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Title:Krioprezervacija rebrinčevolistne hladnikovke (Hladnikia pastinacifolia Rchb.) z optimizacijo inkapsulacijske dehidracije in vitrifikacije
Authors:Ciringer, Terezija (Author)
Ambrožič-Dolinšek, Jana (Mentor) More about this mentor... New window
Files:.pdf EDOK_Ciringer_Terezija_2016.pdf (2,68 MB)
MD5: E03DBA11F587FEBDA21A88BE444DD7D4
 
Language:Slovenian
Work type:Dissertation (m)
Typology:2.08 - Doctoral Dissertation
Organization:FNM - Faculty of Natural Sciences and Mathematics
Abstract:Hladnikia pastinacifolia Rchb. je monotipična endemična vrsta Apiaceae z zelo ozkim območjem razširjenosti, ki se pojavljajo samo v Sloveniji. Shranjevanje z zamrzovanjem (krioprezervacija) je ena od dejavnosti, s katero bi lahko bistveno prispevali k ohranjanju te redke in potencialno ogrožene vrste. Namen naše disertacije je razviti krioprezervacijske postopke za trajno hrambo na ultra nizkih temperaturah (-196 C) v tekočem dušiku in prevetriti ali ta postopek vpliva na mrfologijo in gensko stabilnost te vrste. Razvili smo dva krioprezervacijska postopka: inkapsulacijsko dehidracijo (ID) in inkapsulacijsko vitrifikacijo (IV), optimizirali posamezne faze krioprezervacije in ju preizkusili na več različnih genotipih. Raziskali smo vpliv osmoprotektantov, v predkultivaciji, inkapsulaciji -polimerizaciji, osmoprotekciji, dehidraciji, če je šlo za ID ali vitrifikaciji, če je šlo za IV, na sposobnost preživetja in ponovne rasti po odmrzovanju. Pri vseh testiranih linijah so poganjki najbolje preživeli in se regenerirali po predkultivaciji čez noč v tekočem MS gojišču s 0,4 M saharozo; inkapsulaciji v MS s 3 % Na-alginata z 0,4 M saharozo in 1 M glicerolom in polimerizaciji v MS z 0,4 M saharozo, osmoproteciji z 0,4 M saharozo in 1 M glicerolom pri ID in z 0,4 M saharozo in 2 M glicerolom pri IV, po 150 minutah dehidracije v eksikatorju (ID) ali 85 minutah dehidracije (vitrifikacije) s PVS2 (IV). Po ID je preživelo 44 % in se regeneriralo 33 % vcepkov. Po postopkih IV je preživelo 65 % in se regeneriralo 52 % vcepkov. Nadalnji razvoj in morfologijo poganjkov smo spremljali še eno leto po krioprezervaciji. Zamrznjeni poganjki so v nadalnjem razvoju morfološko podobni nezamrznjenim; oboji podobno regeneriajo poganjke in korenine. Po enem letu smo na in vitro vzgojenem rastlinskem materialu, ki je izviral iz krioprezerviranih poganjkov po postopkih ID in IV in na ne krioprezerviranih poganjkih, ocenili tudi gensko stabilnost z RAPD tehniko. Med nezamrznjenimi in zamrznjenimi poganjki H. pastinacifolia je bila z RAPD označevalci zaznana zelo nizka stopnja variabilnosti. Po analizi AMOVA je večina krioprezerviranih rastlin gensko identična tistim, ki niso bile zamrznjene v TD. Obe metodi sta primerni za dolgoročno hrambo (krioprezervacijo) genskih viriov H. pastinacifolia pri ultra niskih temperaturah (-196).
Keywords:Slovenija, endemična rastlinska vrsta, Hladnikia pastinacifolia Rchb., Apiaceae, kobulnice, tkivna kultura, mikropropagacija, krioprezervacija, inkapsulacijska dehidracija, vitrifikacija, inkapsulacijska vitrifikacija, genska stabilnost, RAPD
Year of publishing:2016
Publisher:T. Ciringer]
Source:[Maribor
UDC:57.086.13/.14:582.795(497.4)(043.3)
COBISS_ID:22390024 New window
NUK URN:URN:SI:UM:DK:LPQU5GRE
Views:2282
Downloads:148
Metadata:XML RDF-CHPDL DC-XML DC-RDF
Categories:FNM
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Secondary language

Language:English
Title:Optimizing cryopreservation of (Hladnikia pastinacifolia Rchb.) by encapsulation-dehydration and vitrification
Abstract: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).
Keywords:Slovenia, endemic plant species, Hladnikia pastinacifolia, Apiaceae, micropropagation, cryopreservation, encapsulation dehydration, encapsulation vitrification, tissue culture, genetic stability, RAPD


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