Psammosilene tunicoides suspension cell culture system and establishment method thereof

A culture system and suspension cell technology, applied in the field of plant cell engineering, can solve problems such as death, tissue hypoxia, and aging

Active Publication Date: 2015-08-19
DALIAN PRACTICAL BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are outstanding problems in the process of hairy root preparation and liquid culture: (1) hairy root induction rate is low, about 20%, which is determined by the inherent characteristics in the interaction process between plant cells and Agrobacterium rhizogenes; (2) Agglomeration in the process of liquid culture causes t

Method used

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  • Psammosilene tunicoides suspension cell culture system and establishment method thereof

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preparation example Construction

[0047] Preparation of reference solution: Accurately weigh about 20mg of oleanolic acid standard substance (dried to constant weight at 120°C under reduced pressure), dissolve it in absolute ethanol, and set the volume in a 50mL volumetric flask to obtain the oleanolic acid reference substance Sample liquid.

[0048] Preparation of the sample solution: Accurately weigh about 30 mg of the crude total saponins of Aurantia chinensis dried to constant weight, dissolve it in absolute ethanol, and set the volume in a 50 mL volumetric flask to obtain the sample solution.

[0049] Determination of the maximum absorption wavelength: Accurately measure 0.6mL of the standard solution and 5mL of the sample solution, place each in a 10mL volumetric flask, evaporate the solvent on a water bath at 75°C, and then add a newly prepared 5% vanillin-glacial acetic acid solution (50mg vanillin dissolved in 10mL glacial acetic acid) 0.4mL, perchloric acid 1.6mL, shake well, heat in a water bath at ...

Embodiment 1

[0054] The high-efficiency induction of embodiment 1 golden iron lock hairy root system

[0055] (1) Take the leaves of Jintiesuo aseptic seedlings, remove the tissue around the leaf edge, and use 10mmol / L CuCl 2 After soaking the explants in the aqueous solution for 10 minutes, dry the excess water on the surface of the explants with sterile filter paper;

[0056] (2) Explants treated with Agrobacterium rhizogenes dipping step (1):

[0057] 2a. Agrobacterium rhizogenes ACCC10060 bacterial liquid was inoculated in YEB solid medium, and cultured in the dark at 25°C ± 1 for 2-3 days to obtain clones of Agrobacterium rhizogenes ACCC10060;

[0058] 2b. Pick the clone of Agrobacterium rhizogenes ACCC10060, inoculate it in YEB medium, and culture it with shaking at 25°C±1; take 1ml of the bacterial solution with an OD value of 0.6-0.8 and place it in 100ml YEB liquid medium and shake it to OD When the value is 0.6, the bacteria liquid is collected, and the bacteria are collected b...

Embodiment 2

[0067] Embodiment 2: the comparison induction of golden iron lock hairy root system

[0068] According to the method of embodiment 1, but omit the CuCl with 10mmol / L in the step (1) 2 The step of immersing the explant in the aqueous solution for 10 minutes is followed by the next step with the cleaned Jintiesu explant to induce the generation of the hairy root of Jintiezio.

[0069] In the culture process of this embodiment, 4-6 days after inoculation, hairy roots will occur at the petiole and leaf veins, and hairy roots will be produced in 5-8 days, and the rooting time is longer than that of Example 1; the induced transformation rate is lower than 30%..

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Abstract

The invention relates to a psammosilene tunicoides suspension cell culture system and an establishment method thereof. The establishment method comprises the steps of inducing callus by utilizing psammosilene tunicoides hairy roots as explants, and establishing the suspension cell culture system. On the basis of efficient induction of the hairy roots, the excellent explants is provided for establishing the system by virtue of efficient hairy root induction and culture, the psammosilene tunicoides leaves are treated at the first time by utilizing CuCl2, the leaves are immersed in rooting agrobacterium, the hairy roots can be generated at leaf veins of a leaf stalk and leaf margins in 2 to 4 days, the rooting time is shortened, the rooting can be generated in 3 to 5 days, and the inducing conversion rate is greatly increased and can reach 60 percent or more. The callus is generated by virtue of screening, dedifferentiation and induction, the suspension cell is prepared by virtue of the pretreatment of calcium oxalate, the suspension cell is cultured in an improved culture medium, the secondary metabolism and the biochemical inheritance of the cultured suspension cell are stable, the cell is unlikely to degrade, and a reliable material source and a reliable material foundation can be provided for extracting a natural product from cells.

Description

technical field [0001] The invention belongs to the technical field of plant cell engineering, and in particular relates to a method for efficiently inducing hairy roots of golden iron locks and a cell suspension culture system based thereon. Background technique [0002] Psammosilene tunicoides (Psammosilene tunicoides W.C.Wu et C.Y.Wu) is a perennial herbaceous plant of the genus Psammosilene tunicoides in the family Caryophyllaceae, also known as Dudingzi, Xiaobawang, Kunming Aphrodisiac, Jinsi Aiduoduo, Tunicoides, etc. It is a unique single-genus species in southwest my country Medicinal plants. The roots of Jintiesuo are mainly used as medicine, and the main medicinal ingredients are triterpenes, triterpene saponins, cyclic peptides and lactams. Pharmacological studies have found that the extracts of Jintiesuo are mainly analgesic and anti-inflammatory, blood stasis and bleeding, anti-tumor, immune regulation, antibacterial It is used for the treatment of bruises, rheu...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12N5/02C12P33/20C12R1/91
Inventor 张宗申程诺金朝霞张聪张孟夏仇南南刘军何连芳
Owner DALIAN PRACTICAL BIOTECH
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