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Vacuum permeation helped soybean in-situ fasciculated bud conversion method

A vacuum infiltration, soybean technology, applied in the fields of biotechnology and modern agriculture, can solve the problems of low efficiency of soybean genetic transformation, and achieve the effects of simple and easy method, improved transformation rate and high transformation rate

Inactive Publication Date: 2004-11-17
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to overcome the difficulty of the low efficiency of the existing soybean genetic transformation, on the basis of the method for the transformation of soybean in situ cluster buds, to provide a method for the transformation of soybean in situ cluster buds assisted by vacuum infiltration, so as to improve the transformation efficiency of soybeans, And the method is simple and easy to implement

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] The experimental varieties were Hefeng 35 and 100 capsules.

[0014] After the seeds were sterilized, they were inoculated on MSB+6-BA0.4mg / L (PH5.8) medium and germinated for 5 days;

[0015] Remove the terminal buds to make wounds to obtain explants;

[0016] Pre-culture on MSB+6-BA10mg / L+IBA0.2mg / L (PH5.8) medium for 1 day;

[0017] Under the condition of 0.06MPa vacuum pressure, infect the Agrobacterium with B.t. gene for 10 minutes (OD 600nm ≈0.5), placed on MSB+6-BA1.0mg / L+IBA0.2mg / L medium for co-cultivation for 3 days;

[0018] Put it on MSB+6-BA0.5mg / L+IBA0.1mg / L+cephalosporin 300mg / L+carbenicillin 300mg / L medium to sterilize for 2 weeks;

[0019] Put it on MSB+kanamycin 80mg / L medium and transfer it every 2 weeks, screen until adventitious buds appear, cut off the cotyledon when it grows to 1-2cm, and grow to 3-4cm;

[0020] The adventitious buds were cut off and placed on 1 / 2MSB+Kanamycin 50mg / L (PH5.8) medium to induce rooting for 15 days; transplanted i...

Embodiment 2

[0022] The test variety is Hefeng No. 35, 100 capsules.

[0023] After the seeds were sterilized, they were inoculated on MSB+6-BA0.4mg / L (PH5.8) medium and germinated for 5 days;

[0024] Remove the terminal buds to make wounds to obtain explants;

[0025] Pre-culture on MSB+6-BA10mg / L+IBA0.2mg / L (PH5.8) medium for 1 day;

[0026] Under the condition of 0.08MPa vacuum pressure, infect the Agrobacterium with gna. gene for 15 minutes (OD 600nm ≈0.5), placed on MSB+6-BA1.0mg / L+IBA0.2mg / L medium for co-cultivation for 3 days;

[0027] Put it on MSB+6-BA0.5mg / L+IBA0.1mg / L+cephalosporin 300mg / L+carbenicillin 300mg / L medium to sterilize for 2 weeks;

[0028] Put it on MSB+kanamycin 80mg / L medium and transfer it every 2 weeks, screen until adventitious buds appear, cut off the cotyledon when it grows to 1-2cm, and grow to 3-4cm;

[0029] The adventitious buds were cut off and placed on 1 / 2MSB+Kanamycin 50mg / L (PH5.8) medium to induce rooting for 18 days; transplanted into large p...

Embodiment 3

[0031] The test variety is Hefeng No. 35, 100 capsules.

[0032] After the seeds were sterilized, they were inoculated on MSB+6-BA0.4mg / L (PH5.8) medium and germinated for 5 days;

[0033] Remove the terminal buds to make wounds to obtain explants;

[0034] Pre-culture on MSB+6-BA10mg / L+IBA0.2mg / L (PH5.8) medium for 1 day;

[0035] Under the condition of 0.07MPa vacuum pressure, infect the Agrobacterium with pta gene for 20 minutes (OD 600nm ≈0.5), placed on MSB+6-BA1.0mg / L+IBA0.2mg / L medium for co-cultivation for 3 days;

[0036] Put it on MSB+6-BA0.5mg / L+IBA0.1mg / L+cephalosporin 300mg / L+carbenicillin 300mg / L medium to sterilize for 2 weeks;

[0037] Put it on MSB+kanamycin 80mg / L medium and transfer it every 2 weeks, screen until adventitious buds appear, and cut off the cotyledon when it grows to 1-2cm, until it grows to 3-4cm;

[0038] The adventitious buds were excised and placed on 1 / 2MSB+Kanamycin 50mg / L (PH5.8) medium to induce rooting for 20 days; transplanted int...

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Abstract

The vacuum infiltratino auxilairy soybean in-situ tufted bud transformation method includes the following steps: placing the disinfected seed on germination culture medium to makeing germination, taking out explant whose terminal bud is removed and placing it on the preculture medium to make preculture, under the condition of vacuum making agroinfection for a certain time, placing it on co-cultivation medium to make co-cultivation, taking out and placing it no bacterium-removing culture medium containing kanamycin to remove bacterium, placing it to bud elongation culture medium to screen advertitions bud, then induced rooting and transplanting. Said invention can raise its transformatino rate, and can obtained more transform plants.

Description

Technical field: [0001] The invention relates to a method for vacuum infiltration assisted transformation of soybean cluster buds in situ, is a biological cultivation process, and belongs to the fields of biotechnology and modern agricultural technology. Background technique: [0002] The most commonly used methods in soybean genetic transformation are the Agrobacterium method and the particle bombardment method (gene gun method), and the most reliable method is the Agrobacterium-mediated method. Conventional soybean transgenic methods have problems such as difficult plant regeneration and low transformation efficiency. Although there have been people using different explants (cotyledonary nodes, leaves, petioles and other tissues) for transformation, the transformation efficiency is generally low. Soybean tissue culture is difficult due to the lack of an efficient soybean regeneration plant system. Bechtold once used the vacuum infiltration technique to inoculate Arabidop...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01H3/00A01H4/00
Inventor 武天龙马晓红邱承祥唐冬梅
Owner SHANGHAI JIAOTONG UNIV