Low-temperature perception and modulations in Ocimum basilicum commercial cultivar CIM-Shishir: Biosynthetic potential with insight towards climate-smart resilience.

The newly released interspecific hybrid variety CIM-Shishir, resulting from a cross between Ocimum basilicum and Ocimum kilimandscharicum claims to be a multicut, lodging resistant, cold tolerant, high essential oil yielding with linalool rich variety. It has a purple-green stem and has a unique feature and advantage of better survival in the winter season than other O. basilicum varieties, illustrating its physiological mechanisms for cold tolerance. In this study, we subjected both the CIM-Shishir variety and a control plant to cold stress to investigate the impact of low temperatures on various physiological, trichome developments, secondary metabolite constitution aspects related to essential oil production, and gene expression. The analysis revealed a significantly higher density and altered morphology of trichomes on the leaf surface of the variety subjected to low temperatures, indicating its adaptation to cold conditions. Furthermore, when comparing the treated plants under low-temperature stress, it was observed that the relative electrolyte leakage and Malondialdehyde (MDA) contents substantially increased in the control in contrast to the CIM-Shishir variety. This finding suggests that CIM-Shishir exhibits superior cold tolerance. Additionally, an increase in proline content was noted in the variety exposed to low temperatures compared to the control. Moreover, the chlorophyll and anthocyanin content gradually increased with prolonged exposure to low-temperature stress in the newly developed variety, indicating its ability to maintain photosynthetic capacity and adapt to cold conditions. The activities of superoxide dismutase (SOD) also increased under low-temperature conditions in the CIM-Shishir variety, further highlighting its cold tolerance behaviour. In our research, we investigated the comprehensive molecular mechanisms of cold response in Ocimum. We analyzed the expression of key genes associated with cold tolerance in two plant groups: the newly developed hybrid variety known as CIM-Shishir Ocimum, which exhibits cold tolerance, and the control plants susceptible to cold climates that include WRKY53, ICE1, HOS1, COR47, LOS15, DREB5, CBF4, LTI6, KIN, and ERD2. These genes exhibited significantly higher expression levels in the CIM-Shishir variety compared to the control, shedding light on the genetic basis of its cold tolerance. The need for climate-smart, resilient high-yielding genotype is of high importance due to varied climatic conditions as this will hit the yield drastically and further to the economic sectors including farmers and many industries that are dependent on the bioactive constituents of Ocimum.

DNA Fingerprinting and Genetic Relationships Similarities Among the Accessions/Species of Ocimum Using SCoT and ISSR Markers System.

Studies on genetic diversity could enhance taxonomic authentication and evolutionary relationship among the species of Ocimum. Therefore, diversity among 36 Ocimum accessions representing species from different regions of world were analyzed using Start Codon-Targeted Polymorphism (SCoT) and inter-simple sequences repeat (ISSR) marker. Marker systems used in this study was potentially targeted the different regions of the genome and included 18 SCoT and 15 ISSR primers, which showed successful amplification profile for Ocimum. Between these two, SCoT revealed the highest mean value of percentage of Polymorphism (84.6%), polymorphic information content (PIC, 0.65), and resolving power (Rp, 8.80), which were higher than ISSR. A total of 140 and 111 amplicons were obtained with SCoT and ISSR marker. The Mantel test indicted a significant correlation (r2 = 0.44) between ISSR and SCoT, which suggested a common genetical background among the accessions. The principal coordinate study showed the selection of different Ocimum genotypes by the cluster analysis. This study will help and support identification, genetic mapping, and molecular ecology to enhance the breeding program's efficiency for developing elite varieties to meet industrial demand globally. The present study is the first report of the genetic diversity, and relationship determination with SCoT-based molecular marker among Ocimum accessions.

Assessing and integrating the transcriptome analysis with plant development, trichomes, and secondary metabolites yield potential in Mentha arvensisL.

Mentha arvensisL. (corn mint) is well known for the production of menthol, a used commodity in flavouring industries, and provides natural fragrances. Glandular trichomes are responsible for producing specific secondary metabolites in vascular plants having species chemistry. Ten cultivars/varieties of M. arvensis, namely, Saksham, Kosi, Himalaya, Gomti, Sambhav, Kalka, Damroo, Kushal, and Shivalik, were used to study the developmental regulation of trichomes, essential oil yield, chemical constituents of essential oil and morphological parameters were estimated with gene expression using a randomized block design. Simultaneously, RNA sequence-based transcriptome analysis was done to reveal the transcription factors and differential gene analysis, which are responsible for the biosynthesis of essential oil as well as trichome development. Plant growth showed the maximum transition between 35 and 50 days stage, while essential oil and its metabolite bioconversion was observed in between 70 and 100 days stage. Glandular trichomes were maximally increased between 50, 70 days, and 100 days stage in var. Kosi followed by var. Saryu which has rapid growth in oil content. Menthol reductase activity was found to be a regulatory element during development, as it follows the inverse trend of menthol content and leads to menthol accumulation in subcuticular spaces. Transcriptional factors, cog, and nonredundant novel genes were identified. The composition of mintessential oils is regulated at multiple levels, including transcript abundance, catalytic properties of enzyme catalysts, and cell type-specific epigenetic processes.

In Silico Identification of miRNA and Targets from Chrysopogon zizanioides (L.) Roberty with Functional Validation from Leaf and Root Tissues.

microRNAs are small non-coding RNA molecule that plays an important role in metabolism. Chrysopogon zizanioides (L.) Roberty is an important aromatic plant used in perfumery industries, soil, water conservation, and agricultural practices. In this study, the transcriptomic sequence of vetiver leaf and root was subjected to miRNA identification by the computational methods. miRNA identification was carried out using a homology-based method by C-mii software with several other online tools. A total of 80 miRNA were identified from both leaf and root sequences. Target identification was done by identified miRNA sets. A total of 25 and 31 miRNA families were identified in both leaf and root, respectively, with ten common families involve in different ontological function. miR169 and miR5021 regulate most of the target in leaf and root. In vetiver, many primary and secondary metabolism elements are regulated by miRNA as photo-system, transcription factor, terpenoid metabolism, etc. Here is the first in silico study revealing the specific miRNAs and their target genes for corresponding root and leaf tissues respectively in C. zizanioides.

Induced polygenic variations through γ -rays irradiation and selection of novel genotype in chamomile (Chamomilla recutita [L.] Rauschert).

Purpose: To develop elite mutants in chamomile (Chamomilla recutita [L.] Rauschert) for increasing the quantity and quality of essential oil rich in acetylenic compound (2Z,8Z)-matricaria acid methyl ester by applying γ -rays irradiation. Molecular and chemical analysis was performed for ithe dentification/differentiation of mutant genotype. Materials and methods: Chamomile (Chamomilla recutita [L.] Rauschert) variety Vallary seeds were irradiated by applying γ -rays irradiation at 100, 200, 300, 400, 500, 600, 700, 800, 900 and1000 Gy doses at a dose rate of 55 Gy/min and mutants were isolated and analyzed for the quantity and quality of essential oil. The oil was found to be rich in acetylenic compound (2Z,8Z)-matricaria acid methyl ester and the results obtained were validated using gas chromatography (GC) coupled with either Flame Ionization detection (GC-FID) or mass spectrometer (GC-MS), and nuclear magnetic resonance (NMR). Results: The selected mutant SELM-1 (Selection Mutant-1) showed the production potential of 7.00-7.50 q ha-1 dry flowers and 6.00-6.50 kg ha-1 essential oil yield. Essential oil of mutant SELM-1 contained in [(2Z,8Z)-matricaria acid methyl ester] (76-80%) useful in cosmetic, perfumery, and pharmaceutical industries. Conclusion:γ -rays irradiation method is a very efficient mutation breeding method for chamomile crop. GC-FID or GC-MS and NMR methods are found to be the most powerful methods for screening of essential oil chemical compounds isolated from the mutants. The novel mutant (SELM-1) is very promising in terms of high flower and essential oil yield rich in acetylenic compound (2Z,8Z)-matricaria acid methyl ester (76-80%), hence, it was released as variety in Council of Scientific & Industrial Research (CSIR)-Central Institute of Medicinal and Aromatic Plant (CIMAP), Lucknow U.P. (India) named as CIM-Ujjwala for commercial cultivation.

Biochemical characterization and spatio-temporal analysis of the putative L-DOPA pathway in Mucuna pruriens.

MAIN CONCLUSION: Transcriptome analysis and biochemical characterization of the putative l-3,4-dihydroxyphenylalanine (l-DOPA) pathway in Mucuna pruriens (L.) DC have been performed. Spatio-temporal quantification of the putative l-DOPA biosynthetic pathway genes and its correlation with respective metabolites was established. l-tyrosine, l-DOPA, and dopamine from all plant parts were quantified. The de novo transcriptome analysis was performed using leaves of the selected M. pruriens mutant T-IV-9 during maturity. The putative L-DOPA pathway and its regulatory genes were retrieved from transcriptome data and the L-DOPA pathway was biochemically characterized. The spatial and temporal gene expression for the L-DOPA pathway was identified with respect to the chemical constituents. L-tyrosine, L-DOPA, and dopamine contents were highest in leaves during maturity (about 170-day-old plants). The polyphenol oxidase (PPO) was highly expressed in tender stems (230-fold higher as compared to seeds) as well as a high L-DOPA content. The PPO gene was highly expressed in leaves (3367.93 in FPKM) with a 79-fold increase compared to control plants during maturity. L-DOPA was found in every part with varied levels. The highest L-DOPA content was found in mature dried seed (3.18-5.8%), whereas the lowest amount was recorded in mature and dried leaves. The reproductive parts of the plant had a higher amount of L-DOPA content (0.9-5.8%) compared to the vegetative parts (0.2-0.91%). Various amino acid transporters and permeases were expressed in M. pruriens. The transcripts of dopa decarboxylase (DDC) were found in almost all parts of the plant, but its higher content was limited to the leaf.

Modulations in primary and secondary metabolic pathways and adjustment in physiological behaviour of Withania somnifera under drought stress.

In general medicinal plants grown under water limiting conditions show much higher concentrations of secondary metabolites in comparison to control plants. In the present study, Withania somnifera plants were subjected to water stress and data related to drought tolerance phenomenon was collected and a putative mechanistic concept considering growth responses, physiological behaviour, and metabolite content and gene expression aspects is presented. Drought induced metabolic and physiological responses as well as drastic decrease in CO2 uptake due to stomatal limitations. As a result, the consumption of reduction equivalents (NADPH2+) for CO2 assimilation via the calvin cycle declines significantly resulting in the generation of a large oxidative stress and an oversupply of antioxidant enzymes. Drought also results in the shifting of metabolic processes towards biosynthetic activities that consume reduction equivalents. Thus, biosynthesis of reduced compounds (isoprenoids, phenols and alkaloids) is enhanced. The dynamics of various metabolites have been discussed in the light of gene expression analysis of control and drought treated leaves. Gene encoding enzymes of pathways leading to glucose, fructose and fructan production, conversion of triose phosphates to hexoses and hexose phosphorylation were up-regulated in the drought stressed leaves. The down-regulated Calvin cycle genes were co-ordinately regulated with the down-regulation of chloroplast triosephosphate/phosphate translocator, cytoplasmic fructose-1,6-bisphosphate aldolase and fructose bisphosphatase. Expression of gene encoding Squalene Synthase (SQS) was highly upregulated under drought stress which is responsible for the diversion of carbon flux towards withanolides biosynthesis from isoprenoid pathway.

Analyzing trichomes and spatio-temporal expression of a cysteine protease gene Mucunain in Mucuna pruriens L. (DC).

Mucuna pruriens is a well-known legume for the itching attributes of the trichome and a valuable medicinal herb that is used for the treatment of Parkinson's disease, sexual debilities, etc. Its cultivation was deprived due to its itching behavior. The wild genotype of M. pruriens have the largest trichome length (2015 ± 29 µm) compared to other genotype and mutants. The white-seeded variety of M. pruriens was found to be the most suitable for large-scale cultivation due to the small trichome size and less trichome density on the pod. The external surface trichomes have protuberance with unknown function. The unicellular trichomes of Mucuna show the flowing fluid or cytoplasm inside the trichome. The unigenes regulating the differentiation and development of the trichome such as GLABRA-1, GLABRA-2, and cpr-5 have been identified in M. pruriens transcriptome of the leaf. The Mucunain shows a higher transcript abundance in the flower and pod cover compared to the seeds. The Mucunain was found in every stage of plant growth, but it was highly expressed during maturity (about 170 days) with a high fragment per kilobase per million value.

Chromosomal Fragmentation: A Possible Marker for the Selection of High Gymnemic Acid Yielding Accessions of Gymnema sylvestre R. Br.

BACKGROUND: Gymnema sylvestre R. Br. a member of family Asclepiadaceae as mentioned in Indian Pharmacopoeia popular among the researchers because of stimulatory effect of its phytoconstituent on pancreatic cells and potential to treat Type I and II type of diabetes. OBJECTIVES: Development of cost-effective marker system for the selection of high gymnemic acid yielding accessions of G. sylvestre. MATERIALS AND METHODS: Presoaked seeds of Brassica campestris treated with different dilutions of gymnemagenin and 10% leaf extract of twenty different accessions of G. sylvestre. Root tips of germinated seeds were fixed, and chromosomal studies were made by root tip bioassay method. RESULTS: Exposure of seeds to treatment solutions promotes various types of chromosomal anomalies in root meristem, and surprisingly, direct correlation between the percentage of chromosomal fragmentation and the percentage of gymnemic acid shared by treatment solution were observed. CONCLUSION: Later finding may be explored for the development of a novel methodology or marker system for the selection of high active principle yielding accessions of G. sylvestre. SUMMARY: An experiment was carried out using root tip bioassay method for the study of effect of different dilutions of standard gymnemic acid and 10% leaf extract of twenty different accessions of Gymnema sylvestre on root tip meristem of Brassica campestris. Various types of chromosomal anomalies were observed. Of which, percentage of chromosomal fragmentation was showed a direct (∞) relationship with the percentage of gymnemic acid shared by treatment solution. This interesting result after more and more exploration and revalidation could be utilized for the development of a novel methodology for the selection of high active principle yielding accessions of G. sylvestre. Abbreviations used: MI: Mitotic index; CP: Condensed prophase; CM: Clumped metaphase; MC: Metaphase cleft; FR: Fragmentation; AP: Anaphase with persistent nucleolous; LA: Laggard, BR: Bridge; BI: Bi-nucleated cell; DA: Disturbed anaphasic polarity.

Inducing mutations through γ-irradiation in seeds of Mucuna pruriens for developing high L-DOPA-yielding genotypes.

PURPOSE: To develop elite genotypes in Mucuna pruriens (L.) DC with high L-DOPA (L-3, 4 dihydroxyphenylalanine) yields, with non-itching characteristics and better adaptability by applying γ-irradiation. Molecular and chemical analysis was performed for screening based on specific characteristics desired for developing suitable genotypes. MATERIALS AND METHODS: Developed, mutant populations were analyzed for L-DOPA % in seeds through TLC (thin layer chromatography), and the results obtained were validated with the HPLC (High performance liquid chromatography). The DNA (Deoxyribonucleic acid) was isolated from the leaf at the initial stage and used for DNA polymorphism. RNA (Ribonucleic acid) was isolated from the leaf during maturity and used for expression analysis. RESULTS: The selected mutant T-I-7 showed 5.7% L-DOPA content compared to 3.18% of parent CIM-Ajar. The total polymorphism obtained was 57% with the molecular marker analysis. The gene expression analysis showed higher fold change expression of the dopadecarboxylase gene (DDC) in control compared to selected mutants (T-I-7, T-II-23, T-IV-9, T-VI-1). CONCLUSION: DNA polymorphism was used for the screening of mutants for efficient screening at an early stage. TLC was found suitable for the large-scale comparative chemical analysis of L-DOPA. The expression profile of DDC clearly demonstrated the higher yields of L-DOPA in selected mutants developed by γ-irradiation in the seeds of the control.

Genetic elaborations of glandular and non-glandular trichomes in Mentha arvensis genotypes: assessing genotypic and phenotypic correlations along with gene expressions.

Mentha arvensis (corn mint) is well known for the production of menthol, a widely used commodity in pharma and flavoring industries and provides natural fragrances and products. Glandular trichomes are specialized hairs found on the aerial surface of vascular plants species producing specific secondary metabolite chemistry. Correlations were established among trichomes, oil yield, and major secondary metabolites. Nine improved, elite cultivars representing different M. arvensis genotypes were used for analysis. Phenotypic coefficient of variation (PCV) and genotypic coefficient of variation (GCV) were estimated; results indicated the presence of considerable amount of genetic variability, thereby emphasizing wide scope of selection. Positive and significant associations were found among glandular trichomes, oil yield, essential oil constituents, and leaf morphology itself, whereas morphological parameters of leaf show positive and negative correlations to average number of trichome and essential oil constituents. Average number of glandular, non-glandular trichomes, their ratios, menthol content, and trichome number showed a good heritability. Trichomes were studied microscopically in leaf parts in all varieties for analyzing their distribution pattern. The trichome number variations showed significant correlation throughout the genotypes with essential oil yield and monoterpenoid constituents. Differential changes were analyzed for Glutathione S-transferases, Glutathione reductase, Malondialdehyde, phenolics, and chlorophyll content. Gene expressions were analyzed for biosynthesis genes and selected transcription factors TRANSPARENT TESTA GLABRA 1 (TTG1), ENOLASE 1, GLABRA 3, GTL 1, NUCLEAR TRANSCRIPTION FACTOR Y SUBUNIT B-6, WRKY transcription factor 22, putative WRKY 33, WRKY 17, WRKY 1, and WRKY 65-like for harnessing their relation with trichome development in M. arvensis genotypes.

Genetic and Chemical Profiling of Gymnema sylvestre Accessions from Central India: Its Implication for Quality Control and Therapeutic Potential of Plant.

BACKGROUND: Gymnema sylvestre, a vulnerable plant species, is mentioned in Indian Pharmacopeia as an antidiabetic drug. OBJECTIVE: Study of genetic and chemical diversity and its implications in accessions of G. sylvestre. MATERIALS AND METHODS: Fourteen accessions of G. sylvestre collected from Central India and assessment of their genetic and chemical diversity were carried out using ISSR (inter simple sequence repeat) and HPLC (high performance liquid chromatography) fingerprinting methods. RESULTS: Among the screened 40 ISSR primers, 15 were found polymorphic and collectively produced nine unique accession-specific bands. The maximum and minimum numbers of amplicones were noted for ISSR-15 and ISSR-11, respectively. The ISSR -11 and ISSR-13 revealed 100% polymorphism. HPLC chromatograms showed that accessions possess the secondary metabolites of mid-polarity with considerable variability. Unknown peaks with retention time 2.63, 3.41, 23.83, 24.50, and 44.67 were found universal type. Comparative hierarchical clustering analysis based on foresaid fingerprints indicates that both techniques have equal potential to discriminate accessions according to percentage gymnemic acid in their leaf tissue. Second approach was noted more efficiently for separation of accessions according to their agro-climatic/collection site. CONCLUSION: Highly polymorphic ISSRs could be utilized as molecular probes for further selection of high gymnemic acid yielding accessions. Observed accession specific bands may be used as a descriptor for plant accessions protection and converted into sequence tagged sites markers. Identified five universal type peaks could be helpful in identification of G. sylvestre-based various herbal preparations. SUMMARY: Nine accession specific unique bandsFive marker peaks for G. sylvestre.Suitability of genetic and chemical fingerprinting Abbreviations used: HPLC: High Performance Liquid Chromatography, ISSR: Inter Simple Sequence Repeats, CTAB: Cetyl Trimethylammonium Bromide, DNTP: Deoxynucleotide Triphosphates.

A cold-tolerant evergreen interspecific hybrid of Ocimum kilimandscharicum and Ocimum basilicum: analyzing trichomes and molecular variations.

Ocimum (Lamiaceae) is an important source of essential oils and aroma chemicals especially eugenol, methyl eugenol, linalool, methyl chavicol etc. An elite evergreen hybrid has been developed from Ocimum kilimandscharicum and Ocimum basilicum, which demonstrated adaptive behavior towards cold stress. A comparative molecular analysis has been done through RAPD, AFLP, and ISSR among O. basilicum and O. kilimandscharicum and their evergreen cold-tolerant hybrid. The RAPD and AFLP analyses demonstrated similar results, i.e., the hybrid of O. basilicum and O. kilimandscharicum shares the same cluster with O. kilimandscharicum, while O. basilicum behaves as an outgroup, whereas in ISSR analysis, the hybrid genotype grouped in the same cluster with O. basilicum. Ocimum genotypes were analyzed and compared for their trichome density. There were distinct differences on morphology, distribution, and structure between the two kinds of trichomes, i.e., glandular and non-glandular. Glandular trichomes contain essential oils, polyphenols, flavonoids, and acid polysaccharides. Hair-like trichomes, i.e., non-glandular trichomes, help in keeping the frost away from the living surface cells. O. basilicum showed less number of non-glandular trichomes on leaves compared to O. kilimandscharicum and the evergreen cold-tolerant hybrid. Trichomes were analyzed in O. kilimandscharicum, O. basilicum, and their hybrid. An increased proline content at the biochemical level represents a higher potential to survive in a stress condition like cold stress. In our analysis, the proline content is quite higher in tolerant variety O. kilimandscharicum, low in susceptible variety O. basilicum, and intermediate in the hybrid. Gene expression analysis was done in O. basilicum, O. kilimandscharicum and their hybrid for TTG1, GTL1, and STICHEL gene locus which regulates trichome development and its formation and transcription factors WRKY and MPS involved in the regulation of plant responses to freezing and cold. The analysis showed that O. kilimandscharicum and the hybrid were very close to each other but O. basilicum was more distinct in all respects. The overexpression of the WRKY coding gene showed high expression in the hybrid as compared to O. kilimandscharicum and O. basilicum and the transcription factor microspore-specific (MPS) promoter has also shown overexpression in the hybrid for its response against cold stress. The developed evergreen interspecific hybrid may thus provide a base to various industries which are dependent upon the bioactive constituents of Ocimum species.

Analysis of differentially expressed genes in abiotic stress response and their role in signal transduction pathways.

The study of abiotic stress response of plants is important because they have to cope with environmental changes to survive. The plant genomes have evolved to meet environmental challenges. Salt, temperature, and drought are the main abiotic stresses. The tolerance and response to stress vary differently in plants. The idea was to analyze the genes showing differential expression under abiotic stresses. There are many pathways connecting the perception of external stimuli to cellular responses. In plants, these pathways play an important role in the transduction of abiotic stresses. In the present study, the gene expression data have been analyzed for their involvement in different steps of signaling pathways. The conserved genes were analyzed for their role in each pathway. The functional annotations of these genes and their response under abiotic stresses in other plant species were also studied. The enzymes of signal pathways, showing similarity with conserved genes, were analyzed for their role in different abiotic stresses. Our findings will help to understand the expression of genes in response to various abiotic stresses. These genes may be used to study the response of different abiotic stresses in other plant species and the molecular basis of stress tolerance.

AFLP marking and polymorphism among progenies of Gymnema sylvestre: an important medicinal plant of India.

The level of polymorphism among twelve selected progenies of Gymnema sylvestre was investigated through AFLP markers by multiplexing PCR reactions using 64 (8x8) primer combinations. Fourteen primer combinations were selected as the most suitable combination for G. sylvestre. Analysis of the 12 progenies with these 14 primer pairs produced 1689 fragments of which 972 (57.5%) were polymorphic and 485 (28.7%) were unique to a particular genotype. The number of fragments produced by individual primer pairs was in the range of 55 to 225. Out of these, polymorphic fragments were in the range of 34 (E-ACC/M-CAC) to 157 (E-AGG/M-CAG) and unique bands observed were 8 (E-ACC / M-CAC) to 69 (E-AGG/M-CAC). Different primer combinations detected different levels of polymorphism, ranging from 33% (E-AGG/ M-CAC) to 69.8% (E-AGG/ M-CAC). From the observations, it appears that the primer combinations E-AGG/M-CAC, E-AGG/CTG, E-AGG/CAG and E-ACA/CAT were the most informative for the detection of polymorphism among the progenies compared with others, since they produced a high number of unique fragments. The similarity coefficient ranged from 0.212 to 0.731. High similarity was observed between progeny S8 and S9 (73%) and high divergence between progenies S3 and S11. Among the selected progeny, S9 was found to be the most similar to the parent (63%), while genotype S11 was the most distant (36.9%).