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Background Anarrhinum pubescens Fresen. (Plantaginaceae) is a rare plant, endemic to the Saint Catherine area, of South Sinai, Egypt. Earlier studies have reported the isolation of cytotoxic and anti-cholinesterase iridoid glucosides from the aerial parts of the plant. The present study aimed to investigate the chemical profiling of the wild plant shoots as well as establish efficient protocols for in vitro plant regeneration and proliferation with further assessment of the genetic stability of the in vitro regenerated plants. Results Twenty-seven metabolites have been identified in wild plant shoots using the Nuclear Magnetic Resonance (NMR) spectroscopy. The metabolites include alkaloids, amino acids, carbohydrates, organic acids, vitamins, and a phenol. In vitro propagation of the plant was carried out through nodal cutting-micropropagation and leaf segment-direct organogenesis. The best results were obtained when nodal cutting explants were cultured on Murashige and Skoog medium with Gamborg B5 vitamins supplemented with 6-benzylaminopurine (BAP) (1.0 mg/L) and naphthaleneacetic acid (NAA) (0.05 mg/L), which gave a shoot formation capacity of 100% and a mean number of shoots of 27.67 ± 1.4/explant. These shoots were successfully rooted and transferred to the greenhouse and the survival rate was 75%. Genetic fidelity evaluation of the micropropagated clones was carried out using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) molecular markers. Jaccard’s similarity coefficient indicated a similarity as high as 98% and 95% from RAPD and ISSR markers, respectively. Conclusions This study provides the chemical profiling of the aerial part of Anarrhinum pubescens. Moreover, in vitro regeneration through different tissue culture techniques has been established for mass propagation of the plant, and the genetic fidelity of the in vitro regenerated plants was confirmed as well. Our work on the in vitro propagation of A. pubescens will be helpful in ex situ conservation and identification of bioactive metabolites.

Introduction. Wild-crafting leads to the local extinction of many medicinal plants that are rich in phenolic substances. In vitro cultivation of cells and organs of higher plants can be the optimal solution to this problem. The research objective was to study the biosynthetic activity of in vitro extracts of wild Siberian plants. Study objects and methods. The study featured callus, cell suspension, and hairy root extracts of such Siberian medicinal plants as Eleutherococcus senticosus, Codonopsis pilosula, Platanthera bifolia, and Saposhnikovia divaricata. They were obtained by in vitro cultivation using modified nutrient media of Murashige and Skoog and Gamborg. The content of secondary metabolites was studied using the methods of thin-layer and high-performance liquid chromatography. A set of in vitro experiments tested the antioxidant and antimicrobial activity of the extracts. Results and discussion. All the samples demonstrated a high content of secondary metabolites of phenolic nature. Flavonoglycosides, apigenin, and rutin were found to be the predominant biologically active substances in the callus extracts. Flavonoglycosides dominated in the suspension extracts. The root extracts contained more caffeic acid, rutin, ecdysteroids, quercetin, apigenin, cardiofolin, and coleofolide than the callus and suspension cultures. The list of prevailing secondary metabolites in the root extracts included rutin, apigenin, coleofolide, and quercetin. All the extracts showed antimicrobial and antioxidant activity. Conclusion. All the extracts demonstrated good antioxidant and antimicrobial properties. Therefore, they can be used for the production of pharmaceuticals and biologically active food supplements as they can be helpful against infectious diseases, as well as oncological, cardiovascular, and neurodegenerative diseases linked to oxidative stress.

Efficient methods for callus induction and the high-frequency plant regeneration of Ruta chalepensis L. were established, and the phytochemical potential and antioxidant activity of a donor plant, ex-vitro-established micropropagated plants, and callus were also studied. Yellowish-green callus was induced with a frequency of 97.8% from internode shoot segments of the donor plant growing in soil in the botanical garden cultured on Murashige and Skoog (MS) medium containing 10 μM 2,4-D (2,4-dichlorophenoxyacetic acid) and 1 μM BA (6-benzyladenine). Adventitious shoots were regenerated from the yellowish-green callus on MS medium containing 5.0 μM (BA) and 1.0 μM 1-naphthaleneacetic acid (NAA), with a regeneration frequency of 98.4% and a maximum of 54.6 shoots with an average length of 4.5 cm after 8 weeks. The regenerated shoots were rooted in a medium containing 1.0 μM IBA (indole-3-butyric acid) and successfully transferred to ex vitro conditions in pots containing normal garden soil, with a 95% survival rate. The amounts of alkaloids, phenolics, flavonoids, tannins, and antioxidant activity of the ex-vitro-established micropropagated plants were higher than in the donor plant and callus. The highest contents of hesperidin and rutin (93.3 and 55.9 µg/mg, respectively) were found in the ex-vitro-established micropropagated plants compared to those obtained from the donor plant (91.4 and 31.0 µg/mg, respectively) and callus (59.1 and 21.6 µg/mg, respectively). The genetic uniformity of the ex-vitro-established micropropagated plants was appraised by the ISSR markers and compared with the donor plant. This is the first report describing the callus-mediated plant regeneration, as well as the production of phenolic compounds and antioxidant activities in R. chalepensis, which might be a potential alternative technique for the mass propagation and synthesis of bioactive compounds such as hesperidin and rutin.

Genetic transformation technology emerged as a useful tool to shorten the breeding time in plants and also allowed the improvement of the plants by only intended character without changing their other characters. To carry out gene transfer studies, the regeneration efficiency of plants must be known. In this study, in vitro regeneration efficiency was investigated in 9 genotypes in 3 different apple species; Malus domestica, Malus sieversii and Malus niedzwetzkyana. After explants shooted in the media, callus culture was applied to sterile leaves. Genotypes' shoot formation ratio was ranged from 8.33% to 75%. The genotype with the highest shoot was 'genotype 8', species of M. domestica, with a rate of 75%. Sterile leaves were taken from shoots for callus culture. The regeneration potential of genotypes ranged from 0% to 90%. While callus formation was not observed in 'genotype 4' and 'genotype 5' (M. domestica), 90% callus formation was observed in 'genotype 3' (M. domestica). With this study, it has been determined that there is a serious variation in the regeneration efficiency of different apple species and genotypes.

Callus cultures from ovary and ovule of Neottia nidus-avis (L.) Rich. were obtained. Dependence of callus genesis on growth regulators composition of nutrient medium it was shown. Cytomorphological analysis of callus cultures demonstrat their high morphogenetic potential.

Ligaria cuneifolia (R. et P.) Tiegh (Loranthaceae), known as liga, muérdago criollo, or Argentinean mistletoe, is a hemiparasitic plant with a broad distribution in central and northern Argentina. Pharmacological studies showed that L. cuneifolia extracts have hypolipemic, antioxidant, antibacterial, and immunomodulatory effects. We have established callus cultures from embryo and haustoria fragments. The highest frequency of callus formation from embryos (85%) was obtained on White medium with 4% (w/v) sucrose and 2.5 µM 1-naphtalene acetic acid and 9.2 µM kinetin as plant growth regulators (PGRs). From haustoria, the best result (35%) was obtained on Gamborg medium with 3% (w/v) sucrose and 0.45 µM 2,4-dichlorephenoxyacetic acid and 0.47 µM zeatin as PGRs. Thin layer chromatography showed that callus methanolic extract (2.5% w/v) had a lower content of flavonoids and proanthocyanins as compared to the wild plant (5% w/v for leaves, stems, and flowers), but a higher content of hydroxycinnamic acids. High performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) showed the presence of quercetin glycosides and phenolic acids in the methanolic extracts both from the parent plant and the callus obtained from embryo. Fil: Ricco, María Valeria. Universidad Maimónides. Facultad de Ciencias de la Salud. Carreras de Farmacia y Bioquímica. Cátedra de Farmacobotánica y Farmacognoscia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bari, Martín León. Universidad Maimónides. Facultad de Ciencias de la Salud. Carreras de Farmacia y Bioquímica. Cátedra de Farmacobotánica y Farmacognoscia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bagnato, Federico. Universidad Maimónides. Facultad de Ciencias de la Salud. Carreras de Farmacia y Bioquímica. Cátedra de Farmacobotánica y Farmacognoscia; Argentina Fil: Cornacchioli, Carolina. Universidad Maimónides. Facultad de Ciencias de la Salud. Carreras de Farmacia y Bioquímica. Cátedra de Farmacobotánica y Farmacognoscia; Argentina Fil: Laguía Becher, Melina. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico. Departamento de Estudios Biomédicos y Biotecnológicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Spairani, Leonardo Ulises. Ministerio de Relaciones Exteriores, Comercio Interno y Culto. Dirección Nacional del Antártico. Instituto Antártico Argentino; Argentina. Universidad Maimónides. Facultad de Ciencias de la Salud. Carreras de Farmacia y Bioquímica. Cátedra de Farmacobotánica y Farmacognoscia; Argentina Fil: Posadaz, Ariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Luis. Facultad de Turismo y Urbanismo; Argentina Fil: Dobrecky, Cecilia Beatriz. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Farmacobótanica; Argentina Fil: Ricco, Rafael Alejandro. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Farmacobótanica; Argentina Fil: Wagner, Marcelo Luis. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Farmacobótanica; Argentina Fil: Alvarez, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Maimónides. Facultad de Ciencias de la Salud. Carreras de Farmacia y Bioquímica. Cátedra de Farmacobotánica y Farmacognoscia; Argentina

In order to optimize direct regeneration of potato cultivars (Solanum tuberosum L.) including Arinda, Agria, Sante, Maradona and Marfona, a factorial experiment was carried out in the form of completely randomized design with three replications. Internode explants were placed on MS medium supplemented with different concentrations of 6-Benzylaminopurine (1, 2, 5, 10 and 15 mg/l) in combination with Kinetin (0.5, 1, 2, 5, 10 and 15 mg/l). Results showed that using 6-Benzylaminopurine (BAP) was more effective than Kinetin in direct regeneration of potato. Low concentrations of BAP had the greatest effect on regeneration and also length of root and shoot but maximum numbers of somatic embryos were obtained in the presence of high a concentration of BAP. Using kinetin only influenced the number and length of regenerated shoots. Although Kinetin had no effect on the rate of regeneration but BAP increased it. There were significant differences among cultivars in all traits. Among tested cultivars, Marfona had the best direct regeneration ability in vitro condition. Therefore, it can be concluded that induction of direct organogenesis in potato is largely dependent on the genotype, and the use of BAP growth regulator plays an important role in inducing direct regeneration in potato cultivars.

In vitro cultures of scarlet flax (Linum grandiflorum L.), an important ornamental flax, have been established as a new possible valuable resource of lignans and neolignans for antioxidant and anti-inflammatory applications. The callogenic potential at different concentrations of α-naphthalene acetic acid (NAA) and thidiazuron (TDZ), alone or in combinations, was evaluated using both L. grandiflorum hypocotyl and cotyledon explants. A higher callus induction frequency was observed on NAA than TDZ, especially for hypocotyl explants, with a maximum frequency (i.e., 95.2%) on 1.0 mg/L of NAA. The presence of NAA (1.0 mg/L) in conjunction with TDZ tended to increase the frequency of callogenesis relative to TDZ alone, but never reached the values observed with NAA alone, thereby indicating the lack of synergy between these two plant growth regulators (PGRs). Similarly, in terms of biomass, NAA was more effective than TDZ, with a maximum accumulation of biomass registered for medium supplemented with 1.0 mg/L of NAA using hypocotyls as initial explants (DW: 13.1 g). However, for biomass, a synergy between the two PGRs was observed, particularly for cotyledon-derived explants and for the lowest concentrations of TDZ. The influence of these two PGRs on callogenesis and biomass is discussed. The HPLC analysis confirmed the presence of lignans (secoisolariciresinol (SECO) and lariciresinol (LARI) and neolignan (dehydrodiconiferyl alcohol [DCA]) naturally accumulated in their glycoside forms. Furthermore, the antioxidant activities performed for both hypocotyl- and cotyledon-derived cultures were also found maximal (DPPH: 89.5%, FRAP 866: µM TEAC, ABTS: 456 µM TEAC) in hypocotyl-derived callus cultures as compared with callus obtained from cotyledon explants. Moreover, the anti-inflammatory activities revealed high inhibition (COX-1: 47.4% and COX-2: 51.1%) for extract of hypocotyl-derived callus cultures at 2.5 mg/L TDZ. The anti-inflammatory action against COX-1 and COX-2 was supported by the IC50 values. This report provides a viable approach for enhanced biomass accumulation and efficient production of (neo)lignans in L. grandiflorum callus cultures.

Thevetia peruviana is an ornamental plant considered source of biologically compounds with cardiac and antimicrobial activity. These compounds are normally extracted from different parts of the fully growth plants. In this work, extracts were obtained from callus and cell suspension cultures of T. peruviana and their antimicrobial activity was evaluated by disk diffusion tests against gram negative ( Salmonella thipimurium and Escherichia coli ) and gram positive ( Staphylococcus aureus and Bacillus cereus ) strains. Ethanol, methanol and hexane extracts from callus and cell suspension cultures showed biological activity. Methanolic cell suspension extract showed activity against B. cereus and S. aureus . Ethanolic cell suspension extract inhibit all the bacteria, especially S. thipimurium while hexanic extract showed resistance activity against S. thipimurium , S. aureus and B. cereus . In terms of the source of the extracts, hexane extracts obtained from cell suspension cultures showed a higher antimicrobial activity compared to callus, while ethanol extracts had an inverse behavior. These results outline in vitro cell culture of T. peruviana as a feasible biotechnological platform for the production of compounds with antimicrobial activity.

Flax ( Linum usitatissimum L.) is an ancient crop that is widely cultivated as a source of oil, fiber, and bioactive compounds. Flax fiber is traditionally used in textile industry, linseed oil is processed for industrial oils, paints, varnishes and bio-petroleum. Flaxseeds are also rich in α-linolenic acid and phytochemicals such as lignans. In addition to the commercial aspects, this species has been used widely and readily in biotechnological, developmental, and plant-pathogen interaction studies. Differences in the levels of endogenous hormones in various cultivars of flax significantly affected the intensity of callogenesis and determined the type and concentration of growth regulators necessary for callus production. The aim of our investigation was to optimize the culture conditions for callus formation and cell proliferation in liquid medium of the Polish cultivar of fiber flax – Modran. In the first step, 4 combinations of phytohormones in the medium were tested to obtain established callus tissue suitable for initiation of suspension culture. Next, we investigated the effect of chosen plant growth regulators on cell divisions, fresh and dry weight, and dispersal of callus cells in liquid medium. Fast growing and friable callus was obtained in a modified MS medium supplemented with 0.5 mg/l BAP and 0.1 mg/l NAA. We determined that for the initiation of cell suspension supplementation with 0.5 mg/l BAP and 0.5 mg/l NAA is optimal. The results obtained indicated that high concentration of cytokinin (BAP) in liquid medium limited cell proliferation and decreased biomass formation.

Arabidopsis tissue culture is valuable for any laboratory working on this model plant. Tissue culture methodology facilitates the production of a large number of plants that are genetically identical over a relatively short growth period. Currently this in vitro regeneration system is a good system to study the mechanism by which plants show regenerative plasticity. Plant regeneration is a key technology for successful stable plant transformation, while cell suspension cultures can be exploited for metabolite profiling, kinetic study and mining. In this paper we report methods for the successful and highly efficient in vitro regeneration of plants and production of stable cell suspension lines from cotyledons of Arabidopsis thaliana. It is an easy and reproducible method of regenerating Arabidopsis plants from callus culture. A combination of 6 benzylaminopurine BAP and a naphthalene acetic acid NAA in a Murashige and Skoogs MS based medium gives a high percentage of shoot formation. Further cell suspension culture were used to study the growth kinetics and also for checking level of antioxidant enzymes at different stages of culture. An expression analysis of antioxidant genes such as superoxide dismutase SOD and ascorbate peroxidase APX was also done at callus, shoot and root regeneration stage. It was found that levels and activity of these antioxidant enzymes were higher at regeneration stage, indicating antioxidant enzyme role in plant morphogenesis. Here we describe a standard protocol for regenerating Arabidopsis plants in tissue culture, and for preparing and observing samples using steriosome and bright field microscopy to study different stages of regeneration. Amrina Shafi | Insha Zahoor | Mudasir A Mir "Invitro Regeneration of Arabidopsis Thaliana from Suspension and Invitro Cultures and Analysis of Regeneration and Antioxidant Enzyme Levels" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-2 | Issue-1 , December 2017, URL: https://www.ijtsrd.com/papers/ijtsrd5843.pdf

Spherical hyaluronan-pectic gel particles (HPGPs) from hyaluronic acid (HA) and low-methyl esterified pectins of callus cultures (CC) of tansy, duckweed, campion and commercial apple pectin were obtained by the method of ionotropic gelation in the presence of calcium ions. We investigated the morphology, swelling and degradation of the obtained HPGPs in the conditions of a simulated gastrointestinal environment and established that the greatest stability in the artificial environment of the digestive tract is achieved with HPGPs obtained from the pectin of tansy CC. HPGPs can be used as potential carriers for drug delivery systems in parts of the small and large intestine.

Anthocyanins, the color compounds of plants, are known for their wide applications in food, nutraceuticals and cosmetic industry. The biosynthetic pathway of anthocyanins is well established with the identification of potential key regulatory genes, which makes it possible to modulate its production by biotechnological means. Various biotechnological systems, including use of in vitro plant cell or tissue cultures as well as microorganisms have been used for the production of anthocyanins under controlled conditions, however, a wide range of factors affects their production. In addition, metabolic engineering technologies have also used the heterologous production of anthocyanins in recombinant plants and microorganisms. However, these approaches have mostly been tested at the lab- and pilot-scales, while very few up-scaling studies have been undertaken. Various challenges and ways of investigation are proposed here to improve anthocyanin production by using the in vitro plant cell or tissue culture and metabolic engineering of plants and microbial culture systems. All these methods are capable of modulating the production of anthocyanins , which can be further utilized for pharmaceutical, cosmetics and food applications.

A somatic embryogenesis protocol was developed from the immature leaves of adult plants of the macaw palm. Leaf explants from different regions of the palm heart were used for callus initiation in a modified Y3 medium, supplemented with 2,4-D or Picloram at 450 μM. Calli were separated from the leaf explants at 6-, 9- and 12-month periods and transferred to a fresh culture medium of the same composition. They were multiplied for up to 120 days. Reduced concentrations of 2,4-D and Picloram were used to differentiate somatic embryos. They were then germinated in a medium without plant growth regulators. Morphological and anatomical analyses were conducted at different stages of the embryogenic process. The best results for callus induction were achieved by Picloram, when explants were maintained for up to 9 months on culture medium (64.9%). The farthest portions of the apical meristem were those that provided the biggest calli formation. The formation of the somatic embryos was observed from the calli multiplication phase. Reduction in concentrations of growth regulators failed to promote the formation of complete plants. Picloram at 450 μM promotes high callogenesis in leaf tissues of macaw palm, with a potential for somatic embryo formation.

Background. One of the alternative sources of obtaining biologically active substances is a culture of plant cells in vitro. The objective of this work is to introduce Artemisia vulgaris in the callus culture. The plant grows in Central Yakutia, known as a species with a high content of various biologically active substances used in traditional medicine and widely spread on a territory researched. Materials and methods. The phytomass of Artemisia vulgaris L. was collected during the expeditions on the territory of Amga’s region of the Republic of Sakha (Yakutia) in June-July 2016–2018. For introduction of Artemisia vulgaris L. into a callus culture, the actual leaves of sterile plants were used as explants. The plants were obtained via cultivating the seeds of a wild plant in controlled conditions. The cultivation was performed on a Murashige-Skoog nutrient medium with the use of two different growth regulators: 2,4-dichlorophenoxyacetic acid and kinetin, 6-benzylaminopurine and α-naphthylacetic acid. Morphological analysis of cells of the obtained calli was performed using a light microscope. The obtained primary calli were selected for further transplantation on a Murashige-Skoog nutrient medium with different concentrations of growth regulators. The growth dynamics of the callus wet mass was studied during one cycle (21 days). Results. The biomass of primary callus Artemisia vulgaris L. in the Murashige- Skoog nutrient medium with supplementation of 2,4-dichlorophenoxyacetic acid and kinetin was light yellow in color and had a dense consistency, and with the addition of 6-benzylaminopurine and α-naphthylacetic acid, it was also pale yellow in color, but with easily separable loose structure. Morphological analysis of cells of primary callus obtained showed the predominance of round-ovoid cells with a distinct nucleus. In a nutrient medium supplemented with 2,4-dichlorophenoxyacetic acid, kinetin, and α-naphthylacetic acid, the peak increase in callus wet mass was observed on day 11 after transplantation. On a nutrient medium supplemented with 2,4-dichlorophenoxyacetic acid, α-naphthylacetic acid, and 6-benzylaminopurine the peak increase in the callus wet mass was observed on the 14th day. Conclusions. The primary callus of Artemisia vulgaris L. were obtained from leaf explants taken from sterile plants. A nutrient medium with growth regulators was optimized to obtain callus and for a further introduction of Artemisia vulgaris in the callus culture. On a nutrient medium supplemented with 2,4-dichlorophenoxyacetic acid, α-naphthylacetic acid, and 6-benzylaminopurine callus biomass of Artemisia vulgaris L. is three times heavier than the biomass of callus obtained on a nutrient medium with 2,4-D, kinetin, and NAA. The obtained callus cultures had a soft, easily separable, loose structure, pale yellow in color.

DNA methylation changes can occur in some loci during callus culture, resulting in somaclonal variations (SVs). In the present study, we applied whole genome bisulfite sequencing to analyze context-specific DNA methylation changes in the pineapple genome between the cutting seedings and 5 SV plants. In general, SV plants exhibited methylation patterns analogous to those of cutting seedlings (CK). A total of 27.98% of the genomic cytosines of CK were methylcytosines, which was higher than that of 5 SV plants. Moreover, mCG and mCHG was hypermethylated, whereas mCHH was hypomethylated among the 5 SV plants genomic when compared with the CK. Most of the variation of DNA methylation was distributed in gene bodies, thus suggesting that phenotypic differences are probably perturbed by genes methylated from callus culture. In addition, the methylated genes were highly enriched for the Gene Ontology (GO) categories of binding and catalytic activity, cell part and organelle, cellular process, abiotic stimulus, and DNA modification. These results suggest that methylation mediates these pathways in the callus culture of pineapple. The results also suggested that the callus culture induced DNA methylation may result in the SV.

DNA methylation changes can occur in some loci during callus culture, resulting in somaclonal variations (SVs). In the present study, we applied whole genome bisulfite sequencing to analyze context-specific DNA methylation changes in the pineapple genome between the cutting seedings and 5 SV plants. In general, SV plants exhibited methylation patterns analogous to those of cutting seedlings (CK). A total of 27.98% of the genomic cytosines of CK were methylcytosines, which was higher than that of 5 SV plants. Moreover, mCG and mCHG was hypermethylated, whereas mCHH was hypomethylated among the 5 SV plants genomic when compared with the CK. Most of the variation of DNA methylation was distributed in gene bodies, thus suggesting that phenotypic differences are probably perturbed by genes methylated from callus culture. In addition, the methylated genes were highly enriched for the Gene Ontology (GO) categories of binding and catalytic activity, cell part and organelle, cellular process, abiotic stimulus, and DNA modification. These results suggest that methylation mediates these pathways in the callus culture of pineapple. The results also suggested that the callus culture induced DNA methylation may result in the SV.

Soybean (Glycine max (L) Merrill) is used in India mostly as a substantial fund of protein and oil, which makes the crop significantly important. Somaclonal variation has been researched as a base of additional variability for drought in soybean. In the present experiment calli/cell clumps/embryoids rose from immature and mature embryonic axis and cotyledons explants were exposed to different concentrations of polyethylene glycol (PEG6000). A discontinuous method proved to be superior as it permitted the calli/embryoids/cell clumps to regain their regeneration competence. A total of 64 (12.21%) plantlets of genotype JS335 and 78 (13.13%) of genotype JS93-05 were regenerated after four consequent subcultures on the selection medium with an effective lethal concentration of 20% PEG6000, and proliferated calli/embryoids/cell clumps were further subcultured on Murashige and Skoog regeneration medium supplemented with 0.5 mgL−1 each of α-napthalene acetic acid (NAA), 6-benzyladenine (BA) and Kinetin (Kn), 20.0 gL−1 sucrose and 7.5 gL−1 agar. Putative drought-tolerant plantlets were acquired from genotype JS93-05 (38) in more numbers compared to genotype JS335 (26). Random decamer primers confirmed the presence of variability between mother plants and regenerated plants from both the genotypes. Since these plantlets recovered from tolerant calli/embryoids/cell clumps selected from the medium supplemented with PEG6000, the possibility exists that these plants may prove to be tolerant against drought stress.

A growing demand for the ecologically pure products brings us for searching novel biotechnological approaches for plant cultivation. One of these approaches is the in vitro cultivation and further acclimatization of valuable plant species. The object of our investigation was Ajugareptance L. ornamental plant which possesses high metabolic activity. In vitro cultivation was carried out applying Murashige-Skoog nutrient medium and its modifications. Acclimatization of in vitro plants was implemented according Hazarika. In the presence of twice higher concentration of cytokinins over auxins and 0.2 mg/ml gibberellins callus culture was formed from the leaf explants. Callus tissue was formed in the presence of 0.2 mg/ml kinetin and 2 mg/ml indole-3-acetic acid which has denser structure than the first one. The shoot formation was observed on callus cultures growing on the same medium approximately after 5th passage. Callus culture growth was supported also by the adding of 2 mg/ml 2,4-dichlorophenoxyacetic acid. For the micropropagation, the already formed shoots were transferred to the nutrient medium which contains only 0.1 mg/ml 1-Naphthaleneacetic acid as a phytohormone. A. reptans culture has high regenerative ability and the micro-propagation index was 104 – 105. In vitro regenerated plants were successfully acclimatized to the soil conditions during two-week period.

International audience; Ocimum basilicum L. (Purple basil) is a source of biologically active antioxidant compounds, particularly phenolic acids and anthocyanins. In this study, we have developed a valuable protocol for the establishment of in vitro callus cultures of O. basilicum and culture conditions for the enhanced production of distinct classes of phenylpropanoid metabolites such as hydroxycinnamic acid derivatives (caffeic acid, chicoric acid, rosmarinic acid) and anthocyanins (cyanidin and peonidin). Callus cultures were established by culturing leaf explants on Murashige and Skoog medium augmented with different concentrations of plant growth regulators (PGRs) [thidiazuron (TDZ), α-naphthalene acetic acid (NAA), and 6-benzyl amino purine (BAP)] either alone or in combination with 1.0 mg/L NAA. Among all the above-mentioned PGRs, NAA at 2.5 mg/L led to the highest biomass accumulation (23.2 g/L DW), along with total phenolic (TPP; 210.7 mg/L) and flavonoid (TFP; 196.4 mg/L) production, respectively. HPLC analysis confirmed the differential accumulation of phenolic acid [caffeic acid (44.67 mg/g DW), rosmarinic acid (52.22 mg/g DW), and chicoric acid (43.89 mg/g DW)] and anthocyanins [cyanidin (16.39 mg/g DW) and peonidin (10.77 mg/g DW)] as a function of the PGRs treatment. The highest in vitro antioxidant activity was determined with the ORAC assay as compared to the FRAP assay, suggesting the prominence of the HAT over the ET-based mechanism for the antioxidant action of callus extracts. Furthermore, in vivo results illustrated the protective action of the callus extract to limit the deleterious effects of UV-induced oxidative stress, ROS/RNS production, and membrane integrity in yeast cell culture. Altogether, these results clearly demonstrated the great potential of in vitro callus of O. basilicum as a source of human health-promoting antioxidant phytochemicals.

Phenolic acids and flavonoids, important bioactive compounds of polyphenols, play a significant role in plants; their impact, mainly as antioxidants, on human health have been of great interest in recent years. The genetically uniform shoots of Eryngium alpinum L. cultured in vitro, developed via axillary buds and regenerated from callus tissue, maintained on the media supplemented with various plant growth regulators, were subjected to the phenolic acids and flavonoids quantitative analysis applying HPLC-DAD technique. In vitro cultures give the opportunity to perform the phytochemical studies on the protected species without harvesting the plant material from natural environment. Depending on the hormonal supplementation, the biomass from the shoot cultures accumulated from 11.41 to 25 times more phenolic acids [the total content ranged from 158.66 to 1817.96 mg/100 g of dry weight (DW)] and from 6.8 to 11.8 times more flavonoids (the total content ranged from 29.30 to 51.30 mg/100 g DW) than the shoots from the soil-grown plant. The polyphenols present in the shoot cultures include two phenolic acids: 3,4-dihydroxyphenylacetic and caffeic, four depsides: caftaric, neochlorogenic, chlorogenic, isochlorogenic, and rosmarinic acids, and flavonoids: aglycone—isoquercetin and glucoside—quercitrin. Most of them (apart from chlorogenic and rosmarinic acids) were detected for the first time in this species cultured in vitro. To our best knowledge, the present report is the first one that discusses establishment of Eryngium alpinum L. in vitro cultures and the shoot and callus biomass capacity to produce two subgroups of polyphenols i.e. phenolic acids and flavonoids. This is the first report on establishment of Eryngium alpinum in vitro cultures and the shoot and callus biomass capacity to produce polyphenols—phenolic acids and flavonoids.

In vitro screening of the spring durum and bread wheat F2 hybrids for water deficit resistance by the direct selection using low molecular mannitol as a stressor was conducted. It is shown that with an increase in the concentration of mannitol from 0.2 to 0.8 M in all genotypes there was the inhibition of the growth of the callus culture that indicates the toxic effect of the stress factor. It was established that the concentration of 0.6 M mannitol allows to differentiate spring wheat genotypes for water deficit.It was found that the F2 hybrid Elehiia myronivska / Krasa Polissia was most resistant to osmotic stress because calli of this genotype under selective conditions are characterized by relatively high morphogenic potential, had the highest survival rate and regenerated plants were obtained only from explants of this hybrid after cultivation on the medium containing mannitol concentration of 0.8 M. The F2 hybrid Zhizel / Lan was most susceptible to osmotic stress because mass necrosis and lack of regenerative ability were observed in its calli, under selective conditions. The genotypic dependence of processes of shoot formation in in vitro culture was observed in the spring wheat forms of studied. The formation of regenerated plants from wheat calli took place through both gemmorizogenesis and somatic embryogenesis.Plant regenerants were obtained from the induced calli and their rearing, rooting and transfer to in vivo conditions were optimized. Genotypic responses to osmotic stress in the callus culture of spring wheat were manifested by different survival rate and different regenerative ability under the action of a stress factor. The hybrid Elehiia myronivska / Krasa Polissia can be used as a valuable material for further breeding of spring wheat. The in vitro tissue culture can be used as a test system for the screening of wheat genotypes for resistance to osmotic stress. The optimized procedure of vigorous regenerated plants production of spring durum and bread wheat in in vitro callus cultures can be used in cell selection and genetic engineering experiments, Методом прямого добору проведено скринінг in vitro гібридів F2 пшениці м’якої та твердої ярої на стійкість до водного дефіциту з використанням низькомолекулярного маніту в якості стрес-чинника. Показано, що зі збільшенням концентрації маніту з 0,2 до 0,8 М у всіх генотипів відбувалося пригнічення росту калюсної культури, що свідчить про токсичний вплив стресового чинника. Встановлено, що концентрація 0,6 М маніту дозволяє диференціювати генотипи пшениці ярої за стійкістю до водного дефіциту.Виявлено, що найбільшою стійкістю до осмотичного стресу характеризувався гібрид F2 Елегія миронівська / Краса Полісся, оскільки калюси цього генотипу за селективних умов відрізнялись підвищеним морфогенетичним потенціалом, мали найбільший рівень виживання і лише з експлантів цього гібриду після культивування на середовищі з манітом концентрацією 0,8 М було отримано рослини-регенеранти. Гібрид F2 Жізель / Лан виявився найчутливішим до осмотичного стресу, так як у його калюсів за селективних умов спостерігали масовий некроз та відсутність регенераційної здатності. У вивчених форм пшениці ярої відмічено генотипову залежність процесів морфогенезу у культурі in vitro. Формування рослин-регенерантів з калюсів пшениці відбувалося шляхом як геморизогенезу, так і соматичного ембріоїдогенезу.З індукованих калюсів отримано рослини-регенеранти, оптимізовано їх дорощування, укорінення та переведення в умови in vivo. Генотипова реакція на осмотичний стрес у калюсній культурі гібридів F2 пшениці ярої виявлялась за різним рівнем виживання та різною регенераційною здатністю за дії стресового чинника. Гібрид F2 Елегія миронівська / Краса Полісся може бути використаний як цінний матеріал для подальшої селекції пшениці ярої. Культуру тканин in vitro можна використовувати як тест-систему для проведення скринінгу генотипів пшениці на стійкість до осмотичного стресу. Оптимізований регламент отримання повноцінних рослин-регенерантів пшениці м’якої та твердої ярої в калюсній культурі in vitro може бути застосований у клітинній селекції та генно-інженерних експериментах, Методом прямого отбора проведено скрининг in vitro гибридов F2 пшеницы мягкой и твердой яровой на устойчивость к водному дефициту с использованием низкомолекулярного маннита в качестве стресс-фактора. Показано, что с увеличением концентрации маннита с 0,2 до 0,8 М у всех генотипов происходило угнетение роста каллусной культуры, что свидетельствует о токсическом влиянии стрессового фактора. Установлено, что концентрация 0,6 М маннита позволяет дифференцировать генотипы пшеницы яровой по устойчивости к водному дефициту.Обнаружено, что наибольшей устойчивостью к осмотическому стрессу характеризовался гибрид F2 Елегія миронівська / Краса Полісся, поскольку каллусы этого генотипа в селективных условиях отличались повышенным морфогенетическим потенциалом, имели самый высокий уровень выживания и только с эксплантов этого гибрида после культивирования на среде с маннитом концентрацией 0,8 М были получены растения-регенеранты. Гибрид F2 Жізель / Лан оказался наиболее чувствительным к осмотическому стрессу, поскольку в его каллусов в селективных условиях наблюдали массовый некроз и отсутствие регенерационной способности. В изученных форм пшеницы яровой отмечена генотипическая зависимость процессов морфогенеза в культуре in vitro. Формирование растений-регенерантов с каллусов пшеницы происходило путем как гемморизогенеза, так и соматического ембриоидогенеза.С индуцированных каллусов получены растения-регенеранты, оптимизировано их доращивание, укоренение и перевод в условия in vivo. Генотипическая реакция на осмотический стресс в каллусной культуре гибридов F2 пшеницы яровой проявлялась по разному уровню выживания и разной регенерационной способностью при действии стрессового фактора. Гибрид F2 Елегія миронівська / Краса Полісся может быть использован как ценный материал для дальнейшей селекции пшеницы яровой. Культуру ткани in vitro можно использовать как тест-систему для проведения скрининга генотипов пшеницы на устойчивость к осмотическому стрессу. Оптимизированный регламент получения полноценных растений-регенерантов пшеницы мягкой и твердой яровой в каллусной культуре in vitro может быть применен в клеточной селекции и генно-инженерных экспериментах

International audience; Fagonia indica is one of the commercially vital medicinal plant species. It is well-known for biosynthesis of anticancer phenolics and flavonoids metabolites. The plant has been exploited for in vitro studies and production of vital phytochemicals, however, the synergistic effects of melatonin and lights remains to be investigated. In current study, we have evaluated the synergistic effects of melatonin and different light emitting diodes (LEDs) in callus cultures of F. indica. Both, light and melatonin play vital role in physiological and biochemical processes of plant cell. The highest Fresh weight (FW: 320 g/L) and Dry weight (DW: 20 g/L) was recorded in cultures under white LEDs. Optimum total phenolics content (11.3 μg GAE/mg), total flavonoids content (4.02 μg QAE/mg) and Free radical scavenging activity (97%) was found in cultures grown under white LED and melatonin. Furthermore, cultures maintained under white light were also found with highest levels of phenolic and flavonoids production (total phenolic production; 226.9 μg GAE/mg, Total flavonoid production; 81 μg QAE/mg) than other LED-grown cultures. However, the antioxidant enzymes; Superoxide dismutase (SOD: 0.53 nM/min/mg FW) and Peroxidase (POD:1.18 nM/min/mg FW) were found optimum in cultures grown under blue LED. The HPLC data showed that enhanced total production of metabolites was recorded in cultures under white LED (6.765 μg/mg DW) than other lights and control. The findings of this study comprehend the role of melatonin and influence of light quality on biomass accumulation and production of phytochemicals in callus cultures of F. indica.

An efficient protocol was developed to control excessive phenolic compound secretion during callus culture of cotton. As cotton is naturally rich in phenolic compounds factors influencing the phenolic compound secretion, callus induction and proliferation were optimized for getting high frequency callus culture. Different carbon sources such as fructose, glucose, sucrose and maltose were tested at various concentrations to control phenolic secretion in callus culture. Among them, 3% maltose was found to be the best carbon source for effectively controlling phenolic secretion in callus induction medium. High frequency of callus induction was obtained on MSB5 medium supplemented with 3% Maltose, 2,4-D (0.90μM) and Kinetin (4.60μM) from both cotyledon and hypocotyl explants. The best result of callus induction was obtained with hypocotyl explant (94.90%) followed by cotyledon explant (85.20%). MSB5 medium supplemented with 2,4-D (0.45μM) along with 2iP (2.95μM) gave tremendous proliferation of callus with high percentage of response. Varying degrees of colors and textures of callus were observed under different hormone treatments. The present study offers a solution for controlling phenolic secretion in cotton callus culture by adjusting carbon sources without adding any additives and evaluates the manipulation of plant growth regulators for efficient callus culture of SVPR-2 cotton cultivar.

Our experiments have confirmed the considerable disposition of leaf explants of Actinidia chinensis Planch. for induction and intensive proliferation of callus cultures, as well as, a possibility to regulate morhogenesis in in vitro conditions. Under specific culture conditions the morphogenic potential of callus cells of Actinidia chinensis was manifested both in organogenesis and somatic embryogenesis. Organogenesis was represented by induction of adventitious buds and regeneration shoots on the modified MS culture medium (Murashige and Skoog 1962) with BAP in combination with GA3 (each 1.0 mg. l-1). Rooting of shoots was successful on modified MS medium containing IBA (0.5-1.0 mg. l-1). Histological studies of callus tissues revealed their structural heterogeneity. Morphogenic processes in the callus were characterized by the appearance of meristematic zones and vascular elements. The formation of apical meristem, leaf primordia and finally shoot development proved de novo regeneration in callus culture. The obtained results demonstrate a possibility of plant regeneration through indirect organogenesis, which can be used for propagation of Actinidia chinensis Planch.

Regenerated plants and callus tissue were obtained by embryo culture and micropropagation of explants from shoots and leaves of Eustoma grandiflorum Shinn. (Gentianaceae), a medicinal plant. This species, which does not naturally occur in this country, but is found in the southwestern regions of North America, can be propagated in vitro in a relatively short time. Regenerated plants were obtained in amounts enabling extensive phytochemical analyses to be conducted.

The effects of different concentrations of copper ions (Cu2+ in the form of CuSO4 × 5H2O) on in vivo cucumber (Cucumis sativus L. ‘Edinstvo’) seedlings as well as on in vitro hypocotyl-derived callus were considered. Callus induction from hypocotyls was more prolific than from roots or cotyledons. Thus, callus obtained from hypocotyls of 7-day-old cucumber plants was cultured for 5 weeks on Murashige and Skoog medium containing 4 mg dm-3 2,4-D + 1 mg dm-3 BA supplemented with 0.01 mM, 0.1 mM or 1.0 mM of Cu2+. Biochemical indices related to oxidative stress were assessed. Cu2+ at 0.01 mM stimulated callus induction but 1.0 mM Cu2+ negatively affected callus formation and growth. LPO intensity was significantly lower than the control at all concentrations of Cu2+ but significantly higher than the control in plants exposed to 0.01 or 0.1 mM Cu2+. A similar trend was observed for the generation of the superoxide radical in both callus and plantlets. Superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APOX) activity increased in both callus and plantlets, but the level of increase in these antioxidant enzyme systems depended on the Cu2+ concentration. Cu2+ ions had a stronger (i.e., more negative) influence on oxidative stress in cucumber seedlings than on cucumber callus.

Conditions for induction of callus formation from root and stem explants and long-term maintenance of Deschampsia antarctica Desv. Tissue culture have been specified. Ability to callus formation depended on mineral composition of nutrient medium, combination of growth regulator concentrations, place of donor-plant vegetation and type of explant. The optimal for callus tissue generation was Gamborg, Eveleigh — B5 nutrient medium, supplemented with 0.9–1.0 mg/l 2.4-dichlorophenyl acetic acid and 0.09–0.1 mg/l of cytokinine benzylaminopurine. Callus formation potency from the root explants considerably exceeded (1.5–2 times) that of from stem ones. The shoots were derived through spontaneous indirect organogenesis. Regeneration efficiency was found to be affected by nutrient medium composition and callus origin. Regenerated shoots were rooted and conditions for growth of regenerated plants in vitro were specified.

Drought is one of the complex environmental factors affecting growth and yield of sorghum in arid and semi-arid areas of the world. Sixteen elite sorghum (Sorghum bicolor (L) Moench) genotypes were evaluated for their genetic potential to drought tolerance at callus induction and plant regeneration stage for drought tolerance. The non-ionic water soluble polymer polyethylene glycol (PEG) of molecular weight 6000 was used as osmoticum to simulate water stress. The factorial experiment was laid down in a completely randomized design which comprised of a combination of two factors (genotypes and five PEG stress level; 0, 0.5, 1.0, 1.5, and 2.0% (w/v) treatments). Data were recorded for callus induction efficiency, callus fresh weight, embryogenic callus percentage and plant regeneration percentage. Significant differences were observed among the genotypes, treatments and their interactions for the evaluated plant traits suggesting a great amount of variability for drought tolerance in sorghum. The correlation analysis also revealed strong and significant association between embryogenic callus percent and plant regeneration percent as well as between embryogenic callus percent and plant regeneration percent. By taking into consideration all the measured traits, Mann Whitney rank sum test revealed that 76T1#23 and Teshale followed by Meko, Gambella-1107 and Melkam showed better drought stress tolerance. Therefore they are recommended to be used as parents for genetic analysis, gene mapping and improvement of drought tolerance while Chelenko, Hormat and Raya appear to be drought sensitive.

The paper described the scheme of sterilization of silver birch leaf explants for callus culture establishment, and the dependence of morphogenesis from the composition of growth regulators and culture conditions. Noticeable effects of long-term maintenance of callus cultures and individual differences between initial forms on its regeneration capacity have shown.

The growth and morphogenesis of cultured plant tissues can be improved by small amounts of some organic elements. In addition to being a natural source of carbon, organic additives may contain natural vitamins, phenols, fiber, hormones and also proteins. Hence, the physiological effects of apricot gum on the regeneration capacity and growth rate of three different plant species i.e. carrot (as a model plant), stevia (as an herbaceous plant), and grapevine (as a woody plant) were examined. The proliferated callus cultures of carrot and in vitro-derived microcuttings of stevia and grapevine were inoculated on their respective standardized proliferation media supplemented with 2.0-6.0 g/l apricot gum. The growth parameters of treated samples were measured and compared to gum-free medium. Earlier callus initiations with greater fresh weight, volume, as well as improved pigmentation were recorded in media fortified with apricot gum. The usefulness of gum application was also obvious in both stevia and grapevine with respect to better shoot multiplication and rooting parameters. Due to positive effects of apricot gum, longer vines with a higher number of lateral shoots, internodes and leaf area were achieved. Overall, the gum at the rate of 4.0 g/L was found to be a logical concentration with respect to encouraging response in all three species. Owing to promising results evolved in the present research, the application of gum in commercial tissue culture protocols is highly recommended. However, further studies are needed to exploit plant derived gums as an alternative carbon source in plant tissue culture media., Advances in Horticultural Science, Vol 30 No 2 (2016)

Callus was inducted on wounded leaf explants from shoot tips of a particular Prunus domestica 'Węgierka Zwykła' clone cultivated in vitro. The improvement of Sweet Common Prune stock callus tissue parameters has been approached by experiments on culture protocols. Either for the induction or maintenance of tissue modified Murashige and Skoog medium, supplemented with different auxins and cytokinins at varying concentrations, was used. The goal was to obtain the highiest possible proliferative capacity of friable tissue without any signs of cell redifferentiation for about 10 weeks. The choice of auxin was an important factor regulating the rate and kind of tissue growth, and for the examined prune clone auxin alone brought a relatively small proportion of cells into division, so advantageous was to combine it with oxygenated cytokinin. Friable tissue was obtained on media supplemented with dicamba or with picloram, but not with 2.4-D neither alone nor combinated with IBA.

Investigations were carried out to determine antimicrobial and antioxidant properties and total phenol content of three wild species of Ephedra compared with their respective callus cultures. Callus induction was performed in a standard Murashige and Skoog (MS) medium with the following hormonal ranges (mg/L) for every species NAA:1.5, Kin:1 for Ephedra strobiliacea, NAA:2, Kin:1 for Ephedra procera and NAA:2, Kin:0.5 for Ephedra pachyclada. These ranges of PGPR (Plant Growth Promote Regulators) were chosen based on callus induction rates, RGR (Relative Growth Rate) and their fresh weights. An antimicrobial test against five gram negative and two gram positive bacteria and two fungi was performed using the disc diffusion method. All methanolic extracts showed antimicrobial activity, but the antimicrobial activity of the callus cultures was lower than those of the wild plants. E. strobilacea showed the highest antimicrobial activity, and all methanolic extracts of the wild plants and callus cultures unexpectedly showed the highest antimicrobial activity against Pseudomonas aeruginosa. A FRAP (Ferric Reducing Antioxidant Power) test was conducted to evaluate extracts for antioxidant activity. E. strobilacea with 1.61 +/- 0.08 mmol eq quercetin/g extract and 0.278 +/- 0.02 mmol eq quercetin/g extract for the wild plant and callus, respectively, showed the highest results.The total phenol content of extracts was measured by a Folin Ciocalteau test. All the chosen species displayed phenol contents but E. strobilacea had the highest amount (504.9 +/- 41.51 micromol eq catechin/g extracts and 114.61 +/- 15.13 micromol eq catechin/g extracts for the wild plants and callus, respectively).

Although several histone variants have been studied in both animal and plant organisms, little is known about their distribution during processes that involve alterations in chromatin function, such as differentiation, dedifferentiation and hormone treatment. In this study we evaluated the ratio of each histone variant in each of the four core histone classes in the three developmental zones of maize (Zea mays L.) root and in callus cultures derived from them, in order to define possible alterations either during plant cell differentiation or dedifferentiation. We also evaluated core histone variant ratios in the developmental zones of roots treated with auxin and gibberellin in order to examine the effects of exogenously applied plant hormones to histone variant distribution. Finally, immunohistochemical detection was used to identify the root tissues containing modified forms of core histones and correlates them with the physiological status of the plant cells. According to the results presented in this study, histone variant ratios are altered in all the cases examined, i.e. in the developmental zones of maize root, in callus cultures derived from them and in the developmental zones of roots treated either with auxin or gibberellin. We propose that the alterations in linker histone variant ratios are correlated with plant cell differentiation and physiological status in each case.

Flavan-3-ol is a secondary metabolite in tea plant that is used as anti obesity agent. The difficulty in obtaining Flavan-3-ol out of tea plant is due to dependency on season, need for large land, need for very intensive care and relatively low production. Therefore flavan 3-ol production needs tobe developed in vitro culture technique. This technique can cope with the handicapes above. It can effectively control production and requires less space. The purpose of this research was to enhance fl avan 3-ol production by modifyingmedium and precursor appropriately. The steps of this process were: (1) Callus induction through cultivating the tea shoot explants on medium fi lled with some growth hormone, (2) fl avan-3-ol induction on callus culture using Cu 2+ elicitor. (3) Observation over callus growth, (4) Observation over qualitative characterizes of flavan 3-ol. The result of the research using Cu 2+ elicitor shows that the production of flavan-3-ol increases by 12.5%.

Callus was stimulated from leaves and stems explants of Pea seedlings on MS media supplemented with growth regulators. Medium containing 1.5 mg/L BA and 2.0 mg/L NAA was the most favorable for callus induction from leaves. Whereas MS medium containing 2.0 mg/L BA and 4.0 mg/L IAA was the best for stem callus. Selection of resistant callus to the virulent strains of Fusarium solani was examined from culture medium that contain the concentration 2.5, 5.0 7.5, 10.0, 12.5. 15.5. 17.5 and 20.0 % of filtrates. The concentration which allowed selection were 10.0 and 12.5 %. Regeneration of pea plants from callus of leaves and stems were resisted the toxic effects of filtrate. In spite of reduction of the regeneration ability of the resistant callus up to 45 and 26% derived from leave and steam respectively compared with control. The chromosome number assay showed no difference between resistant and control plants, Pea plant regenerated from tolerate callus exhibited clear resistance when inoculated with virulent fungi.

Highlights • We have evaluated the role of carbon sources in controlling excessive phenolic secretion from callus culture of cotton. • Maltose was found to control phenolic secretion when it is used as a sole carbon source. • Efficient protocol was developed for high frequency callus induction and proliferation for SVPR-2 cotton cultivar using different plant growth regulators. • Adjustment of carbon source concentrations was found to play an important role in cotton callus culture. • The present work will be helpful for establishing callus culture and getting regeneration to carry out genetic transformation studies in SVPR-2 cotton cultivar., An efficient protocol was developed to control excessive phenolic compound secretion during callus culture of cotton. As cotton is naturally rich in phenolic compounds factors influencing the phenolic compound secretion, callus induction and proliferation were optimized for getting high frequency callus culture. Different carbon sources such as fructose, glucose, sucrose and maltose were tested at various concentrations to control phenolic secretion in callus culture. Among them, 3% maltose was found to be the best carbon source for effectively controlling phenolic secretion in callus induction medium. High frequency of callus induction was obtained on MSB5 medium supplemented with 3% Maltose, 2,4-D (0.90 μM) and Kinetin (4.60 μM) from both cotyledon and hypocotyl explants. The best result of callus induction was obtained with hypocotyl explant (94.90%) followed by cotyledon explant (85.20%). MSB5 medium supplemented with 2,4-D (0.45 μM) along with 2iP (2.95 μM) gave tremendous proliferation of callus with high percentage of response. Varying degrees of colors and textures of callus were observed under different hormone treatments. The present study offers a solution for controlling phenolic secretion in cotton callus culture by adjusting carbon sources without adding any additives and evaluates the manipulation of plant growth regulators for efficient callus culture of SVPR-2 cotton cultivar.

application/pdf, Regenerable callus culture of Dianthus hybrid ‘Telstar Scarlet’ was successfully applied to Agrobacteriummediated genetic transformation. Five days coculture with 5 mM glucose and 50 mM acetosyringone was suitable for Agrobacterium‐infection. Transformed shoots were obtained from inoculated calluses selected on MS medium containing 1.0 mg/l thidiazuron, 0.1 mg/l α‐naphthalene acetic acid, 20 g/l sucrose, 2 g/l gellan gum and 20–80 mg/l G‐418 within 20 weeks from inoculation. Transformation efficiency based on the number of calluses that produced transformed plants, and based on the total number of transformed shoots were 6% and 27% respectively. About 10% of transformants were tetraploid. All diploid transformants grew, flowered normally and produced seeds when crossed with other Dianthus species., 高い再生能力を有するダイアンサス‘テルスタースカーレット’のカルスを用いた形質転換系を検討した。カルスを5mMのグルコースと50mMのアセトシリンゴンだけで湿らせたろ紙上で共存培養することにより、アグロバクテリアの感染が向上した。接種したカルスをG‐418濃度を段階的に20–80mg/lに上げた再生培地上で選抜することにより形質転換体を獲得した。形質転換体の一部はin viroで早期開花を示した。また約10%の転換体が四倍体であった。順化した植物は正常に生育・開花し、さらに他のナデシコ属植物と交配することにより形質転換した後代が得られた。

Four furanocoumarins: bergapten, xanthotoxin, isopimpinellin (linear furanocoumarins) and sphondin (angular furanocoumarin) were isolated for the first time from callus tissues of Pastinaca sativa L.(Apiaceae) cultured in vitro on solid medium. The compounds were identified using spectral methods. They are well-known secondary metabolites of the intact plant. This is the first report on the isolation of sphondin from in vitro plant cultures.

The callus culture of Tectona grandis was previouly reported by us to produce five antibacterial acids which occurred only in very small amounts or were not detected in the native plant. This paper shows the production of these antibacterial compounds in much higher by cell suspension culture of the plant., 著者らは以前、Tectona grandis のカルスが、母植物には少量しか、あるいは全然存在しない抗菌性トリテルペン酸を相当量することを報告した。本報は、確立した細胞懸濁培養系では、これらの抗菌性トリテルペン酸の生産性がカルス培養の100～200倍にも上昇することを述べ、これらの抗菌性化合物を細胞懸濁培養で生産できることを示した。

Four callus lines from immature embryos of a self-crossed maize (Zea mays L.) hybrid cultivar were selected for "high" (two lines) and "low" (two lines) polyamine (PA) levels. Each selected line was exposed to culture media containing no (control) or 1% (0.171 M) NaCl and the relative growth rates were compared after subculture. Low-PA lines appeared to be tolerant to salt stress, while high-PA lines were sensitive. Analysis of PA at the end of the subculture showed that treated calli of sensitive lines had increased their putrescine content in comparison with their control, while putrescine remained constant in tolerant lines. Callus lines were analysed by RAPD (random amplification of polymorphic DNA) markers. One polymorphism (550-bp band) was found, demonstrating a genetic difference between the lines.

The paper described the scheme of sterilization of silver birch leaf explants for callus culture establishment, and the dependence of morphogenesis from the composition of growth regulators and culture conditions. Noticeable effects of long-term maintenance of callus cultures and individual differences between initial forms on its regeneration capacity have shown.

Globe artichoke leaves are highly rich in phenolic acids, in particular chlorogenic acid and dicaffeoylquinic acids. The latter are of particular interest since they can exert a stronger antioxidant activity, due to the presence of two adjacent hydroxyl groups on each of their phenolic rings. Plant tissue and cell cultures have been widely exploited for the production of secondary metabolites. We compared the content of 5-O-caffeoylquinic acid (chlorogenic acid) and 1,5-O-dicaffeoylquinic acid in leaf tissues and calli obtained from leaf explants of two globe artichoke genotypes (SAROM and C3-RR) belonging to the varietal type ‘Romanesco’. HPLC analyses highlighted that chlorogenic acid accumulates preferentially in leaf tissue, while dicaffeoyquinic acid in calli. We previously set up a system, based on UV-C exposure of leaf foliar discs, which was found to induce dicaffeoylquinic acid synthesis. Preliminary results demonstrate that UV-C further increases the content of dicaffeoylquinic acid of 1.5-fold also when applied to callus tissue. Globe artichoke callus culture combined with UV-C radiation may thus represent a promising system for the production of dicaffeoylquinic acids.

The in vitro antimicrobial activity of Rubia tinctorum callus extracts were studied against selected microorganisms by agar well diffusion assay. Calli were extracted using ethanol, n-hegzan and chloroform. Among the three solvents used, callus extracted in ethanol was found to be more effective against some microorganisms with inhibition zone between 10 and 24 mm. Extracts of chloroform showed poor inhibition than other two solvents. No activity was observed against Proteus vulgaris and Enterococcus faecalis. Thus, the positive results suggest that the R. tinctorum callus extracts should be further studied to determine the bioactive chemical compounds.

Although several linker histone variants have been studied in both animal and plant organisms, little is known about their distribution during processes that involve alterations in chromatin function, such as differentiation, dedifferentiation and hormone treatment. In this study, we identified linker histone variants by using specific anti-histone Hl antibodies. Each variant's ratio to total Hl in the three developmental zones of maize (Zea mays L.) root and in callus cultures derived from them was estimated in order to define possible alterations either during plant cell differentiation or during their dedifferentiation. We also evaluated linker histone variants' ratios in the developmental zones of maize roots treated with auxin in order to examine the effects of exogenous applied auxin to linker histone variant distribution. Finally, immunohistochemical detection was used to identify the root tissues containing each variant and correlate them with the physiological status of the plant cells. According to the results presented in this study, linker histone variants' ratios are altered in the developmental zones of maize root, while they are similar to the meristematic zone in samples from callus cultures and to the differentiation zone in samples from roots treated with auxin. We propose that the alterations in linker histone variants' ratios are correlated with plant cell differentiation and dedifferentiation.

Efficiency in transformation of three hybrids of Coleus blumei using wild and mutant strains of Agrobacterium sp. was evaluated. Successful transformation depended on specific bacterial strain-plant genotype combination and co-cultivation treatment. The wild type B6S3 of the bacterial strains employed gave the maximum efficiency. Strains C58C1(pArA4abc), GV3101(pGV2215), 8196 and A281 were also effective. Among several co-cultivation conditions tested, the most efficient treatment was as follows: excised leaf explants were incubated immeadiately in bacterial suspension for 5 min and were co-cultured with Nicotiana tabacum crown gall callus for two days. Plant hormone autotrophy, PCR analyses and hybridization analysis confirmed genetic transformation of the cultures. Sixteen different transgenic callus and cell cultures were maintained for more than four years in the absence of exogenous growth regulators and antibiotics without deterioration in the growth rate., Tri genotipa ukrasne koprive (Coleus blumei Benth.) transformirana su s pomoću bakterija Agrobacterium tumefaciens i A. rhizogenes. Uspješnost transformacije ovisila je o sukultivacijskom postupku lisnih eksplantata i bakterija te o interakciji bakterijskoga soja i biljnoga genotipa. Soj divljeg tipa B6S3 bio je najvirulentniji. Najuspješniji je postupak transformacije bio petminutna inkubacija svježe narezanih lisnih eksplantata s bakterijama te nakon toga dvodnevna sukultivacija s tumorskim tkivom duhana (Nicotiana tabacum). Rast kalusnoga tkiva na hranidbenoj podlozi bez regulatora rasta, PCR analiza te hibridizacijska analiza potvrdili su genetičku transformaciju tkiva. Ustanovljeno je 16 transgeničnih kalusnih linija koje su se međusobno razlikovale po fenotipu i stopi rasta. Fenotipske značajke pojedine linije nisu se mogle povezati s bakterijskim sojem upotrijebljenim u poticanju transformiranih linija niti s biljnim genotipom.

Plant tissue culture is recognised as a disposable tool to generate useful genetic variability for crop improvement. Nine somaclones of tetraloid potato cv. Bintje regenerated from stem, tuber or protoplast-derived callus were analysed in vitro and in the field. All of them were morphological and cytological chimeras. They had alterations in general appearance, leaf morphology and tuber characteristics. The phenotypic variations of the first vegetative progeny (SC2 plants) were in most cases different in comparison with the primary regenerants (SC1 plants). The observed phenotypic instability was in correlation with chromosome mosaicism (chromosomal instability) as aneuploid and polyploid cells were detected at high frequency (57-89%) in root tips of all somaclons investigated.

A primary amine, ethylamine, was isolated, purified and identified by 1H-NMR from perchloric acid-extracts of maize embryogenic callus, where it occurred in high amount (∼2 μmol/g fresh weight, an amount ten-fold higher than that of other well-known amines such as polyamines) while only a faint trace was found in maize plant organs. Ethylamine amount was measured in different phases of callus proliferation and differentiation (somatic embryogenesis). Results indicated that ethylamine mostly accumulates in the proliferation phase. It decreased to very low concentration in cells that have acquired a new competence for differentiation or are already differentiated, except for in the developing somatic embryo phase. Undifferentiated callus appears to be an ethylamine hyper-accumulator.

Callus tissue was induced on root tips of in vitro cultured seedlings of Allium commutatum Guss. cultured on MS medium supplemented with 4.6 mM kinetin and 4.5 mM 2, 4-dichlorophenoxyacetic acid (2, 4-D). Developed calli were transferred to the same basal medium with addition of 1.0, 2.5 and 5.0 mM 2, 4-D or without 2, 4-D. After three weeks in culture adventitious shoot and root induction was observed in compact greenish coloured callus on medium without 2, 4-D. On media supplemented with 1.0 and 2.5 mM 2, 4-D the callus was compact and yellowish with adventitious roots. No organogenesis was noticed on medium with 5.0 mM 2, 4-D and callus tissue was whitish and slimy. For preliminary cytological analyses samples of callus tissue were collected on the 7. and 14. day after the transfer. Fixed calli fragments were hydrolized in 1 N HCl at 60 °C for 11 minutes and stained in toto with basic fuchsin. The slides were prepared according to standard Feulgen squash technique. In each sample 1 000 cells were screened for mitotic activity and for establishing the rate of abnormalities. Relatively low level of genetic alterations was found. Among them micronuclei and polyploid cells were observed.