Full-strength Murashige And Skoog Medium Research Articles

Factors Affecting the Micropropagation of Sapindus trifoliatus from Nodal Explants of Mature Tree

Background:: Explant recalcitrance is one of the major challenges during the tissue culture of a tree species, especially when explants are derived from mature plants. It is mainly as-sociated with the genotypes/species, source and physiological status of explants, seasonal variations, and competency/incompetency of explants towards PGRs for in vitro manipulations. There-fore, to optimize the appropriate conditions and minimize explant recalcitrance, it is necessary to elucidate the different factors influencing the tissue culture of a tree species. Objective:: Several factors influencing in vitro shoot induction from nodal explants derived from mature plants of Sapindus trifoliatus were investigated. Methods:: Nodal segment obtained from mature plants was used as an explant for shoot regeneration. The factors assessed in this study included the types and concentrations of plant growth regulators, nutrient composition of MS (Murashige and Skoog) medium, sucrose concentrations, the position of nodes on their respective branches, seasonal variations, and successive transfers of mother explant. Results:: The performance of nodal explants was better on modified MS medium, i.e., M4 medium (half-strength of MS major salts and full-strengths of MS minor salts, Fe-EDTA and MS organic nutrients each) than the full-strength MS medium. The addition of BAP in the medium resulted in a higher shoot induction rate than other cytokinins i.e., Kin, Zeatin, TDZ. M4 medium supplemented with 3.0 mg l-1 BAP and 2% sucrose was optimum for shoot proliferation. However, elongation of shoots was observed only when induced shoots, along with the mother explant, were subcultured on a medium containing 1.0 mg l-1 BAP. The shoot multiplication was positively affected by the repeated transfer of the mother explant for different passages on the multiplica-tion medium. Mother explant transferred to multiplication medium (0.8% agar-solidified M4 medium + 2% sucrose + 1.0 mg l-1 BAP) proliferated maximum shoots after fourth passages. Regenerated shoots were rooted in vitro and further successfully acclimatized in field conditions. Conclusion:: The in vitro regeneration system developed from an explant obtained from mature trees can be successfully exploited for mass multiplication of Sapindus trifoliatus, a medicinally and commercially important tree.

Establishment of An in Vitro Mass Propagation System for Dendrocalamus asper.

Dendrocalamus asper is a species of bamboo that has high commercial value and is the bamboo of choice for large scale agro-forestry plantations in the tropical regions of the world. Micropropagation using tissue culture is essential to generate uniform clones that are amenable to establishment in industrial agro-forestry projects for bamboo biomass, habitat restoration or in carbon sequestration. This paper reports on the establishment of D. asper invitro using commercially available seeds. The seeds were surface sterilized using three different chemical agents which were Sodium Hypochlorite (20%), Mercuric Chloride (0.1%) and Ethanol (70%) followed by shoot initiation on Murashige and Skoog (MS) medium supplemented with 6-Benzylaminopurine (BAP) with a concentration ranging from 1.0 - 5.0 mg/L. Propagules were multiplied on MS media supplemented with different concentration of IBA Indole-3-Butyric Acid (IBA) and Naphthalene Acetic Acid (NAA), and finally rooted and hardened in peat moss. The findings of our study indicate that the sterilization protocol eliminated all the plant pathogens, resulting in an axenic culture. Full strength MS medium supplemented with 5 mg/L BAP yielded the highest number of shoots (11.46 per explant) after four weeks of inoculation. The highest multiplication rate (3.95 shoots per explant) was obtained on MS medium supplemented with 3 mg/L BAP. The time required from initiation to hardening was 70 to 90 days, following which the plantlets were ready for field trials. The findings of this study will facilitate the establishment of plant tissue culture programmes dedicated to the production of D. asper locally, thus eliminating the need for imports and the possible entry of plant pathogens that can be detrimental to the local agro-forestry industry.

Optimization of Hormonal Compositions of Media for In vitro Propagation of Apple (Malus ×domestica Borkh.) Cultivars

Background: Apple (Malus × domestica Borkh.) is one of the most important fruit crops in the world. Traditionally, vegetative propagation methods (including cutting, budding, and layering) are time-consuming (about three years), with low production rates and low success in obtaining virus-free plants. Objective: The present study was planned to investigate the in vitro propagation of apple (M. domestica) cultivars from nodal segments. Methodology: The Murashige and Skoog (MS) medium supplemented with sucrose and different concentrations of plant growth regulators (PGRs) were used for shoot proliferation and root induction. The optimal concentrations of PGRs in the MS medium were assessed. The effect of full and half-strength MS medium on root induction was investigated. Results: Examination of the effects of MS medium supplemented with various concentrations of indole-3-acetic acid (IAA) and kinetin revealed that the significantly highest shoot response was recorded for the ‘Princess’ cultivar with maximum shoot proliferation rate (65.25%), shoot number per explant (2.57), shoot length (7.28cm), and leaf number per shoot (6.15) after four weeks of culture. The root induction in micro shoots of three apple cultivars was observed after 20 days of culturing. The strength MS medium (full and half) containing 1.5 mg/L IAA significantly affected (at P<0.05, chi-square test) root induction in all three apple cultivars, especially rooting rate. However, there was no significant difference in root number and root length per micro shoot among the apple cultivars. Among the cultivars, significantly the highest rooting rate(48.30%), root number (6.25), and root length (4.15cm) were recorded for cultivar ‘Princess’ on full-strength MS medium. Conclusion: PGR combination of IAA (1.0 mg/L) and kinetin (3.0 mg/L) was found to be the best for shoot proliferation. The shoot responses were found to increase with an increase in kinetin concentration combined with IAA at 1.00 mg/L.

Synthetic seed propagation of the therapeutic-honey plants

Context Increasing demand for therapeutic honey has driven establishment of Leptospermum nectar plantations. Methods for propagation involving synthetic seeds (artificially encapsulated miniature cuttings) may speed production of Leptospermum polygalifolium Salisb. and L. scoparium J.R.Forst. & G.Forst. Aims The study aimed to determine how nutrient strength of the encapsulation solution and the presence of benzyladenine (BA) in the emergence medium affect shoot and root emergence from synthetic seeds of L. polygalifolium and L. scoparium. Methods Nodes from in vitro shoots of three L. polygalifolium clones (P1, P6, P11) and two L. scoparium clones (S6, S12) were encapsulated in 3% sodium alginate with half- or full-strength Murashige and Skoog (MS) medium, and the synthetic seeds were placed on full-strength MS emergence medium containing 0 or 2.2 μM BA. Key results Full-strength MS in the encapsulation solution was effective for shoot emergence of both species. BA increased the percentage of synthetic seeds with shoot emergence in clone P6 but decreased the percentage in clone S12. BA stimulated shoot emergence through callus in clones P1, P6, S6 and S12, and increased the number of shoots per emergent synthetic seed in clones S6 and S12. Surprisingly, the simple use of full-strength MS medium without hormones was highly effective for adventitious rooting, stimulating root emergence and plantlet formation in 26–57% of L. polygalifolium and 100% of L. scoparium synthetic seeds. Conclusions These two Leptospermum species are highly amenable to propagation via synthetic seeds. A simple formulation of hormone-free, full-strength MS medium in the encapsulation solution and emergence medium provides high frequencies of plantlet conversion. Implications Synthetic seeds have potential to assist in mass production of Leptospermum plants for nectar plantations to meet demand for therapeutic honey.

The Efficient and Easy Micropropagation Protocol of Phyllanthus niruri.

Phyllanthus niruri (P. niruri) or Dukung Anak is a herbal plant in the Phyllanthaceae family that has been used traditionally to treat various ailments such as diabetes, jaundice, flu and cough. P. niruri contains numerous medicinal benefits such as anti-tumor and anti-carcinogenic properties and a remedy for hepatitis B viral infection. Due to its beneficial properties, P. niruri is overharvested and wild plants become scarce. This study was conducted to develop an appropriate in vitro culture protocol for the mass production of P. niruri. An aseptic culture of P. niruri was established followed by multiplication of explants using different types of basal medium and its strength and plant growth regulators manipulation. This study also established the induction of in vitro rooting utilizing various types and concentrations of auxin. Treatment of Clorox® with 30% concentration showed the lowest percentage (%) of contamination, 4.44% in P. niruri culture. Nodal segments of P. niruri were successfully induced in full-strength of Murashige and Skoog (MS) basal media with 2.33 number of shoots, 3.11 cm length of shoot and 27.91 number of leaves. In addition, explants in full-strength MS media without any additional cytokinin were recorded as the optimum results for all parameters including the number of shoots (5.0 shoots), the length of shoots (3.68 cm) and the number of leaves (27.33 leaves). Treatment of 2.5 µM indole-3-butyric acid (IBA) showed the highest number of roots (17.92 roots) and root length (1.29 cm). Rooted explants were transferred for acclimatization, and the plantlet showed over 80% of survival rate. In conclusion, plantlets of P. niruri were successfully induced and multiplied via in vitro culture, which could be a step closer to its commercialization.

Culture condition optimization and FT-IR analysis of Polygonum multiflorum Thunb. adventitious root cultures grown in an air-lift bioreactor system

Bioreactor cultures have been used for biomass production and bioactive compounds accumulation in adventitious root cultures of medicinal plants. In this study, we evaluated the effects of different auxin types [indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA)] and concentrations (0.5, 1.0, 2.0 and 4.0 mg·L−1), Murashige and Skoog (MS) medium salt strength (0.25, 0.5, 0.75, 1.0, 1.5 and 2X), and sucrose concentrations (0, 1.5, 3, 5, 7 and 10%) on Polygonum multiflorum Thunb. adventitious root cultures in a 3-L balloon-type bubble bioreactor (BTBB). IBA (1, 2, and 4 mg·L−1) was more effective than NAA in promoting root growth. Additionally, low MS salt strength (0.25 and 0.5X MS) increased the accumulation of total phenolics and flavonoids but reduced biomass accumulation. Four weeks of culture in full-strength MS medium supplemented with 2 mg·L−1 IBA and 5% sucrose resulted in the highest root biomass [98.46 g·L−1 fresh weight (FW); 13.46 g·L−1 dry weight (DW)] and bioactive compounds accumulation (total phenolics compounds, 53.08 mg·g−1 DW; total flavonoids, 25.10 mg·g−1 DW). To determine whether metabolic fingerprinting of whole-cell extracts could be used to compare metabolic equivalence of P. multiflorum root samples, we treated adventitious roots with different culture conditions, and analyzed the treated adventitious roots and natural roots by Fourier transform infrared (FT-IR) spectroscopy. The results showed that the metabolic pattern of adventitious root samples was similar under different culture conditions; however, these samples could be discriminated from each other in pilot-scale bioreactors. Overall, our study provides useful information for industrial-scale cultivation of P. multiflorum adventitious roots. The optimum condition for bioreactor culture of Polygonum multiflorum is full-strength MS medium supplemented with 2 mg·L-1 IBA and 5% sucrose after 4 weeks of culture. FT-IR analysis can rapidly discriminate among root samples based on total metabolite equivalence.

In vitro tissue culture protocol of ancient einkorn (Triticum monococcum ssp. monococum) wheat via indirect shoot regeneration

Einkorn indirect shoot regeneration was obtained from leaf, coleoptile, and root explants cultured on full, ½, and ¼ strength Murashige and Skoog (MS) medium containing 0.5 to 10 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D). Calluses derived from coleoptile and root explants were cultured on full-strength MS medium supplemented with 0.5 to 5 mg L−1 thidiazuron (TDZ) for 6 wk to induce plant regeneration. Only callus cultures derived from coleoptile explants regenerated shoots. For root induction, 1.0 to 10 mg L−1 indole-3-acetic acid (IAA) was used for 4 wk. The highest rate of callus formation (93%) was from root explants cultured on full-strength MS medium with 4 mg L−1 2,4-D. Coleoptile explants had the highest callus formation (100%) after culture on full-strength MS medium with 3 to 6 mg L−1 of 2,4-D. The highest indirect shoot regeneration (7.0 ± 1.1 shoots produced per callus with a 67% shoot formation frequency) was from calluses derived from coleoptile explant cultured on full MS medium supplemented with 5 mg L−1 of TDZ. Of the different IAA concentrations investigated for rooting, the greatest number of roots per explant (7.7 ± 0.088 roots produced per regenerated shoot with a 100% root formation frequency) was observed on MS medium supplemented with 6 mg L−1 IAA. This indirect shoot regeneration protocol for coleoptile-derived einkorn wheat callus will be useful for future wheat genetic and improvement studies.

Micropropagation protocol for Stevia rebaudiana through axillary shoot proliferation

The present investigation was carried out at Anand Agricultural University, Gujarat during the year 2016 to develop a micropropagation protocol for mass multiplication of medicinally rich stevia plants. Axenic culture was established by sequential application of 200 mg/l Kanamycin, 200 mg/l Carbendazim-50% and 1000 mg/l HgCl2. Out of two different basal media, viz. Gamborg (B5) and Murashige and Skoog (MS) used, full strength MS gave highest (98%) culture establishment. Out of three different basal media, viz. Gamborg’s (B5), Linsmair and Skoog (LS), Murashige and Skoog (MS) and four different cytokinins, viz. 6-benzylaminopurine (BAP), Kinetin (Kn), Thiadiazuron (TDZ), Zeatin (Zn) tested for multiple shoot induction, full strength MS medium and 2 mg/l BAP, respectively, promoted maximum shoot formation. In the experiment to study the effect of varying strength of MS media and various plant growth regulators, viz. Indole-3-acetic acid (IAA), Indole-3-butyric acid (IBA), Naphthalene acetic acid (NAA) on in vitro rooting, ½ strength MS and 1 mg/l NAA, respectively, gave superior response. Among the 26 different combinations of potting mixtures used for primary acclimatization of in vitro rooted plants, cocopeat based substrates yielded highest survival rate (75.06%). Secondary acclimatization was carried out in the soil bags in the poly house. Thus, a reproducible and reliable micropropagation protocol for mass multiplication of Stevia rebaudiana (Bertoni) Bertoni using nodal segments has been developed.

In vitro propagation of male and female Hippophae salicifolia D. Don

Hippophae salicifolia D. Don. is a deciduous, temperate, and dioecious tree of great economic and ecological importance. In this study, an in vitro propagation protocol was established for male and female explants of H. salicifolia. The most suitable explants were selected by screening the various types of explants such as dormant and active axillary buds, apical buds, nodal parts, and leaves. The explants were selected every month throughout the year to test the response, and only dormant and active buds were able to generate a response. Accumulation of phenolics was one of the most significant problems in the in vitro propagation of H. salicifolia, which was overcome by using an antioxidant solution of 1500 mg L−1 each ascorbic acid and citric acid followed by a lower temperature treatment, and pre-culture in 1.2% (w/v) agar supplemented with 0.01% (w/v) myo-inositol and 3% (w/v) sucrose. Active buds proved to be the best explant type to propagate H. salicifolia in vitro, either on Murashige and Skoog (MS) or Lloyd and McCown Woody Plant media supplemented with high cytokinin concentrations. Half- and full-strength MS media were used for rooting the in vitro-grown shoots, and the rooting percentage was higher in the half-strength MS media than in the full strength. There were no significant differences in the responses of male and female explants in various culture conditions. Female plants showed better acclimatization than male plants.

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

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.

Micropropagation to Conserve the Endangered Gabal Elba Dragon Tree (Dracaena ombet Heuglin ex Kotschy & Peyr)

Plant tissue culture offers opportunities for the rescue and conservation of endangered plant species. Here, we report the successful in vitro propagation of Dracaena ombet, an endangered plant. Several physical and chemical seed treatments were evaluated to develop a propagation approach. Germination of D. ombet seeds was monitored for 16 weeks by placing them onto Murashige and Skoog (MS) medium. Maximum seed germination (20%) was recorded when seeds were soaked-scarified, whereas all other treatments did not result in seed germination. Fragmented (longitudinally bisected) and intact in vitro shoots were cultured onto MS medium supplemented with various concentrations of 6-benzylaminopurine (BAP) and indole butyric acid (IBA) to induce axillary shoots. Longitudinal fragmentation of explants had a greater effect than the intact explants for shoot proliferation when cultured onto medium containing plant growth regulators. Fragmented shoots cultured onto MS medium supplemented with 2 mg·L−1 BAP and 0.5 mg·L−1 IBA treatment resulted in the highest amount of axillary shoots (seven shoots per explant). The intact shoots had the highest axillary shoots (1.8 shoots per explant) when cultured onto a medium supplemented with a combination of 1 mg·L−1 BAP and 0.5 mg·L−1 IBA. One hundred percent rooting was obtained using half strength MS medium supplemented with 0.5 or 1 mg·L−1 IBA. With full strength MS medium, a maximum rooting of 60% was obtained with 1 mg·L−1 IBA or naphthalene acetic acid (NAA) addition. The plantlets were acclimatized to ex vitro conditions with a 95% survival rate. This study offers a simple method for in vitro propagation of D. ombet, which is valuable to enable conservation of this endangered species.

Nutritional requirements for germination and in vitro development of three Orchidaceceae species in the southern Brazilian Amazon

Tissue culture is an alternative form of producing healthy, vigorous and regular plants on a large scale. The purpose of this study was to evaluate the most efficient culture medium for in vitro plantlet germination and development of three Orchidaceae species. Seeds disinfested of three species were dispersed in distilled water and dripped into basic Murashige and Skoog (MS) medium. The experimental design was completely randomized in a factorial 3 x 4 (three species x four culture media), with 5 replications. Four treatments were established: (1) full-strength MS medium, with the full nutrient concentration (MSØ), (2) full-strength MS medium plus 0.3% activated charcoal (MSØ ACh), (3) half- strength MS medium (½ MS) and (4) half- strength MS medium with 0.3 % activated charcoal (½ MS ACh). Germination was evaluated after 15, 20, 25, 30, and 60 days. The shoot height, leaf number and length, root number and length of plantlets of the three studied species were assessed. In A. variegata, 73% germinated after 60 days in ½ MS ACh medium. In the same period, 100% of E. viparum and S. gloriosa seeds germinated in MSØ ACh medium. The plant height, leaf number and length, root number and length were significantly higher for the species A. variegata and E. viviparum in MSØ ACh medium. The culture media ½ MS and MSØ with addition of activated charcoal favored in vitro germination for the three orchid species of this study.

Organogenesis of seedling explants of Carum copticum

ABSTRACTOrganogenesis of Carum copticum from hypocotyl explants of in vitro seedlings was examined. The highest callus induction was observed from hypocotyl explants on both Murashige and Skoog (MS) and Nitsch and Nitsch (NN) media. A maximum of 92% of hypocotyl explants produced callus on MS medium supplemented with 1.0 mgL–1 N6-benzyladenine (BA)and 0.5 mgL–1 naphthaleneacetic acid (NAA), whereas, 84% of hypocotyl explants produced callus on NN medium with 1.0 mgL–1 BA and 0.5 mgL–1 NAA. Hypocotyl explants-derived light green and friable callus responded to shoot organogenesis on both MS and NN media with plant growth regulators. A maximum of shoot regeneration frequency (76%) was observed on MS with 1.5 mgL–1 BA, whereas, 55% shoot regeneration frequency was observed on NN medium with 1.5 mgL–1 BA. A maximum number of 8.4 shoots per callus explant having a shoot length of 4.05 cm was observed on MS medium with 1.5 mgL–1 BA, whereas, 5.5 shoots per callus explant with a shoot length of 3.5 cm was observed on NN medium with 1.5 mgL–1 BA. Regenerated shoots were cultured on full strength MS and NN medium for root induction. The highest root regeneration frequency of 96% was obtained on MS medium with 0.25 mgL–1 NAA inducing 7.6 roots per shoot with root length of 4.25 cm, whereas, 83% root regeneration frequency was observed on NN medium with 0.25 mgL–1 NAA inducing 6.4 roots per shoot with a root length of 3.81 cm. In vitro derived plantlets were hardened and acclimatized successfully.

Micropropagation of Oregano (Origanum syriacum L.) Through Tissue Culture Technique

This research was carried out at the Plant Tissue Culture Laboratory (PTCL) in Faculty of Environmental Agricultural Sciences, Arish University, Egypt during 2013 to 2016. The aim of this study was using tissue culture technique for the micropropagation of Origanum syriacum L. plant. One node cutting and shoot tip were cultured on Murashige and Skoog (MS), Nitsch and Nitsch (NN) and Gamborg (B5) media. The results showed that shoot tip cultured on the MS medium had the best combination for the establishment stage of mother plants. Shoots cultured on MS medium supplemented with0.50 mgl-1 Kin and 0.05 mgl-1NAA produced the highest shoots developed during multiplication stage. MS medium supplemented with 0.40 mgl-1 thiamine achieved the best shoots development compared with the other additive treatments and control. Addition of 1 mgl-1IBA to the full strength MS medium and 1.00 mgl-1 IAA to the half strength MS had the highest number of main roots and root lengths. Hardening the produced rooted plantlets were done in a greenhouse in pots containing mixture of peatmoss, vermiculite and washed sand at the rate (1:1:1) and after adjustment of mixture pH. Plantlets were successfully acclimated with 93 % survive.

Ultrastructure of somatic embryo development and plant propagation for Lachenalia montana

Embryogenesis for plant propagation by cell suspension culture was achieved for the first time using friable embryogenic callus (FEC) from leaf explants of Lachenalia montana Schltr. ex W.F. Barker. FEC was established with solid (8gL−1 agar) MS (Murashige and Skoog, 1962) medium containing various concentrations and combinations of sucrose and plant growth regulators (PGRs). The somatic embryos (SEs) were developed from FEC in liquid MS (MSL) medium with or without PGRs. A higher number of SEs of different developmental stages (26.0–19.4, globular to cotyledonary-stages respectively) were obtained on MSL medium with 0.5μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1μM thidiazuron (TDZ). The germination frequency (33.7%) was highest in MSL medium containing 1μM 2,4-D and 2μM TDZ. The different stages of SEs germinated (92%) on solid full-strength MS medium containing 15gL−1 sucrose and 10μM phloroglucinol (PG). The plantlets were successfully acclimatized in the greenhouse. Developmental stages and features of embryoids were confirmed by histological and ultrastructural studies using light and transmission electron microscopy (TEM). Many mitochondria, lipid bodies together with starch grains, chloroplasts, Golgi apparatuses, vacuoles and nuclei were detected. The system developed offers controlled large-scale clonal propagation which ensures germplasm conservation, confirms viability of embryogenesis by ultrastructural studies and provides a system for genetic transformation studies for horticultural and ornamental improvement.

In vitro approaches for conservation and reducing juvenility of Zanthoxylum armatum DC: an endangered medicinal plant of Himalayan region

This paper provides authentic information on the micropropagation and genetic fidelity assessment methods, which can be used for conservation and quality assessment of endangered medicinal plant Zanthoxylum armatum. An in vitro propagation protocol for Zanthoxylum armatum (local name—Timur, Family-Rutaceae), an endangered medicinal plant of the Himalayan region, has been developed through culture of nodal explants. Use of Murashige and Skoog (MS) medium, supplemented with 12.0 µM 6-benzylaminopurine (BAP), 0.5 µM indole-3-acetic acid (IAA), and 0.5 µM gibberellic acid (GA3), resulted in good shoot proliferation (4.78 ± 0.18 shoots/explant) and shoot length (3.46 ± 0.05 cm). One hundred percent rooting was achieved through pulse treatment of shoots on full-strength MS medium supplemented with 50.0 µM indole-3-butyric acid (IBA) for 12 h, followed by subsequent transfer to half strength MS medium without plant growth regulators (PGRs). The plant survival was found to be 75% following acclimation. Plants started to flower after 1 year of transfer to soil under nursery conditions. Genetic stability of the regenerated plants was assessed using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers. Results confirmed monomorphic nature of regenerated plants, matching with that of the mother plant, and thus genetic fidelity of the micropropagated plants. This is the first report for micropropagation of Z. armatum. Following further field trials, the protocol can be used for raising quality planting material for commercial cultivation which should also help in its conservation.

Enhancing plant regeneration of Lachenalia viridiflora, a critically endangered ornamental geophyte with high floricultural potential

Lachenalia viridiflora is a critically endangered bulbous plant with high potential on the international floriculture market. In the present study, an efficient protocol for in vitro plantlet regeneration through somatic embryogenesis was developed. Embryogenic callus was established from leaf explants cultured on Murashige and Skoog (MS) basal medium supplemented with various concentrations and combinations of picloram and thidiazuron (TDZ). A high number of somatic embryos (28.5±1.49) at different developmental stages of somatic embryos (SEs: globular embryos, juvenile embryo and cotyledon with bipolar characteristics) was obtained on Murashige and Skoog (1962) medium with 2.5μM picloram, and 1.0μM TDZ. Histological and scanning electron microscope (SEM) analyses confirmed the presence of somatic embryos. Mature somatic embryos germinated and developed into plantlets after 6 weeks on full strength MS medium. High plant regeneration frequency (91.1%) was achieved on full-strength MS medium supplemented with 5μM phloroglucinol (PG). Well-developed healthy plantlets were successfully acclimatized in the greenhouse with a survival rate of 90%. The result of this study is beneficial in the mass propagation of high-quality L. viridiflora plants for the commercial horticultural market and also provides a platform for future genetic transformation studies.

Micropropagation and Genetic Fidelity of the Regenerants of Aglaonema ‘Valentine’ Using Randomly Amplified Polymorphic DNA

The present study reports a simple protocol for in vitro regeneration of Aglaonema ‘Valentine’ using axillary shoot explants for rapid multiplication and production of true-to-type plants. Different concentrations of benzyladenine (BA; 0, 1, 3, 5, and 7 mg·L−1), kinetin (Kin; 0, 1, 3, 5, and 7 mg·L−1), thidiazuron (TDZ; 0, 0.5, 1.0, 1.5, and 2.0 mg·L−1), naphthalene acetic acid (NAA; 0, 0.5, and 1.0 mg·L−1), and indole-3-butyric acid (IBA; 0, 0.5, and 1.0 mg·L−1) were used for shoot regeneration. The highest shoot proliferation (5.0) was obtained on Murashige and Skoog (MS) medium supplemented with 1.5 mg·L−1 TDZ and 1 mg·L−1 NAA. In vitro rooting was easily achieved with 100% at all concentrations of NAA and IBA supplemented to half- or full-strength MS medium. Regenerated plantlets were acclimatized in greenhouse with 100% survival rate. Randomly amplified polymorphic DNA (RAPD) analysis confirmed the genetic fidelity of the regenerated plantlets and mother plant.

Regeneration of plantlets fromin vitroRoot and Leaf culture of Vandaceous orchid,Ascocentrum ampullaceum(Roxb.) Schlter

The paper describes in vitro culture protocol of commercially important Vandaceous orchid from North East India, Ascocentrum ampullaceum using leaves and roots as explants. Leaves and roots of in vitro raised 5–6 months old Ascocentrum ampullaceum were inoculated in Murashige and Skoog (MS) medium supplemented with additive growth hormone (15%) Coconut milk and growth regulators such as NAA, IAA, BAP and Kn (0.5, 1.0, 2.0, 2.5 mgl−1), both singly and in combination. Leaf culture produced highest protocorm like bodies (Plbs) (40%) in the medium supplemented with 0.5 mgl−1 BAP, followed by 30% Plbs supplemented with 15% CM and BAP 2.0 mgl−1, whereas the highest (60%) multiple shoot bud formation was noticed in MS medium supplemented with 15% CM and 0.5 mgl−1 BAP, followed by 30% in 1/2MS medium supplemented with 0.5 mgl−1Kn; Callus formation (30%) was observed in 1/2MS medium supplemented with 2.0 mgl−1 NAA. Roots when cultured in 1/2 MS, only elongation was observed, but in full strength MS medium, it produced both Plbs and shoot buds. The highest Plbs and shoot buds (40%) was observed in the media supplemented in combination of CM (15%) + BAP (0.5 mgl−1) followed by 25% in CM (15%) + BAP (1.0 mgl−1).

Influence of BA and IBA or NAA Combinations on Micropropagation of <i>Cryptolepis sanguinolenta</i>

The study being the first of its kind established an efficient protocol for micropropagation of Cryptolepis sanguinolenta, an important endangered medicinal plant species, used in the treatment of Malaria. For shoot induction, semi hard wood nodal segments were maintained on MS (Murashige and Skoog) nutrient medium supplemented with MS vitamins, 30 g/L sucrose, 3% gelrite and various auxin and cytokinin combinations. Treatments involved 6-benzyladenine (BA) at 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 mg/L in combination with 0.1 mg/L Indole 3-butyric acid (IBA) or Naphthaleneacetic acid (NAA). Control consisted of hormone free MS medium. BA and IBA combinations were found to be more efficient in shoot regeneration than the BA and NAA combinations. Cultures maintained on MS medium supplemented with 3 mg/L BA, in combination with 0.1 mg/L IBA recorded the highest shoot induction (100%), mean shoot length (1.28 cm) and mean number of nodes per explant (2.5). This, however, did not differ significantly from cultures maintained on 2 or 2.5 mg/L BA and 0.1 mg/L IBA supplemented MS medium. Regenerated shoots were transferred onto different media for root induction. Treatments consisted of full strength MS medium augmented with either 0.5 or 0.1 mg/L IBA, hormone free half strength MS medium and half strength MS medium augmented with 0.01 mg/L IBA. After six weeks of culture, no rooting was recorded in all treatments with the exception of half strength MS medium supplemented with 0.01 mg/L IBA, which recorded 60% rooting. Regenerated plantlets were successfully weaned and established in the greenhouse.