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Method for transforming and cultivating citrus transgenic plant by agrobacterium-mediated blade

An Agrobacterium-mediated, transgenic technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, horticultural methods, etc.

Inactive Publication Date: 2012-07-18
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there have been no reports of using leaves as explants to carry out genetic transformation of citrus to obtain transgenic plants at home and abroad.

Method used

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  • Method for transforming and cultivating citrus transgenic plant by agrobacterium-mediated blade
  • Method for transforming and cultivating citrus transgenic plant by agrobacterium-mediated blade
  • Method for transforming and cultivating citrus transgenic plant by agrobacterium-mediated blade

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1) Preparation of explants

[0044] After the seeds of the citrus variety Fulingxia orange (a common cultivated sweet orange variety that matures in summer, from the orchard of the Citrus Research Institute of Huazhong Agricultural University) were taken out from the fruit, they were first treated with 1M sodium hydroxide solution for 20 minutes, and washed with sterile water for 3 minutes. Once again, it is 2.5% sodium hypochlorite solution to sterilize for 5-20 minutes with available chlorine, and the citrus seeds after disinfection are sown on MT (Murashige T and Tucker DPH.Growth factor requirements of Citrus tissue culture.Proc.First Intl.Citrus Symp, 1969, 3:1155-1161) on medium (pH5.8), under light (45μmol m -2 the s -1 Light intensity, light time 16h per day) cultivated for 4-12 weeks to obtain sterile seedlings.

[0045] 2) Preparation of Agrobacterium liquid

[0046] Before transformation, take a small tube of Agrobacterium bacterial liquid and scrape the c...

Embodiment 2

[0065] Example 2: Transformation of citrus leaves to obtain transgenic plants

[0066] 1. Transformation receptor material and culture method

[0067] Fulingxia sweet orange fruits were collected at the National Citrus Breeding Center of Huazhong Agricultural University, Wuhan City, Hubei Province, China. After the seeds were removed from the fruit, they were treated with 1 M sodium hydroxide for 20 min, washed 3 times with sterile water, and then treated with 2.5% available chlorine Sodium hypochlorite solution was sterilized for 5-20 minutes, washed 2-3 times with sterile water, and seeded in MT (Murashige T, Tucker D P H.Growth factor requirements of Citrus tissue culture.Proc. First Intl. Citrus Symp, 1969, 3: 1155-1161) medium (pH 5.8), cultured under light for 4-12 weeks to obtain sterile seedlings.

[0068] Unless otherwise specified, the medium components of the present invention are shown in Table 1.

[0069] The culture medium was sterilized by conventional methods...

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Abstract

The invention belongs to the field of plant transgenic technique, particularly relates to a cultivating method of a citrus transgenic plant and discloses a method for cultivating the citrus transgenic plant by utilizing agrobacterium mediation and taking a citrus test-tube seedling blade as an explant. The method comprises the following steps of genetic transformation, screening and regeneration,PCR (Polymerase Chain Reaction) detection and GFP (Green Fluorescent Protein) fluorescence observation. The method is characterized in that a GFP reporter gene is guided into the receptor citrus blade through agrobacterium mediation, different factors capable of influencing the blade transformation are screened, kanamycin is utilized as a selector to screen a transformed receptor cell, and the transgenic citrus plant is screened and obtained through the PCR detection, the GFP fluorescence observation and other auxiliary methods. The method provides a new technical platform for innovative germplasm resources of citrus genetic transformation.

Description

technical field [0001] The invention belongs to the field of plant transgenic technology. Specifically, it relates to a method for cultivating citrus transgenic plants through leaf genetic transformation mediated by Agrobacterium. Background technique [0002] Citrus is an important fruit tree in my country, which plays an important role in the adjustment of agricultural structure, the poverty alleviation of farmers and the earning of foreign exchange through export. In recent years, my country's citrus production and area have continued to grow, ranking first in the world. However, citrus is also subject to many biotic and abiotic stresses during the development process, so cultivating stress-resistant varieties is crucial for the sustainable and stable development of the citrus industry. However, as a perennial woody plant, citrus has a long life cycle, tall trees, complex genetic background (highly heterozygous genes), and long childhood. mongrel. The current bud muta...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12Q1/68G01N21/64A01H4/00A01H5/00
Inventor 刘继红艾山付行政
Owner HUAZHONG AGRI UNIV
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