Regeneration of plants from callus cultures of Origanum vulgare L.

Investigations were undertaken to achieve rapid multiplication and improvement of Origanum vulgare (a herbaceous, ornamental plant well known for its aromatic and medicinal value) through plant regeneration from callus. The explants (cotyledons, hypocotyl and root segments) excised from 15 d old aseptic seedlings were cultured on Gamborg's B₅ medium supplemented with 2,4-D, NAA and BAP individually and in various combinations (at concentrations of 0,10^–7,10^–6 and 10^–5 M). Best callus induction was noted on medium with 10^–7 M 2,4-D alone. The cotyledonary expiants proved to be the best source for compact and nodulated callus. The subcultured cotyledonary calli showed shoot induction when transferred onto media supplemented with BAP alone orin combination with 10^–7M or 10^–6MNAA. However, 10^–5M NAA completely suppressed the shoot inducing ability of BAP. In general, NAA promoted root induction from all explants used including cotyledonary callus. Best shoot induction was obtained on medium supplemented with 10^–6M BAP+10^–6MNAA. Both IBA and NAA at 10^–6 M proved to be equally effective in induction of roots from the cut ends of 15–20 mm long shoots (excised from callus) in half-strength B₅ liquid medium. Rooted shoots were successfully re-established in soil under controlled conditions.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine     

In vitro Plant Formation from Stem Explants of Rape (Brassica napus cv. Zephyr)

Internodal segments from 6-weeks-old rape plants (Brassica napus L. cv. Zephyr) were induced to differentiate in vitro producing shoots or shoots and roots on synthetic nutrient medium under controlled conditions. Benzyladenine (BA) alone (5 × 10^?6 M) induced multiple shoot formation on all stem explants. Roots were induced on shoots when recultured on nutrient medium supplemented with auxins such as naphthalene-acetic acid (NAA) or indoleacetic acid (1AA) or when planted in vermiculite. Complete plant formation was obtained when NAA (2 × 1^?6, 5 × 10^?6 and 10^?5 M) was employed in conjunction with BA at 5 × 10^?6M. At higher concentrations (10^?5M) NAA retards the shoot development while 1AA suppresses it totally. Lower levels of auxins along with the cytokinin did not retard or inhibit shoot differentiation.     

Shoot Differentiation in Callus Cultures of Datura innoxia

Datura innoxia Mill. callus cultures formed shoots in 2–4 weeks on media containing; a) gibberellic acid, b) indoleacetic acid, c) low concentrations of naphthylacetic acid, d) low concentrations of 2,4-dichlorophenoxyacetic acid, e) benzylaminopurine, f) no growth substance. Benzylaminopurine promoted shoot differentiation. Gibberellic acid inhibited shoot formation weakly, but inhibited proper leaf blade formation. Root differentiation was rare. The callus cultures of Datura innoxia grew rapidly (100-fold in 4 weeks) on a slightly modified Murashige and Skoog medium (0.5 mg/l thiamin · HCl, pH 5.5, no glycine) in light at 30°C. Callus grew well on any single one of the growth substances NAA (10^?5M), 2,4-D (10^?6M) or BAP (3 × 10^?6M). Growth was less and more erratic on GA or IAA. The callus cultures did not grow significantly better when BAP was combined with one of the auxins or with GA.     

Organic nitrogen stimulates caulogenesis from hypocotyl callus of Phyllanthus fraternus

Factors involved in promoting caulogenesis from hypocotyl explants of Phyllanthus fraternus were studied. Hypocotyl explants were cultured on B₅ medium supplemented with 2,4-D or NAA in the presence and absence of BAP (at concentrations 0, 10^–7, 10^–6 and 10^–5M). Adventitious shoots differentiated from callus developed from the cut ends of 12.5% of the hypocotyl segments cultured on medium supplemented with 10^–6M BAP in combination with 10^–6M 2,4-D or 10^–6M NAA. Profuse rooting occurred from the hypocotyl explants on medium supplemented with 10^–6M BAP + 10^–6M NAA. Incorporation of casein hydrolysate in B₅ medium along with 10^–6M BAP + 10^–7M 2,4-D enhanced the frequency of cultures with adventitious shoots upto 68.0%. Glutamine, glutamic acid or proline could partially substitute for the effect of casein hydrolysate. Amongst the hypocotyls from 3–14 d old seedlings, the best caulogenesis was obtained with hypocotyls from 7 d old seedlings both in presence or absence of casein hydrolysate. Best rooting of shoots was achieved on half-strength B₅ medium supplemented with 10^–6M IBA. After hardening, plantlets were successfully transferred to the soil.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2, 4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - CH casein hydrolysate - Arg L-arginine - Glu L-glutamic acid - Gln L-glutamine - Leu L-leucine - Lys L-lysine - Pro L-proline     

An improved method for callus proliferation and regeneration of Fuchsia hybrida

Best callus initiation was obtained when single-node explants of Fuchsia hybrida were incubated in the light on Gamborg B5 medium containing 5×10^-6 M indoleacetic acid and benzylaminopurine at 5×10^-7 M or 10^-6 M. Healthy callus proliferation was maintained in darkness on full-strength B5 medium supplemented with 5×10^-6 M IAA and 5×10^-7 M BAP. Regeneration from callus was obtained in 3 to 6 weeks, using half-strength hormone-free Campbell & Durzan medium.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4 dichlorophenoxyacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - SE standard error     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

Callus Induction in Small Flowered Willow Herb (<Emphasis Type="Italic">Epilobium parviflorum</Emphasis> Schreb)

In order to determine the most suitable in vitro tissue culture and plant regeneration conditions for the small flowered willow herb (Epilobium parviflorum Schreb), various explants were cultured on semi-solid MS media containing factorial combinations of plant growth regulators. Callus induction from hypocotyl, cotyledon, petiole and leaf explants was achieved on media containing 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (KIN). All other growth regulator combinations [□-naphtaleneacetic acid (NAA) ± benzylaminopurine (BAP), NAA ± thidiazuron (TDZ), indol acetic acid (IAA) ± Zeatin (ZEA)] tested failed to respond. The best results with cotyledon- and petiole- derived callus were obtained from MS medium supplemented with 1.0 mg l^?1 2,4-D + 0.1 mg l^?1 KIN and 2.0 mg l^?1 2,4-D + 0.2 mg l^?1 KIN. It was observed that B5 basal medium was more effective than MS basal medium for producing seedling and the most effective seed sterilizing solution was 25 % (v/v) sodium hypochlorite (NaOCl). No plant regeneration was observed in either callus induction or during the subculturing stage. This is the first report on in vitro tissue culture study within the genus Epilobium.     

Growth and Plating of Cell Suspension Cultures of Datura innoxia

Suspension cultures of Datura innoxia Mill, were successfully grown on a modified Murashige and Skoog medium with 2,4–D, NAA or BAP as growth substances, provided the micronutrient levels were reduced to 1/10. Normal amounts of micronutrients were toxic. Attempts to identify the toxic elements did not succeed. Cultures grew exponentially on a shaker at 27°C in the light. Their doubling times varied from 1.1 days on 2,4–D (10^–6M) or NAA (10^?5M)+ 1 g/1 casein hydrolysate to 2.7 days on BAP (3 × 10^?7M) and 5.1 days on supraoptimal levels of 2,4-D (10^?5M). Cultures grew on NH₄⁺-N alone (from ammonium malate) or on NO₃^?-N alone. Dry weight yield was proportional to the amount of nitrate-N added (47 mg/mg N). Filtered suspension cultures containing single cells (plating cultures) could be grown in agar in petri dishes when NAA or 2,4-D were used as growth substances. Cells grew at densities above 500 units/ml in the agar. Most colonies grew from cell aggregates but division in single cells was observed. The highest plating efficiency was about 50% on 10^?6 M 2,4-D + 1 g/1 casein hydrolysate.     

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.     

Regeneration of plants from primary leaves of cowpea

Plants were regenerated from the in vitro cultured explants of primary leaves of cowpea (Vigna unguiculata L. Walp). Primary leaves, including the intact petiole, were excised from three-day-old seedlings and cultured on Gamborg's B5 basal medium containing 8×10^–7 M 2,4,5-trichlorophenoxyacetic acid, 1×10^–2 M L-glutamine and 1×10^–4 M adenine sulfate. Callus formed at the petiole end. Prolific shoot regeneration occurred when this callus was transferred to B5 basal medium containing 5×10^–6 M 6-benzyl-aminopurine (BAP). Regenerated shoots rooted in growth-regulator-free B5 basal medium and were established in soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA 1-napthalene acetic acid - 2,4,5-T 2,4,5-trichloro-phenoxyacetic acid     

Somatic embryogenesis and plant regeneration from callus cultures of Aconitum heterophyllum Wall

Plants were obtained via somatic embryogenesis in callus derived from in vitro raised leaf and petiole explants of Aconitum heterophyllum Wall. Callus was induced on a Murashige-Skoog medium supplemented with either 2,4-dichlorophenoxy acetic acid (2,4-d 1 mg l^-1) and kinetin (KN 0.5 mg l^-1) with coconut water (CW 10% v/v) or naphthalene acetic acid (NAA 5 mg l^-1) and benzylaminopurine (BAP 1 mg l^-1). Somatic embryos appeared after 2–3 months or 2 subculture passages when 2,4-d or NAA induced source of the callus was transferred to a MS medium containing BAP (1 mg l^-1) and NAA (0.1 mg l^-1). For successful plantlet formation, the somatic embryos were transferred to a medium containing 1/4 strength MS nutrient with indole-3-butyric acid (IBA 1 mg l^-1). Alternatively, the somatic embryos were dipped in a concentrated solution of IBA for 5 min and placed on a hormone free medium. Complete plantlets were formed after 4 weeks and were transferred successfully to soil.CIMAP Publication No. 1020.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis> by organogenesis and somatic embryogenesis

In the present study, in vitro regeneration system for a recalcitrant woody tree legume, Leucaena leucocephala (cvs. K-8, K-29, K-68 and K-850) from mature tree derived nodal explants as well as seedling derived cotyledonary node explants was developed. Best shoot initiation and elongation was found on full-strength Murashige and Skoog (MS) medium supplemented with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 100 mg dm⁻³ glutamine, 20.9 μM N ⁶-benzylamino-purine (BAP) and 5.37 μM 1-naphthalene acetic acid (NAA). Rooting was induced in half-strength MS medium containing 2 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 14.76 μM indole-3-butyric acid (IBA) and 0.23 μM kinetin. The cultivar K-29 gave the best response under in vitro conditions. Rooted plantlets were subjected to hardening and successfully transferred to greenhouse. Further, somatic embryogenesis from nodal explants of cv. K-29 via an intermittent callus phase was also established. Pronounced callusing was observed on full-strength MS medium containing 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 40.28 μM NAA and 12.24 μM BAP. These calli were transferred to induction medium and maximum number of globular shaped somatic embryos was achieved in full-strength MS medium fortified with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 15.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.0 μM BAP and 1.0 mM proline. Moreover, an increase in endogenous proline content up to 28^th day of culture in induction medium was observed. These globular shaped somatic embryos matured in full-strength MS medium with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 10.0 μM BAP, 2.5 to 5.0 μM IBA and 0.5 mM spermidine.     

Development of a Polygonum minus cell suspension culture system and analysis of secondary metabolites enhanced by elicitation

Polygonum minus has been reported to contain valuable metabolites and to date, there is no report on using cell culture technique for metabolite production in P. minus. Naphthalene acetic acid (NAA) concentrations in the range of 2–6 mg L^?1 were used in a matrix of combinations with dichlorophenoxyacetic acid (2,4-D) concentrations in the range of 2–10 mg L^?1 as plant growth regulators (PGRs) to induce callus cultures. Media that were supplemented with 2 mg L^?1 2,4-D + 4 mg L^?1 NAA, 2 mg L^?1 2,4-D + 6 mg L^?1 NAA and 6 mg L^?1 2,4-D + 8 mg L^?1 NAA were effective for callus induction (93.3 % of the explants produced callus). To establish cell culture, the best growth was obtained from medium that was supplemented with 1 mg L^?1 2,4-D + 2 mg L^?1 NAA. From a 1-g inoculum size, the fresh weight increases exponentially after 5–10 days of culture, and a 26.71 g maximum fresh weight was obtained after 25 days of culture. The cell culture medium was then analyzed using gas chromatography–mass spectrometry (GC–MS). Jasmonic acid (100, 50, 25 and 5 μM), salicylic acid (100, 50, 25 and 5 μM), yeast extract (500, 250 and 100 mg L^?1) and glass beads were used in this research as elicitors. The cell cultures were then incubated with the different elicitors for 1, 2, 3 and 4 days. Several compounds with high peak area percentages were detected, including 2-furancarboxaldehyde, 5-hydroxymethyl, furfural, and 2-cyclopenten-1-one, 2-hydroxy. These results show the diversity of metabolites released by P. minus cell into the culture medium under control conditions.     

Production of phenolic compounds in callus cultures of tea plant under the effect of 2,4-D and NAA

The effect of hormone-like compounds at different concentrations: 2,4-D (2 × 10^?6; 2 × 10^?5; and 2 × 10^?4M) and 1-NAA (2 × 10^?7; 2 × 10^?6; 2 × 10^?5; 4 × 10^?5, and 6 × 10^?5 M) on the growth and production of phenolic compounds, including flavans and lignin, was investigated in callus culture of tea plant (Camellia sinensis L., a highly productive strain IFR ChS-2). The growth of the culture was vigorous, and production of phenolic compounds therein was efficient in the medium containing 2 × 10^?5 M 2,4-D. Substitution of 1-NAA for 2,4-D in all the cases decelerated the growth of the culture. These changes were more pronounced when 2 × 10^?7 and 2 × 10^?6 M 1-NAA was used; in this case, biomass accumulation decreased by 1.5–2.0 times as compared with control material growing on the medium with 2 × 10^?5 M 2,4-D. In the presence of 1-NAA, the content of total soluble phenolic compounds and flavans in the calli rose by 30% on the average as compared with control material. Accumulation of lignin remained essentially the same. Therefore, the replacement of 2,4-D with 1-NAA in the nutrient medium used for the growing of highly productive strain of tea plant callus did not induce considerable changes in its ability to produce phenolic compounds.     

Variation amongst Brassica juncea cultivars for regeneration from hypocotyl explants and optimization of conditions for Agrobacterium-mediated genetic transformation

Summary Twelve cultivars of Brassica juncea grown in different agroclimatic regions of the world were tested for their ability to regenerate in vitro from hypocotyl explants and, accordingly, were divided into three groups. One group of cultivars regenerated on MS medium supplemented with 2,4-D, BAP and with NAA, BAP combinations; another group regenerated only on MS with 2,4-D, BAP; and the third group showed very low regeneration on both of these combinations. Inclusion of silver nitrate in the medium was essential for high frequency of regeneration. In general, Indian cultivars were more responsive than the cultivars of CIS and Australian origin. Using the media optimal for regeneration and an Agrobacterium-based binary vector carrying hpt and gus-intron genes, conditions for genetic transformation of B. juncea hypocotyl explants were optimized. Transformation frequencies, identified by GUS staining at the initial stages of growth, were lower on MS medium with 2,4-D, BAP than on MS with NAA, BAP. Plants resistant to 20 g/ml hygromycin were regenerated at a frequency of 11–36% from hypocotyl explants and were shown to be transformed by Southern blotting, GUS staining and progeny analysis.     

Somatic embryogenesis and plant regeneration in centipedegrass (Eremochloa ophiuroides [Munro] Hack.)

Centipedegrass (Eremochloa ophiuroides [Munro] Hack.) is an important warm-season turfgrass and pasture grass. To explore the potential use of biotechnical tools in breeding of centipedegrass, we established an efficient plant regeneration system for this species. Four basal media and 24 combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BAP) were examined for their effects on callus induction from mature seed explants. Twenty combinations of naphthaleneacetic acid (NAA) and BAP were tested for their effect on plant regeneration. Results indicated that Murashige and Skoog basal medium supplemented with 4.5 mg l⁻¹ 2,4-D and 1 mg l⁻¹ BAP was the best medium for callus induction, while the combination of 2 mg l⁻¹ BAP and 1 mg l⁻¹ NAA induced the highest rate of regeneration and development of shoots and roots. This work provides a basis for the breeding of centipedegrass through somaclonal variation and genetic transformation.     

Phenol content, acidic peroxidase and IAA-oxidase during somatic embryogenesis in Theobroma cacao L.

Calli were induced in cacao cotyledon explants on a half-strength Murashige and Skoog medium containing 6 × 10-2 g m-3 saccharose and various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) with kinetin (kin), benzylaminopurine (BAP) or 2-isopentenylphosphate (2-iP). Experiments were carried out on two clones of cacao differing in their susceptibility to black pod disease. The highest percentage of explants forming callus and the most rapid callus development were obtained with 10-6 g m-3 2,4-D and 0.5× 10-6 g m-3 kin. Somatic embryogenesis and rhizogenesis were induced by transferring 3-week-old callus in a half strength Murashige and Skoog medium containing 3 × 10-2 g m-3 saccharose and NAA or IBA in the 0 to 5 × 10-6 g m-3 concentration range. No differentiation could be observed when the medium was supplemented with kin or BAP. The conversion of callus into somatic embryos and roots was accompanied by a drop in phenol content and an increase in peroxidase and IAA-oxidase activities. Moreover, cell differentiation was characterized by the persistence in the callus of one acidic soluble isoperoxidase which was not detected in nondifferentiating callus. Although some differences were noticed between the clones, alterations responsible for cell differentiation were the same in both genotypes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Callus and Cell Suspension Cultures of Carnation

Callus cultures of carnation, Dianthus caryophyllus L. ev. G. J. Sim, were grown on a synthetic medium of half strength Murashige and Skoog salts, 3 % sucrose, 100 mg/l of myo-inositol, 0.5 mg/l each of thiamin, HCl, pyridoxin, HCl and nicotinic acid and 10 g/l agar. Optimal concentrations of growth regulators were observed to be 3 × 10^?6M indoleacetic acid (JAA) combined with 3 × 10^?6M benzylaminopurin (BAP) or 10^?6M 2,4-dichlorophenoxy acetic acid (2,4-D) alone. IAA + BAP caused a 100 fold increase in fresh weight over 4 weeks at 25°C. Addition of casein hydrolysate increased growth further. Cell suspension cultures worked best in media containing 2,4-D in which they had a doubling time of about 2 days. Filtered suspensions were successfully plated on agar in petri dishes, but division was never observed in single cells. The cultures initiated roots at higher concentrations of IAA or NAA, but all attempts to induce formation of shoots or em-bryoids gave negative results.     

High regenerative nature ofMentha arvensis internodes

Media and incubation conditions have been defined for highly efficient regeneration of shoots from internode explants of slow and fast growing cultivars ofMentha arvensis. Internodal segments excised from thein vitro raised shoots were inoculated on the MS medium supplemented with combinations of 5 concentrations of l-napthalene acetic acid (NAA) and 3 concentrations of 6-benzyl amino purine (BAP). The media containing 2 μg ml⁻¹ NAA, 10 Μg ml⁻¹ BAP and 1 μg ml⁻¹ NAA, 5 μg ml⁻¹ BAP proved best for shoot regeneration and growth responses on cv Himalaya and cv Kalka explants, respectively. In 12 weeks time, on average one explant of cv Himalaya produced about 200 shoots and that of cv Kalka produced about 180 shoots. The Himalaya explants required higher concentrations of NAA and BAP for high efficiency proliferation as compared to the Kalka explants. The experiments demonstrated that internodal tissue inMentha arvensis can be induced to obtain direct shoot regenerants with high efficiency. The analysis of the RAPD profiles of 100 regenerated plantlets each of cv Himalaya and Kalka showed more than 99.9% homogeneity in bands with respect to the parents.     

An efficient protocol for <Emphasis Type="Italic">in vitro</Emphasis> regeneration and conservation of Shirui lily (<Emphasis Type="Italic">Lilium mackliniae</Emphasis> Sealy): a lab-to-land approach to save the rare endangered Asiatic lily species

An efficient protocol for direct and indirect shoot regeneration and proliferation from bulb scales of Shirui lily (Lilium mackliniae Sealy), an endangered Asiatic lily species endemic to the Shirui hill peak, Manipur, India, has been developed. Bulb scales were isolated from mature bulbs and cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of 6-benzylaminopurine (BAP), kinetin (KIN), or thidiazuron (TDZ). For direct shoot regeneration from bulb scale explants, 0.5 mg L^?1 BAP yielded the highest shoot induction (3.5 shoots per scale; a 96.7% response). For indirect de novo organogenesis, optimum callus induction was achieved with 2.0 mg L^?1 2,4-dichlorophenoxyacetic acid (2,4-D), and shoot organogenesis was higher (16.2) when subcultured onto 0.5 mg L^?1 BAP medium. Multiple shoot regeneration and pseudo-bulb formation protocols were assessed; the highest shoot proliferation (10.1) occurred with 0.5 mg L^?1 BAP and 1.0 mg L^?1 gibberellic acid (GA₃). Rooting response was 96% with 0.5 mg L^?1 1-naphthalene acetic acid (NAA), with multiple roots per shootlet. Plantlet survival was increased to 92.5% during the hardening-off process by using hydroponics with Hoagland’s solution in a mist chamber. Clonal fidelity was assessed through random amplified polymorphic DNA (RAPD) analysis comparing the mother plant and regenerated plantlets. After confirming genetic uniformity, the pseudo-bulblets with four to six leaves and three to four roots were successfully established at the Shirui hills peak. This in vitro regeneration and ex vitro conservation approach could be helpful to save this rare endangered species in a sustainable way.