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Soybean stem apex transformation vacuum permeation helped exogenous gene introduction method

A technology of vacuum infiltration and exogenous genes, applied in the field of biotechnology and modern agriculture, can solve the problem of low transformation efficiency, achieve the effect of increasing the chance of invasion, improving the transformation rate, and improving the infection efficiency

Inactive Publication Date: 2003-04-23
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Someone has successively selected cotyledon nodes, immature embryos, etc. as explants to transform successfully, but the transformation efficiency is not high (0.6%)
Therefore, the main problem at present is to improve the transformation efficiency

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] The experimental variety is Dongnong 43, 50 capsules.

[0017] Take soybean seeds and wash them repeatedly with tap water, soak them in 70% alcohol for 0.5 minutes, turn them into saturated calcium hypochlorite solution and soak them for 15 minutes, rinse them with sterile water for 4 times, and then soak the seeds in sterile water 18 hours. Peel off the seed coat, remove the cotyledons, remove the two original leaves under a dissecting microscope, expose the apical meristem area, take out the shoot tip of about 0.5 cm from it, and place it in the MSB 5 +6-BA3.0mg / L medium, pre-cultured for 24 hours.

[0018] The pre-cultured shoot tip was taken out, placed under 0.06MPa vacuum pressure condition, Agrobacterium infection for 10 minutes (OD 600nm ≈0.5), placed on MSB+6-BA1.0mg / L+IBA0.2mg / L (PH5.8) medium for co-cultivation in the dark for 3 days.

[0019] Transfer to MSB+BA0.5mg / L+cb 500mg / L (pH5.8 liquid) for continuous culture for 3 days, and change fresh medium eve...

Embodiment 2

[0024] The experimental variety is Jilin 27, 50 capsules.

[0025] Take soybean seeds and wash them repeatedly with tap water, soak them in 70% alcohol for 1 minute, turn them into saturated calcium hypochlorite solution and soak them for 15 minutes, wash them with sterile water for 5 times, and then soak the seeds in sterile water 24 hours. Peel off the seed coat, remove the cotyledons, remove the two original leaves under a dissecting microscope, expose the apical meristem area, take out the shoot tip of about 0.5 cm from it, and place it in the MSB 5 +6-BA3.0mg / L medium, pre-cultured for 24 hours.

[0026] The pre-cultivated shoot tip was taken out, placed under 0.08MPa vacuum pressure condition, Agrobacterium infection for 15 minutes (OD 600nm ≈0.5), placed on MSB+6-BA1.0mg / L+IBA0.2mg / L (PH5.8) medium for co-cultivation in the dark for 3 days.

[0027] Transfer to MSB+BA0.5mg / L+cb 500mg / L (pH5.8 liquid) for continuous culture for 3 days, and change fresh medium every da...

Embodiment 3

[0032] The experimental variety is Hefeng 35, 50 capsules.

[0033] Take soybean seeds and wash them repeatedly with tap water, soak them in 70% alcohol for 0.5-1 minute, turn them into a solution containing saturated calcium hypochlorite and soak them for 15 minutes, rinse them with sterile water for 5 times, and then place the seeds in sterile Soak in water for 19 hours. Peel off the seed coat, remove the cotyledons, remove the two original leaves under a dissecting microscope, expose the apical meristem area, take out the shoot tip of about 0.5 cm from it, and place it in the MSB 5 +6-BA3.0mg / L medium, pre-cultured for 24 hours.

[0034] The pre-cultured shoot tip was taken out, placed under 0.07MPa vacuum pressure condition, Agrobacterium infection for 20 minutes (OD 600nm ≈0.5), placed on MSB+6-BA1.0mg / L+IBA0.2mg / L (PH5.8) medium for co-cultivation in the dark for 3 days.

[0035] Transfer to MSB+BA0.5mg / L+cb 500mg / L (pH5.8 liquid) for continuous culture for 3 days, an...

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Abstract

The soybean shoot tip transformation vacuum infiltration supplementary exogenous gene introudction method includes the following steps: in the course of soybean shoot tip transformation by using agrobacterium mediated BT gene adopting soybean seed germination after disinfection, removing terminal bud and resulting in epidermal meristematic cell damage as explant, under the condition of vacuum pressure making agroinfection treatment, making co-cultivation to germination culture meidum, using a certain quantity of kanamycin sulfate and adding it into budding culture medium and rooting culture medium to make screening, so can obtain more regenerative seedlings and transformed soybean plants. Said invention can obvious raise agroinection effect and effectively raise budding rate and number oftransformed seedlings.

Description

Technical field: [0001] The invention relates to an exogenous gene introduction method assisted by vacuum infiltration for the transformation of soybean stem tips, which is an operation method for introducing exogenous genes into soybeans by means of vacuum infiltration assistance, thereby improving the genetic traits of soybeans, belonging to biotechnology and modern field of agricultural technology. Background technique: [0002] The success of plant gene transformation depends on whether it has a good receptor system, that is, whether it has strong plant regeneration ability and high-frequency transformation rate, ("Principles and Technologies of Plant Genetic Engineering" edited by Wang Guanlin, Technology Press). At present, the methods of genetic modification are mainly divided into two categories: 1 direct method, for example, pollen tube passage method, microinjection method, electric shock method, PEG method and gene gun method, 2 vector method, for example, Agrobac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01C1/00A01H1/00A01H4/00C12N15/09
Inventor 武天龙邱承祥马晓红
Owner SHANGHAI JIAO TONG UNIV
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