IN VITRO INTRODUCTION OF GRAPE VARIETIES IN DEMAND IN AGRICULTURE OF THE REPUBLIC OF CRIMEA

ABSTRACT

The paper considers the optimization of the conditions for the introduction of grape rootstockvarieties, which are in demand in agriculture of the Republic of Crimea, into in vitro culture. Investigation aimed to determine the effectiveness of using sterilants to obtain aseptic cultures of grape meristems of ‘Ruggeri 140’, ‘CO 4’ and ‘Kober 5BB’ varieties.

The results showed that the highest percentage of aseptic meristem in vitro (75.39 ± 3.62 %) was achieved by treating the starting materials with 15% hydrogen peroxide for 15 min. The solution of a neutral chlorine-containing preparation made it possible to obtain up to 69.33 ± 3.33 % of the aseptic culture of meristems, while 5 % hydrogen peroxide showed the lowest result (43.67 ± 2.18 %). During the experiment, it was found that meristems cultivated on a modified Murashige-Skoog medium supplemented with 30 g/l sucrose, 2.0 mg/l 6-BAP and 0.5 mg/l IBA increased significantly in size, forming shortened shoots.

The statistical processing of the data did not reveal significant differences in the size of the microshoots formed, depending on the sterilization option.

Keywords: microclonal propagation, grapes, aseptic culture, in vitro.

There is a shortage of own planting materials in the Republic of Crimea. The expansion of vineyard areas in the region requires a significant amount of high-quality planting materials for rootstock and technical grape cultivars, which is possible to obtain using in vitro culture.

Microclonal propagation is the most effective way for fast mass production of genetically identical grape planting materials without pathogenic infections [1]. The first stage of microclonal reproduction is the introduction of plants into culture in vitro, which plays a fundamental role for the further reproduction of healthy plant mericlones. The main goal of this stage is to achieve the maximum number of sterile and viable explants. The success of introducing plants to in vitro conditions depends on many factors, including the age and genotype of the mother plant, the type of explants used, the chemical group and time of exposure to the sterilizing agent, as well as the composition of the basic culture media, the type and concentrations of growth regulators used at the initial stage of microclonal propagation – all of the above conditions are selected individually for each genotype investigated [5].

Grape rootstocks are interspecific hybrids obtained by hybridization of such species as Vitis vinifera L., V. labrusca L., V. riparia Michx., V. amurensis Rupr., V. rupestris Scheele, V. berlandieri Planch. and V. lincecumii Buckley. When introduced into culture, it is necessary to develop individual protocols for sterilization, induction and development of meristems for each specific cultivar [2]. As the patent inquiry on the research topic in the scientific works, data are very scarce on the results of the most difficult methodological stage, over the past decades: introduction into in vitro culture, especially for complex interspecific rootstock hybrids such as ‘Berlandieri × Rupestris Ruggeri 140’, ‘Berlandieri × Riparia Kober 5BB’, ‘Berlandieri × Riparia CO 4’. In this regard, research updating aimed at optimizing the conditions for in vitro introduction of rootstock grape cultivars in demand in agriculture of the Republic of Crimea and the Southern Federal District for their further cultivation, in order to increase the efficiency of microclonal propagation and develop protocols for the mass production of healthy planting materials is necessary both from a theoretical and a practical point of view. The study aimed to optimize the conditions for obtaining a sterile culture of meristems at the stage of their introduction into in vitro culture, i.e. to determine the effectiveness of using sterilants to obtain an aseptic culture of meristems in vitro from hybrid rootstocks of three most popular grape cultivars: ‘Ruggeri 140’, ‘CO 4’, ‘Kober 5BB’.

The research was carried out in the Laboratory of Microclonal Reproduction of the Breeding and Seed-Growing Center of Fruit Crops of V.I. Vernadsky Crimean Federal University.

For in vitro culture, the material was obtained from private and state collection plantations: ‘Ryugzheri 140’ – from “LLC Invest+” (Vilino village); ‘SO 4’ –“Vinogradnaya Loza Estate” (Trudolyubovka village); and ‘Kober 5BB’ – from the collection site of cultivars of the Institute “Agrotechnological Academy” of V. I. Vernadsky Crimean Federal University. Introduction of materials (meristems from buds of a mature vine) into in vitro culture was carried out from September to November in 2024. In laboratory condition, vine apexes (2–3 cm long) were removed, and the vine shoot was cut into nodes (about 5 cm high) with 1–2 buds. To obtain an aseptic culture, three two-stage sterilization options were used, with: a neutral chlorine-containing preparation – a solution of sodium dichloroisocyanuronate (0.7–0.8 % active chlorine in solution) and hydrogen peroxide at concentrations of 5 % and 15 % (15 min). After, Nodes were washed three times with sterile distilled water (5 min), immersed for several seconds in a mixture of alcohols (70 % ethanol containing 60 % isoprapanol and 10 % butanol), and dried in the laminar flow cabinet Neoteric (Lamsystems, Russia) condition  until the alcohol solution signs completely disappear. Meristems with 1–2 leaves primordia (at least 90 pcs for each sterilization variant) were isolated binocular microscope MSP–2 (LOMO-Microsystems, Russia) at magnification 7–10x. 

The meristems were placed on a modified Murashige-Skoog medium (MS) [4], supplemented with 30 g/l sucrose, 2.0 mg/l 6-benzylaminopurine (6-BAP) and 0.5 mg/l indolyl-3-butyric acid (IBA) to induce multiple shoot formation [3]. The pH of the medium was adjusted to 5.8 ± 0.1 using 0.1 N NaOH or HCl before adding agar to a concentration of 0.7 % (Sigma-Aldrich, USA). The autoclaving of the medium was performed at a temperature of 121 °C and a pressure of 1.2 kg/cm² for 40 minutes in an autoclave VK-75-01 (TZMOI, Russia), followed by dispensing into sterile cultivation containers using a peristaltic pump DOSYWEL (Alliance Bio Expertise, France). After sterile filtration (using Merck Millipore syringe filters with a pore diameter of 0.22 μm), 6-benzylaminopurine (6-BAP) and indole-3-butyric acid (IBA) were added to the autoclaved medium. Explants were cultured in test tubes (150 × 24 mm) containing 10 ml of nutrient medium, which were closed with autoclaved polypropylene caps, as well as in glass jars (200 ml capacity) with ventilated polypropylene lids containing 30 ml of nutrient medium. The cultivation of explants was conducted at a temperature of 24 ± 1 °C under light conditions (16-hour photoperiod with illumination from 40-watt cool-white fluorescent lamps) with an intensity of 105–115 μmol PPFD/m²/s.

After initial assessment of the yield of aseptic meristems from the total number of explants (during the first month of cultivation after their introduction into in vitro conditions – to exclude the development of endophytic and other microflora in explants), the meristems were transplanted to MS media in various modifications (indicated below) to study the features of the morphogenesis directivity. Experiments were conducted in triplicate with an average sample of 30 meristems per replicate. The data obtained was statistically processed using the Microsoft Office (Exel) software package. The reliability of the difference between the values of the compared variants was determined by the Student's t-criterion: at p < 0.05 or less, the reliability of the differences between the values of the compared variants is considered reliable. Numerical results were presented as mean and standard error of the mean (M±SE).

The highest percentage of aseptic culture (75.39 ± 3.62 %) was obtained by treating the initial plant material with 15% hydrogen peroxide for 15 min. The yield of aseptic meristem culture when treated with a solution of sodium salt of dichloroisocyanuronic acid was averaged 69.33 ± 3.33 %, while the lowest result was found when treated with 5 % hydrogen peroxide – 43.67 ± 2.18 % aseptic ones of the total number of meristems introduced into in vitro culture.

Our observations show that on a medium containing 2.0 mg/l of 6−BAP and 0.5 mg/l of IBA during the first 30 days of cultivation (zero passage), the meristems increased significantly in size, forming shortened shoots in the shape of a cabbage head (probably indirect morphogenesis processes were underway, but without the formation of primary callus). Obtained shortened shoots were transferred on modifiedMS media (first passage) with 30 g/l sucrose, 2.0 mg/l 6−BAP (option 1), 2.0 mg/l 6−BAP and 0.3 mg/l IBA (option 2), without growth regulators as a control.

After first subcultivation, during month, 15–20% of explants on all investigated media formed microshoots, which later were transferred to media of the same composition.

In the course of the research, it was found that, depending on the sterilization option used for the initial plant materials, we noted subjective differences in estimating the average increase in meristems per passage. Thus, when using a neutral chlorine-containing sterilant (sodium dichloroisocyanuronate), the increase in part of the meristems and the formation of micro-shoots was more intense (meristems formed micro shoots with sizes from 2 mm to 13 mm by the end of the zero passage) than when using hydrogen peroxide with a concentration of 5 % or 15 % (meristems formed micro-shoots with sizes from 1 mm to 7 mm).

Statistical processing showed that there was no significant difference in the sterilization options.

The selection of an effective sterilant was carried out, which makes it possible to obtain at least 70 % of aseptic grapes meristems of the rootstock cultivars ‘Ryuggeri 140’, ‘CO 4’ and ‘Kober 5BB’. Regardless to the sterilization method used, the primary explanted meristems in the zero passage, although they differ by individual sizes, into average do not have reliable differences in the size of the micro shoots formed, depending on the type of sterilization we used. Introduction of plants into in vitro culture on MS medium, supplemented with 30 g/l sucrose, 2.0 mg/l BAP, 0.5 mg/l IBA, followed by passage on MS medium of, supplemented with 30 g/l sucrose, 2.0 mg/l 6-BAP and 0.3 mg/l IBA promotes multiple shoot formation up to 7 pcs per primary explants of the ‘Ruggeri 140’, ‘CO 4’ and ‘Kober 5BB’ varieties.

References:

1. Doroshenko, N. P. Improvement of the technology of clonal micropropagation of grapes / N. P. Doroshenko, V. G. Puzyreva, L. P. Troshin et al. // Magarach Viticulture and Winemaking. – 2022. –№ 2 (120) 24. – P. 102–111 [in Russian].
2. Zmushko, A. A., Krasinskaya, T. A. Introduction into in vitro grape culture – a critical stage of clonal plant micropropagation / A. A. Zmushko, T. A. Krasinskaya // Fruit growing. – 2022. – Vol. 34(1). – P. 228–234 [in Russian].
3. Kataeva, N. V. Clonal micropropagation of plants / N.V. Kataeva, R.G. Butenko. – M.: Nauka, 1983. – 95 p. [in Russian].
4. Murashige, T. A revised medium for rapid growth and bio assays with tobacco tissue cultures / T. Murashige, F. Skoog // Physiologia Plantarum. – 1962. – Vol. 15. – P. 473 – 497.
5. Rebrov, A. Influence of concentration and ratio of macro elements in culture medium on the regeneration of grape meristems in vitro // BIO Web of Conferences – 2020. – Vol. 25. No10. – Article Number 05005.