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Agrobacterium tumefaciens-mediated transformation lettuce tissue culture method

A technique of Agrobacterium-mediated tissue culture, applied in horticultural methods, botanical equipment and methods, plant regeneration, etc., can solve problems such as difficult manual regulation, unfavorable breeding work, long cultivation period, etc., to improve germination rate and rooting The effect of low browning rate, low browning rate and cycle shortening

Inactive Publication Date: 2021-09-17
NORTHWEST A & F UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Therefore, the existing Agrobacterium-mediated lettuce tissue culture process is affected by many factors, such as co-cultivation time, co-cultivation conditions, selection and dosage of antibiotics, etc., which are not easy to be manually regulated, and are likely to have a negative impact on the tissue culture process. And the cultivation cycle is too long, which is not conducive to the breeding work.

Method used

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  • Agrobacterium tumefaciens-mediated transformation lettuce tissue culture method
  • Agrobacterium tumefaciens-mediated transformation lettuce tissue culture method
  • Agrobacterium tumefaciens-mediated transformation lettuce tissue culture method

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preparation example Construction

[0027] The invention mainly relates to the preparation and infection process of the Agrobacterium bacterial liquid. If the concentration of Agrobacterium bacteria is too low, the infection time is too short, and the transformed explants cannot attach enough bacteria, resulting in a low transformation rate; Excessive bacterial infection is not conducive to the survival of explants and the subsequent tissue culture process. The present invention preferably Agrobacterium liquid OD 600 =0.3-0.5, the infection time is 5-10 minutes; further preferred Agrobacterium liquid OD 600 =0.5, infection time 7-9 minutes.

[0028] The invention provides a callus-germination medium for Agrobacterium-mediated transformed lettuce tissue culture, which consists of 25.0-35.0 g / L of sucrose + 7.0-8.0 g / L of agar + 1.0-1.5 mg / L of IAA + 6-BA 0.5-0.9mg / L. In the process of plant tissue culture, exogenous hormones need to be added, and different types and concentrations of exogenous hormones have d...

Embodiment 1

[0037] A method for agrobacterium-mediated transformation of lettuce tissue culture, comprising the following steps:

[0038] (1) Cultivation of aseptic seedlings of lettuce

[0039] Put lettuce seeds in a beaker, soak them in tap water for 2 hours, then disinfect them with 75% ethanol for 30 seconds in an ultra-clean workbench, rinse them twice with sterile distilled water, then disinfect them with 0.1% mercury liter for 4 minutes, rinse them with sterile distilled water 6 times, Blot the water on the seed surface with sterile filter paper. The treated seeds were planted on the culture medium (MS medium, 30g / L sucrose, 7.5g / L agar, pH5.8, 121°C, sterilized at 110Kpa for 25min), and cultivated in a light culture room at a temperature of 25°C , light 16 / 8hrs (light / dark), light intensity 200umol m -2 ·s -1 .

[0040] (2) Agrobacterium infection of explants

[0041] Take aseptic seedlings of lettuce with a seedling age of 7 days, and cut the leaves to a size of 1cm 2 , at ...

Embodiment 2

[0049] A method for agrobacterium-mediated transformation of lettuce tissue culture, comprising the following steps:

[0050] (1) Cultivation of aseptic seedlings of lettuce

[0051] Put the lettuce seeds in a beaker, soak the seeds in tap water for 2 hours, then disinfect them with 75% ethanol for 30 seconds in an ultra-clean workbench, rinse them twice with sterile distilled water, then disinfect them with 0.1% mercury liter for 3 minutes, rinse them with sterile distilled water 6 times, Blot the water on the seed surface with sterile filter paper. The treated seeds were planted on the culture medium (1 / 2MS medium, 30g / L sucrose, 7.5g / L agar, pH5.8, 121°C, sterilized at 110Kpa for 25min), and cultivated in the light culture room, the temperature 24°C, light 16 / 8hrs (light / dark), light intensity 200umol m -2 ·s -1 .

[0052] (2) Agrobacterium infection of explants

[0053] Take aseptic seedlings of lettuce with a seedling age of 5 days, and cut the leaves to a size of 0....

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Abstract

The invention provides an agrobacterium tumefaciens-mediated transformation lettuce tissue culture method, and belongs to the technical field of agrobacterium tumefaciens-mediated plant tissue culture. The method comprises the following steps: infecting explants of aseptic seedlings of the lettuce by using agrobacterium liquid, sequentially putting the infected explants of the lettuce on a callus-germination culture medium and a rooting culture medium for induction, and domesticating and transplanting, wherein the callus-germination culture medium is prepared from the following components: 25.0 to 35.0 g / L of cane sugar, 7.0 to 8.0 g / L of agar, 1.0 to 1.5 mg / L of IAA and 0.5 to 0.9 mg / L of 6-BA. By means of the specific callus-germination culture medium, the co-culture process is omitted, antibiotics do not need to be added, the tissue culture period is shortened, and the high germination rate, the high rooting rate, the low pollution rate and the low browning rate in the lettuce tissue culture process are guaranteed.

Description

technical field [0001] The invention belongs to the technical field of Agrobacterium-mediated plant tissue culture, and in particular relates to a method for Agrobacterium-mediated transformation of lettuce tissue culture. Background technique [0002] Lettuce (Lactuca sativa.L.) belongs to the leaf-use type of Lactuca genus Lactuca in the family Asteraceae. It is a common name for leaf lettuce and is a herbaceous plant of one or two years. Lettuce is very nutritious, containing antioxidants, carotene and vitamin B 1 , B 2 , B 6 and V C , V E , it is also rich in trace elements, such as calcium, phosphorus, potassium, sodium, magnesium and a small amount of copper, iron, zinc, in addition, it also contains dietary cellulose, and the milky juice of stems and leaves also contains a large amount of organic matter, such as sugar, Organic acids, proteins, chicory, etc. [0003] Biotechnology has been widely used in lettuce breeding. Among them, the transgenic methods on let...

Claims

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Application Information

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IPC IPC(8): A01H4/00
CPCA01H4/001A01H4/008
Inventor 武永军张翰卿杨振超梅延豪刘齐
Owner NORTHWEST A & F UNIV
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