Optimized Nutrient Medium Research Articles

Callus induction with 6-BAP and IBA as a way to preserve Prunus ledebouriana (Rosaceae), and endemic plant of Altai and Tarbagatai, East Kazakhstan

Abstract. Orazov A, Myrzagaliyeva A, Mukhitdinov N, Tustubayeva S. 2022. Callus induction with 6-BAP and IBA as a way to preserve Prunus ledebouriana (Rosaceae), and endemic plant of Altai and Tarbagatai, East Kazakhstan. Biodiversitas 23: 3178-3184. This article presents the results of the study of in vitro conditions for the cultivation and induction of callus tissues of a rare plant species Prunus ledebouriana(Schlecht.) Y.Y.Yao (Ledebour's almond), endemic plant to East Kazakhstan. For the first time, protocol and optimal composition of Murashige & Skoog (MS) were developed for obtaining calendemiclus tissues. The main indicator of successful introduction was the frequency of callus tissue formation in vitro. Two types of explants were used: the whole embryos and embryos with a removed root part. The removal of the root part inhibited the organogenesis of the embryonic root and the first shoot, which allowed the accumulation of nutrients for the growth of the callus mass and increased the frequency by 7.79±0.46%. Various exogenous phytohormones such as Kinetin, 6-BAP, GA, IBA were added to the MS culture medium. A decrease in Kinetin concentrations of 0.04 mg/L resulted in a lower intensity, but a higher frequency of formation (72.00±5.66%) and mass gain. The average concentration up to 0.5 mg/L of exogenous phytoregulators, such as GA and 6-BAP, resulted in frequency from 65.38±5.44% to 72.00±5.66%, the concentration of IBA up to 1 mg/L increased the frequency from 46.00±2.83 to 72.00±5.66%. The protocol for obtaining callus from an embryonic explant and the composition of the optimal nutrient medium for the induction of callusogenesis made it possible to obtain P. ledebouriana meristem tissues for further use in the development of callus cryopreservation technology.

ОПТИМИЗАЦИЯ ПРИЕМОВ ВВЕДЕНИЯ САДОВЫХ РАСТЕНИЙ В СТЕРИЛЬНУЮ КУЛЬТУРУ IN VITRO

The stage of introduction into sterile culture plays an important role in clonal micropropagation. The purpose of these studies was to find the optimal sterilizing agent at the stage of introducing blue honeysuckle, red raspberry, garden strawberry, steppe cherry, house plum into in vitro culture, as well as the optimal nutrient medium at this stage for blue honeysuckle and red raspberry. As sterilizing agents with optimal exposure, the following were used: 48% ethyl alcohol (control) 8–10 min., 33% perhydrol 6–8 min., 6% chlorhexidine 8–10 min., 0.1% sublimate 1–2 min., 10.0% Domestos 6–7 min., Amway pursue 10–12 min. Murashige-Skooga modified (NH4 content reduced at 15%) and Woody Plant Medium nutrient media were used to identify the optimal media for blue honeysuckle, and Anderson and Quorin-Leporrier media – for raspberries. All nutrient media had a half dose of macro- and microsalts. The control medium was traditional Murashige-Skooga. The results of the experiment showed that 33.0% perhydrol is the most effective sterilizing agent for processing the initial plant material of all horticultural crops when initiating explants in vitro, since it provided a significantly high yield of sterile apexes of 63.0% on average. A significantly high result was also obtained when using 6% chlorhexidine – 51.9% with 39.1% in control (48% ethyl alcohol). Treatment of plant material with 33.0% perhydrol ensured the absence of vitrified (glassy) explants in all studied cultures, as well as chlorotic explants in cherries and plums. Significantly better results of the survival rate of blue honeysuckle apexes were obtained on the nutrient medium Woody Plant Medium – 76.7%. Cultivation of raspberry apexes on the Kvorin-Leporie nutrient medium significantly increased their survival rate to 56.7% of red raspberry and 36.7% of remontant raspberry.

ECOLOGICALLY SAFE UTILIZATION OF ORGANIC WASTES AND TECHNOLOGY OF PRODUCTION OF THE FUNGUS PLEUROTUS OSTREATUS (THE OYSTER MUSHROOM)

Background. Organic cellulose-containing wastes are represented by various substrates with diverse physicochemichal properties suitable as feedstock for obtaining new products, e.g. mycelium and fruit bodies of edible mushrooms. Industrial mushroom cultivation based on utilization of different kinds of organic wastes is one of the perspective directions in biotechnology and agriculture.
 Improving the nutrient value of substrates and increasing the reliability of mushroom culture is possible at expense of utilizing nutrient supplies originated as wastes of another industry, for example, the use of sparging grain, which is the major waste of brewery. Wet sparging grain has humidity of 70-80%, and contains in average more than 20% of dry residues with high protein level (12-15%), which is approximately three times higher its content than in barley. For that reason, as a supplement to nutrient media and substrates, for rearing mycelium and fruit bodies of the fungus Pleurotus ostreatus, we propose the use of the brewers’ industry, the wet sparging grain.
 Purpose. Evaluation of the possibility of utilising the wastes of agriculture and brewery, for growing mycelium and fruit bodies of the edible mushroom Pleurotus ostreatus.
 Materials and methods. The study was carried out in the years 2017-2021 in the laboratory of the Department of biology and technology of living systems at L.N. Tolstoy TSPU. The research objects were the fungus Pleurotus ostreatus, wet sparging grain, and various agricultural wastes (wheat straw, buckwheat chuff, sunflower seed husk).
 For mycelium extraction, we used the method of seeding superficially sterilized pieces of fruit bodies onto the hard sterile agarized nutrient medium in the Petri dishes. For obtaining the master culture of mycelium and fruit bodies of the fungus Pleurotus ostreatus we utilized substrates of various composition containing the sparging grain and agricultural wastes. The data obtained were statistically processed and analysed.
 Results. In result of the experiments, it was revealed that optimal nutrient media and substrates for obtaining and cultivation of the mycelium are those with addition of the sparging grain and cellulose-containing wastes. For obtaining fruit bodies, we used the substrate composed of the straw with addition of the brewers’ grain. Utilization of nutrient media with similar composition leads to shortening of the period of mycelium adaptation and initiation of its active growth phase when producing the master culture of the fungus. Further, for that reason, increases the speed of colonization by the fungal mycelium the substrate blocks for producing carposomes, and the fungus fruitification occurs more intensively.
 Conclusion. The use of the brewers’ draff for obtaining mycelium and fruit bodies of the edible oyster mushroom creates valuable, ecologically clean and quality nutrition product and facilitates cost effectiveness of its production. Design and application this biotechnology solve the problem of organic waste utilization, which in turn positively stimulates positive changes in the regional ecological situation and contributes partially into the task of import substitution.

Development of a probiotic for animals and aquaculture based on Bacillus toyonensis B-13249 and Bacillus pumilus B-13250 strains

Abstract: This article aims to develop a probiotic for animals and aquaculture based on the Bacillus toyonensis B-13249 and Bacillus pumilus B-13250 strains. The selection of a nutrient medium was conducted for cultivating the inoculum of these microorganisms. Several bacteria fermentations of the Bacillus genus were performed in biological reactors with a capacity of 15 and 250 l. A technology for obtaining a finished probiotic for animals and aquaculture was developed. The results indicate that L-broth is the most optimal nutrient medium for cultivating the studied strains. The cultivation of B. toyonensis B-13249 and B. pumilus B-13250 strains in fermenters revealed that sporulation begins after 4–8 hours of fermentation. In contrast to the vegetative medium, the fermentative medium helped the bacilli develop a higher optical density (the maximum value in the B. pumilus strain – 2.400±0.149), pH value (maximum value in the B. toyonensis strain – 8.483±0.609) and titer (at least 1010 CFU/g). After 20–24 hours of incubation, both strains of bacilli in the fermenter, almost completely pass into endospores, which serve as a signal for the start of biomass centrifugation. This was indicated by the following: from a 15 l fermenter – 83.3±6.1 g of concentrate, from a 250 l fermenter – 499.8±51.4 g. The number of bacilli in a concentrated state was at least 1·1011 CFU/g for both strains. Obtaining a finished preparation required mixing bacterial concentrates with maltodextrin to a titer of at least 1·1010 CFU/g. The number of bacteria in the preparation checked every month during the year, recorded no value less than 1·1010 CFU/g. Thus, L-broth is most favorable for growing the mother culture of the B. toyonensis B-13249 and B. pumilus B-13250 strains, and fermentative nutrient medium – for the cultivation in fermenters. The expiry date of the bacilli-based biological preparation is at least 12 months, during which the drug’s polycomponence, color and consistency are preserved, in addition to the bacteria titer (at least 1·1010 CFU/g) and their viability.

Мікробіом приепітеліальної біоплівки товстої кишки білих щурів з експериментальним тиреотоксикозом

Актуальність. Мікробіом приепітеліальної біоплівки товстої кишки при безпосередньому контакті з організмом взаємодіє з імунною та іншими системами, що підкреслює актуальність його вивчення при різних захворюваннях. Мета дослідження — встановлення таксономічного складу, популяційного рівня, аналітичних мікроекологічних показників і ступеня порушень мікроекології приепітеліальної біоплівки товстої кишки білих щурів із тиреотоксикозом. Матеріали та методи. Проведені експерименти на 25 статевозрілих самцях білих щурів масою 220–240 г, з яких 15 тварин віднесено до контрольної групи (інтактні тварини), 10 щурів належали до основної групи. Моделювали експериментальний тиреотоксикоз шляхом внутрішньошлункового введення L-тироксину 14 днів. У стерильних умовах проводили лапаротомію, брали відрізок (до 3 см) товстої кишки з її вмістом. Відмиту частину кишки гомогенізували з стерильним 0,9% розчином хлориду натрію. Готували серію десятикратних розведень з концентрацією вихідної суміші від 10–2 до 10–7. З кожної пробірки 0,01 мл висівали на тверді оптимальні живильні середовища з наступним виділенням та ідентифікацією мікробів за морфологічними, тинкторіальними, культуральними та біохімічними властивостями. Результати. У частини тварин елімінують біфідобактерії та лактобактерії, а також бактероїди та ешерихії. Встановлено виражений дефіцит не тільки біфідобактерій — на 48,50 % і лактобактерій — на 94,59 %, а й бактероїдів — на 44,85 %. Визначення кількісного домінування кожного таксона показало, що домінуюча роль біфідобактерій у мікробіоценозі знижується на 82,76 %, лактобактерій — у 2,20 раза, а також знижується роль у мікробіоценозі приепітеліальної біоплівки товстої кишки білих щурів з експериментальним тиреотоксикозом бактероїдів — на 43,04 %, кишкової палички — на 7,18 %, але суттєво зростає роль умовно-патогенних ентеробактерій і стафілококів, які контамінували й колонізували слизову оболонку товстої кишки. Висновки. Експериментальний тиреотоксикоз супроводжується частковою елімінацією з біотопу приепітеліальної біоплівки товстої кишки біфідобактерій та лактобактерій, а також бактероїдів. Відбувається елімінація із слизової оболонки товстої кишки в усіх експериментальних тварин пептострептококів, клостридій та ентерококів, а також колонізація біотопу умовно-патогенними ентеробактеріями (протеями, клебсієлами) і стафілококами.

Колонізаційна резистентність слизової оболонки товстої кишки білих щурів з експериментальним гіпотиреозом

Актуальність. Мікроекосистема організму людини тісно взаємодіє з імунною та ендокринною системами, і власне мікробіом приепітеліальної біоплівки слизової оболонки товстої кишки є одним з найчутливіших біотопів, здатних зазнавати змін за різних захворювань. Мета. Встановлення мікробіологічної структури (видовий склад і популяційний рівень мікрофлори) приепітеліальної біологічної плівки слизової оболонки товстої кишки білих щурів з експериментальним гіпотиреозом. Матеріали та методи. Проведено експерименти на 25 статевозрілих самцях білих щурів масою 220–240 г, з яких 15 тварин були віднесені до контрольної групи (інтактні тварини), 10 щурів належали до основної групи. Експериментальний гіпотиреоз моделювали шляхом внутрішньошлункового введення мерказолілу впродовж 14 днів. У стерильних умовах проводили лапаротомію, брали відрізок (до 3 см) товстої кишки з її вмістом. Відмиту частину кишки гомогенізували зі стерильним 0,9% розчином хлориду натрію. Готували серію десятикратних розведень з концентрацією суміші від 10–2 до 10–7. З кожної пробірки 0,01 мл висівали на тверді оптимальні живильні середовища з подальшим виділенням та ідентифікацією мікробів за морфологічними, тинкторіальними, культуральними та біохімічними властивостями. Результати. Колонізаційна резистентність приепітеліальної біоплівки слизової оболонки товстої кишки в експерименті на дослідних тваринах при моделюванні гіпотиреозу зазнає змін: знижується популяційний рівень лактобактерій на 51,58 %, біфідобактерій — на 37,35 %, бактероїдів — на 22,82 %, пептострептококів — на 6,5 %. Приепітеліальна біоплівка контамінується кишковою паличкою та іншими ентеробактеріями (Proteus, Klebsiella), P.niger, стафілококами. Встановлено зростання популяційного рівня кишкових паличок на 10,31 % і клостридій на 8,2 %. Детерміновано дисбіотичні порушення І–III ступеня, що потребує корекції і лікування за допомогою пробіотиків. Висновки. В експерименті на білих щурах при мерказоліл-модельованому гіпотиреозі встановлено елімінацію бактерій роду Bifidobacterium, Lactobacillus, контамінацію і колонізацію біотопу умовно-патогенними мікроорганізмами родів Proteus, Klebsiella, Staphylococcus, Peptococcus на тлі зростання популяційного рівня кишкової палички. Наявні порушення мікробіоценозу у вигляді дисбіозу I–III ступеня.

Features of seed growth and development of sproutings of the russian dereza (Lycium ruthenicum Murr.) under laboratory conditions and in culture in vitro

Russian dereza (Lycium ruthenicum Murr.) is a shrub plant that spreads on the territory of the dried Aral Sea in conditions of high soil salinity, dry and sharply continental climate. The fruits were collected and the features of germination of seeds of the russian dereza halophyte (Lycium ruthenicum Murr.) Under sterile and non-sterile conditions were studied. The optimal temperature and illumination regime for the germination of annual seeds of this species is a 16-hour photoperiod and a temperature of 25 °C, the germination of seeds in nonsterile conditions was: 4-year-old – 46 %; 2-year – 83; one-year – 96 %. It has been shown that L. ruthenicum plants remain viable for up to 4 years or more, on this basis, we attributed the seeds of this species to truly orthodox. When introduced into culture in vitro, it has been shown that multi-stage sterilization significantly reduces the viability of seeds and seedlings of Lycium ruthenicum Murr., which leads to a decrease in seed germination by up to 40 %. The optimal nutrient medium for the stable development of microshoots without anomalies, callus formation and initiation of root formation was MS with the addition of 1.0 mg/l 6-BAP to the nutrient medium, without sucrose. Maintaining the samples in the in vitro collection is carried out on a half-MS medium without hormones, without sucrose at a low positive temperature of 4 °C, illumination of ~500 lx and a photoperiod of 8 hours. The samples of L. ruthenicum in vitro can be used to develop methods of clonal micropropagation; for genotyping of samples and identification of molecular biomarkers of plant resistance to soil salinity; in the ex vitro morpho-biological study of plants resistant to salt stress.

Влияние состава питательной среды на рост и антифунгальную активность бактерий р. Bacillus – основы экспериментальных образцов биофунгицидов для экологизированной системы защиты растений

The article presents the results of the studies of the influence of the nutrient medium composition for the cultivation of Bacillus bacteria. We compared standard microbiological media and optimized nutrient medium according to criteria such as the number of colony forming units in liquid culture and antifungal activity against the test culture of F. graminearum fungus. It was noted that the number of colony forming units of LC based on the B. subtilis BZR 336g strain on the optimized medium turned out to be significantly higher than on King B media and potato-glucose medium and amounted to (8.7±0.66) x 1010 CFU/ml, for B. subtilis BZR 517 strain titer on an optimized medium was (7.2±0.42) x 1010 CFU/ml. Antifungal activity on an optimized nutrient medium for B. subtilis BZR 336g was strain 83.5%, for B. subtilis BZR 517 strain – 61.1%. We found the positive effect of experimental samples of biofungicides obtained with the help of an optimized nutrient medium on the growth and development of winter wheat plants against the background of artificial infection in a climatic chamber. The treatment of winter wheat seeds with experimental samples of biofungicides ensured biological efficiency from 35.4% to 60.8% with the efficiency of the Kinto Duo chemical standard, KS 38.9%, Fitosporin-M biological standard – 28.9%. It was noted that seed germination in the variant with biofungicide treatment based on the optimized medium was 20-30% higher than in the variants with standard media. Seed treatment with experimental samples of biofungicides obtained on various nutrient media showed a statistically significant increase in the biometric parameters of winter wheat seedlings.

Introduction of hyssopus officinalis l . into in vitro culture to optimize the conditions for obtaining callus tissues and microclonal propagation as a promising metod of innovative agrobiotechnologies

The sterilization process of plant explants of H. officinalis was optimized when introduced into an in vitro culture, the most effective sterilization modes, optimal sterilizing agents, their exposure time and concentration were selected. Callus tissues and mini-plants of H. officinalis were obtained in vitro and the most optimal nutrient media were determined both for microclonal propagation and for the induction of callus tissue H. officinalis, which can be further used for mass cultivation of cell and culture and obtaining safe bio-additives with active substances for livestock and crop production as a part of the development of modern agrobiotechnologies.

Оптимизация процесса культивирования клона-продуцента рекомбинантного лизосомального фермента идуронат-2-сульфатазы

This paper describes experiments aimed at developing conditions for suspension culturing of a producer clone of the recombinant lysosomal enzyme iduronate-2-sulfatase based on the CHO cell line. As a result of sequential culturing in batch and fed-batch modes, the optimal nutrient medium composition was established, providing a 2.5-fold increase in the iduronate-2-sulfatase specific activity. Optimization of the feed formulation was carried out, which increased the yield of recombinant iduronate-2-sulfatase. The ambr® 15 Cell Culture System of minibioreactors was used to optimize the cultivation process. Chinese hamster ovary cells, CHO, iduronate-2-sulfatase, idursulfase, lysosomal enzyme, sulfatases, Аmbr Tap Biosystems, ambr® 15 Cell Culture System, mucopolysaccharidosis type II, formyl glycine generating enzyme, optimization of cell culturing conditions, fed-batch culture, batch culture.

ИЗЫСКАНИЕ ОПТИМАЛЬНОЙ ПИТАТЕЛЬНОЙ СРЕДЫ ДЛЯ ПОЛУЧЕНИЯ АНАТОКСИНОВ БАКТЕРИЙ CLOSTRIDIUM PERFRINGENS

The article presents the results of studies on the selection of the optimal nutrient medium for the cultivation of bacteria Cl. perfringens. Meat-peptone broth, Hottinger broth, thioglycolic medi-um, Kitta-Tarozzi medium, meat-liver-casein were tested for these purposes. It has been established that the meat-liver-casein medium is the most optimal nutrient medium for the cultivation of Cl. perfringens bacteria and the production of toxoids. To obtain toxoids, production strains of Cl. perfringens No. 28, 392, 213 were grown under anaerobic conditions of the bioreactor on meat-liver-casein medium for 6–9 h at a temperature of 37–38 °C. Inactivation of bacteria and their toxins was carried out by adding formalin to a culture of 0,7 % final concentration and maintaining at a temperature of 37-38 °C for 10 days. Inactivated biomass of bacteria Cl. perfringens and toxoids was precipitated with aluminum hydroxide. The antigenic properties of the obtained preparation were studied in rabbits by double subcutaneous administration of it in a dose of 2 cm3. It was estab-lished that inactivated bacteria Cl. perfringens and toxoids sorbed on aluminum hydroxide have high antigenic activity and induce the production of specific antibodies in blood serum in rabbits in titers up to 13,84 ± 0,22 log2.

Введение в культуру in vitro Hedysarum gmelinii Ledeb.

Abstract. Hedysarum gmelinii is a perspective species for the introduction into the culture and further use in the agricultural industry and pharmacology. The purpose of this research is to find suitable medium for seed germination. At present, this species is under the threat of extinction in several subjects of the Russian Federation. That is why research of the optimal nutrient medium for the seeds of H. gmeliniiis very relevant. Materials and methods: 100 pcs of mature seeds were selected from the populations of Tatarstan and Bashkortostan. The seed material had been stored at the 4°C before it was used. The Murashige and Skoog (MS) medium was prepared under the protocol with adding of such plant hormones like 6-benzylaminopurine (BAP) and Indole-3-acetic acid (IAA) in four types of concentrations. After that the sterilized seeds were planted. The germination was occurred at 20 °С and the daylight was 16 hours. The results: seeds started to grow up to 15th day of an experiment. Then the plants were transferred on the fresh medium. Young plants were formed after 13 days. Plants which seeds had been taken in Tatarstan showed the most active callus formation. Thus, the originality of this research is that the optimal medium for growing seeds of Hedysarum gmelinii in vitro was found. This medium ensures good viability and gives the opportunity to get the tissues culture for the further studies of the biologically active substances.

Efficiency Indicators Of Phosphorus In The Development And Cultivation Of Potato

In order to determine the effect of the phosphoric element on the growth and development of potatoes, their productivity and efficiency, studies were conducted to determine the optimal nutrient medium in laboratory and field conditions. In nutritional variants with a concentration of P of 16 mg / l and 48 mg / l, the P content is higher than in leaves. In nutritional variants with a concentration of 48 mg / l, there is a smaller number of leaves compared to the 16 mg/l concentration variants and a delay in the flowering period. It was found that the nutrients contain important vital microelements in plant tissue and the output composition in variants of the concentration of P is 16 mg /l.

Features of introduction of sour cherries variety ‘Kseniia’ and cherries variety ‘Vasylysa prekrasna’ into in vitro culture

Purpose. Determine the optimal timing of explant selection, select sterilizing agents and sterilization regimens, as well as the nutrient medium for the introduction of new perspective varieties of sour cherries (Prunus cerasus L.) and cherries (Prunus avium L.) into in vitro culture. Methods. During the work the method of clonal micropropagation of plants and statistical processing of experimental data were applied.Results. The optimal term of explants selection , the duration of exposure during sterilization, the optimal composition of the nutrient medium at the first stage of microclonal reproduction were determined. A 0.1% solution of mercuric chloride and a 3% solution of “Lizoformin 3000” with exposure to sterilization of 5, 6 and 7 minutes were used to determine the optimal sterilization and sterilizing regimen. The highest yield of sterile explants for both ‘Kseniia’ sour cherries and ‘Vasylysa prekrasna’ cherries was obtained with a 7 min sterilization exposure for both sterilizing agents. When using a 0.1% solution of mercury chloride, this figure was higher than in the sterilization with 3% solution of the preparation “Lizoformin 3000” – 71 and 99%, respectively.Conclusions. The efficiency of sterilization and the introduction into in vitro culture of sour cherries ‘Kseniia’ and cherries ‘Vasylysa prekrasna’ explants were influenced by the duration of sterilization, the time of selection of explants, the phytosanitary state of the mother plant, the composition of the nutrient medium. A 0.1% solution of mercuric chloride at 7 min exposure was the most effective in obtai­ning aseptic culture from explants removed from donor plants at rest when sprouting buds under controlled conditions. The use of the preparation “Lizoformin 3000” at a concentration of 3% for 6–7 min while sterilizing explants of the studied cultures contributes to their better survival in the environment. The optimal nutrient medium for the cultivation of sour cherry ‘Kseniia’ explants is the medium with the addition of aloe juice, and for cherries ‘Vasylysa prekrasna’ – MS + phloroglucinol. The highest sterilization efficiency (99% in ‘Vasylysa prekrasna’ and 71% in ‘Kseniia’) was obtained using 0.1% HgCl2 in 7 min exposure. When using the preparation “Lizoformin 3000” with the same sterilization exposure, this indicator was 83 and 52%, respectively. Therefore, we can recommend the use of the prepa­ration “Lizoformin 3000” at 3% concentration with an exposure of 7 min for sterilization of stone cultures

Technologies of Reproduction Terry Varieties of Clematis

The paper presents issues of optimizing the technology of growing popular terry varieties of clematis (Clematis L.) in Russia. Trimming groups, bookmark features of flower buds are described. The issues of reproduction and agricultural technology when laying uterine plantations are considered. A comparative analysis of traditional breeding technology with innovative technology (in vitro). The optimal nutrient medium for the stages of micro propagation, cultivation and rooting, enriched with vitamins and other substances. In experiments on green cuttings, 8 terry varieties were used. Estimated rooting of green cuttings by standard propagation technology. Spring cuttings are recommended for the varieties Bellof Woking (83%), Empress (81%), Blue Light (79%) and give a high yield of planting material from one mother plant. Two varieties with low rooting were selected—Purpurea Plena Elegans, Multi Blue. In vitro breeding technology has been developed for them. Accounting and observation of the development of two terry clematis varieties in the tissue culture: Purpurea Plena Elegans, Multi Blue. The aim of our study is to develop a technology for clonally propagation for these varieties, as well as the adaptation of micro plants of these varieties to non-sterile conditions. Therefore, in vitro propagation is recommended for these two varieties. An optimal substrate composition has been developed for the adaptation of clematis plants propagated in vitro, as well as for cuttings. The article provides recommendations for planting, pruning and caring for uterine plantings of clematis with double flowers in sheltered ground.

Отримання асептичної культури Eucommia ulmoides Oliver

Cultivation of rare flora species in order to preserve their gene pool is one of the main tasks of our time. In vitro methods, in combination with ex situ, are nowadays becoming increasingly important means of supporting and maintaining the level of phytodiversity stability. Eucommia ulmoides Oliver is a deciduous tree, belongs to the Eucommiaccac family. This tree species grows in the territory of the NULES of Ukraine Arboretum in a single copy, is a valuable decorative introducer and has a number of medicinal properties. It is currently in poor condition and needs treatment and reproduction for preservation. The purpose of the study was to develop biotechnology of microclonal propagation of E. ulmoides Oliv plants in the initial stages of reproduction for using healthy plant-materials in landscaping and cultivation in botanical gardens. The relevance of microclonal reproduction of E. ulmoides Oliv is substantiated. Features of introduc tion into culture of E. ulmoides Oliv were established with using various sterilizing agents, types of explants and growing conditions. Different types of shoots 15–25 cm long with apical and lateral buds, selected from the donor plant in February – March were used for the work. Murasighe & Skoog (MS) and Driver (DKW) nutrient media were used for cultivation of Eucomia, with cytokinins and auxins addition to their composition: 6-BA (6-benzylaminopurine), NAC (α-naphthylacetic acid) and IAA (β-indolyl-3-acetic acid) both individually and in combination. Sucrose (0.3 %), mesoinositol (100 mg / l) and agar (0.7 %) were added to the medium, the pH of the medium was 5.7–5.8. The influence of different sterilization variants on the development of Eucommia ulmoides Oliver microshoots has been studied. As a result of the research the technique of sterilization of E. ulmoides Oliv explants with 78 % efficiency due to surface sterilization of 0.1 % HgCl2 on annual shoots has been worked out. Optimal nutrient media were established at the stage of in vitro culture of E. ulmoides Oliv. In particular, for the microclonal reproduction of E. ulmoides Oliv., the nutrient medium MS 1 supplemented with 0.5 mg·l-1 BA and 0.1 mg·l-1 NAA is recommended in the initial stages.

Сирек және жойылып бара жатқан Іле (Berberis iliensis) және Қарқаралы (Berberis karkaralensis) бөріқарақаты түрлерінің in vitro өсіру жағдайларын оңтайландыру

Today, many living organisms are negatively affected by climate change and anthropogenic activities, which leads to a decrease in their numbers. One of these rare and endangered plant species is the Ili barberry (Berberis iliensis) and the Karkaraly barberry (Berberis karkaralensis). This work is devoted to the optimization study of the cultivation conditions for a rare and endangered species of Ili barberry and Karkaralinsky barberry in vitro. To obtain sterile and viable explants, the sterilizing agent was a solution of 0.5% «Domestos» (7 min). For the regeneration of the main shoot of the barberry, the optimal nutrient medium is Murashige and Skoogwith the addition of 6-benzylaminopurine- 0.5 mg/l, gibberellic acid- 1.0 mg/l, indole-3-butyric acid -0.01 mg/l, where regeneration was 80% for the Ili barberry, barberry karkaralinsky - 70%. For the multiplication of Berberis iliensis microshoots, the Quoirin & Lepoivre culture medium with the addition of 0.75 mg/l - benzylaminopurine is optimal; the number of microshoots formed was 3.6 per explant. Root formation is one of the most difficult stages in micropropagation. For the rooting of microshoots of Karkaralinsky barberry, a nutrient medium of ½ Murashige and Skoog was used with the addition of indolylbutyric acid -1.5 mg/l.

Bacterial Cellulose and Pullulan from Simple and Low Cost Production Media

In this study, the production rate of both water-insoluble EPS, bacterial cellulose, and water-soluble EPS, P, was improved through сultivation of their producers on a nutrient media containing industrial wastes, and their material properties were analyzed. The growth rate and productivity of Gluconoacetobacter xylinus C3 strain on media with industrial wastes was investigated. An optimal nutrient medium based on molasses was selected for the bacterial cellulose producer. The nutrient medium contains 2% molasses, 1% yeast extract and peptone in a 1: 1 ratio, 0.3% sodium hydrogen phosphate, 0.1% citric acid and 1% ethanol. Cultivation of Gluconoacetobacter xylinus C3 strain on this medium for 7 days at 25–30 °С ensures its high productivity – 8.21 g/L. The composition of the optimized medium with molasses provides high mechanical properties (tensile strength – 37.12 MPa and relative elongation at break – 3.28%) of bacterial cellulose and does not affect the polymer microfibrillar structure. A modified Czapek-Dox medium with 10% molasses and 1% peptone is preferable for the exopolysaccharide accumulation by A. pullulans C8 strain. The optimized media has an advantage over the traditionally used media in terms of the efficiency of exopolysaccharide accumulation and cost reduction. The pullulan yield in media was 10.08 g/l, that is 1.5 times higher than in a standard Czapek-Dox medium. The surface morphology and microstructure of the pullulan samples obtained on different media showed minor changes. Therefore, the replacement of carbon source for molasses in a Czapek-Dox media for pullulan production did not alter the polymer content and viscosity.

FORMATION OF THE MULTIPLE GRAPE SHOOTS IN CULTURE IN VITRO ON DIFFERENT NOURISHING MEDIA

Semi-liquid nutrient media were used for the multiple grape shoots induction. Aim of the research was to study the agar concentration effect in different nutrient media and define influence of the media consistency on the survivability parameters, growth and development of initial explants, and the multiple grape shoots induction. Methods. Initiation of culture in vitro formation from explants, microclones cultivation, and the method of the meristematic plant regeneration were used. Nutrient media were liquid (without agar as a component), semiliquid (with agar content of 4 g/l), and solid (with agar content of 8 g/l), including an addition of 1 mg/l 6-BAP to all experimental media. The registration of the explants’ survivability, time of the beginning of axillary buds proliferation, and amount of obtained shoots was conducted. Results. Semi-liquid nutrient media were used. The advantage of using semi-liquid media in comparison with solid and liquid nutrient media is revealed. The search of the optimal nutrient medium for multiple grape shoots induction in vitro was successfully done. Modified semiliquid MS was determined as the optimal nutrient media. Its application contributed to better survivability, differentiation, and regeneration of grape meristems. On average in sortes, it formed 9.59 pcs., shoots, which further increases the coefficient of grape multiplication in culture in vitro to 1: 9. Conclusions. The application of the improved method of induction of multiple shoots contributed to a better survivability of the initial explants of grapes of technical varieties in culture in vitro, accelerates the processes of proliferation of the axillary microbuds, and the processes of generating more shoots suitable for subsequent clonal micropropagation.

Improvement of metals leaching optimal parameters from technogenic waste dumps with using mesophilic association of acidophilic chemolithotrophic bacteria

The results of improving the standard parameters of the process of bacterial leaching of metals, which allow to shorten the term of carrying out technological process and increase the efficiency of the extraction of germanium and other useful components are provided in article. To achieve the goal the mineralogical and chemical analysis of waste dumps have been made and the composition and leaching potential of the main groups of microorganisms of waste dumps were established also. Bacterial leaching was carried out using the mesophilic association of acidophilic chemolithotrophic bacteria, which are characterized by a high level of leaching activity. To determine the optimal parameters of the biotechnological process were compared results obtained with used: standard 9K medium with ferrous iron as an energy source; at constant elevated values of the oxidation-reduction potential at the level of 0.650 V; with ferric iron as an oxidizing agent, and using an optimized nutrient medium (OPM), the composition of which has been determined on the basis of mathematical optimization. The obtained results showed, that the using of ferric iron, OPM and elevated values the oxidation-reduction potential allow to shorten the term of leaching process and increase the recovery of germanium and other metals by 2 to 5 times. Moreover, the advantage of leaching with OPM is the absence of iron ions, which are an impurity and interfere with further sorption recovery of germanium from polymetallic solutions to produce germanium concentrate. Thus, optimum process parameters of bacterial leaching of metals, allows using them depending on the conditions and needs receiving of the targets metals from waste dumps.