Method for converting lucerne by vacuum penetrating auxiliary agrobacterium-mediated

An Agrobacterium-mediated, vacuum infiltration technology, applied in the fields of biotechnology and modern agriculture, can solve the problems of only 50% high, 12-month transformation period, and low transformation efficiency.

Inactive Publication Date: 2009-06-17
SHANDONG FOREST SCI RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In 2003, Wang Tao et al. invented the patent (Patent No.: 200310102540.2) of "a molecular breeding method for quickly obtaining a large number of new varieties of transgenic plants". The highest resistance callus reached only 50%, and the transformation period was as long as 12 months
In 2007, Liu Lixia and others invented the patent of "Agrobacterium-mediated g

Method used

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  • Method for converting lucerne by vacuum penetrating auxiliary agrobacterium-mediated
  • Method for converting lucerne by vacuum penetrating auxiliary agrobacterium-mediated
  • Method for converting lucerne by vacuum penetrating auxiliary agrobacterium-mediated

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The plant material is the No. 1 seed of Medicago sativa (purchased from the Institute of Animal Husbandry, Chinese Academy of Sciences), which is packed into a 1.5ml centrifuge tube, washed with tap water for 2 minutes, sterilized with 70% ethanol for 60 seconds, and then sterilized with 0.1% mercuric chloride. Sterilize for 6 minutes, then wash 6 times with sterile water, inoculate the seeds on MSB + 2.0% sucrose + 0.45% agar, pH 5.8 medium after sterilization, cut the seedlings after 7 days and continue to transfer to the above medium for differentiation After culturing for 18 days, the young true leaves were cut off.

[0048] The seed germination and differentiation culture conditions are: day and night light cycle culture, light intensity 40μmol·m -2 ·s -1 , light time 14h·d -1 , day temperature 25 ℃, night temperature 18 ℃.

[0049]Select a single colony inoculation of Agrobacterium tumefaciens LBA4404 strain carrying the binary vector pBin438 (the vector contai...

Embodiment 2

[0070] Inoculate the sterilized alfalfa seeds on the germination medium (MSB+2.0% sucrose+0.5% agar, pH value 5.8), cut the seedlings after 10 days and continue to transfer them to the above-mentioned medium for 15 days, then cut off the young true leaves , culture condition with embodiment 1.

[0071] Pick a single colony of Agrobacterium tumefaciens GV3101 carrying the binary vector pCAMBIA3301 (the vector contains the CaMV35s promoter, the rd29A gene of Arabidopsis thaliana, the selectable marker gene bar and the reporter gene 35sGUS) and inoculate in 5ml containing Kan 50mg L -1 In the YEB liquid medium, cultivate for 24 hours (28°C, 180rpm), take 1ml and transfer it to 50ml of antibiotic-free YEB medium and culture it for 16 hours under the same conditions, then take 15ml of bacterial liquid and add it to 50ml of medium I , cultured for 4 hours to OD at a shaking speed of 200rpm 600 0.7 spare.

[0072] Put the young true leaves of alfalfa tissue-cultured seedlings in th...

Embodiment 3

[0082] Inoculate the sterilized alfalfa seeds on the germination medium (MSB+0.1mg / L6-BA+3.0% sucrose+0.55% agar, pH 5.8), cut the seedlings after 8 days and continue to transfer to the above medium for differentiation and culture for 20 days , cut off the young true leaves.

[0083] Select the single colony of Agrobacterium tumefaciens EHA105 strain (its carrier contains CaMV35s promoter, SOS2+3 gene of Arabidopsis thaliana, selectable marker gene bar and reporter gene 35sGUS) carrying the binary vector pCAMBIA3301 to inoculate in 5ml containing Kan 50mg. L -1 In the YEB liquid medium, cultivate for 28 hours (28°C, 190rpm), take 1ml and transfer it to 50ml of YEB medium without antibiotics and culture it for 10 hours under the same conditions, then take 20ml of bacterial liquid and add it to 50ml of medium I , cultivated for 2 hours to OD at a shaking speed of 220rpm 600 0.4 spare.

[0084]Put the young true leaves of alfalfa tissue-cultured seedlings in the prepared Petr...

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Abstract

The invention discloses a method of mediated transformation of an alfalfa by vacuum permeability auxiliary agrobacterium comprising (1) the seed sterilization and the test-tube plantlet culture, (2) the agrobacterium tumefaciens containing objective genes culture, (3) the vacuum permeability auxiliary agrobacterium infection, (4) the bacteriostasis and resistant plants selection and culture, (5) the resistant plants rootage selection, (6) the transformed plants hardening-seedling and the field transplantation or the like; the invention employs a transformation method by using the agrobacterium mediated transformation of the alfalfa young and tender true leaves and the vacuum permeability method, thereby greatly improving the transformation efficiency. The secondary resistance rootage of the resistance plants reduces the later detection workload and obtains a plurality of transformation plants. The method of the invention overcomes the difficulties of low genetic transformation efficiency and long transformation period of the conventional alfalfa, improves the alfalfa transformation efficiency by more than 95, shortens the transformation period 2-3 months and has important meaning for the theoretic research at the alfalfa genetic engineering aspect and the genetic breeding practical.

Description

technical field [0001] The invention relates to a method for transforming alfalfa mediated by agrobacterium, in particular to a method for transforming alfalfa mediated by vacuum infiltration, and belongs to the field of biotechnology and modern agricultural technology. Background technique [0002] Low temperature, drought and salinity have seriously affected agricultural production in my country and the world. my country is one of the countries where droughts occur frequently. According to statistics, the average area of ​​drought in my country from 1991 to 2005 was 26.02 million hectares per year, of which the drought area in 1998 The smallest area was 14.24 million hectares, while the largest drought area was 40.54 million hectares in 2000, and it tended to increase year by year. Water shortage and soil salinization are currently a global problem restricting agricultural production. So far, 20% of the world's arable land is affected by salinization, and 43% of the arable ...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H1/00A01H4/00A01G31/00
Inventor 夏阳梁慧敏燕丽萍王太明刘德玺
Owner SHANDONG FOREST SCI RES INST
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