Cryopreservation of Digitalis obscura Selected Genotypes by Encapsulation-Dehydration

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

Key words

Digitalis obscura - Scrophulariaceae - cryopreservation - encapsulation-dehydration - RAPDs - shoot tips

References

• 1 Nebauer S G, Del Castillo-Agudo L, Segura J. Cardenolide variation within and among natural populations of Digitalis obscura .  Journal Plant Physiology. 1999;  154 426-30
• 2 Nebauer S G, Del Castillo-Agudo L, Segura J. RAPD variation within and among natural populations of outcrossing willow-leaved foxglove (Digitalis obscura L.)  Theoretical and Applied Genetics. 1999;  98 985-94
• 3 Vela S, Gavidia I, Pérez-Bermúdez P, Segura J. Micropropagation of juvenile and adult Digitalis obscura and cardenolide content of clonally propagated plants.  In Vitro Cellular and Developmental Biology. 1991;  27P 143-6
• 4 Diettrich B, Haack U, Luckner M. Cryopreservation of Digitalis lanata cells grown in vitro. Precultivation and recultivation.  Journal Plant Physiology. 1986;  126 63-73
• 5 Diettrich B, Wolf T, Bormann A, Popov A S, Butenko R G, Luckner M. Cryopreservation of Digitalis lanata shoot tips.  Planta Medica. 1987;  53 359-63
• 6 Goldner E M, Seitz U, Reinhard E. Cryopreservation of Digitalis lanata Ehrh. cell cultures. Preculture and freeze tolerance.  Plant Cell Tissue and Organ Culture. 1991;  24 19-24
• 7 Moran M, Cacho M, Fernández-Tárrago J. A protocol for the cryopreservation of Digitalis thapsi L. cell cultures.  Cryo Letters. 1999;  20 193-8
• 8 Zhao Y, Wu Y, Engelmann F, Zhou M, Zhang D, Chen S. Cryopreservation of apple shoot tips by encapsulation-dehydration: effect of preculture, dehydration and freezing procedure on shoot regeneration.  Cryo Letters. 1999;  20 103-8
• 9 Paulet F, Engelmann F, Glaszmann J C. Cryopreservation of apices of in vitro plantlets of sugarcane (Saccharum sp. hybrids) using encapsulation/dehydration.  Plant Cell Reports. 1993;  12 525-9
• 10 Vandebussche B, De Proft M P. Cryopreservation of in vitro sugar beet shoot tips using the encapsulation-dehydration technique: development of a basic protocol.  Cryo Letters. 1996;  17 137-40
• 11 Martínez D, Tamés R S, Revilla M A. Cryopreservation of in vitro-grown shoot tips of hop (Humulus lupulus L.) using encapsulation dehydration.  Plant Cell Reports. 1999;  19 59-63
• 12 Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture.  Physiologia Plantarum. 1962;  15 473-97
• 13 Tukey J W. Some selected quick and easy methods for statistical analysis.  Transactions of the New York Academy of Science Series II. 1953;  16 88-97
• 14 Del Castillo-Agudo L, Gavidia I, Pérez-Bermúdez P, Segura J. PEG precipitation, a required step for PCR amplification of DNA of wild-growing plants of Digitalis obscura L.  Biotechniques. 1995;  18 766-8
• 15 Panis B, Totté N, Van Nimmen K, Withers L S, Swennen R. Cryopreservation of banana (Musa spp.) meristem cultures after preculture on sucrose.  Plant Science. 1996;  121 95-106
• 16 Gavidia I, Del Castillo Agudo L, Pérez-Bermúdez P. Selection and long-term cultures of high-yielding Digitalis obscura plants: RAPD markers for analysis of genetic stability.  Plant Science. 1996;  121 197-205
• 17 Nebauer S G, Del Castillo-Agudo L, Segura J. An assessment of genetic relationships within the genus Digitalis based on PCR-generated RAPD markers.  Theoretical and Applied Genetics. 2000;  100 1209-16
• 18 Goto S, Thakur R C, Ishii K. Determination of genetic stability in long-term micropropagated shoots of Pinus thumbergii Parl. using RAPD markers.  Plant Cell Reports. 1998;  18 193-7
• 19 De Verno L L, Park Y S, Bonga J M, Barrett J D. Somaclonal variation in cryopreserved embryogenic clones of white spruce (Picea glauca (Moench) Voss.)  Plant Cell Reports. 1999;  18 948-53
• 20 Fourré J L, Berger P, Niquet L, André P. Somatic embryogenesis and somaclonal variation in Norway spruce: morphogenetic, cytogenetic and molecular approaches.  Theoretical and Applied Genetics. 1997;  94 159-69