Krioprezervacija rebrinčevolistne hladnikovke (Hladnikia pastinacifolia Rchb.) z optimizacijo inkapsulacijske dehidracije in vitrifikacije : doktorska disertacija

slovenian

doktorska dela

2016

Univerza v Mariboru, Fakulteta za naravoslovje in matematiko

Hladnikia pastinacifolia Rchb. is a monotypic endemic Apiaceae with an extremely narrow distribution area, occurring only in Slovenia. Cryopreservation could be one of several conservation activities which would help conserve this rare and potentially endangered species. The aim of this work was to successfully cryopreserve Hladnikia pastinacifolia Rchb. (Apiaceae), using encapsulation-dehydration (ED) and encapsulation-vitrification (EV). The effect of osmoprotectants in encapsulation/polymerization and osmoprotection, dehydration or vitrification on viability and regrowth were investigated. The morphology, the regeneration capacity and genetic stability of in vitro plant material originating from cryopreserved and non-cryopreserved shoots were evaluated one year after treatment. RAPD markers were used to estimate genetic variation. For all tested lines, the highest rates of shoot regeneration and multiplication were obtained after preculture overnight in 0.4 M sucrose, encapsulation/polymerized with 3 % Na-alginate in MS with 0.4 M sucrose and 1 M glycerol following polymerization in MS with 0.4 M sucrose and osmoprotection with 0.4 M sucrose and 1 M glycerol for ED and with 0.4 M sucrose and 2 M glycerol for EV, ending with dehydration for 120 minutes in exsiccator (ED) vitrification for 85 minutes in PVS2 (EV). Under these conditions, 53 % regrowth was achieved after ED, and 65 % regrowth after EV. Both methods could be applicable for the long-term cryopreservation of H. pastinacifolia germplasm. The cryopreserved shoots were morphologically similar to non-cryopreserved shoots, with a similar regeneration capacity in both shoots and roots. Between the cryopreserved and non-cryopreserved shoots of H. pastinacifolia, only a low level of RAPD variability was exhibited. After analysis by AMOVA, most cryopreserved plants proved genetically identical to those that were frozen in liquid nitrogen (LN). Both, ED and EV methods are suitable for long-term storage of genetic material from Hladnikia pastinacifolia Rchb.freeze at ultra-low temperatures (-196).

08.08.2023