Clonal Micropropagation Research Articles

Polyploidy in tissues of plants in vitro of grape somaclones

In vitro experimental plants obtained by clonal micropropagation of 9 grape somaclones of 5 original forms were the material for cytogenetic research. A biological microscope XSP-146TP was used for cytogenetic analysis. 823 cases of deviation from diploidy were observed in total. Significant tissue ploidy was observed in the meristematic tissue of in vitro plants of grape somaclones obtained by colchicine treatment of proembryogenic cells of various varieties. The significant direct correlation was found between the frequency of polyploidy in meristem tissues of in vitro plants and the number of chloroplasts in the stomata of grape somaclones. The reverse correlation was found between the frequency of polyploidy and the number of stomata on the leaf area. Somaclone No. 72, obtained as a result of regeneration from colchicinated proembryogenic cells of the Ruta grape variety and identified as a tetraploid (2n = 4x = 76), is recommended for use in the polyploid creation program.

Stock Plant Material of St John’s Wort (Hypericum perforatum L.) Based on Biotechnology

Biotechnology applied in the production of medicinal plant biomass will facilitate the multiplication and use of high-quality renewable medicinal materials without destroying natural resources. The study was aimed at the progeny plants of Hypericum perforatum L., Zolotodolinsky cultivar, morphological and anatomical evaluation to identify differences between conventional plant material and the one obtained through biotechnological methods, its biochemical analysis. The technique was developed for clonal micropropagation of Hypericum perforatum L., Zolotodolinsky cultivar. The paper presents a microscopic analysis of St. John’s wort material grown in tissue culture. The key microscopic signs that have diagnostic value are the structure and size of the epidermis, veins, and also 3 types of floral cups. Macro- and microdiagnostic signs were found to be identical in the intact and progeny plants. A phytochemical analysis suggested the presence of phenolic compounds in ethanol extracts (70%) from H. perforatum progeny plants. The content of extractives was established to make up 12.8±0.6%, a total of flavonoids – 5±1%, the amount of tannins – 6.40±0.01% per ADM of progeny plants. For the first time, Hypericum perforatum L. was established to have the content of xanthones 0.20±0.01% per ADM in terms of mangiferin.

МОРФО-БИОЛОГИЧЕСКИЕ ОСОБЕННОСТИ ФОРМИРОВАНИЯ ДИАФРАГМЫ У IN VITRO И EX VIRO РАСТЕНИЙ ВИНОГРАДА МЕЖВИДОВОГО ПРОИСХОЖДЕНИЯ

Clonal micropropagation of grape varieties of interspecific origin Moscow white (Vitis amurensis Rupr. × Vitis vinifera L.) showed that on the 40th day of the animation stage, a monopodial type of branching is typical for micro-plants, and there is no diaphragm in all nodes of micro-shoots. On the 120th day of growing in containers, the researchers revealed that the nodes of the plant shoots, from the 1st to the 6th, have a monopodial branching type. Parenchymal cells which form an incomplete diaphragm are present in the seventh node. A fully formed diaphragm appears from the eighth node, and the branching type switches to a sympodial-monopodial one.

Размножение сортов сливы амурской селекции в культуре in vitro

Results are presented on clonal micropropagation for three cultivars selected in Amur Region: 'Blagoveshchenskii chernosliv', 'Lyudmila', and 'Oranzhevaya rannyaya'. Tips of growing young shoots are preferable explants for the tissue culture production as compared to lateral buds of the same shoots. The Quoirin-Lepoivre (QL) agar medium supplemented with sorbitol (20 g/L) and 6-benzylaminopurine (BA, 3 mg/L) was used for the establishment of explants and tissue culture initiation. Shoot proliferation was conducted on a QL agar medium with a modified microelement and vitamin composition and supplemented with sucrose (30 g/L), BA (0.2, 0.5, or 2.0 mg/L) and indole-3-butyric acid (IBA, 0.04 mg/L). Optimal shoot proliferation was noted to be achieved by alternate cultivation cycles on the medium with various BA concentration: 2 mg/L for an increased proliferation rate and 0.2–0.5 mg/L for an increased microshoot length. Higher values of shoot proliferation (the proliferation rate and microshoot length) were found for cv. 'Blagoveshchenskij chernosliv' (1.6 and 1.9 respectively) as compared to cv. 'Lyudmila' (1.3 and 1.5) and cv. 'Oranzhevaya rannyaya' (1.4 and 1.2). In vitro rooting was achieved after a preliminary incubation of microshoots in the aqueous solution of IBA (15 mg/L) with the subsequent cultivation on the growth regulator-­free QL agar medium. The cultivar 'Blagoveshchenskij chernosliv' had higher values of rooting (5.8 roots per shoot and 11.6 cm of total root length) as compared to cv. 'Lyudmila' (4.6 and 6.6 respectively) and cv. 'Oranzhevaya rannyaya' (5.3 and 7.4). The protocol was used for production of own­rooted plants of the plum cultivars.

Optimization of the technology of clonal micro-propagation of cherry varieties and clonal rootstocks at the initial stages of in vitro cultivation

The article presents the results of studying some components of the in vitro technology of clonal rootstocks and common cherry varieties, which allow to increase the efficiency of its application to the tested cherry varieties and clonal rootstocks. Differences in the viability and intensity of the development of clonal cherry rootstocks and varieties explants depending on the timing of their introduction into the crop, the characteristics of genotypes and the composition of nutrient media were established. The influence of nutrient medium composition, content and ratio of growth regulators and genotype in it on the multiplication coefficient of cherry varieties and clonal rootstocks was revealed. The optimal calendar dates for the introduction of cherry varieties and clonal rootstocks explants into the crop in vitro for the autumn-winter-spring period are established. The optimal composition of the culture medium for the initial stages of clonal micropropagation is established. The reproductive potential of some clonal rootstocks and cherry varieties in crop in vitro was revealed.

Optimization of stages of clonal micropropagation of garden strawberry varieties Nelly and Kemiya breeding of NCFSCHVW

The production of strawberry planting material using the method of clonal micropropagation of plants is promising, modern and already widely used. This paper presents the results of assessing the breeding potential of garden strawberry varieties Nelly and Kemiya (selection of FSBSI NCFSCHVW) in in vitro culture. Apexes from growing rosettes of strawberries served as the starting material for the initiation. The best survival rate of explants was noted during the initiation in the second decade of August, which were in Nelly variety - 90%, Kemiya - 83.3%. At the stage of proliferation, 6-Benzylaminopurine (6-BAP) was injected into the culture medium at various concentrations (0; 0.5; 1.0 and 1.5 mg / L). To increase the efficiency of micropropagation of strawberries Nelly and Kemiya varieties, it is advisable to add 6-BAP into the culture medium with the amount of 1.0 mg / l. In this variant, 10.6 shoots were formed in the explants in Nelly variety, 5.9 in Kemiya variety. With an increase of the concentration of 6-BAP to 1.5 mg / l, the number of vitrified shoots were raised. According to the results of three subcultures, it was noted that the Nelly variety has a higher reproduction potential compared to the Kemiya variety. At the stage of rhizogenesis, the addition of auxins to the medium was not required, since the roots of regenerated plants formed independently.

The effectiveness of the use of the silicon-containing preparation Siliplant in the case of clonal micropropagation of the rose Reine Sammut

Clonal micropropagation of plants is a modern popular method of vegetative reproduction, which allows to obtain high-quality planting material in a large volume in a short time. The research is devoted to the technology of clonal micropropagation of the rose Reine Sammut, in particular, optimization of the nutrient medium composition. To increase the efficiency of micropropagation, the silicon-containing preparation Siliplant was added to the composition of the nutrient medium according to the Murashige-Skoog (MS) recipe as an alternative to the complex of trace elements. At the stage of the actual micropropagation with the combined use of 6-benzylaminopurine (6-BAP) at doses of 1.0; 2.0 and 3.0 mg/l and Siliplant at doses of 1.0; 2.0 and 3.0 ml/l, no large differences were found. Nevertheless, the inclusion of Siliplant (1.0 ml/l) and 6-BAP (3.0 mg/l) in the medium contributed to an increase in the reproduction factor by 3.1 pcs./cuttings at LSD05= 1.0 pcs. In addition, Siliplant at a dose of 1.0 ml/l as part of a hormone-free nutrient medium MS contributed to a significant increase in shoot growth (by 4.2 mm at LSD05= 3.6), and at a dose of 3.0 ml/l stimulated the development of standard micro-plants of roses.

Creation and maintanance of in vitro collections of ornamental cultures from the Lomonosov MSU Botanical Garden’s funds

The article contains the data on creating and maintaining the collections in vitro of some ornamental plants – lilac, roses, tree peonies. The analysis of morphogenetic processes during microclonal propagation of lilac cultivars demonstrates well-defined correlation between susceptibility of this culture to mineral mixture of nutrient medium and plant genotype. Rose cultivars from different garden groups keep high growth capacity during 12 subculturing cycles on Murashige Skoog medium (MS) supplemented with 0.5 mg/l BAP. For all culturing tree-peonies cultivars Woody Plant Medium (WPM) with double content of Ca2+ ions and total exception of Cl- ions turns out to be more suitable. The opportunity of 10 lilac cultivars and 4 rose cultivars preservation in slow growth cultures was appreciated. The correlation between long-term cultivation in low temperature and genotype of ornamental plants cultivars was shown. Thus, 18 ºС turns out to be suitable for creation of slow-growth collection of all studied rose cultivars, and 14 ºС – only for 'Nina Weibull'. Maintaining of lilac microshoots at 10 ºС during 5 months practically did not act negatively on their viability.

<i>In vitro</i> propagation as one of the promising methods of conservation of species <i>Cotoneaster melanocarpus</i> Fisch. ex. Blytt.

Cotoneaster melanocarpus Fischer ex Blytt is rare species and included in Red Books of Russia (8 Regions) and Belarus, it is included in the list of wild rare and endangered plants of the Kostanay Region in Kazakhstan. C. melanocarpus is used as an ornamental, medicinal, honey-bearing plant, but it is difficult to propagate both seed and vegetative methods. Microclonal propagation of plants is a promising method of propagation and conservation of this species, but there are no researches in this direction in the world scientific literature for C. melanocarpus . The article presents the conditions of in vitro propagation for other species of the genus Cotoneaster , studied at different times by scientists from France, Korea, China, Russia and Iraq. It is shown how the application of various types of explants, sterilization modes, nutrient media, plant growth regulators affect on features of the morpho- and rhizogenesis of Cotoneaster in culture in vitro . This review was conducted for the first time and can serve as a basis for the development of a method for microclonal propagation of C. melanocarpus , which will conserve its gene pool and obtain plant material for practical use.

ФЕНОЛОГІЧНА ОЦІНКА СТВОРЕНИХ СОМАКЛОНАЛЬНИХ ЛІНІЙ РИЖІЮ ЯРОГО СТІЙКИХ ДО СОЛЬОВОГО І ОСМОТИЧНОГО СТРЕСІВ

The biological features of сamelina sativa make it possible to grow it in different soil and climatic conditions with high economic efficiency, to obtain environmentally friendly products and make full use of the natural potential of the region. Camelina oil is used for the production of varnishes, paints, soaps, plastics, biodiesel. It is also used for dietary and medical nutrition. The introduction of high-yielding adaptive varieties is the main condition for increasing the production of camelina sativa. Biotechnological methods are used to increase the efficiency of the selection process. Plant forms obtained by biotechnological methods must be evaluated according to a set of economically valuable traits. The length of the growing season is one of the main breeding characteristics of crop varieties. The aim of the work was to analyze the duration of the vegetation period and phenological phases of development of the created somaclonal lines of, resistant to salt and osmotic stress. Phenological evaluation of somaclonal lines of сamelina sativa obtained from explants of Stepovyi 1, Peremoha, Klondike and Yevro 12 varieties was performed during 2018–2020. Created by cell selection, stress-resistant plant material (sodium chloride, mannitol) plant material after microclonal propagation, rooting and adaptation was grown in the research areas of the Department of Genetics, Plant Breeding and Biotechnology, Uman National University of Horticulture. The analysis of the duration of the vegetation period and phenological phases of development of somaclonal lines of сamelina sativa resistant to salinity and osmotic stress was performed. It is established that the lack of moisture and elevated air temperatures contribute to the acceleration of the phases of ontogenesis and the reduction of the total growing season. The duration of «sowing-germination» period plants over the years of research, depending on the genotype, varied from 9 to 13 days, the formation of the rosette — from 8 to 13 days, the period of stalking and budding — from 18 to 20 days, the duration of flowering — from 8 to 27 days, the phase of the green pod — from 13 to 15 days, the period from the beginning of pod browning to the onset of full ripeness of seeds — from 26 to 31 days. The vegetation period of the created samples of сamelina sativa in 2018 averaged 80 days, in 2019 — 89 days and in 2020 — 88 days. According to the Methodology of examination of сamelina sativa for difference, homogeneity, stability the obtained data make it possible to rank the created somaclonal plant lines by the duration of the growing season on medium- ripening – C-234-8, C-326-9, C-402-6, С-419-6, С-586-7, П-46-2, П-46-5, П-202-6, П-202-7, П-248-8, П-485-4, П-618-6, П-646-3, П-658-8, Є-405-5, Є-405-8, K-478-2, K-480-2, K-480-4 and late-ripening – C-87-4, C-87-7, C-121-2, C-121-11, C-384-4.

INFLUENCE OF THE STERILIZATION METHOD AND THE TIME OF INTRODUCTION INTO IN VITRO CULTURE ON THE VIABILITY OF EXPLANTS OF THE CLONAL APPLE STOCK 54-118

The stage of introducing explants into a sterile culture is difficult in the technology of clonal micropropagation of plants. The article shows the possible ways of sterilization and the introduction terms of explants of the clonal apple stocks 54-118 into in vitro culture in order to reduce planting infection and increase the yield of sterile viable explants. The best time for introducing clonal apple stocks 54-118 into sterile in vitro culture is the period of active shoot growth. Sterilization of explants with ethyl alcohol (70,0 %, 1 min) in combination with hydrogen peroxide (33,0 %) for 7 minutes and ethyl alcohol (70,0 %, 1 min) in combination with diacide (0,1 %) within 6 minutes contributed to the production of 63,0 % and 60,0 % of viable sterile explants.

Аналіз застосування біотехнологій щодо одержання високоякісного садивного матеріалу рослин родини Salicaceae Mirb. in vitro для створення біоенергетичних плантацій

In conditions of a sharp reduction in the stocks of traditional fuels, it is important to find new efficient and renewable carbon-neutral energy sources. One of the most promising sources of renewable energy is the biomass of woody plants, in particular the family Willow (Salicaceae Mirb.). Traditionally, Salicaceae plants are propagated by generative and vegetative methods. Microclonal propagation, in contrast to traditional methods of reproduction, allows to obtain genetically homogeneous healthy planting material throughout the year. A significant number of biotechnological publications focus on the development of the optimal protocol for the reproduction of plants of the family Salicaceae, the study of the morphogenetic potential of tissues and optimizing their growth. However, the authors note the individually determined regenerative ability of plant material in vitro, which depends on a number of factors. The purpose of the study is to analyze the results of biotechnological research on the effectiveness (possibility) of obtaining high-quality planting material of plants of the family Salicaceae by tissue in vitro. For this purpose, we used the results of biotechnological studies of plant tissues of the family Salicaceae in vitro by foreign and domestic authors published in professional journals during 2010−2020. Research methods − analysis, comparison, synthesis, generalization. The analysis revealed that the sterilization regime of Salicaceae plant material depends on the type of explant, phenological phase and age of the donor plant. Step-by-step sterilization using mercury chloride, sodium hypochlorite and silver nitrate effectively neutralizes the exogenous biota of woody explants. The stage of active vegetation is the optimal period of isolation of explants. To obtain virus-free regenerating plants, it is advisable to use apical meristems, callus tissue − leaf blades, active regeneration − microshoots. Stable regeneration system, its type, multiplication factor of Salicaceae plants are genetically determined. For the introduction of plants in vitro, active proliferation, rooting, microclonal propagation, it is advisable to use nutrient media according to WPM (McCown & Lloyd, 1981) and MS (Murashige & Skoog, 1962). For regeneration of plants by direct morphogenesis and activation of growth of existing meristems of an explant to apply the environment with cytokinins (BAP (6-benzylaminopurine), kinetin or 2-isopentenyladenine (2-ip), for rooting − with auxins NAA (α-naphthylacetic), IBA (3-indolylbutyric acid) and IAA (β-indolyl-3-acetic acid). Further research is aimed at optimizing the propagation protocols of Salicaceae plants in vitro.

PERSPECTIVES OF USING BACTERIA FOR CELL AND TISSUE PLANT CULTURE

The review presents data from modern literature according the interaction of bacteria and plants. Peculiarities of coexistence of plants and epiphytic and endophytic microorganisms in natural conditions and in aseptic culture are given. The advantages of the interaction of plants and bacteria and the problems of lack of microbiota in plant seedlings during microclonal propagation are highlighted. The experience of using microorganisms in the culture of plant cells and tissues is described in detail. The processes of bacterial inoculation on plant microclones are described. In vitro plant culture is also considered as a model of interaction between bacteria and plants. The growth-stimulating and antagonistic properties of bacteria of the genus Bacillus, which can potentially have a beneficial effect on plants during adaptation to ex vitro conditions, are highlighted. Prospects for the use of bacteria of the genus Bacillus at the stage of acclimatization of plants to ex vitro conditions are considered. Examples of successful use of bacteria of the genus Bacillus to stimulate plant growth and to protect against pathogens are given.

Оптимизация питательных сред для клонального микроразмножения сорта-подвоя винограда Кобер 5ББ

Сорт винограда Кобер 5 ББ (Берландиери x Рипариа Кобер 5ББ) - один из основных подвоев, используемых в питомниководстве для получения привитых саженцев, поэтому в настоящее время крайне актуально создание маточников данного подвоя посадочным материалом категории «оригинальный». Это объясняет необходимость проведения исследований, связанных с оптимизацией условий культивирования сорта Кобер 5ББ, для повышения эффективности массового клонального микроразмножения с сохранением его генетической однородности и стабильности. Целью исследования являлась оптимизация и подбор питательных сред для клонального микроразмножения сорта-подвоя Кобер 5 ББ на этапе тиражирования (микрочеренкования). Материалом для исследований служили растения in vitro сорта подвоя Кобер 5ББ, свободные от основной патогенной инфекции (по результатам тестирования). Исследования проводили на средах: МS; WPM; DKW; PG (контроль). В качестве регуляторов роста использовали GA (гиббереллиновая кислота) в концентрациях: 0,2; 0,6; 1; 1,4 мг/л в сочетании с NAA (α-нафтилуксусная кислота) 0,05 мг/л. Показано, что растения на среде WPM, содержащей NAA-0,05 мг/л, по биометрическим показателям превосходили развившиеся на среде PG с аналогичным гормональным составом. Проведенные исследования по оптимизации среды культивирования для ускорения ростовых процессов позволили по результатам биометрических показателей выделить блок сред с основой WPM для размножения сорта-подвоя Кобер 5 ББ на этапе микрочеренкования. После проведения дополнительных исследований с расширенной выборкой среду WPM можно будет рекомендовать для клонального микроразмножения винограда на этапе микрочеренкования. The grape variety ‘Kober 5 BB’ (‘Berlandieri x Riparia Kober 5BB’) is one of the main rootstocks used in rootstock-growing farming to obtain grafted seedlings. Currently it is a hot issue to create nurseries for the rootstock grapevine with planting material of the "original" category. This explains the need for research related to optimization of cultivation conditions of the variety ‘Kober 5BB’ to increase the efficiency of mass clonal micropropagation while retaining its genetic homogeny and stability. The aim of the study was to optimize and select nutrient media for clonal micropropagation of the rootstock variety ‘Kober 5 BB’ at the stage of tiraging (micropropagation by cutting). The material of research was the in vitro plants of ‘Kober 5BB’ rootstock variety, free from basic pathogenic infection (according to the test results). The studies were carried out on media: MS; WPM; DKW; PG (control). Gibberellic acid (GA) was used as a growth regulator at concentrations: 0.2; 0.6; 1; 1.4 mg/l in combination with NAA (α-naphthyl acetic acid) 0.05 mg/l. Plants on WPM medium containing NAA-0.05 mg/l in biometric parameters were superior to those grown on PG medium with a similar hormonal composition. According to the results of biometric parameters the studies on optimization the culture medium for accelerating growth processes made it possible to isolate a group of media with a WPM base for propagation of the rootstock variety ‘Kober 5 BB’ at the stage of microcutting. After additional studies with expanded selection, the WPM medium can be recommended for clonal micropropagation of grapes at the stage of microcutting.

Развитие генетико-селекционных методов повышения продуктивности лесов

Представлена история становления института и основные направления работ ВНИИЛГИСбиотех за 50 лет научной и производственной деятельности. Основное внимание уделено инновационным разработкам и перспективам развития института. The article presents the history of the All-Russian Research Institute of Forest Genetics, Breeding and Biotechnology, the principal directions of its activity through over 50 years of scientific and production work. It focuses on innovations and the development perspective of the Institute. The Institute’s priority research areas are forest genetics and biotechnology. The Institute scientists were among the first in Russia to conduct research on the tissue culture methods for the plants genetic diversity and their future genetic improvement. The Institute has developed clonal micropropagation techniques for valuable tree biotypes, they have been put into forestry practice and are now 18 – 26 years old. Also, the Institute has developed the long-term in vitro storage method with the use of hormone-free nutrient solutions for valuable deciduous tree species. Based on this method, in 1991 our scientists began to create the collection of deciduous tree plants, which currently includes more than 70 clones of valuable, resistant, and fast-growing birch, poplar, aspen and willow genotypes. Throughout its existence the Institute has established a large experimental facility which includes seed plantations of Scots pine, pedunculate oak and other species. All experimental units are being closely monitored. Through over 50 years of scientific work we have developed the multi-year breeding program, selected the economically valuable clones and forms, obtained and patented new varieties of tree species. Our scientists are now focused on obtaining new genotypes of forest trees, increasing productivity and selective value of the main forest-forming species, studying their ecological and genetic parameters, creating new breeding facilities, conserving the plant genetic resources, etc.

Clonal micropropagation of decorative cereal <i>Molinia caerulea</i> (L.) Moench

The research is devoted to the development of clonal micro-propagation technology of the decorative cereal Molinia caerulea (L.) Moench. The concentration of 2.0 mg /l of cytokinin 6-benzylaminopurine (6-BAP) contributed to obtaining the maximum number of microshoots: 6.3 and 7.9 pcs. on Anderson's and Murashige-Skoog's (MS) media, respectively, which exceeded the control (by 4.7 and 6.3, respectively, with LSD05 = 2.3). The length shoots were observed on hormone-free media, this indicator significantly decreased with an increase in 6-BAP content. On Anderson and MS media with 1.0 mg/l 6-BAP, the shoot length averaged 21.5 and 26.4 mm, respectively, which made it possible to transplant them for rooting, bypassing planting on a medium for elongation. The inclusion of the Siliplant micro-fertilizer in the MS medium at doses of 1.0 and 2.0 ml/l contributed to a significant increase in shoot size, by 16.7 and 10.7 mm (LSD05 = 8.9), respectively, in comparison with the control (MS). It is recommended to use Anderson's medium and 0.5 mg/l of indole-3-acetic acid as a medium for rhizogenesis: after two weeks of cultivation, the regenerants had a standard appearance with developed roots suitable for planting for adaptation. At the adaptation stage, watering the substrate with the biofungicide «Trichoderma veride» according to the instructions and a cereals single spraying with the micro-fertilizer «Siliplant» at a dose of 1.5 ml/l contributed to their 100 % survival rate.

Conservation and clonal micropropagation of rare species Gladiolus palustris Gaudin

Conservation and clonal micropropagation of rare species <i>Gladiolus palustris</i> Gaudin

Reproduction methods of Iris Spuria varieties in the forest steppe of the Altai Territory

The subgenus of beardless Iris Spuria (SPU) includes 19 species. The entire spectrum of flower colors of Iris Spuria species selected in Russia and abroad is used for gardening in the forest steppe of the Altai Territory. A low vegetative reproduction coefficient of Iris Spuria is reported: 1-4 for three-year-old plants, and 1-6 for four-year-old plants. A suggested production method of Iris Spuria planting material involving the clonal micro-propagation and adaptation in aeroponics conditions is commercially applicable, furthers the growing high quality young plants and simplifies technological stages. Using this cultivation technique, one mature plant may supply 2048 young plants in a year given that a reproduction coefficient is 2.0. The tissue culture reduces labor and financial costs and production areas.

Development of clonal micropropagation technique for Gloxinia hybrida hort. “Strawberry Ice-Cream”

The reported paper presents outcomes of clonal micropropagation of ornamental variety Gloxinia hybrida hort. “Strawberry Ice-Cream”. In the study conditions supporting in vitro implanting leaf blade fragments are developed with an outcome of 97 % viable explants. An optimal composition of the Murashige and Skoog growth medium is suggested; it contains BAP (0.5 mg/l) and IAA (0.2 mg/l) and brings about efficient adventive sprout regeneration. Implantation takes place in a growth medium; no root stimulating hormones are applied. The survival of regenerative plants is reported to be high after transplanting ex vitro.

Determination of an effective method of Citrus aurantifolia lime and Citrus lemon varieties plant explants sterilization for in vitro introduction into the culture

Potted citrus plants are in constant demand, but the propagation of such plants requires the use of a winter greenhouse and several well-developed mother plants to obtain planting material. The use of microclonal propagation can accelerate the production of seed material though it requires detailed development of in vitro cultivation techniques. The aim of the research was to study the effectiveness of sterilizing substances and methods of sterilization on the yield of viable micro shoots of lime plants (Citrus aurantifolia) as well as Meyer and Jubilee lemon varieties and their growth in vitro. The research was conducted in the interdepartmental "Biotechnological Laboratory" of the Agrobiotechnological Faculty of Bila Tserkva NAU. The source material was microshoots from lime plants (Citrus aurantifolia), and lemon varieties (Citrus lemon) Meyer and Jubilee. The objects of the study were selected according to the diversity of genotypes, development type and varietal characteristics. The possibility of using different parts of plants for reproduction is a feature of in vitro culture. We used microshoots with a bud in our research. To neutralize the exogenous bacterial and fungal microflora, we used 70 % ethanol solution C2 H5 OH, sodium hypochlorite 5 %, 15 % solution of hydrogen peroxide H2 O2 , sulema (0.1) and washed the material in sterile water (for 5–10 min) under several schemes. The study revealed the influence of different sterilization regimes for obtaining sterile explants and the introduction into plant culture of explants of the species of lime Citrus aurantifolia as well as Meyer and Jubilee lemon varieties. It was found that the use of step sterilization using 2.5 % solutions of sodium hypochlorite gives a high percentage of sterile viable material – 27.7 %, which is significantly higher than in other variants of the experiment. Key words: microshoots, lime, sterile material, micropropagation, sterilization, hydrogen peroxide, viability of explants.