Cryopreservation of an endangered Hladnikia pastinacifolia Rchb. by shoot tip encapsulation-dehydration and encapsulation-vitrification
The objective of the present study was the cryopreservation of monotypic endemic Hladnikia pastinacifolia Rchb. shoot tips from an in vitro culture, via encapsulation-dehydration (ED) or encapsulation-vitrification (EV). For all tested genotypes, the highest rates of shoot regrowth and multiplication were obtained after overnight preculture in 0.4 M sucrose, encapsulation in Murashige and Skoog (MS) medium with 0.4 M sucrose and 1 M glycerol, followed by polymerization in 3% (w/v) Na-alginate in MS with 0.4 M sucrose. Optimal osmoprotection was achieved for ED with 0.4 M sucrose plus 1 M glycerol and for EV with 0.4 M sucrose plus 2 M glycerol. The best dehydration time for ED was 150 min in a desiccation chamber with silica gel, and the best vitrification time for EV was 85 min in plant vitrification solution 2 (PVS2). For ED, dehydration for 150 min resulted in explant water content of 22%. When the encapsulation method was combined with ED, 53% regrowth was achieved, and when it was combined with EV, 64% regrowth was achieved. Both methods could become applicable for the long-term cryopreservation of H. pastinacifolia germplasm, although EV was faster and resulted in better final regrowth success. Genetic stability analysis of cryopreserved plant samples was carried out for two genotypes, using random amplified polymorphic DNA (RAPD) markers to compare the two different cryopreservation protocols. Significant genetic differences between the genotypes were detected and a low level of genomic variation was observed.
KeywordsShoot tip cryopreservation Hladnikia pastinacifolia Rchb. Apiaceae Encapsulation-dehydration Encapsulation-vitrification
The Slovene Ministry of Higher Education, Science, and Technology supported this research within the program “Research to Ensure Food Safety and Health” with the Grant No. P1-0164, led by D. Škorjanc.
- Čušin B (2004) Hladnikia pastinacifolia Rchb. – rebrinčevolistna hladnikija, hladnikovka. In: Čušin B, Babij V, Bačič T, Dakskobler I, Frajman B, Jogan N, Kaligarič M, Praprotnik N, Seliškar A, Skoberne P, Surina B, Škornik S, Vreš B. (eds) Natura 2000 v Sloveniji, Rastline. Založba ZRC, ZRC SAZU, Ljubljana (ISBN 961-6500-66-X), pp 107–113 (Slovenian language)Google Scholar
- Hirai D, Sakai A (2000) Cryopreservation techniques. Cryopreservation of in vitro grown meristems of potato (Solanum tuberosum L.) by encapsulation–vitrification. JIRCAS Int Agric Ser 8:205–211Google Scholar
- Panis B, Lambardi M (2005) Status of cryopreservation technologies in plants (crops and forest trees). The role of biotechnology, Villa Gualino, Turin, Italy – 5-7 March pp.43–54Google Scholar
- Rohlf FJ (1992) NTSYS-PC: numerical taxonomy and multivariate analysis system. Exeter Software, New YorkGoogle Scholar
- Šajna N, Kavar T, Šuštar-Vozlič J, Kaligarič M (2012) Population genetics of the narrow endemic Hladnikia pastinacifolia Rchb. (Apiaceae) indicates survival in situ during the Pleistocene. Acta Biol Cracov Ser Bot 54:1–13Google Scholar
- Shatnawi M, Engelmann F, Frattarelli A, Damiano C, Withers LA (1999) Cryopreservation of apices from in vitro plantlets of almond (Prunus dulcis). CryoLetters 20:13–20Google Scholar