Evaluación del cultivo in vitro de variedades nacionales de lúpulo (Humulus lupulus) como una alternativa a la propagación agámica tradicional

Abstract

The hop (Humulus lupulus L.) is a plant of the Cannabaceae family that is characterised by being a climber, with a tall growth. The optimum geographical distribution in both hemispheres for its growth is between latitudes 35° and 55°, a region in which the city of Viedma, in the province of Río Negro, is located. Since the attraction of the crop lies in the production of cones (female inflorescences), which are mainly used as flavouring and aromatizing in beer production, the industry concentrates its demand on the female plants, which excludes the possibility of propagation via seeds. As a result, hops are almost exclusively multiplied asexually, through rhizomes or nodal cuttings, which represents a disadvantage for local producers in terms of material quality and production costs, because these practices, sustained over time, favour the spread and accumulation of viral and fungal infections. On the other hand, at present, the cultivation of national varieties has not only been relegated, but also, together with imported varieties, they don’t fulfill demands and needs of the national market. In this context, the present project aimed to evaluate the responses of three national varieties of hops, Mapuche, Nahuel and Traful to in vitro tissue culture, in order to provide the first knowledge bases on their propagation through plant biotechnology, and to meet the current demands of the brewing sector. Thus, the responses to PTC of two types of explants were evaluated: meristems and nodal sections. An efficient methodology for disinfection of plant material was established which allowed the cultivation of meristems in Murashige and Skoog (MS) nutrient medium, at half the original concentration, added with different combinations and concentrations of growth regulators. Although cytokinin-added (BAP 0.88 μM) media were used to induce multiple shoot formation, this morphogenic response was not obtained. However, this medium allowed the differentiation of meristems into shoots, which then elongated when transferred to medium with gibberellins (AG3 2.89 μM). Additionally, rooting of these shoots was achieved in two culture stages: first with IBA (2.46 μM) and BAP (0.88 μM), and then with IBA (9.84 μM) and 2,4-D (0.45 μM). However, auxin media were not essential for root formation, as some rhizogenesis events were also recorded on auxin-free media. Also, in the meristem culture, it was observed that, as incubation time increased, and with it the exposure of the tissues to different growth regulators, callus formation increased, as well as the events of indirect organogenesis. On the other hand, although it was not possible to adjust an efficient disinfection protocol for the nodal sections, between 5 and 20% of explants were obtained free of contamination, which allowed us to advance with tissue culture, and to have sterile material, which was then successfully multiplied. Thus,in the cultivation of microstakes from in vitro material, between 30 and 70% more nodal sections rooted in medium free of growth regulators were observed than in auxin medium, which depended on the variety. The presence of auxins in the culture medium promoted callus formation; however, in contrast to the response observed in the rooting of shoots derived from differentiated meristems, direct organogenesis prevailed in this case. Additionally, synthetic seeds were made from the encapsulation of meristems, and the influence of different storage times at 4°C was evaluated prior to their cultivation under controlled light and temperature conditions (photoperiod of 16 h light/ 8 h dark and 21 ± 2°C). The culture medium used for conversion was MS at half the original concentration, supplemented with BAP (0.88 μM) and AG3 (2.89 μM). Although the three hop varieties responded favourably to this biotechnique, without having obtained callus formation, Mapuche was the one that registered the highest conversion percentages, and some rooting events, which made it possible to obtain complete plants. On the other hand, the process of acclimatisation and rustication of the vitroplants obtained from the different trials carried out during the research was initiated, achieving 100% survival. In conclusion, this work constitutes the first report on the establishment of in vitro cultivation of the Argentinean hop varieties Mapuche, Nahuel and Traful. It was also possible to establish a protocol for obtaining synthetic seeds by encapsulation of meristems, which allowed progress to be made towards the establishment of new methodologies for agamic propagation and conservation of these varieties. However, new trials should be designed to optimise the disinfection of explants, especially for nodal sections from the field, and to increase the conversion and rooting percentages of seedlings obtained from synthetic seeds. Keywords: plant tissue culture (PTC), organogenesis, synthetic seeds, growth regulators.