Murashige-Skoog Medium Research Articles

Biotization of in vitro calli and embryogenic calli of oil palm (Elaeis guineensis Jacq.) with diazotrophic bacteria Herbaspirillum seropedicae (Z78)

A continuous discovery of novel practices for in vitro plant culture is essential to promote better plant growth. An alternative approach to the conventional micropropagation practice could be achieved through biotization by supplying selected diazotrophs to the plant materials. Due to this considerable potential, an artificial symbiosis was created between the Herbaspirillum seropedicae strain Z78 (ATCC 35893) and in vitro oil palm (Elaeis guineensis Jacq.). This experiment was carried out with the aim of studying the effects of H. seropedicae inoculation on growth of oil palm calli and embryogenic calli. A total of 4 treatments were tested for calli and embryogenic calli of oil palm on modified Murashige–Skoog medium, namely: T1: 100 % nitrogen source + 100 % 2,4D; T2: 25 % nitrogen source + 10 % 2,4D; T3: Z78 sonicated cell + 25 % nitrogen source + 10 % 2,4D and T4: Z78 pellet cell + 25 % nitrogen source + 10 % 2,4D. The ability of the inoculum to produce phytohormone indole-3-acetic acid was detected by ultra-performance liquid chromatography. Scanning electron microscopy and transmission electron microscopy showed successful artificial symbiosis of calli and embryogenic calli-bacterium. This study has demonstrated that Z78 pellet cell and sonicated cell have the potential to promote growth of in vitro calli and embryogenic calli of oil palm under symbiosis conditions.

Influence of biogenic metals nanoparticles on the morphogenetic processes effectiveness of mint peppery (Mentha piperita L.) in the culture in vitro

Investigation of the influence of nanoparticles of copper and cobaltous on the micropropagation of plants of peppermint (M. piperita L.) in such varieties as «Mama», «Chornolist» and «Lydia» on the basis of a complex of culture methods of isolated tissues and organs in vitro is carried out. According to the research, it can be stated that there is the positive influence of the cobaltous and copper nanoparticles on the morphogenesis of peppermint explants in culture in vitro. After 28 days of cultivation in MurashigeSkoog growth medium (MS) with nanoparticles of copper with concentration of 0.8 mg/l there was a significant increase in the quantity of the bines of such varieties as «Mama» for 50%, «Сhernolistnaya» — 43%, in growth medium with addition of copper with a concentration of 1.2 mg/l the plants of peppermint of variety «Lydia» has the enlargement of 48% (p<0.05). The number of nodes in a nutrient medium with copper nanoparticles with a concentration of 0.8 mg/l was larger in comparison with controlled medium of variety «Mama» for 29% (p<0.05), «Сhernolistnaya» — 31% (p<0.05), and in the medium with copper nanoparticles with a concentration of 1.2 mg/l of mint plants of such variety as «Lydia» for 43% (p<0.05). The optimum density of cobaltous in the MS growth medium was identified. It is 0.4 mg/l and the optimum concentration of copper is 0.8–1.2 mg/l. The usage of such modification of Murashige-Skoog growth medium allows to receive more (an average 27%) regenerated peppermint by plants. After 28 days the coefficient of reproduction of peppermint plants of variety «Mama» was 1:14, «Lydia» — 1:13, «Сhernolistnaya» — 1:15.

A CORRECTION HAS BEEN PUBLISHED:Casein hydrolysate as a sole source of nitrogen for in vitro grown tobacco plantlets

We tested the capability of plants to utilize protein as the exclusive source of nitrogen. The aim of this study was to find out how such a nutrition affected plantlet growth, photosynthetic performance, and N assimilation metabolism in tobacco (Nicotiana tabacum L., cv. Petit Havana SR1) grown in vitro. Plantlets grown in a casein-supplemented (CA) medium were compared to plantlets grown in a complete Murashige-Skoog (MS) medium, plantlets grown in an ammonium-deficient medium (N1), or plantlets grown in a nitrate-reduced medium (N2). In addition, the plantlets were grown in the presence or absence of 1.5 % (m/v) saccharose as an additional carbon source. Casein, similarly as inorganic N limitation, reduced generally plantlet growth, whereas no significant effects were observed on photosynthetic parameters evaluated by chlorophyll a fluorescence. Although addition of saccharose stimulated the plantlet growth particularly in the MS, it showed a rather negative influence both on the growth and on the photochemical efficiency of photosystem II in the plantlets grown in the CA and N1. The activities of enzymes involved in N assimilation, such as nitrate reductase (NR) and glutamine synthetase (GS), were lower in the plantlets grown in the CA, N1, and N2, both in leaves and in roots. On the other hand, glutamate synthase and glutamate dehydrogenase were employed by the plantlets grown in the CA. The presence of saccharose in the growth medium stimulated mainly NR and GS activities in the MS grown plantlets, whereas enzyme activities of the plantlets grown on the N1, N2, and CA were not significantly influenced. We proved that the tobacco plantlets can utilize casein as the sole source of N particularly during their photoautotrophic cultivation. Contrary to positive effects of photomixotrophic nutrition for the MS grown plantlets, exogenous sugar seemed to diminish the ability of the casein-supplemented plantlets to utilize efficiently the additional C source.

Effect of La(NO3)3 and Ce(NO3)3 on shoot induction and seedling growth of in vitro cultured anoectochilus roxburghii

Anoectochilus roxburghii, a highly valuable medicinal plant species, is threatened in its native habitat. To ensure the sustainability of this useful resource, we studied the effect of La(NO3)3 and Ce(NO3)3 on plant tissue culturepropagated A. roxburghii. Apical buds, mid-stem segments, and basal rhizome segments were taken from aseptic seedlings of A. roxburghii and cultured in vitro on half-strength Murashige-Skoog medium containing different concentrations (up to 5.0 mg/L) of La(NO3)3 or Ce(NO3)3. After 100 d of culturing, average heights of plantlets derived from apical buds were 6.0 cm with 5.0 mg/L La(NO3)3 and 6.2 cm with 2.0 mg/L Ce(NO3)3, which respectively increased by 28.0% and 32.0% as compared with that in the non-treated control group. The optimum concentration for shoot induction from mid-stem segments was 1.0 mg/L Ce(NO3)3 which had a better proliferation times of 1.5-fold and an average length of 3.0 cm compared with 1.0-fold and 2.2 cm in the control group. Optimum growth from basal rhizome segments was achieved on media supplemented with 3.0 mg/L Ce(NO3)3, which provided a better proliferation times of 5.1-fold and an average shoot length of 4.5 cm compared with corresponding control values of 2.0-fold and 3.5 cm. Our results showed that La(NO3)3 and Ce(NO3)3 can accelerate A. roxburghii regeneration, which was probably due to the effect on chlorophyll contents, enzymes activity (superoxide dismutase, catalase and peroxidase) and malonldialedhyde contents caused by the addition of La(NO3)3 or Ce(NO3)3.

Activity of seaweed extracts and polysaccharide-enriched extracts from Ulva lactuca and Padina gymnospora as growth promoters of tomato and mung bean plants

Although marine seaweeds have been used as biostimulants since the beginning of modern agriculture, studies have only recently focused on the ability of seaweed extracts and their polysaccharides to enhance growth of plants. In this work, two bioassays were carried out to study the growth-promoting activity of seaweed extracts and polysaccharide-enriched extracts from Ulva lactuca and Padina gymnospora, obtained in neutral and alkaline conditions. Initially, the effect of seaweed extracts and polysaccharide-enriched extracts on seed germination and growth-promoting activity on tomato (Solanum lycopersicum cv. Rio Grande) plants under in vitro conditions was studied. Half-strength Murashige-Skoog (MS) medium with or without sucrose was supplemented with different concentrations of seaweed extracts (2, 4, and 10 mg mL−1) or polysaccharide-enriched extracts (0.2, 0.4, and 1.0 mg mL−1). The parameters evaluated were germination percentage, radicle and shoot length, and dry weight. In a second experiment, polysaccharide-enriched extracts at 1.0 mg mL−1 and indole-3-butyric acid as the control were studied for root inducer activity in mung bean (Vigna radiata). The majority of seaweed extracts had an inhibitory effect on seed germination. However, a significant effect (P ≤ 0.05) on tomato seedling growth (except for dry weight) was shown with seaweed extracts at 2 mg mL−1 included in half-strength MS medium with sucrose (30 g L−1). Moreover, 10 mg mL−1 neutral and alkaline seaweed extracts had an inhibitory effect on the parameters evaluated. In contrast, polysaccharide-enriched extracts obtained from U. lactuca and P. gymnospora promoted germination and stimulated growth of tomato plants compared to the controls. Additionally, treatment of mung bean hypocotyl cuttings with polysaccharide-enriched extracts of U. lactuca and P. gymnospora induced rooting more rapidly and in greater number compared to the controls. These results provide evidence that polysaccharide-enriched extracts act as an effective growth-promoting treatment.

Trace elements’ uptake and antioxidant response to excess of manganese in in vitro cells of sensitive and tolerant wheat

Manganese, a microelement important for plant metabolism, when accumulated at higher doses, may act as a stress factor. Such action of this element is not fully recognized and is currently being intensively studied. The influence of manganese, at high (1, 2 and 3 mM) concentrations, on the induction of oxidative stress in wheat cells was studied under in vitro conditions. Calli of two wheat cultivars, different in terms of stress tolerance, were cultured for 7 days on Murashige–Skoog media with or without auxin (2,4-D) and with additional Mn supplementation. Changes of malondialdehyde (MDA) concentration and the activity of enzymes (SOD, CAT and POX) involved in oxidative response as well as the accumulation of Mn and elements essential for plant development (Mn, Mg, S, Ca, K, P, Na, Zn, Mo, Cu, Fe) were detected. Moreover, proline and carbohydrate contents were determined to check the induction of osmotic stress. An increase of lipid peroxidation (expressed by MDA content), induced by ROS generation, was smaller in the tolerant (‘Parabola’) cultivar than in the sensitive one (‘Raweta’). The activation of antioxidative enzymes was more effective in the cells of tolerant wheat, where a lower quantity of Mn was accumulated. Mn uptake was correlated with a decrease of an amount of almost all the investigated elements. Auxin presence in the culture media accelerated the stressogenic effect of Mn.

Glucosinolate biosynthesis as influenced by growth media and auxin in hairy root cultures of Kale (Brassica oleracea var. acephala)

This study aimed to investigate the effects of various growth media and three types of auxins on glucosinolate biosynthesis in hairy root cultures of kale. Four different glucosinolates (4-hydroxyglucobrassicin, glucobrassicin, 4-methoxyglucobrassicin, and neoglucobrassicin) were used in this study. The accumulation of glucosinolates was influenced by both media and auxin treatments. Of the media treatments, full-strength Gamborg’s B5 medium (B5) supported the highest accumulation of total glucosinolates, followed by full-strength Murashige–Skoog medium (MS), whereas the lowest glucosinolates accumulation was in the half-strength MS medium treatment. The accumulation of glucobrassicin was very high, followed by 4-methoxyglucobrassicin, irrespective of the growth medium used. The highest content of glucobrassicin was measured in cultures in B5 medium, while MS medium resulted in the highest accumulation of 4-methoxyglucobrassicin. Half-strength B5 medium resulted in the highest content of neoglucobrassicin, and Schenk and Hildebrandt medium (SH) supported the highest content of 4-hydroxyglucobrassicin. The total and individual levels of glucosinolates in hairy root cultures of kale were all influenced by exposure to the nine auxin treatments, with the exception glucobrassicin. In general, levels of glucosinolate decreased with increasing concentrations of auxins. Treatment with the auxin indole-3-butyric acid (IBA) 0.1 resulted in the highest total concentration of glucosinolates, measuring 1.83 times higher than that of hairy root cultures treated with naphthalene acetic acid (NAA) 1.0, which resulted in the lowest glucosinolate concentrations. Of the four glucosinolates, glucobrassicin and 4-methoxyglucobrassicin contents were considerably higher. The auxin IBA 0.1 promoted the highest 4-methoxyglucobrassicin accumulation in the hairy root cultures of kale. The auxin treatment indole-3-acetic acid (IAA) 0.1 resulted in the highest amount of 4-hydroxyglucobrassicin and neoglucobrassicin. Hairy root cultures could be a valuable alternative approach for the production of glucosinolate compounds from kale.

Asymbiotic Germination of <i>Phalaenopsis</i> cv. ‘Dublin’ Seeds in Relation to Pollination Months and Nutrient Media

Orchids are one of the most popular flowers in the world and Phalaenopsis genus shares maximum sales in global floral industry among them. Successful pollination, followed by capsule formation and in vitro seed germination, is a powerful tool for mass propagation of orchids. In the present study, flowers of Phalaenopsis cv. ‘Dublin’ were self-pollinated artificially during the months of November, December, January, February, March and April. The influence of pollination month over capsule formation was assessed and it was observed that pollination during December resulted as maximum with 86% capsule formation, followed by 72% during January. However, only 40% successful capsule formation occurred following pollination during April. Seeds from mature capsules developed through artificial pollination in different months were cultured on three different culture media (modified Chen, ½Murashige Skoog and Vacin-Went medium) for in vitro germination. A maximum of 97% germination of seeds (collected from capsules developed through pollination in January) was recorded on modified Chen medium. Nevertheless, the earliest initiation of germination occurred around 12 days of culture when seeds were harvested from capsules (derived through pollination in December) and inoculated on modified Chen medium. The plantlets developed following germination were rooted on ½MS supplemented with 2.5 g/l activated charcoal. Subsequently, well-rooted plantlets were acclimatized on potting mixture that contained cocopeat, charcoal, industrial cartridge and the bites of yonolit (1:1:2:4; v/v) with a very high rate of success. A seed culture protocol that crops a bulk number of seedlings in a short time span is advantageous and the present report eventually provides such simple yet proficient method that can be further tested.

PENGARUH NAA DAN BAP TERHADAP EKSPLAN PEGAGAN (Centella asiatica (L.) Urb.)

Pegagan ( Centella asiatica (L.) Urb.) representing one of theplant which good of as drug. Plantof pegagan ( Centella asiatica (L.) Urb.) good of to launch the urine, degrading blood pressure and quicken to heal the hurt. Ready of the seed for the crop of drug which require to be paid attention by quality from itself seed. One of the alternative to get the uniform seed and a flash in the pan is with the technique of tissue culture. Tissue culturelaboratory research was conducted Researchand Development Center for Medicinal Plantsand Traditional MedicineTawangmangu. Research Method use the Random Device of Complete Group at MS (Murashige Skoog) media with the treatment ofplant growth regulator the NAA concentration 0, 1, 3 and 5 mg / l and BAP concentration 0, 1, 2, 3 and 4 mg / l). Result of research show the combination of giving of NAA 1 until 3 mg / l and BAP 1 until 5 mg / l of is condition of explan experience of the change become the callus. Treatment of combination NAA 3 mg / l and BAP 4 mg / l give the best result to callus forming with the quicker callus forming time that is 25 day. Keywords : Pegagan, Centella asiatica (L.) Urb., tissue culture, NAA, BAP

Salinity effects on pigments of Turnera diffusa (Willd.) in vitro

Salinity is an unfavourable factor to plant growth, development, and quality because high salinity increases oxidative stress and damage in plants. Damiana (Turnera diffusa Willd.) plants grow in arid zones. They are socio-economically important as they have industrial potential. Currently, no reports on their tolerance to salinity are available. This study was performed to determine the effect of salinity in damiana plants in an in vitro model under controlled conditions by applying NaCl in three levels (0.1, 1.0, and 1.5%) in Murashige-Skoog medium. To prove decrease in salinity stress, exogenous salicylic acid (SA) treatments (1 and 10 ppm) were tested, and biomass and water content were determined. Photosynthetic pigments and their degradation were determined with high performance liquid chromatography to assess the degree of salinity damage. To define the adaptation mechanism to salinity, specific peroxidase (POX) activity was quantified by spectrophotometric assay. Results showed that the determined parameters (chlorophyll a, chlorophyll b, s-carotenes, pheophytin, violaxanthin, and zeaxanthin contents) reduced progressively with increase in salinity level. Chlorophyllide a and pheophorbide a contents were not modified by salinity stress but responded to SA presence. POX activity increased in all treatments with plants supplemented with NaCl. Treatments by SA did not modify the negative effects of NaCl on the photosynthetic pigments, but even so significantly enhanced POX activity compared with the untreated stressed plants (NaCl without SA). The results showed that damiana plants were moderately salt-tolerant and could be grown in soils with such characteristics.

Biofertilization with Azospirillum brasilense improves in vitro culture of Handroanthus ochraceus, a forestry, ornamental and medicinal plant

Biofertilization with plant growth-promoting rhizobacteria is a potential alternative to plant productivity. Here, in vitro propagation of Handroanthus ochraceus (yellow lapacho), a forest crop with high economic and environmental value, was developed using the Azospirillum brasilense strains Cd and Az39 during rhizogenesis. Epicotiles of in vitro plantlets were multiplied in Woody Plant Medium (WPM). For rooting, elongated shoots were transferred to auxin-free Murashige-Skoog medium with Gamborg's vitamins and WPM, both at half salt concentration (½MSG and ½WPM), and inoculated with Cd or Az39 at the base of each shoot. Anatomical studies were performed using leaves cleared and stained with safranin for optical microscopy and leaves and roots metalized with gold-palladium for scanning electron microscopy (SEM). In ½WPM auxin-free medium, A. brasilense Cd inoculation produced 55% of rooting, increased root fresh and dry weight (45% and 77%, respectively), and led to lower stomata size and density with similar proportion of open and closed stomata. Both strains selectively increased the size or density of glandular trichomes in ½MSG. Moreover, bacteria were detected on the root surface by SEM. In conclusion, the difference in H. ochraceus response to A. brasilense inoculation depends on the strain and the plant culture media. Cd strain enhanced rooting in auxin-free ½WPM and produced plantlets with features similar to those expected in ex vitro plants. This work presents an innovative in vitro approach using beneficial plant-microorganism interaction as an ecologically compatible strategy in plant biotechnology.

Morphogenic Response of Leaf and Petiole Explants of Broccoli Using Thidiazuron

Broccoli (Brassica oleracea var. italica) is an important nutritionally rich vegetable cole crop grown in the world. Environmental stress, pests, and diseases cause enormous yield losses because of a limited gene pool. Genetic manipulation is becoming an important method for broccoli improvement. The objective of present study was to evaluate the potency of thidiazuron (TDZ) as a plant growth regulator in evoking morphogenic responses in leaf and petiole explants of broccoli. An efficient, reproducible, and high frequency plant regeneration protocol has been standardized in broccoli cv. Solan green head. Leaf and petiole explants were cultured on Murashige-Skoog (MS) medium, supplemented with a wide range of TDZ concentrations. The following treatments were designed for efficient in vitro shoot regeneration: TDZ alone, TDZ with adenine, TDZ with naphthalene acetic acid (NAA), and TDZ with indole acetic acid (IAA). Among the 36 combinations of growth regulators used, the highest percentage of leaf explants producing shoot (89.25%) was recorded on MS medium containing 1.0 μM TDZ and 0.107 μM NAA. The multiple shoot regeneration response of petiole explant producing shoots (91.55%) was obtained on MS medium containing 2.0 μM TDZ and 0.107 μM NAA. Shoot multiplication and elongation were obtained on the same medium. For root regeneration in in vitro regenerated shoots, different concentrations of NAA were applied. High frequency (100%) root regeneration response with healthy and vigorous roots was observed on MS medium supplemented with 0.54 μM NAA. The regenerated plantlets with well-developed shoots and root system were transferred to pots containing cocopeat and successfully acclimatized. We recommend 1.0 μM TDZ with 0.107 μM NAA and 2.0 μM TDZ and 0.107 μM NAA combinations for adventitious shoot regeneration from leaf and petiole explants in broccoli cv. Solan green head respectively. This is the first report on high frequency organogenesis from leaf and petiole explants of broccoli cv. Solan green head using thidiazuron.

Effect of genotype and medium culture content on microspore-derived embryo formation in Chinese cabbage (Brassica rapa ssp. chinensis) cv. Lastochka

The influence of different factors on microspore embryogenesis in Chinese cabbage (B. rapa ssp. chinensis) was studied. A genotype dependence for embryo formation was observed. The majority of embryos and plants were obtained from microspores isolated from flower buds (2-2.9 mm in length) and cultured in the NLN liquid medium with 13% sucrose (w/v) supplemented with 24-epibrassinolide and 1% activated charcoal. Embryos cultured on the 1/2 Murashige-Skoog culture medium with 2% sucrose (w/v), 0.1 mg/L benzylaminopurine, and 3g/L Phytagel stimulated the formation of secondary embryos that resulted in development of large number of doubled haploid plants.

Azospirillum brasilense increased salt tolerance of jojoba during in vitro rooting

Jojoba and Azospirillum brasilense can survive at high NaCl concentrations but with some undesirable effects. The aim of this work was to evaluate whether A. brasilense inoculation reduced the negative effects of salinity on several clonal lines of the jojoba plant. Shoots were induced in half-strength Murashige–Skoog medium with 24.6μM or 49.2μM indole-3-butyric acid for 6 days and grown in an auxin-free medium with 0, 40, 80, 120 or 160mM NaCl and inoculated or not with 107cfu of A. brasilense Cd or Az39 for 45 days. Rooting percentage, shoot and root biometric parameters, and root index were determined. Regressions of rooting percentage and root index with respect to NaCl concentration were performed for each combination of bacterization and indole-3-butyric acid induction, and the 50% inhibitory concentration (IC50) was determined. A. brasilense Cd and Az39 promoted rhizogenesis and increased root index and IC50, reducing the undesirable effects of NaCl on jojoba rooting. Both strains were successfully reisolated at the end of the experiment. Here, we establish a useful model for evaluating plant-bacteria interactions and selecting strains that promote plant growth under saline conditions.

Agrobacterium-mediated transformation of tomato (Lycopersicon esculentum L. cv. Hezuo 908) with improved efficiency

We developed an efficient and rapid Agrobacterium-mediated tomato transformation protocol by using cotyledon and hypocotyl as explants. The transgenic nature of the regenerants was confirmed through β-glucuronidase activity staining, polymerase chain reaction (PCR), quantitative real-time PCR (qPCR) analysis and northern blot analysis. In the used protocol, the optimized experimental conditions of the tomato genetic transformation were Agrobacterium liquid concentration, equivalent to optical density (OD600) of 1.0, 20 min (cotyledon) or 30 min (hypocotyl) infection time and 2 d of co-culturing period. The optimized medium for shoot induction was Murashige & Skoog (MS) medium + 0.5 mg/L indole-3-acetic acid (IAA) + 0.5 mg/L 6-benzylaminopurine (BAP) for cotyledon and MS + 0.5 mg/L IAA + 2.0 mg/L BAP for hypocotyl. The root induction rate was the highest on the MS medium, supplemented with 0.1 mg/L IAA. The transformation frequency reached 40% per explant. The received results are a base for a larger scale development of transformation procedure.

HERITABILITY OF TRAITS RELATED TO GERMINATION AND MORPHOGENESIS IN VITRO IN ORNAMENTAL PEPPERS

The species Capsicum annuum L. has a low seed viability and is recalcitrant in vitro. Therefore, studies on the genetic control of traits favorable for in vitro development obtained from a diallel analysis might be interesting. Here we investigated the heritability of germination percentage and plant morphogenesis in in vitro seedlings of ornamental peppers. Four lines of ornamental pepper (UFPB 132, UFPB 134, UFPB 137, and UFPB 390) and their crosses were used. The seeds were germinated in half-strength MS (Murashige Skoog) medium. On the 7th day of cultivation, the percentage of germinated seeds (GS) was analyzed. After 30 days, the percentage of deformed plants (PD); presence of root (PR); length (RL) and number of roots (NR); hypocotyl length (HL) and diameter (HD); and number (NL), length (LL) and width of leaves (LW) were analyzed. Data were subjected to analysis of variance and Hayman’s diallel analysis. The effects of treatment (parents, F1 hybrids, and -reciprocals) were significant at the level of 1% for all traits, indicating existence of genetic variability. In the sufficiency test of the additive-dominant model, variables GS, NR and NL showed non-significant values for the regression coefficients. This demonstrated the validity of the imposed assumptions and indicated the feasibility of this model for the study of these genetic traits. Correlations Wi+Vi and Yii were negative for GS, PR and RL, ranging from -0.3137 to -0.9760, indicating these traits were controlled by dominant alleles. Positive correlations for other traits were seen, suggesting that the observed increase in the traits’ values are mostly caused by recessive alleles. Additive effects were more important than dominant effects for all traits, except NL, which indicates that these traits will be transmitted to the next generations.

In Vitro Development of Meristematic Centers in Cyclea peltata—A Pharmaceutically Important Plant

The callus derived from nodal explant of Cyclea peltata on Murashige Skoog medium supplemented with α-naphthalene acetic acid + benzyl amino purine (5.4 μM + 0.44 μM) was used for indirect shoot proliferation. The stem derived calli of 50–60 d old were inoculated to half strength and full strength Murashige Skoog media with different growth regulators at varying concentrations. The callus, upon subculture to the medium supplemented with either naphthalene acetic acid or indole acetic acid alone or in combination with cytokinins, led to the development of shiny, small, globular cells. Histological studies revealed the induction of callus from the cambial cells of the explant and the presence of numerous, actively dividing, darkly stained meristematic centers in the organogenic callus.

Вплив розміру експлантів та фітогормонального складу живильного середовища на відновлення меристем часнику після кріоконсервування методом вітрифікації

Investigation of the effect of explants dimensions and composition of nutritive medium on morphometric parameters of devitrified garlic meristems will enable a significant optimizing of the cryopreservation techniques intended to establish a collection of its cultivars at low temperature bank. Apexes were subjected to dehydration with plant vitrification solution (1M sucrose + 2M glycerol + 2.5 M ethylene glycol based on Murashige-Skoog salt medium) for 120 min, placed into a polypropylene container and immersed into liquid nitrogen. Thawing was performed in a water bath at 40°C, washing of cryoprotectants was carried-out by a two-fold transfer into the medium with 0.3 Msucrose. It has been shown that the solution under study was non-toxic to garlic meristems and allowed the obtaining of a high level of viability. After cryopreservation by means of vitrification of garlic meristems of ‘Manuylivskyy’ spring variety the number of regenerated plants reached (56.3 ± 11.2)%, and ‘Duchess’ winter variety did (44.8 ± 3.7)%. For the cultivation of the cryopreserved garlic meristems as the phytohormone the kinetin is advisable to be used in concentration of 0.5 mg/l. It has been shown that after cryopreservation by vitrification the garlic meristems of 2–3 mm had the highest viability (60–70%). Larger meristems (3.5–4 mm) had a high regenerative potential but low viability (below 25%). In smaller meristems (1–1.5 mm) the viability rates were reduced (30–47%) as well as a regenerative potential. In the meristems of 0.5 mm a regenerative potential was absent. Probl Cryobiol Cryomed 2015; 25(1): 3-12.

Optimization of factors affecting efficient shoot regeneration in chrysanthemum cv. Shinma

Factors affecting shoot regeneration were optimized in order to develop an efficient protocol for shoot regeneration in leaf explants of the chrysanthemum (Chrysanthemum morifolium Ramat) cultivar Shinma. The maximum number of shoots per explant (9.7) was regenerated in leaf explants of 6-week-old donor plants cultured on Murashige–Skoog medium containing a combination of 0.5 mg L−1 6-benzyladenine and 0.5 mg L−1 α-naphthaleneacetic acid with initial dark treatment for 7 days. Among the different auxins tested, indolebutyric acid was the most effective for root induction and development, whereas the presence of ethylene inhibitors strongly suppressed shoot regeneration. Ploidy levels were analyzed by flow cytometry: there was no ploidy variation between the regenerated plants and the mother plant grown under greenhouse conditions. The new protocol will facilitate genetic transformation and micropropagation of chrysanthemum cv. Shinma.

High-efficiency propagation of Chinese water chestnut [Eleocharis dulcis (Burm.f.) Trin. ex Hensch] using a temporary immersion bioreactor system

Several studies have reported different propagation methods for the Chinese water chestnut [Eleocharis dulcis (Burm.f.) Trin. ex Hensch]. To date, none of these methods have efficiently achieved large-scale plantlet production using minimal labor input. The present study investigated the propagation of Chinese water chestnut shoot tips derived from corm and creeping rhizomes in a temporary immersion bioreactor system (TIBS) compared with propagation using conventional semi-solid medium methods. We evaluated the regeneration capacity of the corm and creeping rhizome shoot tips of E. dulcis Guiti No. 2 sp. nov. and found that culturing under optimized TIBS conditions (15 min of immersion into nutrient medium every 8 h) produced the highest multiplication rate (36.5 times) and shoot-forming capacity (SFC, 68.9), and reduced the vitrification rate. Creeping rhizome shoot tips had slightly higher SFC than corm shoot tips; the highest shoot multiplication rate of 43.7 times in TIBS was obtained using Murashige–Skoog (MS) medium containing 4 mg L−1 6-benzylaminopurine and 0.5 mg L−1 1-naphthaleneacetic acid (NAA). These optimized conditions were applied to corm shoot tips of E. dulcis Guiti No. 1 var. nov., E. dulcis Guiti No. 2 sp. nov., and E. dulcis Guifengti No. 1 sp. nov. Root formation was observed during the shoot multiplication stage on semi-solid MS medium; however, root formation was not noted during the shoot multiplication stage in TIBS. While TIBS produced a higher multiplication rate and SFC indices, there were no significant differences in average number of shoots per explant between TIBS and semi-solid medium. We also evaluated the effects of different immersion frequencies and NAA concentrations on TIBS propagation of E. dulcis Guiti No. 2 sp. nov. shoot tips by measuring the rooting percentage, average number of roots (length ≥ 3 cm) per explant, maximum root length, and root-forming capacity (RFC). The results showed that the medium containing 2.0 mg L−1 NAA at the rooting stage produced the best-developed root systems with the highest rooting percentage, maximum root length, and highest RFC (75.8 %, 8.5 cm, and 3.8, respectively); it also reduced the immersion frequency. The optimized conditions produced plantlets that had a greater capacity to acclimatize after transfer to the greenhouse. E. dulcis Guiti No. 1 var. nov. plantlets had the highest survival rate (95 %), largest diameters, and highest number of creeping rhizomes. The results of this study demonstrate that TIBS is a reliable and efficient method for large-scale propagation of Chinese water chestnut plantlets.