Micropropagation of Withania somnifera from germinating seeds and shoot tips
TL;DR: Shoot multiplication was achieved in vitro from shoot tips of aseptically germinated seedlings of Withania somnifera L. with direct multiple shoot initiation from germinating seeds in the presence of BA alone.
Abstract: Shoot multiplication was achieved in vitro from shoot tips of aseptically germinated seedlings of Withania somnifera L. using low concentrations of 6-benzyladenine (BA), viz. 2.2, 4.4 and 8.9 μM. Maximum number of shoots were obtained when 2.3 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or 2.5 μM indolebutyric acid (IBA) was added to medium containing 4.4 μM BA during initiation of shoot multiplication, but not when added later. Direct multiple shoot initiation was also obtained from germinating seeds in the presence of BA alone. Rooting was successful in excised shoots grown on growth regulator-free MS medium. Rooted shoots were successfully established in soil in a greenhouse.
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TL;DR: In this paper, an efficient large-scale clonal propagation protocol has been described for Withania somnifera (L.) Dunal, a valuable medicinal plant, using cotyledonary nodes derived from axenic seedlings.
Abstract: An efficient large-scale clonal propagation protocol has been described for Withania somnifera (L.) Dunal, a valuable medicinal plant, using cotyledonary nodes derived from axenic seedlings. Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) (MS) medium supplemented with 1.0 mg l−1N6-benzyladenine (BA) was found to be optimum for production of multiple shoots (100 % shoot proliferation frequency and 16.93 shoots per explant). Successive shoot cultures were established by repeatedly sub-culturing the original cotyledonary node on a fresh medium after each harvest of newly formed shoots. Multiple shoot proliferation was also achieved from nodal segments derived from in vitro raised shoots on MS medium augmented with 1.0 mg l−1 BA. Regenerated shoots were best rooted (95.2 %, 38.7 roots per shoot) in half-strength MS medium supplemented with 1.0 mg l−1 indole-3-butyric acid. The plantlets were successfully acclimated and established in soil. Random amplified polymorphic DNA and inter-simple sequence repeats analysis revealed a homogeneous amplification profile for all micropropagated plants analyzed validating the genetic fidelity of the in vitro regenerated plants.
56 citations
Cites background or methods from "Micropropagation of Withania somnif..."
...…and Singh 1991; Kulkarni et al. 2000; Sivanesan and Murugesan 2008; Sivanandhan et al. 2011), axillary meristems (Roja et al. 1991), shoot tips (Sen and Sharma 1991; Furmanowa et al. 2001; Ray and Jha 2001), apical shoot buds (Sivanesan 2007), somatic embryogenesis (Sharma et al. 2010),…...
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...1991), shoot tips (Sen and Sharma 1991; Furmanowa et al. 2001; Ray and Jha 2001), apical shoot buds (Sivanesan 2007), somatic embryogenesis (Sharma et al....
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...…per cotyledonary node, which is higher than the earlier reports of W. somnifera micropropagation using meristems (145 shoots/shoot tips explant; Sen and Sharma 1991), (24 shoots/node; Kulkarni et al. 2000), (38 shoots/shoot tip; Ray and Jha 2001), (120 shoots/shoot tips; Furmanowa et al.…...
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...propagation using meristems (145 shoots/shoot tips explant; Sen and Sharma 1991), (24 shoots/node; Kulkarni et al....
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TL;DR: An efficient in vitro plant regeneration protocol for Swertia chirata Buch.-Ham was developed using shoot tip explants derived from in vitro grown seedlings and hardening was successfully attained under controlled conditions inside the plant tissue culture room.
Abstract: An efficient in vitro plant regeneration protocol for Swertia chirata Buch.-Ham. ex Wall (Gentianaceae), a critically endangered Himalayan medicinal herb, was developed using shoot tip explants derived from in vitro grown seedlings. Media with 2% sucrose and various types of hormones markedly influenced in vitro propagation of S. chirata. An in vitro shootlet production system using Murashige and Skoog (MS) medium with various hormones such as BAP, KN and TDZ was established. BAP at 1.0 mg/l and KN, 0.1 mg/l induced highest number of multiple shoots (42.16 ± 1.05) per explant. Micro-proliferated shoots were transferred to elongation medium amended with GA3 (0.1 mg/l) and hormone free basal medium, after which they were transferred to rooting medium. The highest frequency of rooting (22.48 ± 1.08) was obtained in half-strength MS medium supplemented with NAA, 0.1 mg/l after testing with different auxins at various concentrations within 4 weeks of transfer to the rooting medium. Hardening was successfully attained under controlled conditions inside the plant tissue culture room. This method could effectively be applied for the conservation and clonal propagation to meet the pharmaceutical demands.
54 citations
TL;DR: The influence of cytokinins and culture conditions including medium volume, harvest time and elicitation with abiotic elicitors (SA/MeJ) have been studied for the optimal production of biomass and withanolides in the multiple shoot culture of Withania somnifera as mentioned in this paper.
Abstract: The influence of cytokinins and culture conditions including medium volume, harvest time and elicitation with abiotic elicitors (SA/MeJ) have been studied for the optimal production of biomass and withanolides in the multiple shoot culture of Withania somnifera. Elicitation of shoot inoculum mass (2 g l−l FW) with SA at 100 μM in the presence of 0.6 mg l−l BA and 20 mg l−l spermidine for 4 h exposure time at the 4th week in 20 ml liquid medium recorded higher withanolides production (withanolides A [8.48 mg g−l DW], withanolides B [15.47 mg g−l DW], withaferin A [29.55 mg g−l DW] and withanone [23.44 mg g−l DW]), which were 1.14 to 1.18-fold higher than elicitation with MeJ at 100 μM after 5 weeks of culture. SA-elicited cultures did not exhibit much variation in biomass accumulation when compared to control. This cytokinin induces and SA-elicited multiple shoot culture protocol provides a potential alternative for the optimal production of biomass and withanolides utilizing liquid culture.
52 citations
Cites background or methods from "Micropropagation of Withania somnif..."
...Various explants (shoot tip, single bud, leaf, axillary bud, node and cotyledon) were used by many authors using solid medium in W. somnifera (Sen and Sharma 1991; Kulkarni et al. 1996, 2000; Furmanowa et al. 2001; Sivanandhan et al. 2011)....
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...Sen and Sharma (1991) recorded 145 shoots/ shoot tip and 120 shoots/shoot tip on BA ?...
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TL;DR: High fidelity micro-propagated system for efficient and rapid micro- Propagation protocol of this important medicinal plant and great use in conserving without risk of genetic instability is provided.
Abstract: An efficient plant propagation system through nodal explants was established in Ocimum gratissimum L, a medicinally important herbaceous perennial herb belonging to the family Lamiaceae. Axillary shoot bud proliferation was initiated from nodal explants cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of N6-benzyladenine (BA) (0.5 - 3.0 mg/l), Kinetin (KN) (0.5 - 3.0 mg/l) and 2-isoPentenyladenine (2-iP) (0.5 - 3.0 mg/l). Maximum numbers of shoots (5.17 ± 0.04) with average length (2.50 ± 0.07) were induced on medium containing 1.0 mg/l BA. Shoot multiplication was maintained by repeated subculturing the original nodal explants on shoot multiplication medium after each harvest of newly formed shoots. Histological study shows that the organogenesis occurs directly, without callus formation on epidermal and sub epidermal layer of the explants. Rooting of shoots was achieved on half strength MS medium supplemented with 1.5 mg/1 Indole-3-butyric acid (IBA) and 2% sucrose. Well-developed complete plantlets were transferred to plastic pots containing a mixture of (1:1) soil and vermiculite showed 82.5 % survival rate. Genetic fidelity was assessed by chromosome analysis and DNA fingerprinting using random amplified polymorphic DNA (RAPD) of in vitro and in vivo plants. Nine arbitrary decamers displayed same banding profile showed no genomic alterations, indicating homogeneity among the tissue culture regenerates and genetic uniformity with that of donor plants. The present study provides high fidelity micro-propagated system for efficient and rapid micro-propagation protocol of this important medicinal plant and great use in conserving without risk of genetic instability.
50 citations
Cites background from "Micropropagation of Withania somnif..."
...Reduction in the number of shoots generated from each node at BA concentration higher than the optimal level was also reported for several medicinal plants [22,33]....
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TL;DR: This review presents a consolidated account of the phytochemistry, pharmacology and biotechnology involving in vitro propagation, genetic transformation and metabolite profiling in W. somnifera and W. coagulans.
Abstract: Withania (Family: Solanaceae) is a highly acclaimed genus in the Indian Ayurvedic system of medicine. In Ayurveda, Withania is known to promote physical and mental health and used to treat almost all the disorders that affect human health. Withania somnifera andWithania coagulans are the two most esteemed species of this genus having high medicinal significance. These species are natural source of withanolides (steroidal lactones) which are used as ingredients in many formulations prescribed for a variety of diseases. Many pharmacological studies have been conducted to investigate the properties of Withania as a multi-purpose medicinal agent. Advances in biotechnology, especially in vitro culture techniques, molecular biology and metabolite profiling provided new insights for conservation and management of plant genetic resources and better harvesting of drugs from medicinal plants. This review presents a consolidated account of the phytochemistry, pharmacology and biotechnology involving in vitro propagation, genetic transformation and metabolite profiling in W. somnifera and W. coagulans.
Key words: Withania, phytochemistry, pharmacology, withanolides, micropropagation, metabolite profiling.
49 citations
Cites background from "Micropropagation of Withania somnif..."
...Sen and Sharma (1991) examined regeneration from cultured shoot-tip and nodal explants of W. somnifera....
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References
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TL;DR: In vivo redox biosensing resolves the spatiotemporal dynamics of compartmental responses to local ROS generation and provide a basis for understanding how compartment-specific redox dynamics may operate in retrograde signaling and stress 67 acclimation in plants.
Abstract: In experiments with tobacco tissue cultured on White's modified medium (basal meditmi hi Tnhles 1 and 2) supplemenk'd with kiticthi and hidoleacctic acid, a slrikin^' fourlo (ive-told intTease iu yield was ohtaitu-d within a three to Tour week j^rowth period on addition of an aqtteotis exlrarl of tobacco leaves (Fi^'ures 1 and 2). Subse(iueutly it was found Ihiit this jnoniotiou oi' f^rowih was due mainly though nol entirely to inorj^auic rather than organic con.stitttenls in the extract. In the isolation of Rrowth factors from plant tissues and other sources inorj '̂anic salts are fre(|uently carried along with fhe organic fraclioits. When tissue cultures are used for bioassays, therefore, il is necessary lo lake into account increases in growth which may result from nutrient elements or other known constituents of the medium which may he present in the te.st materials. To minimize interference trom rontaminaitis of this type, an altempt has heen made to de\\eh)p a nieditmi with such adequate supplies of all re(iuired tnineral nutrients and cotntnott orgattic cottslitueitls that no apprecial»le change in growth rate or yield will result from the inlroduclion of additional amounts in the range ordinarily expected to be present in tnaterials to be assayed. As a point of referetice for this work some of the culture media in mc)st common current use will he cotisidered briefly. For ease of comparis4)n Iheir mineral compositions are listed in Tables 1 and 2. White's nutrient .solution, designed originally for excised root cultures, was based on Uspeuski and Uspetiskaia's medium for algae and Trelease and Trelease's micronutrieni solution. This medium also was employed successfully in the original cttltivation of callus from the tobacco Iiybrid Nicotiana gtauca x A', tanijadorffii, atitl as further modified by White in 194̂ ^ and by others it has been used for the
63,098 citations
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16,251 citations
01 Jan 1985
TL;DR: From Metabolism and Osmotic Work to Totipotency and Morphogenesis: A Study of Limitations Versus Multiple Interactions.
Abstract: I From Metabolism and Osmotic Work to Totipotency and Morphogenesis: A Study of Limitations Versus Multiple Interactions.- II Primary Metabolism.- Photosynthetic Carbon Metabolism in Photoautotrophic Cell Suspension Cultures of Chenopodium rubrum L..- On the Photosynthetic System and Assimilate Metabolism of Daucus and Arachis Cell Cultures.- Regulation of Carbon and Nitrogen Assimilation Pathways in Tobacco Cell Suspension Cultures in Relation with Ultrastructural and Biochemical Development of the Photosynthetic Apparatus.- Application of Gas Analysis to Continuous Culture.- Carbohydrate Source, Biomass Productivity and Natural Product Yield in Cell Suspension Cultures.- Nitrogen Metabolism of Leaf and Microspore Callus of Betula pendula.- III Secondary Metabolism.- 4-Coumarate: CoA Ligase in Wild Carrot Cell Culture Clones Which Accumulate Different Amounts of Anthocyanin.- Induction of Anthocyanin Synthesis in Relation to Embryogenesis in a Carrot Suspension Culture - A Model System for the Study of Expression and Repression of Secondary Metabolism.- Metabolism of Quinolizidine Alkaloids in Plants and Cell Suspension Cultures: Induction and Degradation.- Production of Alkaloids by Ergot (Claviceps fusiformis Lov.) on Pennisetum typhoides (Burm.) Stapf and Hubb. in Vitro.- Compartmentation of Alkaloids in a Cell Suspension of Catharanthus roseus: A Reappraisal of the Role of pH Gradients.- Studies on Variability of Plant Tissue Cultures for Alkaloid Production in Catharanthus roseus and Papaver somniferum Callus Cultures.- Biosynthesis and Accumulation of Indole Alkaloids in Cell Suspension Cultures of Catharanthus roseus Cultivars.- Formation of Cardenolides in Cell and Organ Cultures of Digitalis lanata.- Metabolism of Caffeoyl Derivatives in Plant Cell Cultures.- Metabolic Relationships of Putrescine, GABA and Alkaloids in Cell and Root Cultures of Solanaceae.- Metabolism and Degradation of Nicotinic Acid in Plant Cell Cultures.- Plant Cell and Tissue Culture of Cinchona Species.- The Production of Pyrethrins by Chrysanthemum cinerariaefolium (Trev) Bocc..- Biosynthesis of Chorismate-Derived Quinones in Plant Cell Cultures.- The Role of Leucine in Terpenoid Metabolism: Incorporation of Leucine into Sesquiterpenoids and Phytosterols by Andrographis Tissue Cultures.- Accumulation of Antineoplastic Agents by Plant Tissue Cultures.- Induction of Enzymes of Phytoalexin Synthesis in Soybean Cells by Fungal Elicitor.- Protoplast Fusion of Secondary Metabolite-Producing Cells.- Conventional and New Approaches to Increase the Alkaloid Production of Plant Cell Cultures.- Multiple Shoot Cultures: A Viable Alternative in Vitro System for the Production of Known and New Biologically Active Plant Constituents.- IV Fermentation and Cryopreservation.- Large-Scale Production of Rosmarinic Acid from Plant Cell Cultures of Coleus blumei Benth..- Immobilised Plant Cell Culture Systems.- Biotransformation of Cardiac Glycosides by Digitalis Cell Cultures in Airlift Reactors.- Cryopreservation of Plant Cell Cultures.- V Herbicides.- Altered Amino Acid Biosynthesis in Amino Acid Analog and Herbicide-Resistant Cells.- Acetohydroxyacid Synthase Inhibitors as Herbicides.- A Glyphosate-Tolerant Plant Tissue Culture.- VI Plant Cell Culture - Future Perspectives.
321 citations
TL;DR: In this paper, the inheritance characteristics of various substituents on the withanolide skeleton were analyzed based on the occurrence in per cent of each substituent in relation to the total withanolides content in the hybrid plants and their respective parents.
43 citations
TL;DR: Of the cytokinins tested, benzylaminopurine (BAP) was more effective than either 2-isopentenyladenine (2iP) or kinetin (Kin) and produced maximum proliferation when used at 5×10-6M.
Abstract: Successful vegetative propagation of seedling jackfruit (Artocarpus heterophyllus Lam.) has been achieved by in vitro methods. Proliferation from nodal explants was greater than from shoot tips. Of the cytokinins tested, benzylaminopurine (BAP) was more effective than either 2-isopentenyladenine (2iP) or kinetin (Kin) and produced maximum proliferation when used at 5×10-6M. Shoot proliferation was optimal at 30°C with a 12 h photoperiod. Optimal rooting of shoots in vitro was obtained with indolebutyric acid (IBA) at 10-6M. The number and length of roots was significantly increased in 12 h light as compared with the dark.
40 citations