Molecular genetic divergence analysis amongst high curcumin lines of Golden Crop (Curcuma longa L.) using SSR marker and use in trait-specific breeding.

Curcuma longa L., is recognized worldwide as a medicinally and economically important plant species due to its curcumin content which is an industrially important compound. In this study, a total of 329 accessions were collected from four states of India and planted in the experimental farm of CSIR-NEIST, Jorhat, India, in augmented design. Among these, 152 high curcumin (> 1.50%) accessions were screened for molecular divergence study using 39 SSR primers. The primers showed the most efficient outcome with 2-8 allele/ loci and a total 163 number of alleles with 100% polymorphism. Cluster analysis revealed the construction of three clusters, out of which one cluster was geographically dependent, and germplasm was particularly from Assam state. Jaccard's pairwise coefficient showed maximum genetic dissimilarity of (0.75) between accession RRLJCL 3 and RRLJCL 126, indicating high variation as it was from two different states viz Arunachal Pradesh and Nagaland respectively and minimum genetic dissimilarity of (0.09) between RRLJCL 58 and RRLJCL 59 indicating significantly less variation as the two accessions were from same state, i.e., Arunachal Pradesh. Analysis of Molecular Variance (AMOVA) revealed high molecular variation within the population (87%) and significantly less variation among the population (13%). Additionally, Neighbour Joining dendrogram, Principal Component Analysis (PCA), and bar plot structure revealed similar clustering of germplasm. This diversity assessment will help in selecting the trait-specific genotypes, crop improvement program, conservation of gene pool, marker-assisted breeding, and quantitative trait loci identification. Moreover, to the best of our knowledge, it is the first molecular diversity report among 152 high curcumin lines of C. longa from North East India using 39 SSR primers.

Estimation of genetic variation in yield, its contributing characters and capsaicin content of Capsicum chinense Jacq. (ghost pepper) germplasm from Northeast India.

Capsicum chinense Jacq. (ghost pepper), a naturally occurring chili species of Northeast India is known throughout the world for its high pungency and a pleasant aroma. The economic importance is due to the high capsaicinoid levels, the main source for pharmaceutical industries. The present study focused on identifying important traits necessary for increasing the yield and pungency of ghost pepper and to determine the parameters for the selection of superior genotypes. A total of 120 genotypes with more than 1.2% capsaicin content (>1,92,000 Scoville Heat Unit, w/w on dry weight basis) collected from different northeast Indian regions were subjected to variability, divergence and correlation studies. Levene's homogeneity test of variance studied for three environments did not show significant deviation and so homogeneity of variance was reasonably met for analysis of variance study. Genotypic and phenotypic coefficient of variation was highest for fruit yield per plant (33.702, 36.200, respectively), followed by number of fruits per plant (29.583, 33.014, respectively) and capsaicin content (25.283, 26.362, respectively). The trait number of fruits per plant had maximum direct contribution to fruit yield per plant and the trait fruit yield per plant towards capsaicin content in the correlation study. High heritability with high genetic advance, which is the most favored selection criteria was observed for fruit yield per plant, number of fruits per plant, capsaicin content, fruit length and fruit girth. The genetic divergence study partitioned the genotypes into 20 clusters, where fruit yield per plant contributed maximum towards total divergence. Principal components analysis (PCA) studied to determine the largest contributor of variation showed 73.48% of the total variability, of which the PC1 and PC2 contributed 34.59% and 16.81% respectively.

Stability estimation through multivariate approach among solasodine-rich lines of Solanum khasianum (C.B. Clarke): an important industrial plant.

Solanum khasianum is a medicinally important plant that is a source of steroidal alkaloids 'solasodine.' It has various industrial applications, including oral contraceptives and other pharmaceutical uses. The present study was based on 186 germplasm of S. khasianum, which were analyzed for the stability of economically important traits like solasodine content and fruit yield. The collected germplasm was planted during Kharif 2018, 2019, and 2020 in RCBD with three replications at the experimental farm of CSIR-NEIST, Jorhat, Assam, India. A multivariate approach for stability analysis was applied to identify stable germplasm of S. khasianum for economically important traits. The germplasm was analyzed for additive main effects and multiplicative interaction (AMMI), GGE biplot, multi-trait stability index, and Shukla's variance which were evaluated for three environments. The AMMI ANOVA revealed significant GE interaction for all the studied traits. The stable and high-yielding germplasm was identified from the AMMI biplot, GGE biplot, Shukla's variance value, and MTSI plot analysis. Lines no. 90, 85, 70, 107, and 62 were identified as highly stable fruit yielders while, lines no. 1, 146, and 68 were identified as stable high solasodine lines. However, considering both traits, i.e., high fruit yield and solasodine content, MTSI analysis was performed which showed that lines 1, 85, 70,155, 71, 114, 65, 86, 62, 116, 32, and 182 could be used in a breeding program. Thus, this identified germplasm can be considered for further varietal development and could be used in a breeding program. The findings of the present study would be beneficial for the S. khasianum breeding program.

Identification and Registration for High-Yielding Strain through ST and MLT of Curcuma caesia Roxb. (Jor Lab KH-2): A High-Value Medicinal Plant.

(1) Background: Curcuma caesia Roxb. is a high valued crop which is extensively used in pharmaceuticals, flavour and fragrances. C. caesia is recognised as an endangered species due to its extensive collection from the wild through human intervention. Therefore, to prevent the species from extinction, it is very necessary to conserve and cultivate this plant species for the sustainable availability of the raw material. (2) Methods: In the present plant breeding programme, a multi-year study was performed for the identification of superior genotypes which will help in conservation. To fulfil this objective, a total of 135 accessions of C. caesia were collected from different regions of India and were set up for experimental selection trial for three years (2016-2018). After proper evaluation of the genotypes based on six agronomical traits, five high-yielding genotypes were identified which underwent multilocation trial for two years (2019 and 2020). The stability analysis using the Eberhart-Russell method, AMMI and GGE biplot were used to study the consistency of the genotypes in varied environments compared with the check variety. (3) Results: Analysis of variance indicated significant genotype and environment interaction for the yield traits, i.e., dry rhizome recovery, rhizome yield and essential oil yield. The coefficient of variation (CV) was highest for tillers per plant (21.76) and lowest for the plant height (4.93). All the results clearly demonstrated Jor Lab KH-2 as the highest yielding and stable genotype in varied environments compared with the check variety and other selected genotypes. (4) Conclusions: This genotype was then submitted to ICAR-NBPGR, New Delhi, for germplasm registration and received its confirmation vide registration number INGR 21159. This genotype will greatly benefit the breeders and will also help in the conservation of this endangered species. This is the first report on the identification and registration of a high-yielding variety of C. caesia.

Anethole rich Clausena heptaphylla (Roxb.) Wight & Arn., essential oil pharmacology and genotoxic efficiencies.

Anethole, a widely used industrial flavoring agent is majorly sourced from anise and star anise. The present study is aimed to the in-depth pharmacological analysis i.e. anti-diabetic, skin whitening, neurodegenerative disorder inhibitory activities of anethole-rich Clausena heptaphylla leaf essential oil (ARCHEO) (88.59%) as revealed by the Gas Chromatography/Mass Spectrometry (GC/MS) analysis and further confirmed by proton nuclear magnetic resonance 1H-NMR as well as to compare with standard compound anethole. ARCHEO (ABTS EC50 6.97 ± 0.004 µg/mL; Protease assay 4.51 ± 0.004 µg/mL) outperformed the standard compound anethole (ABTS EC50 9.48 ± 0.048 µg/mL; Protease assay EC50 22.64 ± 0.016 µg/mL) in antioxidant and anti-inflammatory experiments. ARCHEO was also shown to be more effective than the reference compound anethole in terms of anti-diabetic activity (EC50 22.35 ± 0.121 µg/mL), tyrosinase inhibitory activity (EC50 16.45 ± 0.012 µg/mL), and anti-cholinesterase activity (EC50 22.32 ± 0.016 µg/mL). However, ARCHEO exhibited lower antimicrobial activity towards all the tested microbes compared to standard compound anethole and as for the MIC, ARCHEO was effective only towards Salmonella typhimurium (60 µg/mL), Streptococcus mutans (20 µg/mL), and Aspergillus fumigatus (75 µg/mL). ARCHEO (11.11%) and anethole (12.33%) showed no genotoxic effect based on Allium cepa assay mitotic index value. Thus, ARCHEO could be a commercially viable and widely available cheaper source of anethole, which has buoyant demand in the field of food flavoring, fragrance, and pharmaceutical industries.

Evaluation of Genetic Diversity Based on Microsatellites and Phytochemical Markers of Core Collection of Cymbopogon winterianus Jowitt Germplasm.

Cymbopogon winterianus Jowitt is an industrially important crop due to its value in the aromatic, perfumery and pharmaceutical industries. In this study, 72 accessions of C. winterianus were selected for molecular diversity analysis using SSR markers. It revealed a total of 65 polymorphic alleles showing an average of 68.10% polymorphism. The best SSR primer with competency in discriminating the germplasm was 3CM0506 with PIC (0.69), MI (0.69) and Rp (3.12). Genetic variation was studied between Assam, Manipur, Meghalaya and Arunachal Pradesh populations. A dendrogram based on the Neighbour-Joining Method showed clustering of germplasm on the collection site. A total of six relevant genetic populations were identified through a structure harvester software analysis. Moreover, a dendrogram based on similarity, complete linkage and Euclidean distance was also elucidated differentiating the genotypes with respect to the major phytochemical constituents of the essential oil. GC-FID and GC-MS analyses of the essential oil of the 72 germplasms revealed citronellal content from 2.58-51.45%, citronellol from 0.00-26.39% and geraniol from 0.00-41.15%. This is the first molecular diversity report with 72 accessions of C. winterianus collected from the NE region using 28 SSR primers as well as their diversity based on phytochemical markers. This diversity computation will help with acquisition of the knowledge and relationship among each individual accession leading to the development of improved and essential oil component-rich cultivars.

Induced variations by gamma radiation and EMS on the agronomic traits, essential oil yield with its quality and their exploitation in Java citronella (Cymbopogon winterianus Jowitt).

PURPOSE: The slips of Cymbopogon winterianus (Java citronella) are used to propagate this industrially important crop. It does not have a mating system that allows it to create new variability or develop superior progenies, which could be addressed by using induced mutation caused by physical or chemical agents. Physical treatments, rather than chemical treatments, are believed to be the most incredible alternative for mutation breeding. As a result, this study aims to assess the effects of gamma radiation and EMS treatment on the development of better C. winterianus progenies. MATERIALS AND METHODS: The three superior lines, i.e. JC-62 (IC-0627007), JC-55 (IC-0627000), and JC-71 (IC-0627018) based on high herbage, essential oil yield and citronellal content were selected through clonal selection during three-year evaluation. These were then irradiated with 60Co gamma rays (50 Gy, 100 Gy, 150 Gy, 200 Gy, 250 Gy, and 300 Gy) and treated with EMS dose (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1%) separately. Ten plants of each dose were mutated and planted in the experimental field to obtain the MV2 progenies. After 4 months, the plants were harvested, and the slips were prepared and planted to get the MV3 and subsequently MV4 progenies. All morphological and essential oil data have been recorded and analyzed statistically. The essential oil was extracted through the Clevenger apparatus for all the progenies, followed by GC and GC-MS analysis. RESULTS: The morpho-chemical study of the 112 healthy mutants has led to the identification of six superior progenies in MV4 progenies, M17, M63, M77, M179, M233, and M392. In terms of the economic yield, it was observed that mutant M77 treated with 0.20% EMS dose increased the herbage yield and citronellal content tremendously. In contrast, the progeny M17 irradiated with γ-100Gy increased essential oil yield from 1.32% to 1.50%. The EMS treatment at 0.10% and 0.20% doses showed an increasing trend in plant height, leaf length, tillers/bush, leaves/bush, herbage yield, and citronellal content compared to the base variety. The LD50 value of C. winterianus for gamma radiation and EMS treatment was averaged at 173 Gy and 0.72%, respectively. CONCLUSION: In C. winterianus, both gamma rays and EMS treatment successfully created superior progenies with desired characteristics. The 0.20 percent EMS treatment significantly improved economic features, including herbage yield and citronellal content. Simultaneously, gamma radiation at 100 Gy produced the highest essential oil yield, which is a highly desirable characteristic in C. winterianus breeding.

Use of stability statistics in the selection of Clausena heptaphylla (Roxb.) Wight & Arn for novel anethole rich strain (Jor Lab CH-2).

Introduction: Anethole is an industrially important compound which is extensively used in pharmaceuticals, cosmetics, perfumery, food and confectioneries. Anethole is primarily obtained from fennel, anise, and star anise which is highly expensive. Therefore, a study was performed to identify a cost-effective and natural anethole rich strain of Clausena heptaphylla through selection and confirmed through multilocation trial. Methods: The study was conducted using 23 accessions collected from North eastern region of India from 2014-2018 (initial evaluation trial) and 2018-2022 (multilocation trial). The initial trial was conducted in the experimental farm of CSIR-NEIST, Jorhat, Assam using Complete Randomized Block Design with three replications. Five agronomical traits (plant height, leaf length, leaf width, number of stem branching, herbage yield per plant per cutting) along with essential oil yield and anethole content were evaluated which led to the identification of anethole rich strain of C. heptaphylla. This identified strain was further evaluated along with the two check genotypes for stability based on three yield parameters viz. herbage yield, essential oil yield and anethole content at four multi-locations (Imphal, Jorhat, Runne and Madang) for four years using the same experimental design. Results and discussion: The identified superior line (Jor Lab CH-2) showed consistent performance for the studied yield parameters across all the environments maintaining its superiority. The identified strain exhibited average herbage yield of 1.2 Kg/plant/cutting and essential oil yield of 1.22%. The GC-MS analysis of the essential oil depicted trans anethole as the major constituent (93.25%) followed by estragole (4.85%) while benzene, 1,2-dimethoxy-4-(1-propenyl Isoeugenol methyl ether and cis-anethole were the trace components. This is the first novel report of anethole rich variant of C. heptaphylla which has undergone multilocational trial over the years. Jor Lab CH-2 strain will open a new scope for the industries to isolate anethole from a different source in a cost-effective approach.