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Method for inducing dandelion to produce hairy roots by means of Agrobacterium rhizogenes

A technology of Agrobacterium rhizogenes and dandelion, which is applied in the biological field to achieve the effect of easy material, high synthesis ability and strong synthesis ability

Pending Publication Date: 2019-01-11
XINXIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports of successful transformation of dandelion hair roots at home and abroad

Method used

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  • Method for inducing dandelion to produce hairy roots by means of Agrobacterium rhizogenes
  • Method for inducing dandelion to produce hairy roots by means of Agrobacterium rhizogenes
  • Method for inducing dandelion to produce hairy roots by means of Agrobacterium rhizogenes

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Experimental program
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Effect test

preparation example Construction

[0043] Step 1. Preparation of explants

[0044]Soak dandelion seeds in warm water, select plump dandelion seeds and place them in a container, after sterilizing, rinsing and drying the residual water on the surface of the seeds with sterile paper, transfer dandelion seeds to 1 / 2MS solid medium and store them at 21°C Cultivate for 30 to 35 days in an artificial climate chamber at ~23°C and a photoperiod of 8h to 16h. When the sterile seedlings grow to a certain height, take dandelion explants for infection experiments;

[0045] Step 2, activation of Agrobacterium rhizogenes

[0046] Take out the Agrobacterium rhizogenes R1601 stored at low temperature, inoculate the bacteria solution in LB solid medium, and culture it under the condition of 25℃~30℃ for 2~3 days. colonies, and transfer them into centrifuge tubes containing LB liquid medium, and culture them on a shaker at a temperature of 25°C-30°C and a rotation speed of 150-200r / min for 18-20h until the bacterial liquid becom...

Embodiment 1

[0068] Embodiment 1: the influence of preculture time on the induction of dandelion hairy roots

[0069] Appropriate pre-cultivation time is very important for the infection transformation experiment. As shown in Table 1, when the pre-culture time is 1 to 2 days, a higher rooting rate can be obtained, but the average number of roots in the 2-day pre-cultivation is different from the long-term root growth rate. Better than pre-incubating for 1 day. When the pre-cultivation is 0 day or 3 days, the rooting time of dandelion is longer than that of 2-day pre-cultivation, and the average root number and average root length are not 2-day pre-cultivation.

[0070] Table 1 Effect of pre-cultivation time on hairy roots induced by dandelion

[0071]

Embodiment 2

[0072] Embodiment 2: the influence of bacterial solution concentration on the induction of dandelion hairy roots

[0073] The concentration of bacterial solution has a great relationship with the rooting rate of dandelion explants. As shown in Table 2, when the bacterial concentration is slightly low, the rooting rate of the explants increases with the increase of the bacterial concentration. When the OD600 exceeds this range, the rooting rate of the explants is significantly reduced, the browning is intensified, and the subsequent sterilization operation is difficult, causing the explants to be covered with colonies and subsequent operations cannot be performed. And when the bacterial liquid OD600=0.7, the rooting rate is high, and the average number of roots is also large. Therefore, the bacterial solution concentration OD600=0.7 is more suitable for the induction experiment of dandelion hairy roots.

[0074] Table 2 The effect of bacterial concentration on the induction o...

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Abstract

The invention discloses a method for inducing dandelion to produce hairy roots by means of Agrobacterium rhizogenes, which comprises the following steps: preparation of explant, activation of Agrobacterium rhizogenes, Agrobacterium rhizogenes infection and co-culture, hairy eradication bacteria and subculture. The invention successfully induces dandelion to produce hairy root after subculture, andhas stronger potential of producing secondary metabolite. The hairy root obtained by inducing the hairy root has the advantages of fast growth speed and good genetic stability. The invention not onlyhas the advantages of simple operating, convenience and high inducing rate, but also has the potential of large-scale production of substitute resources of medicinal ingredients of industrial dandelion.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for inducing and producing dandelion hairy roots by using Agrobacterium rhizogenes, and preliminarily establishes a dandelion hairy root in vitro culture system. Background technique [0002] Agrobaeterium rhizogenes (Agrobaeterium rhizogenes) is a Gram-negative soil Agrobacterium belonging to the genus Agrobacterium (Agrobactefium) of the family Rhizobiaceae (Rhi-zobiaceae), which can infect most dicotyledonous plants and a few monocotyledonous plants. Agrobacterium infects plants, transfers and integrates a piece of DNA (T-DNA) on the Ri plasmid it contains into the plant genome, and induces hairy roots. [0003] Dandelion, as a food and medicine homologous plant, is a plant resource with broad development and application prospects. Chinese scholars and clinical medical workers have confirmed that dandelion has the functions of anti-oxidation, antibacterial, l...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/06A01H6/14
CPCC12N15/8205
Inventor 徐小博徐萍庄静静金振锐王利平牛娇娇王选年任敏赵天宇
Owner XINXIANG UNIV
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