Agrobacterium rhizogenes-mediated transgene Stevia rebaudiana genetic transformation method

A technology of Agrobacterium rhizogenes and genetic transformation, applied in biochemical equipment and methods, horticultural methods, genetic engineering, etc., can solve problems such as the establishment of Stevia rebaudiana transgenic system, and achieve easy mastery of operation technology, convenient material acquisition, and equipment simple effect

Active Publication Date: 2014-03-26
ANHUI UNIVERSITY
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  • Application Information

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Problems solved by technology

At present, the following problems exist in the production of stevia across the country: due to the high glycoside content of Morita No. 2 and Morita No. 3 cultivated in Japan, they have lo

Method used

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  • Agrobacterium rhizogenes-mediated transgene Stevia rebaudiana genetic transformation method
  • Agrobacterium rhizogenes-mediated transgene Stevia rebaudiana genetic transformation method

Examples

Experimental program
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Embodiment 1

[0030] Example 1 Extraction of the recombinant pRI 101 AN-DNA plasmid carrying the target gene UGT76G1

[0031] After activation, a single colony was selected and cultured in 5 ml LB liquid medium containing 50 mg / mL Kan at 220 r / min at 37 °C overnight. The recombinant pRI 101 AN-DNA plasmid was extracted by conventional alkaline lysis method, and after the purified recombinant pRI 101 AN-DNA plasmid was obtained, the concentration of the plasmid DNA solution was measured with an ultraviolet spectrophotometer, and the concentration of the plasmid DNA solution was adjusted to 1.0 μg / L .

[0032] After Sal I and EcoR I double enzyme digestion, take 5u1 digestion solution, electrophoresis in 1.5% agarose gel plate containing ethidium bromide, TAE electrophoresis buffer, voltage 5V / cm. The agarose gel plate was placed under the gel imager, and two DNA fragments of about 11Kb and 1600bp were observed ( figure 1 ), indicating the target gene carried by the recombinant pRI 101 AN-D...

Embodiment 2

[0033] Example 2 Genetic transformation and plant regeneration mediated by Agrobacterium rhizogenes

[0034] (1) Pre-cultivation of Stevia rebaudiana stem tip explants

[0035] Cut shoot tips 1-1.5 cm long from test-tube plantlets with a height of about 5 cm, inoculate them on the pre-cultivation medium, and inoculate them in a place with a temperature of 25±1°C, light intensity of 40001x, and a photoperiod of 14 hours of light and 10 hours of darkness. After 1-3 days of culture under the condition, it is used as the recipient of genetic transformation.

[0036] The pre-culture medium is: MS + 0.1 mg / L 6-BA + 0.05 mg / L NAA + 30g / L sucrose + 6 g / L agar, pH 5.6-5.8.

[0037] (2) Genetic transformation of stem tip explants of Stevia rebaudiana

[0038] Transfer the activated engineering bacteria solution into a sterile centrifuge tube, centrifuge at 4000g at room temperature for 5 minutes, remove the supernatant, resuspend and dilute to OD with fresh MS liquid medium 600 When ...

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Abstract

The invention relates to an Agrobacterium rhizogenes-mediated transgene Stevia rebaudiana genetic transformation method. The method comprises: pre-cultured Stevia rebaudiana stem apex explant is immersed into an Agrobacterium rhizogenes solution of pRI101-ANDNA plasmid carrying target gene, treatments such as infection, co-culture and sterilization are performed, the hairy root is cultured in a kanamycin-containing screening culture medium, the anti-kanamycin hairy root is formed on the explant through induction, the hairy root is induced to form callus, differentiation of adventitious bud is performed, the adventitious bud continuously grows to form the test-tube plantlet, and a PCR amplification method is adopted to identify the transgene plant carrying out the target gene. With the technical scheme, the target gene can be transformed into the Agrobacterium rhizogenes, such that the steps of separation from the protoplast and culture are eliminated compared with genetic transformation of polyethylene glycol and other chemical methods; and compared with genetic transformation of gene gun and other physical methods, the method of the present invention has characteristics of no requirement of expensive equipment, and simple operation technology.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering in plant biotechnology, in particular to a method for genetic transformation of transgenic stevia mediated by Agrobacterium rhizogenes mediated by Agrobacterium rhizogenes . Background technique [0002] Genetic transformation of plant cells is a key link in plant genetic engineering. Since Abel et al. (1986) introduced tobacco mosaic virus (TMV) capsid protein cDNA into tobacco cells and obtained transgenic tobacco plants resistant to TMV virus, many plant genetic transformation methods and technologies have been developed successively. [0003] Stevia rebaudiana was introduced into my country by the Chinese Academy of Agricultural Sciences from Japan in 1978 and has spread all over the country. Its leaves contain steviol glycosides (SGs), a natural sweetener, and are a new type of sugar crop. my country has become the world's largest producer of stevia and exporter of ste...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H5/00A01H4/00
Inventor 王钰陈达伟汤其坤万田胡同华
Owner ANHUI UNIVERSITY
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