The Cardamom Genome (Book)

 Overview

Editors: K. K. Sabu, Chittaranjan Kole

• Written by globally reputed experts on cardamom genetics, genomics, transcriptomics, and breeding
• Is the first one compiling advances in genomics and metabolic profiling of cardamom
• Presents an overview of advanced techniques of biotechnology and genome profiling in cardamom

About this book

This book is the first comprehensive compilation of elucidation of the genome of cardamom (Elettaria cardamomum Maton) and its genetic improvement. It includes reviews on the genetic resources, genetics and breeding and in vitro culture in cardamom and molecular characterization of phylogenetic relationship of cardamom of Columbia and Costa Rica.  This book presents deliberations on the whole genome sequencing and SSR markers of small cardamom. It also enumerates on the genetics and genomics of biotic stress and host-pathogen interaction, genomics of flavors, and transcriptomics resources and their implications and finally functional regulation of mRNAs in cardamom. Altogether, the book contains about 150 pages over 10 chapters authored by globally reputed experts on the relevant field in this crop. The book is useful to the students, teachers, and scientists in the academia and relevant private companies interested in classical genetics and breeding, molecular genetics, structural genomics, functional genomics, plant pathology and plant physiology of cardamom.

Visit book page online at https://link.springer.com/book/10.1007/978-3-031-71829-8

Growth promoting effects of endophytic fungus Piriformospora indica in small cardamom (Elettaria cardamomum Maton)

Piriformospora indica, an endophytic fungus of Sebacinales, colonizes the roots of a wide range of host plants and provides various benefits to the plants. Cardamom (Elettaria cardamomum Maton) is an economically valuable spice crop of the tropics. In this work, we describe differentially expressed transcripts responding to P. indica root colonization in small cardamom for elucidation of molecular basis of growth and development. During the study, a wild genotype of cardamom was propagated under in vitro conditions using full strength Murashige and Skoog medium supplemented with 1 mg l⁻¹ BAP (for shoot induction) and basal MS liquid medium (for root induction). Cardamom plantlets were co-cultivated with P. indica. Microscopic observation confirmed the presence of P. indica inside the roots of cardamom plantlets. Growth parameters of control and P. indica colonized plantlets were observed for three months at an interval of 15 days. P. indica colonization resulted in a significant increase in the morpho-physiological traits of the host plant. The growth enhancement was visible after 15 days of co-culture. There was a significant increase (p < 0.05) in the number and length of leaves, height of the plant and chlorophyll content in P. indica colonized plants compared to non-colonized control plant. In addition to this, the expression levels of auxin, nitrate reductase, vegetative storage protein and phosphate transporter genes were upregulated by 3.45, 3.26, 1.62 and 1.19 times respectively by the co-cultivation of P. indica in cardamom plantlets.

Anjana H. N., K. N. Anith, Sabu K K. 2023. Growth promoting effects of endophytic fungus Piriformospora indica in small cardamom (Elettaria cardamomum Maton). 2023. Symbiosis. DOI: 10.1007/s13199-023-00949-1

Expression profiling of stress responsive genes in cell suspension of Elettaria cardamomum (L.) Maton under abiotic stress

Cardamom is an economically important spice, valued for its multiple utility from culinary to medical purposes. The plant is highly susceptible to both abiotic and biotic stresses. Despite the fact that several studies focusing on stress in cardamom have been conducted; a molecular analysis at the cellular level has not been reported. This study highlights the molecular response of homogenous population of cardamom cell suspension following the temperature and drought stresses for a short period of time (20 mins). Temperature stress at 30, 35, and 40°C caused a significant increase in the transient expression of genes, sHSP 17.8 and sHSP 17.9, which are molecular chaperones involved in protein folding coping with the heat stress response of plants. Drought stress with various concentrations of PEG 6000 has demonstrated only a little increase in the expression of transcription factors, WRKY 35 and WRKY 71. The study implies that sHSP 17.8 and sHSP 17.9 play a crucial role during heat stress, which is a major limiting factor for the cultivation of cardamom in lower altitudes where atmospheric temperature is usually high. But WRKY 35 and WRKY 71 genes are found not to have a high impact at the cellular level in response to drought stress in cardamom when it is exposed to a brief duration of drought. Understanding the molecular mechanism underlying abiotic stress response in cardamom will aid in developing elite varieties adaptable to lower altitudes and to cope with the frequent climatic variations.

Cite this article

Soumya S. Dharan and K.K. Sabu. Expression profiling of stress responsive genes in cell suspension of Elettaria cardamomum (L.) Maton under abiotic stress. Journal of Plantation Crops 2023, 51(1): 23-30. https://doi.org/10.25081/jpc.2023.v51.i1.8188

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Data on Large cardamom transcriptome associated with Chirke disease

Mary Mathew K, Renjanan Reshma, M Geethu, Varghese Rithin, Swapna Sasidharan, P P Gouthaman, K K Sabu, F Nadiya, Noushad Muhammad Ali, Dharan S Soumya, R Prakashkumar, A B Remashree. 2019. Data on Large cardamom transcriptome associated with Chirke disease. Data in Brief 29: 10504. https://doi.org/10.1016/j.dib.2019.105047

Abstract
Large cardamom (Amomum subulatum Roxburg), is an ancient spice native to North-Eastern India and Southeast Asia, which belongs to the family Zingiberaceae under the order Scitaminae. Large cardamom is mostly affected by a viral disease termed Chirke caused by Large Cardamom Chirke Virus (LCCV). These disease has spread due to drastic changes in the ecosystem, inadequate rain in dry months and absence of good agricultural practices by the farmers resulting in aphid infestations. In the present study, using HiSeq™ 2000 RNA sequencing technology transcriptome sequencing was performed for both control (disease not expressed) and diseased large cardamom leaf tissues. RNA-seq generated 77260968 (7.72 GB) and 72239708 (7.22 GB) paired raw reads for large cardamom control and diseased samples respectively. The raw data were submitted to the NCBI SRA database under the accession numbers SRX2529373 and SRX2529372 and the assembled transcriptomes were submitted to TSA under the accession numbers GIAV01000000 and GIAW01000000 for the control and diseased samples respectively. The raw reads were quality trimmed and assembled de novo using TRINITY assembler which created 156822 (control) and 148953 (diseased) contigs with N50 values 2107 (control) and 2182 (diseased). The data were used to identify the significantly differentially expressed genes between control and diseased samples.

Data on small cardamom transcriptome associated with capsule rot disease

Mary Mathew K, Renjanan Reshma, M. Geethu, Varghese Rithin, K.K. Sabu, F. Nadiya, Muhammad Ali Noushad, Soumya S. Dharan, Y.S. Rao, A.B. Remashree. 2019. Data on small cardamom transcriptome associated with capsule rot disease. Data in Brief 27: 104625. https://doi.org/10.1016/j.dib.2019.104625

Abstract
Small cardamom (Elettaria cardamomum (L.) Maton, also known as the ‘Queen of Spices’ is a rhizomatous herbaceous monocot from the family Zingiberaceae. In the present study, using HiSeq™ 2000 RNA sequencing technology, transcriptome sequencing was performed for both control and disease stressed small cardamom leaf tissues. RNA-seq generated 46,931,637 (101 base) and 31,682,496 (101 base) raw reads and totally 9.93GB and 6.63GB of sequence data for cardamom control and stressed samples respectively. The raw data were submitted to NCBI SRA database of under the accession numbers SRX2512359 and SRX2512358 for the control and diseased samples respectively. The raw reads were quality filtered and assembled using TRINITY de novo assembler which created 1,11,495 (control) and 91,096 (diseased) contigs with N50 values 3013 (control) and 2729 (stressed). The data was further used to identify significantly differentially expressed unigenes between control and stressed samples. Assembled unigenes were further annotated and evaluated in silico to predict the function using publicly available databases and gene annotation tools.

Identification and characterization of drought responsive microRNAs and their target genes in cardamom

Anjali, N, Nadiya, F, Thomas, J, and K. K. Sabu. 2018. Identification and characterization of drought responsive microRNAs and their target genes in cardamom (Elettaria cardamomum Maton). Plant Growth Regulation. doi: 10.1007/s10725-018-0462-9 Online First: 2018-12-06

Abstract
Plant miRNAs are found to be present throughout the genome and they regulate gene expression either by cleaving mRNA or inhibiting the translational process at the post transcriptional level. Drought is one of the major limiting factors that negatively affect productivity of plants. Cardamom cultivation is having good production potential but the plants are vulnerable to biotic and abiotic stress factors. To date, nothing is known about the regulatory roles of miRNAs in response to drought stress in cardamom. Ion Torrent sequencing of two small RNA libraries prepared from control (C) and treated (T) plants raised under well irrigated and drought stressed treatments respectively created 3,938,342 and 4,083,181 primary reads. A total of 150 conserved and 20 novel microRNAs were identified from both the control and treated libraries. Discovery of 17 differentially expressed miRNAs under drought stress suggests that these miRNAs might have involved in various biological processes to improve plant tolerance to water stress. Several target genes for drought stress regulating miRNAs were identified including miR156l and miR169c which cleave the target mRNA involved in response to water deprivation. miR530b and miR156a target mRNAs which respond to water deprivation and inhibit the translational process. The expression patterns of some of the miRNAs and their targets were validated by qRT-PCR. This study is the first report of drought responsive miRNAs and their targets in cardamom. The outcome of this research could provide insights into the miRNA based regulatory mechanisms in response to drought stress in monocot plants.

See at Journal website - https://link.springer.com/article/10.1007%2Fs10725-018-0462-9

Nadiya, F., N. Anjali, Jinu Thomas, A. Gangaprasad, K. K. Sabu. 2018. Deep sequencing identified potential miRNAs involved in defense response, stress and plant growth characteristics of wild genotypes of cardamom. Plant Biology. Published online: 11 August 2018 https://doi.org/10.1111/plb.12888

Abstract

• Cardamom has long been used as a food flavouring agent and in ayurvedic medicines for mouth ulcers, digestive problems and even depression. Extensive occurrence of pests and diseases adversely affect its cultivation and result in substantial reductions in total production and productivity. Numerous studies revealed the significant role of miRNAs in plant biotic stress responses.
• In the current study, miRNA profiling of cultivar and wild cardamom genotypes was performed using an Ion Proton sequencer.
• We identified 161 potential miRNAs representing 42 families, including monocot/tissue‐specific and 14 novel miRNAs in both genotypes. Significant differences in miRNA family abundance between the libraries were observed in read frequencies. A total of 19 miRNAs (from known miRNAs) displayed a twofold difference in expression between wild and cultivar genotypes. We found 1168 unique potential targets for 40 known miRNA families in wild and 1025 potential targets for 42 known miRNA families in cultivar genotypes. The differential expression analysis revealed that most miRNAs identified were highly expressed in cultivars and, furthermore, lower expression of miR169 and higher expression of miR529 in wild cardamom proved evidence that wild genotypes have stronger drought stress tolerance and floral development than cultivars.
• Potential targets predicted for the newly identified miRNAs from the miRNA libraries of wild and cultivar cardamom genotypes involved in metabolic and developmental processes and in response to various stimuli. qRT‐PCR confirmed miRNAs were differentially expressed between wild and cultivar genotypes. Furthermore, four target genes were validated experimentally to confirm miRNA–mRNA target pairing using RNA ligase‐mediated 5′ Rapid Amplification of cDNA Ends (5′RLM‐RACE) PCR.

Keywords: Cardamom, microRNA, next generation sequencing, RNA sequencing

Transcriptome mining for EST-INDELs and development of EST-SSR markers in turmeric

Sabu K. K., M. D. Shehenaz and J. Amrutha. 2018. Transcriptome mining for EST-INDELs and development of EST-SSR markers in turmeric (Curcuma longa L.). International Journal of Agriculture, Environment and Biotechnology 11(3): 487-492. http://ndpublisher.in/countpdfdownload.php?id=3040&pdf=IJAEBv11n3i.pdf

ABSTRACT
Curcuma longa L. commonly known as turmeric is used as a culinary spice in India and many Asian countries. Turmeric has anti-fungal, anti-bacterial, anti-malarial and anti-cancer properties to mention a few. We have analyzed the transcriptome of C. longa retrieved from NCBI SRA database (SRR495223) for the development and validation of EST-SSR markers and identification of EST-InDels to be used for C. longa and related genera. A total of 337 primers were developed and 20 primers having the rating of 100% with the help of NetPrimer were selected and used for PCR validation in C. longa, Amomum subulatum Roxb. (large cardamom) and Elettaria cardamomum (L.) Maton (small cardamom). It was found that around 50% primers generated PCR products in both types of cardamom and 85% in C. longa. The developed primers worked with curcuma, large cardamom and small cardamom plants at varying levels. Besides, the transcriptome analysis detected high amount of deletion and 18 addition of bases which could be screened through the development of CAPS markers using the tool SNP2CAPS and it was found that 93 restriction enzyme can be used for screening these InDels.

Highlights

• Transcriptome sequence of Curcuma longa was used for the development of EST-SSR markers.
• The EST-SSR primers were validated in Curcuma longa and its related taxa such as Amomum subulatum and Elettaria cardamomum
• EST-InDels were also successfully identified in the transcriptome sequences of Curcuma longa.

Keywords: Curcuma longa, EST-SSR, EST-InDel, PCR, ClustalW, WebSat, NetPrimer, SNP2CAPS

Agri Gene

Volume 8, June 2018, Pages 18-27

Genome-wide differential expression profiling in wild and cultivar genotypes of cardamom reveals regulation of key pathways in plant growth and development

Author links open overlay panelNadiyaF.a  AnjaliN.a   JinuThomasa  Gangaprasad A.b  SabuK.K.a

https://doi.org/10.1016/j.aggene.2018.03.002

Highlights

•

Transcriptome sequencing data of five cultivars and a wild genotype of cardamom was used to find the differential expression profiling of the cultivar and wild genotypes of cardamom.

•

Differential expression profiling of the cultivar and wild genotypes of cardamom exhibited 132 unigenes as differentially expressed (log2>4) with 105 up regulated and 27 down regulated genes in cultivars comparing with its wild genotype.

•

This work represents the first report on differential expression profiling and functional annotation of wild and cultivar genotypes which will enhance the genomic resources of the species.

Abstract

Cardamom (Elettaria cardamomum Maton) is an economically valuable spice crop and the essential oil of cardamom possesses splendid medicinal properties. To get insight into the domestication related traits and varied gene regulation, differential expression profiling of wild and cultivar cardamom was performed by analyzing the transcriptome data available for cardamom. Functional annotation using seven different publicly available databases identified significant genes coding for enzymes participating in monoterpenoid biosynthesis. Differential expression profile of cultivar and wild genotypes of cardamom exhibited 132 unigenes as differentially expressed (log2 > 4) with 105 up regulated and 27 down regulated genes in cultivars comparing with its wild genotype. Expression analysis showed wild cardamom has increased drought stress tolerance, defense response, and various plant growth regulations. Most of the genes coding for enzymes participating in flavanoid biosynthesis were up regulated, while resistance related genes (cinnamoyl CoA reductase) and few genes involved in the monoterpenoid biosynthetic pathway were down regulated in cultivars when compared to its wild progenitor. Transcriptome data were validated and correlated using qPCR using 10 randomly selected differentially expressed genes. Our study is the first application of next generation RNA-seq to explore the genes involved in various metabolic pathways and quantification of transcript expression levels in cultivar and wild cardamom genotypes.

Keywords

Cardamom, 

Transcriptome, 

Differential expression, 

Up regulation, 

Down regulation, 

Essential oil biosynthesis

Discovery of MicroRNAs in Cardamom (Elettaria cardamomum Maton) under Drought Stress

Dataset Papers in Science
Volume 2017 (2017), Article ID 9507485, 4 pages
https://doi.org/10.1155/2017/9507485

Dataset Paper

Discovery of MicroRNAs in Cardamom (Elettaria cardamomum Maton) under Drought Stress

N. Anjali, F. Nadiya, Jinu Thomas, and K. K. Sabu

Jawaharlal Nehru Tropical Botanic Garden and Research Institute (JNTBGRI), Palode, Thiruvananthapuram 695562, India

Correspondence should be addressed to K. K. Sabu

Received 31 December 2016; Revised 21 March 2017; Accepted 19 April 2017; Published 31 July 2017

Academic Editor: Michael Hackenberg

Copyright © 2017 N. Anjali et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

MicroRNAs (miRNAs) are endogenous, 19–24-nucleotide-long, noncoding RNAs found in animals, plants, and some viruses. Drought is one of the major limiting factors that negatively affect productivity of plants. To date, nothing is known about the regulatory roles of miRNAs in response to drought stress in cardamom. Cardamom collected from the natural forest area in Therakkudi in the Idamalayar forest range was selected for the study. Total RNA was isolated from leaves and stems of control and drought treated cardamom plant, which was used for library construction. Ion torrent sequencing of the two small RNA libraries prepared from plants raised under well irrigated and drought stressed treatments, respectively, created 3,938,342 and 4,083,181 primary reads. A total of 150 conserved and 20 novel microRNAs were identified from both the control and the treated libraries. Discovery of 17 differentially expressed miRNAs under drought stress suggests that these might have been involved in various biological processes to improve plant tolerance to water stress. This study is the first report of drought responsive miRNAs and their targets in cardamom. The outcome of this research could provide insights into the miRNA based regulatory mechanisms in response to drought stress in monocot plants.

Development and Cross-genera Transferability of Ginger EST-SSR Markers for Cardamom

Background: Cardamom (Elettaria cardamomum Maton) is an important commodity spice that comes under the ginger family (Zingiberaceae). Several genetic markers are widely being used to analyze plant genomes. However, genetic mapping of cardamom has never been attempted owing to the lack of sufficient number of high quality genetic markers and other pertinent genome information.

Objective: The goals of the present study were to design SSR markers from the EST sequences of ginger (Zingiber officinale) and to validate the same in cardamom for demonstrating cross generic transferability. Method: 38,116 expressed sequence tags of ginger downloaded from the NCBI dbEST database were used to develop and validate co-dominant, multi-allelic SSR markers.

Results: A total of 1214 SSRs including mono, di, tri, tetra and hexa repeats were identified in the study. The validation through SSR-PCR followed by agarose gel electrophoresis was carried out for the developed markers. Genetic analysis of the SSR markers showed polymorphism and it clearly differentiated wild genotypes from cultivars and wild escapes from the plantations. Large cardamom, Amomum subulatum Roxb. was used as an outgroup and the newly developed EST-SSR markers were amplified well in this species.

Conclusion: The newly developed EST-SSRs could be useful as reproducible markers for cardamom genetic studies.

See https://benthamscience.com/journals/current-bioinformatics/article/147712/

Transcriptome profiling of Elettaria cardamomum (L.) Maton (small cardamom)

Elettaria cardamomum (L.) Maton, known as ‘queen of spices, is a perennial herbaceous monocot of the family Zingiberaceae, native to southern India. Cardamom is an economically valuable spice crop and used widely in culinary and medicinal purposes. In the present study, using Ion Proton RNA sequencing technology, we performed transcriptome sequencing and de novo transcriptome assembly of a wild and five cultivar genotypes of cardamom. RNA-seq generated a total of 22,811,983 (92 base) and 24,889,197 (75 base) raw reads accounting for approximately 8.21GB and 7.65GB of sequence data for wild and cultivar genotypes of cardamom respectively. The raw data was submitted to SRA database of NCBI under the accession numbers SRX1141272 (wild) and SRX1141276 (cultivars). The raw reads were quality filtered and assembled using MIRA assembler resulted with 112,208 and 264,161contigs having N50 value 616 and 664 for wild and cultivar cardamom. The assembled unigenes were functionally annotated using several databases including PlantCyc for pathway annotation. This work represents the first report on cardamom transcriptome sequencing. In order to generate a comprehensive reference transcriptome we further assembled the raw reads of wild and cultivar genotypes which might enrich the plant transcriptome database and trigger advanced research in cardamom genomics. 

Citation
Nadiya, F., N. Anjali, Jinu Thomas, A. Gangaprasad, K. K. Sabu. 2017. Transcriptome profiling of Elettaria cardamomum (L.) Maton (small cardamom). Genomics Data Vol 11: 102–103. doi: 10.1016/j.gdata.2016.12.013

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Genome Size Estimation Using Flow Cytometry in Elettaria cardamomum Maton

The relative 2C genome size and total number of base pairs of small cardamom (Elettaria cardamomum Maton) was determined using flow cytometer. Seven accessions of cardamom were included in the present study. Zea mays L. CE-777 (2C=5.43 pg) was found to be the most suitable reference standard. Samples for analysis were prepared following the two step procedure described by Otto and involving Propidium iodide staining. The fluorescence intensity of 5000 particles was recorded. The mean amount of 2C nuclear DNA of the cardamom sample was calculated as 2.84 pg. Conversion between DNA content and genome size (1 pg DNA=980 Mbp) indicate that the diploid genome size of cardamom is 2783 Mbp. This is the first report of DNA content and genome size in cardamom. Low variation in genome size has been observed for various germplasm accessions including wild, released varieties and landraces.

Citation
Anjali, N., Nadiya, F., Shefeek, S. and K. K. Sabu. 2016. Genome Size Estimation Using Flow Cytometry in Elettaria cardamomum Maton. Indian Forester 142 (9), 878-881

Assessment of genomic diversity in cardamom by flow cytometry, cytological studies and ISSR analysis

Intraspecific variations in cardamom (Elettaria cardamomum Maton): Assessment of genomic diversity by flow cytometry, cytological studies and ISSR analysis

Article in SpringerPlus 5(1) · September 2016
DOI: 10.1186/s40064-016-3226-x

Background
The main goal of the work was to analyse intraspecific variation in Elettaria cardamomum Maton (cardamom) using genome size, cytological studies and molecular marker data. Nuclear DNA content and molecular marker details furnish data on genome size and genetic diversity respectively among the studied accessions and both complement each other for evolutionary and taxonomic studies.

Results
The relative 2C genome size and total number of base pairs of cardamom was determined through flow cytometric analysis using propidium iodide staining. The nuclear DNA content was estimated in various sections of the species representing individuals from wild and cultivar genotypes following Zea mays L. CE-777 (2C = 5.43 pg) as internal reference standard. Chromosome number from growing root tip was examined following standard protocols. Twenty-six ISSR primers that generated polymorphic bands were used for genetic diversity analysis of the thirty accessions of cardamom. Estimated nuclear 2C DNA content ranged from 2.57 to 3.22 pg demonstrating 1.25-fold variation. The mean amount of 2C nuclear DNA of the cardamom was calculated as 2.87 pg which is equivalent of 2806 Mbp as the diploid genome size. The chromosome number was found to be 2n = 48. Among the thirty accessions of cardamom studied using ISSR markers, C53 (feral from Bonacaud) showed a very prominent level of genetic diversity and was lowest for C96 (Avinash-I, a released variety from Indian Institute of Spices Research, Kozhikode).

Conclusion
These analyses revealed the existence of genetic variability within the studied cardamom accessions. The plant specimens also differed significantly in their genome size. However, the genetic variability parameters did not show any correlation with genome size.

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Development of EST-SSR Markers to Assess Genetic Diversity in Elettaria Cardamomum Maton

Elettaria cardamomum Maton is one of the most ancient and valuable spice crops. Cardamom is cultivated following intensive pesticide usage where alleles present in the wild cardamom genotypes could positively contribute towards genetic improvement of the cultivars. However, the genetic map or whole-genome sequence of E. cardamomum is not available and very limited information on simple sequence repeat (SSR) markers are publicly available. We have tested whether SSRs from Curcuma longa can be used to analyze genetic diversity E. cardamomum.

Int J Appl Sci Biotechnol, Vol 3(2): 188-192

N Anjali, Sowmya S Dharan, F Nadiya, K K Sabu

DOI: http://dx.doi.org/10.3126/ijasbt.v3i2.12380  

Keywords: EST-SSR; microsatellite; genetic diversity; Elettaria cardamomum; cardamom; marker development

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Genetic diversity among cardamom germplasm accessions assessed by SSR and ISSR analysis

Abstract: Cardamom (Elettaria cardamom Maton; Zingiberacea), the ‘Queen of spices’ is one of the most important and highly priced spices. The important areas of cultivation in Kerala are Nelliampathy, Wynad and Idukki. The origin of cardamom is believed to be in the Western Ghats, where presumably rich and untapped genetic diversity of the cardamom still resides. Biodiversity present in the wild relatives of crop plants in Kerala have not yet been addressed properly (Nayar, 2011). The present study aims to collect and evaluate genetic diversity among wild cardamom populations in comparison to the cultivated counterparts.

Citation

Ashitha J P, Anija T A, Anjali, N and K K Sabu. 2013. Genetic diversity among cardamom germplasm accessions assessed by SSR and ISSR analysis. Proc of the 25th Kerala Science Congress held at Technopark at Thiruvananthapuram during 29th January to 1st February 2013, pp. 263-265 (03-27).

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