Induced redifferentiation method for improving paclitaxel content in calluses of Taxus wallichiana var. mairei

A southern yew, callus technology, applied in horticultural methods, botanical equipment and methods, plant regeneration, etc., can solve the problems of not being widely used in commercialization, restricting the commercial application of technology, and metabolic genetic instability.

Inactive Publication Date: 2014-03-26
JISHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0027] In summary, there have been reports on the effects of the disclosed basic composition of the medium, supplemented plant growth regulators, elicitors and precursors, etc. on the yew cell suspension system, callus culture, and the synthesis and accumulation of paclitaxel. Cedar hairy root culture production paclitaxel technology and yew tissue culture test tube seedling rapid propagation technology etc., though have certain relevance with the present invention, but so far, yew callus, cell suspension culture, hairy root culture technology all Not widely used c

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  • Induced redifferentiation method for improving paclitaxel content in calluses of Taxus wallichiana var. mairei
  • Induced redifferentiation method for improving paclitaxel content in calluses of Taxus wallichiana var. mairei
  • Induced redifferentiation method for improving paclitaxel content in calluses of Taxus wallichiana var. mairei

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Experimental program
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Effect test

Embodiment 1

[0045] (1) Explant pretreatment: take mature young stems of Taxus chinensis in the same year, cut off the leaves, rinse with tap water for 2-4 hours, dry the surface water, cut into small sections about 5 cm long, and place them in a triangular flask. Soak in 75% alcohol for 15 s in the ultra-clean workbench, then wash with 0.1% HgCl 2 Soak for 20 min, rinse with sterile water for 6-8 times, and then aseptically cut into 0.5-1 cm long pieces for inoculation.

[0046] (2) Callus induction culture: Aseptically inoculate stem segments in MS medium supplemented with 1.2mg / L 2,4-D, 4mg / L NAA, and 2.8mg / L 6-BA for induction culture; culture conditions All were cultivated in artificial climate chambers, with temperature (20±5) ℃, humidity (80±5)% dark and temperature (25±5) ℃, humidity (85±5)%, light 1000-1200 Lx respectively 12 h alternately.

[0047] (3) Subculture of callus: In step (2), after induction of yellow-green callus, the yellow-green callus was transferred to supplemen...

Embodiment 2

[0055] (1) Explant pretreatment: take mature young stems of Taxus chinensis in the same year, cut off the leaves, rinse with tap water for 2-4 hours, dry the surface water, cut into small sections about 5 cm long, and place them in a triangular flask. Soak in 75% alcohol for 15 s in an ultra-clean workbench, then soak in 0.1% HgCl2 for 20 min, rinse with sterile water 6-8 times, and cut into 0.5-1 cm long pieces for inoculation.

[0056] (2) Callus induction culture: Stem segments were aseptically inoculated in MS medium supplemented with 1 mg / L 2,4-D, 3 mg / L NAA, and 2 mg / L 6-BA for induction culture; the culture conditions were Cultivated in an artificial climate box, with temperature (20±5) ℃, humidity (80±5)% dark and temperature (25±5) ℃, humidity (85±5)%, light 1000-1200 Lx for 12 h each process alternately.

[0057] (3) Subculture of callus: In step (2), after induction of yellow-green callus, the yellow-green callus was transferred to supplemented with 1.5 mg / L 2,4-D,...

Embodiment 3

[0065] (1) Explant pretreatment: take mature young stems of Taxus chinensis in the same year, cut off the leaves, rinse with tap water for 2-4 hours, dry the surface water, cut into small sections about 5 cm long, and place them in a triangular flask. Soak in 75% alcohol for 15 s in an ultra-clean workbench, then soak in 0.1% HgCl2 for 20 min, rinse with sterile water 6-8 times, and cut into 0.5-1 cm long pieces for inoculation.

[0066] (2) Callus induction culture: Stem segments were aseptically inoculated in MS medium supplemented with 1.5 mg / L 2,4-D, 5 mg / L NAA, and 3 mg / L 6-BA for induction culture; the culture conditions were For culturing in an artificial climate box, and temperature (20±5) ℃, humidity (80±5)% dark and temperature (25±5) ℃, humidity (85±5)%, light 1000-1200 Lx each 12 h alternately.

[0067](3) Subculture of callus: In step (2), after induction of yellow-green callus, the yellow-green callus was transferred to supplemented with 2.0 mg / L 2,4-D, 4 mg / L ...

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Abstract

The invention provides a technology for inducing the redifferentiation of calluses of Taxus wallichiana var. mairei to improve or maintain the genetic stability of the cellular metabolism of the Taxus wallichiana var. mairei and improve the content of paclitaxel as a secondary metabolite of the Taxus wallichiana var. mairei. The technology comprises four steps of explant preprocessing, induced culture of calluses, calluses subculture and calluses redifferentiation induction. In the step of induced culture of calluses, subculture is performed four times with 20-35 days for each culture, the calluses with good color and right texture are transferred to an improved MS (Murashige & Skoog) culture medium in which 0.05-0.20mg/L6 of BA, 0.2-2.0mg/L of NAA, 0.1mg/L of KT and 0.5mg/L of CH are added for redifferentiation induction culture, after 45-75 days of the induction culture, tiny sprouts grow from the calluses of the Taxus wallichiana var. mairei, the calluses are harvested at the time, after samples are subjected to drying, weighing and a series of preprocessing, HPLC (High Performance Liquid Chromatography) is adopted to measure the content of the paclitaxel, and a result shows that the content of the paclitaxel is one time higher than that of the calluses which are not subjected to differentiation. In the improved MS culture medium, ammoniacal nitrogen is 2000-2600 mg/L and the nitrate nitrogen is 800-1000mg/L.

Description

technical field [0001] The invention belongs to the field of producing medicinal secondary metabolites by plant cell culture, in particular, relates to a method for increasing paclitaxel content in the callus of Taxus chinensis by inducing redifferentiation, more specifically the invention provides a The method for increasing the paclitaxel content in the southern yew callus in the technology of producing paclitaxel from the taxus chinensis callus doubles the paclitaxel content in the callus. Background technique [0002] Paclitaxel is a drug for the treatment of ovarian cancer approved by the US Food and Drug Administration (FDA) in 1992. In recent years, clinical studies have found that paclitaxel also has good curative effects on metastatic breast cancer, lung cancer, head and neck tumors, malignant melanoma and lymphosarcoma, and has certain curative effects on other diseases such as rheumatoid arthritis. [0003] At present, paclitaxel is mainly extracted and isolated ...

Claims

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Application Information

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IPC IPC(8): A01H4/00
Inventor 杜亚填
Owner JISHOU UNIVERSITY
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