Agrobacterium tumefaciens-mediated petunia hybrida 'Mellin' efficient genetic transformation system

A genetic transformation system and Agrobacterium-mediated technology are applied in the field of establishing an Agrobacterium-mediated high-efficiency genetic transformation system of petunia 'Merlin' to achieve the effects of convenient material collection, high regeneration rate and avoidance of variation.

Inactive Publication Date: 2021-08-13
NORTHEAST FORESTRY UNIVERSITY
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Problems solved by technology

At present, there is no report on the genetic transformation of this variety. Establish an efficient genetic transformation system, obta...

Method used

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  • Agrobacterium tumefaciens-mediated petunia hybrida 'Mellin' efficient genetic transformation system
  • Agrobacterium tumefaciens-mediated petunia hybrida 'Mellin' efficient genetic transformation system
  • Agrobacterium tumefaciens-mediated petunia hybrida 'Mellin' efficient genetic transformation system

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Embodiment Construction

[0019] The construction of the Agrobacterium-mediated genetic transformation system for petunia 'Melin' leaves is further illustrated by the following examples. The Agrobacterium tumefaciens strain used in the examples is EHA105, which contains the plant expression vector PBI121 and carries the green fluorescent protein gene GFP.

[0020] 1. Dark cultivation of petunia 'Merlin' leaves

[0021] Before the transformation, the cut petunia ‘Meilin’ leaf discs were respectively placed on the leaf differentiation medium for dark culture for 1, 2, 3, 4 d and then used for infection (with diluted bacterial solution (OD 600 =0.5) infection for 3 min), the infected leaves were inoculated on the leaf differentiation medium, co-cultivated in the dark at 28°C for 36 h, and then transferred to -1 Cephalosporin (Cef) and 50mg·L -1 Kanamycin (Kana) leaf differentiation medium. Subculture once every 15 days until resistant callus or resistant shoots grow. The resistant callus differentiati...

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Abstract

The invention relates to establishment of an efficient genetic transformation system of petunia hybrida 'Mellin'. The method comprises the following steps: (1) dark culture of leaves; (2) preparation of an infection solution; (3) infection; (4) co-culture; (5) screening and culturing; (6) resistant plant acquisition; and (7) positive plant acquisition. According to the method, the genetic transformation rate of petunia hybrida 'Mellin' is improved through dark culture of leaves, bacterial liquid infection time, kanamycin sensitivity detection, antibiotic concentration screening and the like, the problems of low transformation rate and differentiation rate, long breeding time and the like are solved, the rooting rate of resistant plants reaches 66.24% by the system, and positive transgenic plants can be obtained within 2.5 months.

Description

technical field [0001] The invention relates to a petunia genetic transformation method in the field of biotechnology, in particular to a method for establishing an Agrobacterium-mediated high-efficiency genetic transformation system for petunia 'Mei Lin' (abbreviated as 'Mei Lin') using leaves as receptors. Background technique [0002] Petunia (Petunia hybrida) is a perennial herbaceous plant of Solanaceae, which is often cultivated as an annual in Northeast China. It has been widely used in gardens all over the world for a long time because of its rich flower colors and strong adaptability, and is known as the king of flower beds; because of its short life cycle, clear genetic background, and easy gene transfer mediated by Agrobacterium tumefaciens, etc. Advantages It becomes a model plant in the study of flowering molecular biology, and provides favorable conditions for verifying gene functions and creating new varieties in plant genetic engineering. At present, domesti...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/65A01H4/00A01H5/12A01H6/82
CPCC12N15/8205C12N15/65A01H4/005A01H4/008
Inventor 张彦妮燕浩黄洪峰王依萍
Owner NORTHEAST FORESTRY UNIVERSITY
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