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Subject: Plant Biotechnology × Author: KAU × End date: 2012 × Start date: 2012 × Type: Thesis × Thesis Type: M.Sc ×
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    ThesisItemOpen Access
    Morphological and molecular analysis of genetic stability in micropropagated banana (Musa spp) var. Nendran
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2012) Amar Ramesh, Kadam; KAU; Nazeem, P A
    Banana is an important fruit crop widely grown throughout the world. With the increasing demand and vast export potential coupled with the farmers desire to grow banana on a large area, in vitro propagated plants are becoming increasingly important as planting material for rapid multiplication of economically important commercial varieties. In vitro propagation has many advantages, such as higher rates of multiplying clean (pest and disease-free) planting material and the small amount of space required to multiply large number of plants. A major problem associated with micropropagation is the occurrence of genetic variation resulting from in vitro cultures, i.e., somaclonal variation amongst sub-clones of one parental line. The molecular basis of such variation is not well explained till date. The study entitled “Morphological and molecular analysis of genetic stability in micropropagated banana (Musa spp) var. Nendran” was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara, during the period from 2010 to 2012 with the objective of evaluation and characterization of the variation in tissue culture derived banana plants regenerated in different subcultures through in vitro organogenesis. Micropropagation of banana was carried out using male bud and sucker as explant source. Adventitious shoots were induced from both the sucker derived and male bud derived explants. The culture establishment rate and multiplication frequency were significantly low in male bud derived explants. The plantlets derived in specific subcultures (3, 6, 8, 10, 12, 14 and 16) were planted out and subjected for further evaluation. Considering vegetative characters during the field performance; plants derived from later subcultures were found inferior to others in both male bud and sucker derived plants. The fruit characters of the male bud derived plants were comparable to the mother plant though bunch weight and size was less in later subculture (10th). The CTAB procedure reported by Rogers and Benedich (1994) for the extraction of nucleic acids was used for the isolation of genomic DNA from tissue culture derived banana plants. The young unfurled leaves from healthy plants were collected early in the morning and used for the genomic DNA isolation. The RNA contamination was completely removed through RNase treatment. Good quality DNA with UV absorbance ratio (A260/A280) 1.80 - 1.89 was used for further analysis. The PCR conditions were optimized for Inter Simple Sequence Repeats (ISSR) assay. DNA isolated from each subculture were bulked and amplified using 10 selected primers. The amplification pattern was scored and analysed for quantifying the variation among the plants derived from different subcultures using male bud and sucker as explant. The computer package NTSYS-pc was used for cluster analysis. Plants derived from different subcultures were found grouped into two distinct clusters. Plants derived from upto 12th subculture were grouped in first cluster (<10 % variation) and those derived from 12th subculture onwards were found grouped in second cluster. The maximum genetic variation detected through ISSR assay was 15% for both male bud and sucker derived plants and the variation was relatively more in the plants derived in later subcultures (12th onwards). Changes in DNA methylation (addition of -CH3 to cytosine) has been hypothesized as an underlying mechanism of tissue culture induced variation due to the high frequency of quantitative phenotypic variation, the activation of transposable elements, heterochromatin-induced chromosome breakage events etc. An attempt was made to detect the extent of methylation pattern in micropropagated banana plants using Methylation Sensitive Amplification Polymorphism (MSAP) assay. In the study, four MSAP primer combinations were used to amplify the fragments cleaved by restriction enzymes MspI and HpaII (isoschizomers). For male bud derived plants a total of 39.4 percent methylation sites were detected while for sucker derived plants it was only 23.3 percent. Variation in methylation pattern was observed more in later subcultures (10th onwards) than initial. Percentage of internal methylation was relatively more compared to hemimethylation for both male bud and sucker derived plants. From the study, it was observed that the plants derived from initial subcultures showed lesser variation (<10 %) compared to later subcultures (12th onwards). The methylation pattern detected through MSAP assay was observed as an important factor inducing variation in tissue cultured banana. The ISSR and MSAP primers which detected polymorphism could be further utilized in quality control of tissue culture derived banana plants. Since the banana genome is sequenced recently, the information generated could be exploited to detect the hotspots in banana genome.
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    ThesisItemOpen Access
    Transcriptome analysis of drought induced systemic tolerance in rice (Oryza sativa L) medicated by plant growth promoting rhizobateria
    (Department of plant biotechnology, College of horticulture, Vellanikkara, 2012) Tirthkar Meera, Baburao; KAU; Abida, P S
    Plant-growth-promoting rhizobacteria (PGPR) are associated with plant roots and augment plant productivity and immunity; however, recent work by several groups shows that PGPR also elicit so-called ‘induced systemic tolerance’ to salt and drought. Some PGPR also elicit physical or chemical changes related to plant defense, a process referred to as ‘induced systemic resistance’ (ISR). ISR elicited by PGPR has suppressed plant diseases caused by a range of pathogens in both the greenhouse and field . However, fewer reports have been published on PGPR as elicitors of tolerance to abiotic stresses, such as drought, salt and nutrient deficiency or excess (Yang et al., 2009) . The present study on “ Transcriptome analysis of drought induced systemic tolerance in rice mediated by plant growth promoting rhizobacteria “ was undertaken at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2010-2012 with the objective to identify the differentially expressed genes and signal tranduction pathways operating in rice (Oryza sativa.L) during water stress conditions mediated by plant growth promoting bacteria. The Mattathriveni, a popular rice variety of Kerala which is susceptible to drought was used as the experimental material as its performance in upland is also very poor. The pot experiment was carried out under water logged condition and three treatments were given. In T1, control plants were maintained in water logged condition, in T2 water was with held for 48 hours and in T3 plants which were treated with Psedomonas fluorescens Pf 1 (KAU strain) water was with held for 48 hours. Observations on biometric and physiological parameters were taken. The physiological parameters were taken using Infra red gas analyzer. Enhanced growth promotional effect and photosynthetic efficiency was noticed in Pf treated plants. Also plants maintained lower leaf temperature and lower stomatal conductance in Pf treated stressed plants which is a water saving mechanism to cope up with water stress. The mechanisms for abiotic stress tolerance are based on activation and regulation of specific set of stress-related genes which are involved in signaling, transcriptional control, and protection of membranes and proteins. The technique used to analyse the transcriptome was the differential display-RTPCR allows extensive analysis of gene expression among several cell populations (Sturtevant, 2000). The gene expression analyses requires good quality RNA which was isolated by Trizol reagent method with utmost care to prevent its degradation by nucleases. About 1μg of total RNA from all the treatments were taken for DD-RT-PCR analysis. The first strand cDNA was synthesized from the above RNA samples using HT11C (AAGCTTTTTTTTTTTC). Each first strand cDNA was used for the second strand amplification with 8 different arbitrary primers. The PCR product was resolved on 6% denaturing urea polyacrylamide gels and visualized after silver staining. Tthe gel was dried overnight for elution and further analysis. The up regulated ,down regulated and differentially expressed cDNA fragments were retrived from the gel and reamplified with the same set of primers as in the initial DD-RT-PCR reaction and analyzed electrophoretically. The agarose gel electrophoresis showed that the transcript derived fragments(TDFs) obtained were relatively short (150-700bp). TDFs were cloned. The vector used was pJET which used less ligation time and gave only white colonies of recombinants. The clones were sequenced at Sci Genome Cochin. The sequence data obtained were analysed by in silico tools. The sequence from differentially expressed TDFs showed homology to SOS1 regulatory proteins which is involed in ion homeostasis in abiotic stress signaling which is salint finding. The other major genes identified were cytochrome oxidase, ATP synthase, heat shock proteins, MYB transcription factor which are all operating in major signal transduction pathways. The gene expression were confirmed by e Northern analysis, which is an in silico tool to validate the gene expression during abiotic and biotic stress related studies by comparing with the Arabidopsis gene expression data base available at Botany array resources.
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    ThesisItemOpen Access
    Tagging of bacterial wilt resistance gene in Solanum melongena var.insanum by molecular markers
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2012) Chavan Pradeep, Uttamrao; KAU; Valsala, P A
    The study entitled ‘Tagging of bacterial wilt resistance gene in Solanum melongena var. insanum by molecular markers’ was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara during the period 2009-2011. Bacterial wilt caused by Ralstonia solanacearum (Smith 1896, Yabuuchi et al., 1995) is an important problem in cultivation of tropical and subtropical crops like potato, brinjal, chilli, tomato etc. World wide approach to control the disease is to use resistant varieties. Gopimony (1983) reported Solanum melongena var. insanum (wild variety) is resistant to bacterial wilt and controlling gene is monogenic and dominant. This investigation was taken up for tagging of bacterial wilt resistance gene in Solanum melongena var. insanum by RAPD through bulked segregant analysis as reported by Michelmore et al., (1991). The genotypes used for the study were resistant variety Solanum melongena var. insanum I.C. number 421463, susceptible variety Pusa Purple Long and segregating F2 population for bacterial wilt incidence. To raise segregating F2 population F1 was raised by controlled crossing of Solanum melongena var. insanum I.C. number 421463 with pollen grains of Pusa Purple Long. Then F1 plant was selfed to get F2 population.The parents and F2 genotypes were screened for bacterial wilt incidence by stem puncture method of artificial inoculation using R. solanacearum inoculum. In all cases death of plants were confirmed by ooze test. The genotypes were classified against bacterial wilt incidence according to classification of Mew and Ho (1976). The variety Solanum melongena var. insanum I. C. number 421463 was resistant, Pusa Purple Long and F2 were susceptible. Ratio of susceptible to resistant in F2 generation was 2.7:1. So it can assume that bacterial wilt resistant character in resistant parent can be homozygous and recessive.Genomic DNA for RAPD analysis was isolated by modified CTAB method (1994). Good quality DNA with an absorbance ratio of 1.8-2.0 was used for RAPD analysis. PCR reaction mixtures and conditions for DNA amplification were standardized. Ninty eight primers belonging to series A, OPA, OPAG, OPAH, OPB, OPC, OPF, OPP, OPU, RY and RN were initially screened with DNA of resistant and susceptible parents to select primers with polymorphism. Out of ninty eight primers tested thirty, were reported as bacterial wilt specific. Seventeen primers were selected for BSA based on polymorphism. None of wilt specific primers showed polymorphism.Bulked segregate analysis was done with seventeen selected primers for polymorphism. The genotypes used for the study were susceptible parent, resistant parent, five resistant and five susceptible F2 progenies. Among the tested primers only OPP-14 has recorded polymorphism between resistant and susceptible genotypes in BSA. It has produced 470 bp polymorphic amplicon in resistant parent and resistant bulk. Co-segregation analysis was done with OPP-14 primer with individuals of susceptible and resistant bulk. In co-segregation analysis OPP-14 specific amplicon got amplified not only in resistant parent, resistant bulked but also in three susceptible F2s also.The polymorphic band produced by OPP-14 primer in resistant parent and resistant bulk was eluted from resistant parent and cloned in pGEM-T vector, and was transformed into E. coli JM 109 cells. Recombination of the insert was confirmed through colony PCR reaction with universal T7 and SP6 primers. The cloned fragment was sequenced to obtain the nucleotide sequence information and was named as W-3.The sequence obtained after vector screening was named as “Sol-3”, was subjected to Blastn search. Blastn search revealed significant levels of homology with AC237888 Solanum tuberosum clone RH183L22 present in NCBI database. The sequence was also analysed using EMBOSS Transeq and Blastp. Analysis revealed homology with ABC948993 polyprotein (Oryza australiesis). ORF analysis revealed the longest ORF of 108 bp was on +3 frame which revealed significant level of homology with putative retrotransposon protein in Solanum demissum.Two sets STS primers were designed using Primer3Plus. The efficiency of STS primer set BWRGB-2 to distinguish resistant and susceptible genotypes was tested and it amplified a fragment of 200 bp in all the genotypes tested.
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    ThesisItemOpen Access
    Metagenomic approach to assess diversity of bacterial community in saline pokkali habitats of kerala
    (College of Horticulture, Vellanikkara, 2012) Sarveshwar, Sah; KAU; Girija, D
    The use of traditional microbiological culturing methods for the study of microbes has its limitations. It has been estimated that 99 per cent of microbes cannot be cultured easily. Over the past decade, “Metagenomics,” which is culture-independent genomic analysis of microbes, has been developed to overcome the drawbacks in culture-based analysis of microbial communities. Soil is considered to be a complex environment and a major reservoir of microbial genetic diversity. Metagenomics is a powerful tool in the analysis of diversity of microbial communities in specific environments. An attempt was made to analyse the diversity of bacterial community in the acid saline Pokkali soil from Vyttila, through Metagenomic approach. Environmental DNA was isolated from the soil by direct method. 16S rDNA which encodes 16S rRNA found in the 30S subunit of prokaryotic ribosome is used as the barcode for bacterial identification. 1500bp fragment of 16S rDNA was amplified using specific primers through polymerase chain reaction (PCR). The amplicons were ligated in plasmid vector pGEMT and cloned in E. coli, to construct a Metagenomic library. Sequence analysis of 30 randomly picked colonies revealed that 23 clones (76.7%) were of unculturable bacteria and 7 clones (23.3%) were culturable. The most predominant phylum was Proteobacteria, which included Ectothiorhodospiraceae, Azospira sp., Stenotrophomonas maltophilia, Thiobacillus prosperus, Levilinea saccharolytica, Desulfobacteraceae, Thioalkalivibrio sp. and Rhodocylales. Other phyla included Chloroflexi, Acidobacteria and Cynobacteria. A range of bacteria including acidophiles, extreme halophiles, denitrifier, S oxidizers, sulphate reducers, aerobes, strict anaerobes, thermophiles, mesophiles, photosynthetic bacteria and even human pathogens were obtained, of the 23 unculturable bacteria detected, six did not show homology with any bacterial sequence available in NCBI database, indicating the possibility that these could be novel. The phylogenetic tree based on partial 16S rRNA gene placed 30 clones from Pokkali soil in three major cluster- Proteobacteria, Chloroflexi and Acidobacteria. Three predominant bacteria isolated by the traditional method belonged to the genus Bacillus. Metagenomic approach successfully revealed the composition and diversity of bacterial community in Pokkali soil. A function-derived strategy could be used for bioprospecting of gene related to acid and salt tolerance genes.
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    ThesisItemOpen Access
    Diversity and population structure analysis in coconut (Cocos nucifera L) using molcular markers
    (College of Horticulture, Vellanikkara, 2012) Renju, S; KAU; Sujatha, R
    Assessment of genetic diversity and thus understanding the population structure is the mainstay for any crop improvement programmes. With the advent of molecular markers and suitable software for statistical analysis, this became a routine work in plant breeding. The technique is especially helpful in breeding of perennial heterozygous crops such as coconut. Hence the present study on ‘Diversity and population structure analysis in coconut (Cocos nucifera L.) using molecular markers’ was undertaken with the objective to estimate the genetic diversity among the selected cultivars using three marker systems viz., Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeats (ISSR) and Simple Sequence Repeats (SSR). The experiment material included six tall cultivars (Laccadive Ordinary, Tiptur Tall, Komadan, Kuttiadi, Malappuram, Kasaragode), three dwarf cultivars (Chowghat Green Dwarf, Chowghat Orange Dwarf and Malayan Yellow Dwarf) and one intermediate (Gangabondam). Five palms from each cultivar were used for the study. The DNA isolated from each cultivar was amplified using selected primers for RAPD, ISSR and SSR. The DNA isolation procedure for coconut was standardized as it was difficult for isolating DNA from coconut due to the high polysaccharide and polyphenol content of its leaves. Several DNA isolation procedures were tried and among them, the protocol reported by Porebski et al. (1997) with suitable modifications yielded good quality DNA in sufficient quantity. The ten selected random primers for RAPD (after screening 35) generated 65 markers with an average of 40 percent polymorphism. The polymorphism information content (PIC) of selected primers ranged from 0.67 (RN4) to 0.89 (RN5, RN8). For ISSR assay, eleven primers were selected (after screening 41) which generated ninety amplicons. The average polymorphism per cent was 44.44 and the PIC value of selected primers ranged from 0.78 (UBC 855, UBCS2) to 0.90 (UBC 834). Ten sets of primers selected for the SSR analysis (after screening forty three) produced a total of 13 amplicons with 30.76 per cent polymorphism. Altogether, there were 168 markers produced by RAPD, ISSR and SSR assay of the 10 genotypes of which 70 bands (41.66%) were polymorphic with an average of 2.29 polymorphic markers per primer. The presence or absence of data was entered into a binary data matrix and was used for calculating the similarity coefficient using Dice coefficient (Nei and Li, 1979) using software DARwin (Version5.0) and Jaccard’s coefficient (Jaccard, 1908) using software NTSYS-PC (Rohlf, 1993). Cluster analysis was done using the UPGMA method and dendrograms (using DARwin and NTSYS) were constructed by neighbor joining. The genetic diversity values were calculated using the software, DARwin as the dendrogram based on DARwin showed more accuracy according to morphological characters. The dendrogram generated (using DARwin) from sixty five RAPD markers, grouped the cultivars into three major clusters. Cluster I consisted of four tall cultivars namely Kuttiadi, Tiptur Tall, Malappuram and Kasaragode. Cluster II included three dwarf cultivars viz., Chowghat Green Dwarf, Chowghat Orange Dwarf, Malayan Yellow Dwarf and the intermediate type, Gangabondam. Cluster III included the two tall cultivars, Laccadive Ordinary and Komadan. The dendrogram (using DARwin) based on ISSR markers grouped the cultivars into three major clusters which was slightly different from that of RAPD. Cluster I comprised of four tall cultivars viz., Kuttiadi, Tiptur Tall, Malappuram but instead of cv. Kasaragode in RAPD dendrogram, Laccadive Ordinary was included in first cluster of ISSR. The tall cv Kasragode was separated out as the third cluster. Cluster II is similar to that in RAPD except for the presence of one tall cultivar, Komadan. Based on all the 168 markers, dendrograms were constructed using DARwin and NTSYS software which grouped the cultivars into three clusters (Cluster I and III for Talls and Cluster II for Dwarfs and intermediate) by the former and into two (ClusterI for talls, Cluster II- Dwarfs and intermediate) by the latter. In the present study it was found that combination of three markers produced more accurate classification of genotypes. Based on 168 markers from three marker systems the overall genetic diversity observed among the three morphological groups viz.,Talls, Dwarfs and intermediate type palms in the selected population of coconut seed farm at Vellanikkara is comparatively less (GD=0.13). The highest genetic diversity was between Talls and Dwarfs (GD=0.14). The tall population consisting of six tall cultivars among which four are ecotypes of WCT showed the lowest diversity (GD-0.06). Within Talls, Kuttiadi is found to be similar to Tiptur Tall as well as Malappuram with least diversity (GD=0.04). The diversity observed within the Dwarf population of present study showed a higher diversity index (GD=0.09). This is mainly contributed by the dwarf cultivar Malayan Yellow Dwarf collected from RARS Pilicode which is found to be genetically most distant from the rest of the nine cultivars (average GD=0.16). The intermediate type Gangabondam is genetically diverse from Talls and is grouped under the cluster for dwarfs. Within this cluster it was found genetically closer to Malayan Yellow Dwarf. The two cultivars (Tiptur Tall and Malayan Yellow Dwarf) were identified for the production of a T x D hybrid based on the study as they were the most divergent among the cultivars selected.