Biocollection of pharmaceutical parasitic strains of Claviceps purpurea – base for selection of new lines producing ergoalkaloids in vitro

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Abstract

Relevance. Ergot Claviceps purpurea is a very important source of pharmaceutical raw materials (ergoalkaloids).

Aim of the study – for biocollection maintenance two parasitic strains of Claviceps purpurea (Fries) Tulasne were reproduced by in planta cultivation on winter rye: ergotoxin A-6, ergotoxin VKM-F-2450-D.

Material and methods. Spectrophotometric determination of total content of indole alkaloids (with the van Urk reagent) and qualitative-quantitative selective determination of the alkaloid composition by thin-layer chromatography (TLC) with densitometric scanning of chromatograms were used.

Results. Maternal sclerotia contained from 0.6 to 1 g of alkaloids per 100 g of sample for VKM-F-2450-D, from 0.5 to 0.7 g per 100 g of sample for A-6. VKM-F-2450-D and A-6 contained from 30% to 40% of the sum of α-ergocryptine and ergocornine and approximately 20% of β-ergocryptine. New generation sclerotia contained from 0.31 to 0.76 g of alkaloids per 100 g of sample for VKM-F-2450-D, from 0.21 to 0.62 g per 100 g of sample for A-6. VKM-F-2450-D and A-6 contained from 40% to 62% and from 21% to 66% of the sum of α-ergocryptine and ergocornine, from 13% to 22% and from 13% to 36% of β-ergocryptine respectively.

Conclusion. Two stable lines (ergotoxin A-6-S, ergotoxin ВKM-F-2450-D-S) with in vitro biosynthesis of ergoalkaloids with purple pigmentations features were detected, isolated and subcultivated by morphological selection in axenic culture. Accumulation and retention of Claviceps genetic resources is very actual: biocollection of parasitic strains is a good reserve for selection of new saprophytic lines.

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About the authors

A. A. Volnin

FSBSI All-Russian Scientific Research Institute of Medicinal and Aromatic Plants

Author for correspondence.
Email: volnin.a@mail.ru

Ph.D. (Biol.), Leading Research Scientist, Biotechnology Laboratory

Russian Federation, Moscow

N. S. Tsybulko

FSBSI All-Russian Scientific Research Institute of Medicinal and Aromatic Plants

Email: volnin.a@mail.ru

Ph.D. (Pharm.), Engineer, Biotechnology Laboratory

Russian Federation, Moscow

P. S. Savin

FSBSI All-Russian Scientific Research Institute of Medicinal and Aromatic Plants

Email: volnin.a@mail.ru

Ph.D. (Biol.), Leading Research Scientist, Biotechnology Laboratory

Russian Federation, Moscow

S. B. Myasnikova

FSBSI All-Russian Scientific Research Institute of Medicinal and Aromatic Plants

Email: volnin.a@mail.ru

Research Scientist, Biotechnology Laboratory

Russian Federation, Moscow

R. I. Bobyleva

Email: volnin.a@mail.ru
Russian Federation

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2. Fig. 1. Scheme for obtaining infectious conidial material by the surface method (on rye grain): 1 - mature sclerotium; 2 - superelite infectious material in test tubes on slanted agar medium; 3 - cloned infectious material in test tubes on slanted agar medium; 4 - infectious material in flasks-mattresses on rye grain

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3. Fig. 2. Parasitic culture of ergot: 1 - conidial infectious material, conidiospores are shown by an arrow; 2 - infectious material on boiled rye grain; 3 - drops of "honey dew" on rye inflorescences; 4 - mature sclerotia on rye inflorescences

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4. Fig. 3. Results of qualitative TLC analysis of the mycelium of two lines producing ergoalkaloids on the 30th day of cultivation and sclerotia of the parasitic culture: on the left, in vitro culture; on the right, sclerotia of parasitic cultures; A - ergotoxin A-6; B – ergotoxin VKM-F-2450-D; B - standard sample; 1 - the sum of ergocornine and α-ergocryptine (in the case of in vitro cultivation, presumably); 2 - β-ergocriptine (in the case of in vitro cultivation, presumably)

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