Showing papers on "Murashige and Skoog medium published in 2019"

Differential Production of Phenylpropanoid Metabolites in Callus Cultures of Ocimum basilicum L. with Distinct In Vitro Antioxidant Activities and In Vivo Protective Effects against UV stress.

Abstract: 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 confirm...

Meta-topolin Improves In Vitro Morphogenesis, Rhizogenesis and Biochemical Analysis in Pterocarpus marsupium Roxb.: A Potential Drug-Yielding Tree

Abstract: Meta-topolin (mT), a benzyladenine analog [N 6-(3-hydroxybenzylamino) purine] is a highly active cytokinin. The present study evaluates the efficiency of two aromatic cytokinins, mT and BA for inducing in vitro regeneration in a woody legume Pterocarpus marsupium (Roxb.) using cotyledonary node (CN) explants. Of the two cytokinins tested, mT-derived cultures resulted in better shoot multiplication and rhizogenesis than BA. Among the different doses of mT, maximum shoot (9.58 ± 0.30) induction per explant and average shoot length (4.12 ± 0.05 cm) were recorded on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium containing 7.5 µM mT, after 6 weeks of culture. The combined effect of cytokinin and auxin was tested, auxin was mixed with optimum doses of BA or mT separately and the effect of combination was studied. Among the cytokinin–auxin combinations, the highest number of shoots (17.44 ± 0.25) per explant and average shoot length (5.72 ± 0.18 cm) were achieved on MS medium containing 7.5 µM mT with 1.0 µM αnaphthalene acetic acid in 85% of the cultures after 12 weeks. Meta-topolin alone or in combination with auxin has shown an increase in the quality and number of shoots in comparison to BA. In vitro rhizogenesis in individually regenerated microshoots was carried out on half-strength MS medium augmented with 1.0 µM indole-3-butyric acid via a two-step procedure method. After 4 weeks, 7.35 ± 0.11 roots per shootlet with an average root length of 4.54 ± 0.10 cm were recorded in mT-derived microshoots. The well-developed plantlets were acclimatized in a separate batch of single CN explant-derived plantlets. About 80% survival rate was recorded for mT-derived plantlets. Biomass and photosynthetic pigments were also improved in mT-derived plantlets, when compared with the BA derived. Analysis of genetic homogeneity of ten micropropagated plantlets was done through RAPD. Out of 40 RAPD primers, 29 primers produced clearly scorable monomorphic bands, thus exhibiting complete genetic uniformity among in vitro regenerated plantlets.

Multi-walled carbon nanotubes improved growth, anatomy, physiology, secondary metabolism, and callus performance in Catharanthus roseus: an in vitro study.

Abstract: This study was conducted to monitor the physiological and molecular responses of Catharanthus roseus (rose periwinkle) to multi-walled carbon nanotube (MWCNT) incorporation into the culture medium. The seeds were grown on hormone-free MS medium supplemented with 0, 50, 100, and 150 mgL-1of MWCNT. The supplementations of culture medium with MWCNTs led to significant increases in plant growth indexes such as leaf width, leaf area, leaf fresh weight, root length, and total plant biomass). Slight increases were also observed in chlorophyll a (Chla), Chlb, and carotenoid contents (mean = 18.6%) in MWCNT-treated seedlings. Protein concentrations increased by an average of 34% relative to the control. The application of MWCNT resulted in twofold increases in the catalase and peroxidase activities. A similar trend was also observed in the phenylalanine ammonia lyase activities (by an average of 36.5%), soluble phenols (by 23%), and alkaloids (by 1.7-fold). Moreover, upregulations (mean = 37-fold) in the transcriptions of the DAT gene resulted from the MWCNT supplementations. Exposure to MWCNT improved cell sizes and xylem conducting tissue in treated seedlings. The applications of MWCNTs also stimulated the callus initiation and performance, implying their effects on proliferation and possible differentiation. This study has provided evidence of role MWCNT play in improving plant performance and production of pharmaceutical secondary metabolites.

Meta-topolin improved micropropagation in Syzygium cumini and acclimatization to ex vitro conditions

Abstract: An efficient micropropagation system was developed for a recalcitrant woody tree Syzygium cumini (L.) Skeels using nodal explants excised from 15-d-old aseptic seedlings. The explants were employed on an Murashige and Skoog (MS) medium supplemented with different concentrations (1.0 - 10.0 μM) of cytokinins, such as benzyladenine (BA), kinetin (Kin), meta-topolin (mT), or 2-isopentyl adenine (2ip), either alone or in combination with different concentrations (1.0 - 3.0 μM) of auxins, such as indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or α-naphthalene acetic acid (NAA). Of the cytokinins tested, mT proved to be best for shoot bud induction and proliferation. Among the tested combinations, a maximum regeneration (90 %) with a mean shoot number of 25.33 ± 0.33 and a shoot length of 5.20 ± 0.11 cm were recorded on the MS medium containing 5.0 μM mT + 2.0 μM NAA after 12 weeks of incubation. Further 4-week incubation on optimum 5.0 μM mT before transfer to a secondary medium consisting of MS + 5.0 μM BA + 2.0 μM NAA yielded up to 51 microshoots with an average length (6.53 cm). For in vitro rooting, healthy shoots (about 5 cm) were excised and incubated on the half or full strength MS medium enriched with different concentrations (1 - 7.5 µM) of NAA. A substantial increase in rhizogenic competency (15 %) was observed in shoots raised on a medium with mT with a mean root number of 6.33 ± 0.10 and a mean length of 5.13 ± 0.21 cm on the half MS supplemented with 5.0 µM NAA after 4 weeks. A maximum of 95 % plantlets regenerated on the medium with mT was successfully acclimatized and established in earthen pots under field conditions. The consistent increases in activities of superoxide dismutase, catalase, glutathione reductase, and ascorbate peroxidase during acclimatization indicate that mT raised plantlets response well to the stress induced by ex vitro conditions.

Effects of light intensity and plant growth regulators on callus proliferation and shoot regeneration in the ornamental succulent Haworthia.

Abstract: Haworthia are desert succulents belonging to the Asphodelaceae family. Haworthia species are cultivated commercially as ornamentals and some rare species are quite valuable at retail market but growth slowly and difficult to propagation. However, an efficient micropropagation protocol was remained insufficient. The organogenic cultures obtained from inflorescence explants were cultured on Murashige and Skoog (MS) medium supplemented with various combinations of 6-benzylaminopurine (BA) and α-naphthalene acetic acid (NAA) under a light intensity of 10 μmol m−2 s−1 or 45 μmol m−2 s−1. The highest callus proliferation index (93.15%) with 1.0 mg L−1 BA + 0.1 mg L−1 NAA under a light intensity of 10 μmol m−2 s−1. The best shoot proliferation rates were on media with either 1 mg L−1 BA + 0–0.4 mg L−1 NAA (65.57–81.01%) under a light intensity of 45 μmol m−2 s−1. The highest root length (15.57 mm) and the highest rooting frequency (17 roots per shoot) were obtained when adventitious shoots were inoculated on MS medium with 0.4 mg L−1 NAA + 0.4 mg L−1 IBA. The survival rate of the transplanted plantlets was about 100%. The efficient micropropagation protocol proliferated Haworthia regenerate plants from inflorescence within 11 weeks. The present study determined the best combination of light intensity and plant growth regulators (PGRs) for improved organogenesis of Haworthia during propagation by tissue culture. This optimized protocol showed light intensity is an important factor for efficient callus or shoot regeneration. These results indicate that it will be useful to optimize the light conditions for future commercial cultivation, germplasm conservation, genetic engineering and molecular biology research of this ornamental plant.

The effect of selected growth regulators and culture media on regeneration of Daphne mezereum L. ‘Alba’

Abstract: Tissue cultures are the only effective way to proliferate this valuable species (Daphne mezereum). These researches may help in solving the problem of reproduction of other woody plants. Daphne mezereum L. commonly known as February daphne or mezereon is a decorative shrub from the family Thymelaeaceae. It is a valuable nursery plant due to its high ornamental values, but its propagation by traditional methods is ineffective, therefore, little profitable. Micropropagation may be a good alternative way to produce this ornamental shrub. The experiment aimed to examine the effect of cytokinins (meta-Topolin, benzyladenine, and zeatin) and other growth regulators (gibberellic acid GA3 and auxin 1-naphthalene acetic acid NAA) added to the Murashige and Skoog (MS) and Woody Plant Medium (WPM) nutrients on regeneration of explants of D. mezereum ‘Alba’. In the all treatments containing plant growth regulators (PGR), 100% regeneration of explants has been observed. The highest number of shoots, after 6 weeks of culture, was produced on MS medium with 1 mg·L−1 BA and 0.1 mg·L−1 NAA. The longest shoots were produced on MS medium with 0.1 mg·L−1 of gibberellic acid (GA3) and 1 mg·L−1 of meta-Topolin (mT). Generally, the type and concentration of plant growth regulators had an essential effect on regeneration and growth of shoots of Daphne mezereum ‘Alba’ in the in vitro culture.

Elicitation of Stevia Glycosides Using Salicylic Acid and Silver Nanoparticles Under Callus Culture

Abstract: Development of biosynthesis of phytochemicals, especially medicinal products, is highly important due to their broad bioactivity properties. In this study, optimization of callus growth was initially carried out using various combinations of plant growth regulators. Callus with the highest fresh weight was produced on Murashige and Skoog (MS) medium containing 1 (mg/l) naphthalene acetic acid + 0.5 (mg/l) benzyl aminopurine. The effect of different concentrations of salicylic acid (SA) (0.25, 0.5 and 0.75 mg/l) and silver nanoparticles (Ag NPs) (15, 30, 45 and 60 mg/l) on callus growth as well as the possibility of stevia glycosides (SGs) production in callus culture was subsequently evaluated. The SA elicitation, at a concentration of 0.75 (mg/l), resulted in the highest level of callus growth rate (0.1 cm/day), callus diameter (0.79 cm) and relative callus fresh weight (0.085). Likewise, 45 (mg/l) of Ag NPs led to the highest amount of stevioside (32.34 mg/g dry weight callus). The addition of 0.25 (mg/l) of SA to the MS medium led to production of the highest amount of rebaudioside A (3.40 mg/g dry weight callus). The results of this study may enhance the commercial application of important glycosides prevalent in Stevia by highlighting that the nano-elicitors and SA should be utilized at optimized concentrations.

The influential role of polyamines on the in vitro regeneration of pea ( Pisum sativum L.) and genetic fidelity assessment by SCoT and RAPD markers

Abstract: A proficient and reliable in vitro plant regeneration protocol was established for pea by utilizing cotyledonary node as an explants, which excised from 3-days old imbibed seeds. The present research work explains the positive role of polyamines (PAs) accompanied by plant growth regulators (PGRs) on the multiple shoot induction and rooting in pea plant regeneration. The highest multiple shoots (65.1 shoots/explant) was attained from the cotyledonary node explant on Murashige and Skoog (MS) medium accompanied with 20 mg l−1 spermidine (SPD) along with 1.5 mg l−1 6-benzyladenine (BA). Moreover, the highest elongation of multiplied shoots (10 cm length/shoot) was noted on MS medium enriched with 1 mg l−1 of gibberellic acid (GA3). The elongated shoots produced the highest number of roots (33.66 roots/shoot) on MS medium augmented with 30 mg l−1 putrescine (PUT) along with 0.6 mg l−1 1-naphthaleneacetic acid (NAA). Rooted plants were hardened and acclimatized in the greenhouse with an existence rate of 98%. The standardized procedure with the use of PAs has enhanced the multiple shoot induction to threefold higher than the plants raised from PGRs treatments. The regenerated pea plants revealed the fivefold enhanced photosynthetic pigment, twofold enhanced antioxidant profile and the substantial growth in chloroplast count have been achieved in optimized SPD and BA supplemented MS medium compared to control plant. Start Codon Targeted polymorphism and Random Amplified Polymorphic DNA molecular markers recognized the genetic purity of the in vitro regenerated pea plants for their true type of nature. An enhanced and consistence in vitro regeneration protocol was established for pea, by the application of polyamines along with PGRs, which significantly improved the multiple shooting, shoot elongation, rooting, total chlorophyll and antioxidant profile of regenerated plants, further not hampering their genetic fidelity.

Meta-Topolin (mT) enhances the in vitro regeneration frequency of Sesamum indicum (L.)

Abstract: An efficient micropropagation protocol developed for sesame using cotyledonary node explants derived from 7-day-old in vitro grown seedlings. The efficacy of meta-Topolin (mT) was evaluated during different stages of regeneration. Multiple shoots were initiated and proliferated on Murashige and Skoog (MS) medium supplemented with various concentrations of mT and N6–Benzyladenine (BA), individually. Multiple shoots induced in the medium containing mT, responded well for shoot elongation, rooting and in vitro acclimatization. mT at a concentration of 6.21 μM in MS medium induced maximum number of shoots (23.36 shoots per explant) from 90.66% of cotyledonary node explants. About 73.33% of shoots induced on medium containing mT were elongated (5.93 cm per shoot) in liquid MS medium fortified with 5.77 μM GA3, and 95.66% of them developed profuse roots (8.62 roots per shoot; 13.82 cm in length) in liquid MS medium containing 2.46 μM IBA. All the plantlets with roots were hardened and successfully acclimatized in the greenhouse with a survival rate of 94.33%. Multiple shoots obtained from the cotyledonary node in the medium containing BA showed significantly higher H2O2 content, enhanced antioxidant enzyme (SOD, APX, and CAT) activities, and lower chlorophyll than for the shoots derived from the medium containing mT. SCoT and RAPD markers revealed that all the regenerated plants from the medium amended with either BA or mT showed similar banding pattern to that of the in vivo grown plant, thus confirming the genetic stability of regenerated sesame plants.

Plant Regeneration via Somatic Embryogenesis in Mature Wild Olive Genotypes Resistant to the Defoliating Pathotype of Verticillium dahliae.

Abstract: Regeneration capacity, via somatic embryogenesis, of four wild olive genotypes differing in their response to defoliating Verticillium dahliae (resistant genotypes StopVert, OutVert, Ac-18 and the susceptible one, Ac-15) has been evaluated. To induce somatic embryogenesis, methodologies previously used in wild or cultivated olive were used. Results revealed the importance of genotype, explant type, and hormonal balance in the induction process. Use of apical buds obtained from micropropagated shoots following a methodology used in cultivated olive (4 days induction in liquid 1/2 MS medium supplemented with 30 µM TDZ-0.54 µM NAA, followed by 8 weeks in basal 1/2 MS medium) was adequate to obtain somatic embryos in two genotypes, StopVert and Ac-18, with a 5.0 and 2.5% induction rates, respectively; however, no embryogenic response was observed in the other two genotypes. Embryogenic cultures were transferred to basal ECO medium supplemented with 0.5 µM 2iP, 0.44 µM BA, and 0.25 µM indole-3-butyric acid (IBA) for further proliferation. Somatic embryos from StopVert were maturated and germinated achieving a 35.4% conversion rate. An analysis of genetic stability on StopVert, using Simple Sequence Repeats (SSRs) and Random Amplified Polymorphic DNA (RAPDs) markers, was carried out in embryogenic callus, plants regenerated from this callus and two controls, micropropagated shoots used as explant source, and the original mother plant. Polymorphism was only observed in the banding pattern generated by RAPDs in 1 of the 10 callus samples evaluated, resulting in a variation rate of 0.07%. This is the first time in which plants have been regenerated via somatic embryogenesis in wild olive.

Efficient plant regeneration and Agrobacterium-mediated transformation via somatic embryogenesis in purslane (Portulaca oleracea L.): an important medicinal plant

Abstract: Portulaca oleracea is an important medicinal plant, which is a source of pharmacologically active molecules such as β-Carotene, ascorbic acid, and Omega-3 fatty acids. The present research focuses on the development of an efficient protocol for micropropagation and Agrobacterium-mediated genetic transformation of P. oleracea. Callus induction, somatic embryogenesis, and plant regeneration from stem and leaf explants were investigated at various concentrations of kinetin (Kin) and 6-Benzylaminopurine (BAP) alone or in combination with indole-3-acetic acid, 1-Naphthaleneacetic acid and 2,4-Dichlorophenoxyacetic acid (2,4-D). Direct differentiation of somatic embryos from leaf explants occurred on the MS medium supplemented with 1.5 mg/L BAP under dark conditions. The embryos were transferred to the same medium without growth regulators under 16 h light/8 h dark cycles. In this medium, germinated somatic embryos rapidly developed into healthy plantlets with shoots and roots. Several parameters such as pre-culture of explants, co-cultivation period, wounding of explants, type of explants and bacterial strains were studied to optimize transformation efficiency. Different kanamycin concentrations were assessed for the selection of transgenic plants. Agrobacterium tumefaciens strains LBA4404 and GV3101, harbouring the GUS gene on pBI121 binary vector, were used for plant transformation and strain LBA4404 was found to be more efficient. The results indicated that use of leaf as explant, pre-culture of explants for 7 days, co-cultivation period for 4 days at 25 ± 2 °C and wounding of leaf explants produced the best transformation results. Expression, integration and inheritance of GUS reporter gene were confirmed by histochemical and molecular analyses.

Phenolic compounds, antioxidant capacity and antimutagenic activity in different growth stages of in vitro raised plants of Origanum vulgare L.

Abstract: Efficient micropropagation procedure was developed for Origanum vulgare, a high-value culinary herb, and the phytochemicals, phenolic content, antioxidant and antimutagenic activity of leaf and stem, derived from different growing stages were analyzed. The agar solidified Murashige and Skoog (MS) medium supplemented with a combination of 6-benzylaminopurine and α-naphthaleneacetic acid was optimized as best shoot-multiplication-medium. Shoots were rooted best on 1/2 strength MS medium supplemented with 50 µM indole-3-butyric acid (IBA). The plantlets were successfully acclimatized ex vitro in a soil, sand and farmyard manure mixture (2:1:1 v/v/v) with 100% survival rate in greenhouse. The total anthocyanin and total phenolic content were observed significantly higher in leaves of in vitro-raised plants. However, total tannin, flavonoid and antioxidant activity remained higher in leaves of mother plant maintained under ployhouse condition. All the plant extracts have shown significant antimutagenic activity except in vitro-growing plants. A total of 13 polyphenolic compounds were detected in different extracts using high performance liquid chromatography. Among these, catechin was detected maximum in in vitro-growing cultures and chlorogenic acid in leaves of mother plant. These findings will help the farmers, medicinal plant growers, and industries for mass multiplication and effective extraction of phytochemicals from O. vulgare.

Does somaclonal variation play advantageous role in conservation practice of endangered species?: comprehensive genetic studies of in vitro propagated plantlets of Viola stagnina Kit. (Violaceae)

Abstract: In vitro regeneration of Viola stagnina Kit., endangered in most part of its European distribution range, was successfully obtained based on the newly developed protocol. Adventitious shoots via direct and indirect organogenesis were induced on leaf blade and petiole fragments on solidified Murashige and Skoog (MS) medium supplemented with 0.5 or 1 mg l−1 thidiazuron, respectively. Shoots were rooted on half-strength MS medium with 2% sucrose and 0.5 mg l−1 indole-3-acetic acid (IAA), and plantlets were successfully acclimatized. Sixty-five of the regenerated plants (72% of isolated shoots cultured on rooting medium) survived at the experimental plot conditions. Among recovered via organogenesis plants, individuals ‘true-to-type’ derived from initial plant G1 and also plants genetically distant from initial plants were detected by ISSR markers. In all groups of clones genetic indices (number of genotypes, polymorphic markers, gene diversity, total gene diversity, mean gene diversity) were lower than in natural populations. Regenerated plantlets had the same genome size estimated by flow cytometry as initial material and plants from natural populations. They developed chasmogamous flowers with highly viable pollen grains (over 90%), cleistogamous flowers, and set seeds from both flower types in the first and second seasons cultivated at experimental plots. This is the first report of a successfully developed micropropagation protocol of V. stagnina, and the first detailed genetic analysis of recovered plants with the use of ISSR markers and genome size measurements allowing to discuss the advantageous role of somaclonal variation in ex situ plant conservation with the use of in vitro micropropagation.

Efficient and reproducible somatic embryogenesis and micropropagation in tomato via novel structures - Rhizoid Tubers.

Abstract: A dual in vitro regeneration system consisting of indirect organogenesis and somatic embryogenesis (SE), applicable to several varieties of tomato-Solanum lycopersicum (cv. Riogrande, cv. Roma, hybrid 17905 and model cv. M82) has been established. This system is both improved and highly reproducible compared to current methods. Callus initiation, plant regeneration and SE was developed for one-week-old cotyledon explants. Indirect organogenesis via callus induction (CI) was developed for all four varieties of tomato used in this study. One-week-old tomato seedlings were used as a source of cotyledon and hypocotyl segments as explants. The explants were subsequently cultured on Murashige and Skoog (MS) medium supplemented with different combination and concentrations of plant growth regulators (PGRs). Substantial trends in regeneration and propagation response were observed among the varieties and treatments. For commercial varieties cvs. Riogrande and Roma, maximum CI was observed at 2 weeks in CIMT9 (0.5 mg/L NAA, 1 mg/L BAP) and CIMT12 (2 mg/L IAA, 2 mg/L NAA, 2 mg/L BAP, 4 mg/L KIN). However, cv. M82 responded after 4 weeks to a combination of treatments CIMT9 (0.5 mg/L NAA + 1 mg/L BAP) and CIMT13 (2 mg/L IAA + 2 mg/L NAA + 2 mg/L BAP + 4 mg/L ZEA) for the production of calli. Subsequent shoot and root organogenesis were optimized for all four varieties. Cv. Riogrande, exhibited fastidious in vitro regeneration potential and selected for induction of somatic embryos via SE involving novel structure: rhizoid tubers (RTBs). Numerous fine hair like rhizoids (~23/explants) were first developed from cotyledon and hypocotyl explants cultured on MS medium supplemented with 0.5 or 2 mg/L NAA at pH 4.0 in dark conditions. Further incubation of each rhizoid under light conditions on MS media supplemented with 5 mg/L TDZ or BAP at pH 4.0 led to the formation of a novel structure-rhizoid Tubers (RTBs). Thus, as evident from histology, SE in Riogrande tomato species requires a medium with pH of (4.0) and higher concentration of cytokinins (BAP/TDZ) to form on average 40-45 RTBs from both explants. Histological and morphological studies revealed that RTBs develop through different stages of embryogenesis to multiple plantlets, on MS medium with 5 mg/L TDZ/BAP at normal pH (5.8). The results obtained indicated that the induced somatic embryos of tomato with lower pH are a more efficient mode of propagation than the organogenesis with or without callus formation. The RTBs led to a complete plantlets regeneration in 45 days compared to indirect organogenesis at 60 days.

Improved plant regeneration in callus cultures of Sorghum bicolor (L.) Moench

Abstract: Sorghum bicolor is a recalcitrant species for tissue culture regeneration and genetic transformation. Browning of explants is one of the factors limiting organ and tissue cultures. To overcome this, callus tissue was initiated from the shoot tips of in vitro germinating seeds (S. bicolor cv. Rona 1), and then cultured on modified MS media (Murashige and Skoog in Physiol Plant 15:473–497, 1962). In the first experiment, we tested callus induction on several media supplemented with casein hydrolysate, polyvinylpyrrolidone, honey, and sucrose. The best callus induction was recorded for the medium with honey and sucrose (80.0%) and for control medium (79.8%). Shoot regeneration was tested on the MS medium with 6-benzylaminopurine (BAP) supplemented with honey and sucrose at a 1:1 ratio (by weight) or with sucrose only. The highest percentage of calluses regenerating shoots was noted for those induced on the medium with sucrose and honey—approx. four times higher when compared to the control. Rooted plantlets were acclimatized with a 92% survival rate. In the second experiment, we analyzed culture responses to various ways of honey application to the induction media: honey (autoclaved or filtered) in presence or absence of sucrose. Supplementation of the medium with fructose, glucose, and maltose at a proportion typical for honey was also investigated. The explant and callus survival rates were similar to those of the honey–sucrose combination in the first experiment. Only presence of both sucrose and honey in the induction medium improved the total regeneration rate to 37.9% over the control (18.8%). Sucrose and honey appear to act synergistically for shoot regeneration in callus cultures of sorghum.

An efficient in vitro shoot regeneration through direct organogenesis from seedling-derived petiole and leaf segments and acclimatization of Ficus religiosa

Abstract: Prolific and rapid in vitro plant organogenesis via direct regeneration has been obtained from axenic seedling-derived petiole and leaf explants of Ficus religiosa in Murashige and Skoog (MS) medium containing different concentrations of cytokinins in combination with indole-3-butyric acid (IBA). MS medium with 1.5 mg/l 6-benzylaminopurine plus 0.15 mg/l IBA produced the highest shoot induction frequency with an average of 6.26 and 10.13 shoots per leaf and petiole explants, respectively. After 4 weeks, the highest root formation frequency (96.7%), root number (5.73), and root length (4.76 cm) were with MS medium containing 2.0 mg/l IBA plus 0.1 mg/l α-naphthalene acetic acid. In addition, the effect of four sodium nitroprusside (SNP) treatments on acclimatization was also studied. Highest morphological traits such as survival rates, fresh and dry root weights as well as antioxidant enzymatic activities such as superoxide dismutase, peroxidase, and catalase was achieved with 125 ppm SNP. The α-amino acid, proline, content was highest with this treatment while the highest H2O2 (hydrogen peroxide) was in the controls. This study introduces a cost-effective, prolific, and efficient in vitro multiplication system to supply pharmaceutical and ornamental needs. It is the first report of an in vitro organogenesis protocol for F. religiosa by direct regeneration through axenic seedling-derived petiole and leaf explants, which can be efficiently employed for the utilization of active biomolecules.

High frequency in vitro plantlet regeneration in Solanum trilobatum L., an important ethno-medicinal plant and confirmation of genetic fidelity of R1 plantlets by using ISSR and RAPD markers

Abstract: An efficient and reproducible protocol was developed for in vitro clonal propagation of Solanum trilobatum L., an ethno-medicinally important plant. Leaf, stem and cotyledon explants were used for callus induction and shoot regeneration via indirect organogenesis. Initially, maximum amounts (mg) of green friable callus (312.86 ± 0.50, 285.3 ± 0.40 and 305.13 ± 0.62) was induced from leaf, stem and cotyledon explants, respectively, on Murashige and Skoog (MS) medium supplemented with 13.57 μM L−1 of 2,4-dichloro phenoxy acetic acid (2,4-D) and 2.21 μM L−1 of 6-benzylaminopurine (BAP), after 4 weeks of culture. Highest mean number of shoots (69.2 ± 0.73, 34.1 ± 0.62 and 57.1 ± 0.62) were differentiated de novo from leaf, stem and cotyledon calluses, respectively, when cultured on MS medium amended with 2.27 μM L−1 of Thidiazuron (TDZ) and 2.68 μM L−1 of Naphthaleneacetic acid (NAA), after 4 weeks of culture. Maximum rooting response (97%) with mean number of roots (31.7 ± 0.61 roots per shoot) was observed from shoots when cultured on half strength MS medium supplemented with 4.90 μM L−1 of indole-3-butyric acid (IBA), after 4 weeks of culture. In vitro raised plantlets (R1) of S. trilobatum were hardened in plastic pots, acclimatized in green house and successfully transferred to field conditions with 82% survivability. ISSR and RAPD based PCR banding profile of the acclimated R1 plantlets was confirmed as synonymous to the mother plant.

In Vitro Propagation of Endangered Orchid, Vanda pumila Hook.f. through Protocorms Culture

Abstract: The Vanda pumila is a monopodial orchid with beautiful flowers that are native to Thailand but now found across South Asia. The immature seeds of Vanda pumila were used for in vitro culture and then the protocorms developed were used as explants for seedling development and mass propagation. Protocorms were cultured on 1/2 MS (Murashige and Skoog, 1962) medium fortified separately with Kinetin (Kn), 6-Benzyl amino purine (BAP) and Gibberellic Acid (GA3) each in different concentrations as (0.5 mg/L, 1.0 mg/L and 2.0 mg/L) well as each on each concentrations of each medium supplemented with 5% and 10% coconut water (CW) respectively. The greatest number of shoots (9.50 ± 0.29 shoots per culture) was developed on 1/2 MS medium fortified with 1.0 mg/L Kn plus 10% CW and the longest shoots (0.78 ± 0.07 cm per culture) developed on 1/2 MS medium fortified with 2.0 mg/L BAP plus 10% CW. The shoots derived from protocorms were then developed on 1/2 MS medium fortified with three different rooting hormones viz. Indole-3-acetic acid (IAA), Indole-3-butyric acid (IBA) and α-Naphthalene acetic acid (NAA), each in four concentrations (0.5 mg/L, 1.0 mg/L, 1.5 mg/L and 2.0 mg/L) as well as 1.0 mg/L of each hormone supplemented with 10% CW. The 1/2 MS medium fortified with 0.5 mg/L IAA was found to be the most effective condition for the development of maximum number of root (5 ± 0.0 roots per culture) and root length (0.93 ± 0.07 cm). Hence, the present study could be useful for standardizing the protocol for mass propagation of the endangered orchid V. pumila.

In vitro micrografting using three diverse indigenous rootstocks for the production of Citrus tristeza virus- free plants of Khasi mandarin

Abstract: The present work reports the prospective applicability of three indigenous rootstocks belonging to different species viz. Nemutenga, Tayum and Tasi cultivars of north eastern Himalayan region for producing Citrus tristeza virus (CTV; a viral species of the Closterovirus genus) free quality planting materials of Khasi mandarin (Citrus reticulata Blanco) through micrografting or shoot-tip grafting (STG). The development of disease-free plants through STG is essential as CTV is in vivo transmissible through infected bud sticks. The technique of STG along with the diverse culture parameters like effect of sucrose, plant growth regulators, pre-treatment of scion and stock, effect of scion size after thermotherapy and grafting method was analysed. Among the three indigenous tested rootstocks, Nemutenga was found superior showing maximum STG success of 58.5%. STGs using scion size ranging from 0.3 to 0.5 mm, survival was up to 42.0% and completely virus-free. Pre-treatment of scion and stock with kinetin (1.0 mg L−1) was found more suitable than N6-benzylaminopurine (BAP) and further increased the rate of micrografts. For further growth of micrografts, Murashige and Skoog medium fortified with 0.5 mg L−1 BAP and 0.1 mg L−1 indole-3-acetic acid (IAA) along with 5% sucrose resulted in maximum (56.8%) response. Considering the two techniques applied for STG, cleft grafting was found more suitable than inverted T grafting. Viral assessment with enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR) revealed negative results for all plants from 0.3 to 0.5 mm scion while 0.7-mm scion-raised micrografts showed 60% negative with RT-PCR for CTV.

Micropropagation and Quantification of Bioactive Compounds in Mertensia maritima (L.) Gray.

Abstract: The goal of this study was to establish an efficient protocol for the large-scale propagation of Mertensia maritima (L.) Gray, and evaluate the carotenoid, fatty acid, and tocopherol contents in the leaves of in vitro regenerated shoots. Surface-disinfected node and shoot tip explants were placed on semisolid Murashige and Skoog (MS) medium with 0-16 µM N6-benzyladenine (BA), kinetin, (KN), and thidiazuron (TDZ) alone, or in combination with, 1 or 2 µM α-naphthaleneacetic acid (NAA). Of the three different cytokinins employed, TDZ elicited the best results for axillary shoot proliferation. A maximum frequency of shoot initiation above 84%, with a mean of 8.9 and 4.8 shoots per node and shoot tip, respectively, was achieved on the culture medium supplemented with 4 µM TDZ. A combination of TDZ + NAA significantly increased the percentage of multiple shoot formation and number of shoots per explant. The best shoot induction response occurred on MS medium with 4 µM TDZ and 1 µM NAA. On this medium, the node (93.8%) and shoot tip (95.9%) explants produced an average of 17.7 and 8.6 shoots, respectively. The highest root induction frequency (97.4%) and number of roots per shoot (25.4), as well as the greatest root length (4.2 cm), were obtained on half-strength MS medium supplemented with 4 µM indole-3-butyric acid (IBA). The presence of six carotenoids and α-tocopherol in the leaf tissues of M. maritima was confirmed by HPLC. Gas chromatography-mass spectrometry analysis confirmed the presence of 10 fatty acids, including γ-linolenic acid and stearidonic acid in the leaf tissues of M. maritima. All-E-lutein (18.49 μg g-1 fresh weight, FW), α-tocopherol (3.82 μg g-1 FW) and α-linolenic acid (30.37%) were found to be the significant compounds in M. maritima. For the first time, a successful protocol has been established for the mass propagation of M. maritima with promising prospects for harnessing its bioactive reserves.

De novo in vitro shoot morphogenesis from shoot tip-induced callus cultures of Gymnema sylvestre (Retz.) R.Br. ex Sm

Abstract: Gymnema sylvestre is a medicinal woody perennial vine known for its sweetening properties and anti-diabetic therapeutic uses in the modern and traditional medicines. Its over-exploitation for the therapeutic uses and to meet the demand of pharmaceutical industry in raw materials supply for the production of anti-diabetic drugs has led to considerable decline in its natural population. An efficient system of shoot bud sprouting from nodal segment explants and indirect plant regeneration from apical meristem-induced callus cultures of G. sylvestre have been developed on Murashige and Skoog (MS) medium amended with concentrations of cytokinins. Of the three growth regulators tested, N6-benzylaminopurine (BAP) was the most efficient and 2.0 mg L−1 gave the best shoot formation efficiency. This was followed by thidiazuron (TDZ) and kinetin (Kin) but, most of the TDZ-induced micro shoots showed stunted growth. Multiple shoot formation was observed on medium amended with BAP or TDZ at higher concentrations. The produced micro shoots were rooted on half strength MS medium amended with auxins and rooted plantlets acclimatized with 87% survival of the regenerates. The developed regeneration system can be exploited for genetic transformation studies, particularly when aimed at producing its high yielding cell lines for the anti-diabetic phytochemicals. It also offers opportunities for exploring the expression of totipotency in the anti-diabetic perennial vine.

Somatic Embryogenesis in the Family Gentianaceae and Its Biotechnological Application

Abstract: The family Gentianaceae consists of 1736 species, which play an important role in human being existence due to their pharmacological and horticultural values. Many species accumulate bitter iridoid substances used medicinally and in flavorings, while others are cultivated because of beauty of their flowers showing a wide range of colors and patterns. Out of 99 genera belonging to the gentian family, process of somatic embryogenesis (SE) was reported for 5. The first reports, aimed at micropropagation of ornamental cultivars and production of secondary metabolites, concerned Centaurium erythraea Rafn., Eustoma russellianum Grieseb. and Exacum affine Balf. Somatic embryos were induced on different explants cultured in the liquid Murashige and Skoog medium supplemented with auxins and cytokinins. In the 1990s of the last century, significant progress in the exploration of the phenomenon of SE and its biotechnological application was made for the genus Gentiana. The process was induced on various explants and studied at the structural and ultrastructural levels. Regenerated plants were screened for genetic stability using flow cytometry, chromosome counting, and molecular markers. Besides typical indirect SE, the use of leaf fragments enabled to obtain single-cell origin of somatic embryos. On the other hand, proliferation of embryogenic callus in liquid medium resulted in the establishment of long-term embryogenic cell suspension cultures, paving the way not only to study the formation of somatic embryos and the development of regenerants but also to preserve the morphogenic potential of cell aggregates by cryopreservation. Cell suspensions re-established after storage in liquid nitrogen maintained their embryogenic character and allowed to obtain somatic embryo-derived regenerants that were true-to-type at both genetic and epigenetic levels. Another application of SE was related to genetic manipulation purposes. Efficient protocols of plant regeneration from callus-, cell suspension-, or leaf mesophyll-derived protoplasts allowed engaging procedures of somatic hybridization or protoplast electroporation for gentian genome modifications. Also, high embryogenic potential existing in the numerous gentian species enabled successful Agrobacterium-mediated transformation of G. cruciata L. and G. dahurica Fisch.