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Agrobacterium mediated wild pepper transgene method

An Agrobacterium-mediated, transgenic technology, applied in the biological field, can solve the problem of lack of a tissue culture regeneration system of prickly ash

Inactive Publication Date: 2008-06-11
GUIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of an efficient tissue culture regeneration system for Zanthoxylum bungeanum, there are no domestic and foreign reports on the genetic transformation and transgenic breeding of Zanthoxylum bungeanum

Method used

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  • Agrobacterium mediated wild pepper transgene method
  • Agrobacterium mediated wild pepper transgene method
  • Agrobacterium mediated wild pepper transgene method

Examples

Experimental program
Comparison scheme
Effect test

experiment example

[0034] Experimental example: Antibiotic susceptibility experiment on petiole and stem of Zanthoxylum bungeanum

[0035] In this experiment, the construction of Zanthoxylum bungeanum genetic transformation contains kanamycin resistance gene, so kanamycin was used to screen transgenic plants. In addition, cephalosporin was used as Agrobacterium in the process of tissue culture of Zanthoxylum bungeanum. Inhibitors. In the pepper induction medium, add 0mg / L kanamycin, 5mg / L kanamycin, 10mg / L kanamycin, 20mg / L kanamycin, 30mg / L kanamycin, 50mg / L kanamycin and 100mg / L kanamycin, 150mg / L cephalosporin. Cut petioles and stems of about 0.5 cm into sections, inoculate them into the above medium, and count the differentiation and survival of the materials after 25 days. The results showed that 150mg / L cephalosporin had little effect on the differentiation of Zanthoxylum bungeanum hypocotyl and cotyledon, so we used 150mg / L cephalosporin to inhibit bacteria during screening. The petio...

Embodiment

[0037](1) Material disinfection method: take the explants in the field and wash them in running water for 2 hours, brush the surface of the material with a small amount of washing powder, then fully wash the washing powder with running water, disinfect the surface with 75% alcohol for 45 seconds, and rinse with sterile water for 1 Disinfect with 0.1% mercuric chloride for 7 minutes, rinse with sterile water for 5 times, cut 0.5-1cm stems with axillary buds and inoculate them in the induction medium

[0038] (2) Preparation of Agrobacterium: Dip the Agrobacterium containing the pBin19 recombinant plasmid (the recombinant plasmid contains the isopentenyl transferase gene ipt and the recombinase gene flp) frozen at -80°C with an inoculation needle, and culture it on the YEP solid Scrape a plate on the substrate, and culture at a constant temperature of 28°C for 24-48h until a suitable size of plaque grows; pick a single colony of Agrobacterium with a toothpick and add it to 5ml of...

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Abstract

The invention discloses a method for agrobacterium mediated pepper transgene. The invention comprises the following steps: firstly, a young and tender stem section with an axillary bud and a stem tip are inoculated in an induction culture medium, and a leaf stalk and the stem section of the sprouting axillary bud for 20 days are used as a dip-dyeing explant; secondly, the single colony of agrobacterium is picked with toothpicks and added into a YEP liquid culture medium to cultivate shaking culture till an OD value reaches 0.4 to 0.7, thallus is centrifugally collected, and agrobacterium liquid is prepared by the weight dropping of a WPM weigh dropping culture medium; thirdly, the leaf stalk and the stem section are cut into about 0.5 centimeters explants, inoculated with the induction culture medium to perform pre-incubation in dark, the cultivation time needs 48 to 72 hours, the explants are thrown into the agrobacterium liquid of the step b, to ensure that the explants are adequately contacted with the agrobacterium, the explants are taken out, shifted on the common culture medium to be cultivated for 48 to 72 hours, and then shifted in the sieving medium to induce buds to be regenerated, finally material is inoculated in the rooting culture medium to induce root regenerating. The invention can cultivate transgene pepper with insect resistance, low temperature resistance, drought resistance, high essential oil content, high medicinal value, and no thorn.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an Agrobacterium-mediated transgenic method for prickly ash. Background technique [0002] Zanthoxylum piperitum DC var.inerme Makino is a deciduous shrub of the Rutaceae Zanthoxylum. Zanthoxylum bungeanum is widely used and is an important economic tree. Its fruit can not only be used as medicine and oil extraction, but also the main seasoning for people's daily cooking. Modern studies have shown that the chemical components of plants of the genus Zanthoxylum mainly include alkaloids, amides, lignans, coumarins, and fatty acids, which have strong pharmacological activities on the cardiovascular system, digestive system, immune function, blood coagulation function, and tumors. Studies have found that Zanthoxylum bungeanum has a high inhibitory ability to reverse transcriptase and DNA polymerase activity of cancer cells. Therefore, the research on anticancer active substa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H4/00
Inventor 赵德刚曾晓芳李岩
Owner GUIZHOU UNIV
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