In vitro razmnožavanje virusima zaraženih genotipova vinove loze u postupku ozdravljivanja krioterapijom

master's thesis

• Author: Iva Šikuten
• Mentor: Jasminka Karoglan-Kontić (mentor)
• Committee member: Jasminka Karoglan-Kontić (član povjerenstva)
• Committee member: Edi Maletić (član povjerenstva)
• Committee member: Darko Preiner (član povjerenstva)
• Committee member: Zvjezdana Marković (član povjerenstva)
• Granter: University of Zagreb; Faculty of Agriculture; (Department of Viticulture and Enology); Zagreb
• Defense date and country: 2016-04-15, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Agronomy; Viticulture and Enology

Abstract

Vinova loza jedna je od poljoprivrednih kutura na kojoj je utvrđena zaraza velikim brojem virusa. Stoga je za očuvanje unutarsortne varijabilnosti i proizvodnju sadnog materijala nužno njihovo ozdravljivanje. Za ozdravljivanje se najčešće primjenjuje kultura meristema in vitro (sa ili bez termoterapije), a u novije vrijeme se razvijaju i protokoli za krioterapiju, gdje se biljni materijal izlaže niskim temperaturama tekućeg dušika. Za provođenje ovih metoda potrebno je biljni materijal uvesti u kulturu in vitro, a zatim ga multiplicirati kako bi se dobila dovoljna količina.
Ciljevi ovoga rada bili su utvrditi razlike među sortama korištenih u istraživanju (Cabernet Franc, Cabernet Sauvignon, Grenache, Merlot, Babić) zaraženih različitim virusima u početnoj kulturi in vitro, zatim odabrati sortu s najboljim rezultatima u početnoj kulturi in vitro za provedbu protokola krioprezervacije, te usporediti uspješnost preživljavanja i regeneracije odabrane sorte sa sortom Portan, nakon provedenog protokola krioprezervacije.
Vrlo loše rezultate u kuluri in vitro su pokazale sorte Cabernet Sauvignon i Grenache, te su iste eliminirane iz daljnjeg istraživanja. Sorte Cabernet Franc i Merlot su nakon uspostave početne kulture in vitro bile multiplicirane i subkultivirane na različite hranjive medije. Mediji su odabirani prema vizualnoj procjeni biljaka, te su u njih dodavani fitohormoni s određenim ciljevima- IBA za poticanje zakorijenjavanja ili kombinacija auksina i citokinina (IBA+NAA) za poticanje biljaka u rastu i razvoju.
Sorta Babić je pokazala najbolje rezultate početne kulture in vitro, te najbolji rast biljaka in vitro, te je stoga odabrana kao sorta na kojoj će se provesti protokol krioprezervacije. Uz inficirani genotip koji je uvođen u kulturu in vitro, za provedbu protokola krioprezervacije još su odabrane biljke zdravog genotipa sorte Babić i kontrolna sorta Portan, koje su dobivene iz ranijih istraživanja. Postotak preživljavanja je bio podjednak kod nesmrzavanih ekpslantata, dok su smrznuti eksplantati dali znatno lošije rezultate. Regeneracija je dala loše rezultate- pokazalo se kako nekrotizirano biljno tkivo koči rast i razvoj eksplantata, te su potrena daljnja istraživanja na ovom području.

Abstract (english)

The grapevine is one of the horticultural crops on which we can find a large number of viruses. To preserve genetic variability among Vitis genus and to produce healthy plant material it is important to eliminate the viruses. The most common technique to eliminate viruses is in vitro meristem culture (with or without thermotherapy). Lately there are protocols for crypreservation where the plant material is exposed to extra low temperatures of liquid nitrogen. To conduct these methods it is necessary to establish in vitro culture and use multiplication to gain enough plant meterial.
The aims of these study were to determine differences between cultivars used in experiments (Cabernet Franc, Cabernet Sauvignon, Grenache, Merlot, Babić) infected with different viruses in the initial in vitro culture and then choose the cultivar with the best results in initial in vitro culture to carry out the cryopreservation protocol. After the crypreservation compare the success of survival and regrowth of chosen cultivar with cv. Portan.
Cultivars Cabernet Sauvignon i Grenache showed very bad results of in vitro culture and were eliminated from further research. After inital in vitro culture, cultivars Cabernet Franc and Merlot were multiplied and subculutred on different mediums. The mediums were selected by visual evaluation of plantlets and accordingly the fitohormones were added with certain aim- IBA for rooting or combination of auksins and cytokinins (IBA+NAA) to stimulate growth.
The best results of in vitro culture and plantlets growth gave cultivar Babić, therefore it was selected for crypreservation protocol. Besides the infected plantlets, in the crypreservation protocol were included and healthy plantlets of Babić along with plantlets of Portan, wich was control, and were obtained from earlier experiments. The percentage of survival of explants that were not exposed to liquid nitrogen was similar, while explants exposed to nitrogen gave bad results- it was shown that necrotized tissue prevents the growth of explants, therefore the further research is necessary.

• Keywords: vinova loza; zaraza virusima; metode ozdravljivanja; autohtona sorta; krioprezervacija
• Keywords (english): grapevine; virus infection; elimination methods; cryopreservation
• Language: croatian
• URN:NBN: urn:nbn:hr:204:865053
• Study programme: Title: Horticulture; Study programme type: university; Study level: graduate; Academic / professional title: magistar/ magistra inženjer/inženjerka hortikulture (magistar/ magistra inženjer/inženjerka hortikulture)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Repository: Repository Faculty of Agriculture University of Zagreb
• Created on: 2016-05-11 13:06:45