells. Microphotographs of embryogenic callus cells of Chinese fir figure 1 shown.

[0032] (2) Take 50mL Chinese fir liquid culture medium and add it into the Erlenmeyer flask, place the material in a constant temperature shaker at 23°C and 85r / min for shaking culture. Chinese fir liquid medium is DCR basic medium, supplemented with 10mg / L VC, 0.1-2.0mg / L GA, 2-5mg / L ABA, 0.5g / L CH, 30g / L maltose.

[0033] (3) Subculture once every 7 days, and it can be used for Agrobacterium infection 3 days after the third subculture (that is, from the third day of the fourth generation).

[0034] The receptor system in the process of plant gene transformation refers to the explants used for transformation through tissue culture ...

Embodiment 2

[0037] Example 2 Agrobacterium liquid preparation.

[0038] (1) Melt the EHA105 bacterial liquid with 35S:GUS gene (for transformation detection) and NPT-II gene (for screening) stored at -70°C on ice, dip a small amount of bacterial liquid with an inoculation loop, and inoculate Put it on the LB medium containing appropriate amount of antibiotics (kanamycin 50mg / L, streptomycin 30mg / L), and place it in a constant temperature incubator at 28°C for culture.

[0039] (2) After a single colony grows, use an inoculation loop to pick a single colony, inoculate it on the LB medium containing an appropriate amount of antibiotics (50 mg / L kanamycin, 30 mg / L streptomycin), and activate it again. Then they were cultured in a constant temperature incubator at 28°C.

[0040] (3) After a single colony grows, use a sterilized pipette tip to pick a single colony and inoculate it into a conical flask containing 10 mL of LB liquid medium with an appropriate amount of antibiotics, culture at 2...

Embodiment 3

[0047] Example 3 Infestation.

[0048] Shake the bacterial solution evenly, draw 3mL of the diluted bacterial solution and add it to the Chinese fir suspension cells (Example 1) that have been subcultured for 3 days. After standing for 1-3 minutes, place it on a constant temperature shaker at 23°C and 85r / min to continue shaking Cultivate, that is, co-cultivate.

[0049] Infection refers to the inoculation of engineered bacteria on the surface of the receptor material. The method is to add the prepared Agrobacterium liquid into the culture medium of the receptor material, soak for a certain period of time, and carry out co-cultivation. During the infection process, mastering the infection time will help reduce the possible pollution during the later cultivation process and reduce the poisonous effect of bacteria on plants. The dipping time is too short to allow enough Agrobacterium to attach to the explant wound, thereby reducing the frequency of genetic transformation. If t...

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Abstract

The invention discloses a genetic transformation method of a Chinese fir. The genetic transformation method comprises the steps of: establishment of a transformation receptor, preparation of agrobacterium liquid, infection, bacterium removal culture and screening culture. According to the genetic transformation method of the Chinese fir disclosed by the invention, a Chinese fir suspension cell system is taken as the receptor and a b-glucuronidase (GUS) gene is used as a report gene to implement a genetic transformation through an agrobacterium mediated method, and the plant regeneration is realized through a somatic embryogenesis way, and a stable and efficient Chinese fir genetic transformation system is established. Due to the utilization of the technical system, genetic improvement of the Chinese fir with comparatively low cost can be achieved; and anti-disease, stress-resistant, wood formation and other related genes obtained from a basic and theoretical research are subjected to the genetic transformation so as to obtain a high-quality Chinese fir variety having characteristics of rapid growth, strong anti-disease and stress-resistant performances, and the like.

Description

technical field [0001] The invention relates to a method for genetic transformation of Chinese fir, in particular to a genetic transformation method mediated by Agrobacterium with a Chinese fir suspension cell line as a receptor. Background technique [0002] Chinese fir is a unique timber tree species in my country and one of the most important afforestation tree species in the southern provinces. Chinese fir grows fast, has high yield, good properties, many uses, and good sales. It is very important for forestry production to select and breed excellent Chinese fir plants and clones with fast growth, high fecundity and strong stress resistance. [0003] However, coniferous trees have a long growth cycle, tall trees, and because of long-term outcrossing, they have a high degree of heterozygosity and a large amount of genetic load. Therefore, the traditional breeding cycle is long, and it is difficult to meet the needs of genetic improvement of fast-growing trees. Forest t...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H4/00A01H5/00
Inventor 施季森王丹陈金慧周小红
Owner NANJING FORESTRY UNIV
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