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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.

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.

The callus cultures of Fagonia indica could prove as factories for the production of important phytochemicals when triggered through different types of stress. In this study, we initiated callus cultures from healthy stem explants in the presence of iron-doped zinc oxide nanoparticles (Fe-ZnO-NPs). We performed experiments with the callus cultures of F. indica to determine the impact of Fe-ZnO-NPs in concentrations (15.62–250 µg/mL) on biomass accumulation, production of important phenolic and flavonoids, and antioxidative potential. Our results showed that maximum callus biomass [Fresh weight (FW) = 13.6 g and Dry weight (DW) = 0.58 ± 0.01] was produced on day 40 when the media was supplemented with 250 μg/mL Fe-ZnO-NPs. Similarly, maximum total phenolic content (268.36 μg GAE/g of DW) was observed in 40 days old callus added with 125 μg/mL Fe-ZnO-NPs. Maximum total flavonoid content (78.56 μg QE/g of DW) was recorded in 20 days old callus grown in 62.5 μg/mL Fe-ZnO-NPs containing media. Maximum total antioxidant capacity (390.74 µg AAE/g of DW) was recorded in 40 days old callus with 125 μg/mL Fe-ZnO-NPs treated cultures, respectively. Similarly, the highest free radical scavenging activity (93.02%) was observed in callus derived from media having 15.62 µg/mL Fe-ZnO-NPs. The antioxidant potential was observed to have positive correlation with TPC (r = 0.44). HPLC analysis showed that Fe-ZnO-NPs produced compounds (e.g., Epigallocatechin gallate) that were either absent or in lesser quantities in the control group. These results showed that Fe-ZnO-NPs elicitors could increase the biomass and activate secondary metabolism in F. indica cells. Supplementary Information The online version contains supplementary material available at 10.1007/s11240-021-02123-1., Key Message Nanomaterial supplementation engineers the cells in an effective way to trigger biomass and secondary metabolism for the production of important phenolic compounds in plant cells. Supplementary Information The online version contains supplementary material available at 10.1007/s11240-021-02123-1.

The removal of textile dyes from wastewater by using plant peroxidases (PODs) offers environmentally effective solutions. Therefore, researching the availability and applicability of PODs from different sources becomes important area for the sustainable process design. In this article, PODs were produced in vitro from white and red cabbage callus cultures for evaluating their potential usage to decolourization of the Reactive Blue 19 (RB-19) and Acid Blue 25 (AB-25) textile dyes for the first time. The PODs with high activity were purified 133-fold with a specific activity of 45,083 U mg−1 and 134-fold with a specific activity of 26,785 U mg−1 from produced white and red cabbage calluses by single-step affinity chromatography. The molecular weights of purified red cabbage callus peroxidase (r-POD) and white cabbage callus peroxidase (w-POD) were found to be as 43 and 44 kDa, respectively. In the assays to determine the r-POD and w-POD dye decolourization potential, noteworthy degradation levels were recorded for RB-19 (>85) and AB-25 (>88) dyes in the reactions catalysed by w-POD. Also, r-POD exhibited maximum decolourization efficiency of % 68 for RB-19 and %73 for AB-25 dyes under the conditions at 50 °C, pH 5.5 and in the presence of 4 mM H2O2. © 2021 Informa UK Limited, trading as Taylor & Francis Group.

Cell cultures derived from strawberry fruit at different developmental stages have been obtained to evaluate their potential use to study different aspects of strawberry ripening. Callus from leaf and cortical tissue of unripe-green, white, and mature-red strawberry fruits were induced in a medium supplemented with 11.3 &micro, M 2,4-dichlorophenoxyacetic acid (2,4-D) under darkness. The transfer of the established callus from darkness to light induced the production of anthocyanin. The replacement of 2,4-D by abscisic acid (ABA) noticeably increased anthocyanin accumulation in green-fruit callus. Cell walls were isolated from the different fruit cell lines and from fruit receptacles at equivalent developmental stages and sequentially fractionated to obtain fractions enriched in soluble pectins, ester bound pectins, xyloglucans (XG), and matrix glycans tightly associated with cellulose microfibrils. These fractions were analyzed by cell wall carbohydrate microarrays. In fruit receptacle samples, pectins were abundant in all fractions, including those enriched in matrix glycans. The amount of pectin increased from green to white stage, and later these carbohydrates were solubilized in red fruit. Apparently, XG content was similar in white and red fruit, but the proportion of galactosylated XG increased in red fruit. Cell wall fractions from callus cultures were enriched in extensin and displayed a minor amount of pectins. Stronger signals of extensin Abs were detected in sodium carbonate fraction, suggesting that these proteins could be linked to pectins. Overall, the results obtained suggest that fruit cell lines could be used to analyze hormonal regulation of color development in strawberry but that the cell wall remodeling process associated with fruit softening might be masked by the high presence of extensin in 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 lignans and neolignans impart health benefits, particularly in treating different types of cancers, due to their strong phytoestrogenic and antioxidant properties. The present study enhances the comprehension on the biosynthesis of antioxidant lignans and neolignans in root-derived in vitro cultures of flax (both callus and adventitious root). The results presented here clearly showed that the adventitious root culture efficiently produced a higher amount of lignans (at day 40) and neolignans (at day 30) than callus culture of flax. High performance liquid chromatography (HPLC) analysis revealed that the accumulations of secoisolariciresinol diglucoside (SDG, 5.5 mg g&minus, 1 DW (dry weight)) and dehydrodiconiferyl alcohol glucoside (DCG, 21.6 mg/g DW) were 2-fold higher, while guaiacylglycerol-&beta, coniferyl alcohol ether glucoside (GGCG, 4.9 mg/g DW) and lariciresinol glucoside (LDG, 11.9 mg/g DW) contents were 1.5-fold higher in adventitious root culture than in callus culture. Furthermore, the highest level of total phenolic production (119.01 mg/L), with an antioxidant free radical scavenging activity of 91.01%, was found in adventitious root culture at day 40, while the maximum level of total flavonoid production (45.51 mg/L) was observed in callus culture at day 30 of growth dynamics. These results suggest that adventitious root culture can be a good candidate for scaling up to industrial level to commercially produce these pharmacologically and nutritionally valuable metabolites.

Bromus auleticus (Trin.) is a grass native to the southern cone with important agronomical potential as fodder. Different breeding programs have been initiated with this grass, but plant tissue culture techniques could not be used because B. auleticus is recalcitrant. The aim of the present study was to develop a micropropagation protocol in the genus Bromus and to investigate if the association between B. auleticus and Epichloë endophytes affected in vitro culture and growth of micropropagated plantlets. In different micropropagation stages, better results were obtained with endophyte-infected (E+) seeds compared to endophyte-free (E−) seeds. The E+ seeds presented higher percentages of in vitro germination (82 ± 5 vs. 57 ± 6%), callus induction (72 ± 6 vs. 37 ± 6%), and plant regeneration from callus (89 ± 5 vs. 13 ± 5%). We also compared the biomass of shoot complexes and regenerated plantlets. After 4 weeks of culture, shoot complexes obtained from E+ seeds reached greater weight than the ones regenerated from E− seeds (173 ± 24 vs. 74 ± 9 mg). More than the 80% of the regenerated shoot complexes were rooted ex vitro and acclimated, regardless of their origin (E+ or E−). Finally, after 4 weeks of acclimatization, the plantlets regenerated from E+ seeds reached a greater weight than the ones from E− seeds, (461 ± 64 vs. 172 ± 25 mg). These results indicate that the use of endophyte-infected (E+) seeds enhances significantly B. auleticus micropropagation and promotes growth of the regenerated plantlets. Fil: Regalado González, Jose Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Berdion Gabarain, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Vignale, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Novas, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Pitta Alvarez, Sandra Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Iannone, Leopoldo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina

Spherical calcium-pectic gel particles (CaPGPs) were obtained from pectins of callus cultures (CC) of campion Silene vulgaris (silenan) and duckweed Lemna minor (lemnan), as well as from commercial apple and citrus pectins by the method of ionotropic gelation. We studied the morphological characteristics of the obtained gel particles and found that the largest gel particles were formed from citrus pectin (CP), and the densest gel particles were formed from lemnan pectin (LP). The swelling and degradation of CaPGPs were comparatively evaluated by incubation in a simulated gastrointestinal environment. The swelling and degradation of CaPGPs formed from pectins of the silenan and lemnan callus cultures were studied at different concentrations of pectinase (0.4, 0.8 and 1.7 mg/ml) in the simulated fluid of the colon. It has been established that the CaPGPs obtained from lemnan are more resistant to degradation in the simulated colon fluid than CaPGP obtained from silenan. It was shown that the concentration of pectinase in the simulated fluid of the colon and the type of pectin affect the degradability of CaPGPs formed on the basis of pectins of callus cultures.

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

Italian ryegrass (Lolium multiflorum) is an annual grass considered as one of the most important temperate forage grasses in the world. However, it is recalcitrant to plant tissue culture techniques hindering its genetic manipulation. Epichloë occultans is an endophytic fungus associated with L. multiflorum. This symbiosis causes improvements in physiological and ecological traits of the host plants. The objective of this work was to study the effect of E. occultans on L. multiflorum micropropagation. We compared the response of endophyte-infected (E+) and endophyte-free (E−) seeds in different micropropagation stages. The E+ seeds were more successful than E− seeds in in vitro germination (83 ± 5 vs. 63 ± 6%), callus induction (78 ± 5 vs. 57 ± 6%), callus proliferation (average diameter of 21.5 ± 1.3 mm in two subcultures vs 17.3 ± 0.8 mm in three subcultures) and plant regeneration from callus (83 ± 7 vs. 30 ± 8%). These results indicate that E. occultans enhances significantly L. multiflorum micropropagation. The use of endophyte-infected (E+) seeds can be a solution to make this grass more amenable to different biotechnological tools, such as the genetic transformation. Fil: Regalado González, Jose Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Vignale, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Novas, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Pitta Alvarez, Sandra Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Iannone, Leopoldo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina

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.

Brassica juncea is a crucial cultivated mustard species and principal oilseed crop of India and Madhya Pradesh, grown for diverse vegetables, condiments, and oilseeds. Somaclonal variation was explored as a probable source of additional variability for the manipulation of fatty acids, especially low erucic acid contents that may be valuable for this commercially important plant species. The plantlets regenerated from tissue cultures (R0), their R1 generation and respective parental lines were compared for morpho-physiological traits and fatty acid profile for the probable existence of somaclonal variations. The first putative somaclone derived from genotype CS54 contained 5.48% and 5.52% erucic acid in R0 and R1 regenerants, respectively, compared to the mother plant (41.36%). In comparison, the second somaclone acquired from PM30 exhibited a complete absence of erucic acid corresponding to its mother plant (1.07%). These putative somaclones present a source of variation for exploitation in the development of future mustard crops with low erucic acid content.

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.

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.

Bitkiler uzun yıllardan beri farklı amaçlarla kullanılmaktadır. Tez kapsamında çalışılan Boraginaceae familyası üyesi Echium L. cinsi de, içermiş olduğu metabolitlerin sayısının fazlalığı nedeniyle endüstride kullanılabilir bitkilerdendir. E. italicum L.'de bulunan şikonin ve türevleri ile yağ asitlerinin üretimi ve artırımı için farklı kültürler (kotiledon yaprağı ve kök süspansiyon ile saçak kök kültürleri) oluşturulmuştur. Oluşturulan kültür hatlarına elisitasyon (+4 °C, MeJA, DMSO, ABA, AgNO3, SC Ekstraktı) uygulanarak metabolit üretiminin artırımı hedeflenmiştir. Çalışma sonucunda, en yüksek miktarda şikonin eldesi 10 µM MeJA'nın 24 saat uygulandığı saçak kök kültürlerinde tespit edilirken, en yüksek düzeylerde yağ asidi üretimi ise herhangi bir elisitasyon uygulamasının olmadığı kontrol grubu kotiledon yaprağı süspansiyon kültürlerinde elde edildi. Bununla birlikte, saçak köklere MeJA uygulaması sonucunda üretilen kültürlerden saflaştırılan Şikonin Türev-3 ekstraktı, [(İzovaleril/2-Metil Bütiril Şikonin) elisite edilmiş saçak kök kültürleri kaynaklı] meme kanseri hücre hatlarından MCF-7 ve MDA-MB-231'e uygulandı. Düşük miktarlarda denenen Şikonin Türev-3 fraksiyonu, MCF-7 ve MDA-MB-231 kanser hücre hatlarında antiproliferatif etki göstermiştir. E. italicum L.'nin in vitro kültür hatlarına metabolit üretimi için uygulanan yöntemlerin, Echium L. cinsinin yanı sıra diğer türlerde de mevcut biyoaktif bileşiklerin endüstriyel ölçekte kitlesel üretimleri için kullanılabilme potansiyeli bulunmaktadır. Plants have been used for different purposes for many years. The genus Echium L. from Boraginaceae family examined in the present study are among the plants that can be used in industry as it containes various secondary metabolites. Different cultures (cotyledone leaves, root suspension and hairy root cultures) for the production and elevation of fatty acids and shikonin with its derivatives have been initiated in E. italicum L.. Moreover, elicitation (+4 °C, MeJA, DMSO, ABA, AgNO3, SC Extract) is also carried out to increase metabolite production in these cultures. The highest amount of shikonin was determined in the hairy root cultures with 10 µM MeJA applied for 24 hours, while the highest concentrations of fatty acids was observed in the control group of cotyledon leaf suspension cultures without any elicitation. However, purified Shikonin Derivative-3 [Isovaleryl/2-Methyl Butyryl Shikonin (derived from elicited hairy root cultures)] was applied on MCF-7 and MDA-MB-231 cancer cell lines. Low amounts of shikonin Derivative-3 fraction was examined antiproliferative effect in MCF-7 and MDA-MB-231 cancer cell lines. The methods used for the production of metabolites to in vitro culture lines of E. italicum L. have the potential for the mass production of bioactive compounds found in the genus Echium L. as well as other species. 229

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.

International audience; Fagonia indica is valuable for its multiple uses such as anticancer, antimicrobial and antioxidant throughout the world. Various biotechnological approaches need to be employed for the sustainable production of plant biomass and its phytochemical content. Elicitation has been shown to be a very potent tool for enhanced production of secondary metabolites in different in vitro cultures. The current study involves the application of various concentrations of chitosan and salicylic acid as elicitors in callus cultures keeping Thiadizuron induced callus as a control. The main aim was to enhance the accumulation of biomass and secondary metabolite contents. The results show that maximum biomass; Fresh weight (FW: 38.0 g/100 mL) and secondary metabolites [Phenolic content (16.9 mu gGAE/mg; Flavonoid content (2.2 mu gQE/mg)] were observed in CHT treated cultures. The SA treated calli also showed better accumulation of biomass (FW: 37.5 g/100 mL) and phytochemicals [Phenolic content (12.29 mu gGAE/mg); Flavonoid content (1.73 mu gQE/mg)] as compared with control. The antioxidant potential was found in correlation with the production of PC/FC and found higher in CHT (94.3%) cultures than SA (90.2%) and control. For instance, the antioxidative enzyme activities [peroxidase (POD: 4.41) and superoxide dismutase (SOD: 0.42 nM/min/mg FW)] were found optimum in CHT treated samples. HPLC analyses revealed significant accumulation (5.5 +/- 0.004 mu g/mg DW) and (5.2 +/- 0.001 mu g/mg DW) of pharmacologically active components in CHT and SA treated samples, respectively. The results showed that both elicitors have potential to enhance the biomass accumulation and polyphenols in callus cultures of Fagonia indica.

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.

Application of such biotechnological method as a callus culture, allowed developing the somaclonal variants of cucumber, six of which were selected as a regenerated lines for the breeding program. New forms are characterized by early-ripeness, percent of parthenocarpy, good quality, yield and fruit weight. These lines can be used as an initial material for heterosis breeding and for improvement of donor cultivars of cucumber.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) has become a frequently deadly infection due to increasing antimicrobial resistance. This serious issue has driven efforts worldwide to discover new drugs effective against Mtb. One research area is the synthesis and evaluation of pyrazinamide derivatives as potential anti-TB drugs. In this paper we report the synthesis and biological evaluations of a series of ureidopyrazines. Compounds were synthesized by reacting alkyl/aryl isocyanates with aminopyrazine or with propyl 5-aminopyrazine-2-carboxylate. Reactions were performed in pressurized vials using a CEM Discover microwave reactor with a focused field. Purity and chemical structures of products were assessed, and the final compounds were tested in vitro for their antimycobacterial, antibacterial, and antifungal activities. Propyl 5-(3-phenylureido)pyrazine-2-carboxylate (compound 4, MICMtb = 1.56 μg/mL, 5.19 μM) and propyl 5-(3-(4-methoxyphenyl)ureido)pyrazine-2-carboxylate (compound 6, MICMtb = 6.25 μg/mL, 18.91 μM) had high antimycobacterial activity against Mtb H37Rv with no in vitro cytotoxicity on HepG2 cell line. Therefore 4 and 6 are suitable for further structural modifications that might improve their biological activity and physicochemical properties. Based on the structural similarity to 1-(2-chloropyridin-4-yl)-3-phenylurea, a known plant growth regulator, two selected compounds were evaluated for similar activity as abiotic elicitors.

The main purpose of this publication was to obtain the S-enantiomer of indan-1-ol with high enantiomeric excess and satisfactory yield. In our research, we used carrot callus cultures (Daucus carota L.), whereby the enzymatic system reduced indan-1-one and oxidized indan-1-ol. During the reaction of reduction, after five days, we received over 50% conversion, with the enantiomeric excess of the formed S-alcohol above 99%. In turn, during the oxidation of racemic indan-1-ol after 15 days, 36.7% of alcohol with an enantiomeric excess 57.4% S(+) remained in the reaction mixture. In addition, our research confirmed that the reactions of reduction and oxidation are competing reactions during the transformation of indan-1-ol and indan-1-one in carrot callus cultures.

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.

This is the first attempt towards an efficient regeneration protocol for an endangered and valuable medicinal plant, Thymus persicus using in vitro callus induction and indirect organogenesis. Callus induction was performed on MS medium supplemented with different concentrations of NAA and 2,4-D, alone or in combination with BAP and KN. Maximum callus induction (100%) was achieved from internode explants cultured on MS medium fortified with 2.0 mg L-1 NAA and 0.5 mg L-1 KN. The highest frequency of shoot multiplication (96%) was observed with 2.0 mg L-1 BAP+1.0 mg L-1 NAA. The maximum number of rootlets (16.6 ± 1.4) was induced on half-strength MS medium with 0.5 and 1.0 mg L-1 IBA. Rooted plantlets were then successfully grown and acclimatized in the greenhouse with a 70-85% survival rate. The benefits of the protocol described here include all-year-round application, germplasm conservation, suitability for commercial production and also for the biotechnological production of pentacyclic triterpenoids.

Trigonelline (N-methylnicotinate) biosynthesized from nicotinate is one of the metabolically active pyridine alkaloid, widely distributed in plant kingdom. In the present study trigonelline has been isolated from various plant parts and callus cultures of Moringa oleifera Lam., Moringaceae, and was identified using TLC, GLC, GC-MS, which was comparable to that of the standard trigonelline. The trigonelline recovery was found to be maximum in the pods and minimum in flowers. In order to enhance the production of trigonelline in vitro grown cultures, different treatment doses of nicotinic acid (250, 500 and 750 mg L-1) were supplemented in the medium as precursor. Maximum increase (up to 1.10 fold) was observed in the treatment dose of 500 mg L-1 of nicotinic acid.

Many populations of high-mountainous relic dioecious willows in Central Europe only consist of female individuals and are thus limited in their reproductive potential. We completed micropropagation experiments with shoot apexes and nodal segments of common and endangered willow (Salix) species, which can help to reintroduce autochthonous genotypes to their natural sites. Until recently, cultivation of green young shoot apexes of S. alba and S. lapponum showed the highest percentage of regeneration. We successfully applied the two-times-sterilisation due to high contamination of natural explants. The OK medium was the most efficient culture medium. In vitro propagation of willows with unisexual catkins, anther and ovule cultures were tested and optimised. Isolated anthers were cultivated on selected media and then microcallus and calluses of S. caprea and calluses of S. viminalis were formed on the A medium. Among various tested and optimised media for the ovule culture, the CP medium was the most efficient one. In this case, only the microcalluses of S. viminalis were observed. We developed biotechnological procedures that can be useful in conserving fragmented populations of high-mountainous willows.

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.

Preliminary work on Passiflora alata leaves failed to detect harmane alkaloids using LC. The aim of this work was to investigate the production of harmane alkaloids through the cell culture of P. alata, inducing its precursor (L-tryptophan). The leaf explants presented satisfactory results after disinfection, and the callus formation was initiated in MS media with adequate quantities of phytohormones. Sixty days after inoculation, calli were inoculated in the optimized semi-solid MS media, with and without the addition of L-tryptophan (50, 100, 200 mg/L) and kept in standard conditions for 90 days. Calli were collected on days 6, 16, 26, 36, and 90, followed by acid-base extraction, and analysed by LC. The results showed an absence of harmane, harmin, harmol, harmalol, and harmaline. With L-tryptophan feeding, two peaks were detected, collected and analysed through positive mode electrospray [ESI(+)-MS] and sequential analysis in tandem ESI(+)-MS/MS. The spectra obtained were very similar, with a repetition of the more intense ions, and consecutive loss of 68 Da units, attributed to the heterocycle pyrazole. It appeared that this transformation was not related to any enzymatic pathway previously described for the plant from L-tryptophan, and the biosynthesis of β-carboline alkaloids in callus culture of P. alata were not observed in this work.As folhas de varias espécies de Passiflora são utilizadas como ansioliticas e sedativas. Passiflora alata Curtis, Passifloraceae consta em três edições da farmacopéia brasileira, porem não há muitos estudos sobre sua composição química. No passado, enfatizava-se a ação conjunta de alcalóides e flavonóides. Em trabalho anterior, não foi detectada a presença de alcalóides harmanicos através de CLAE. Assim, decidiu-se investigar a produção dos mesmos através de cultivo celular, introduzindo seu precursor metabólico L-triptofano. Os explantes foliares apresentaram resultados satisfatorios para germinação apos assepsia, e a formação de calo foi iniciada em meio MS com quantidades adequadas de fitohormonios, previamente determinadas. Sessenta dias após a inoculação os calos foram repicados para meio semi-solido com e sem L-triptofano (50, 100, 200 mg/L), mantidos por 90 dias em condições padrão. Amostras foram coletadas com 6, 16, 26, 36, e 90 dias, realizada extração acido-base e o extrato analisado por CLAE. Os resultados mostraram a ausência de harmana, harmina, harmol, harmalol e harmalina. Dois picos presentes nas amostras com L-triptofano foram coletados e analisados através de espectrometria de massas, electrospray modo positiva [ESI(+)-MS] e analise em tandem ESI(+)-MS/MS. Os espectros correspondentes foram similares, mostrando a perda consecutiva de 68 Da, atribuídos ao pirazol. Este fato aponta para uma transformação não enzimática, não relacionada a uma biossintese previamente descrita para alcalóides β-carbolínicos.

A protocol was established to produce bioactive compounds in a callus culture of Ageratina pichinchensis by using 1 mg L−1 NAA with 0.1 mg L−1 KIN. The phytochemical study of the EtOAc extract obtained from the callus biomass, allowed the isolation and characterization of eleven secondary metabolites, of which dihydrobenzofuran (5) and 3-epilupeol (7), showed important anti-inflammatory activity. Compound 5 inhibits in vitro the secretion of NO (IC50 = 36.96 ± 1.06 μM), IL-6 (IC50 = 73.71 ± 3.21 μM), and TNF-α (IC50 = 73.20 ± 5.99 μM) in RAW (Murine macrophage cells) 264.7 macrophages, as well as the activation of NF-κB (40% at 150 μM) in RAW-blue macrophages, while compound 7 has been described that inhibit the in vivo TPA-induced ear edema, and the in vitro production of NO, and the PLA2 enzyme activity. In addition, quantitative GC-MS analysis showed that the anti-inflammatory metabolites 5 and 7 were not detected in the wild plant. Overall, our results indicated that A. pichinchensis can be used as an alternative biotechnological resource for obtaining anti-inflammatory compounds. This is the first report of the anti-inflammatory activity of compound 5 and its production in a callus culture of A. pichinchensis.

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).

Cytisus aeolicus Guss. ex Lindl. (Fabaceae family, subfamily Faboideae) is an endangered endemic species of the Aeolian Islands, Sicily. In vitro multiplication of C. aeolicus shoots was described in this work and cell cultures were established from cotyledons and hypocotyls to investigate their potential production of isoflavones. Aseptically germinated seeds, cultivated on LS modified basal medium, gave the initial explants used both to induce axillary propagation and callus cultures. The LS (Linsmaier and Skoog) basal medium, supplemented with 0.1 mg L−1 of 6-benzylaminopurine were used to induce axillary propagation. The callus induction was performed using the basal medium added with 5 mg L−1 2,4-dichlorophenoxy acetic acid and 5 mg L−1 kinetin (control medium). Basal medium was also added with 2000 mg L−1 casein hydrolysate (CH) or 900 mg L−1myo-inositol (MI). C. aeolicus callus cultures on CH and MI media produced an unique compound, the isoflavone genistein 7-O-ß-D-glucopyranoside (genistin), which has not previously been isolated from wild plants. Callus cultures grown on the medium containing myo-inositol produced the greatest amount of genistin. C. aeolicus tissue culture procedures could provide suitable plant material both for germplasm preservation (by micropropagation) and for biotechnological selective isoflavone production (by callus culture).

On long-term introduction to rats in dosage 100 mg/kg polyphenol complexes from heartwood and callus culture of Maackia amurensis Rupr. et Maxim. had a marked influence on the activity of free radical oxidation. Under conditions of induced oxidant stress both polyphenol complexes inhibited prooxidant activity directly affecting the products of free radical oxidation. Inhibiting influence on the production of oxygen active forms that is characteristic for both phytocomplexes is determined by inhibition of mechanisms both enzymatic and non-enzymatic defense.

Substituted pyrazinecarboxamides markedly influenced production of flavonolignans in Silybum marianum callus and suspension cultures. In this study the effect of two compounds, N-(3-iodo-4-methylphenyl)pyrazine-2-carboxamide (1) and N-(3-iodo-4-methylphenyl)-5-tert-butyl-pyrazine-2-carboxamide (2), as abiotic elicitors on flavono-lignan production in callus culture of S. marianum was investigated. Silymarin complex compounds have hepatoprotective, anticancer and also hypocholesterolemic activity. In vitro flavonolignan concentration in cells is very low and the elicitation is one of the methods to increase production. Elicitors were tested at three concentrations and at different culture times. In the case of elicitation with 1, the greatest increase of flavonolignan and taxifoline production was observed at concentration c(1a) after 6-hours of elicitation and after 24 and 72-hours at concentration c(1b). However, increased production of silychristin, one of the compounds in the silymarin complex, was achieved after only 6-hours elicitation with c(1a) (2.95 x 10(-4) mol/L). The content of silychristin was 2-times higher compared to the control sample. An increased production of silychristin was reached with compound 2 at the concentration c(2) (2.53 x 10(-3) mol/L) after 72 h of elicitation. The production of silychristin in this case was increased 12-times compared to control.

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.

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.

Callus cultures from olive (Olea europaea L.) were used to study characteristics of desaturation in this oil-rich tissue. The incorporation of [1-(14)C]oleate and [1-(14)C]linoleate into complex lipids and their further desaturation was followed in incubations of up to 48 h. Both radiolabelled fatty acids were rapidly incorporated into lipids, especially phosphatidylcholine and triacylglycerol. Radiolabelling of these two lipids peaked after 1-4 h, after which it fell. In contrast, other phosphoglycerides and the galactosylglycerides were labelled in a more sustained manner. [1-(14)C]Linoleate was almost exclusively found in the galactolipids. With [1-(14)C]linoleate as a precursor, the only significant desaturation to linolenate was in the galactolipids. Monogalactosyldiacylglycerol was the first lipid in which [1-(14)C]linoleate and [1-(14)C]linolenate appeared after incubation of the calli with [1-(14)C]oleate and [1-(14)C]linoleate, respectively. The presence of radioactivity in the plastidial lipids shows that both [1-(14)C]oleate and [1-(14)C]linoleate can freely enter the chloroplast. Two important environmental effects were also examined. Raised incubation temperatures (30-35 degrees C) reduced oleate desaturation and this was also reflected in the endogenous fatty acid composition. Low light also caused less oleate desaturation. The data indicate that lysophosphatidylcholine acyltransferase is important for the entry of oleate and linoleate into olive callus lipid metabolism and phospholipid:diacylglycerol acyltransferase may be involved in triacylglycerol biosynthesis. In addition, it is shown that plastid desaturases are mainly responsible for the production of polyunsaturated fatty acids. Individual fatty acid desaturases were differently susceptible to environmental stresses with FAD2 being reduced by both high temperature and low light, whereas FAD7 was only affected by high temperature.

Bu çalışmada, Türkiyenin Batı Anadolu Bölgesinde doğal olarak yetişen Campanulaceae familyasına ait, kritik düzeyde tehlike altında olan endemik bitki Campanula tomentosa türüne ait tohumların çimlendirilmesi ve kallus kültürü eldesi için en uygun besin ortamının tespiti amaçlanmıştır. Tohumdan üretilerek yapılan doku kültürü araştırmalarına başlandığında yetiştirilen steril fidelerden alınan eksplantlar öncelikle kallus oluşturma ve daha sonra da bu kallusların rejenerasyona yönlendirilmeleri şeklinde olacaktır Araştırmanın birinci aşamasında; Temel besin ortamlarının çimlenme üzerine etkisinin araştırıldığı çalışmalarda besin ortamlarının tohumların çimlenme oranları üzerinde farklı etkileri vardır.Bundan dolayı tohumların çimlenme oranları arasındaki farkı karşılaştırmak için materyal olarak kullanılan tohumlar besi ortamlarına ekilmeden önce yüzey sterilizasyonları yapılan steril tohumların bitki büyüme düzenleyicisi (BBD) içermeyen 4 farklı besi ortamına ( yüksek tuz oranı içeren MS ve ½ MS , yüksek nitrat azotu içeren B5 ve olgunlaşmamış embriyoların kültüründe kullanılan N6) kültüre alınarak çimlendirilmesi sağlanmış ve bu şekilde besi ortamlarının etkisi ayrı ayrı test edilmiştir. Fitohormon yokluğunda çimlenmenin en yüksek olduğu ortam ½ MS (%33 çimlenme başarısı) olmuştur. İkinci aşamada : Steril fidelerden alınan yaprak eksplantları kallus başlatma çalışmaları için büyüme düzenleyicilerinden oksin türevi olan 1α-Naftalen asetikasit (NAA)'nın farklı konsantrasyonlarını (0.1 mg/l ve 3 mg/l) ve sitokinin türevi olan 6-Benzilaminopürin (BAP)'ın farklı konsantrasyonlarını (1 mg/l ve 4 mg/l) içeren MS, ½ MS , N6 ve B5 besi ortamlarına aktarılarak besi ortamlarında kültüre alınmıştır.Besi ortamı ve bitki büyüme hormonlarının kallus gelişimine olan etkisine bakılmıştır.Yaprak eksplantları için 0.1mg/L NAA + 4 mg/L BAP destekli B5 VE 0,1 mg/L NAA+4 mg/L BAP ve 3 mg/L NAA + 1 mg/L BAP ile desteklenmiş MS besi ortamlarının en iyi kallus gelişim görüldüğü ortamlar olduğu belirlenmiştir. Sürgünlerin morfolojik gelişiminin iyi olmadığı kallus yapısının gevşek ve soluk yeşil- kahverengi olduğu (2.tip kallus) , 0.1mg/L NAA ve 4 mg/L BAP içeren MS ve B5 besin ortamlarında gelişen kallusların daha canlı ,yapıca sert ve sağlıklı (1. tip kallus) olduğu gözlenmiştir. Çalışmanın üçüncü aşamasında; Besi Ortamlarına göre I. Tip ve II. Tip kallus oluşum oranları (%) ve farklı ortamların kallus kalitesine etkisi araştırılmıştır. Çalışmanın dördüncü aşamasında; Proliferasyon çalışmaları NAA ve BAP içerikleri sabit tutulan 4 farklı ortamda kültür hazırlanmıştır. Sürgün proliferasyonu için en uygun besi ortamı sürgün gelişiminin daha hızlı ve sağlıklı olması açısından MS'tir., İn this research it is aimed to determine establishment the most suitable nutrition media to get callus culture from seeds of Campanula tomentosa,that was critically endangered endemic plant belongs to Campanulaceae family which naturally grown in Turkey's West Anatolia region. In the first phase of study; Studies that searching effect of basic medias on germination, nutrition medias were different effects over seeds germination rate.Therefore compare the differrences between seeds germination rate on 4 basic nutrition medias seeds of Campanula tomentosa, used as a material.Sterile seeds were germinated based culture on MS ,1/2 MS , N6 , B5 medium free plant growth regulators (PGRs) .The highest germination rate was seen in ½ MS (%33success) media without plant growth regulator. In the second phase of study; İn tissue culture if study is about producing callus , using seeds to obtain explants from sterilized and healthy seedlings will be success to rejenerate. To produce callus ,explants transfer and cultured in MS, ½ MS , N6 and B5 medias with plant growth regulators in different concentrations (0.1 mg/l ve 3 mg/l)of 1α-Nafthalene aseticacid(NAA) that are used as a auxin and different concentrations (1 mg/l ve 4 mg/l) of benzylaminopurine (BAP) that are used as a cytokinin.Although more shoots were obtained in 1 mg/l BAP medium,growth of shoots were not good, callus structure was not compact and the colour of callus was yellowish or brown( 2nd type of callus) .In addition , 4 mg/l BAP of medium , growth of shoots were good , strong,compact and faded green-white colour(1st type of callus). In the third phase of study; effects of different nutrition medias on callus structure and colour and producing 1st type of callus and 2nd type of callus (%) In the fourth phase of study; Four different types of medium was prepared to investigate effects of NAA and BAP to proliferation processes. The best media for shoot development was MS.

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.

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.