Assessment Of Genetic Fidelity Research Articles

Optimization of in vitro propagation protocol of Dendrobium heterocarpum Wall. ex. Lindl. and clonal genetic fidelity assessment of the regenerates: An orchid of horticultural and medicinal importance

Dendrobium heterocarpum Wall. ex Lindl. (Orchidaceae) is an orchid having medicinal as well as ornamental values. The natural population of the species is downsized due to different anthropogenic activities. The present study was undertaken to standardize in vitro propagation technique of the species through immature embryo culture and optimizing different factors. Further, the study demonstrates the use of low cost substrata for hardening of in vitro regenerates. Immature embryos of 5–8 months after pollination (MAP) were cultured on three different media enriched with different organic carbon sources and four plant growth regulators at varied concentrations. The embryos of 8 MAP registered ∼95% germination on MS medium fortified with sucrose (3%, w/v), kinetin (3 μM/L). The resultant PLBs differentiated to young plantlets on the same initiation medium. Subsequently, the young plantlets proliferated and as many as ∼18 shoots were produced on MS medium containing sucrose and kinetin and α-naphthalene acetic acid (3 + 12 μmol/L) in combination. The regenerates were primary hardened by culturing on low cost substrata (coconut husk + wood bark at 1:1 ratio) fortified with 1/4th MS salt solution for one wk, subsequently maintaining to normal room temperature under 75% diffused light for ∼3–4 wk before transferring to potting mix. About 87% transplants survived post transfer. Clonal fidelity of the regenerants was also evaluated using RAPD, DAMD and SCoT markers. High degree of monomorphic percentage (96.89%) and low polymorphism (3.11%) was recorded between the mother plant and in vitro regenerates; thus, showing genetic homogeneity of the in vitro raised plants.

The effect of polyamines and silver thiosulphate on micropropagation of date palm followed by genetic stability assessment.

There are some limitations in date palm micropropagation. These include low multiplication efficiency, low rooting rate, and high mortality experienced by in vitro raised plantlets during laboratory to soil transfer. The objective of the study was to determine the effect of the polyamines and Silver Thiosulphate (STS) on the enhancement of shoot multiplication and genetic stability of in vitro cultures of date palm cultivar Quntar. Media supplemented with 75mg L-1 SPD in combination with 10 mg L-1 STS gave the highest percentage of callus producing buds (83.34%) and average bud formation (16.3) per jar. The addition of PUT and STS to the medium was most effective on root formation and the number of roots per shoot, where the best result, 91.67% and 6.37 roots per shoot, respectively, were obtained using 75 mg L-1 PUT and 10 mg L-1 STS, resulting in fast-growing plantlets during acclimatization phase, reaching 80% of plant survival. The genetic fidelity assessment of plants derived from micropropagation was confirmed by RAPD analysis. Four operon primers were used, and all of them showed amplified unambiguous (OPA02, OPC-04, OPD-07, and OPE-15). All generated bands were monomorphic and had no variation among the tissue culture-derived plants tested. Accordingly, these results indicate that adding polyamines and silver thiosulfate to the nutrient medium of date palm cv. Quntar was beneficial to improving shoot organogenesis, rooting, and production of genetically stable date palm plants.

Direct in vitro regeneration of medicinally important Indian and exotic red-colored ginger (Zingiber officinale Rosc.) and genetic fidelity assessment using ISSR and SSR markers

Direct in vitro regeneration of medicinally important Indian and exotic red-colored ginger (Zingiber officinale Rosc.) and genetic fidelity assessment using ISSR and SSR markers

Micropropagation and assessment of genetic fidelity of tissue-cultured plants in potato (Solanum tuberosum L. ‘Kufri Lima’)

Micropropagation and assessment of genetic fidelity of tissue-cultured plants in potato (Solanum tuberosum L. ‘Kufri Lima’)

M-brigde- and elicitor-assisted enhanced post-storage germination of Rauvolfia serpentina synthetic seeds, their genetic fidelity assessment and reserpine estimation

The present study reports an amelioration of synthetic seed germination efficiency with a simple intervention of filter paper (M-bridge) drenched in ½Murashige and Skoog liquid (½MSL) medium in Rauvolfia serpentina. Synthetic seeds were developed from in vitro shoot tips using 3% (w/v) sodium alginate and 75 mM calcium chloride. Media for synthetic seed gemination were tested in cotton bed drenched with sterile water (SW), cotton bed drenched with ½MSL medium, M-bridge drenched with ½MSL medium, and ½MS semisolid (½MSS) medium alone. Synthetic seeds inoculated on ½MSL or SW or ½MSS media, exhibited slower germination and inferior performance; however, with the involvement of M-bridge, fastest germination (5.6 ± 0.4 days) and maximum proliferation (4.8 ± 0.7 shoots with 42.8 ± 2.4 mm length, and 19.8 ± 2.7 leaves/synthetic seed after 42 days of inoculation) were recorded. Among the tested elicitors (silver nitrate, salicylic acid and sodium nitroprusside) at their different concentrations supplemented in ½MSL medium (with M-bridge), 50 µM silver nitrate resulted in 100% germination frequency and resulted in germination within 5 days. Further, the synthetic seeds were stored for 42 days at − 20 °C, 8 °C, and 25 °C. Maximum germination efficiency was obtained in case of seeds stored at 8 °C with respect to earliness in germination (6.9 ± 0.3 days), germination percentage (71.8 ± 3.4%) and subsequent proliferation (4.9 ± 0.6 shoots with 18.9 ± 0.9 mm length, and 8.9 ± 0.7 leaves/synthetic seed). Clonal fidelity analysis of the in vitro regenerants performed using RAPD markers exhibited monomorphic banding pattern. The reserpine content of the regenerants was found to be maximum following 42 days-storage at 8 °C (126.9 µg/g), followed by a substantial decline in the synthetic seed-derived regenerants following 42-days-storage at 25 °C (120.7 µg/g) or − 20 °C (121.8 µg/g).

Axenic rhizome culture and genetic fidelity assessment of Eulophia dabia (D. Don) Hochr: an endangered terrestrial orchid species

Axenic rhizome culture and genetic fidelity assessment of Eulophia dabia (D. Don) Hochr: an endangered terrestrial orchid species

Flow cytometry and start codon targeted (SCoT) genetic fidelity assessment of regenerated plantlets in Tylophora indica (Burm.f.) Merrill.

Tylophora indica (Burm.f.) Merrill. is a pharmacologically important plant, popular for alkaloidal and non-alkaloidal richness. Large scale propagation of T. indica is difficult in the wild as the seeds are small and the frequency of germination is very poor. In the present study, the genome size estimation of in vitro regenerated (indirect, direct and somatic embryo mediated) T. indica was made by flow cytometric method. Clonal fidelity of the regenerants was assessed using a start codon targeted (SCoT) molecular marker. Initially, the explants were inoculated on Murashige and Skoog basal medium supplemented with various concentrations of plant growth regulators like 2,4-dichlorophenoxy acetic acid (2,4-D), Kinetin, 6-benzyl amino purine (BAP) and 1-naphthalene acetic acid either singly or in combinations. The highest callus induction frequency (87.75%) was obtained in 6.7 µM 2,4-D added MS medium which metamorphosed into progressive stages (globular, heart, torpedo, and cotyledonary) of embryos. Mature and healthy somatic embryos efficiently germinated into plantlets on 8.8 µM BAP + 1.4 µM GA3 enriched MS medium. Histological and scanning electron microscopic study confirmed the above developing stages. The regenerated shoots were rooted best in 2.45 µM Indole-3-butyric acid supplemented solid MS medium. The plants were hardened and acclimatized with 90% survivability. The flow cytometric 2C DNA content of indirect, direct and somatic embryo derived plants was 1.896 pg, 1.940 pg and 1.926 pg respectively, very similar to the mother plant (1.928 pg). SCoT marker generated a high percentage of monomorphic bands (94%) revealing similarity with the mother plant, thus ensuring genetic fidelity. To the best of our knowledge, this is perhaps the first ever report of 2C DNA content estimation and SCoT marker based genetic homogeneity study in T. indica.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11240-022-02254-z.

Genetic clonal fidelity assessment of rhizome-derived micropropagated Acorus calamus L. – A medicinally important plant by random amplified polymorphic DNA and inter-simple sequence repeat markers

Background: Acorus calamus – a critical medicinal plant, is overexploited, leading to population reduction. Establishing an efficient in vitro protocol is essential for the large-scale production of genetically identical plants. Objectives: Development of fast and reliable in vitro regeneration protocol for A. calamus and clonal fidelity assessment of the regenerants using molecular markers. Materials and Methods: Plants were regenerated on Murashige and Skoog medium with different concentrations of growth regulators in two phases – shooting and rooting. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to evaluate the genetic stability of in vitro clones. Results: 6 Benzylaminopurine (BAP) at 1.6 and 2.4 mgL−1 was effective for shoot induction, while root induction was superior in indole-3-butyric acid-incorporated medium at 2.5 mgL−. Thirteen RAPD and 16 ISSR primers produced 59 and 96 clear, unambiguous, and reproducible bands, respectively. Both the markers revealed a high monomorphism of 96.79% and 95.63% among the regenerants. Nei's genetic distance analysis disclosed a close genetic association (0.000–0.068) among the genotypes. Conclusion: ISSR was better than RAPD markers in clonal fidelity assessment of the regenerants. The in vitro protocol developed is reliable and suitable for the rapid propagation of true-to-type A. calamus plants.

Genetic Fidelity Assessment of in Vitro Regenerated Plants of Ceropegia Thwaitesii Through Rapd and Scot Markers

Genetic Fidelity Assessment of in Vitro Regenerated Plants of Ceropegia Thwaitesii Through Rapd and Scot Markers

In vitro tuberization, genetic, and phytochemical fidelity assessment of Dioscorea deltoidea

Dioscorea deltoidea Wall. ex Griseb. (family Dioscoreaceae) has been traditionally used as a remedy for various human ailments. In modern medicine, it is used as an efficacious remedy against diabetes and cardiovascular diseases. Dioscorea deltoidea tubers are known to be a rich reservoir of diosgenin. A consistent and reproducible protocol is needed for the production of tubers for its conservation as well as for the production of diosgenin in vitro. Therefore, the current investigation was aimed to develop an in vitro protocol for tuberization as well as for production of diosgenin with genetic and phytochemical fidelity assessments. Murashige and Skoog (MS) medium fortified with 6-benzyl amino purine (BAP) exhibited tuberization in D. deltoidea; however BAP at 0.9 mg L−1 significantly increased the tuber number (5.8), weight (3.4% FW) and size (9.3 mm). In combinations, BAP (0.15 mg L−1) + naphthalene acetic acid (NAA) (1.0 mg L−1) + kinetin (Kn) (0.25 mg L−1) favoured tuberization in D. deltoidea cultures. Increased concentrations of sucrose (4–8%) in the optimized culture medium with 0.9 mg L−1 BAP had significant effects on tuberization. However, MS medium fortified with 6% sucrose and 0.9 mg L−1 BAP was found to be optimum for tuberization producing maximum tubers (7.5). Genetic fidelity of the regenerated bulbs were analysed using six inter simple sequence repeats (ISSR) markers. In vitro raised D. deltoidea plants exhibited dissimilar banding pattern to the mother plant thus demonstrated the genetic variability in the newly regenerated plants. A phytochemical analysis by liquid chromatography-mass spectrometry (LC-MS) showed diosgenin content in the range of 0.000571% DW to 0.000732% dry weight (DW) in in vitro raised tubers. The developed protocol for in vitro tuberization of D. deltoidea is cost-effective and can be proficiently used for the germplasm conservation as well as for industrial production of diosgenin.

Biotechnological interventions of in vitro propagation and production ofvaluable secondary metabolites in Stevia rebaudiana.

Plant cell and tissue culture makes provision of a sustainable and nature-friendly strategy for the production of secondary metabolites, and modern progress in gene editing and genome engineering provides novel possibilities to improve both the qualitative and quantitative aspects of such phytochemicals. The ever-expanding quest for plant-based medicine to treat diabetes facilitates large-scale cultivation of Stevia rebaudiana to enhance the yield of its much-coveted low-calorie sweetener glycosides. The potential to process stevia as a "natural" product should enhance the acceptance of steviosides as a natural calorie-free sweetener especially suitable for use in diabetic and weight control drinks and foods. Besides sweetener agents, S. rebaudiana is a potent source of many antioxidant compounds and is used to cure immunodeficiencies, neurologic disorders, inflammation, diabetes mellitus, Parkinson's disease, and Alzheimer's disease. This comprehensive review presents the research outcomes of the many biotechnological interventions implicated to upscale the yield of steviol glycosides and its derivatives in in vitro cell, callus, tissue, and organ cultures with notes on the use of bioreactor and genetic engineering in relation to the production of these valuable compounds in S. rebaudiana. KEY POINTS: • Critical and updated assessment on sustainable production of steviol glycosides from Stevia rebaudiana. • In vitro propagation of S. rebaudiana and elicitation of steviol glycosides production. • Genetic fidelity and diversity assessment of S. rebaudiana using molecular markers.

Meta-Topolin mediated improved micropropagation, foliar micro-morphological traits, biochemical profiling, and assessment of genetic fidelity in Santalum album L.

An improved in vitro propagation system has been developed for Santalum album L. using meta-Topolin for the first time. Direct organogenesis (100 %) was achieved by induction of axillary buds and a maximum of 8.2 shoots with 4.0 cm length was developed from nodal explants on Murashige and Skoog’s (MS) medium containing 1.0 mg L−1 meta-Topolin (mT). The MS medium supplemented with 0.5 mg L-1 mT and 0.25 mg L−1 α-naphthalene acetic acid (NAA) exhibited maximum shoot proliferation potential (135 shoots with 7.9 cm length per explant) after the 3rd subculture. Further, the combination of mT and NAA resulted in the production of higher levels of free amino acids [33.6 mg g-1 dry weight (DW)], soluble sugar (37.4 mg g-1 DW), and reducing sugar (45.0 mg g-1 DW) in the leaves than the BAP and NAA combinations. The efficiency of mT has also been highlighted in the foliar structural developments. Comparative anatomy of leaves showed the presence of structural variations influenced by the type of cytokinins used. BAP and NAA-derived leaves revealed the presence of under-developed cuticle, unorganized tissue systems, undifferentiated mesophyll tissues, and under-developed mechanical and vascular tissues. Whereas, mT and NAA-derived leaves exhibited comparatively thick cuticle, collenchymatous hypodermis, well-differentiated mesophylls, and increased vascular elements. Roots were induced ex vitro under the greenhouse conditions when the shoot-bases were treated with 500 mg L−1 indole-3-butyric acid (IBA) for 10 min. These plantlets were hardened in soilrite® and cocopeat (1:1) mixture and a 100 % survival rate was achieved in the field. Additionally, the Start codon targeted (SCoT) markers analysis demonstrated that the genetic homogeneity was maintained during in vitro development of plantlets. The application of mT can be an effective growth regulator for in vitro propagation of S. album, as it favoured enhanced and quick production of genetically uniform clones.

Correction to: Efficient in vitro plant regeneration from leaf-derived callus and genetic fidelity assessment of an endemic medicinal plant Ranunculus wallichianus Wight & Arnn by using RAPD and ISSR markers

Correction to: Efficient in vitro plant regeneration from leaf-derived callus and genetic fidelity assessment of an endemic medicinal plant Ranunculus wallichianus Wight & Arnn by using RAPD and ISSR markers

Efficient in vitro plant regeneration from leaf-derived callus and genetic fidelity assessment of an endemic medicinal plant Ranunculus wallichianus Wight & Arnn by using RAPD and ISSR markers

Ranunculus wallichianus is a medicinally important plant and an endemic species to the Western Ghats of South India. An efficient and reliable indirect regeneration protocol system for R. wallichianus was developed from leaf explants in the present investigation. Leaf explants were cultured on both full-strength and half-strength MS (Murashige & Skoog) medium supplemented with different concentrations (0.5 mg L− 1 to 3.0 mg L− 1) of 2,4-D and NAA. Among the different concentrations tested, the highest percentage of yellowish-green compact nodular callus formation was observed on a half-strength MS medium with 2.0 mg L− 1 of 2, 4-D. Then, the in vitro raised organogenic callus was cultured on a half-strength MS medium containing various concentrations (0.5 mg L− 1 to 3.0 mg L− 1) of BA, KIN, and TDZ with 0.5 mg L− 1 NAA and 10 % CW for in vitro shoot regeneration. The highest percentage of regeneration response (97 %) and a maximum number of shoots formation (11.1 ± 0.13 shoots/culture with 9.2 ± 0.21 cm mean shoot length) were obtained from MS medium containing 2.5 mg L− 1 BA with 0.5 mg L− 1 NAA and 10 % CW. The well elongated in vitro raised shoots were effectively rooted in a half-strength MS medium with 2.5 mg L− 1 IBA + 250 mg L− 1 activated charcoal. The well-rooted plantlets were successfully hardened and acclimatized with a survival rate of 94 %. Clonal fidelity of in vitro raised plantlets was assessed by using DNA-based RAPD and ISSR molecular markers. A total of 56 and 47 monomorphic bands were obtained from RAPD and ISSR markers respectively. This present in vitro propagation protocol system could be effective for the conservation of R. wallichianus with their genetic purity and its further investigations.

Meta-Topolin-induced enhanced biomass production via direct and indirect regeneration, synthetic seed production, and genetic fidelity assessment of Bacopa monnieri (L.) Pennell, a memory-booster plant

meta-Topolin-induced enhanced biomass production via direct and indirect regeneration, synthetic seed production, and genetic fidelity assessment of Bacopa monnieri (L.) Pennell, a memory-booster plant

Molecular marker based assessment of genetic homogeneity within the in vitro regenerated plants of Crocus sativus L. – a globally important high value spice crop

The global market for spice is steadily increasing and saffron is one of the costliest spices in the arena. However, there is a huge gap in the availability of quality planting materials (QPM) of saffron which can only be effectively redressed by appropriate application of plant tissue culture protocols. Nevertheless, one of the major embargoes in the sustainable utilization of in vitro technologies is occurrence of cryptic clonal variabilities defined as somaclonal variations. In the present research, we report a reproducible, cost effective and simple molecular marker mediated assessment of genetic fidelity within the in-vitro propagated shoots of saffron in order to ensure genetic uniformity during cormlet production. The in vitro propagated shoots of Crocus sativus were raised from field grown corms using an earlier reported method. The genetic stability of in vitro propagated shoots was then assessed using molecular markers namely, Start Codon Targeted Polymorphism (SCoT), Amplification of Minisatellite DNA (DAMD) and Inter Retro Transposon Amplified Polymorphism (IRAP). The isolated genomic DNA samples (mother and regenerated plants) were subjected to PCR amplification with 22 best reproducible primers (14 SCoT, 4 DAMD and 4 IRAP). The combined assay of molecular markers revealed a clonal variability of 7.81% thereby, indicating largely stable clonal lines. The present research documents a fast, reproducible and cost effective methodology for assessment of genetic variability amongst the regenerants of saffron and also ensures the true-to-type nature of the micropropagated plants with a target goal of sustainable propagation.

Plant regeneration from root segments of Anthurium andraeanum and assessment of genetic fidelity of in vitro regenerates

A highly efficient regeneration system of Anthurium andraeanum Linden was established using root segments as explants of four genotypes, Arizona, SWD, OYH, and Mississippi. The root explants were firstly incubated on callus induction media containing NH4NO3 level of 206 mg L−1 or 825 mg L−1, and the calluses were formed from both ends of the root segments after 1 mo of culture. Root-derived calluses were then transferred to shoot regeneration media with NH4NO3 level of 412 mg L−1 or 1650 mg L−1, and numbers of shoot were successfully produced from the surface of calluses after 2 mo of culture. Medium containing 206 mg L−1 NH4NO3 induced more callus compared to 825 mg L−1. In contrast, the number of shoots per callus was the same between the above two shoot regeneration media indicating that NH4NO3 level had little effect on shoot regeneration. Long-termin vitro maintenance of the callus-bearing shoot initials was possible without compromising shoot regeneration and subsequent rooting. Histological analysis showed that early cell division was initiated from the inner cortical parenchyma cells near the vascular tissue and from the vascular parenchyma cells adjacent to the xylem vessel. The primary callus mainly comprised inner parenchyma cells with scattered procambial-like cells and xylem elements and two different meristematic zones beneath epidermal layers. Transfer of callus to the regenerative medium caused meristemoid formation and subsequent shoot primordia production. Calcium oxalate crystals and tannin-like deposits were visible on the outer callus layer or within the cotyledon-like structure of shoot primordium throughout callus development. Starch grains were abundant in root explants, but disappeared in the profusely growing primary callus, and were mobilized in the shoot primordium, indicating energy requirements of these two developmental stages. Flow cytometry analysis demonstrated that ploidy levels were maintained during in vitro cultures, including long-term regenerated plants. The inter-simple sequence repeat analysis indicated genomic DNA stability of Arizona and OYH but occasional genetic variation in primary regenerates of SWD and Mississippi.

Organogenesis from Leaf Tissue of Spondias pinnata (L. f.) Kurz, SEM study and Genetic Fidelity Assessment by ISSR and ScoT

In vitro raised plantlets were obtained from nodal tissue through direct organogenesis and they served as donor plants for the collection of leaf explants. Leaf explants were inoculated on Murashige and Skoog’s medium with different concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D). Hundred percent callogenesis was observed on medium supplemented with 5 mg l−1 2,4-D. For the multiplication of cells (proliferation), calli were transferred to either basal medium or media containing different types and concentrations of cytokinins. Proliferation was observed maximum on media containing 0.5 mg l−1 BAP or 1.0 mg l−1 BAP. Shoot differentiation from calli took place on media supplemented with BAP in combinations with TDZ or ZN. Initiation of organogenesis was observed in calli within two weeks of their subculture on differentiation medium. Shoot differentiation was maximum, when calli proliferated on medium having 1 mg l−1 BAP were transferred to the medium containing 1 mg l−1 BAP and 0.5 mg l−1 TDZ. Organogenic responses after four weeks of subculture on above differentiation medium were as such: number of shoots per explants (25.33 ± 0.88), number of shoots per calli replicate (3.67 ± 0.33) and maximum shoot length (3.43 ± 0.20 cm). Medium supplemented with 2.5 mg l−1 NAA was most responsive for rooting of shoots (54.16 ± 1.39%). About 62.5% plantlets survived after hardening and 54.17% plantlets got acclimatized. All acclimatized plants were transferred to field condition successfully. Formation of unipolar shoots and their multicellular attachment with callus were observed by scanning electron microscopy. To confirm the genetic fidelity of micropropagated plants, five micropropagated plants derived from different leaf explants and two mother plants (randomly selected from micropropagated plants raised from nodal explants) were subjected to molecular analysis. The genetic fidelity of in vitro regenerated plants was assessed by using SCoT and ISSR molecular markers. 12.5% polymorphism was reported in both studies, which may be due to callus mediated regeneration of shoots. Indirect organogenesis from leaf tissues of Spondias pinnata can be used both for conservation as well as the improvement of plants.

Micropropagation of an ornamental shrub Disanthus cercidifolius Maxim. and assessment of genetic fidelity of regenerants using ISSR and flow cytometry

Micropropagation of an ornamental shrub Disanthus cercidifolius Maxim. and assessment of genetic fidelity of regenerants using ISSR and flow cytometry

DNA barcoding and genetic fidelity assessment of micropropagated Aenhenrya rotundifolia (Blatt.) C.S. Kumar and F.N. Rasm.: a critically endangered jewel orchid.

Aenhenrya rotundifolia is a critically endangered terrestrial jewel orchid. It is monotypic and endemic to evergreen forests of southern western ghats of India. In the present study, identification of this plant species is validated with DNA barcoding using matK and rbcL chloroplast markers. Further, germ-free juvenile axillary bud explants were cultured on Mitra medium supplemented with different kinds of cytokinins like 6-benzyladenine, 6-furfurylaminopurine, N6-(Δ2-isopentyl) adenine, thidiazuron, zeatinandmeta-topolin as well as auxins such as α-naphthaleneacetic acid, indole-3-acetic acid and indole-3-butyric acid at different concentrations and combinations for successful proliferation and establishment in vitro. After 12weeks of culture, axillary bud explants produced an average of 30.12 ± 0.71 shoots per explant, 3.87 ± 0.06cm shoot length, 1671 ± 2.82mg fresh mass of proliferated shoots with a proliferation frequency of 100% on Mitra medium supplemented with 6.20µM meta-topolin and 2.25µM thidiazuron. No root formation was observed in in vitro proliferated microshoots. However, tiny hair like projections were observed in some elongated shoots on Mitra medium pertaining to5.37µM NAA. The tiny hair like structure bearing plantlets were hardened and acclimatized with 100% survival rate in the polytunnel chamber. After 8-10months of establishment ex vitro, flowering was observed. Additionally, the genetic fidelity of in vitro derived plants was tested with ISSR and SCoT marker profiling. The test results revealed that the plants derived from the protocol has 99% genetic similarity to that of the donor mother plant. This study can be applied in forensic interventions of this species, describes the maintenance of germplasm in vitro and establishment of new viable population in its original habitats by restoring existing sites of this critically endangered jewel orchid.