Comparative study of different surface sterilization treatments and optimal month for establishment of aseptic cultures of raspberry cultivars

Abstract

Removing contaminants from plant material with minimal damage to plant cells using different chemical solutions represents very important and one of the most critical steps in establishing plant tissue culture in vitro. Our study deals with the optimisation of the protocol for successful surface sterilization of initial explants in two raspberry cultivars, ‘Meeker’ and ‘Willamette’ in two experimental years. The main objective during the first experimental year was to examine the protocols for surface sterilization which included different combinations of disinfectants (mercuric chloride or commercial bleach solution each in combination with 70% ethanol). In the following year, the impact of the two most effective sterilization treatments on the establishment of aseptic cultures over the four-month period (May, June, July, and August) was investigated in both raspberry cultivars. Aseptic cultures were established on Murashige and Skoog medium (MS) containing 2.0 mg/l N6-Benzyladenine (BA), 0.5 mg/l indole-3-butyric acid (IBA) and 0.1 mg/l gibberellic acid (GA3). The highest percentage of explants initiation in ‘Meekerʼ (79.3%) was achieved with surface sterilization in 70% ethanol (1 min) combined with 10% (v/v) commercial bleach solution (20 min), whereas sterilization in 70% ethanol (1 min) followed by HgCl2 (3 min) gave the best results (38.3%) in ‘Willametteʼ. The most optimal month for the establishment of aseptic culture was May for ‘Meeker’, while June was the most suitable month for the ‘Willametteʼ cultivar.

Eliminacija zaraze sa biljnog materijala uz minimalno oštećenje biljnih ćelija primenom različitih hemijskih rastvora je izuzetno važan i jedan od najkritičnijih koraka u postupku uspostavljanja in vitro kulture biljnog tkiva. U radu se ispituje optimizacija protokola uspešne površinske sterilizacije početnih eksplanata kod dve sorte maline, ‘Meekerʼ i ‘Willametteʼ, tokom dve eksperimentalne godine. U prvoj eksperimentalnoj godini, ispitivan je protokol za uspešnu površinsku sterilizaciju, uključujući varijacije dezinficijenata poput živinog hlora i varikine, svaki u kombinaciji sa 70% etanolom. Sledeće eksperimentalne godine, ispitivan je uticaj dva najefikasnija tretmana sterilizacije na uspešnost uspostavljanja aseptične kulture tokom četvoromesečnog perioda od maja do avgusta meseca. Aseptična kultura je uspostavljena na Murashige i Skoog podlozi (MS) koja sadrži 2.0 mg/l N6-benziladenina (BA), 0,5 mg/l indol-3-buterne kiseline (IBA) i 0,1 mg/l giberelinske kiseline (GA3). Najviši procenat inicijacije eksplantata kod sorte ‘Meekerʼ (79,3%) postignut je pri površinskoj sterilizaciji u 70% etanolu (1 min) u kombinacijii sa 10% (v/v) varikinom (20 min), dok je sterilizacija u 70% etanolu (1 min) praćena HgCl2 (3 min) dala najbolje rezultate (38,3%) kod sorte ‘Willametteʼ. Najoptimalniji mesec za uspostavljanje aseptične kulture bio je maj za sortu ‘Meekerʼ, dok je jun najpogodniji mesec za sortu ‘Willametteʼ.