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20172
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Subject: Plant Biotechnology × Author: KAU × End date: 2017 × Start date: 2017 × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    Metabolite Profiling and gene expression analysis for gingerol production in selected somaclones of ginger (zingiber officinale rosc.)
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2017) Sreeja, S; KAU; Shylaja, M R
    Ginger (Zingiber officinale Rosc.) is one of the oldest known spices and is much valued for its medicinal properties. Ginger rhizome is a source of active biological compounds which are responsible for its medicinal properties. Gingerol is the major pungent polyphenol in ginger which has got a wide array of pharmacological properties. Research on somaclonal variation done at Kerala Agricultural University could locate ginger somaclones with high gingerol content and in the course of investigations high somaclonal variation was observed for quality components. Ginger has a very big genome of 23,618 Mbp which is little exploited and reports on the genomic base of gingerol biosynthesis are scanty. The present investigations hence aim at profiling the metabolites in selected ginger somaclones using high throughput analytical platforms and to analyze the gene expression with respect to gingerol production. One released ginger variety from KAU (Athira), two selected ginger somaclones (B3 and 132M) and parent cultivar (Maran) formed the experimental materials for the study. Studies were carried out at Centre for Plant Biotechnology and Molecular Biology, Distributed Information Centre of College of Horticulture and Arjuna Natural Extracts Pvt. Ltd., Aluva during August 2013 to July 2017. The profiling of aroma principles using Gas Chromatography-Mass Spectrometry (GC-MS) and pungency principles using High Performance Liquid Chromatography (HPLC) at various growth stages viz. five months after planting (5MAP), six months after planting (6MAP) and seven months after planting (7MAP), revealed that aroma and pungency principles accumulated in ginger rhizomes at the rhizome formation stage (5MAP). Clone to clone variation was observed in the number and quantity of aroma and pungency principles accumulated in the rhizome. Total gingerol content in somaclone B3 (19.07%) was high when compared to the control cultivar Maran (17.49%) irrespective of the growth stages. Gene expression for Chalcone synthase in selected somaclones done using real time PCR assay showed highest gene expression in somaclone B3 when the control cultivar Maran was set as calibrator. Somaclone B3 recorded 54 per cent increase in Chalcone synthase gene expression over the control cultivar Maran. Suppression subtractive hybridization done to identify differentially expressed genes in somaclone B3 and control cultivar Maran could prepare Expressed Sequence Tag (EST) libraries both for rhizome and leaf. Analysis of EST sequences (25 rhizome ESTs and 19 leaf ESTs) using various bioinformatic tools revealed that there were no differentially expressed genes for gingerol production in rhizome ESTs. But eleven differentially expressed proteins involved in signaling response, protein trafficking, photosynthesis, ATP formation and transposon mediated mutation were observed in rhizome ESTs. The analysis of leaf ESTs showed differential gene expression in somaclone B3 for 3-ketoacyl CoA thiolase (ACAA1) gene which is involved in gingerol biosynthetic pathway. Hence the higher expression of 3-ketoacyl CoA thiolase gene is responsible for the high gingerol content in somaclone B3 as compared to control cultivar Maran. Eighteen other differentially expressed proteins involved in biological processes like transportation of plant secondary metabolites and their intermediates, mobilization of sucrose into pathways involved in metabolism, lipid biosynthesis, transportation of cellular material to microtubules, biogenesis of metabolic pathways in Calvin cycle were observed in leaf ESTs. The differentially expressed gene (ACAA1) can be further validated using northern blotting and quantitative real time PCR by designing specific primers from the ESTs. Expressed sequence tags and corresponding differentially expressed proteins can be used as molecular markers. Post translational modification in differentially expressed proteins can be used to study the mechanism of gingerol production. Forty four sequences deposited at NCBI form the base sequences for further research.
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    ThesisItemOpen Access
    Validation of apomixis and transcriptome analysis for detection of the genes related to apomixis in black pepper (piper nigrum L.)
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2017) Rohini Rajkumar, Bansode; KAU; Valsala, P A
    Black pepper (Piper nigrum L.) universally honoured as “Black Gold” and also known as “King of Spices” is one of the most important spices in the world. It is a perennial climber belonging to the family Piperaceae and is valued throughout the world for its spice value and medicinal properties. Black pepper is grown in Southern India mainly in Kerala. Here productivity is declining due to various reasons and continuous vegetative propagation leads to accumulation of diseases and finally results in unproductive vines. Therefore, use of quality planting material of improved varieties is necessitated for the enhancement of productivity. Apomixis is a mode of asexual reproduction where the sexual organs are utilized, but the seeds develop without fertilization. It combines the advantage of seed propagation and vegetative propagation, and can be utilized for developing disease free planting material without losing the clonal integrity. The present study on “Validation of apomixis and transcriptome analysis for detection of the genes related to apomixis in black pepper (Piper nigrum L.)” was undertaken at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2011-2014 with the objective to validate apomixis in black pepper varieties, Panniyur-1 and Panniyur-2 through controlled pollination studies and to identify differentially expressed genes associated with apomixis through transcriptome analysis. Studies were conducted on bush pepper plants of the selected varieties maintained in the green house. Floral biology of Panniyur-1 and Panniyur-2 were studied. The female phase in Panniyur-1 started 8th day after spike initiation and completed on 27th day. Stigma became receptive on 9th day and receptivity was indicated by creamy white colour. Beginning of male phase indicated by anther emergence on either side of the ovary occurred on 19th day of spike emergence and active dehiscence of anthers occurred on 20th day. In Panniyur-2, beginning of female phase started on 14th day after spike initiation and stigma became receptive on 15th day. Beginning of male phase occurred on 22nd day of spike emergence and active dehiscence of anthers occurred on 23rd day. So in Panniyur-1 and Panniyur-2 the active female and male phase is separated by 10 and 8 days, respectively. Attempts were made to confirm apomixis in Panniyur-1 and Panniyur-2 varieties by allowing berry development in controlled condition by bagging of spike initials. Berry development occurred under bagged condition. In this study, upper six berries were considered as apomictic and lower six berries were considered as pollinated. Efforts were made to regenerate apomictic and non- apomictic progenies under in vitro and ex vitro. In vitro embryo culture resulted in embryo germination and multiple shoot induction in SH medium with hormones. But plants were lost due to microbial contamination and phenolic interference. So ex vitro germinated seedlings were used for further studies. Histological examination of pollinated and unpollinated ovaries was done through microtomy. The results revealed that in case of pollinated berries, the sexual fertilization occurs and embryo develops inside the embryo sac in the micropylar end. Whereas, in apomictic embryo sac more than one aposporous initial cells were observed which were arising from a somatic cell located in the nucellus. So, it can be concluded that facultative apomixis exists in black pepper varieties P1 and P2 and the embryo develops parthenogenetically. The molecular characterization of apomictic and non-apomictic seedlings was done through Inter Simple Sequence Repeats (ISSR) and Simple Sequence Repeats (SSR) assay. Isolation of good quality genomic DNA from apomictic, pollinated progenies and mother plant was carried out from the young leaves using modified Rogers and Bendich (1994) as reported by Mogalayi (2011). In ISSR and SSR assay certain primers showed polymorphism among mother plant, apomictic and pollinated progenies. Similarity matrix was calculated and corresponding dendrogram was also constructed for both markers using UPGMA cluster analysis for P1 and P2 varieties. In ISSR assay the similarity coefficient between P1 mother plant and apomictic progenies ranged from 69 to 89 per cent while it was 64 to 66 per cent between mother plant and pollinated progenies. In P2 variety similarity coefficient between mother plant and apomictic progenies ranged from 76 to 90 per cent while it was 62 to 69 per cent between mother plant and pollinated progenies. In SSR assay similarity coefficient between P1 mother plant and apomictic progenies ranged from 50 to 100 per cent while it was 71 to 100 per cent between pollinated progenies and mother plant. Similar results were obtained for P2 variety also. So apomictic progenies may differ from mother plant in certain characters. RNA mediated transcriptome analysis of the apomictic and pollinated berries was done to detect differentially expressed genes. Good quality RNA was isolated by modified LiCl precipitation method. Total RNA from the apomictic and pollinated berries were taken for DDRT-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 in 6 per cent denaturing urea polyacrylamide gel and visualized after silver staining. The differentially expressed cDNA fragments were retrieved from the gel and reamplified and electrophoresed. The agarose gel electrophoresis showed 7 transcript derived fragments (TDFs) ranging from 200-600 bp. TDFs were cloned using pGEMT vector and were sequenced by outsourcing. The sequence data were analysed by BLASTn and BLASTx. The sequences showed homology to NADH dehydrogenase subunit J, acetyl-CoA-benzylalcohol acetyltransferase and purine permease 4 but no significant sequence similarities for apomictic genes deposited in NCBI database were found.