Method for agrobacterium tumefaciens-mediated genetic transformation of sugarcane

A genetic transformation method and technology of Agrobacterium tumefaciens is applied in the field of sugarcane genetic transformation mediated by Agrobacterium tumefaciens to achieve the effects of increasing quantity, improving efficiency and good dispersion.

Inactive Publication Date: 2011-08-17
广西作物遗传改良生物技术重点开放实验室
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, the integration of the transgene on the recipient chromosome is random, and the genetic stability and expression activity of the transgene vary greatly among different transformants. If ...

Method used

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  • Method for agrobacterium tumefaciens-mediated genetic transformation of sugarcane
  • Method for agrobacterium tumefaciens-mediated genetic transformation of sugarcane
  • Method for agrobacterium tumefaciens-mediated genetic transformation of sugarcane

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Preparation of embryogenic callus

[0029] Choose the plant tail tip of No. 25 sugarcane variety Taitang, which grows robustly, disinfect the surface with 70% ethanol, get the young leaf tissue about 10 cm away from the top growth point as explants, and cut it into 0.1-0.2 cm thick Thin slices were inoculated on embryogenic callus induction medium MS+2,4-D 2mg / L, cultured in the dark at 26°C-28°C for 40 days, the leaves were covered with white dense callus, and type I embryogenic callus.

[0030] Pick the primary embryogenic callus and insert it into the embryogenic callus medium MS+2,4-D1mg / L for subculture, culture in the dark at 26°C-28°C, subculture once every 20-30 days; These embryogenic callus further induced a large number of shiny, light yellow, well-dispersed and loose type II embryogenic callus.

Embodiment 2

[0031] Embodiment 2 preparation of embryogenic callus

[0032] Choose the plant tail tip of No. 25 sugarcane variety Taitang, which grows robustly, disinfect the surface with 70% ethanol, take the young leaf tissue about 10 cm away from the top growth point as explants, and cut it into 0.1-0.2 cm thick Thin slices were inoculated on embryogenic callus induction medium MS+2,4-D 3mg / L, cultured in the dark at 26°C to 28°C for 20 days, the leaves were covered with white dense callus, and type I embryogenic callus.

[0033] Pick the primary callus and insert it into the embryogenic callus medium MS+2,4-D 1mg / L for subculture, culture in the dark at 26°C-28°C, subculture once every 20-30 days; The embryogenic callus further induced a large amount of lustrous, light yellow, loose type II embryogenic callus with good dispersion.

Embodiment 3

[0034] Example 3 Antibiotic Screening

[0035] Callus subculture stage: set six gradient concentrations (0, 10.0, 20.0, 30.0, 40.0, 50.0 mg / L) of hygromycin (Hyg). Choose the type II embryogenic callus (approximately 1 cm in size) prepared in vigorous growth Example 1 2 ) as the experimental material, inoculated on the subculture medium MS+2,4-D1.0 mg / L containing hygromycin, cultured in the dark at 26°C to 28°C, and transferred once every 20 days Fresh culture medium, inoculate 5 culture dishes for each concentration, pick up 10 pieces of material in each dish, observe the test results every 10 days, record the growth of callus, and count the results after 40 days. The mass concentration of Hyg was determined according to the growth of the callus.

[0036] Adventitious bud differentiation culture stage: set six gradient concentrations of hygromycin (Hyg) (0, 10.0, 20.0, 30.0, 40.0, 50.0 mg / L). Choose the type II embryogenic callus (about 1 cm in size) prepared by the vigor...

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Abstract

The invention discloses a method for agrobacterium tumefaciens-mediated genetic transformation of sugarcane. The method comprises the steps: leading agrobacterium tumefaciens liquor carrying plant expression vectors to infect embryogenic callus of the sugarcane; and selecting and proliferating resistant calli to induce the resistant calli to obtain the resistant buds of the sugarcane, wherein matrix attachment region sequences are connected at two sides of a gene expression box of the plant expression vector. According to the method, MARs (metal artifact reduction) sequences are built at two sides of the gene expression box of the plant expression vector, thus greatly improving the exogenous gene transformation efficiency and seedling rate of the sugarcane, and being simple in process flow and low in cost.

Description

technical field [0001] The invention belongs to the technical field of plant transgenesis, and in particular relates to a sugarcane genetic transformation method mediated by Agrobacterium tumefaciens. Background technique [0002] Sugarcane is the main sugar crop. High yield, high sugar, high resistance and high economic benefit are the ultimate goal of sugarcane cultivation and the main goal of sugarcane breeding. However, the main economic traits of sugarcane (such as cane stem yield, sucrose content and growth characteristics, etc.) and stress resistance are mostly quantitative traits, which are controlled by micro-effect polygenes, greatly affected by the environment, and have many linkage groups. It is very difficult to combine all the good traits of the parents. In particular, it is difficult to introduce resistance genes targetedly into elite sugarcane varieties. Modern cultivated sugarcane is a compound heterozygous descendant of multiple species, which is highly a...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 邓智年魏源文李楠胡春锦郭文峰李杨瑞
Owner 广西作物遗传改良生物技术重点开放实验室
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