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Method for agrobacterium tumefaciens-mediated genetic transformation of dried radix rehmanniae

A genetic transformation method, Agrobacterium-mediated technology, applied in biochemical equipment and methods, horticultural methods, botanical equipment and methods, etc., can solve the problems of lagging Ri plasmid transformation technology, complicated transformation procedures, slow differentiation of regenerated buds, etc. , to achieve the effect of short culture period, simplified transformation procedure and short transformation period

Inactive Publication Date: 2013-12-25
HENAN AGRICULTURAL UNIVERSITY
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Problems solved by technology

However, because the transgenic plants transformed by Agrobacterium rhizogenes generally show shortened internodes in the above-ground part and fibrous roots in the underground part (Zhang Yinlin et al., Chinese Journal of Traditional Chinese Medicine, 1997, 5: 274-275), and the Ri plasmid transformation technology lags behind, limitations application in the genetic modification of plant genomes
Park et al. (Biotechnology letters, 2002, 7: 547-550) in South Korea reported the first Agrobacterium tumefaciens-mediated transformation of Rehmannia glutinosa plants, however, the germplasm of Rehmannia glutinosa as the transformation recipient is unknown, and the transformation procedure is complicated
Zang Yachao (Master's Thesis of Hebei Agricultural University, 2012) also obtained Rehmannia glutinosa transgenic plants, but due to the high concentration of antibacterial antibiotics (ampicillin 600 mg / L), the differentiation of regenerated shoots was slow and the transformation efficiency was also very low.

Method used

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  • Method for agrobacterium tumefaciens-mediated genetic transformation of dried radix rehmanniae
  • Method for agrobacterium tumefaciens-mediated genetic transformation of dried radix rehmanniae
  • Method for agrobacterium tumefaciens-mediated genetic transformation of dried radix rehmanniae

Examples

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Effect test

Embodiment 1

[0033] Agrobacterium-mediated genetic transformation method of rehmannia glutinosa, the steps are as follows:

[0034] 1) Wash the root tubers of Huai Di Huang fine variety “Wen 85-5” dug in the field, rinse with tap water for 12 h, disinfect the surface with 70% alcohol for 30 s, rinse with sterile water for 3 times, and then rinse with 0.1% HgCl 2 Disinfect for 10 min, rinse with sterile water 6 times, and place on sterile filter paper to dry. Cut the part with bud eyes into 1.5 cm long pieces and put them into MS basic medium, 14 h / d, 4000 lux light, culture at 25±1°C, and transfer the regenerated buds to new MS basic medium after 15 days , subculture once every 30 days.

[0035] 2) Streak and activate Agrobacterium tumefaciens LBA4404 containing vector pCAMBIA1301 on YEB solid medium, culture in dark for 48 hours at 27°C, pick single clone colony and inoculate into 20 mL YEB liquid medium, and inoculate at 27°C Under 180 rpm shaking culture for 24 h, 100 μL of bacterial ...

Embodiment 2

[0041] Repeat Example 1 with the following differences: in step 3), the leaf discs were soaked in the prepared infection solution for 10 min, and in step 4), the concentration of hygromycin in the screening medium was 15 mg / L.

Embodiment 3

[0043] Example 1 was repeated with the following differences: step 2) the plasmid vector contained in Agrobacterium was pBi121, and step 4) kanamycin was used as the screening agent in the screening medium at a concentration of 50 mg / L.

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Abstract

The invention belongs to the technical field of plant biology, and particularly relates to a method for agrobacterium tumefaciens-mediated genetic transformation of dried radix rehmanniae. A leave disc formed in a shearing mode after blades of aseptic dried radix rehmanniae seedlings are unfolded initially and veins are removed serves as the infection receptor of agrobacterium tumefaciens, and a transgenetic seedling of the radix rehmanniae is finally acquired through the processes of agrobacterium infection, co-culture, resistant callus induction, resistance bud initiation, and resistance bud rooting. The method is short in culture period and high in transformation efficiency, successfully achieves genetic transformation of the dried radix rehmanniae, lays the foundation of stable expression of an exogenous gene in the dried radix rehmanniae, and has significance in radix rehmanniae genetic improvement and the molecular biology study.

Description

[0001] technical field [0002] The invention belongs to the field of plant biotechnology, in particular to a method for genetic transformation of rehmannia glutinosa mediated by Agrobacterium. Background technique [0003] Rehmannia glutinosa ( Rehmannia glutinosa L.) is a perennial herbaceous plant of Scrophulariaceae, and its roots are used as medicine. It is one of the famous "four medicines for pregnant women". Because of its long history of medication and definite clinical efficacy, it has become one of the bulk medicinal materials recommended for research and utilization in the construction of my country's modern Chinese medicine industry chain (Huang Luqi et al., China Science and Technology Investment, 2010, 5: 67-69). At present, the planting area in Jiaozuo, Henan Province has exceeded 15,000 hectares, with an output value of several billion yuan. However, since rehmannia root is mostly used for asexual reproduction in production, it is more susceptible to path...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H4/00A01H5/00
Inventor 王丰青张重义魏荷陈新建孙红正高致明李娟杜家方周艳
Owner HENAN AGRICULTURAL UNIVERSITY
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