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Method for gene transformation for barbadosnut by utilizing kanamycin resistant gene as screening mark

A kanamycin and selection marker technology, applied in the field of tissue culture and genetic engineering of Jatropha curcas, can solve the problems of impracticality and low success rate, avoid a large number of molecular detection work in the later stage, and achieve a high differentiation rate. , to ensure the effect of normal growth and differentiation

Inactive Publication Date: 2014-05-28
SINO FOREST CHINA INVESTMENTS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is to obtain transgenic plants without adding kanamycin in the screening stage. However, only one of the 120 regenerated plants takes root, and the success rate is too low to be practical.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0078] This example uses Agrobacterium to transfer the GUS gene into wild Jatropha curcas in Yuanmou, Yunnan. The method created by the present invention is described in detail. Please refer to Table 1 for the total number of samples and the number of survivors after each processing step.

[0079] Step (1), specific operations include:

[0080] 1. Collect wild Jatropha seeds from Yuanmou, Yunnan. Pick the plump seeds, peel off the shell, soak in 75% ethanol for 30 seconds, rinse with sterile water twice, then disinfect in 0.1% mercuric chloride for 3 minutes, rinse with sterile water for 5 times, and then soak in sterile water After 2 hours the endosperm is peeled off. The complete embryos and cotyledons were inoculated in MS medium, 2 seeds per bottle, cultured in the dark at 23°C for 2 days, and then transferred to light culture for 10 days to obtain sterile seedlings for the experiment.

[0081] 2. Pick a single clone of Agrobacterium EHA105 (containing the expression vec...

example 2

[0091] This example uses Agrobacterium to transfer the GUS gene into wild Jatropha curcas in Yuanmou, Yunnan. The method created by the present invention is described in detail. Please refer to Table 2 for the total number of samples and the number of survivors after each treatment step.

[0092] Step (1), including the following specific operations:

[0093] 1. Collect the wild jatropha seeds in Yuanmou, Yunnan, pick the plump seeds, peel off the shell, soak in 75% ethanol for 30 seconds, rinse with sterile water for 3 times, and then sterilize in 0.1% mercuric chloride for 5 minutes, aseptic Rinse with water 7 times, then soak in sterile water for 4 hours and then peel off the endosperm. The complete embryos and cotyledons were inoculated in MS medium, 3 seeds per bottle, cultured in the dark at 26°C for 4 days, and then transferred to light culture for 12 days to obtain sterile seedlings for the experiment.

[0094] 2. Pick a single clone of Agrobacterium GV3101 (containin...

example 3

[0104] In this example, Agrobacterium-mediated transfer of the GUS gene into wild Jatropha curcas in Yuanmou, Yunnan, specifically describes the method created by the present invention. Please refer to Table 3 for the total number of samples and the number of survivors after each processing step.

[0105] Step (1), including the following specific operations:

[0106] 1. Collect wild Jatropha seeds from Yuanmou, Yunnan. Pick the plump seeds, peel off the shell, soak in 75% ethanol for 30 seconds, rinse with sterile water for 3 times, then disinfect in 0.1% mercury chloride for 4 minutes, rinse with sterile water for 6 times, and then soak in sterile water After 3 hours the endosperm is peeled off. Take complete embryos and cotyledons, inoculate them in MS medium, pick 3 seeds per bottle, culture in the dark at 24°C for 3 days, and then transfer to light culture for 11 days to obtain sterile seedlings for the experiment.

[0107] 2. Pick a single clone of Agrobacterium LBA440...

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Abstract

The invention relates to a method for gene transformation for barbadosnut by utilizing a kanamycin resistant gene as a screening mark. The method comprises the following steps: preparing a barbadosnut explant and an agrobacterium liquid, and carrying out cocultivation on the explant after impregnation by agrobacterium; cultivating the explant after the explant is subjected to cocultivation and bacteria removal and put in a screening culture medium C13KC, thus resistance callus is obtained; transferring the resistant callus to a differential medium SR13KC, and differentiating to obtain resistant adventitious buds; and transferring the resistant adventitious buds to a rooting medium R2KC, thus a transgenosis plant is obtained. Through utilizing the method provided by the invention, the genetic transformation operation to the barbadosnut by utilizing agrobacterium mediation is simple and convenient, the cost is low, and the method is used for establishing a favorable foundation for the barbadosnut genetic modification by an imported-function gene and the acquisition of high-yield high-oil and chill-proof novel products and the like.

Description

technical field [0001] The invention relates to the technical field of tissue culture of Jatropha curcas and the field of genetic engineering, in particular to a method for gene transformation of Jatropha curcas by using kanamycin as a selection marker. Background technique [0002] The energy crisis is a huge challenge facing the world today. China's oil reserves only account for 2% of the world's total, but its consumption ranks second in the world. Therefore, there is an urgent need to find a new type of energy to replace the currently widely used fossil energy. [0003] Compared with fossil resources, growing energy plants to obtain biodiesel has the advantage of being renewable, which is the fundamental way to solve the energy crisis. The energy plants that have been discovered so far are mainly concentrated in Apocynaceae, Euphorbiaceae, Romoaceae, Compositae, Myrtaceae, and Fabaceae. [0004] Jatropha ( Jatopha curcas L.), also known as Jatropha, Diesel tree, belo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 王梅珍孙怀娟许文钊潘文欢李耿光
Owner SINO FOREST CHINA INVESTMENTS LTD
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