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Efficient and stable genetic transformation method for strawberries mediated by agrobacterium tumefaciens

A genetic transformation method, the technology of Agrobacterium tumefaciens, is applied in the fields of biochemical equipment and methods, horticultural methods, and botanical equipment and methods, and can solve the problems of screening difficulties, reducing the false positive rate of regenerated buds, and low transformation efficiency. Achieve the effect of reducing false positive rate, improving strawberry transgenic efficiency and improving genetic transformation efficiency

Pending Publication Date: 2017-05-31
SICHUAN AGRI UNIV
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Problems solved by technology

[0004] In order to overcome the above-mentioned technical problems existing in the prior art, the present invention provides a highly efficient and stable genetic transformation method of strawberry mediated by Agrobacterium tumefaciens, which optimizes the steps of genetic transformation, thereby greatly improving the transgenic efficiency of strawberry and solving the problem of The traditional Agrobacterium-mediated method has solved the problems of low transformation efficiency, easy generation of chimeras, difficulty in screening, and heavy workload. It has greatly improved the efficiency of genetic transformation and significantly reduced the false positive rate of regenerated shoots.

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  • Efficient and stable genetic transformation method for strawberries mediated by agrobacterium tumefaciens
  • Efficient and stable genetic transformation method for strawberries mediated by agrobacterium tumefaciens
  • Efficient and stable genetic transformation method for strawberries mediated by agrobacterium tumefaciens

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example 1

[0048] Example 1, taking 'Hongyan' strawberry as an example, adopt the genetic transformation method provided by the present invention to test its genetic transformation effect.

[0049] 1) Pre-cultivation: take 10-30-day-old aseptic tissue-cultured seedlings of 'Hongyan' strawberry, punch out a leaf disk with a diameter of about 5 mm, and place it in a petri dish containing 25 ml of M1 medium. The abaxial surface of the leaves is in contact with the culture medium, put 16 pieces in each petri dish, seal, and culture in the dark at 25°C for 3 days; figure 2 Shown in a is the effect diagram of the genetic transformation of 'Hongyan' strawberry in the pre-cultivation stage.

[0050] 2) Agrobacterium activation: pick the positive monoclonal of Agrobacterium GV3101 in 25 ml of YEB liquid medium containing antibiotics (50 mg / L kanamycin sulfate + 20 mg / L rifampicin), 28 ° C under dark conditions, 150 rpm Centrifuge at 6,000 rpm for 5 minutes to harvest the bacteria, resuspend the...

example 2

[0058] Example 2, taking 'Fengxiang' strawberry as an example, using the genetic transformation method provided by the present invention to test its genetic transformation effect.

[0059] 1) Pre-cultivation: take 10-30-day-old aseptic tissue-cultured seedlings of 'Fengxiang' strawberry, use a puncher to punch out a leaf disc with a diameter of about 5 mm, and place it in a petri dish containing 25 ml of M1 medium , with the abaxial surface of the leaves in contact with the medium, put 16 in each petri dish, seal, and culture in the dark at 25°C for 3 days;

[0060] 2) Agrobacterium activation: pick Agrobacterium LBA4404-positive single clones in 25 ml of YEB liquid medium containing antibiotics (50 mg / L kanamycin sulfate + 20 mg / L rifampicin), 28 ° C under dark conditions, 150 rpm Centrifuge at 6,000 rpm for 5 minutes to harvest the bacteria, resuspend the bacterial pellet in 25 ml of M2 medium, culture at 28°C, 150 rpm, and shake for 3-4 hours to OD 600 =0.3 (about 10 8 c...

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Abstract

The invention relates to an efficient and stable genetic transformation method for strawberries mediated by agrobacterium tumefaciens. The method comprises the steps: preculturing aseptic seedlings of strawberries, activating agrobacteria, infecting, co-culturing, delaying screening, and screening and rooting. According to the efficient and stable genetic transformation method, the genetic transformation steps are optimized, so that the transgenosis efficiency of the strawberries is increased by a large margin; the problems that a traditional agrobacterium-mediated method is low in transformation efficiency, chimeras are easy to generate, the screening is difficult and the workload is great are solved; the genetic transformation efficiency is improved enormously; and meanwhile, the false positive rate of regeneration buds is obviously lowered.

Description

technical field [0001] The invention relates to the technical field of plant biology, in particular to an efficient and stable genetic transformation method of strawberry mediated by Agrobacterium tumefaciens. Background technique [0002] The Agrobacterium tumefaciens-mediated method is an important method for the genetic transformation of strawberry. It uses strawberry leaves, petioles, protoplasts and callus as explants, and after being infiltrated with Agrobacterium, the regeneration is achieved through antibiotic selection and organogenesis. Plants, of which the "leaf disc method" is the most commonly used. [0003] The traditional "leaf disk method" has problems such as low genetic transformation efficiency, serious bacterial contamination, difficulty in regeneration of adventitious buds, and easy generation of chimeras and false positive plants, which lead to heavy screening workload and difficulty in obtaining transgenic plants. With the deepening of strawberry geno...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H4/00A01H5/00
CPCA01H4/00C12N15/8205
Inventor 汤浩茹江雷雨陈品文肖婕宋霞陈清王小蓉罗娅张勇孙勃
Owner SICHUAN AGRI UNIV
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