In vitro Propagation of Endemic Species Mahonia Jaunsarensis Ahrendt Through Callus Culture.

Mahonia jaunsarensis Ahrendt (Family Berberidaceae) an endemic species was successfully propagated in vitro. An efficient propagation protocol has been developed first time. The callus cultures were established from leaf explants on Murashige and Skoog (MS) medium supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D; 1 µM) and resulted 70% callus induction with green compact callus. When callus was transferred to MS medium containing Thidiazuron (TDZ; 0.75 µM), maximum average number of shoot (3.06) produced but shoot length (3.37 cm) and average leaf number (2.87) was increased upon transfer to MS medium containing N6-benzylaminopurine (BA; 6.0 µM) plus α-naphthalene acetic acid (NAA; 0.5 µM). In MS medium containing indole-3-butyric acid (IBA; 0.01 µM), the maximum rooting percentage (56%) and average root number (2.56) per shoot and root length (3.33 cm) were recorded. The rooted plantlets transferred in vermiculite + garden soil + farmyard manure (1:1:1) with maximum (55%) survival percentage under greenhouse condition. The phytochemical analysis of leaves obtained from tissue culture-raised plants revealed significantly higher levels of alkaloids (berberine and palmatine) than those obtained from wild plants. Similar trends were observed for antioxidant and antimutagenic activities. Results of this study offer a baseline for the conservation and sustainable utilization strategies for M. jaunsarensis.

Elevation determines the productivity of large cardamom (Amomum subulatum Roxb.) cultivars in Sikkim Himalaya.

Large cardamom (Amomum subulatum Roxb.) is an economically important cash crop that provides a livelihood option for the rural communities in Sikkim Himalaya. However, its production has declined drastically over the past few decades due to climate change and other factors affecting the livelihood of marginal cardamom-dependent farmers in the region. Climate change causes a shift in elevational distributions of mountain species, and it is pivotal to understand its effect on yield and yield-related traits for economically important plant species like large cardamom. For this, we randomly studied 41 large cardamom cultivation sites in Sikkim (India) with elevations ranging between 975 and 2069 m asl and evaluated the yield-related traits (number of capsules per spike, capsule length, capsule width, fresh capsule weight, dry capsule weight, number of seeds per locule, fresh seed weight, and dry seed weight) in five cultivars (Dzongu Golsey, Sawney, Seremna, Ramsey, and Varlangey). We observed a significant variability (P < 0.05) for morphometric traits in each of the five cultivars cultivated in different elevations. The cultivation of low-elevation cultivars like Seremna and Dzongu Golsey (suitable in elevation < 975 m) has shifted upward to mid (975-1515 m) and high-elevation (> 1515 m), while cultivation of high-elevation Ramsey cultivar (suitable in elevation > 1515 m) has shifted downward (< 1515 m). The Dzongu Golsey, Sawney, and Seremna cultivated in mid-elevation (975-1515 m) performed better in terms of yield-related traits than the same cultivars cultivated in high-elevation (> 1515 m) and showed moderate to high negative correlation between elevation and yield-related traits, indicating the negative effect of elevation on their yield. Likewise, Ramsey and Varlangey cultivated in high elevation (> 1515 m) performed better than the one cultivated in mid-elevation (975-1515 m) and depicted moderate to high positive correlation between elevation and yield-related traits, suggesting a positive influence of elevation on their yield. Although there is an elevational shift in the cultivation of large cardamom cultivars, the elevation influences the performance of the large cardamom cultivars, and it also suggests cultivating the cultivars in their suitable elevation range for better productivity.

Maling bamboo (Yushania maling) overdominance alters forest structure and composition in Khangchendzonga landscape, Eastern Himalaya.

The Khangchendzonga Landscape (KL), a part of 'Himalayan Biodiversity Hotspot', is known for its unique biodiversity assemblage. In recent years, the KL is experiencing threats to biodiversity due to the biological overdominance of native Maling bamboo (Yushania maling). In the present study, we investigated the impacts of the overdominance of Y. maling on the forest composition of Singalila National Park (SNP), Eastern Himalaya, India. Elevational habitats 2400 to 3400 m asl were sampled by laying 69 (10 m × 10 m) forest plots including 51 bamboo plots and 18 non-bamboo plots. Bamboo plots showed significantly (p < 0.05) low species richness and density in both shrub and herb layers which further manifested the low seedling density. Generalized Additive Model (GAM) estimated a significant (p < 0.0001) decline in species richness and density with increasing bamboo density in SNP. Our study projects the overdominance of Y. maling has a significant negative impact on forest structure and composition. Therefore, management of invasiveness of Y. maling is essential through its optimized removal from the protected areas and utilization in making handicrafts, paper industries etc. to create ecological and economic benefits. Further long-term studies assessing the impacts of Y. maling overdominance on forest ecosystems and soil dynamics are recommended.

Predicting the distributional range shifts of Rhizocarpon geographicum (L.) DC. in Indian Himalayan Region under future climate scenarios.

Himalaya, the highest mountain system in the world and house of important biodiversity hotspot, is sensitive to projected warming by climate change. Rhizocarpon geographicum (map lichen), a crustose lichen, grows in high mountain ranges, is a potential indicator species of climate change. In the present study, MaxEnt species distribution modeling algorithm was used to predict the suitable habitat for R. geographicum in current and future climate scenarios. Nineteen bioclimatic variables from WorldClim database, along with elevation, were used to predict the current distribution and three representative concentration pathway (RCP) scenarios by integrating three general circulation models (GCMs) for future distribution of species covering years 2050 and 2070. Furthermore, we performed change analysis to identify the precise difference between the current and future distribution of suitable areas of the species for delineating habitat range expansion (gain), habitat contraction (loss), and stable habitats. The final ensemble model obtained had average test value 0.968, and its predicted ~ 27.5% of the geographical area in the Indian Himalayan Region is presently climatically suitable for the species. The predicted highly suitable area for R. geographicum is observed to be declining in Northwestern Himalaya, and it is shifting towards the higher elevation areas of the Eastern Himalaya. The projected distribution in future under the RCP scenarios (RCP 4.5, 6.0, and 8.5) showed the range expansion towards higher elevations, and it is more pronounced for the extreme future scenarios (RCP 8.5) than for the moderate and intermediate climate scenarios (RCP 4.5 and RCP 6.0). However, assuming that species can migrate to previously unoccupied areas, the model forecasts a habitat loss of 10.86-16.51% for R. geographicum, which is expected due to increase in mean annual temperature by 1.5-3.7 °C. The predictive MaxEnt modeling approach for mapping lichen will contribute significantly to the understanding of the impact of climate change in Himalayan ecosystems with wide implications for drawing suitable conservation plans and to take adaptation and mitigation measures.

Optimized extraction of polyphenolic antioxidants from the leaves of Himalayan Oak species.

In this study heat-assisted extraction conditions were optimized to enhance extraction yield of antioxidant polyphenols from leaves of Himalayan Quercus species. In initial experiments, a five-factor Plackett-Burman design including 12 experimental runs was tested against the total polyphenolic content (TP). Amongst, XA: extraction temperature, XC: solvent concentration and XE: sample-to-solvent ratio had shown significant influence on yield. These influential factors were further subject to a three-factor-three-level Box-Wilson Central Composite Design; including 20 experimental runs and 3D response surface methodology plots were used to determine optimum conditions [i.e. XA: (80°C), XC:(87%), XE: (1g/40ml)].This optimized condition was further used in other Quercus species of western Himalaya, India. The High-Performance Liquid Chromatography (HPLC) revealed occurrence of 12 polyphenols in six screened Quercus species with the highest concentration of catechin followed by gallic acid. Amongest, Q. franchetii and Q. serrata shared maximum numbers of polyphenolic antioxidants (8 in each). This optimized extraction condition of Quercus species can be utilized for precise quantification of polyphenols and their use in pharmaceutical industries as a potential substitute of synthetic polyphenols.

Species richness and ß-diversity patterns of macrolichens along elevation gradients across the Himalayan Arc.

Understanding the species richness and ß-diversity patterns along elevation gradients can aid in formulating effective conservation strategies particularly in areas where local anthropogenic stresses and climate change are quite significant as in the Himalaya. Thus, we studied macrolichen richness and ß-diversity along elevational gradients at three sites, namely Kashmir (2200 to 3800 m a.m.s.l), Uttarakhand (2000-3700 m a.m.s.l) and Sikkim (1700 to 4000 m a.m.s.l) which cover much of the Indian Himalayan Arc. In all, 245 macrolichen species belonging to 77 genera and 26 families were collected from the three sites. Only 11 species, 20 genera and 11 families were common among the three transects. Despite the differences in species composition, the dominant functional groups in the three sites were the same: foliose, fruticose and corticolous forms. The hump-shaped elevation pattern in species richness was exhibited by most of the lichen groups, though an inverse hump-shaped pattern was also observed in certain cases. ß-diversity (ßsor) based on all pairs of comparisons along an elevation gradient varied from 0.48 to 0.58 in Kashmir, 0.03 to 0.63 in Uttarakhand and 0.46 to 0.77 in Sikkim. The contribution of turnover to ß-diversity was more than nestedness at all the three transects. Along elevation ß-diversity and its components of turnover and nestedness varied significantly with elevation. While species turnover increased significantly along the elevation in all the three transects, nestedness decreased significantly in Kashmir and Sikkim transects but increased significantly in the Uttarakhand transect. Except for the Kashmir Himalayan elevation transect, stepwise ß-diversity and its components of turnover and nestedness did not vary significantly with elevation. The present study, the first of its kind in the Himalayan region, clearly brings out that macrolichen species richness, ß-diversity, and its components of turnover and nestedness vary along the elevation gradients across the Himalayan Arc. It also highlights that contribution of turnover to ß-diversity is higher in comparison to nestedness at all the three transects. The variations in species richness and diversity along elevation gradients underpin the importance of considering elevational gradients in planning conservation strategies.

Anthocyanins, multi-functional natural products of industrial relevance: Recent biotechnological advances.

Anthocyanins, the color compounds of plants, are known for their wide applications in food, nutraceuticals and cosmetic industry. The biosynthetic pathway of anthocyanins is well established with the identification of potential key regulatory genes, which makes it possible to modulate its production by biotechnological means. Various biotechnological systems, including use of in vitro plant cell or tissue cultures as well as microorganisms have been used for the production of anthocyanins under controlled conditions, however, a wide range of factors affects their production. In addition, metabolic engineering technologies have also used the heterologous production of anthocyanins in recombinant plants and microorganisms. However, these approaches have mostly been tested at the lab- and pilot-scales, while very few up-scaling studies have been undertaken. Various challenges and ways of investigation are proposed here to improve anthocyanin production by using the in vitro plant cell or tissue culture and metabolic engineering of plants and microbial culture systems. All these methods are capable of modulating the production of anthocyanins , which can be further utilized for pharmaceutical, cosmetics and food applications.

Phytopharmacology and Clinical Updates of Berberis Species Against Diabetes and Other Metabolic Diseases.

The incidences of diabetic mellitus and other metabolic diseases such as hypertension and hyperlipidemia are increasing worldwide; however, the current treatment is not able to control the rapidly increasing trend in diabetes mortality and morbidity. Studies related to the effectiveness of extracts and pure compounds obtained from plants have shown promising responses in preclinical and clinical studies related to these metabolic diseases. Plants belonging to the genus Berberis (Family: Berberidaceae) are widely distributed with nearly 550 species worldwide. Extracts and compounds obtained from Berberis species, especially Berberine alkaloid, showed effectiveness in the management of diabetes and other metabolic diseases. Various pharmacological experiments have been performed to evaluate the effects of Berberis extracts, berberine, and its natural and chemically synthesized derivatives against various cell and animal disease models with promising results. Various clinical trials conducted so far also showed preventive effects of Berberis extracts and berberine against metabolic diseases. The present review focuses on i) research updates on traditional uses, ii) phytopharmacology and clinical studies on Berberis species, and iii) active metabolites in the prevention and treatment of diabetes and other metabolic diseases with a detailed mechanism of action. Furthermore, the review critically analyzes current research gaps in the therapeutic use of Berberis species and berberine and provides future recommendations.

Optimized microwave assisted extraction (MAE) of alkaloids and polyphenols from Berberis roots using multiple-component analysis.

Berberis, one of the major sources of berberine and polyphenols, is widely accepted genus for its medicinal properties. The inclusion of these phytochemicals in different health formulations has widened its scope in pharmaceuticals and nutraceuticals. In the present study, multi-component analysis (MCA) has been used to extract these nutraceutical compounds from Berberis jaeschkeana roots under microwave-assisted extraction (MAE) conditions. To determine the optimum extraction condition, different factors, including, microwave power, sample to solvent ratio, irradiation time, solvent pH and solvent concentration were tested under 42 experiments. The MCA includes, Plackett-Burmen and Central Composite Design and analyzes model fitness, regression coefficient (ß), analysis of variance (ANOVA) and 3D response curve. The results showed significant model fitness and involvement of linear, quadratic and interactive effect of different factors. Under optimized MAE condition, [i.e. 1 g of sample extracted through 70 mL of a solution (100% methanol pH 2.0), provided microwave power of 598 W for 2 min of irradiation time], the berberine and palmatine contents were recorded as 46.38 mg g-1 and 20.54 mg g-1 respectively. Under optimized condition, the yield of alkaloids were found closer to the models' predicted value. Similarly, total phenolic content and antioxidant activities were also found closer to the models' predicted value. To test the suitability of the optimized MAE condition for other species i.e., Berberis asiatica, extraction of alkaloids and polyphenolics was conducted and recorded higher yield to the previous records. Moreover, under optimum extraction condition, six and seven polyphenolic compounds from B. jaeschkeana and B. asiatica were quantified respectively. The proposed MAE optimization design using MCA contributes towards faster and greener extraction of alkaloids and polyphenolics with higher yield. Moreover these greener approaches could sustainably utilize species during extract preparation and harnessing its nutraceutical and pharmaceutical potential. This study design could also be replicated on other valuable species or compounds for effective extraction of nutraceutical components and sustainable utilization of natural products.

Trends of polyphenolics and anthocyanins accumulation along ripening stages of wild edible fruits of Indian Himalayan region.

Wild fruits are important food resources that provide health promoting nutraceutical components, which vary with ripening stages. In present study, five wild edible fruits of Indian Himalayan Region i.e., Myrica esculenta, Berberis asiatica, Rubus ellipticus, Pyracantha crenulata and Morus alba were examined for their nutraceutical potential at different ripening stages. The results of present study showed that polyphenolic concentration decreased whereas anthocyanin level increased with fruit ripening, however few species and compounds showed different trends. Among the tested fruit species, unripe fruits of B. asiatica followed by M. esculenta were found to be the best for harvesting polyphenolics (especially catechin), while ripen fruits of M. esculenta followed by B. asiatica were found the best for anthocyanin (cyanin and delphinidin) extraction. The results from this study can be effectively used by the harvesters, consumers, traders and food and nutraceutical industries to harness maximum nutraceutical potential depending on the preferred compounds and ripening stages of these species.

Phenolic compounds, antioxidant capacity and antimutagenic activity in different growth stages of in vitro raised plants of Origanum vulgare L.

Efficient micropropagation procedure was developed for Origanum vulgare, a high-value culinary herb, and the phytochemicals, phenolic content, antioxidant and antimutagenic activity of leaf and stem, derived from different growing stages were analyzed. The agar solidified Murashige and Skoog (MS) medium supplemented with a combination of 6-benzylaminopurine and α-naphthaleneacetic acid was optimized as best shoot-multiplication-medium. Shoots were rooted best on 1/2 strength MS medium supplemented with 50 µM indole-3-butyric acid (IBA). The plantlets were successfully acclimatized ex vitro in a soil, sand and farmyard manure mixture (2:1:1 v/v/v) with 100% survival rate in greenhouse. The total anthocyanin and total phenolic content were observed significantly higher in leaves of in vitro-raised plants. However, total tannin, flavonoid and antioxidant activity remained higher in leaves of mother plant maintained under ployhouse condition. All the plant extracts have shown significant antimutagenic activity except in vitro-growing plants. A total of 13 polyphenolic compounds were detected in different extracts using high performance liquid chromatography. Among these, catechin was detected maximum in in vitro-growing cultures and chlorogenic acid in leaves of mother plant. These findings will help the farmers, medicinal plant growers, and industries for mass multiplication and effective extraction of phytochemicals from O. vulgare.

Timberline structure and woody taxa regeneration towards treeline along latitudinal gradients in Khangchendzonga National Park, Eastern Himalaya.

With main purpose of developing a coherent baseline information of timberline forests of Sikkim, Eastern Himalaya, we investigated, (i) forest structure and composition, (ii) regeneration status of dominant timberline tree species between timberline and treeline, (iii) influence of environmental variables in species composition, and (iv) relationship between environmental variables and ecological attributes. The study was conducted along the timberline stretch of Dzongri landscape nested within the Khangchendzonga National Park (UNESCO's World Heritage Site, 2016), a core zone of Khangchendzonga Biosphere Reserve (in UNESCO WNBRs, 2018), Sikkim, India. We employed quadrat method to sample 9 contiguous sites to capture all possible variations in timberline composition. Transect method was used to study the regeneration of woody taxa between timberline to treeline. In total, 20 woody species belonging to 10 genera and 6 families were recorded. Among these, Abies densa, Rhododendron lanatum and Sorbus microphylla exhibited higher dominance, comprising of 50% of the total importance value index (IVI) weightage. Betula utilis the common treeline species in much of the western and central parts of Himalaya was absent here. Tree density in studied timberline was significantly higher than its western Himalayan counterparts of Indian Himalayan region. Environmental variables viz., elevation, slope, and humus were observed determinants of species composition across the study area. The species dominance correlated negatively (p<0.01; n = 9) with species diversity and richness. We observed an irregular spatial pattern of timberline across the 9 study sites, and the extent of timberline elements (seedling; sapling; live tree or dead tree) ranged between 5.3m to 187.7m higher than the current timberline at different sites. The present trends suggest that upslope advancement is unlikely to occur in near future, while treeline densification is anticipated. Further investigations are suggested to develop a holistic understating of these timberline patterns across the Indian Himalayan region.