Biotechnological approaches for in vitro propagation, conservation and secondary metabolites production in Bulbophyllum, an endangered orchid genus: a review.

Bulbophyllum represents the largest genus in the family Orchidaceae. The orchid species of this genus are widely used in the traditional medicine systems in different Asian countries such as China, India, Indonesia and Thailand. Studies on the secondary metabolites of Bulbophyllum have revealed the presence of important phytochemicals such as phenols, flavonoids, alkaloids, tannins, triterpenoids, sesquiterpenoids, steroids and glycosides. Some species of Bulbophyllum are reported to be of horticultural importance for their unique flowers. Habitat destruction and unsustainable utilization of different species of Bulbophyllum have led to a decline in the natural populations. The present review provides insights into the phytochemistry and ethnomedicinal uses of different species of Bulbophyllum, and highlights the biotechnological approaches developed for its conservation and sustainable utilization. Overall, the details provided in the present review can potentially be used for genome editing and biotechnological advances to develop plants with improved traits, which will be essential for the judicious utilization of the Bulbophyllum species so as to conserve and save the populations in the wild.

Genome-wide identification and analysis of the PAL genes from the orchids Apostasia shenzhenica, Dendrobium catenatum and Phalaenopsis equestris.

Phenylalanine ammonia-lyase (PAL) is a key gateway enzyme that connects the phenylpropanoid pathway to primary metabolism. The phenylpropanoid pathway plays a vital role in the growth and environmental adaptation of many plants leading to the production of valuable bioactive compounds with industrial and medical applications. In the present study, nine putative PAL genes from three orchids were identified; five in Apostasia shenzhenica and two each in Dendrobium catenatum and Phalaenopsis equestris. Eighteen motifs and four major conserved functional domains were identified as reported in PAL proteins of other species. All the nine PALs were stable based on their computed physicochemical properties and localized in the cytoplasm. The three-dimensional structures of PALs revealed a homo-tetrameric structure consisting of four identical subunits. A total of 21 cis-regulatory elements with known functions were identified from the promoter regions of all PALs which are responsible for various plant responses to light, stress and growth regulators like auxins, gibberellins and abscisic acid. Phylogenetic analysis showed that the studied PAL proteins clustered in two major clades (clade I and II), placing dicot and monocot PALs in two separate monophyletic clades. In silico gene expression of the identified PALs in different vegetative and reproductive tissues revealed the differential expressions based on tissue type and disclosed that the expression of PAL genes was upregulated in all the tissues examined with an exception of PePAL leaf samples where no expression was detected, however, the same being highly expressed in reproductive tissues (PePAL1-labellum; PePAL2-sepal). In case of AsPALs, the expression was found to be highest in reproductive tissues (AsPAL4-maximum in inflorescence). On the other hand, the expression of DcPALs was found to be highest in vegetative tissues (DcPAL2-maximum in root). Based on the medicinal importance of orchids and the significant role of PAL genes in synthesis of bioactive compounds, the functional characterization of PAL genes can be further exploited in genetic improvement of medicinal orchids.Communicated by Ramaswamy H. Sarma.

Contrasting Reproductive Strategies of Two Nymphaea Species Affect Existing Natural Genetic Diversity as Assessed by Microsatellite Markers: Implications for Conservation and Wetlands Restoration.

Nymphaea, commonly known as water lily, is the largest and most widely distributed genus in the order Nymphaeales. The importance of Nymphaea in wetland ecosystems and their increased vulnerability make them a great choice for conservation and management. In this work, we studied genetic diversity in a collection of 90 N. micrantha and 92 N. nouchali individuals from six different states of India, i.e., Assam, Manipur, Meghalaya, Maharashtra, Goa, and Kerala, using simple sequence repeat (SSR) markers developed by low throughput Illumina sequencing (10X coverage of genome) of N. micrantha. Nymphaea nouchali is native to India, whereas N. micrantha is suggested to be introduced to the country for its aesthetic and cultural values. The study revealed extensive polymorphism in N. nouchali, while in N. micrantha, no apparent genetic divergence was detected prompting us to investigate the reason(s) by studying the reproductive biology of the two species. The study revealed that N. micrantha predominantly reproduces asexually which has impacted the genetic diversity of the species to a great extent. This observation is of immense importance for a successful re-establishment of Nymphaea species during restoration programs of wetlands. The information generated on reproductive behaviors and their association with genotypic richness can help in strategizing genetic resource conservation, especially for species with limited distribution. The study has also generated 22,268 non-redundant microsatellite loci, out of which, 143 microsatellites were tested for polymorphism and polymorphic markers were tested for transferability in five other Nymphaea species, providing genomic resources for further studies on this important genus.

Looking for a way forward for the cryopreservation of orchid diversity.

The family Orchidaceae, with over 25,000 species, includes five subfamilies and nearly 700 genera. Loss of plants in the wild has resulted from clearing of forests and excessive collection for various purposes. Moreover, the requirement of symbiotic association during seed germination under natural conditions adds a certain level of difficulty in retaining the orchid resources in the wild. Cryopreservation is an important arena in conservation science due to its potential of storing genetic resources without altering the genetic makeup. Cryopreserved orchids are a very small percentage of the species, and are also not representative of most genera. Finding effective protocols for the various explant types is of prime importance in conserving orchid diversity. Seed is the most commonly stored and directly useful explant, and direct plunging in liquid nitrogen or PVS2 vitrification appear to be suitable for most tested species. The myriad of other species should be screened as they become available, with special emphasis on seed maturity and moisture content. Studies of protocorms and protocorm-like bodies mostly employ desiccation, PVS2 vitrification or encapsulation-dehydration. Pollinia are generally stored successfully following desiccation or slow cooling. There are too few examples of shoot tip cryopreservation to make a determination, however vitrification techniques are likely the most useful for a range of genera. A systematic and coordinated effort is needed to screen all available species in as many taxa as possible, initially with seed, protocorms and pollinia. It is a charge to the orchid research community to organize this effort and fill in the required data for the large number of untested taxa. In addition, providing stored samples to established orchid cryo collections would greatly increase preservation of these endangered treasures.

Molecular cloning and characterization of chalcone synthase gene from Coelogyne ovalis Lindl. and its stress-dependent expression.

Chalcone synthase (CHS, EC 2.3.1.74) is one of the key and rate-limiting enzymes of phenylpropanoid pathway which plays superior roles in the production of secondary metabolites. In the present study a full-length cDNA of CHS gene was isolated and characterized from Coelogyne ovalis, an orchid of ornamental and medicinal importance. The CHS gene sequence from C. ovalis (CoCHS) was found to be 1445 bp and comprised an open reading frame of 1182 bp, encoding for 394 amino acid residues. Further, the sequence alignment and phylogenetic analysis revealed that CoCHS protein shared high degree of similarity with CHS protein of other orchid species. It also confirmed that it contained all four motifs (I to IV) and signature sequence for the functionality of this gene. Structural modeling of CoCHS based on the crystallographic structure of Freesia hybrida indicated that CoCHS had a similar structure. Quantitative polymerase chain reaction (qPCR) disclosed that CoCHS was expressed in all tissues examined, with the highest transcript being in leaves, followed by pseudobulbs and roots. CoCHS expression was also evaluated in the in vitro-raised plantlets under the abiotic stress (dark, cold, UV-B, wounding, salinity). mRNA transcript expression of CHS gene was found to be positively enhanced and regulated by the different stress types. A correlation between the CoCHS transcript expression with flavonoid and anthocyanin contents revealed that a positive correlation existed between metabolites' content and CoCHS expression within the in vivo as well as in the in vitro-raised plant parts.

Antioxidants and improved regrowth procedure facilitated cryoconservation of Paphiopedilum insigne Wall. Ex. Lindl. - An Endangered Slipper Orchid.

Orchids and their sustainability are very important issues that need global conservation efforts. Paphiopedilum insigne (an endangered orchid), is one of the most excessively exploited species of orchids and is mentioned in the IUCN Red List and Appendix I of CITES. The prospect for conservation and commercialization of this species would be strengthened with the development of improved cryopreservation techniques. This study reports on successful cryopreservation of protocorms of P.insigne after cryopreservation using vitrification (Vit) and encapsulation-vitrification (E-Vit) techniques. The study compared the addition of four antioxidants to the pretreatment and recovery stages, three growth media, and agitated vs. semisolid culture medium for initial recovery. Recovery after cryopreservation for the control was 27% for Vit and 37% for E-Vit. In both cases agitated culture produced improved recovery by about 10%, but with significantly better recovery with E-Vit. The best recovery (51.2 ±â€¯0.9%) was recorded for 0.5 M sucrose precultured encapsulated protocorms treated for 45 min with PVS2 and recovered in ½ MS (L/S) liquid medium for 10 days under agitation, followed by transfer to semi-solid medium. This recovery was further enhanced (62.7 ±â€¯0.5%) with the incorporation of 30 µM glutathione in both liquid preculture and the liquid and semisolid regrowth medium. This new protocol improved the E-Vit cryopreservation recovery from the initial 37%-63%, providing a suitable technique for storage of this threatened orchid.

Secondary metabolite profiling, cytotoxicity, anti-inflammatory potential and in vitro inhibitory activities of Nardostachys jatamansi on key enzymes linked to hyperglycemia, hypertension and cognitive disorders.

ETHNOPHARMACOLOGICAL RELEVANCE: Nardostachys jatamansi (D. Don) DC., 'Spikenard' or 'Jatamansi', a highly valued, aromatic herb from alpine Himalayas has a long history of use as ethnomedicine and dietary supplements in Ayurveda, Unani and Chinese system of medicine since Vedic ages (1000-800 BC). In Ayurveda and traditional system of medicine, the species is used as stimulant, sedative, brain tonic or mind rejuvenator, antidiabetic, cardio tonic, and in the treatment of various neurological disorders such as insomnia, epilepsy, hysteria, anxiety and depression. It is considered as Sattvic herb in Ayurveda and is now commercially marketed either as single or poly-herbal formulations by many companies in national and international markets. AIM OF THE STUDY: The species has become threatened in its natural habitats due to over exploitation and illegal trade of its rhizomes for drug preparation in herbal and pharmaceutical industries. Considering the increasing demand and tremendous medicinal importance of this threatened plant species, a detailed study was undertaken to evaluate its antioxidant potential, secondary metabolite profiling, cytotoxicity, anti-inflammatory potential and in vitro enzyme inhibitory activities on key enzymes linked to hyperglycemia, hypertension and cognitive disorders in different plant parts of wild and in vitro-raised plants with respect to different solvent systems for its sustainable utilization. MATERIALS AND METHODS: Anti-cholinesterase activity of leaves and rhizome of wild and cultured plant extracts was investigated against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes. In vitro anti-hyperglycemic (α-amylase and PTP1B), anti-hypertensive (angiotensin-converting enzyme), anti-tyrosinase and anti-inflammatory potential (5-lipoxygenase and hyaluronidase) of different plant parts of wild and in vitro-raised plants with respect to different solvent systems were also evaluated. In vitro cytotoxic effect of rootstock extracts of wild and in vitro-derived plants were against cancer (HCT-116, MCF-7 and OE33) and two normal (HEK and MEF) cell lines. Secondary metabolite profiling of rhizome segments of wild and in vitro-derived plants was carried out by quantitative gas chromatography-mass spectrometry (GC-MS). RESULTS: In vitro-raised plantlets showed comparative higher yield of various secondary metabolites with a significantly high antioxidant activity as compared to the wild plants. Methanolic rootstock extracts of both wild and in vitro-derived plants of N. jatamansi exhibited significant AChE (IC50 36.46 ±â€¯2.1 and 31.18 ±â€¯2.6 µg/ml, respectively) and BuChE (IC50 64.6 ±â€¯3.5 and 60.12 ±â€¯3.6 µg/ml, respectively) inhibitory potential as compared to standard inhibitor galanthamine (IC50 0.94 ±â€¯0.03 and 4.45 ±â€¯0.5 µg/ml). Methanolic rootstock extract of in vitro-derived plants showed significant α-amylase (IC50 90.69 ±â€¯2.1 µg/ml), PTP1B (IC50 24.56 ±â€¯0.8 µg/ml), angiotensin-converting enzyme (IC50 42.5 ±â€¯3.6 µg/ml) and tyrosinase (IC50 168.12 ±â€¯3.6 µg/ml) inhibitory potential as compared to standard acarbose (IC50 52.36 ±â€¯3.1 µg/ml), ursolic acid (IC50 5.24 ±â€¯0.8 µg/ml), captopril (IC50 32.36 ±â€¯2.5 µg/ml) and kojic acid (IC50 = 54.44 ±â€¯2.3 µg/ml). Both the methanolic rootstock and leaf extracts of tissue culture-derived plants exhibited promising anti-5-LOX and anti-hyaluronidase activities against the known inhibitor of 5-LOX and hyaluronidase. Furthermore, methanolic rootstock extracts of both wild and in vitro-derived plants exhibited promising cytotoxic effects to HCT-116, MCF-7 and OE33 cell lines as compared to the normal HEK and MEF after 12 h of treatment. Secondary metabolite profiling of wild and in vitro-derived plants by quantitative GC-MS analysis revealed the presence of different classes of terpenoids and phenolic acids might be responsible for its effective biological activities. CONCLUSION: In vitro-derived plants revealed a substantial anti-cholinesterases, anti-hyperglycemic anti-inflammatory, anti-hypertensive and anti-tyrosinase potential with higher yield of various bioactive metabolites and significantly higher antioxidant activity which substantially explain medicinal importance of N. jatamansi in traditional medicine, used for centuries in different Ayurvedic formulations. The present findings suggest that cultured plants could be a promising alternative for the production of bioactive metabolites with comparative biological activities to the wild plants.

In silico characterization and transcriptional modulation of phenylalanine ammonia lyase (PAL) by abiotic stresses in the medicinal orchid Vanda coerulea Griff. ex Lindl.

Phenylalanine ammonia lyase (PAL) is the first enzyme of phenylpropanoid pathway. In the present study, a full-length PAL transcript from Vanda coerulea Griff. ex Lindl. (Family: Orchidaceae) was isolated and characterized. It was found that complete PAL transcript of V. coerulea (VcPAL; Gene Bank no. MG745168) contained 2175 bp with the open reading frame (ORF) of 2112 bp, encoding 703 amino acid residues. The multiple sequence alignment showed that VcPAL protein had 81% identity with that of the orchid, Bromheadia finlaysoniana. Phylogenetic analysis also disclosed that VcPAL shared the same evolutionary relationship with PAL proteins of other orchid species and to be closely related to that of other angiosperm species as well. The three-dimensional structure of VcPAL was found to be homo-tetrameric in nature consisting of four identical subunits with a molecular mass of 75 kDa per subunit. In silico characterization revealed the deduced protein to be a stable protein, comprising three major functional domains as reported in PAL proteins of other species. The transcription profiling of VcPAL exhibited the highest expression level to be present in the in vitro - raised leaf and root samples as compared to that of the ex vitro plant. The differential expression of VcPAL transcript was observed to be up-regulated by different types of abiotic stresses like wounding, cold, UV-B, salinity, and down-regulated by dark treatment. The study also exhibited that the VcPAL enzyme activity was directly proportional to the gene expression after the tissues were subjected to salinity and wounding stresses wherein a 1.7- fold increase in the enzyme activity was recorded in the leaf tissues exposed to salinity stress. A positive correlation could be found between the enzyme activity and the accumulation of phenylpropanoids such as total phenolic and flavonoid contents with R2 = 0.85 and 0.842 respectively.

Insights into nuclear DNA content, hydrogen peroxide and antioxidative enzyme activities during transverse thin cell layer organogenesis and ex vitro acclimatization of Malaxis wallichii, a threatened medicinal orchid.

Malaxis wallichii (Lindl.) Deb, a small, perennial, monopodial, terrestrial orchid, is endemic to tropical Himalayas at an altitude of 1200-2000 m asl. The pseudobulbs are important ingredients of century old drug 'Ashtavarga' and a polyherbal energetic tonic 'Chyavanprash'. An efficient genetically stable in vitro propagation protocol using transverse thin cell layer culture system was established for M. wallichii. In the present report, meta-topolin alone proved to be three times more beneficial compared to other routinely used cytokinins in inducing highest number of shoot buds, plant height and growth of regenerated shoots. The highest regeneration frequency (89%) along with maximum number of adventitious shoots per explant (22.5 ± 0.6) was observed in MS medium supplemented with 1.0 mg/l meta-topolin and 0.5 mg/l α-naphthalene acetic acid. Highest rooting frequency with highest number of roots (8.66 ± 0.3) was achieved in half-strength MS medium fortified with 1.0 mg/l indole acetic acid. Clonal stability of in vitro-derived plantlets was evaluated and compared to donor plant using intron splice junction (ISJ) markers and flow cytometry. ISJ markers revealed 4.76% clonal variability indicating high degree of genetic stability amongst the in vitro-derived regenerants. The nuclear DNA content of M. wallichii (2n) was found to be 2C = 2.760 ± 0.02 pg and therefore, 1349.64 Mbp (1C). Flow cytometry analysis of actively growing young and mature leaves from donor as well as in vitro-derived plantlets revealed presence of three peaks corresponding to 2C, 4C and 8C, while 2C was the most abundant. In the present investigation, there was no significant difference in the 2C DNA content between the mother and in vitro-derived plants; however, the frequency of endopolyploid cells varied in young and adult plants. An increased H2O2 content as well as lipid peroxidation activities were observed during early stages of acclimatization which declined afterwards. The enhanced activities of antioxidant enzymes like superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase in acclimatized plantlets as compared to in vitro-grown ones revealed their active involvement in growth and development against oxidative stress under external adverse environment.

Studies on secondary metabolite profiling, anti-inflammatory potential, in vitro photoprotective and skin-aging related enzyme inhibitory activities of Malaxis acuminata, a threatened orchid of nutraceutical importance.

Malaxis acuminata D. Don., a small, terrestrial orchid, is endemic to tropical Himalayas at an altitude of 1200-2000m asl. The dried pseudobulbs are important ingredients of century old ayurvedic drug 'Ashtavarga' and a polyherbal immune-booster nutraceutical 'Chyavanprash', known to restore vigour, vitality and youthfulness. Considering tremendous medicinal importance of this threatened orchid species, a detailed study was undertaken for the first time to address its antioxidant potential, secondary metabolite contents and biological activities against skin-aging related enzymes (anti-collagenase, anti-elastase, anti-tyrosinase and xanthine oxidase) and anti-inflammatory activity (5-lipoxygenase and hyaluronidase) in different plant parts of wild and in vitro-derived plants of M. acuminata. Methanolic leaf and stem extracts were further evaluated for in vitro photoprotective activity against UV-B and UV-A radiations. Furthermore, secondary metabolite profiling of various plant parts was carried out by Gas Chromatography Mass Spectrometry (GC-MS). A significantly higher antioxidant potential (DPPH, metal chelating and ABTS•+) with a comparative higher yield of secondary metabolites was observed in in vitro-derived plantlets as compared to the wild plants. Among various solvent systems used, methanolic leaf and stem extracts showed promising inhibitory activity against major skin aging-related enzymes and anti-inflammatory potential. Methanolic leaf and stem extracts of both wild and in vitro-derived plants showed promising photoprotective activity against UV-B and UV-A radiations in vitro with comparatively higher sun protection factor (SPF). Furthermore, GC-MS analysis of methanolic extracts of leaves and stems of wild as well as in vitro-derived plantlets revealed presence of many bioactive metabolites such as, dietary fatty acids, α-hydroxy acids, phenolic acids, sterols, amino acids, sugars and glycosides which substantially explain the use of M. acuminata as one of the potential rejuvenator and anti-aging ingredient in many Ayurvedic formulations.

In vitro regeneration of Drosera burmannii Vahl.: a carnivorous plant of north-east India.

An efficient in vitro regeneration protocol has been developed from shoot tips of Drosera burmannii Vahl., a carnivorous plant of north-east India. Various plant growth regulators were used to study their efficacy in the induction of multiple shoots and roots. Of the various treatments, the maximum number of shoots (28.8 ± 1.5) and roots (9.7 ± 0.6) was observed in one-fourth strength standard medium (MS with 50 mg/l citric acid and 10 mg/l ascorbic acid) supplemented with 4 mg/l 6-benzylaminopurine (BAP) and 4 mg/l α-naphthalene acetic acid (NAA) followed by 26.8 ± 1.4 shoots in one-fourth strength SM fortified with 4 mg/l kinetin (KN) and 4 mg/l NAA. The well-developed plantlets with shoots and roots were potted in small plastic glasses filled with a mixture of sand and farmyard manure (3:1); these plantlets when transferred to a glasshouse for hardening and acclimatization showed 90% survival.

Genetic diversity and molecular evolution of Naga King Chili inferred from internal transcribed spacer sequence of nuclear ribosomal DNA.

Sequences of the Internal Transcribed Spacer (ITS1-5.8S-ITS2) of nuclear ribosomal DNAs were explored to study the genetic diversity and molecular evolution of Naga King Chili. Our study indicated the occurrence of nucleotide polymorphism and haplotypic diversity in the ITS regions. The present study demonstrated that the variability of ITS1 with respect to nucleotide diversity and sequence polymorphism exceeded that of ITS2. Sequence analysis of 5.8S gene revealed a much conserved region in all the accessions of Naga King Chili. However, strong phylogenetic information of this species is the distinct 13 bp deletion in the 5.8S gene which discriminated Naga King Chili from the rest of the Capsicum sp. Neutrality test results implied a neutral variation, and population seems to be evolving at drift-mutation equilibrium and free from directed selection pressure. Furthermore, mismatch analysis showed multimodal curve indicating a demographic equilibrium. Phylogenetic relationships revealed by Median Joining Network (MJN) analysis denoted a clear discrimination of Naga King Chili from its closest sister species (Capsicum chinense and Capsicum frutescens). The absence of star-like network of haplotypes suggested an ancient population expansion of this chili.

Biotechnological enhancement of capsaicin biosynthesis in cell suspension cultures of Naga King Chili (Capsicum chinense Jacq.).

Cell suspension cultures were initiated from hypocotyl derived callus to induce capsaicin biosynthesis in suspension cultures of Naga King Chili (Capsicum chinense Jacq.). Efficient capsaicin production with high growth index (GI) was obtained by exposing cells to salicylic acid (SA) and calcium channel modulators in suspension cultures. The time course of capsaicin formation is related to the cell growth profile in a batch culture. Cells cultivated in the standard medium (SM) initially showed low level of capsaicin yield during active growth. When the cells approached stationary phase, cell growth and cell viability decreased whereas capsaicin production increased continuously. In the fed-batch cultures, the highest capsaicin yield (567.4 ± 8.1 µgg(1) fresh weight) (f.wt) was obtained by feeding the cells with 1 mM SA. However, SA feeding during cultivation repressed the cell growth. Enhanced cell growth (3.1 ± 0.1 GI/culture) and capsaicin yield (534 ± 7.8 µgg(-1)f.wt) were obtained when the cells were fed with calcium ionophore A23187 (0.5 mM) on day 25 as compared to the control. Addition of the calcium channel blocker verapamil hydrochloride (100 mM) inhibited cell growth and capsaicin production in Naga King Chili suspension cell cultures.

Applicability of ISSR and DAMD markers for phyto-molecular characterization and association with some important biochemical traits of Dendrobium nobile, an endangered medicinal orchid.

Dendrobium nobile is an important medicinal orchid having profound importance in traditional herbal drug preparations and pharmacopeias worldwide. Due to various anthropogenic pressures the natural populations of this important orchid species are presently facing threats of extinction. In the present study, genetic and chemical diversity existing amongst 6 natural populations of D. nobile were assessed using molecular markers, and the influence of genetic factors on its phytochemical activity especially antioxidant potential was determined. Molecular fingerprinting of the orchid taxa was performed using ISSR and DAMD markers along with the estimation of total phenolics, flavonoids and alkaloid contents. Antioxidant activity was also measured using DPPH and FRAP assays which cumulatively revealed a significant level of variability across the sampled populations. The representatives from Sikkim in Northeast India revealed higher phytochemical activity whereas those from Mizoram showed lesser activity. Analysis of molecular variance (AMOVA) revealed that variation amongst the populations was significantly higher than within the populations. The data generated by UPGMA and Bayesian analytical models were compared in order to estimate the genetic relationships amongst the D. nobile germplasm sampled from different geographical areas of Northeast India. Interestingly, identical grouping patterns were exhibited by both the approaches. The results of the present study detected a high degree of existing genetic and phytochemical variation amongst the populations in relation to bioclimatic and geographic locations of populations. Our results strongly establish that the cumulative marker approach could be the best suited for assessing the genetic relationships with high accuracy amongst distinct D. nobile accessions.

Genetic fidelity assessment in micropropagated plants using cytogenetical analysis and heterochromatin distribution: a case study with Nepenthes khasiana Hook f.

Rapid clonal propagation of selected genotypes has been one of the most extensively exploited approaches of biotechnology. However, inclusion of somaclonal variations in tissue-culture-derived plants results in the production of undesirable plant off-types which limits its applications in tissue culture industry. Therefore, the most critical concern has been the maintenance of genetic uniformity of micropropagated plants. Assessment of genetic fidelity in tissue-culture-raised plants of three consecutive regenerations of Nepenthes khasiana has been successfully carried out using chromosome counts and heterochromatin distribution pattern wherein changes in the number of chromosomes and the distribution of AT and GC base pairs were recorded. The cells studied in the plantlets of the first regeneration (23.33 %) showed deviant number of chromosome which was increased to 33.33 % and 40 % in the plantlets of the second and the third regenerations, respectively. Also, 4',6-diamidino-2-phenylindole (DAPI)(+) and chromomycin A3 (CMA)(+) binding sites, on an average of 5.74 ± 0.47 and 5.00 ± 0.30, were observed in the plantlets of the first regeneration. Subsequently, DAPI(+) binding sites were increased to 6.61 ± 0.39 and 6.74 ± 0.57 in the plantlets of the second and the third regenerations, respectively, with a corresponding decrease in the CMA(+) binding sites (4.63 ± 0.45 and 4.16 ± 0.47 CMA(+) sites in the plantlets of the second and the third regenerations, respectively). The study reveals an increase in cytological variations in the morphologically similar micropropagated plants of N. khasiana with the subsequent regenerations which further necessitate the determination of genetic integrity of micropropagated plants.

Single primer amplification reaction (SPAR) methods reveal subsequent increase in genetic variations in micropropagated plants of Nepenthes khasiana Hook. f. maintained for three consecutive regenerations.

The genetic fidelity of in vitro-raised plants of three successive regenerations of Nepenthes khasiana Hook. f. was assessed using three different single primer amplification reaction (SPAR) methods, viz., random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and direct amplification of minisatellite DNA region (DAMD) markers. Out of 80 RAPD primers screened, 14 primers reflected a genetic variation of 4.1% in the first regeneration which was increased to 9.4% in the third regeneration. In the case of ISSR, out of 36 primers screened for assessment of genetic homogeneity of the regenerated plantlets, 12 primers showed an increase of genetic variation from 4.3% to 10% from the first to the third regenerations. In DAMD profiling, 15 primers were used for the evaluation of genetic fidelity where 8.47% of polymorphism was observed in the first regeneration which was increased to 13.33% in the third regeneration. The cumulative analysis reflected a genetic variation of 5.65% in the first regeneration which increased subsequently to 7.77% in the second regeneration and 10.87% in the third regeneration. The present study demonstrates SPAR technique to be an efficient tool for the assessment of clonal fidelity of in vitro-raised plants.

Manipulation of culture strategies to enhance capsaicin biosynthesis in suspension and immobilized cell cultures of Capsicum chinense Jacq. cv. Naga King Chili.

Manipulation of culture strategies was adopted to study the influence of nutrient stress, pH stress and precursor feeding on the biosynthesis of capsaicin in suspension and immobilized cell cultures of C. chinense. Cells cultured in the absence of one of the four nutrients (ammonium and potassium nitrate for nitrate and potassium stress, potassium dihydrogen orthophosphate for phosphorus stress, and sucrose for sugar stress) influenced the accumulation of capsaicin. Among the stress factors studied, nitrate stress showed maximal capsaicin production on day 20 (505.9 ± 2.8 µg g(-1) f.wt) in immobilized cell, whereas in suspension cultures the maximum accumulation (345.5 ± 2.9 µg g(-1) f.wt) was obtained on day 10. Different pH affected capsaicin accumulation; enhanced accumulation of capsaicin (261.6 ± 3.4 µg g(-1) f.wt) was observed in suspension cultures at pH 6 on day 15, whereas in case of immobilized cultures the highest capsaicin content (433.3 ± 3.3 µg g(-1) f.wt) was obtained at pH 5 on day 10. Addition of capsaicin precursors and intermediates significantly enhanced the biosynthesis of capsaicin, incorporation of vanillin at 100 µM in both suspension and immobilized cell cultures resulted in maximum capsaicin content with 499.1 ± 5.5 µg g(-1) f.wt on day 20 and 1,315.3 ± 10 µg g(-1) f.wt on day 10, respectively. Among the different culture strategies adopted to enhance capsaicin biosynthesis in cell cultures of C. chinense, cells fed with vanillin resulted in the maximum capsaicin accumulation. The rate of capsaicin production was significantly higher in immobilized cells as compared to freely suspended cells.

Genetic stability and phytochemical analysis of the in vitro regenerated plants of Dendrobium nobile Lindl., an endangered medicinal orchid.

An efficient genetically stable regeneration protocol with increased phytochemical production has been established for Dendrobium nobile, a highly prized orchid for its economic and medicinal importance. Protocorm like bodies (PLBs) were induced from the pseudostem segments using thidiazuron (TDZ; 1.5 mg/l), by-passing the conventional auxin-cytokinin complement approach for plant regeneration. Although, PLB induction was observed at higher concentrations of TDZ, plantlet regeneration from those PLBs was affected adversely. The best rooting (5.41 roots/shoot) was achieved in MS medium with 1.5 mg/l TDZ and 0.25% activated charcoal. Plantlets were successfully transferred to a greenhouse with a survival rate of 84.3%, exhibiting normal development. Genetic stability of the regenerated plants was investigated using randomly amplified polymorphic DNA (RAPD) and start codon targeted (SCoT) polymorphism markers which detected 97% of genetic fidelity among the regenerants. The PIC values of RAPD and SCoT primers were recorded to be 0.92 and 0.76 and their Rp values ranged between 3.66 and 10, and 4 and 12 respectively. The amplification products of the regenerated plants showed similar banding patterns to that of the mother plant thus demonstrating the homogeneity of the micropropagated plants. A comparative phytochemical analysis among the mother and the micropropagated plants showed a higher yield of secondary metabolites. The regeneration protocol developed in this study provides a basis for ex-situ germplasm conservation and also harnesses the various secondary metabolite compounds of medicinal importance present in D. nobile.

Start Codon Targeted (SCoT) marker reveals genetic diversity of Dendrobium nobile Lindl., an endangered medicinal orchid species.

Genetic variability in the wild genotypes of Dendrobium nobile Lindl. collected from different parts of Northeast India, was analyzed using a Start Codon Targeted (SCoT) marker system. A total of sixty individuals comprising of six natural populations were investigated for the existing natural genetic diversity. One hundred and thirty two (132) amplicons were produced by SCoT marker generating 96.21% polymorphism. The PIC value of the SCoT marker system was 0.78 and the Rp values of the primers ranged between 4.43 and 7.50. The percentage of polymorphic loci (Pp) ranging from 25% to 56.82%, Nei's gene diversity (h) from 0.08 to 0.15 with mean Nei's gene diversity of 0.28, and Shannon's information index (I) values ranging from 0.13 to 0.24 with an average value of 0.43 were recorded. The gene flow value (0.37) and the diversity among populations (0.57) demonstrated higher genetic variation among the populations. Analysis of molecular variance (AMOVA) showed 43.37% of variation within the populations, whereas 56.63% variation was recorded among the populations. Cluster analysis also reveals high genetic variation among the genotypes. Present investigation suggests the effectiveness of SCoT marker system to estimate the genetic diversity of D. nobile and that it can be seen as a preliminary point for future research on the population and evolutionary genetics of this endangered orchid species of medicinal importance.

Compatible fungi, suitable medium, and appropriate developmental stage essential for stable association of Dendrobium chrysanthum.

Establishment of symbiotic association at the appropriate developmental stage helped maintain continued growth which is vital for the long-term ex vitro survival of the orchid. In the present study, symbiotic association was carried out using different developmental stages of Dendrobium chrysanthum and pathogenic Rhizoctonia isolates (obtained from orchids and non-orchid hosts) in different culture media. Isolate 2162 supported highest symbiotic germination on OMA-S (oat meal agar medium without nutrients + sucrose), whereas, stable symbiotic association with plantlets was obtained with isolate 4634 on OMA-NC (oat meal agar medium + cellulose). Isolate Dc-2S2 obtained from the host plant did not promote seed germination nor did it form association with protocorms or plantlets. This study, for the first time identifies a combination of compatible fungal isolate, suitable culture medium, and appropriate developmental stage at which symbiotic association in vitro can be deemed successful for the medicinally important orchid, D. chrysanthum.