1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)

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    DNA fingerprinting of released varieties and selected superior somaclones of ginger (Zingiber officinale Rosc)
    (Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2013) Pujaita Ghosh; Shylaja, M R
    Ginger (Zingiber officinale Rosc.), one of the widely cultivated and consumed spices worldwide, is well known for its medicinal properties also. As the natural variability stands limited in the crop, induction of variability through tissue culture techniques was attempted at College of Horticulture, Vellanikkara from 1996 onwards. After indepth investigations on the somaclones regenerated, two varieties viz., “Athira” and “Karthika” were released during 2010 and four clones viz. B3, 292R, 88R and 478R were selected as superior somaclones. For the newly released ginger varieties and selected superior somaclones in pipeline for release, no fingerprint data are available for genotype identification and protecting the plant varieties / clones. The investigations on “DNA fingerprinting of released varieties and selected superior somaclones of ginger (Zingiber officinale Rosc.)” were carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Kerala Agricultural University, Thrissur during the period from January 2012 to March 2013. The objectives of the study were to characterize two released varieties and four selected superior somaclones using molecular markers and to develop a DNA fingerprint specific to each variety / somaclone. Morphological characters like growth habit and size and shape of the rhizomes were found to vary in the varieties / somaclones studied. The somaclone 292R could be distinguished based on its dwarf plant stature and dark green leaves. The variety Athira has bold and flat rhizomes while the variety Karthika has medium bold and round rhizomes. Quantitative clustering for vegetative and rhizome characters attempted as per Mahalanobis D2 analysis could group the varieties and somaclones into three separate clusters. Of the seven vegetative characters analysed, plant height and number of tillers showed more divergence. The number of fingers, girth of primary and secondary fingers, thickness of flesh and inner core were the characters which exhibited more divergence for the rhizome characters. For molecular characterization, good quality genomic DNA extracted from ginger varieties / somaclones using CTAB (Rogers and Bendich, 1994) method was used. Thirty five RAPD and thirty ISSR primers were screened for amplification of genomic DNA and ten RAPD and eleven ISSR primers were selected based on the amplification pattern. DNA fingerprints of the varieties / somaclones were developed utilizing the clear, distinct bands generated in RAPD and ISSR profiles and size of the bands. Different colour codes were assigned for sharing of bands between varieties / clones to generate specific fingerprints. The RAPD marker system could bring out unique bands in the variety Karthika and somaclones B3, 292R and 478R. The RAPD primer, OPA 12 produced unique band in Karthika and B3, the primer OPA 04 in 292R and the primer OPA 28 in 478R. ISSR marker system could also bring out unique band in the variety Athira with primer ISSR 06. The RAPD, ISSR and combined fingerprints developed for each variety / somaclone were unique. Variability in the somaclones and the extent of variability from source parent cultivars were analysed using cluster analysis. The dendrogram seperated Maran and Rio-de-Janeiro somaclones in two separate clusters. Somaclones derived from cultivar Maran exhibited more variability than somaclones from Rio-de- Janeiro. The variety Athira was more diverse from the source parent cultivar Maran. Similarly, the somaclone 292R was more diverse from the source parent cultivar Rio-de-Janeiro. The Resolving Power (Rp) of RAPD and ISSR primers ranged from 6.00 to 16.25, indicating the ability of the selected primers to distinguish the varieties / clones most efficiently. The Polymorphic Information Content (PIC) ranged from 0.67 to 0.88, indicating the suitability of the selected primers for DNA fingerprinting. RAPD, ISSR and combined fingerprints developed specific for the ginger varieties / somaclones could be utilized for registration, documentation of varieties and for settling IPR issues.
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    DNA barcoding in momordica spp.
    (Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2014) Girme Aoudumbar Ramesh; Deepu Mathew
    The genus Momordica comprises of 59 species, among which 7 are of Indian origin. Though the vegetables belonging to this genus are nutritionally rich with medicinal properties, their taxonomy remains confusive. The botanical names are often used incorrectly and interchangeably. Different taxonomic classification approaches have resulted in controversies about the number of species that exist and the phylogenetic relationships among these species. This situation necessitates an accurate, sensitive and simple alternate for the traditional taxonomy. DNA barcoding is a novel system designed to provide rapid, accurate, and automatable species identification using short, standardized genomic regions as internal species tags. DNA barcoding is based on the variation on the sequences identified genomic regions, which can distinguish individuals of a species because genetic variation between species exceeds that within species. Species identification through barcoding is usually achieved by the retrieval of a short DNA sequence i.e. the „barcode‟ from a standard part of the genome (i.e. a specific gene region either from chloroplast, mitochondria or nuclear genome). The barcode sequence from each unknown specimen is then compared with a library of reference barcode sequences derived from individuals of known identity. The study entitled “DNA barcoding in Momordica spp.” was done at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara. The objective of this study was to develop the barcodes for the 7 Momordica spp. which were found in India. For this study, 25 accessions of seven Momordica species M. charantia, M. balsamina, M. dioica, M. sahyadrica, M. cochinchinensis, M. subangulata subsp. renigera and M. cymbalaria and two accession of Luffa has been taken. Total genomic DNA isolated using CTAB method (Rogers and Bendich, 1994) was subjected to PCR assay using various combinations of universal barcode primers for three loci matK, ITS2 and trnH-psbA. As ITS2 and trnH-psbA gave the bands of 600 and 300bp, respectively, and which are not sufficient to develop the complete barcodes, these loci were not used in the study. Thus the bands generated using the matK primers from all the 25 Momordica and 2 Luffa were used for sequencing. Phylogenetic analysis using ClustalW has discriminated the various species under Momordica, except dioica and sahyadrica. Kimura 2 Parameter (K2P) model using MEGA software. A wide level of molecular diversity detected with both the method which shows the high level of genetic variation among the species of 25 accessions of Momordica of Indian origin and Momordica and Luffa. Barcoding gap, a position in the sequence at which unique nucleotide is present in all the members of a particular species, was assessed for all the Momordica species and the Luffa, and these gaps were used to generate the barcodes for that species. From this study, barcodes were successfully generated for M. charantia, M. subangulata, M. cochinchinensis, M. cymbalaria, M. balsamina and Luffa acutangula. The BLAST analysis had shown that matK is 95 per cent efficient for species discrimination in Momordica. From the base sequence of matK generated from this study, barcoding primers were designed for Momordica and were successful in all the 25 accessions. The matK barcodes developed in this study could be successfully used to solve the taxonomic confusion in Momordica.
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    Variability in Chakkarakolli (Gymnema sylvestrwe R.Br.) using morphological, biochemical nad molecular markers
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2005) Smitha Nair; Keshavachandran, R
    Plants represent an unparalleled source for drug development. Plant based natural products play a dominant role in the pharmaceutical industry. Plant based remedies are available for a number of different health problems. In today’s scenario, diabetes is one of the most common non-communicable diseases globally and is also the fourth major cause of death in most developed countries. So the demand for natural alternatives to blood sugar control is ever increasing. Several indigenous medicinal plants have been indicated in the Ayurveda system of medicine to possess antidiabetic activity. Native to the forests of South India, Gymnema sylvestre R.Br. is known for its hypoglycaemic property from the time immemorial. Practitioners of Ayurveda first used Gymnema to treat diabetes almost 2000 years ago. Keeping in view the importance of this medicinal plant, a study was conducted to identify the variability in Gymnema accessions collected and maintained at the Centre for Plant Biotechnology and Molecular Biology. The present study was undertaken at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2003-2005. The objectives of the study were to characterise the variability in Gymnema germplasm accessions using morphological, biochemical and molecular markers. The morphological characterisation of 93 Gymnema accessions based on vegetative characters indicated the existence of a wide variation. All the 93 accessions were then characterised using biochemical markers. The total saponin content in the leaves was chosen as the marker constituent since saponins are the major bioactive components in Gymnema. The saponins were estimated based on TLC-Densitometry technique. The techniques for detection of saponins through TLC could be standardised. The saponin content in the leaves of different accessions ranged between 0.6 per cent and 5.4 per cent. Based on the morphological and biochemical characters, variants were identified in Gymnema germplasm collection. Eighteen such plants showing high variation were subjected to molecular characterisation using isozyme and RAPD markers. Isozyme analysis was carried out using three enzyme systems viz. malate-dehydrogenase, esterase and RUBISCO. Eight isozyme loci could be clearly identified of which five were polymorphic. Ten alleles were identified over the five polymorphic loci giving an average of two alleles per polymorphic locus. A mean observed heterozygosity of 19.8 per cent and a mean expected heterozygosity of 17.2 per cent were obtained over the heterozygous loci studied. The cluster analysis grouped the 18 accessions in to two major clusters of 17 and one accession. Molecular characterisation using RAPD markers was conducted to appraise the extent of diversity among the 18 accessions of Gymnema. The protocol for genomic DNA isolation from Gymnema was standardised. The protocol suggested by Rogers and Bendich (1994) with slight modification was found to be most appropriate. The protocol for RAPD assay in Gymnema was standardised. Sixty random primers were screened and 15 were selected for RAPD profiling of Gymnema accessions. The primer OPAH 12 was found to have the highest resolving power. A total of 123 amplification products were generated by 15 primers, of which 90 were polymorphic. In the dendrogram, the 18 accessions were grouped in to two major clusters of 16 and two accessions. The accessions Peringottukurushi 137 and Kuzhalmannon 89, occurring in the third sub-cluster were the most closely related with 85 per cent similarity. A combined dendrogram was also derived from the results of morphological and molecular studies. The present study revealed the existence of sufficient genetic variation in the Gymnema germplasm collection. This variability can be used to identify useful genotypes that could be used as cultivars for the extraction of standard drugs. More precise techniques like HPLC and AFLP could further be used to get a better idea about the extent of variability in Gymnema.