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Process for obtaining transgenosis plant by ovary drop

A technology for transgenic plants and ovaries, applied in the field of bioengineering, can solve the problems of difficult unification of operation procedures, difficult operation, and great influence of environmental conditions, and achieves the effect of reducing complexity, improving transformation efficiency, and wide application.

Inactive Publication Date: 2005-06-08
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the only way for exogenous gene introduction is the pollen tube, the tiny tube that extends toward the ovule during pollen germination, there are many problems in actual operation: for example, the working time of the target plant for exogenous gene transformation is limited by the natural flowering period. At the same time, it is necessary to fully understand the time course of flowering and fertilization of each plant; the operation in the natural field is greatly affected by environmental conditions; because the transgene is integrated into the recipient chromosome genome in a random manner, the transgenic engineered plant The genetic situation of the offspring is relatively complicated; the operation of the pollen tube passage method is very empirical and requires certain technical exploration and skills; Difficult, so work efficiency is also low [Ni Wanchao, Guo Sandui, Jia Shirong, Cotton genetic transformation mediated by pollen tube passage method, China Agricultural Science and Technology Herald, 2000(2): 27-31]
In addition, due to the poor controllability of this technology, the difficulty in unifying the operation procedures, the unstable transformation effect, and poor reproducibility, there are still debates about the feasibility of this method in the world (Shou HX, Palmer RG, Wang K, Irreproducibility of the Soybean Pollen -Tube Pathway Transformation Procedure, Plant Molecular Biology Reporter, 2002, 20: 325? 34)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1: Application of dyeing agent to simulate the research of ovary instillation method transformation process

[0016] Maize inbred lines Shen 137, C8605-2, 16 female and 9818 were provided by Liaoning Dongya Seedling Company, L87 was provided by Dalian Academy of Agricultural Sciences, LD009 and LD002 were provided by Shenyang Longdi Seed Industry Co., Ltd. The soybean varieties were Tiefeng 30 and Tiefeng 31, provided by Liaoning Soybean Research Institute. The neutral red dye solution was prepared according to a conventional method, and the experimental material instilled with the neutral red dye was observed with a solid dissecting microscope. Fluorescent staining agent hoechst 33258 was purchased from Biyuntian Biotechnology Research Institute. The stock solution was prepared with distilled water to a concentration of 1 mg / ml, diluted with PBS to a final concentration of 10 μg / ml when in use, and observed with a fluorescent microscope. Experimental Materi...

Embodiment 2

[0020] Embodiment 2: the impact of ovary instillation method transformation on plant seed setting rate and transformation rate

[0021] The soybean variety was Tiefeng 30, provided by Liaoning Soybean Research Institute. Transform according to the aforementioned method, for pistils of different lengths, including complete removal of style (0mm removal of ovary), removal of part of ovary 0-1mm, and removal of part of ovary 1-2mm), instill 0.1×SSC solution at the incision respectively , to study the effects of different treatments on the pod rate of transformed materials; in order to detect the transformation efficiency of the ovary instillation method, 100 μl of the prepared linear transformation element solution containing only regulatory sequences and exogenous genes (DNA concentration is 100ng / μl) Instilled in the ovary incision, the control plants were only transformed with 0.1×SSC solution.

[0022] Maize varieties were inbred lines Shen 137, C8605-2, 16 female and 9818 p...

Embodiment 3

[0026] Example 3: Effect of transformed soybeans on pod morphology by ovary instillation method

[0027] The soybean variety was Tiefeng 30, provided by Liaoning Soybean Research Institute. The method of operation is the same as above, using scissors to remove pistils of different lengths, including completely removing the style (removing the ovary 0mm), removing part of the ovary 0-1mm, and removing part of the ovary 1-2mm), taking untreated soybeans as For the control, 5 μl of 0.1×SSC was instilled respectively to study the effects of different treatments on the pod growth state and pod setting rate of the transformed material. The results showed (Table 1) that the apex of the pods without any treatment was sharp; the apex of the pods with completely excised style was obviously blunt; 2 mm obviously affected the normal development of the first and even the second kernel in the pod.

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Abstract

A process for preparing the transgenic plant by ovary dripping includes such steps as pollination, germination of pullen particles, male nucleus coming in embryonary sac via pollen tube, cutting off style and 0-1.0 mm of ovary, and dripping exogenous DNA to direct transform reproductive cells or oospores.

Description

technical field [0001] The invention belongs to the field of biological engineering. The invention relates to a gene transformation method for obtaining transgenic plants through ovary dripping, which can be applied to flowering plants without going through genetic transformation processes such as protoplast culture, cell culture, tissue culture and regeneration plants. Background technique [0002] For a long time, in order to introduce exogenous functional genes into plants, people have explored a variety of transformation methods (Zhu Shengwei et al., Research progress in direct introduction of exogenous DNA into recipient plants, Bulletin of Botany, 2000, 17, 11-16), At present, two transformation methods mediated by gene gun and Agrobacterium are relatively perfect and widely used. In addition, the pollen tube passage method established by Chinese scholars [Zhou GY, Weng J, Introduction of exnogenous DNA into cotton embryos, Method in Enzymology, 1983, (101): 433-448] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87
Inventor 安利佳王长海苏乔杨君高晓蓉栾雨时吴畏刘明
Owner DALIAN UNIV OF TECH
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