Huperzia serrata hairy root system preparation and cultivation method

A cultivation method, the technology of the Melaleuca tower, which is applied in the field of preparation and cultivation of the hairy root system of the Melaleuca tower, to achieve the effect of alleviating market demand and solving the problem of dependence on imports

Inactive Publication Date: 2012-10-31
DALIAN POLYTECHNIC UNIVERSITY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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At present, there is no relevant literature report on the preparatio...

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  • Huperzia serrata hairy root system preparation and cultivation method
  • Huperzia serrata hairy root system preparation and cultivation method
  • Huperzia serrata hairy root system preparation and cultivation method

Examples

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Embodiment 1

[0025] Embodiment 1 Mannopine paper chromatographic analysis

[0026] (1) Materials: Melaleuca hairy roots (NTR) newly induced by the present invention; subcultured hairy roots (TR) of the present invention; organ roots (NR), leaves (L) or callus (C) ; Agrobacterium rhizogenes A4 bacterial liquid;

[0027](2) Extraction of agrobasin: Take 1 g of the above samples and add 1 ml (0.1 M) HCl to grind at room temperature, centrifuge at 12,000 rpm for 5 min, take 10 μl of the supernatant, and spot the samples with a micro-injector.

[0028] (3) Chromatography: Preparation of chromatographic filter paper: Cut the chromatographic filter paper into 8×8 (cm) size, soak it in 0.4mol / L HCl solution for 24h, then rinse with distilled water, then use ethanol and ether in turn After washing, lay flat on a tray, dry at 30°C, and set aside.

[0029] (4) Sample spotting: draw a straight line 1 to 1.5 cm from the bottom edge of the chromatography filter paper, and mark the spotting lines at eq...

Embodiment 2

[0034] (1) Cut the young stems of the Melaleuca tower into 2cm pieces, and use HgCl in the aseptic operation table. 2 Treat for 7min, soak in alcohol for 6s, rinse with sterile water for 4 times and then transfer to B 5 +6-BA0.5mg / L+NAA0.3mg / L medium, cultured in the dark at 25℃±2 for 12d to obtain Melaleuca callus.

[0035] (2) The A. rhizogenes ACCC10060 strain was diluted with 1 μL of sterile water and then applied to YEB solid medium, and cultured in the dark at 25° C.±3 for 4 days to grow a single clone to obtain A. rhizogenes A4.

[0036] (3) Pick A , collect the bacterial liquid, and centrifuge at 3000 rpm for 7 min to collect the bacterial cells.

[0037] (4) Place the thalli collected in step (3) in 100 ml of B containing 100 umol / L acetosyringone and 0.7% agar 5 in solid medium and mix well.

[0038] (5) infiltrate the callus of the Melaleuca tower with the bacterial solution obtained after mixing in step (4) for 12 minutes, absorb the excess bacterial solution w...

Embodiment 3

[0042] (1) Cut the young stems of Melaleuca Pagoda into 4cm pieces, and use HgCl in the sterile operating table. 2 Treated for 10min, soaked in alcohol for 8s, rinsed with sterile water for 7 times, then transferred to B5+6-BA0.5mg / L+NAA0.3mg / L medium, cultivated in dark at 25℃±2 for 30d, and obtained Melaleuca tower healing injured tissue.

[0043] (2) Dilute the A. rhizogenes ACCC10060 strain with 1 μL of sterile water, spread it on YEB solid medium, cultivate in the dark at 25° C.±3 for 5 days to grow a single clone, and obtain A. rhizogenes A4.

[0044] (3) Pick A , collect the bacterial liquid, and centrifuge at 3000 rpm for 10 min to collect the bacterial cells.

[0045] (4) Place the thalli collected in step (3) in 100 ml of B containing 100 umol / L acetosyringone and 0.7% agar 5 in solid medium and mix well.

[0046] (5) infiltrate the callus of the Melaleuca tower with the bacterial solution obtained after mixing in step (4) for 25 minutes, absorb the excess bacter...

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Abstract

The invention discloses a huperzia serrata hairy root system preparation and cultivation method, and belongs to the biological cell engineering technology. The method comprises the following steps: tender stems of huperzia serrata is adopted for dedifferentiation treatment of an explant to acquire huperzia serrata calluses; the callused and agrobacterium rhizogenes (DL 1968) containing Ri plasmid are co-cultivated and transferred to an induced medium for induced cultivation after residual fungus liquid is extracted by aseptic paper, and then hairy roots grow at the huperzia serrata calluses; and the explant with the hairy roots is placed in an expansion medium for expanded cultivation of the hairy roots after bacteriostatic cultivation. According to the invention, the biological cell engineering technology is utilized to build a huperzia serrata hairy root system cultivation system to prepare a huperzia serrata hairy root system, so that standardized production of huperzia serrata is realized, wild resources are replaced, the ever-increasing market demand for huperzia serrata is relieved, and the problem of dependency on huperzia serrata import is solved. Therefore, the huperzia serrata hairy root system preparation and cultivation method has great significance on industrialized and commercialized development of medicinal plant hairy roots.

Description

technical field [0001] The invention relates to a method for preparing and culturing a hairy root system of a plant, in particular to a method for preparing and culturing a hairy root system of a Melaleuca tower, which belongs to a biological cell engineering technology. Background technique [0002] Huperzia serrata (Thunb.ex Muray) Trev., also known as Melaleuca, Sheepfoot, Pagoda Grass, etc., is a fern of Huperzia genus Huperzia, mainly distributed in Northeast China, the Yangtze River Basin and Fujian, Guangdong and Guangxi , Yunnan, Guizhou and other provinces. Melaleuca is a perennial herb, the whole is dark green, slightly shiny, the upper part of the stem has branches again, and the height is 10-15cm. Root-like, rhizome brown, cross-section round or sub-round, diameter 2 ~ 3cm. Stems are cylindrical, greenish-brown on the surface, 2-3cm in diameter. The leaves are green-brown, opposite, the leaves are shriveled, curled or broken, the complete ones are flattened an...

Claims

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Application Information

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IPC IPC(8): A01H4/00C12N15/82
Inventor 张宗申刘同祥于振艳叶乾堂
Owner DALIAN POLYTECHNIC UNIVERSITY
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