A method of increasing the genetic transformation efficiency of medicago sativa

A technology of genetic transformation efficiency and alfalfa, which is applied in the field of improving the genetic transformation efficiency of alfalfa, can solve the problems of low frequency of genetic transformation of alfalfa, difficulty in meeting genetic engineering technology, difficult differentiation of explants, etc., to achieve improved transformation efficiency, Efficiently obtain and promote the effect of regeneration

Inactive Publication Date: 2017-07-04
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to factors such as low regeneration frequency and difficult differentiation of explants after Agrobacterium infection, the frequency

Method used

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  • A method of increasing the genetic transformation efficiency of medicago sativa
  • A method of increasing the genetic transformation efficiency of medicago sativa
  • A method of increasing the genetic transformation efficiency of medicago sativa

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Embodiment 1

[0026] Embodiment one: a kind of method for improving the genetic transformation efficiency of alfalfa disclosed by the present invention comprises the following steps, (1) sowing and cultivating alfalfa: adopt existing technology, for example, alfalfa seeds in 75% ethanol Sterilize for 1 min, then sterilize in 14% sodium hypochlorite solution for 12 min, rinse with sterile water 5 times, inoculate into MS medium, place in a light intensity of 2000lx, (25±1)°C and 16h / 8h photoperiod Cultivate in tissue culture room;

[0027] (2) Prepare the bacteria solution: pick a single colony of Agrobacterium containing the Medtr4g107010.1 gene, the sequence of the Medtr4g107010.1 gene is shown in SEQENCE1, place the colony in YEP medium for overnight culture, when OD 600 When the value reaches 0.6-0.9, centrifuge to collect the bacterial liquid precipitate, and use MS liquid medium to suspend the collected Agrobacterium liquid to OD 600 up to 0.8-0.9;

[0028] (3) Genetic transformation...

Embodiment 2

[0031] Embodiment two: a kind of method for improving the genetic transformation efficiency of alfalfa provided by the present invention comprises the following steps, (1) sowing and cultivating alfalfa: adopt existing technology, for example, put alfalfa seeds in 75% ethanol Sterilize for 1 min, then sterilize in 14% sodium hypochlorite solution for 12 min, rinse with sterile water 5 times, inoculate into MS medium, place in a light intensity of 2000lx, (25±1)°C and 16h / 8h photoperiod Cultivate in tissue culture room;

[0032](2) Prepare the bacteria solution: pick a single colony of Agrobacterium containing the Medtr4g107010.1 gene, the sequence of the Medtr4g107010.1 gene is shown in SEQENCE1, place the colony in YEP medium for overnight culture, when OD 600 When the value reaches 0.6-0.9, centrifuge to collect the bacterial liquid precipitate, and use MS liquid medium to suspend the collected Agrobacterium liquid to OD 600 up to 0.8-0.9;

[0033] (3) Genetic transformati...

Embodiment 3

[0044] Embodiment three: a kind of method for improving the genetic transformation efficiency of alfalfa provided by the present invention comprises the following steps, (1) sowing and cultivating alfalfa: adopt existing technology, for example, put alfalfa seeds in 75% ethanol Sterilize for 1 min, then sterilize in 14% sodium hypochlorite solution for 12 min, rinse with sterile water 5 times, inoculate into MS medium, place in a light intensity of 2000lx, (25±1)°C and 16h / 8h photoperiod Cultivate in tissue culture room;

[0045] (2) Prepare the bacteria solution: pick a single colony of Agrobacterium containing the Medtr4g107010.1 gene, the sequence of the Medtr4g107010.1 gene is shown in SEQENCE1, place the colony in YEP medium for overnight culture, when OD 600 When the value reaches 0.6-0.9, centrifuge to collect the bacterial liquid precipitate, and use MS liquid medium to suspend the collected Agrobacterium liquid to OD 600 up to 0.8-0.9;

[0046] (3) Genetic transform...

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Abstract

The invention discloses a method of increasing the genetic transformation efficiency of medicago sativa. The method includes (1) a step of seeding and culturing medicago sativa; (2) a step of preparing a microbe solution, namely a step of picking a single agrobacterium colony containing an Medtr4g107010.1 gene, subjecting the colony to overnight culture in a YEP medium, performing centrifugation, collecting microbe solution sediment, and suspending the collected agrobacterium solution until OD<600> is 0.8-0.9 by utilizing an MS liquid medium; and (3) a step of genetic transformation, namely a step of cutting off blades of medicago sativa aseptic seedlings, putting the obtained blades into an MS medium, culturing the blades in a tissue culture room, then infecting explants in the microbe suspension, then transferring the explants to an MS medium, performing dark culture, then transferring the explants to a selective medium containing 250 mg/L of piperacillin, and culturing the explants in the tissue culture room. The method is advantaged by significantly promoting regeneration of adventitious buds, rapidly and efficiently preparing transgenic plants and increasing the transformation efficiency.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering utilization, in particular to a method for improving the genetic transformation efficiency of alfalfa. Background technique [0002] Alfalfa is a perennial high-quality leguminous forage widely planted in the world. It has high protein content and high feeding value, and has the reputation of "the king of forage". At present, conventional breeding is the main way of alfalfa breeding, which has made great contributions to the breeding of alfalfa in my country. However, compared with many crops, the genetic background of alfalfa is complex, the degree of heterozygosity is high, the genetic mechanism of many traits is unknown, and the analysis of traits is very difficult. The transgenic technology can introduce good genes into alfalfa, and improve the herbicide resistance, drought resistance, salt-alkali tolerance and other traits of alfalfa, which greatly shortens the breeding cycle of ...

Claims

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Application Information

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IPC IPC(8): C12N15/82
Inventor 仪登霞李聪郑兴卫
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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