Optimization of protocol for virus elimination from grapevine by meristem tip culture

Republika Hrvatska bogata je autohtonim sortama vinove loze. Međutim, samo je mali broj sorata u uzgoju. Prepreku revitalizaciji ovih sorata predstavlja visoka stopa zaraze virusima, odnosno, ne postojanje zdravih matičnih biljaka za proizvodnju certificiranog sadnog materijala. Sorte odabrane u ovom istraživanju bile su autohtone hrvatske sorte 'Bilan', 'Debit', 'Glavinuša', 'Ljutun', 'Mladenka', 'Plavac mali sivi' i 'Žumić', a sav biljni materijal prikupljen je iz Nacionalne kolekcije hrvatskih autohtonih sorata na pokušalištu “Jazbina“ Agronomskog fakulteta Sveučilišta u Zagrebu. Cil ovog rada bio je usporediti četiri termina uzorkovanja biljnog materijala (u zimskom mirovanju te tri uzorkovanja tijekom vegetacije), podrijetlo meristema (vegetacijski vršak ili pup) te veličinu vegetacijskog vrška (1 mm ili > 1 mm) kako bi se odredio „model uzorkovanja“ za daljnja istraživanja na eliminaciji virusa iz autohtonih hrvatskih sorata. Virološka analiza pokazala je kako je GLRaV-3 najzastupljeniji virus među testiranim uzorcima, a nije utvrđeno da je infekcija jednim ili više virusa utjecala na rast vegetacijskih vršaka i pupova u kulturi tkiva. Biljke razvijene iz uzoraka prikupljenih u zimskom mirovanju slabo su se razvijale te nije bilo moguće postići očekivanu populaciju biljaka. Došlo je i do snažne pojave kalusa te su, nakon provedenih subkultivacija, u konačnici svi uzorci eliminirani iz daljnjeg istraživanja. Prilikom uzorkovanja početkom vegetacije količina biljnog materijala bila je ograničena. Rezultati dobiveni u ovom terminu uzorkovanja nisu se bitno razlikovali od onih u zimskom mirovanju te iako su dobivene biljke za aklimatizaciju, one nisu preživjele ovaj korak. Ni iz vegetacijskih vršaka uzorkovanih prije cvatnje biljke u kulturi meristema nisu se uspješno razvijale. U uzorkovanju nakon cvatnje meristemi izolirani iz vegetacijskih vršaka podjeljeni su u dvije kategorije, oni veličine 1 mm i veći od 1 mm. U ovom uzorkovanju uspješno su dobivene biljke za aklimatizaciju koje su rasle i nakon 7 mjeseci, a najviše uspješno razvijenih biljaka dobiveno je od genotipa 'Debit' 2. Na osnovu usporedbe uspjeha početne kulture, regeneracije biljaka i postupka aklimatizacije biljaka utvrđeno je da je najpovoljniji termin za uzorkovanje biljnog materijala za uspostavu kulture meristema onaj nakon cvatnje te se on može preporučiti za daljnja istraživanja.Croatia is rich in indigenous grapevine varieties. However, only a small number of them is grown commercially. The obstacle to the revitalization of these varieties represents a high rate of virus infection, ie, the absence of healthy mother plants for the production of certified planting material. The varieties selected in this study were the indigenous Croatian varieties 'Bilan', 'Debit', 'Glavinuša', 'Ljutun', 'Mladenka', 'Plavac mali sivi' and 'Žumić'. All of the plant material was collected from the National Collection of Croatian indigenous varieties in the “Jazbina“ experimental station of the Faculty of Agriculture, University of Zagreb. The aim of this paper was to compare four plant material sampling terms (during the winter period and three samplings during vegetation), the origin of the meristem (shoot tip or bud) and the shoot tip size (1 mm or > 1 mm) in order to determine the „sampling model“ for further research on the elimination of viruses from indigenous Croatian grapevine varieties. Virological analysis showed that GLRaV-3 was the most common virus among the tested samples, and it has not been established that the infection by one or more viruses had affected the growth of shoot tips and buds in the tissue culture. Plants developed from samples collected during the winter period developed poorly and it was not possible to obtain the expected plant population. There was also a strong emergence of calf and, after subcultures were performed, ultimately all samples were eliminated from further research. During sampling at the beginning of vegetation, the amount of plant material was limited. Results obtained in this sampling term did not differ significantly from those in the winter period, and although plants for acclimatization were obtained, they did not survive this step. Likewise, plants in the meristem culture did not successfully develop from shoot tips sampled in the sampling term before flowering. In the sampling term after flowering, the meristems isolated from shoot tips were divided into two categories, those 1 mm in size and those larger than 1 mm. In this sampling term, plants for acclimatization were successfully obtained, which grew even after 7 months, and the most successfully developed plants were derived from the genotype 'Debit' 2. Based on the comparison of the success of the initial culture, plant regeneration and plant acclimatization, it was found that the most favorable term for plant material sampling to establish the meristem culture is the one after flowering and this sampling term can be recommended for further research