Method for rapid propagation of genetically modified sweet wormwood by hydroponics

A transgenic, Artemisia annua technology, applied in the fields of botanical equipment and methods, cultivation, and application, can solve the problems of environmental pollution, propagation failure, and artemisia annua explant pollution, so as to improve yield and maintain genetic stability. Effect

Active Publication Date: 2012-06-20
SHANGHAI JIAO TONG UNIV SUBEI RES INST
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

However, rapid propagation of transgenic Artemisia annua plants by microadventitious bud technology requires detoxification of transgenic Artemisia annua explants, induction of transgenic Artemisia annua microadventitious buds, elongation of transgenic Artemisia annua adventitious buds, and rooting of transgenic Artemisia annua adventitious buds As well as multiple steps such as transplanting of transgenic Artemisia annua regenerated plants, the process is relatively complicated, and many steps require aseptic operation, and a

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Construction of Plant Binary Expression Vectors Containing Endogenous Genes of Artemisia annua

[0018] 1. Construction of the intermediate vector pCAMBIA2300::p35S-gus-nos

[0019] The binary plant expression vector pCAMBIA2300::p35S-gus-nos was constructed by selecting pBI121 and pCAMBIA2300 as basic elements. Specifically, pBI121 and pCAMBIA2300 plasmids were digested with HindIII and EcoRI; the gus expression cassette of pBI121 and the large fragment of pCAMBIA2300 were recovered; the recovered products were ligated, transformed and screened, and verified by plasmid digestion.

[0020] 2. Construction of plant expression vector pCAMBIA2300::p35S-artemisia annua endogenous gene-nos

[0021] The pCAMBIA2300::p35S-gus-nos was used as the expression vector, and the gus gene on it was replaced with the endogenous gene of Artemisia annua. Specifically, BamHI / SacI double digestion of pGEMT-easy+ endogenous gene of Artemisia annua and pCAMBIA2300::p35S-gus-nos, recovery o...

Embodiment 2

[0024] Agrobacterium tumefaciens mediated genetic transformation of Artemisia annua endogenous genes to obtain transgenic Artemisia annua plants

[0025] 1. Obtaining the engineering bacteria of Agrobacterium tumefaciens containing the binary plant expression vector of the endogenous gene of Artemisia annua

[0026] In Example 1, the plant binary expression vector containing the endogenous gene of Artemisia annua was transformed into Agrobacterium tumefaciens (such as EHA105, which is a publicly available biological material in the market, which can be purchased from CAMBIA Company in Australia, and the strain number is Gambar 1) , and validated by PCR. The results showed that the plant binary expression vector containing the endogenous gene of Artemisia annua has been successfully constructed into the strain of Agrobacterium tumefaciens.

[0027] 2. Agrobacterium tumefaciens mediated transformation of Artemisia annua endogenous gene

[0028] 2.1. Preculture of explants

[...

Embodiment 3

[0038] Determination of artemisinin content in transgenic Artemisia annua by HPLC-ELSD

[0039] 1. HPLC-ELSD conditions and system suitability and preparation of standard solutions

[0040] HPLC: adopt water alliance 2695 system, chromatographic column is C-18 reverse phase silica gel column (SymmetryShieldTM C18, 5 μm, 250 * 4.6mm, Waters), mobile phase is methanol: water, the volume ratio of methanol: water is 70: 30, The column temperature is 30°C, the flow rate is 1.0mL / min, the injection volume is 10μL, the sensitivity (AUFS=1.0), and the number of theoretical plates is not less than 2000 based on the artemisinin peak.

[0041] ELSD: Water alliance 2420 system is adopted, the drift tube temperature of the evaporative light scattering detector is 40°C, the amplification factor (gain) is 7, and the carrier gas pressure is 5bar;

[0042] Accurately weigh 2.0 mg of artemisinin standard (Sigma Company) and dissolve it completely in 1 mL of methanol to obtain a 2 mg / mL artemis...

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Abstract

A method for rapid propagation of genetically modified sweet wormwood by hydroponics belongs to the technical field of hydroponic planting and includes: placing explants of genetically modified sweet wormwood in hydroponic induction culture solution for light-tight cultivation to obtain adventitious roots which are transplanted to transplant matrix; and obtaining a great amount of genetically modified sweet wormwood plants through domestication. By the method, a great amount of genetically modified sweet wormwood seedlings can be propagated in a short time, genetic stability of the sweet wormwood is retained, and significance to large-scale popularization of genetically modified sweet wormwood, guarantee of sufficient high-quality sweet wormwood seedlings and increase of sweet wormwood yield is realized.

Description

technical field [0001] The invention relates to a method in the technical field of hydroponic planting, in particular to a method for rapidly propagating transgenic Artemisia annua using hydroponic technology. Background technique [0002] Artemisia annua L. is an annual herb belonging to the family Asteraceae. Artemisinin (artemisinin) is a sesquiterpene lactone compound containing a peroxide bridge structure isolated from its aerial part. It is currently the most effective drug for treating malaria in the world, especially for cerebral malaria and anti-malaria Chloroquine malaria has the characteristics of quick action and low toxicity. Currently, the most effective treatment for malaria recommended by the World Health Organization is artemisinin combination therapy (ACTs). In addition, with the deepening of pharmacological research on artemisinin, scientists have discovered that artemisinin and its derivatives also have anti-inflammatory, anti-schistosome, anti-tumor, a...

Claims

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Application Information

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IPC IPC(8): A01G31/00
Inventor 唐克轩陆续张凌吴韶龑江伟民张芳源沈乾
Owner SHANGHAI JIAO TONG UNIV SUBEI RES INST
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