Estimation of genetic diversity and population genetic structure in Gymnema sylvestre (Retz.) R. Br. ex Schult. populations using DAMD and ISSR markers.

BACKGROUND: Gymnema sylvestre (Retz.) R. Br. ex Schult. is a well-known medicinal plant against diabetes in India. There is as such no organized cultivation in India, and the plant is still being collected from the wild for their therapeutic uses. It is, therefore, important to estimate the genetic diversity and population genetic structure of G. sylvestre to ascertain the genetically diverse germplasm. The present study, therefore, was undertaken to analyze the genetic variability in 118 accessions belonging to 11 wild populations of G. sylvestre using directed amplification of minisatellite-region DNA (DAMD) and inter simple sequence repeats (ISSR). RESULTS: The present genetic analyses of 11 populations with 25 markers (8 DAMD and 17 ISSR) revealed significant genetic diversity (H = 0.26, I = 0.40, PPL = 80.89%) at a species level, while the average genetic diversity at the population level was low. Among the 11 populations studied, PCH and UTK populations showed maximum genetic diversity, followed by KNR and AMB, while TEL population revealed the lowest genetic diversity. AMOVA and Gst values (0.18) revealed that most of the genetic variations are found within populations and very less among populations, and higher gene flow (Nm = 2.29) was found to be responsible for the genetic homogenization of the populations. The clustering pattern resulting from the UPGMA dendrogram was in congruence with STRUCTURE and PCoA, segregating all the 11 populations into two main genetic clusters: cluster I (populations of North and Central India) and cluster II (populations of South India). The clustering patterns obtained from all three statistical methods indicate that the genetic structure in G. sylvestre populations corresponds to the geographical diversity of the populations and represents a strong genetic structure. CONCLUSION: The genetically diverse populations identified during the present study could be a potential genetic resource for further prospecting and conserving this important plant resource.

Characterization of Gladiolus Germplasm Using Morphological, Physiological, and Molecular Markers.

Estimation of variability and genetic relationships among breeding materials is one of the important strategies in crop improvement programs. Morphological (plant height, spike length, a number of florets/spike), physiological (chlorophyll content, chlorophyll fluorescence, and rapid light curve parameters) and Directed amplification of minisatellite DNA (DAMD) markers were used to investigate the relationships among 50 Gladiolus cultivars. Cluster analysis based on morphological data, physiological characteristics, molecular markers, and cumulative data discriminated all cultivars into seven, five, seven, and six clusters in the unweighted pair-group method using arithmetic mean (UPGMA) dendrogram, respectively. The results of the principal coordinate analysis (PCoA) also supported UPGMA clustering. Variations among the Gladiolus cultivars at phenotypic level could be due to the changes in physiology, environmental conditions, and genetic variability. DAMD analysis using 10 primers produced 120 polymorphic bands with 80% polymorphism showing polymorphic information content (PIC = 0.28), Marker index (MI = 3.37), Nei's gene diversity (h = 0.267), and Shannon's information index (I = 0.407). Plant height showed a positive significant correlation with Spike length and Number of florets/spike (r = 0.729, p < 0.001 and r = 0.448, p = 0.001 respectively). Whereas, Spike length showed positive significant correlation with Number of florets/spike (r = 0.688, p < 0.001) and Chlorophyll content showed positive significant correlation with Electron transport rate (r = 0.863, p < 0.001). Based on significant morphological variations, high physiological performance, high genetic variability, and genetic distances between cultivars, we have been able to identify diverse cultivars of Gladiolus that could be the potential source as breeding material for further genetic improvement in this ornamental crop.

Isolation, Characterization and Anticancer Potential of Cytotoxic Triterpenes from Betula utilis Bark.

Betula utilis, also known as Himalayan silver birch has been used as a traditional medicine for many health ailments like inflammatation, HIV, renal and bladder disorders as well as many cancers from ages. Here, we performed bio-guided fractionation of Betula utilis Bark (BUB), in which it was extracted in methanol and fractionated with hexane, ethyl acetate, chloroform, n-butanol and water. All six fractions were evaluated for their in-vitro anticancer activity in nine different cancer cell lines and ethyl acetate fraction was found to be one of the most potent fractions in terms of inducing cytotoxic activity against various cancer cell lines. By utilizing column chromatography, six triterpenes namely betulin, betulinic acid, lupeol, ursolic acid (UA), oleanolic acid and ß-amyrin have been isolated from the ethyl acetate extract of BUB and structures of these compounds were unraveled by spectroscopic methods. ß-amyrin and UA were isolated for the first time from Betula utilis. Isolated triterpenes were tested for in-vitro cytotoxic activity against six different cancer cell lines where UA was found to be selective for breast cancer cells over non-tumorigenic breast epithelial cells (MCF 10A). Tumor cell selective apoptotic action of UA was mainly attributed due to the activation of extrinsic apoptosis pathway via up regulation of DR4, DR5 and PARP cleavage in MCF-7 cells over non-tumorigenic MCF-10A cells. Moreover, UA mediated intracellular ROS generation and mitochondrial membrane potential disruption also play a key role for its anti cancer effect. UA also inhibits breast cancer migration. Altogether, we discovered novel source of UA having potent tumor cell specific cytotoxic property, indicating its therapeutic potential against breast cancer.

Composition and in vitro cytotoxic activities of essential oil of Hedychium spicatum from different geographical regions of western Himalaya by principal components analysis.

The rhizome of Hedychium spicatum has been widely used in traditional medicines. The present study deals with the evaluation of the cytotoxic potential of rhizome essential oils from four different regions of the Western Himalaya (India) along with comparative correlation analysis to characterise the bioactive cytotoxic component. The essential oils were coded as MHS-1, MHS-2, MHS-3 and MHS-4, and characterised using GC-FID and GC-MS. The main volatile compounds identified were 1,8-cineol, eudesmol, cubenol, spathulenol and α-cadinol. In vitro cytotoxic activities were assessed against human cancer cell lines such as, the lung (A549), colon (DLD-1, SW 620), breast (MCF-7, MDA-MB-231), head and neck (FaDu), and cervix (HeLa). MHS-4 is significantly active in comparison to other samples against all cancer cell lines. Sample MHS-4 has major proportion of monoterpene alcohol mainly 1,8-cineol. Principal components analysis was performed for the experimental results and all four samples were clustered according to their percentage inhibition at different doses.

Assessment of genetic diversity in indigenous turmeric (Curcuma longa) germplasm from India using molecular markers.

Curcuma longa L., commonly known as turmeric, is one of the economically and medicinally important plant species. It is predominantly cultivated in the tropical and sub tropical countries. India is the largest producer, and exporter of turmeric in the world, followed by China, Indonesia, Bangladesh and Thailand. In the present study, Directed Amplification of Minisatellite DNA (DAMD) and Inter Simple Sequence Repeats (ISSR), methods were used to estimate the genetic variability in indigenous turmeric germplasm. Cumulative data analysis for DAMD (15) and ISSR (13) markers resulted into 478 fragments, out of which 392 fragments were polymorphic, revealing 82 % polymorphism across the turmeric genotypes. Wide range of pairwise genetic distances (0.03-0.59) across the genotypes revealed that these genotypes are genetically quite diverse. The UPGMA dendrogram generated using cumulative data showed significant relationships amongst the genotypes. All 29 genotypes studied grouped into two clusters irrespective of their geographical affiliations with 100 % bootstrap value except few genotypes, suggesting considerable diversity amongst the genotypes. These results suggested that the current collection of turmeric genotypes preserve the vast majority of natural variations. The results further demonstrate the efficiency and reliability of DAMD and ISSR markers in determining the genetic diversity and relationships among the indigenous turmeric germplasm. DAMD and ISSR profiling have identified diverse turmeric genotypes, which could be further utilized in various genetic improvement programmes including conventional as well as marker assisted breeding towards development of new and desirable turmeric genotypes.

Physiological and genetic effects of chromium (+VI) on toxitolerant lichen species, Pyxine cocoes.

Chromium is a highly toxic non-essential metal, which causes a variety of metabolic activities in plants. Pyxine cocoes a well known toxitolerant lichen species was considered to evaluate the possible physiological, biochemical, and genetic changes that occur due to chromium Cr (+VI) stress. The physiological (chlorophyll a, chlorophyll b, total chlorophyll, carotenoid, protein, and Fv/Fm) and genetic (ISSR-PCR and ITS) parameters were used to estimate the changes in P. cocoes. Different concentrations of Cr (+VI) (0, 10, 25, 50, 75, and 100 µM) for 10, 20, 30, and 45 days were employed on transplanted lichen species. The results revealed that the exposure of Cr (+VI) for 10, 20, 30, and 45 days under controlled conditions caused a significant decline in physiological processes with increasing metal stress. Amino acid profile at different concentrations on the 45th day too indicated prevailing stress condition as proline content significantly increased at 100 µM concentration. Inter-simple sequence repeat (ISSR) and internal transcribed spacer (ITS) techniques were used to evaluate the genotoxicity induced by chromium stress. ISSR profiles showed a consistent increase in appearance and disappearance of bands with increasing concentration of the chromium. ISSR technique, therefore, is more sensitive and reproducible to study polymorphism induced by environmental stress. The present study revealed that the physiological and genetic changes induced by the Cr (+VI) can be used as a tool to study environmental stress and polymorphisms due to genotoxicity. To the best of our knowledge, application of ISSR-PCR and ITS sequences in toxitolerant species (P. cocoes) appears to be the maiden attempt to evaluate the genotoxicity.

ISSR and DAMD markers revealed high genetic variability within Flavoparmelia caperata in Western Himalaya (India).

Flavoparmelia caperata (L.) Hale is medicinally very important and possesses antifungal and antibacterial activities. F. caperata is the only species found in India. Inter simple sequence repeat (ISSR) and Directed amplification of minisatellite DNA (DAMD) methods were used to analyze the genetic variability within F. caperata from the Western Himalayan region of India. Eleven ISSR and 10 DAMD primers produced 139 and 117 polymorphic bands, and detected 91.44 and 82.34 % polymorphisms, respectively. Cumulative band data generated for ISSR and DAMD markers resulted in 86.86 % polymorphism across all the accessions of F. caperata. The average Polymorphic information content (PIC) value obtained with ISSR, DAMD, and cumulative band data were 0.28, 0.27, and 0.27, respectively. The clustering of the F. caperata accessions in the UPGMA dendrogram showed that these accessions are intermingled with each other in different subclusters irrespective of their geographical affiliations. The pattern of genetic variations within F. caperata accessions could be due to free exchange of spores that might have taken place among these accessions in the wild. ISSR and DAMD markers efficiently and reliably resulted in discrete banding patterns and polymorphic profiles. These markers despite targeting different regions of genome, revealed almost similar levels of polymorphism across all the accessions. The wide range of genetic distance and high level of polymorphism detected by ISSR and DAMD reflected a high genetic variability among the different accessions of F. caperata.

Molecular and chemical profiling of 'sweet flag' (Acorus calamus L.) germplasm from India.

In the present study, molecular (DAMD and ISSR) and chemical (α and ß-asarone contents) markers were used to characterize the A. calamus genotypes procured from different parts of India. The cumulative analysis carried out for both DAMD and ISSR markers revealed 24.71 % polymorphism across all genotypes of A. calamus. The clustering patterns of the genotypes in the UPGMA tree showed that the genotypes are diverse, and did not show any specific correlation with their geographical provenances, reflecting the low level of genetic diversity and a high genetic differentiation among the genotypes from the same localities. All the 27 genotypes of A. calamus were also analyzed for α and ß-asarone contents, and percentage of essential oil. The genotype (Ac13) from Kullu (Himachal Pradesh) showed maximum (9.5 %) percentage of oil, whereas corresponding minimum (2.8 %) was obtained from the genotypes from Pangthang (Sikkim). Similarly, the highest α and ß-asarone contents (16.82 % and 92.12 %) were obtained from genotypes from Renuka (Himachal Pradesh) and Udhampur (Jammu & Kashmir), while lowest α and ß-asarone contents (0.83 % and 65.96 %) resulted from Auranwa (Uttar Pradesh) and Pangthang (Sikkim) genotypes, respectively. A. calamus harbours tremendous economic value, and it is therefore, important to identify the genotypes with low α and ß-asarone contents for its commercial utilization. Further, this study will help in evaluation and documentation of a large number of diverse genotypes for their value traits.

Genetic relationships among wild and cultivated accessions of curry leaf plant (Murraya koenigii (L.) Spreng.), as revealed by DNA fingerprinting methods.

Murraya koenigii (L.) Spreng. (Rutaceae), is an aromatic plant and much valued for its flavor, nutritive and medicinal properties. In this study, three DNA fingerprinting methods viz., random amplification of polymorphic DNA (RAPD), directed amplification of minisatellite DNA (DAMD), and inter-simple sequence repeat (ISSR), were used to unravel the genetic variability and relationships across 92 wild and cultivated M. koenigii accessions. A total of 310, 102, and 184, DNA fragments were amplified using 20 RAPD, 5 DAMD, and 13 ISSR primers, revealing 95.80, 96.07, and 96.73% polymorphism, respectively, across all accessions. The average polymorphic information content value obtained with RAPD, DAMD, and ISSR markers was 0.244, 0.250, and 0.281, respectively. The UPGMA tree, based on Jaccard's similarity coefficient generated from the cumulative (RAPD, DAMD, and ISSR) band data showed two distinct clusters, clearly separating wild and cultivated accessions in the dendrogram. Percentage polymorphism, gene diversity (H), and Shannon information index (I) estimates were higher in cultivated accessions compared to wild accessions. The overall high level of polymorphism and varied range of genetic distances revealed a wide genetic base in M. koenigii accessions. The study suggests that RAPD, DAMD, and ISSR markers are highly useful to unravel the genetic variability in wild and cultivated accessions of M. koenigii.

Genetic diversity in inter-simple sequence repeat profiles across natural populations of Indian pomegranate (Punica granatum L.).

Pomegranate (Punica granatum L.), in the monogeneric family Punicaceae, is found in Iran, Afghanistan, India and Mediterranean countries. Iran is considered to be its primary centre of origin. In India, pomegranate occurs naturally only in the Western Himalayan regions of Jammu and Kashmir, Himachal Pradesh and Uttarakhand States. However, there is no information about genetic variation in wild pomegranate at population level. In this paper, we describe genetic diversity across natural populations of Indian pomegranate based on inter-simple sequence repeat (ISSR) markers. Forty-nine accessions representing eight populations from two regions were analysed using ISSR. Seventeen ISSR primers resulted in 268 polymorphic bands, with 87.01% polymorphism throughout the accessions. Pair-wise population genetic distances ranged from 0.05 to 0.45, with a mean of 0.25 between populations. amova and Nei's genetic diversity analyses revealed higher genetic variation within populations than among populations. A higher genetic differentiation (G(ST)) was observed between the spatially distant populations, indicating a low level of genetic exchange (Nm) among these populations. However, clustering of populations was not in accordance with their geographical affiliations in the tree. The results indicate that the ISSR method is sufficiently informative and powerful to assess genetic variability in pomegranate, and that patterns of genetic variability observed among populations of wild pomegranate from the Western Himalaya differ. Estimation of genetic variation reported here provides a significant insight for in situ conservation and exploitation of genetic resources for this economically important species as potential breeding material.

SPAR profiles and genetic diversity amongst pomegranate (Punica granatum L.) genotypes.

We are interested in studying the distribution and range of diversity amongst the pomegranates in India. Single Primer Amplification Reaction (SPAR) profiling using Random Amplified Polymorphic DNA (RAPD) and Directed Amplification of Minisatellite DNA (DAMD) methods enabled the determination of the genetic diversity amongst a total of 64 Indian pomegranate genotypes including 15 wild, 34 semi-wild and 14 cultivated types. SPAR profile data were scored for the computation of pairwise distances as well as a Neighbour Joining (NJ) tree of all the genotypes. Eight RAPD and four DAMD primers showed discrete polymorphic patterns amongst these genotypes. From the profiles obtained with all the 12 primers considered together, 259 bands were scored. The NJ tree generated after a 1000 bootstrap test using Jaccard coefficient showed separation of Lagerstroemia speciosa used as the out-group taxon, while the pomegranate genotypes were resolved into distinct genetic lineages such that all the cultivated (except CBd70), and wild genotypes (except W101) clearly separated from other genotypes in distinct sub clusters while the semi-wild genotypes were resolved into three sub-clusters. The greatest and least distances detected between genotypes were 0.94 and 0.12, 0.97 and 0.24 and 0.95 and 0.38, amongst the cultivated, semi-wild and the wild genotypes respectively. The results indicate the high levels of genetic diversity present amongst the genotypes. Significantly, the wild genotypes also have a reasonably good range of diversity. A good germplasm collection, especially including the wild genotypes will enable a better pomegranate improvement program. Both SPAR methods, RAPD and DAMD, are found to be useful for studying the genetic diversity of pomegranate.

Kinetics of humoral immune response in pigs vaccinated against foot and mouth disease.

The present investigation was conducted to study the foot and mouth disease virus (FMDV)-specific humoral immune response (HIR) in pigs, following vaccination with oil adjuvanted foot and mouth disease (FMD) vaccine, upto 90 days post vaccination (dpv). For this, 40 Large White Yorkshire (LWY) pigs (20; one-year old female (gilts) and 20; three-month old piglets) were vaccinated @ 2 ml/animal, subcutaneously. Sera samples were collected at fortnight interval from all the animals. The log10 SN50 antibody titres against all the serotypes (Type O, A and Asia-1) were detected in both gilts and piglets from day 7 to 90 dpv indicating the persistence of HIR up to the last day of sampling. The maximum antibody titres were observed on 28 dpv, thereafter, titres started declining, but were present till 90 dpv against all the three FMDV serotypes. HIR was more pronounced in piglets in comparison to gilts, as group mean SN antibody titres against all the three FMDV serotypes were found to be more maintained and significantly higher in piglets.

Long-term survival of allogeneic donor cell-derived corneal epithelium in limbal deficient rabbits.

PURPOSE: To investigate the capability of cultivated allogeneic epithelial stem cells to restore a functional ocular surface in a limbal deficient cornea; to verify the long term survival of epithelial allograft; and to examine the host immune response to heterologous cell transplant in a rabbit model. METHODS: Limbal deficiency was established by performing limbectomy on rabbits (n = 100). Corneal epithelial stem cells were obtained from the limbus and replicated in vitro without a supporting layer. The cell (3 x 10(5)) suspension was then transplanted via topical application as eye drops. Animals were divided into allograft, autograft, and control groups. Females were used as recipients and males as donors for the allograft. Corneas were collected at 7, 14, 21, 40 days as well as 2, 3, 7 and 8 months after cell transplantation. Experimental corneas were evaluated by histology, immunofluorescence, immunohistochemistry and Y chromosome analysis. RESULTS: A well-differentiated corneal epithelium was recognized at 14 to 40 days after cell transfer overlying an infiltrated corneal stroma. Corneal re-epitheliazation was confirmed in 31 of 36 allograft corneas. No significant immune rejection was noted. Stromal abnormality caused by previous limbal deficiency was mostly resolved three months after the regeneration of corneal epithelium. CONCLUSIONS: Transplanted corneal epithelial stem cells were able to differentiate into normal corneal epithelium in vivo without the use of membrane scaffolding. This non-autologous donor cell-derived corneal epithelium survived up to 8 months without immunosuppression and was able to reverse the stromal scarring. Thus, cultivated epithelial stem cells have great potential as an alternative to multiple-surgical procedures in the treatment of limbal deficiency states.