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Populus genetic transformation method

A genetic transformation method and poplar technology, applied in horticultural methods, botanical equipment and methods, introduction of foreign genetic material using vectors, etc. problem, to achieve a good effect of cell state uniformity

Inactive Publication Date: 2013-08-28
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the transgenic research of poplar is generally based on the Agrobacterium-mediated genetic transformation method using the leaf disc as the recipient material. Due to the poor consistency of the physiological and biochemical state of the leaf cells, the genetic transformation conditions are difficult to unify, and the transformation efficiency is not high, which affects the research on the gene function of poplar.

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The petioles, stems, and leaves of Nanlin 895 were cultured in the dark on T medium for 20-30 days at a temperature of 25±2°C. Calluses appeared at the incisions of the petioles and stems, but the leaves gradually became transparent. A thin layer of callus is formed on the surface of the leaves, which cannot be subcultured. The shape and color of the callus grown on the petiole and the stem segment are different, figure 1 It is the result of callus induction of young stem segments and leaf discs. The callus with light yellow color and loose texture was selected and cultured on the medium with different concentrations of hormones, and it was found that 2,4-D had a significant effect on the growth of callus. When the concentration of 2,4-D is less than 1mg / L, the callus grows slowly and the color is dim; Viscous substance secretion; when the concentration of 2,4-D is between 2 and 3 mg / L, the callus is light yellow, small granular, loose in texture, easy to disperse, a...

Embodiment 2

[0027] For the suspension culture of callus, the proportion of hormones in the medium, the number of revolutions of the shaker, the amount of callus inoculum and the volume of liquid medium all have an effect on the establishment of the suspension system. Take about 2 g of Nanlin 895 Populus callus of Example 1, which has been subcultured for 10-15 days and has the same state and good dispersibility, and put it in a 100 mL sterile Erlenmeyer flask, add 30 mL of suspension medium to be T2: MS+2, 4-D 2.0mg / L+NAA 0.2mg / L+KT 0.1mg / L+hydrolyzed milk protein 500mg / L+sucrose 30g / L, the suspension culture was carried out on a shaker at a constant temperature (28°C) at 100rpm. Subculture once every 4-6 days according to the growth status of the culture. During the first few subcultures, suck up about 2 / 3 of the supernatant with a pipette, and add 30 mL of liquid culture base to suspend cells, such as figure 2 shown.

Embodiment 3

[0029] The Nanlin 895 poplar cell suspension culture line that had been cultured for 2 to 3 months was placed in the bud differentiation medium shown in Table 1, and the germination rate was counted after 30 days and 45 days of culture to study the culture medium with different hormone ratios. Effects on bud differentiation of cell suspension lines. The culture conditions for bud induction are temperature 25±2°C, light 3000-4000 lux, light time 16-18h / d.

[0030] Table 1 Suspension cell line bud induction medium

[0031]

[0032] Note: The basic components of the medium are: MS + sucrose 30g / L + crystal agaric 1.8g / L + kan20mg / L, pH: 5.8, and the unit of hormone concentration is mg / L.

[0033] The experimental results are shown in Table 2. Combined with the experimental results, it is finally determined that the most suitable medium for bud induction of the Nanlin 895 poplar callus suspension system is: MS+6-BA1.0mg / L+NAA0.1mg / L+sucrose 30g / L+crystal Agar 1.8g / L+kan20mg / L...

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Abstract

The invention discloses a populus genetic transformation method which comprises a step of establishing a genetic transformation technology system taking a populus suspension cell line as a receptor from the aspects of explant selection, callus induction, suspension cell line establishment, suspension cell plant regeneration, culture medium screening, hormone blending, antibiotic sensitivity, culture conditions and the like. By taking the suspension cell line as the receptor of agrobacterium mediation and establishing the suspension cell line with fast growth and good uniformity of cell states, the method disclosed by the invention realizes a simple, convenient and rapid populus genetic transformation system and provides a new way for populus transgenosis operation.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a poplar genetic transformation method mediated by a suspension cell line as a receptor Agrobacterium. Background technique [0002] Poplar ( Populus ) as a model plant in woody plants, and also the first woody plant whose whole genome was sequenced. Using transgenic technology to study the gene function of poplar has become an important content of post-genomics research after the completion of poplar genome sequencing. At present, the transgenic research of poplar is generally based on the Agrobacterium-mediated genetic transformation method using the leaf disc as the recipient material. Due to the poor consistency of the physiological and biochemical state of the leaf cells, the genetic transformation conditions are difficult to unify, and the transformation efficiency is not high, which affects the research on the gene function of poplar. . The suspe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 诸葛强郑小姣潘惠新尹佟明孙伟博杨贞刘铃
Owner NANJING FORESTRY UNIV
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