Optimization of microclonal propagation and adaptation to ex vitro conditions for the hop (Humulus lupulus L.) variety Slov’ianka

Abstract

Aim. The development of a well-balanced microclonal propagation technology for the Slov’ianka hop variety is of importance for the industry since this is a widely used variety. Therefore, the aims of our research were 1) to investigate the influence of growth medium, disinfection procedures and means, hormones and other growth factors on the success of micropropagation and further multiplication of this variety and 2) to advice a standard protocol for the micropogation of the variety Slov’ianka. Methods. The research was conducted during 2023–2025 at the Department of Virology, Health Improvement and Reproduction of Fruit and Berry Crops of the Institute of Horticulture of the NAAS of Ukraine. Plant material for the experiments was selected in the Khmelnytskyi region and tested for the absence of pathogens in accordance with the EPPO PM4/016(2) standard: Certification scheme for hop. Various, culture media and variations of the standard MS culture medium, using different iron and carbon sources, auxins and cytokinins in early stages of micropropagation, use of perlite, coconut and peat and two biostimulants Helprost and Radifarm in later stages. Statistical processing of the experimental data was performed using ANOVA and correlation analysis was conducted in Excel. Results. For the disinfection the use of 70% ethanol for 30 seconds and a 9% sodium hypochlorite solution diluted to 0.25% for 20 minutes appeared to be the most effective. From five culture media an adapted Murashige-Skoog (MS) medium was found to be optimal for regeneration with minimum callus growth. To improve the overall condition of the plants, a dose of iron chelate FeNaEDTA (40 mg/l), and glucose (30 g/l) as a carbon source and furthermore benzylaminopurine (BAP) at a concentration of 0.1 mg/l the medium gave best results. Despite the concentrations of respectively 2.0 mg/l and 1.0 mg/l of thidiazuron and kinetin in the nutrient medium during the shoot proliferation stage, their effectiveness was non-significant and can be deleted. This was also true for gibberellic acid since the maximum concentration of 1.0 mg/l led to an increase in the distance between internodes, thereby reducing the propagation coefficient. During the induction stage of rhizogenesis in hop microshoots, the auxins indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), naphthalene acetic acid (NAA), were tested at concentrations of 0.5, 1.0, 1.5, and 2.0 mg/l, compared to a nutrient medium without growth regulators. Remarkably a 100% rooting of Slov’ianka variety in a hormone-free medium with high root system quality indicators — the mean number of roots was 7.9, with a mean length of 2.1. However, among the auxins under study, IBA was the most effective at concentrations of 0.5 and 1.0 mg/l, promoting rooting rates of 58% and 52%, respectively, whereas IAA and NAA resulted in rooting rates of less than 43%. IAA and NAA at 2.0 mg/l, were totally inhibitory for root formation. A high level of adaptation of rooted plants was achieved when using peat substrate «Domoflor» as a substrate — 98%; whereas the use of a two-component substrate (peat and perlite in a 3:1 ratio) slightly reduced the adaptation to 72%, and the use of perlite and coconut substrate alone, supplemented with MS macro- and micronutrients, the survival was only 27–30%. During the plant growth stage, Helprost fertilizer, containing amino acids, B vitamins, polysaccharides, and chelated microelements for root feeding after transplantation was most effective, which subsequently resulted in a 97% yield of standard plants. Conclusions. A balanced alternative protocol for the microclonal propagation of Slov’ianka hop variety has been developed, which includes efficient disinfection with ethanol and sodiumhypoclorite, optimized composition of the culture medium, methods for adapting microshoots to ex vitro conditions, and the growing of seedlings in plastic containers until they reach standard sizes.