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Strawberry genetic transformation system construction method

A technology of a genetic transformation system and a construction method, which is applied in the field of tissue culture regeneration and genetic transformation of 'Shenqi' strawberry leaves, can solve the problems of narrow breeding range, long time consumption, difficulty in directional improvement of beneficial traits, etc., and achieve less pollution. effect of the problem

Pending Publication Date: 2021-11-02
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the polyploidy and high heterozygosity of strawberry chromosomes, traditional breeding is limited by the difficulty of targeted improvement of beneficial traits, narrow breeding range, and long time consumption.

Method used

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  • Strawberry genetic transformation system construction method
  • Strawberry genetic transformation system construction method
  • Strawberry genetic transformation system construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 A new strawberry variety 'Shenqi' was obtained aseptic seedlings rapidly propagated from shoot tips.

[0065] The new strawberry variety 'Shenqi' is a new variety bred by the Shanghai Academy of Agricultural Sciences in 2018. This variety has large fruit, anthracnose resistance, and strong continuous fruiting ability. It has been widely promoted in the upper triangle area. The system of the present invention has established a new variety 'Shenqi' shoot tip rapid propagation technology includes shoot tip induction, shoot tip differentiation induction, seedling proliferation culture and rooting culture.

[0066] Stem tip disinfection treatment: select a stolon tip that is free from pests and diseases and has a length of about 4 cm and rinses it under running water for 1.5-2 hours ( image 3 ), sterilized with 75% alcohol for 30s on the ultra-clean bench, then sterilized with 6% sodium hypochlorite for 6 minutes, and finally washed with sterile water for 5 times,...

Embodiment 2

[0071]Example 2 Effects of different concentrations of exogenous hormone 6-BA on callus and adventitious bud induction on leaves and petioles of a new strawberry variety 'Shenqi'.

[0072] Select the leaves of ‘Shenqi’ with a leaf age of about 35 days for the third generation of subculture, remove the leaf margins and main leaf veins, and cut into 0.3-0.5 cm 2 Place the small pieces face down, cut the stems into 0.5cm and fit them on the adventitious bud induction medium with different concentrations of cytokinin 6-BA for cultivation; the ratio of hormones can be seen from Table 1, the medium is replaced once every 20 days, and cultured for 40 days Then the callus grade was counted, and the adventitious bud induction rate was counted after 8 weeks of culture.

[0073] Table 1 Concentration of different hormones

[0074]

[0075] Under the induction of different '6-BA' concentration gradients, the callus induction was statistically counted at 40 days of culture, and it was ...

Embodiment 3

[0078] Embodiment 3 Agrobacterium tumefaciens activation operation method

[0079] Before carrying out Agrobacterium GV3101 and 'Shenqi' leaf or petiole dipping, after the Agrobacterium is activated, it is conducive to the dipping of the bacteria liquid and improving the transformation efficiency. The specific activation method is as follows:

[0080] (1) Use a 200 μL pipette tip to pick up Agrobacterium stored at -80°C on a clean bench, and culture it in a streak on LB+Kan 50mg / L+Gen 50mg / L+Rif 50mg / L solid medium, 28 ℃, 12-48h, grow a single colony.

[0081] (2) Pick up a single colony with a sterile pipette tip, streak and save it, and at the same time use the pipette tip to pick up a single colony and inoculate it in 5ml LB +Kan 50mg / L+Gen50 mg / L+Rif 50mg / L liquid medium, 28 Cultivate at 200 rpm for 20 hours to obtain the bacterial liquid required in step (3).

[0082] (3) Take 10 μL of the bacterial solution cultured in (2) in 20 ml of the same liquid medium, culture a...

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Abstract

The invention discloses a genetic transformation system of a new strawberry variety 'Shenqi'. An agrobacterium tumefaciens-mediated 'Shenqi' leaf and petiole genetic transformation system is established through a leaf and petiole in-vitro regeneration method, and the method comprises the following steps: preparing a sterile leaf; screening the optimal exogenous hormone concentration to induce the leaves and petioles of the strawberries' Shenqi 'to regenerate adventitious buds; analyzing the sensitivity of 'Shenqi' leaf regeneration on antibiotics; activating and culturing agrobacterium tumefaciens; inducing and culturing adventitious buds of the leaves and the petioles; soaking leaves with agrobacterium and performing co-culturing; screening positive strains with antibiotics; and detecting a transformed strain and the like. According to the method disclosed by the invention, adventitious buds are induced by utilizing strawberry leaves and petioles, the leaves are impregnated by agrobacterium, and transformed strains are identified by methods such as antibiotic screening, fluorescence screening and PCR (Polymerase Chain Reaction) detection after co-culture, so that the method is suitable for tissue culture efficient regeneration of the strawberry 'Shenqi' leaves and petioles and agrobacterium-mediated genetic transformation application; the method has important significance on genetic engineering of a new variety of 'Shenqi' strawberries.

Description

technical field [0001] The invention relates to a method for regeneration of 'Shenqi' strawberry leaves and the establishment of an Agrobacterium-mediated genetic transformation system, belonging to the technical field of tissue culture regeneration and genetic transformation of 'Shenqi' strawberry leaves. Background technique [0002] Strawberry (Fragaria×ananassa) is a perennial herbaceous plant of the genus Fragaria in the Rosaceae family. It is one of the important fresh fruit crops and a model species for the study of important functional genes of Rosaceae plants. Due to the polyploidy and high heterozygosity of strawberry chromosomes, traditional breeding is limited by the difficulty of targeted improvement of beneficial traits, narrow breeding range, and long time consumption. In the 1980s, many scholars at home and abroad began to study the transgenic methods of strawberries. In 1990, Michael et al. used the in vitro regeneration system of strawberries to successfull...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00A01H6/74
CPCC12N15/8205A01H4/002A01H4/008
Inventor 邹小花黄系伦刘海婷高清华
Owner SHANGHAI ACAD OF AGRI SCI
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