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Open Access
QTL mapping for yield traits in vegetable cowpea
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2017) Ashwin Varghese, V; KAU; Deepu, Mathew
Cowpea [Vigna unguiculata (L.) Walp.] is one of the most cultivated pulse crops in the semi-arid tropics of Asia, Africa, Southern Europe, and other parts of the world. It is used for both vegetable and fodder purpose. In India, kharif crop of vegetable cowpea is cultivated in an estimated area of 0.5 million hectares in states like Kerala, Karnataka, Tamil Nadu and Madhya Pradesh. Studies aimed at increased yield among crops were always challenged by the quantitative nature of traits. These quantitative traits are generally governed by multiple genes present in regions of the genome called quantitative trait loci (QTL). With the advent of molecular markers it is possible to localize the QTL with the help of linked markers, a process now widely known as QTL mapping. QTL mapping depicts the relative positioning of different markers on the chromosomes and their linkage to a specific trait. In cowpea, even though there has been few mapping efforts for traits such as resistance to Thrips tabaci and Frankliniella schultzei, flowering time, pod length and seed weight, an elaborate QTL map for yield and related traits is missing. Hence, the study “QTL mapping for yield traits in vegetable cowpea” was undertaken with the objective of mapping the SSR markers and identifying the quantitative trait loci for yield components in the genome of vegetable cowpea at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, during February 2016 to June 2017. F3 plants maintained at CPBMB, derived from the cross of Sharika which is a pole type, long poded, high yielding but anthracnose and cowpea mosaic virus susceptible cultivar with Kanakamony which is a semi-trailing, medium-long poded, low yielding, anthracnose immune and cow pea mosaic virus resistant cultivar, were used to raise the F4 mapping population. Morphological observation for traits pod length, individual pod weight (IPW), pod number, days taken for first flowering (DTFF), total dry pod yield (TDPY), grains per pod, branch number, root length, plant height, plant weight, and response to anthracnose and cowpea mosaic virus diseases were recorded. High quality DNA was isolated from the parents and mapping population using the protocol standardized in this study. One hundred SSR primer pairs reported in cowpea were screened among the parental DNA for polymorphism. Thirty polymorphic primer sets were carried forward to genotype the F4 mapping population. The morphological and genotypic data were used to construct a linkage map using software ICIMapping. Two linkage groups, one having eight SSR markers distributed across 637 cM and another one having five SSR markers distributed across 271 cM were obtained. Two approaches, Single Marker Analysis (SMA) and Inclusive Composite Interval Mapping (ICIM) otherwise called Additive Linkage Mapping were followed for QTL mapping. LOD value threshold of 3.0 was used to determine the significance of QTL and linked markers. Multiple QTL hotspots were observed for different traits under study. An anchored marker, CLM0083 has been identified which was significantly linked to traits individual pod weight and total dry pod yield. The region between 25 cM to 125 cM on linkage group 1 had QTL hotspots harboring genes governing traits DTFF, TDPY, root length, plant length and plant height. This entire region was bracketed by two markers, CLM0244 at 24.25 cM and CLM0177 at 126.86 cM with an anchored marker CLM0008. This marker combination could be potentially used in marker assisted selection for the traits DTFF, TDPY, root length, plant length and plant height. Fine mapping of the QTL for these traits with large number of markers would provide more insights into the genes and hot spots involved in the yield contributing traits in cowpea.
Open Access
Development of an in vitro regeneration system and validation of genetic stability in phalaenopsis hybrid winter spot with molecular marker
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Asha Amal, Raj; KAU; Lissamma, Joseph
Phalaenopsis “Moth Orchids” are among the most beautiful flowers in the world. This genus has economic value for pot plant and cut flower production and is distributed throughout Southeast Asia. Most popular method of propagation for orchid is through in vitro propagation, as it produces large number of clones in relatively short duration. Despite its potential to produce numerous plants from a single leaf segment, it is liable to unpredictable mutations or somaclonal variation during the process of multiplication. Variation can arise due to many reasons such as type of media, plant growth regulators and its concentration, type of explants and number of subculture cycles. The percentage of the variation can range from 0-100% depending on varieties with an average of 10% among Phalaenopsis (Tokuhara and Mii, 1993). So the present investigation on “Development of an in vitro regeneration system and validation of genetic stability in Phalaenopsis hybrid Winter Spot with molecular marker” was taken up at the Center for Plant Biotechology and Molecular Biology, College of Horticulture from 2013-2016. Flowering mother plants of Phalaenopsis hybrid Winter Spot were used as explant source. Among the explants namely inflorescence node, transverse thin cell layer of leaf and root segments used for tissue culture study in this orchid, inflorescence node was the best with respect to culture response. The best surface sterilization treatment for leaf explants identified was treatment 0.1% bavistin + prill 2 drops (30 min) and 0.1 per cent HgCl 2 ( 8 min) which give maximum per cent of culture survival and minimum contamination rate. The best surface sterilization treatment for inflorescence node identified was treatment with 0.1% bavistin + 2 drops prill (30 min) , one minute dip in 70 per cent ethanol and 0.1% HgCl 2 (7 min).From different basal media (full MS and 1⁄2 MS) tried, response was observed only in the medium of Full MS for inflorescence node. Among the different growth regulators tried, MS medium supplemented with BA and TDZ was found to give good shoot regeneration from inflorescence node explants. MS +2mgl -1 TDZ recorded highest percentage (80%) of culture establishment, followed by MS + 4.5 mgl -1 of BA (55%) per cent of sprouting. Among the explants tried, only inflorescence node responded with sprouting. Root segment remained as such without any change, whereas leaf explants remained green up to 2 weeks, thereafter started drying in all the growth regulators combination. For induction of multiple shoot, MS medium supplemented with 4.5 mgl -1 BA resulted in the highest average number of multiple shoot (4.15). Elongation and rooting was observed in MS medium supplemented with BA 4.5mgl-1 +IAA 1mgl -1 with 80 % rooting. Root initials were observed 50 days after inoculation. The potting media, charcoal, brick pieces and sphagnum moss in the ratio of 1:1:1 was found ideal for hardening of Phalaenopsis hybrid winter spot with 100% survival. Genetic stability studies using RAPD marker were carried out with the mother plants along with three regenerants each. Six primers were selected based on DNA amplification pattern. In RAPD assay, M1 mother plant recorded the highest average polymorphism of 19.7% and M3 mother plant recorded the least average polymorphism of 8.18%. Using NTSYS software, the similarity coefficients for first, second and third plant between M1 mother plant, M2 mother plant and M3 mother plant and corresponding regenerants were 0.91, 0.92 and 0.93 respectively. In fourth plant, the similarity coefficient exhibited 100% similarity between mother plant, the first clone C1 and third clone C3. The established micropropagation protocol can be used with suitable modification for large scale production of other Phalaenopsis varieties.
Open Access
Molecular characterization of katte mosaic virus of cardamom ( Elettaria cardmomum Manton )
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture,Vellanikkara, 2016) Abida, P S; KAU; Manglam Arya
Cardamom ( Elettaria cardamomum Maton) considered as “Queen of Spices” belongs to the family Zingiberaceae. Due to its aroma, cardamom is one among the most expensive spices in the world. Cardamom is infected by several fungal, bacterial and viral diseases. Katte or Cardamom mosaic disease is the most destructive viral disease affecting cardamom plantations worldwide. The disease is caused by Katte mosaic virus or Cardamom mosaic virus (CdMV) and spreads through infected suckers or through aphid Pentalonia nigronervosa Coq. The loss in yield due to katte disease ranges from 38 to 69 per cent and when infection occurs at early stage, the loss is complete. Management of katte disease totally depends on the use of disease free planting materials. As the infected plant often remains symptomless, identification and diagnosis of the virus becomes difficult at early stages. The present study was undertaken to develop serological and PCR based methods for identification and characterization of CdMV of cardamom. The infected and healthy samples were collected from four locations of Idukki and two locations of Wayanad districts of Kerala. The viral protein was isolated and purified from infected samples and SDS-PAGE analysis of purified protein from infected plants revealed the presence of 37 kDa band of viral coat protein. The purified protein from the infected plants was further used as antigen for the production of polyclonal antibody against CdMV in 6-9 month old rabbit. The rabbit was immunized with 5 mg of purified protein. The blood sample from the immunized rabbit was collected and the antibody was purified. The ODD (Ouchterlony Double Diffusion) assay was performed to standardize the titre of the antibody and results had shown that antigen-antibody complex was formed in 1:10, 1:100 and 1:150 dilutions of primary antibody. The indirect ELISA was carried out with 1:10, 1:100 and 1:150 dilutions of primary antibody and 1:200 dilution of secondary antibody. It was found that virus was easily detected in the crude extract of infected leaves with 1:100 dilution of primary antibody. Indirect ELISA was also performed for investigating the serological relationship of Katte mosaic virus with other viruses. The result of indirect ELISA revealed that the crude sap of infected cardamom leaves cross reacted with the antibody specific for Banana Bract Mosaic Virus (BBrMV) which also belongs to same potyvirus group. For the detection of CdMV through RT-PCR, the total RNA was isolated from the infected and healthy plants using Trizol and converted to cDNA. A total of 11 primers were designed for the amplification of coat protein gene of the virus using Primer 3 software. The primers designed were used for synthesis of second strand of cDNA and presence of virus was detected. Out of 11 primers, 8 primers were able to amplify the coat protein gene of the virus in infected plants. The band size of 250, 650, 750 and 950 base pairs were observed in infected plant but not in healthy plant samples. The viral amplicons of 250, 950, 750, 650 base pairs were generated with primers CP-2, CP-9, CP-10 and CP-11 respectively. These amplicons were further eluted, reamplified and sequenced. The nucleotide sequence annotated using bioinformatics tools BLASTn and BLASTx. The result of BLASTn showed 90 to 100 per cent nucleotide sequence similarity with CdMV whereas; the result of BLASTx revealed that the sequence had 55-100 per cent similarity with the coat protein of CdMV. The phylogenetic analysis was performed using 950 bp products generated with CP-9 primer. The phylogenetic tree was developed using MEGA.7 software by utilizing neibhourhood joining method. The result of phylogenetic analysis revealed that the isolates of Ambalavayal, Meppadi and Myladumpara are closely related and also related to isolates of Irettyar and Paravalam whereas, Pampadumpara isolate showed more variation to the above isolates. The methods developed in the present study are useful in detecting the Katte mosaic virus or Cardamom mosaic virus in the infected leaf samples of cardamom. The method will be useful in virus indexing, quarantine management in germplasm exchange, germplasm management, supply of disease free planting materials to the farmers and also for selecting the resistant line or cultivar for large scale production or incorporation in breeding programme for crop improvement.
Open Access
Identification of duplicates in the germplasm of sweet potato (Ipomoea batatas (L.) Lam.) using morphological and molecular markers
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2017) Babitha Babu; KAU; Shirly Raichal, Anil
The study entitled “Identification of duplicates in the germplasm of sweet potato (Ipomoea batatas (L.) Lam.) using morphological and molecular markers” was carried out at the Division of Crop Improvement, ICAR-Central Tuber Crops Research Institute, Sreekariyam, Thiruvananthapuram during 2016-2017. The objective of the study was to identify duplicates in the sweet potato germplasm based on morphological and molecular markers. Identification and elimination of these common redundant materials will enhance the germplasm viability. Fifty accessions were selected for the study. The study was divided into two phases - morphological and molecular analysis. Morphological analysis was performed by using twenty descriptors as provided by IPGRI (CIP et. al., 1991). The recorded data were analyzed statistically by various tools such as PCA and cluster dendrogram. Cluster dendrogram identified three sets of morphological duplicates and the accessions were separated into six principal clusters and two outliers at a Euclidean distance of 1. The PCA analysis revealed predominant vine colour and secondary vine colour, abaxial vein pigmentation and petiole pigmentation as the major factors that contributed to the clustering of the sweet potato accessions. After morphological analysis, molecular analysis was performed. The genomic DNA was isolated using CTAB method which gave good quality DNA. 11 ISSR primers were used for screening of fifty accessions. After the final PCR using selected primers, the product was resolved in 2% agarose and polymorphic bands were obtained. All the primers showed 100% polymorphism and the number of bands ranged from 9 to 18 with a mean value of 14.7 bands per primer. Using the molecular scoring data, UPGMA clustering was done and the whole fifty accessions were divided mainly into two principal clusters and one outlier. The first principal cluster comprised of 40 accessions which were grouped into many subclusters and there was lot of intraclusteral variation. The second principal cluster consisted of 9 accessions and this principal cluster comprised of two true duplicates which were also found similar in morphological characterization. The outlier was different from all the other accessions and may be due to the peculiar leaf shape which is not seen in other accessions selected in the study. SD-29 was different from all the remaining accessions by a similarity coefficient of 0.61.The similarity between the different accessions ranged between 52-100%. The duplicates S-236 and S-256 were 100% similar. The least similar accessions were SD-39 and S-298 (52%). Thus it can be inferred that a 48% variability or diversity existed within the selected accessions which can be considered as a moderate diversity. The hexaploid nature of the crop, self incompatibility, along with the out crossing nature together might have contributed to the high variation observed among the accessions. Only two duplicates were identified. In future more specific markers may be used for core collection development and to eliminate duplicates.
Open Access
Identification and characterization of Suppressor of Overexpression of Constans1 (SOC1) gene in Black Pepper (Piper nigrum L.)
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2016) Manu, K Venu; KAU; Lekha Sreekantan
The present study entitled“Identification and characterization of Suppressor of Overexpression of Constans1 gene in Black Pepper (Piper nigrum L.)”was conducted at the Integrated Biotechnology Block, College of Agriculture, Vellayani, during 2015-2016.The study envisagedisolation and sequencing ofSOC1, a flowering integrator gene in black pepper (variety - Karimunda) and functional characterization of the gene by studying the expression patterns. Degenerate primers were designed for the above said gene based on the gene sequences from NCBI database (SOC1 forward and reverse primers) which were used to isolate and identify the gene. Total RNA of black pepper was isolated using modified CTAB method followed by synthesis of cDNA using AMV RT (Avian myeloblastosis virus reverse transcriptase). PCR (Polymerase chain reaction) with degenerate primers was done using cDNA as the template. However no amplifications were observed after the first reactions. Therefore nested PCR reactions were done using the PCR products of the first reaction as the template. Two bands of size 640 bp and 330 bp were produced in the nested reactions. Sequencing of the product yielded four sequences with each of the sequence showing similarity to the SOC1 gene, when done sequence analysis, thus making it the first flowering integrator gene to be identified in black pepper. Microscopy studies were carried out to see the floral characters of black pepper in detail. Microscopy studies were done using FAA fluid as fixative, sectioning the tissues and staining with safranin and fast green were carried out to see the changes occurring in different development stages of spikes from immature spike to complete spike with berries.
Open Access
Characterization and validation of microsatelite markers for resistance to vascular streak dieback disease in cocoa (Theobroma cacao L.)
(Centre for Plant Biotechnolgy and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Waghmare Sandesh, Tulshiram; KAU; Deepu Mathew
Cocoa is the third important plantation crop next to coffee and tea. The global production and consumption of cocoa is 27.00 lakh MT. Among the fungal diseases, Vascular Streak Dieback (VSD) caused by Ceratobasidium theobromae is the main constraint in cocoa growing countries, causing heavy losses in mature trees as well as seedlings. The VSD disease cannot be effectively controlled by chemicals and hence breeding for the development of resistant varieties is the best strategy to tackle the disease. In order to confirm the transfer of a desired gene into the offspring, conventional breeding methods rely on the field screening which will be highly influenced by the environmental factors. Marker assisted selection is an alternate where the tightly linked molecular markers will be employed to confirm the presence of the gene of interest in the selected plants. Five ISSR and one SSR markers linked to VSD resistance were identified at Kerala Agricultural University (Chandrakant, 2014). The present study was undertaken with the objective of validating the identified SSR and ISSR markers and to characterize the ISSR markers to identify the corresponding SSR markers. For validation and characterization, twenty VSD resistant hybrids and four susceptible clones were used. For molecular analyses, good quality genomic DNA was isolated from twenty four genotypes and ISSR markers UBC 811, UBC 815, UBC 826, UBC 857, UBC 866 and SSR marker mTcCIR 42 were screened. ISSR analysis had shown that all the primers are capable to differentiate resistant and susceptible genotypes. The SSR assay has also differentiated the resistant and susceptible genotypes. The distinct markers generated in resistant genotypes using UBC 811, UBC 826 and UBC 857 were eluted, cloned to pGEMT vector and sequenced. The nucleotide sequences were annotated using BLAST, ORF finder and SSR finder. The BLASTn of UBC811A and UBC811D nucleotide sequence have shown that this resistance locus lie in the chromosome V of Theobroma cacao genome. BLASTn of UBC826A, UBC826B and UBC857 has positioned these loci in chromosome III. ORF1 and ORF3 in UBC811D are shown to code for aflatoxin biosynthesis regulatory protein and NAD(P)H dehydrogenase quinine, respectively. ORF1 in UBC826B and ORF5 in UBC857-2 code for potassium transporter 27 (0sHAK-27) and structural polyprotein precursor of VP2, capsid protein VP2, respectively. All these proteins are identified to have definite roles in defence pathways. The frequency and distribution of SSR motifs, dimmers to decamers, in these ISSR markers and the corresponding primers were identified. The reported ISSR and SSR markers were validated and found to be successful in differentiating resistant and susceptible genotypes of cocoa; thereby these markers can be used in marker assisted breeding for VSD resistance.
Open Access
Molecular cloning and characterisation of coat protein gene of banana bract mosaic virus
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Darshan Gowda, M R; KAU; Anita Cherian, K
Banana (Musa spp.), identified as ‘tropical treasure’ is grown extensively in the tropical and sub tropical regions of the world. Diseases, especially those caused by viruses are major constraints for the profitable cultivation of banana. Among the viral diseases, banana bract mosaic is one of the most important, which leads to an yield reduction ranging from 52 to 75 per cent. This disease is caused by Banana bract mosaic virus (BBrMV) which is a member of genus Potyvirus and family Potyviridae. In case of any viral disease, early diagnosis is very important since symptomless hosts carry the viral inoculum. Development of molecular clones of viral genome has immense application in the field of disease diagnostics and management. Hence, the present study was carried out with the objective to develop molecular clones of coat protein (CP) gene of BBrMV and to characterize it.The infected suckers were collected from Banana Research Station (BRS), Kannara and maintained in the insect proof net house of Department of Plant Pathology. The symptoms developed on different plant parts under natural field conditions were documented which included longitudinal, irregular, reddish streaks of varying sizes on the base of pseudostem, mosaic pattern on bracts, fan like orientation of leaves, spindle shaped lesions on leaves, reduced bunch size and malformed fingers.The serodiagnostic technique namely, Direct Antigen Coating-Enzyme linked immuno sorbent assay (DAC-ELISA) was validated by determining the antibody titre with different dilutions of primary antibody viz., 1:100, 1:200, 1:300, 1:500 and it was found that BBrMV could be best detected at 1:200 dilution along with 1:500 dilution of secondary antibody. Later, the presence of virus particles in the samples were confirmed by DAC-ELISA using the standardized combination of primary and secondary antibody dilutions. Dot Immuno Binding Assay (DIBA) was validated to detect BBrMV and showed positive reaction for infected leaf sample which was detected by the development of purple coloured spots on nitrocellulose membrane.The genome of BBrMV is RNA and hence, molecular detection of virus was standardized by Reverse Transcription- PCR (RT-PCR). Total RNA was isolated by two different protocols using different reagents. Among the two methods, the one with Ambion Purelink RNA Reagent was the most appropriate for RNA isolation from banana since it provided highest quality and quantity of RNA compared to the protocol with TRIzol reagent. The isolated RNA was converted into complementary DNA (cDNA) using First Strand cDNA synthesis kit. RTPCR amplification of coat protein gene was standardized using gene specific reported primer (B1/B2) and designed primer (BCPF1/R1) which yielded amplicons of approximate size, 605 bp and 850 bp respectively. The molecular cloning of CP gene was done in Escherichia coli (DH5- alpha). The presence of gene insert in transformed colonies were confirmed by colony PCR using plasmid specific primer (T7 and SP6) which yielded amplicons of expected band size of 1150 bp. The amplified colony PCR products were sequenced to obtain CP gene sequence of BBrMV. The characterization of cloned CP gene of BBrMV was carried out by in silico analysis. The blast analysis revealed that the CP gene sequence of the virus showed maximum homology of 99 per cent to KER2 isolate from Kasargod, Kerala (Accession no. KF385491). The sequence exhibited significant nucleotide identity (99 to 96 per cent) and amino acid identity (95 to 83 per cent) with other nucleotide and protein sequences of BBrMV available in the database of Genbank. The phylogenetic analysis by the alignment of CP gene sequences of selected 22 isolates also revealed that the present isolate was more similar to KER2 isolate and the Indian isolates did not show any relationship based on geographical origin.The recombinant clones developed in the present study could be applied in serodiagnosis and genetic engineering. This could be also used as disease diagnostic probes for more sensitive molecular diagnostic techniques like Nucleic acid spot hybridization.
Open Access
Association mapping for cassava mosaic disease (cmd) resistance in cassava using SSR marker
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2017) Aparna, T K; KAU; Mohan, C
The present study” Association mapping for Cassava Mosaic Disease (CMD) resistance in cassava using SSR marker” Was conducted to identify the marker which are closely associated with CMD resistance in cassava. 30 CMD resistant and 25 CMD susceptible cassava varieties were analyzed with 2n selected SSR primers. Genomic DNA isolated from the leaves and PCR amplification was done with SSR primers. PCR products were separated by PAGE. Polymorphic bands were used to assign loci for each primer and score as presence (1) or absence (0) of bands. RMEI primer showed double banding pattern in 700bp size for all the resistant accessions. But in case of susceptible accessions only single bands were observed. TASSEL and STRUCTURE software were used ro analyses the data obtained from the PAGE.. Cladogram was constructed using genotypic data. Different clusters were formed. Clusters clearly distinguished CMD resistant and CMD susceptible varieties of cassava. CMD resistant varieties sub divided into three sub clusters. Dissimilarity matrix constructed by the software used for the diversity study of collected cassava accessions. Two markers were associated with CMD resistance was identified by the software. SSR36 are the two markers associated with CMD resistance. Result showed that there are eight sub populations in the cassava population used. Based upon the output obtained from Evanno’s method a bar diagram was constructed. Which shows the distribution of genotypes in different sub populations.
Open Access
Scientific validation of antiinflammatory, antinociceptive and antioxidant potential of malavirinji (Actinodaphne bourdillonii Gamble)
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2017) Adarsh Prathap; KAU; Suja, S R
The study entitled “Scientific validation of antiinflammatory, antinociceptive and antioxidant potential of malavirinji (Actinodaphne bourdillonii Gamble)” was conducted at the Ethnomedicine and Ethnopharmacology Division of Jawaharlal Nehru Tropical Botanical Garden (JNTBGRI), Palode, Thiruvananthapuram, during the year 2016 to 2017. Objective of the study was to scientifically evaluate the antiinflammatory, antinociceptive, antioxidant potential of leaves of an ethnomedicinal plant Actinodaphne bourdillonii Gamble. Phytochemical examination revealed the presence of various phytoconstituents like alkaloids, flavonoids, saponins, alkaloids, carbohydrates and phenols. In in vitro antioxidant method the ethanolic extracts of leaf showed higher free radical scavenging activity compared to standards with IC50 of DPPH, NO Scavenging Activity, Hydroxyl Free Radical Scavenging Activity, Superoxide radical scavenging activity, Total antioxidant activity and Ferric reducing antioxidant potential (FRAP) assay. Toxicity of the ethanolic extract of leaves of A. bourdillonii were tested by acute toxicity study in mice with four doses 25, 100, 400, 1600 mg/kg body weight. And the mice were cage side observed for fourteen days and no toxic effect were seen in the tested animals. In the detailed in vitro pharmacological studies for antiinflammatory and antinociception were conducted. Antiinflammatory activity was determined by Carrageenan (Acute inflammation) and formalin (sub chronic inflammation) induced paw oedema on hind limb in rats with three different doses 50, 150 and 450 mg/kg. At the dose of EAB 450mg/kg give 83.63% of inhibition in carrageenan induced paw oedema and in the formalin induced paw oedema (sub-acute) study a dose of EAB 150mg/kg gives maximum inhibition of 70.23% and 78.36% of inhibition in the first and seventh day respectively. Antinociception activity was determined by Eddy Hot Plate method and Acetic acid writhing method in mice with three different doses 50, 150 and 450 mg/kg. At the dose of EAB 450mg/kg gives maximum inhibition 87.76% of inhibition in Eddy’s hot plate method and in the acetic acid induce writhing study a dose of EAB 450mg/kg gives maximum inhibition of 71.55% of inhibition.

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