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Vacuum infiltration transgenic method for adventitious buds of Chinese rose flower

A vacuum infiltration and transgenic technology, applied in the field of genetic engineering, can solve the problems of inability to meet the needs of large-scale transgenic operations, difficulty in inducing somatic embryos, and low transformation efficiency, and achieve improved transformation efficiency, low false positive rate, and reproductive cycle. short effect

Active Publication Date: 2017-06-20
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of using buds produced from stems for gene transformation are low multiplication coefficient, each bud needs to be scratched, and the process is cumbersome; the biggest disadvantage of using somatic embryos for transgenic operations is that it is difficult to induce somatic embryos, the cycle is long, and the transformation efficiency Low
It can be seen that neither of the above two transgenic methods can meet the needs of large-scale transgenic operations.

Method used

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  • Vacuum infiltration transgenic method for adventitious buds of Chinese rose flower
  • Vacuum infiltration transgenic method for adventitious buds of Chinese rose flower
  • Vacuum infiltration transgenic method for adventitious buds of Chinese rose flower

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] I Cut the stem tips of the sterile seedlings of Chinese rose'Yueyuefen', and cultivate the stems on MS+6-BA1.0mg / L+NAA0.1mg / L+agar 6.5g / L+sucrose 30g / L basic proliferation medium for 4 weeks. The tip produces adventitious buds, and prepare about 100.

[0025] II. Cut the adventitious buds with 2 to 4 leaflets about 0.5 cm and continue to pre-culture for 3 days on the proliferation medium, and then transfer to the Agrobacterium GV3101 transformation solution of the pFAST-R05 plasmid carrying the FT gene (provided by Ghent University, Belgium) (MS+sucrose 30g / L, OD 600 =0.5)), transfer to a sterile syringe and repeatedly extract and infiltrate for 5 minutes, and gently shake it until the leaves become dark green and fully wetted.

[0026] III. The adventitious buds after the sterilization solution are transferred to the propagation medium and co-cultivated in the dark for 3 days, and then the adventitious buds thoroughly washed with sterile water are transferred to the antibac...

Embodiment 2

[0032] I Cut the young shoot tips of the sterile seedlings of Chinese rose'Yueyuefen' and cultivated on MS+6-BA1.5mg / L+NAA0.05mg / L+agar 6.5g / L+sucrose 30g / L basic proliferation medium for 3 weeks Induce the shoot tip to produce adventitious buds, and prepare about 100.

[0033] II. Cut the adventitious buds with 2~4 leaflets about 0.5cm and continue to pre-culture for 3 days on the proliferation medium, and then transfer them to the Agrobacterium EHA105 transformation solution of the pBGWL7 plasmid carrying the KSN gene promoter (provided by Ghent University, Belgium) (MS+sucrose 30g / L, OD 600 =0.5), transfer to a sterile syringe and repeatedly extract and infiltrate for 7 minutes, and shake gently until the leaves become dark green and fully wetted.

[0034] III. The adventitious buds after sterilization are transferred to the growth medium and co-cultured in the dark for 3 days, and then the adventitious buds thoroughly washed with sterile water are transferred to the bacteriosta...

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Abstract

The invention discloses a vacuum infiltration transgenic method for adventitious buds of Chinese rose flower. The vacuum infiltration transgenic method comprises the following steps: cutting off young and tender tips of aseptic seedlings of the Chinese rose flower, and inducing on a proliferation culture medium to generate a large amount of the adventitious buds; cutting off the adventitious buds, preculturing on the proliferation culture medium for 3 days, then transferring the precultured adventitious buds into an agrobacterium transformation liquid carrying a target gene, carrying out vacuum infiltration for 5 to 10 minutes and sufficiently infiltrating; transferring the adventitious buds subjected to bacterial liquid removal to the proliferation culture medium and carrying out co-culturing under the dark condition for 3 days; then transferring the adventitious buds which are thoroughly cleaned by sterile water into a bacteriostatic culture medium and culturing for 5 days; transferring the cultured adventitious buds to a selective culture medium according to a selective marker of a gene expression carrier carrying the target gene and carrying out gradient screening according to the sequence of low to high, and transferring obtained resistant adventitious buds to a rooting culture medium for inducted rooting; collecting leaf blades of positive plants and carrying out PCR (Polymerase Chain Reaction) identification and reporting gene detection, wherein the positive plants are transgenic plants. The vacuum infiltration transgenic method disclosed by the invention has the advantages of high value-added coefficient, short reproduction cycle, high gene transformation rate and low false positive rate.

Description

Technical field [0001] The invention relates to a vacuum infiltration transgenic method for adventitious buds of rose, which belongs to the technical field of genetic engineering. Background technique [0002] Rose is the first of the “four major cut flowers” ​​in the world, but the current transgenic system is still immature, which seriously hinders the development of its molecular breeding. At present, the related patents that are published and authorized mainly include two gene transformation methods using buds (publication number CN103146748A) and somatic embryos (publication number CN102220372B). The disadvantage of using the bud points produced by stem segments for gene transformation is that the multiplication coefficient is low, each bud point needs to be scratched, and the process is cumbersome; the biggest disadvantage of using somatic embryos for transgenic operations is the difficulty in somatic embryo induction, long cycle, and transformation efficiency. low. It ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00
CPCA01H4/00C12N15/8205C12N15/8212
Inventor 王长泉刘金义陆俊任浩然白梦娟
Owner NANJING AGRICULTURAL UNIVERSITY