Planta Med 2001; 67(9): 833-838
DOI: 10.1055/s-2001-18861
Original Paper
Biochemistry, Physiology, in vitro-cultures
© Georg Thieme Verlag Stuttgart · New York

Cryopreservation of Digitalis obscura Selected Genotypes by Encapsulation-Dehydration

Ester Sales1 , Sergio G. Nebauer2 , Isabel Arrillaga1 , Juan Segura1
  • 1 Departamento de Biología Vegetal, Facultad de Farmacia, Universidad de Valencia, Burjassot (Valencia), Spain
  • 2 IVIA, Moncada (Valencia), Spain
Further Information

Publication History

December 8, 2000

April 28, 2001

Publication Date:
06 December 2001 (online)

Abstract

Shoot-tips from several genotypes of the cardenolide-producing perennial shrub Digitalis obscura L. were successfully cryopreserved using the encapsulation-dehydration technique. Precultivation on MS medium containing 0.5 M sucrose, followed by 2.5 h dehydration (final weight 30 %) induced shoot regrowth in 42 % of cryopreserved shoot-tips. Cold-hardening of the in vitro cultures before sucrose treatment dramatically increased shoot recovery up to 86 %. The optimized cryopreservation protocol was then employed using different shoot cultures from five D. obscura genotypes. Responses to cryopreservation depended mainly on the genotype, best results being obtained when shoot tips from HU3 and LL11 were used. Prolonged subcultures reduced proliferation rates in both control and cryopreserved HU3 shoot-tips, whereas long-term storage in LN did not affect the shoot recovery rate of the genotype. RAPD markers were employed to evaluate possible somaclonal variation occurring in shoots regenerated through successive subcultures and after cryopreservation. The band patterns revealed differences between the original parent plant and the shoots grown in vitro, especially after a prolonged subculture (84.9 % of matches for HU3 after 16 subcultures vs. 93.4 % for AY3 after 2 subcultures). Nevertheless, match percentages were higher (98.6 % to 99.5 %) when band patterns from subcultured shoots were compared to those obtained from their respective control or frozen progenies indicating that cryopreservation ensure genetic stability of selected Digitalis obscura genotypes.

Abbreviations

MS:Murashige and Skoog medium [12]

LN:liquid nitrogen

BA:benzyladenine

ABA:abscisic acid

NAA:naphthaleneacetic acid

DMSO:dimethyl sulfoxide

RAPD:random amplified polymorphic DNA

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Prof. Juan Segura

Depto. Biología Vegetal

Facultad de Farmacia

Avda. Vicent A. Estellés s/n

46 100 Burjassot (Valencia)

Spain

Email: juan.segura@uv.es

Fax: ++34 963 864 926

Phone: Tel.: ++34 963 864 922