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Agrobacterium tumefaciens-mediated genetic transformation method for toona ciliata

A genetic transformation method, Agrobacterium-mediated technology, applied in the field of Agrobacterium-mediated red toon genetic transformation, can solve the problems of intractable browning of explants, long growth cycle, and difficult genetic transformation, and achieve the promotion of industrialization Developmental, repeatable and stable results

Active Publication Date: 2021-08-06
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as a woody plant, Toona sinensis has the characteristics of long growth cycle, high heterozygosity, and poor regeneration ability, which makes genetic transformation difficult and special, and the biological characteristics of Toona sinensis itself lead to its in vitro tissue During the cultivation process, there are technical problems such as difficult disinfection and thorough endophyte contamination, and browning of explants is difficult to overcome, which seriously affects the successful construction of the genetic transformation system of Toona sinensis, and has led to the research on the genetic transformation system of Toona sinensis so far. Not yet reported
Based on this, there is currently no suitable genetic transformation system that can be used to transfer resistance genes, etc.

Method used

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  • Agrobacterium tumefaciens-mediated genetic transformation method for toona ciliata
  • Agrobacterium tumefaciens-mediated genetic transformation method for toona ciliata
  • Agrobacterium tumefaciens-mediated genetic transformation method for toona ciliata

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Embodiment 1

[0076] The present embodiment provides a method for Agrobacterium-mediated genetic transformation of red toon, comprising the following steps:

[0077] 1. Acquisition and pre-cultivation of sterile seedlings and explants

[0078] 1.1 Remove the wings of the red toon seeds, soak them in sterile water with an initial temperature of 45°C for 4 hours, and remove the shriveled seeds floating on the top.

[0079] 1.2 After soaking the seeds for 4 hours, sterilize them with 75% alcohol solution by volume on a sterile ultra-clean bench for 1 min, rinse them once with sterile water, and sterilize them with 10% sodium hypochlorite solution by mass percentage for 18 min. Rinse with bacterial water 4 times, each time for 5 minutes.

[0080] 1.3 Then sow the seeds in a blank MS medium for culture, and the seeds will start to germinate after 3 days of cultivation. After 35 days, the true leaves of the aseptic seedlings will grow. Implant; in this step: the culture conditions are culture t...

Embodiment 2

[0112] The present embodiment provides a method for Agrobacterium-mediated genetic transformation of red toon, comprising the following steps:

[0113] 1. Acquisition and pre-cultivation of sterile seedlings and explants

[0114] 1.1 Remove the wings of the red toon seeds, soak them in sterile water with an initial temperature of 45°C for 3 hours, and remove the shriveled seeds floating on the top.

[0115] 1.2 The seeds soaked for 3 hours were sterilized on a sterile ultra-clean bench with 75% alcohol solution by volume for 1 min, rinsed once with sterile water, and sterilized with 10% sodium hypochlorite solution for 15 min. Rinse with bacterial water 3 times, each time for 5 minutes.

[0116] 1.3 Then sow the seeds in the blank MS medium for culture, and the seeds will start to germinate after 3 days. After 30 days, the true leaves of the aseptic seedlings will grow. Implant; in this step: the culture conditions are culture temperature 25±2°C, culture in the dark until th...

Embodiment 3

[0149] The present embodiment provides a method for Agrobacterium-mediated genetic transformation of red toon, comprising the following steps:

[0150] 1. Acquisition and pre-cultivation of sterile seedlings and explants

[0151] 1.1 Remove the double wings of the seeds of Toona sinensis, soak them in sterile water with an initial temperature of 45°C for 5 hours, and remove the shriveled seeds floating on the top.

[0152] 1.2 After soaking the seeds for 5 hours, sterilize them with 75% alcohol solution by volume on a sterile ultra-clean bench for 1 min, rinse them once with sterile water, and sterilize them with 10% sodium hypochlorite solution by mass percentage for 20 min. Rinse with bacterial water 5 times, each time for 5 minutes.

[0153] 1.3 Then sow the seeds in a blank MS medium for culture, and the seeds will start to germinate after 3 days. After 40 days, the true leaves of the aseptic seedlings will grow, and the leaves will be cut as explants; in this step:

[0...

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Abstract

The invention relates to an agrobacterium tumefaciens-mediated genetic transformation method for toona ciliata. The method comprises the following steps: taking leaves of toona ciliata as an explant, infecting the explant with a bacterial solution of agrobacterium tumefaciens carrying a plant expression vector; inoculating the infected explant into a co-culture medium, and carrying out co-culture; and carrying out induction culture on the co-cultured explant, wherein the induction culture comprises induction culture of callus, induction culture of adventitious buds, induction culture of buds and induction culture of roots; the bacterial solution contains acetosyringone, and the co-culture medium contains acetosyringone. By adopting the method, agrobacterium tumefaciens-mediated toona ciliata genetic transformation is successfully realized, genetic transformation rate is kept at about 10%, and the method is good in repeatability and stability, suitable for batch seedling culture and beneficial to agricultural industrialization development.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and genetic engineering, and relates to an agrobacterium-mediated genetic transformation method of red toon. Background technique [0002] Red toon (Toona ciliata) is a broad-leaved tree species of the genus Toona in the family Meliaceae. In my country, the natural population of red toon is characterized by sporadic distribution, mainly distributed in South China, Central China, East China and Southwest China. Because of its good material, bright color and beautiful pattern, the core material is reddish brown, and the red toon is crowned with " Known as "Chinese mahogany", it is an excellent furniture material and is widely used in the fields of furniture design, vehicle and ship manufacturing and interior decoration; its tree shape is beautiful and can also be planted as a street tree; red toon is also a medicinal material that has received widespread attention in recent years Plants, ro...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N1/20A01H5/00C12R1/01
CPCC12N15/8205C12N1/20
Inventor 毛文迈宋慧云李悦李培王悦阳林慧娟姚驰陈晓阳
Owner SOUTH CHINA AGRI UNIV
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