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A method for genetic transformation of transgenic Stevia rebaudiana mediated by Agrobacterium rhizogenes

A genetic transformation method, the technology of Agrobacterium rhizogenes, which is applied in the field of genetic transformation of stevia rebaudiana mediated by transgenic Agrobacterium rhizogenes, can solve the problems that there is no stevia transgenic system established yet, and achieve easy mastery of operation technology and reduce costs , the effect of simple equipment

Active Publication Date: 2017-10-31
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 long dominated the stevia cultivars and once monopolized the cultivation of stevia across the country
At present, there is no report on the establishment of Stevia rebaudiana transgenic system

Method used

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  • A method for genetic transformation of transgenic Stevia rebaudiana mediated by Agrobacterium rhizogenes
  • A method for genetic transformation of transgenic Stevia rebaudiana mediated by Agrobacterium rhizogenes

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 5min, remove the supernatant, resuspend and dilute to OD with fresh MS liquid medium 600 When the v...

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Abstract

A method for the genetic transformation of transgenic Stevia rebaudiana mediated by Agrobacterium rhizogenes, in which the stem tip explants of Stevia rebaudiana after precultivation are immersed in the Agrobacterium rhizogenes bacterium liquid carrying the pRI 101-AN DNA plasmid of the gene of interest, After infection, co-cultivation and sterilization treatment, the hairy roots were cultured on the selection medium containing kanamycin, and induced the formation of kanamycin-resistant hairy roots on the explants, and induced the formation of hairy roots The callus differentiates into adventitious buds, and the adventitious buds continue to grow to form test-tube plantlets, and PCR amplification method is used to identify transgenic plants carrying the target gene. By adopting the technical scheme provided by the invention, the target gene can be introduced into Stevia rebaudiana, and compared with the genetic transformation of chemical methods such as polyethylene glycol, the link of isolation and cultivation from protoplasts is reduced; and the genetic transformation of physical methods such as gene guns In contrast, it does not require expensive instruments and equipment, and the operation technique is simple.

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 steviol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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