High frequency multiple shoot induction from nodal segments and rhinacanthin production in the medicinal shrub Rhinacanthus nasutus (L.) Kurz

High frequency multiple shoots have been induced from nodal segments of Rhinacanthus nasutus (L.) Kurz., a potent anticancerous ethnomedicinal plant. For initiation of cultures, nodal segments were cultured on MS medium supplemented with various concentrations (1.0–5.0 μM) of 6-benzyladenine or thidiazuron (TDZ) alone or in combination with α-naphthalene acetic acid (NAA 0.5–1.0 μM). The optimum frequency of response (85 %) and shoot number (3.3) was observed on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The shoots developed on initiation media were excised and nodal segments were subcultured on MS medium supplemented with TDZ (4.0 μM) and NAA (0.5–1.0 μM). This subculturing process was repeated thrice, each with 45 days of duration and the multiple shoot formation was recorded at the end of every subculture stage. The highest frequency of response (100 %) and number of multiple shoots (24.1) per explant were recorded at the end of the third subculture passage on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The optimum rooting of shoots was observed on ½ MS medium fortified with 3.0 μM indole-3-butyric acid. The rooted plants were successfully transplanted to soil. The estimation of rhinacanthin (RC) content in shoots and roots was carried out in 6-month-old ex vitro plants (i.e., plants regenerated via in vitro culture) and field grown natural plants by high performance liquid chromatography. Both shoots and roots of naturally grown plants showed slightly higher RC content than ex vitro grown plants. The highest RC content (4.6 mg/g DW RC-C, 0.14 mg/g DW RC-D and 0.10 mg/g DW RC-N) was recorded in roots of naturally grown plants.     

Plant regeneration of non-toxic Jatropha curcas—impacts of plant growth regulators, source and type of explants

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel plant, however, oil and deoiled cake are toxic. A non-toxic variety of J. curcas is reported from Mexico. The present investigation explores the effects of different plant growth regulators (PGRs) viz. 6-benzyl aminopurine (BAP) or thidiazuron (TDZ) individually and in combination with indole-3-butyric acid (IBA), on regeneration from in vitro and field-grown mature leaf explants, in vitro and glasshouse-grown seedlings cotyledonary leaf explants of non-toxic J. curcas. In all the tested parameters maximum regeneration efficiency (81.07%) and the number of shoot buds per explants (20.17) was observed on 9.08 μM TDZ containing Murashige and Skoog’s (MS) medium from in vitro cotyledonary leaf explants. The regenerated shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with 2.25 μM BAP and 8.5 μM IAA. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA and NAA for four days followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg/l activated charcoal. The rooted plants could be established in soil with more than 90% survival rate.     

High-frequency plantlet regeneration and multiple shoot induction from cultured immature seeds of Rhynchostylis retusa Blume., an exquisite orchid

Seeds of an exquisite orchid, Rhynchostylis retusa, germinated in vitro on ½ Murashige and Skoog (MS) medium supplemented with different concentrations of coconut milk (CM). Of the different concentrations of CM employed for seed germination, 15% gave optimum response. On this medium a maximum of 93% cultures produced seedlings 90 days after inoculation. Individual seedlings with a length of about 0.5 cm were subcultured on MS medium supplemented with various concentrations of 6-benzylaminopurine (BA) and α-naphthalene acetic acid (NAA), with or without activated charcoal (AC), for further growth. Seedling growth was maximum on MS medium supplemented with 6 μM BA, 0.2 μM NAA, and 1 g L^?1 AC. Here a maximum seedling length of 2.3 cm was observed after 1 month of culture. The seedlings were subcultured on MS medium supplemented with kinetin (Kn) or thidiazuron (TDZ), in the presence or absence of AC, for multiple shoot induction. A maximum multiple shoot number of 8.2 was observed on MS medium supplemented with 2 μM TDZ in the presence of AC. The shoots were rooted on ½ MS medium supplemented with 2 μM indole-3-butyric acid (IBA) and successfully transplanted to soil. Of the 45 plantlets transferred to soil 40 survived. The reproducible protocol standardized here will enable rapid propagation and conservation of this precious orchid.     

Thidiazuron enhances shoot organogenesis from leaf explants of Saussurea involucrata Kar. et Kir

An efficient protocol for the in vitro micrpropagation of Saussurea involucrata Kar. et Kir, an endangered Chinese medicinal plant, was developed. Shoot organogenesis was obtained following culture of leaf explants on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ). After 28 d of culture, 15.6?±?1.4 shoots were regenerated per leaf explant on MS medium containing 0.5 ??M TDZ. After transfer of shoots to a medium containing 5.0 ??M indole-3-acetic acid, approximately 80% of the regenerated shoots formed roots and whole plantlets. After transfer of rooted shoots to the greenhouse, 83% of the regenerated plantlets survived and grew vigorously. The regeneration protocol developed in this study provides a basis for germplasm conservation and for the production of plant material necessary to study the medicinally active components of S. involucrata.     

Multiple shoot induction and regeneration of whole plants from cotyledonary node and nodal explants of Sterculia urens Roxb., a gum yielding tree

Plant regeneration from the nodal explants of 1-month-old in vitro grown plants and cotyledonary node explants of 15-days-old seedlings of Sterculia urens is reported. Nodal explants were grown on MS medium supplemented with various growth regulators like BA, KIN and TDZ. For shoot induction 13.3 μM BA, 0.9 μM TDZ and 9.3 μM KIN were found optimum. Among the three growth regulators 0.90 μM TDZ was used for the growth of cotyledonary node explants. An average of 8.6 shoots per node and 11.2 shoots per cotyledonary node were observed in 4 to 5 weeks. These shoots were subsequently rooted in vitro on half strength MS medium containing various concentrations of auxins like IBA and NAA. The best concentrations for rooting of shoots were 19.7 μM IBA and 16.1 μM NAA. Plantlets were acclimatized to ex vitro conditions and established in the field.     

In vitro morphogenic response of different explants of Gentiana kurroo Royle from Western Himalayas—an endangered medicinal plant

Micropropagation offers a great potential to produce millions of clonal individuals through tissue culture via induction of morphogenesis. The aim of this work was to obtain an efficient protocol for callus regeneration for Gentiana kurroo Royle. The morphogenic response of different explants (leaves, petioles, roots) varied and responded differently for regeneration according to combinations of growth regulators. The petiole explants were best responding for callus induction and subsequently for indirect and direct regeneration. The callus induction was achieved on MS basal + 1.0 mg/l benzyladenine (BA) and 3.00 mg/l naphthalene acetic acid (NAA). MS medium supplemented with 0.10 mg/l NAA and 1.0 mg/l thidiazuron (TDZ) was recorded as the best medium for indirect regeneration. However, for direct regeneration the maximum number of shoot emergence was observed on MS basal fortified with 0.10 mg/l NAA + 0.75 mg/l TDZ. Half strength MS basal supplemented with indole-3-butyric acid (IBA) 1.00 mg/l gave best response for root induction. Subsequently, the plantlets were transferred and 100 % survival rate was recorded only on autoclaved cocopeat. No morphological variations were recorded in the callus regenerated plantlets.     

In vitro organogenesis from internode derived callus cultures of Capsicum annuum L.

An efficient protocol of shoot organogenesis and plant regeneration from internode derived callus has been developed for Capsicum annuum. Optimal callus was developed from internodal segments on Murashige and Skoog (MS) medium supplemented with 10 μM 2,4-dichlorophenoxy acetic acid (2,4-D) and 2.0 μM 6-benzyladenine (BA). Shoot differentiation was achieved from the surface of callus when transferred on shoot induction medium containing BA and thidiazuron (TDZ) alone or in combination. The highest number of de novo adventitious shoots (25.4?±?1.42) and shoot length (4.6?±?0.37 cm) was recorded on MS medium supplemented with 5.0 μM BA and 2.5 μM TDZ. The individual elongated shoots were rooted well on MS medium supplemented with 1.0 μM Indole-3-butyric acid (IBA). The in vitro raised plantlets with properly developed shoot and roots were acclimatized successfully and grew well in the greenhouse. All the regenerated plants appeared normal with respect to morphology and growth characteristics with 85% survival rate.     

An efficient callus proliferation protocol and rhaponticin accumulation of Rheum franzenbachii Munt., a medicinal plant

An efficient callus proliferation system for Rheum franzenbachii Munt., a rare medicinal plant, has been developed. Callus induced from leaf explants incubated on Murashige and Skoog (MS) medium with appropriate supplements of plant growth regulators. In the 6-benzylaminopurine (6-BAP) in combination with α-naphthalene acetic acid (NAA) treatments, different concentrations of NAA showed different induction effects on explants. When concentration of 6-BAP was as high as 2.0 mgl^?1 in combination with 0.5 mgl^?1 NAA, the callus induction rate reached 58.3%. N-phenyl-N’-1,2,3-thiadiazol-5-ylure (TDZ) in combination with NAA was very suitable for callus proliferation compared to TDZ in combination with 2,4-dicholorophenoxy acetic acid (2,4-D) or TDZ in combination with indole-3-acetic acid (IAA). Fresh and dry weight of callus cultured on MS medium supplemented with 0.5 mgl^?1 TDZ in combination with 0.2 mgl^?1 NAA increased 26.3 and 15.0 times within 35 days culture, respectively. Quantitative analysis of rhaponticin by HPLC showed that the phytochemical profile of callus was similar to that of wild plants, and the content of rhaponticin in callus cultured on MS medium supplemented with 0.5 mgl^?1 TDZ and 0.2 mgl^?1 NAA was 16.6 mgg?¹DW compared to that of 4.0 mgg^?1 DW in wild plants.     

Optimization and quality assessment of adventitious roots culture in Panax quinquefolium L.

In this study, adventitious roots of Panax quinquefolium L. have been successfully established. The highest induction rate of roots was obtained in MS medium containing 3 mg L^?1 IBA after 4 weeks of culture. The culture conditions of adventitious root were optimized and evaluated with response surface methodology. The best culture conditions for root growth seemed to be 0.75 salt strength MS medium, 4.70 g L^?1 inoculum size and 40 days of culture. The active component contents of adventitious roots were compared with those of native roots. The total saponins content was found to be 16.28 mg g^?1 in native root and 4.64 mg g^?1 in adventitious root. The polysaccharide content of the adventitious root was 1.5 times higher than that in the native P. quinquefolium (30.54 vs. 20.28 mg g^?1).     

In vitro propagation, proscillaridin A production and antibacterial activity in Drimia robusta

Drimia robusta is a threatened traditional medicinal plant extensively used in South Africa. Rapid in vitro mass propagation of the species was developed for commercial cultivation from leaf explants using various concentrations and combinations of plant growth regulators and organic elicitors. The highest number of regenerated shoots per explant (14.6 ± 0.54) was obtained on Murashige and Skoog (MS) medium supplemented with a combination of 2.27 μM thidiazuron (TDZ), 2.22 μM benzyladenine (BA) and 20 μM glutamine. Adventitious shoots were rooted and the plantlets were successfully acclimatized (100 %) in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Proscillaridin A (PsA) content and the antibacterial activity of in vitro and ex vitro regenerated plants were evaluated in different tissues in comparison to naturally-grown plants. The highest content of PsA (19.68 μg mg^?1 DW) was recorded in roots of ex vitro plants which were grown on MS medium containing 2.27 μM TDZ, 2.22 μM BA and 100 mg l^?1 casein hydrolysate. In vitro regenerated plants grown on MS medium containing 2.27 μM TDZ, 2.22 μM BA and 50.8 μM MBZ gave high antibacterial activity (MIC of 0.156 mg ml^?1) against both Gram-positive and Gram-negative bacteria. Using this protocol the regenerated plants can be used in traditional medicine as an alternative to naturally collected plants.     

In vitro propagation of <Emphasis Type="Italic">Cymbidium goeringii</Emphasis> Reichenbach fil. through direct adventitious shoot regeneration

The influence of 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and thidiazuron (TDZ) on direct rhizome induction and shoot formation from rhizome explants of Cymbidium goeringii was explored. Rhizome segments obtained from in vitro seed cultures of C. goeringii were placed on Murashige and Skoog (MS) medium incorporated with 5, 10, 20, or 40 µM 2,4-D and 1, 2, 4, or 8 µM BA or TDZ alone or in combination with 20 µM 2,4-D. The explants developed only rhizomes on MS medium with or without 2,4-D. The highest percent of rhizome formation (100%) was obtained on MS medium incorporated with 20 μM of 2,4-D. The morphology and number of rhizomes varied with the level of 2,4-D in the medium. Direct adventitious shoot formation was achieved on medium incorporated with BA or TDZ. The adventitious shoots produced per explant significantly increased with the supplementation of 2,4-D to cytokinin-containing medium. The highest mean of 21.8 ± 1.8 shoot buds per rhizome segment was obtained in medium fortified with 20 μM 2,4-D and 2 μM TDZ. The greatest percent of root induction (100%) and the mean of 5.3 ± 1.1 roots per shoot were achieved on ½ MS medium incorporated with 2 μM of α-naphthaleneacetic acid. About 97% of the in vitro-produced plantlets acclimatized in the greenhouse. An efficient in vitro propagation protocol was thus developed for C. goeringii using rhizome explants.     

Effect of seaweed extracts and plant growth regulators on high-frequency in vitro mass propagation of Lycopersicon esculentum L (tomato) through double cotyledonary nodal explant

An efficient and reproducible two-step in vitro propagation system for tomato (Lycopersicon esculentum) was developed by using the combinations of seaweed biostimulant (Gracilaria edulis and Sargassum wightii) extracts and plant growth regulators (PGRs). Double cotyledonary nodal (DCN) explants of Co-3 cultivar were initially cultured on Murashige and Skoog (MS) and Gamborg’s medium (B5) containing thidiazuron (TDZ) and 6-benzylaminopurine (BA); the best responding cytokinin was tested in combinations with different auxins (NAA, IAA and IBA), and seaweed extracts (G. edulis and S. wightii) of about basal MS medium +10–70% was used for shoot proliferation. The best organogenic culture response was obtained on MS medium fortified with 1.5 mg L^?1 TDZ and 1.5 mg L^?1 IBA. Up to 24 shoots per explants were formed at an optimal duration of exposure to 35 days. Mini shoots of about 3–4 cm were transferred to medium supplemented with MS + iP, MS + zeatin, MS + G. edulis and MS + S. wightii at different concentrations. High frequency of shoot elongation was observed in the medium supplemented with 30% G. edulis (15.2 cm), and profuse rooting was observed in the medium supplemented with 50% S. wightii of about 16.1 cm. Shoot elongation and rooting were observed in the medium supplemented with seaweed extracts. The plantlets were transferred to the plant growth chamber (70% of relative humidity and 9 light cycles) and maintained in it for a week, and then they were transferred to a greenhouse condition. The plant growth chamber to green house transferred plantlets showed an increase in the survival rate from 70 to 85%. Thus a two-step regeneration protocol was developed in this study with a combination of seaweed extracts and PGRs, which provides a basis for the production of transgenics with high frequency and survivability of tomato plants.     

Factors affecting plantlet regeneration from in vitro cultured immature embryos and cotyledons of Prunus mume “Xue mei”

Using immature embryos and cotyledons as explants, a successful immature embryo culture and efficient plant direct regeneration via organogenesis from cotyledons, which showed different patterns, was established for the “Xuemei” cultivar of Prunus mume. For immature embryo culture, high frequency plantlet forming (89.5%) from embryo axis was obtained on half-strength Murashige and Skoog (½MS) medium supplemented with 13.2 μM 6-benzyladenine (BA) and 2.7 μM 1-naphthaleneacetic (NAA). At the same time, shoots direct differentiation from cotyledons with the embryo axis development was also observed on ½MS medium containing 2.2 μM BA together with different combinations of NAA (2.7, 5.4 μM) and indole-3-butyric acid (IBA) (0, 2.5, 5.0 μM). Better results were achieved when embryo axes were removed from cotyledons and cultured on ½MS medium supplement with 13.2 μM BA, 2.7 μM NAA (72.9%) or 2.2 μM BA, 2.2 μM thidiazuron (TDZ), and 2.7 μM NAA (84.2%), respectively. Regenerated shoots were successfully rooted on ½MS or Woody Plant medium (WPM) supplemented with 2.5–5.0 μM IBA. The effect of embryo axes, BA and TDZ, on cotyledons’ regeneration were investigated in detail. The rooted plantlets were transferred to soil successfully with normal morphology.     

Thidiazuron-induced Shoot Multiplication and Plant Regeneration in Bamboo (Dendrocalamus strictus Nees)

Thidiazuron (TDZ) stimulated shoot proliferation from different seedling explants (i.e., shoot, basal node, node and apical segment) of bamboo (Dendrocalamus strictus) when incorporated in half-strength Murashige and Skoog (MS) medium having 2% (w/v) sucrose. All the concentrations of TDZ (0.01 to 1.0 mg l^?1) tried were effective in shoot proliferation. Maximum shoots (14.8 ± 1.0) were obtained from the shoot explants cultured in 0.5 mg l^?1 TDZ supplemented halfstrength MS liquid medium for 21 days and subsequently transferred to the same medium devoid of TDZ. The longer culture period (i.e. 28 and 35 days) in TDZ medium caused reduction in shoot proliferation. The shoots regenerated with lower concentrations of TDZ treatment (i.e. 0.01 to 0.1 mg l^?1) rooted in half-strength MS liquid medium. The shoots formed with 0.5 mg l^?1 TDZ treatment did not root in basal medium and required auxin supplementation in the medium for rooting and about 55% shoots produced roots in 1.0 mg l^?1 IBA supplemented medium. The shoots formed with 1.0 mg l^?1 TDZ did not root even after auxin treatment. The well rooted shoots transplanted to plastic pots filled with sand and garden soil (1:1) mixture showed 98% establishment.     

Multiple shoot formation from cotyledonary node segments of Eastern redbud

A procedure for multiple shoot formation from cotyledonary node explants of Eastern redbud (Cercis canadensis L.) cultured on DKW medium containing benzyladenine (BA) and thidiazuron (TDZ) was developed. Explants on medium with TDZ in combination with BA produced higher numbers of shoots than with either cytokinin alone. The highest number of shoots (7.8 to 9.8 shoots per explant) was obtained when explants from 4 to 10 day-old seedlings were treated with a combination of 10 or 15 μM BA and 0.5 or 1.0 μM TDZ for 20 days before being transferred to the same medium without TDZ. The number of shoots formed was increased from 5.8 to 7.2 shoots per explant by cutting through the cotyledonary node prior to culture. Histological studies indicated that the shoots were formed from actively dividing cells located at the axillary bud region. Shoots formed roots in half strength woody plant medium (WPM) supplemented with 10 to 200 μM indole-3-butyric acid (IBA) cultured for 15 days prior to transfer to greenhouse medium.     

Regeneration of daylily (<Emphasis Type="Italic">Hemerocallis</Emphasis>) from young leaf segments

Calli were initiated from leaf segments (~0.5 × 0.5 cm) of daylily incubated on Murashige and Skoog (MS) (1962) medium supplemented with 2,4-dichlorophenoxyacetic acid (2.4-D), and either benzyladenine (BA) or thidiazuron (TDZ). The highest frequency of callus induction was observed on medium with 6.79 μM 2,4-D plus either 4.55 or 6.81 μm TDZ. A period of callus maintenance on medium containing 5.37 μM naphthaleneacetic acid (NAA) plus 2.22 or 4.44 μM BA was necessary following induction to improve the quality of the callus, and significantly increase the frequency of embryogenic-like callus formation and shoot regeneration once calli were transferred to light. Over 70% of the regenerated shoots produced roots on ½ strength MS medium lacking plant growth regulators. The regenerated plantlets were successfully transferred into soil and acclimatized in growth rooms. This is the first report showing that leaf segments can be used for daylily regeneration.     

Influence of thidiazuron (TDZ) pretreatment of shoot tips on shoot multiplication and ex vitro acclimatization of Harpagophytum procumbens

The effects of thidiazuron (TDZ) pretreatment of shoot tips on Harpagophytum procumbens shoot proliferation and successive stages of micropropagation, i.e. rooting of regenerated shoots and acclimatization of plantlets to ex vitro conditions, were described in the present study. The best response in terms of shoot proliferation (about seven shoots/explant) and shoot length (3.2 ± 0.4 cm) was obtained when explants pretreated with 25 µmol L^?1 TDZ for 6 h were cultured on Schenk and Hildebrandt medium containing indole-3-acetic acid (IAA) (0.57 µmol L^?1) and 6-benzylaminopurine (BAP) (8 µmol L^?1). Under these conditions, a 330 % increase in shoot multiplication over TDZ non-pretreatment culture was achieved and TDZ pretreatment shoots were longer compared to those in control culture (2.6 ± 0.3 cm). The TDZ pretreatment did not affect the percentage of rooted shoots, length of roots and number of roots formed per shoot. The rooted plantlets were transplanted from in vitro to pots with soil and grown during 1 year in the greenhouse. The hardening process was difficult and time-consuming. We found that the plants developed from the TDZ pretreated culture were superior to plants from non-pretreated culture in terms of survival rate and morphological features, such as shoot length, leaf size, flowering and earlier root tuberisation. Random amplified polymorphic DNA and inter-simple sequence repeat analyses of pretreatment with TDZ plants showed genetic similarity to non-pretreatment plants. We conclude that applying the strategy of initial explant pretreatment with TDZ may be valuable for the improvement in H. procumbens in vitro propagation.     

Production of salidroside and tyrosol in cell suspension cultures of Rhodiola crenulata

Salidroside and its aglycone tyrosol are important compounds found in Rhodiola plants. In this study, callus derived from Rhodiola crenulata was induced and grown when explants were incubated on a Murashige and Skoog (MS) medium containing various concentrations of 6-benzyaldenine (BA), naphthalene acetic acid (NAA) and thidiazuron (TDZ). Callus was easily initiated from juvenile leaves in half strength MS medium supplemented with 0.5 mg/L BA and 3.0 mg/L NAA, while full strength MS containing 0.5 mg/L TDZ and 0.5 mg/L NAA was the best for callus subculture and subsequent cell suspension culture. The activities of l-phenylalanine ammonia lyase (PAL) and β-d-glucosidase, two key enzymes in salidroside synthesis, increased at first and subsequently decreased in cell suspension cultures. The salidroside and tyrosol levels in the cell suspension cultures were determined using high-performance liquid chromatography. High levels of salidroside and tyrosol were detected in cell suspension cultures of R. crenulata extracted with 75 % methanol, demonstrating that the biotechnological production of these compounds using plant cell suspension cultures derived from R. crenulata may be an attractive alternative to harvest-based production.     

Establishment of Gymnema sylvestre hairy root cultures for the production of gymnemic acid

Gymnema sylvestre is an important medicinal plant that bears bioactive compound namely gymnemic acid. In the present study, G. sylvestre was transformed by Agrobacterium rhizogenes. Seedling explants namely roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain KCTC 2703. Transformed (hairy) roots were induced from cotyledons and leaf explants. Six transgenic clones of hairy roots were established and confirmed by polymerase chain reaction (PCR) and RT-PCR using rolC specific primers. Hairy roots cultured using MS liquid medium supplemented with 3 % sucrose showed highest accumulation of biomass (97.63 g l^?1 FM and 10.92 g l^?1 DM) at 25 days, whereas highest accumulation of gymnemic acid content (11.30 mg g^?1 DM) was observed at 20 days. Nearly 9.4-fold increment of biomass was evident in suspension cultures at 25 days of culture and hairy root biomass produced in suspension cultures possessed 4.7-fold higher gymnemic acid content when compared with the untransformed control roots. MS-based liquid medium was superior for the growth of hairy roots and production of gymnemic acid compared with other culture media evaluated (B5, NN and N6), with MS-based liquid medium supplemented with 3 % sucrose was optimal for secondary metabolite production. The current results showed great potentiality of hairy root cultures for the production of gymnemic acid.     

Micropropagation and in vitro flowering of endemic and endangered plant Ceropegia attenuata Hook

Factors affecting in vitro propagation were evaluated for Ceropegia attenuata Hook., an endemic and endangered plant having ornamental potential but a limited reproductive capacity. Rapid shoot multiplication from nodal explants was established using varying concentrations of cytokinins and auxins either alone or in combinations. The highest frequency of shoot induction was achieved when nodal explants were inoculated on Murashige and Skoog (MS) medium supplemented with 13.31 μM 6-benzylaminopurine with a mean of 12.9?±?0.5 shoots per explant. High concentrations of TDZ (6.81–11.35 μM) and KN (6.78–11.61 μM) resulted in stunted and vitrified shoots. Factors implicated in the promotion of floral transition of the C. attenuata have been identified which are 4-amino-3, 5, 6-trichloropicolinic acid (picloram), 6-benzylaminopurine, sucrose and photoperiod. The highest frequency of flowering (100%) was obtained when axillary shoot explants were transferred to MS medium supplemented with picloram (4.14 μM) within 4 weeks of culture. Transfer of in vitro regenerated shoots to half strength MS medium with 2.46 μM indole-3-butyric acid (IBA) showed maximum root induction. The in vitro grown plantlets were successfully acclimatized in the glasshouse with 85% of survival and showed normal development. The developed protocol provided a simple, cost-effective approach for the conservation of endangered plant C. attenuata for replenishing its declining populations.