AGRIS - 

2021

Kexiu Wang | Mingxia Tang | Jianjun Hu | Wei He | Wenting Yang | Zaag, P.V.

A good quality planting material for a highly productive minituber production in aeroponic systems is important. This study was conducted to determine if the age and source of the planting materials used in the aeroponic beds would influence tuber productivity. Repeated harvesting of unrooted apical cuttings from hydroponic plants would substantially reduce cost of the planting material by more than 50%. Four different planting materials were evaluated in two experiments in each of two seasons: 28 day old in vitro plants; 28 day old in vitro plants place in a hydroponic system for 14 days; unrooted apical cuttings taken from 42 and 56 day old hydroponic plants. These were evaluated using three cultivars (cvs) in an aeroponic system in the spring season and autumn seasons in a semi tropical setting in Sichuan. All four sources of planting material had 'juvenile' simple rounded leaves when transplanted to aeroponic beds. Whole hydroponic plants had the head start compared to in vitro plants and out yielded in vitro plants in the spring season for cv Mira and Chuanyu 117. Apical cuttings for cv Favorita and Chuanyu 117 from hydroponically grown mother plants rooted in four to seven days grew vigorously and yielded higher tuber weight compared to the younger whole hydroponically sourced plants during the spring season. Autumn season results had generally lower yields and no statistically significant differences observed between the four types of planting material. There was a positive correlation between mid season stolon number and final tuber number. The most economical and productive planting material are the 1st and 2nd unrooted apical cuttings taken from hydroponically grown mother plants.

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University Library, University of the Philippines at Los Baños - Philippines

2021

Kovalev, N.N. | Leskova, S.E. | Mikheev, E.V. | Pozdnyakova, Yu.M. | Esipenko, R.V., Far Eastern State Technical Fishery Univ., Vladivostok

Обосновано применение салициловой кислоты как стимулятора роста микроводорослей. Проведена оценка влияния широкого диапазона концентраций салициловой кислоты на динамику роста Tetraselmis suecica в накопительной культуре. Культивирование осуществляли в монокультуре. Прирост биомассы водорослей находили по увеличению числа клеток, просчитанных в каждом опыте в 3 камерах Горяева под световым микроскопом. Продолжительность экспериментов составляла 14 дней. Показано, что салициловая кислота в концентрациях 0,4-1,9*10Е-5 моль ингибирует рост водоросли. Рост контрольной культуры имеет 2 выраженных пика численности на 4 и 12 день эксперимента. Внесение салициловой кислоты в концентрациях 0,44-1,9*10Е-5 моль сопровождалось изменением характера кривых роста: максимум количества клеток отмечался на 12 день эксперимента. Более высокая концентрация фитогормона (3,75*10Е-5 моль) обеспечивала рост плотности культуры на 414% за 14 дней эксперимента. Рост культуры T. suecica в контрольной группе составил 332%. Рассчитаны значения удельной скорости роста T. suecica в различные периоды культивирования. После 14 дней эксперимента проведена оценка биохимического состава биомассы микроводорослей, которая показала стимуляцию салициловой кислотой в концентрации 3,75*10Е-5 моль накопления углеводов. Содержание углеводов в экспериментальной группе было на 14% больше по сравнению с контрольной группой. Высокая концентрация фитогормона подавляла накопление в культуре белка, липидов и хлорофилла и стимулировала накопление углеводов. Сделано предположение, что возможным механизмом разнонаправленного действия салициловой кислоты является ее влияние на синтез и катаболизм через ингибирование синтеза и метаболизма эндогенных гормонов растений. | The use of salicylic acid as a stimulator of microalgae growth substantiated. The influence of a wide range of salicylic acid concentrations on the growth dynamics of Tetraselmis suecica in enrichment culture has been evaluated. Cultivation was carried out in monoculture. An increase in algal biomass was measured by the increasing number of cells counted in each experiment in 3 Goryaev chambers under a light microscope. The duration of the experiments was 14 days. It is shown that salicylic acid in concentrations of 0.4-1.9*10Е-5 M inhibits the growth of algae. The growth of the control culture has 2 pronounced peak numbers on 4th and 12th days of the experiment. The introduction of salicylic acid in concentrations of 0.44-1.9*10Е-5 M was accompanied by a change of the growth curves: the maximum number of cells was observed on 12-th day of the experiment. A higher concentration of phytohormone (3.75*10Е-5 M) provided an increase in crop density by 414% over 14 days of the experiment. The growth of T. suecica culture in the control group was 332%. The values of the specific growth rate of T. suecica were calculated for different periods of cultivation. After 14 days of the experiment, the biochemical composition of the microalgae biomass was evaluated, which showed stimulation with salicylic acid at a concentration of 3.75*10Е-5 M carbohydrate accumulation. The high concentration of phytohormone suppressed the accumulation of protein, lipids and chlorophyll in the culture and stimulated the accumulation of carbohydrates. It has been suggested that a possible mechanism for the multidirectional action of salicylic acid is its effect on synthesis and catabolism through inhibition of the synthesis and metabolism of endogenous plant hormones.

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Central Scientific Agricultural Library - Russian Federation

2021

Chantal, Isabelle | Minet, Cécile | Berthier, David

Animal African Trypanosomosis (AAT or Nagana) is a severe vector-borne disease caused by protozoan parasites belonging to the Trypanosomatidae family and is usually cyclically transmitted by blood-sucking tsetse flies. AAT remains a major problem in sub-Saharan Africa. Among the main AAT causative agents, Trypanosoma congolense (T. congolense or Tc) is one of the most important trypanosome species, in terms of economic and animal health impacts, infecting cattle and a wide range of animal hosts as well. To advance in AAT prevention and control, it is essential to better understand trypanosome biology and pathogenesis using bloodstream form (BSF) in vitro culture. The in vitro cultivation of T. congolense IL3000 BSF strain is already well established and widely used in research studies and drug activity assays. However, it may probably no longer truly reflect the reality of field trypanosome strains, due to decades of use and subsequent modifications. Here, we propose a novel culture protocol that supports the long-term in vitro growth of the animal-infective BSFs of three Savannah and Forest types of T. congolense strains, including T. congolense clone IL1180, which is not only a field strain but also a commonly-used reference strain in experimental animal assays. We established a homemade culture medium which made it possible to sustain T. congolense IL1180 growth from infected mouse blood for 18 days in axenic conditions. Moreover, we developed an efficient freezing/thawing system that allowed, for the first time, T. congolense IL1180 BSF growth within 30 days after thawing. Our results on T. congolense adaptation to in vitro culture are encouraging for future gene studies using new molecular tools or for new therapeutic drug assays.

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Centre de coopération internationale en recherche agronomique pour le développement - France

2021

Loseva, A.I. | Pozdnyakova, A.V. | Prosekov, A.Yu. | Ostapova, E.V. | Al'tshuler, O.G.

Одним из востребованных в фармакологической сфере растением является Orchis maculata L., содержащий такие биологически активные вещества, как алкалоиды, флавоноиды, каротиноиды, антоцианы и стерины. На территории России данный вид занесен в Красную книгу, следовательно, актуально использовать биотехнологические методы выращивания in vitro. Проводили подбор фитогормонов в питательной среде для получения максимального прироста биомассы каллусной культуры in vitro. Объектом исследования стала 28-дневная каллусная культура Orchis maculata L., выращенная на различных питательных средах: основа среда Мурасиге-Скуга, варьирование количеством фитогормонов (от 0,1 мг до 2,0 мг): цитокинов (кинетин, 6-БАП) и ауксинов (ИМК, 2,4-Д). Из каллусных клеток, обладающих наибольшим сухим остатком, получали экстракт (экстрагент – 70% этанол, продолжительность 60 мин, температура 60 град. С, соотношение сырье:экстрагент – 1:10). Спиртовой экстракт подвергался анализу на определение качественного и количественного состава БАВ с помощью ВЭЖХ (Shimadzu LC-20 Prominence) и ТСХ (Sorbfil ПТСХ-АФ-А). Наибольший прирост биомассы каллуса наблюдается при соотношении цитокин:ауксин 1:1. На 28-е сут культивирования индекс роста 11,71+-0,01 наблюдался при использовании 6-БАП+2,4-Д (1,0 мг + 1,0 мг на 1 л), на питательной среде № 29. Наибольшее содержание сухих веществ (23,81+-0,02%) наблюдалось при использовании среды № 13: кинетин (1,5 мг)+2,4-Д (1,5 мг). Методом ТСХ в этанольном экстракте каллусных клеток (выращенных на среде № 13) было обнаружено присутствие кверцетина и кверцетин-3,7-O-бета-D-глюкопиранозидов в трисахаридной и дисахаридной форме, а методом ВЭЖХ – рутина (73,07+-3,65 мг/кг), кверцетина (15,08+-0,75 мг/кг), апигенина (23,63+-1,18 мг/кг), колеофолида (12,53+-0,63 мг/кг). На разработанной питательной среде можно получать каллусную культуру Orchis maculata L. для дальнейшего извлечения из нее фармакологически значимых биологически активных веществ. | Orchis maculata L. is a medicinal plant of high demand that contains alkaloids, flavonoids, carotenoids, anthocyanins, and sterols. This plant is listed in the Red Book of Russia, therefore, domestic pharmacology needs in vitro biotechnological methods for industrial cultivation of Orchis plant. The research objective was to select phytohormones in a nutrient medium that would increase the callus biomass of Orchis maculata L. The study featured callus cultures grown on various nutrient media and such phytohormones as cytokines (kinetin, 6-BAP) and auxins (BCI, 2,4-D). The amount of the phytohormones varied from 0.1 mg to 2.0 mg. The callus cells with the highest dry residue were subjected to extraction with 70% ethanol at 60 deg. С for 60 min; the ratio of raw materials : extractant was 1:10. The extract was analyzed for the qualitative and quantitative composition of biologically active substances using HPLC (Shimadzu LC-20 Prominence) and TLC (Sorbfil PTSKh-AF-A). The greatest increase in callus biomass was observed when the ratio of cytokine: auxin was 1:1. On cultivation day 14, the growth index was 7.71+-0.01 when using 6-BAP + 2.4-D (1.0 mg + 1.0 mg per 1 L) on nutrient medium No. 29. Medium No. 13, kinetin (1.5 mg) + 2,4-D (1.5 mg), provided the highest amount of dry substances (10.47+-0.02). The TLC method (medium No. 13) revealed the content of quercetin and quercetin-3,7-O-beta-D-glucopyranosides in trisaccharide and disaccharide forms. The HPLC method detected rutin (73.07+-3.65 mg/kg), quercetin (15.08+-0.75 mg/kg), apigenin (23.63+-1.18 mg/kg), and coleofolide (12.53+-0.63 mg/kg). The resulting nutrient medium made it possible to obtain enough callus Orchis maculata L. to extract biologically active substances for medicinal purposes.

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Central Scientific Agricultural Library - Russian Federation

2021

Asyakina, L.K. | Fedorova, A.M. | Dyshlyuk, L.S.

The most effective extractants were selected and the optimal parameters of the extraction process of biologically active substances from the dry biomass of root cultures in vitro of medicinal plants were determined: Baikal skullcap (BS), common skullcap (CS) and white cinquefoil (WC). Methanol, ethyl acetate, acetone, isopropanol, diethyl ether and 70% ethanol were tested as extractants. Maximum yield of total extract in processing biomass of root crops BS and CS were observed when using 70% ethanol (10.32 and 10.37%), and from WC – diethyl ether (16.37%). The following extraction parameters were optimized: duration (10, 30, 60, 120, 180 and 360 min), temperature (25, 40, 60 and 100 deg. C) and water duty (1:1, 1:2, 1:5, 1:10 and 1:20). It has been found that the maximum yield of dry extract from the dried roots of CS, BS and WC is observed with extraction duration of 60 min. The optimal water duty for extracting BAS from CS and BS is 1:10, and from WC – 1:20. The optimal temperature level of the process of extracting BAS from CS, BS and WC is temperatures of 60, 50 and 40 deg. C, respectively. The maximum yield of dry extract from CS, BS and WC, subject to optimal process parameters, is 16.38; 18.68 and 17.57%, respectively. It is concluded that the optimization of extraction parameters will allow more economically using valuable raw materials and preserving the natural populations of medicinal plants of Siberia. | Подобраны наиболее эффективные экстрагенты и определены оптимальные параметры процесса экстракции биологически активных веществ из сухой биомассы корневых культур in vitro лекарственных растений: шлемника байкальского (ШБ), шлемника обыкновенного (ШО) и лапчатки белой (ЛБ). В качестве экстрагентов испытывали метанол, этилацетат, ацетон, изопропанол, диэтиловый эфир и 70%-ный этанол. Максимальный выход тотального экстракта при обработке биомассы корневых культур ШБ и ШО наблюдали при использовании 70%-го этанола (10,32 и 10,37%), а из ЛБ – диэтилового эфира (16,37%). Оптимизировали следующие параметры экстракции: продолжительность (10, 30, 60, 120, 180 и 360 мин), температура (25, 40, 60 и 100 град. C) и гидромодуль (1:1, 1:2, 1:5, 1:10 и 1:20). Установили, что максимальный выход сухого экстракта из высушенных корней ШО, ЩБ и ЛБ наблюдается при продолжительности экстракции 60 мин. Оптимальный гидромодуль при экстрагировании БАВ из ШО и ШБ составляет 1:10, а из ЛБ – 1:20. Оптимальным температурным уровнем процесса экстрагировании БАВ из ШО, ШБ и ЛБ является температуры 60, 50 и 40 град. C соответственно. Максимальный выход сухого экстракта из ШО, ШБ и ЛБ при соблюдении оптимальных параметров процесса составляет 16,38; 18,68 и 17,57% соответственно. Сделан вывод, что оптимизация параметров экстракции позволит экономней расходовать ценное сырье и сохранять естественные популяции лекарственных растений Сибири.

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Central Scientific Agricultural Library - Russian Federation

2021

Shornikov, D.G. | Matushkina, O.V. | Pronina, I.N. | Shamshin, I.N. | Lyzhin, A.S. | Matushkin, S.A. | Kruzhkova, L.V.

Исследования проводили с целью оценки влияние модифицирующего действия питательной среды и условий культивирования на геном микрорастений плодовых и ягодных культур. В качестве биологических объектов исследования использовали линии клоновых подвоев яблони – 54-118, 62-396, культивируемые in vitro 1, 3, 6, 10 лет, а также каллусные ткани и микропобеги, полученные методом адвентивного органогенеза и методом регенерации из апикальных меристем, линии и каллусные ткани клонового подвоя груши ПГ-12, культивируемые in vitro в течение 4, 10, 18 лет, линии cортов малины Яркая и Оранжевое чудо, культивируемые в условиях in vitro 1, 2, 3 года. Анализ генетического полиморфизма культивируемых in vitro микрорастений осуществляли с использованием 8 микросателлитных маркеров CH03d07, CH03d01, CH03d08, CH02c02b (яблоня и груша), RhM001, RhM011, RiM015, RiM019 (малина). Для клонового подвоя яблони 54-118 идентифицировано 9 фрагментов, из которых 3 (33,3%) оказались полиморфными. Наибольшее количество полиморфных фрагментов отмечено для локуса CH03d07 (4 фрагмента). Для клонового подвоя яблони 62-396 суммарно выявлено 14 фрагментов по 4 изучаемым локусам. Из них полиморфными были 4 фрагмента (28,6%). Наибольшее количество полиморфных фрагментов отмечено для локуса CH03d08 (4 фрагмента). Для клонового подвоя груши ПГ-12 общее количество фрагментов по изучаемым локусам было равно 11, в том числе 3 полиморфных (27,3%). Для сортов малины Оранжевое чудо и Яркая идентифицировано 10 фрагментов. При этом уникальных фрагментов не выявлено. Результаты анализа SSR-локусов у плодовых и ягодных растений свидетельствуют о высоком уровне генетической стабильности в процессе культивирования in vitro независимо от использованной модели размножения и сроков культивирования. | The paper aimed to assess the modifying effect of nutrient medium and cultivation conditions on the genome of microplants of fruit and berry crops. As biological objects of the study, we used lines of apple clonal rootstocks 54-118 and 62-396 cultivated in vitro for 1, 3, 6, and 10 years, as well as callus tissues and microshoots obtained by the method of adventive organogenesis and by the method of regeneration from apical meristems; lines, and callus tissues of PG-12 pear clonal rootstock cultivated in vitro for 4, 10, and 18 years; and lines of raspberry varieties Yarkaya and Oranzhevoye Chudo, cultivated in vitro for 1, 2, and 3 years. Genetic polymorphism of microplants cultivated in vitro was analyzed using eight microsatellite markers CH03d07, CH03d01, CH03d08, CH02c02b (apple and pear), RhM001, RhM011, RiM015, and RiM019 (raspberry). For 54-118 clonal rootstock of apple tree, we identified nine fragments, three of which (33.3%) turned out to be polymorphic. The largest number of polymorphic fragments was observed for CH03d07 locus (four fragments). For 62-396 clonal rootstock of apple tree, we identified 14 fragments at the four studied loci; four fragments were polymorphic (28.6%). The largest number of polymorphic fragments was noted for CH03d08 locus (four fragments). For PG-12 pear clonal rootstock, the total number of fragments at the studied loci was 11, including three polymorphic fragments (27.3%). For raspberry varieties Oranzhevoye Chudo and Yarkaya, we identified 10 fragments. At the same time, we did not identify any unique fragments. The results of the analysis of SSR loci in fruit and berry plants indicated a high level of genetic stability during in vitro cultivation, regardless of the reproduction model used and the cultivation time.

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Central Scientific Agricultural Library - Russian Federation

2021

Kononenko, A.N. | Ivakhnova, O.F. | Loginova, Yu.N.

Изучали влияние применения оригинальных микробиопрепаратов (МБП) на основе бактерии Bacillus (штаммы Black-01, Bis-88, TS-3) на урожайность семенного материала картофеля раннеспелых сортов Удача и Импала. Опыт закладывали с растениями in vitro, далее их пересаживали в вегетационные сосуды объемом 5 л. Обработки МБП проводили трижды согласно схеме опыта. Перед посадкой в каждую лунку вливали 50 мл 10%-ного водного раствора МБР, через 3 нед. после посадки и затем через 5 нед. растения опрыскивали 1%-ным раствором. Растения выращивали в защищенном грунте. Получены данные по среднему количеству клубней и их массе с одного растения, а также по фракционному составу картофеля при применении МБП. В результате проведенных исследований выявлено, что применение МБП в целом положительно влияет на продуктивность семенного картофеля. Установлено, что на исследуемых сортах картофеля большее количество миниклубней и, соответственно, большая средняя масса миниклубней с одного растения были получены при применении МБП Black-01 и Bis-88. Массу миниклубня удалось повысить с 252 до 380 г (Удача) и с 187 до 483 г (Импала). | The use effect of original microbial preparations (MBP) based on Bacillus bacteria (strains Black-01, Bis-88, TS-3) on the yield of seed material of early ripening potato varieties 'Udacha' and 'Impala' was studied. The experiment was laid with plants in vitro, and then they were transplanted into vegetative vessels with a volume of 5 l. MBP treatment was carried out three times, according to the scheme of the experiment. Before planting, 50 ml of a 10% aqueous solution of MBR was poured into each well. The plants were sprayed with 1% solution of MBP in 3 weeks after planting and then after 5 weeks again. The plants were cultivated in protected ground. Data were obtained on the average number of tubers and tuber weight per one plant, as well as on the fractional composition of potatoes when used MBP. As a result of the conducted studies, it has been revealed that the use of MBP in general has a positive effect on the productivity of seed potatoes. It was found that on the studied potato varieties, a larger number of mini-tubers and, accordingly, a larger average weight of mini-tubers per one plant were obtained when using MBP Black-01 and Bis-88. The weight of a mini-tuber could be increased from 252 to 380 g ('Udacha') and from 187 to 483 g ('Impala').

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Central Scientific Agricultural Library - Russian Federation

2021

Rouinsard, Alexandre | Institut de Recherche en Horticulture et Semences (IRHS) ; Université d'Angers (UA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut Agro Rennes Angers ; Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro) | Agrocampus Ouest | Agnès Grapin

The micropropagation of variegated cultivars (several colors on the same leaf) can generate off-type plants to the original phenotype, particularly in the case of monocotyledonous plants. However, because of their highly ornamental character, these cultivars have a strong commercial potential. The objective were to determine the micropropagation potential of variegated monocots in an industrial and commercial context, to define the stability of their phenotypes, and to understand the origin of off-types. Observations of leaf sections under a confocal laser scanning microscope on the four studied cultivars: Yucca gloriosa 'Variegata' (YgVAR), Yucca flaccida 'Golden Sword' (YflGS), Phormium tenax 'Jessie' (PtJE), and Cordyline australis 'Pink Passion' (CaPP) revealed that all cultivars were periclinal chimeras, i.e., a variegation bound to stratified meristem with several different genotypes.In micropropagation, YgVAR showed high compliance rates, PtJE moderate rates and CaPP low rates. Histological analyses on vitroplants revealed major differences in the development and in vitro propagation behavior of these three species of the order Asparagales: YgVAR developed only axillary meristems (AxM), PtJE mainly AxM and some adventitious meristems (AdM), and CaPP both AxM and AdM. While preformed axillary meristems maintain the chimeric structure, adventitious meristems maintain little. Thus, the variegation stability of these chimeras depends on their propensity to propagate by adventitious meristems. Trials to lower apical dominance and stimulate axillary meristems were conducted, as well as transcriptome analysis of different variegated tissues in YflGS. Industrial production protocols adapted to each cultivar are finally proposed. | La micropropagation des variétés à feuillage panaché (plusieurs couleurs sur une même feuille) peut générer des plantes non conformes au phénotype d’origine, particulièrement dans le cadre de plantes monocotylédones acaules. Or, par leur caractère fortement ornemental, ces variétés présentent un fort potentiel commercial. L’objectif était de déterminer les potentialités de micropropagation des monocotylédones panachées dans un contexte industriel et commercial, de définir la stabilité de leurs phénotypes, et de comprendre l’apparition des plantes hors-type. Les observations de coupes foliaires au microscope confocal à balayage laser sur les quatre cultivars étudiés Yucca gloriosa ‘Variegata’ (YgVAR), Yucca flaccida ‘Golden Sword’ (YflGS), Phormium tenax ‘Jessie’ (PtJE) et Cordyline australis ‘Pink Passion’ (CaPP) ont révélé que tous les cultivars étaient des chimères périclines, soit une panachure liée à un méristème stratifié avec plusieurs génotypes différents. En micropropagation,YgVAR a présenté des taux de conformité élevés, PtJE des taux modérés et CaPP de faibles taux. Des analyses histologiques sur les vitroplants ont mis en évidence des différences majeures dans le développement et le comportement de multiplication in vitro de ces trois espèces de l’ordre des Asparagales : YgVAR n'a développé que des méristèmes axillaires (AxM), PtJE principalement des AxM et quelques méristèmes adventifs (AdM), et CaPP à la fois des AxM et des AdM. Alors que les méristèmes axillaires préformés maintiennent la structure chimérique, les méristèmes adventifs la maintiennent peu. Donc la stabilité de la panachure de ces variétés dépend de leur propension à se propager par méristèmes adventifs. Des essais pour abaisser la dominance apicale et stimuler les méristèmes axillaires ont été menés, ainsi qu’une analyse du transcriptome des différents tissus panachés chez YflGS. Des protocoles de production industrielle adaptés à chaque cultivar sont finalement proposés.

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Institut national de la recherche agronomique - France

2020

Erma Safitri

Background and Aim: Octamer-binding transcription factor 4 (OCT4) and sex-determining region Y-box 2 (SOX2) are transcription factors whose functions are essential to maintain the pluripotency of embryonic stem cells. The purpose of this study was to derive stem cells for in vitro culture and to maintain their viability and pluripotency, with the goal to obtain a cell line for transplantation in patients with degenerative diseases or injuries. This research focused on examining the effect of low oxygen tension on the ability of bone marrow-derived mesenchymal stem cells (BM-MSCs) to express OCT4 and SOX2 in vitro.Materials and Methods: BM-MSCs were obtained from femurs of 2000 to 3000 g New Zealand male rabbits. BM-MSCs were divided into three groups to test different culture conditions: A control group under hyperoxia condition (21% O2) and two treatment groups with low oxygen tension (1% and 3% O2). We characterized the BM-MSCs using flow cytometric measurement of cluster differentiation 44 (CD44) and cluster differentiation 90 (CD90) expression. The expression of OCT4 and SOX2 was measured by immunofluorescence staining after 48 h of incubation in chambers with normal or low oxygen tension with controlled internal atmosphere consisting of 95% N2, 5% CO2, and 1% O2 (T1) and 3% O2 (T2). We considered OCT4 and SOX2 as two markers of pluripotency induction. All immunofluorescence data were subjected to a post hoc normality Tukey's honestly significant difference test; all differences with p<5% were considered significant.Results: BM-MSCs were positive for CD44 and CD90 expression after isolation. Oxygen tension culture conditions of 1% and 3% O2 led to OCT4 and SOX2 expression on culture days 2 and 4 (p<0.05), respectively, as compared to the hyperoxia condition (21% O2).Conclusion: Based on the OCT4 and SOX2 immunofluorescence data, we conclude that the stem cells were pluripotent at low O2 tension (at 1% O2 on day 2 and at 3% O2 on day 4), whereas under 21% O2 the OCT4 and SOX2 were not expressed.

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Directory of Open Access Journals - Sweden

2020

Aspuria, E.T. | Bernardo, E.L. | Tayangona, M.A.D.

A protocol for the establishment of embryogenic cell suspension in abaca was developed based on the International Network for the Improvement of Banana and Plantain (INIBAP) procedure for Musa. Meristematic buds from shoot cultures were exercised and cultured on P5 medium for several cycles until 'scalps' formed. These scalps were then used to produced embryogenic complexes (ECs) in 2,4-D containing media. Discrete primary somatic embryos (SEs) were observed on ECs 3-4 wk after initial culture. After 6 mo, 6% of the 150 inoculated scalps formed into ECs that had at least 10 SEs. These were then used to commence cell suspensions. Nine liquid media formulations were tested, and only M2 medium produced cell lines that had a characteristics bright yellow suspension indicative of embryogenic potential. However, doubling time during the initiation phase with M2 medium was lower than the doubling time in the rest of the media tested. In several months, cell lines in M2 medium stabilized wiyh an average doubling time of about 4 wk. The old medium was replenished with fresh medium every 4-7 d, replacing at least 80% of the old medium. Maintenance of fine homogenous suspension was done at monthly intervals by transferring 2 mL aliquots with a settled cell volume (SCV) of 0.2-0.3 mL from a 1-mo-old suspension culture to an 8 mL fresh medium in 125-mL Erlenmeyer flasks. As for embryo development, regeneration media trials showed that Murashige and Skoog (MS) and M2 media promoted the formation of yellow, nodular calli, or pro-embryogenic masses (PEMs). MS and M2 medium supplemented with 1 ppm AgNO3 (with or without ascorbic acid) could enhance PEM formation, especially if the cells were pre-induced to form PEMs by gradual reduction of 2,4-D while at the cell suspension stage. Sucrose or glutamine does not seem to have any promotive effect on PEM induction during the regeneration phase. These treatments, however, were not favorable for the conversion of PEMs into mature somatic embryos. Likewise, M3 regeneration media promoted PEM formation but failed to induce somatic embryogenesis in a separate experiment. PEMs derived from the 2,4-D reduction experiment were proliferated in M2 medium and transferred in P5 for embryo development. PEMs bearing mature somatic embryos in P5 medium were transferred to abaca shoot proliferation medium where fully developed shoots were obtained in 6 mo. Although the protocol requires validation, our results clearly demonstrate that abaca could undergo in vitro somatic embryogenesis (SE) and offer a transformation platform for modern genetic improvement work.

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University Library, University of the Philippines at Los Baños - Philippines