Gamma Radiation Induced In-Vitro Mutagenesis and Isolation of Mutants for Early Flowering and Phytomorphological Variations in Dendrobium 'Emma White'.

In vitro mutagenesis offers a feasible approach for developing new orchid cultivars through genetic manipulation. In the present study, protocorm-like bodies (PLBs) were exposed to gamma rays (10, 20, 40, 60, 80 Gy) to study in vitro growth responses and induction of mutants in Dendrobium 'Emma White'. Both proliferation and regeneration of PLBs decreased progressively with increasing doses, except for a significantly enhanced growth response at 10 Gy. The optimal dose of gamma radiation for mutagenesis was found in the range 10 to 25 Gy based on the growth reduction curve. Analysis using a high-throughput cell analyzer revealed a significant reduction in nuclear DNA content at > 40 Gy doses. At 10 Gy treatment, the growth attributes, such as root length, plant height and leaf number, were significantly increased by 36%, 26% and 20%, respectively, compared to the control. This increase was significant over other tested doses as well. Testing of random amplified polymorphic DNA markers revealed the presence of detectable polymorphism among gamma mutant plantlets with a polymorphism information content value at 0.41. The gamma-ray-induced earliness in flower development was observed within 294 days post ex vitro growth of 10 Gy mutant compared to the control plants flowered after 959 days. Our results highlight the significance of gamma radiation in inducing enhanced growth, morphological variations and early floral initiation in Dendrobium, providing a basic framework for mutation breeding and improvement of orchids.

First De novo whole genome sequencing and assembly of mutant Dendrobium hybrid cultivar 'Emma White'.

The Dendrobium hybrid cultivar 'Emma White' is an ornamental, successfully commercialised orchid. We used a gamma ray-induced early flowering mutant and the Illumina HiSeqX10 sequencing platform to generate the first draft de novo whole genome sequence and assembly. The draft sequence was 678,650,699 bp in length, comprising 447,500 contigs with an N50 of 1423 and 33.48% GC content. Comparing 95,529 predicted genes against the Uniprot database revealed 60,741 potential genes governing molecular functions, biological processes and cellular components. We identified 216,232 simple sequence repeats and 138,856 microsatellite markers. Chromosome-level genome assembly of Dendrobium huoshanense was used to RagTag-scaffold available contigs of the mutant, revealing a total length of 687,254,899 bp with an N50 of 2096. The longest final contiguous length was 18,000,059 bp from 30,571 bp. BUSCO genome completeness was 93.6%. This study is valuable for investigating the mechanisms of mutation, and developing Dendrobium hybrid cultivars using mutation breeding.

High-Performance Thin-Layer Chromatography Method for Simultaneous Determination of Antipsychotic and Medicinally Important Five ß-Carboline Alkaloids.

This is the first report on the development and validation of high-performance thin-layer chromatography (HPTLC) method for simultaneous analysis of five antipsychotic and medicinally important ß-carboline alkaloids (ßCAs), namely, harmalol, harmaline, harmine, harmane and norharmane. These ßCAs occurs in both plant and animal system including human being. In the present investigation, their best separation was achieved using an optimized mobile phase, chloroform: methanol: glacial acetic acid (7.8:2.2:0.2, v/v/v) on aluminum TLC plates precoated with silica gel 60 F254. The quantification was performed by densitometric scanning in fluorescence mode at 366 nm. The calibration curves were drawn using linear regression, plotted over the range 25-250 ng band-1 of standard ßCAs with correlation coefficient (R2) between 0.97 and 0.992. Accuracy in terms of recovery (83.95-112.40%), repeatability of application (0.61-2.42%), repeatability of measurement (1.94-3.05%) and intermediate precision (0.62-11.16%) of developed method were simultaneously determined. The limit of detection and limit of quantification were between 4.95-6.59 and 16.50-21.93 ng band-1, respectively. The method was validated according to ICH guidelines and was simple, cost-effective, precise, sensitive and specific for the determination of ßCAs in herbs, Fagonia schweinfurthii, Peganum harmala and Tribulus terrestris. The developed HPTLC method would have importance in forensic and industrial chromatographic analysis and fingerprinting of various herbs and drug formulations containing ßCAs.

In vitro propagation and cell cultures of memory tonic herb Evolvulus alsinoides: a best source for elicited production of scopoletin.

Evolvulus alsinoides L. is used for preparation of 'Shankhapushpi', an important popular ayurvedic drug that contributes considerably to the improvement of memory power. The improvement is attributed to the presence of furanocoumarin scopoletin, a metabolite with a wide range of biological activities. This report describes, for the first time, an in vitro culture system for propagation and enhanced production of scopoletin. Different concentrations of auxins and cytokinins individually and in combination were used in Murashige and Skoog (MS) medium to induce shoot regeneration in cotyledonary nodal explants and callus formation in leaf explants. The best response was achieved in MS medium fortified with 5.0 µM 6-benzyladenine (BA) in which 96 % of cultures produced 7.6 ± 0.6 shoots per explant. Regenerated shoots were rooted on MS medium with 5.0 µM indole-3-acetic acid (IAA). Plantlets were successfully acclimatized and established in soil. MS medium fortified with 10 µM BA + 5.0 µM IAA showed maximum growth and accumulation of scopoletin in cell cultures. Cell cultures could be maintained over 24 months. The influences of auxins, cytokinins, organic acids, amino acids, and fungal-derived elicitors on production of scopoletin were studied. Presence of either L-arginine, sodium pyruvate, or yeast extract highly promoted scopoletin production as compared with control and achieved 75.02-, 72.13-, and 57.98-fold higher accumulation, respectively. The results presented herein have laid solid foundation for large-scale production of scopoletin and further investigation of its purification and utilization as a novel pharmaceutical drug.

In vitro propagation and production of cardiotonic glycosides in shoot cultures of Digitalis purpurea L. by elicitation and precursor feeding.

Digitalis purpurea L. (Scrophulariaceae; Foxglove) is a source of cardiotonic glycosides such as digitoxin and digoxin which are commercially applied in the treatment to strengthen cardiac diffusion and to regulate heart rhythm. This investigation deals with in vitro propagation and elicited production of cardiotonic glycosides digitoxin and digoxin in shoot cultures of D. purpurea L. In vitro germinated seedlings were used as a primary source of explants. Multiple shoot formation was achieved for three explant types (nodal, internodal, and leaf) cultured on Murashige and Skoog (MS) medium with several treatments of cytokinins (6-benzyladenine-BA; kinetin-Kin; and thidiazuron-TDZ) and auxins (indole-3-acetic acid-IAA; α-naphthaleneacetic acid-NAA; and 2,4-dichlorophenoxy acetic acid-2,4-D). Maximum multiple shoots (12.7 ± 0.6) were produced from nodal explants on MS + 7.5 µM BA. Shoots were rooted in vitro on MS containing 15 µM IAA. Rooted plantlets were successfully acclimatized. To further maintain the multiple shoot induction, mother tissue was cut into four equal parts and repeatedly sub-cultured on fresh shoot induction liquid medium after each harvest. On adaptation of this strategy, an average of 18 shoots per explant could be produced. This strategy was applied for the production of biomass and glycosides digitoxin and digoxin in shoot cultures on MS medium supplemented with 7.5 µM BA and several treatments with plant growth regulators, incubation period, abiotic (salicylic acid, mannitol, sorbitol, PEG-6000, NaCl, and KCl), biotic (Aspergillus niger, Helminthosporium sp., Alternaria sp., chitin, and yeast extract) elicitors, and precursors (progesterone, cholesterol, and squalene). The treatment of KCl, mycelial mass of Helminthosporium sp., and progesterone were highly effective for the production of cardenolides. In the presence of progesterone (200 to 300 mg/l), digitoxin and digoxin accumulation was enhanced by 9.1- and 11.9-folds respectively.

In vitro culture, plant regeneration and clonal behaviour of Sesuvium portulacastrum (L.) L.: a prospective halophyte.

An efficient plant regeneration protocol using axillary shoots of the salt accumulator halophyte, Sesuvium portulacastrum (L.) L. was established and in vitro responses of six Sesuvium clones were studied. The shoot and root induction responses to cytokinins and auxins in clone MH (Maharashtra) were concentration specific. Significantly the highest number of shoots, average shoot elongation and percent shoot regeneration per explant were observed on MS medium supplemented with 40 µM 2-isopentenyl adenine (2iP) followed by 20 µM benzyladenine (BA). Higher cytokinin (60 µM), however, inhibited shoot induction and shoot length. The lower concentrations (5 or 10 µM) of α-napthaleneacetic acid (NAA) proved more effective for root induction, number of roots and average root length. Well-developed plantlets were successfully hardened and established in the field with more than 85 % survival rate. In vitro response of six Sesuvium clones cultured on MS + 20 µM BA revealed higher multiplication rate in clone MH and KA (Karnataka, India) compared to other clones. The results offer the prospect of selecting clones of this species with characteristics desirable for utilization and/or restoration in specific ecological zones.