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Item Exopolysaccharide producing bacteria from plant microbiome for drought stress mitigation in tomato(Department of Molecular Biology and Biotechnology, College of Agriculture,Vellayani, 2025) Ameera Vappukannu Ashraf; Chitra, NThe study entitled “Exopolysaccharide producing bacteria from plant microbiome for drought stress mitigation in tomato” was carried out at the Department of Molecular Biology and Biotechnology and Department of Agricultural Microbiology, College of Agriculture, Vellayani, in the year 2023–2024 with the objective to isolate and characterise exopolysaccharide producing bacteria from rhizosphere and phyllosphere of tomato and to evaluate them for drought stress mitigation. The leaf samples and rhizosphere soil were collected from tomato varieties cultivated in different locations for the isolation of exopolysaccharide (EPS) producing bacteria. Isolation was carried out by serial dilution and plating on EPS selective medium amended with sucrose, from which mucoid colonies were selected, purified, and used for further studies. On quantification of EPS production of the isolates, rhizosphere bacterial isolate TRE15 (0.36 ± 0.06g) and phyllosphere isolate TPE10 (0.19 ± 0.01g) produced significantly higher EPS. The isolates were screened for their ability to withstand water stress induced by higher concentrations (0%, 10%, and 20%) of Polyethylene Glycol (PEG) 6000 and their growth was assessed by measuring OD600. Among them rhizosphere isolates TRE15 (0.38±0.02) and phyllosphere isolate TPE8 (0.08±0.00) showed better growth in 20% PEG 6000 compared to other isolates. In oil spread assay rhizosphere isolates showed better biosurfactant property compared to phyllosphere isolates, with TRE8 showing maximum oil dispersion diameter of 3.90±0.14 cm. Microtiter plate assay for determining the biofilm forming potential of the isolates revealed that TRE33 produced significant amount of biofilm (0.13±0.02), where as in phyllosphere isolates TPE16 (0.14±0.02) was the best. Most of the phyllosphere isolates showed lesser biofilm production compared to rhizosphere isolates. Based on these results six isolates each were selected from the rhizosphere and phyllosphere for further assessment of plant growth promotion traits. Rhizosphere isolates TRE 33 (32.69±0.86 mg L-1) and TRE39 (107.87±8.29 mg L-1) showed 203 significantly higher extracellular ammonia production whereas all selected phyllosphere isolates except TPE7 produced extracellular ammonia. Among rhizosphere bacteria, maximum indole acetic acid (IAA) production was observed in TRE8 (41.10±0.61 mg L-1) in medium containing tryptophan and TRE39 (107.87±8.29 mg L-1) in medium without tryptophan. Phyllosphere isolate TPE13 (67.68±8.27 mg L-1) showed higher concentration of IAA in medium containing tryptophan and TPE8 (65.82±1.84 mg L-1) produced maximum IAA in the absence of tryptophan in medium. TRE33 (187.21±4.06 mg L-1) and TPE9 (413.93±9.34 mg L-1) produced significantly higher concentration of gibberellic acid. The isolates exhibiting plant growth promotion traits were selected for further in vivo studies. Growth promotion potential of the selected isolates was analysed in tomato seeds by roll towel assay. Based on the germination percentage, seedling vigour index and survival percentage, the rhizosphere isolates TRE4, TRE8, and TRE21, and phyllosphere isolates TPE8, TPE9, and TPE13 were selected for preparation of consortium and further evaluation in tomato plant. The selected isolates were characterised morphologically, biochemically and identified by 16S rRNA gene sequencing as Preistia aryabhatti (TRE4), Enterobacter cloacae (TRE8), Bacillus pumilus (TRE21), Bacillus stercoris (TPE8), Stenotrophomonas pavanii (TPE9), and Pantoea dispersa (TPE13). Separate consortium of rhizosphere isolates (RC) and phyllosphere isolates (PC) were used for pot culture experiment in tomato. Three treatments were included viz., seed priming with RC, foliar spray with PC at 20 days after transplanting and a combination of both seed priming (RC) and foliar spray (PC), along with control. Ten days after foliar spray, drought stress was induced by withholding irrigation. The stress induced plants were analysed for cell membrane integrity, proline and malondialdehyde (MDA) concentration, and for stress related enzymes viz., super oxide dismutase (SOD) and peroxidase (PO) activity, when the relative water content in stressed control plants reached 60–65%. The results indicate that level of stress factors was less in the plants which received both seed priming with RC and foliar spray with 204 PC, showing high cell membrane integrity (39.995±0.503), reduced proline content (20.000±0.555 mg L-1), reduced MDA content (1.0872±0.002 μmol g-1 tissues), higher SOD (1.165±0.013 activity g-1), and lower PO (103.183±0.212 activity g-1min-1) activity. Expression analysis of stress inducing genes, namely SlNCED3, SlAREB, and SlERF024 in leaf samples of tomato was conducted by real-time PCR. The SlAREB showed an upregulation of 1.16 fold and 4.64 fold in control plants after 3 and 6 hours of drought induction, while in the treatment with seed priming and foliar spray, it was downregulated up to 6 hours. In the case of SlERF024 gene, it showed downregulation up to 6 hours after drought induction. SlNCED3 demonstrated that plants treated with seed priming and foliar spray was drought tolerant as they were upregulated after 6 hours of drought induction. The present study reveals that seed priming of tomato with EPS producing rhizosphere consortium of Preistia aryabhatti (TRE4), Enterobacter cloacae (TRE8), and Bacillus pumilus (TRE21) and foliar spray with 2% solution of EPS producing phyllosphere consortium of Bacillus stercoris (TPE8), Stenotrophomonas pavanii (TPE9), and Pantoea dispersa (TPE13) can induce drought stress tolerance in tomato plants. 2Item Induction of embryogenic calli from immature ovaries in coconut (cocos nucifera L.)(Department of Molecular biology and biotechnology , College of Agriculture, Vellayani, 2024-02-01) Vyshnavi, K; Anuradha, TThe study entitled " Induction of embryogenic calli from immature ovaries in coconut (Cocos nucifera L.)" was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2022-23. The objective of the study was induction of embryogenic calli from immature ovary in coconut variety West Coast Tall (WCT) and effect of sodium butyrate in callus induction through histone deacetylation. In the present study, immature ovaries from explants of maturity indices 4 to -6 were extracted and surface sterilized using 3% sodium hypochlorite, 0.2% mercuric chloride, and 70% ethanol. They were inoculated into basal Y3 medium containing different plant growth regulators such as 2,4-D, TDZ, and picloram. The results showed that out of all the maturity indices tried (-4 to -6), inflorescence of maturity index -4 showed a maximum response in callus induction, and among different callus induction medium (CIM) tried CIM with 100 μM 2,4-D showed maximum response (47.28%) to embryogenic calli initiation. The time taken for embryogenic calli-like structure development was 45 days. To analyze the effect of sodium butyrate (SB) in callus initiation, the excised immature ovaries were inoculated into standardized CIM (Y3 + 100 μM 2,4-D) with different concentrations of SB. The results displayed that, compared with the CIM, the induction rates of embryogenic calli were significantly increased (66%) and the time taken for embryogenic calli-like structure development was significantly reduced (20 days) with the addition of 100 μM SB. The gene expression analysis shows that gene expression of the HDA6 gene was reduced in CIM with SB when compared to CIM. The increased embryogenic response of immature ovaries to CIM with SB as well as the reduced time taken for the embryogenic response can be correlated with the downregulation of the HDAC gene.Item Molecular cloning of key genes involved in piperine biosynthesis pathway from piper sp.(Department of Molecular biology and biotechnology , College of Agriculture, Vellayani, 2023-10-03) Devika Babu.The study entitled “Molecular cloning of key genes involved in piperine biosynthesis pathway from Piper sp.” was carried out at Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani during the year 2022-2023. The objective of the study was molecular cloning and sequence comparison of piperic acid CoA ligase and piperine synthase genes involved in piperine biosynthesis from selected Piper sp. Bush pepper plants of Piper nigrum varieties Panniyur 1 and Panniyur 4; Piper longum and Piper colubrinum were used for the study. The total RNA was isolated from the leaf and immature fruits of Panniyur 1, Panniyur 4, P. longum and P. colubrinum plants. Piperine synthase and piperic acid CoA ligase were amplified from each cDNA samples using designed primers of Piperine synthase and piperic acid CoA ligase gene. Amplicons of size approximately 1000 bp and 150 bp were obtained for leaves of Panniyur-1, Panniyur-4 and Piper colubrinum, whereas only 150 bp amplicon was obtained for leaves and immature fruit of Piper longum and spikes of Panniyur-1, Panniyur-4 and Piper colubrinum. Amplicon of approximately 1000 bp size was obtained from the leaves of Panniyur-1, and Panniyur-4. No bands were obtained for the leaves and immature fruits of Piper colubrinum and Piper longum and spikes of Panniyur-1, Panniyur-4 and Piper longum. Since intact bands of expected size was not obtained on the amplification of cDNA from Piper longum using designed primers, it is concluded that, there could be variation in the primer binding site of Piper longum genomic sequence, which was later confirmed by analysing the transcript sequence. PCR product was cloned into pGEM®-T vector and sequenced. The sequence obtained was subjected for in silico analysis. Nucleotide sequence of piperine synthase and piperic acid CoA ligase was found to contain 1018 bp and 970 bp respectively. The BLAST analysis of piperine synthase nucleotide sequence showed 99% similarity with piperine synthase reference gene in NCBI database. Amino acid sequence from Panniyur 1, Panniyur 4 and P. colubrinum was subjected for BLAST analysis and showed a similarity of 48 % with benzyl alcohol O-benzoyltransferase in Coffea arabica. BLAST analysis of amino acid sequence from Panniyur 1 and Panniyur 4 showed 65% similarity with acyl-CoA ligase of Macadamia integrifolia. Eventhough protein sequences of reference gene and query sequence of piperine synthase and piperic acid CoA ligase are similar, there is a substitution of amino acids at distinct regions. Meanwhile, P. colubrinum showed distinct sequence variation at the active sites of piperine synthase gene. The phylogenetic analysis of piperine synthase gene from different Piper sp. showed relation with Zingiber sp. and Rhodamnia sp. Phylogenetic tree with piperic acid CoA ligase from Piper nigrum varieties formed a separate lineage from CoA ligase enzyme from Camellia sp, Telopia sp and Macadamia sp. with a common branch point. Tertiary structure predicted were similar for Panniyur 1 and Panniyur 4, whereas differed for P. colubrinum due to sequence variation. Piperic acid CoA ligase protein from Panniyur 1 and Panniyur 4 were found to be similar. The stability of the predicted tertiary structure was determined using Ramachandran plot, which was found to be stable. The present study revealed the sequence information of piperine synthase gene from two cultivated varieties Panniyur 1, Panniyur 4 and a wild variety, P. colubrinum and piperic acid CoA ligase gene from Panniyur 1 and Panniyur 4. Although the sequences are similar to the reference sequence of Indonesian black pepper cultivar, distinct coding variants and single nucleotide polymorphisms that lead to substitution of certain amino acids was detected in both Indian cultivars and wild sp. As these variations affects the protein function and activity, future work will be directed towards the recombinant expression and further validation of the protein.Item Nano-PCR for the detection of tomato leaf curl virus(Department of Department of Molecular biology and biotechnology , College of Agricultureand botechnology , College of Agriculture,Vellayani, 2023-09-21) Devika, P P.The study entitled "Nano-PCR for the detection of Tomato leaf curl virus (ToLCV)" was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2022-23. The objective of the study was to evaluate the improvement of detection efficiency of Tomato leaf curl virus (ToLCV) in Solanum lycopersicum L. by NanoPCR by the inclusion of magnesium oxide and silver nanoparticles. ToLCV – partial CP gene cloned in plasmid DNA (pMD 20-T) (Athira et al., 2022) and maintained in E. coli cells (strain DH5-α) was used for standardization of concentration of nanoparticles for viral detection in PCR. Plasmid DNA was isolated from overnight grown cultures of E. coli using alkaline lysis method (Birnboim and Doly, 1979), and the good quality was confirmed using nanodrop spectrophotometric analysis. A universal primer for geminiviruses designed using the CP gene was used for viral detection (Deng et al., 1994). The minimum concentration of plasmid DNA at which virus amplification is obtained using PCR was standardized as 1.5ng and was used for further treatments using nanoparticles in PCR. Silver nanoparticles (AgNPs) of size 20 nm (Sigma Aldrich, USA) and magnesium oxide nanoparticles (MgONPs) of size ≤ 50 nm (Sigma Aldrich, USA) was used for the study. In treatments 1-4, different concentration of AgNPs (1 ng/µL, 2.5 ng/µL, 3 ng/µL, 3.5 ng/µL and 4 ng/µL) alone was used in PCR. In treatments 5-8, different concentration of MgONPs (100 ng/µL, 150 ng/µL, 200 ng/µL, and 250 ng/µL) was used in PCR. Combinations of AgNPs and MgONPs (3 ng/µL +200 ng/µL, 3 ng/µL +150 ng/µL, 2.5 ng/µL +200 ng/µL, 2.5 ng/µL +150 ng/µL) were used for treatments 9-12. In treatments 13-16, different concentrations of MgONPs (100 ng/µL, 200 ng/µL, 250 ng/µL, 275 ng/µL, and 300 ng/µL) by replacement of magnesium chloride (MgCl2) in PCR buffer were tried. Combinations of AgNPs and MgONPs (3 ng/µL +275 ng/µL, 3 ng/µL +250 ng/µL, 2.5 ng/µL +275 ng/µL, 2.5 ng/µL +250 ng/µL) by the replacement of MgCl2 in PCR buffer were used for treatments 17-20. PCR was performed at 95⁰C for 3 min followed by 34 cycles of 95⁰C for 30s, 53⁰C for 90s, 72⁰C for 90s, and final extension at 72⁰C for 10 min. Control was kept without any nanoparticles. Three 100 replications were done. The efficiency and specificity of PCR were checked by analyzing the intensity of the expected amplicon (500bp) in agarose gel electrophoresis and comparing the band area with that of the control using Image Lab/ImageJ software. The inclusion of AgNPs and MgONPs in PCR reaction mix at concentrations of 3 ng/µL and 200 ng/µL respectively, exhibited a 4-fold and 7.6-fold increase in the intensity of the band. Simultaneous inclusion of both AgNPs and MgONPs at concentrations of 3 ng/µL and 200 ng/µL respectively in PCR exhibited a 4.5-fold increase in the intensity of the band. The inclusion of MgONPs (275 ng/µL) replacing the MgCl2 in PCR buffer resulted in a 13-fold increase in band intensity and the simultaneous inclusion of both AgNPs and MgONPs replacing the MgCl2 in PCR buffer exhibited a 12-fold increase in band intensity. The inclusion of nanoparticles in PCR resulted in the production of the visible band even at 25 cycles, thereby reducing the duration of PCR by 26%. The results were further confirmed by using genomic DNA isolated from ToLCV-infected tomato plants as the template for PCR, by the inclusion of MgONPs (275 ng/µL) replacing MgCl2 in PCR buffer with optimized PCR cycles (25 cycles). Leaf samples for the isolation of genomic DNA of ToLCV-infected tomato plants were collected from Instructional Farm, College of Agriculture, Vellayani. The modified cetyltrimethylammonium bromide (CTAB) method was performed for isolating genomic DNA and the good quality was confirmed using nanodrop spectrophotometric analysis. To conclude, the inclusion of MgONPs at a concentration of 275 ng/µL replacing MgCl2 in PCR buffer, exhibited maximum improvement (13-fold increase) in the sensitivity of PCR. The cycle number in PCR is reduced to 25 cycles, thereby decreasing the duration of PCR by 26%. Evaluation of ToLCV-infected samples at different stages can be studied by challenge inoculation to make detection possible at the earliest stage for diagnostic purposesItem Identification of simple sequence repeat (SSR) markers linked to high temperature tolerance in rice (Oryza sativa L.) by bulked segregant analysis(Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, 2023-08-24) Aparna, K.; Beena, RThe study entitled “Identification of Simple Sequence Repeat (SSR) markers linked to high temperature tolerance in rice (Oryza sativa L.) by bulked segregant analysis” was conducted at the Department of Molecular Biology and Biotechnology and the Department of Plant Physiology, College of Agriculture, Vellayani, Thiruvananthapuram during 2022-23. The objective of the study was to evaluate the F3 population of Uma X NERICA-L-44 for identifying SSR markers linked to high temperature tolerance in rice by bulked segregant analysis and to establish the role of the genes associated with them in heat tolerance. Seeds collected from 46 F2 segregants (23 tolerant and 23 susceptible) of Uma X NL-44 along with their parents were raised and transplanted to pots after 18 days of sowing in the Kharif season of 2022. These 48 lines were kept under normal conditions up to the maximum tillering stage and then transfered to polyhouse conditions where it was subjected to a high temperature of 38-40 ⁰C. Phenotypic evaluation was done for plant height, tiller number, productive tiller number, days to flowering, time of anthesis, Pollen viability, Spikelet fertility, and 100 seed weight. Based on spikelet fertility percentage, 10 extremely tolerant and 10 extremely susceptible lines were selected. DNA was extracted from the selected 10 heat tolerant and 10 susceptible lines along with the parents by the modified Cetyltrimethylammoniumbromide (CTAB) method of DNA extraction. The quality and quantity of extracted DNA were checked by using agarose gel electrophoresis and spectrophotometric analysis. The DNA samples were screened by using 55 SSR primers distributed across the rice genome. Out of 55 SSR primers, 18 of them showed polymorphism between the parents. Then equal quantity of DNA was pooled to make heat tolerant and susceptible bulks. The bulked DNA samples were screened using the 18 SSR primers that have shown polymorphism between parents. The polymorphic markers between the tolerant and susceptible bulks were used to study the segregation of the alleles in the individual lines constituting the tolerant and susceptible bulks. Through bulked segregant analysis (BSA), 10 SSR markers were found polymorphic between tolerant and susceptible bulks and their individual lines. It revealed the possible presence of genetic loci for heat tolerance in those locations. Out of the 10 SSR markers identified in the BSA (RM337, RM10793, RM242, RM5749, RM6100, RM490, RM3475, RM470, RM473, and RM556), nine of the markers have been previously reported for heat tolerance traits. RM337 is newly identified in the present study. The genes in the 200 kb vicinity of the RM markers were retrieved from the Rice Annotation Project database. To get a deeper insight into how these genes participate in heat tolerance, gene annotation, gene ontology (GO) enrichment analysis, and trait ontology (TO) were performed for all the significant markers. Upon screening of the loci in the proposed region, genes LOP1 (LOC_Os08g01330) and LOP2 (LOC_Os08g0112) were found to be associated with RM337. LOP1 is a NAC transcription factor that is reported to be involved in the regulation of cellulose synthesis, secondary wall biosynthesis (Os08t0103900-01), and inflorescence development. LOP2 is also known as OsMOT1, which is a molybdate transporter involved in the uptake and translocation of molybdate (Os08t0101500-01). Hence their expression was analyzed in the two rice varieties under both control and high temperature conditions. LOP1 was found to be significantly upregulated in the NL-44 variety under high temperature condition compared to the normal temperature conditions and susceptible variety, Uma. On the other hand, the gene LOP2 was found to be upregulated in both varieties under the higher temperature condition compared to their respective controls. However, the relative expression in Uma was higher than in NL-44. In the present study, the phenotypic evaluation and bulked segregant analysis using 55 SSR primers in F3 generation of Uma X NL-44 revealed that 10 SSR markers (RM222, RM242, RM337, RM470, RM473, RM490, RM556, RM5749, RM6100, RM10793) are linked to high temperature tolerance in rice. The newly identified SSR marker RM337 and associated gene LOP1 is also linked to high temperature tolerance in rice. The results demonstrate that BSA using SSR markers and gene annotation and enrichment analysis is useful in identifying genomic regions and genes that contribute to thermotolerance respectively. Also, these F3 lines can be used for the development of high temperature tolerant rice varieties and these markers can be used for marker assisted selection (MAS) in rice.Item Biotization using piriformospora indica for induction of in vitro floral primordia in saffron(crocus sativus L.)(Department of molecular biology and biotechnology, College of agriculture , Vellayani, 2023-09-25) Midhukrishna; Smitha BhasiThe study entitled "Biotization using Piriformospora indica for induction of in vitro floral primordia in saffron (Crocus sativus L.)" was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2022-23. The objective of the study was to evaluate the effect of biotization using Piriformospora indica for culture establishment and induction of in vitro floral primordia in saffron. The explants namely corms collected from farmer’s field, Jammu were used for the study. The pure cultures of P. indica were collected from the Department of Agricultural Microbiology, College of Agriculture, Vellayani. The effect of biotization of corms using P. indica was evaluated both in potting medium (Treatment 1) as well as in Murashige and Skoog (MS) medium (Treatment 2). Non-biotized corms were used as control (Treatment 3). Corms were planted in sterile potting medium (perlite and compost in 1:1 ratio) during June and kept in shade conditions for 3 months. However, no sprouting or root induction was noticed and later the corms got dried due to fungal contamination. Another strategy attempted was inducing in vitro rooting and further biotization. For this, the corms were kept at 16ºC during August-October. 100% sprouting was observed in 50 days of treatment and the sprouts thus induced in vivo were maintained at 18ºC for elongation. Further, 120-day-old elongated sprouts were cultured in basal MS medium for root induction. Healthy roots appeared in 3 days and in vitro rooted plants were transferred to potting media incorporated with P. indica mycelium. However, roots of all samples got dried in a week. For attempting biotization in vitro, full corms and 1cm3 cut corms were used for initiating sterile cultures. Sprouts were initiated within 15 days from 1cm3 cut corms cultured in MS medium supplemented with 1.5ppm BAP (6 benzylaminopurine). Further, the sprouts induced both in vitro and in vivo were subjected for elongation in basal MS medium. Significant increase in shoot length was noticed in sprouts induced in vivo (kept at 16ºC) compared to sprouts induced in vitro. Flower buds were induced from elongated sprouts by sub culturing in MS 81 medium and 50% sprouts that were induced in vivo, developed in vitro floral buds. However, no floral buds were observed in elongated sprouts induced in vitro. Biotization of in vitro cultures (Treatment 2) was carried out using P. indica agar discs and derivatives of P. indica. 130-day-old elongated sprouts having in vitro roots were inoculated with P. indica agar discs in MS medium for colonization. However, colonization was not observed under microscopic analysis. 165-day-old elongated sprouts were also treated with derivatives of P. indica and compared with control. Elongated sprouts treated with derivatives of P. indica exhibited a significant increase in plant height compared to control. These sprouts were further sub-cultured for a period of 7 weeks in MS medium for inducing multiple shoots. 165-day-old elongated plantlets treated for 80 days with derivatives of P. indica produced more number of multiple shoots per corm compared to control. Further, the multiple shoots developed were cultured in MS medium supplemented with 9% sucrose for inducing in vitro cormlets. Multiple shoots pre-treated with P. indica produced larger and more number of cormlets compared to control. To conclude, sterile potting media (perlite-compost) was found not suitable for the establishment and growth of corms under field conditions of Vellayani region. Storage of corms at 16ºC during dormant stage is found critical for in vitro growth, rooting and flowering in saffron. In vitro treatment using derivatives of P. indica in saffron showed positive responses with respect to plant height, number of multiple shoots, number, and size of in vitro cormlets. Hence, derivatives of P. indica can be used for better in vitro establishment of cultures, multiple shooting, and in vitro cormlet production in saffron.Item Phytochemical profiling and genetic diversity analysis of Mangifere indica Linn. varieties of Kerala(Department of Biotechnology, College of Agriculture, Vellayani, 2020) Bona Sajimon; Rameshkumar, K BThe study entitled “Phytochemical profiling and genetic diversity analysis of Mangifera indica Linn. varieties in Kerala” was carried out at the Phytochemistry and Phytopharmacology Division of Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram during the academic year 2019 – 2020. The objective of the study was to map and authenticate selected local varieties of Mangifera indica in Kerala through phytochemical profiling of leaf volatile chemicals and the genetic diversity analysis using SCoT marker polymorphisms. Essential oils were obtained from the mature leaves of ten local mango varieties through hydrodistillation using a Clevenger-type apparatus and were analysed using GC-MS. A total of 55 volatile compounds ranging from 92.2 % to 98.0 % were identified from the ten varieties studied. Monoterpenes and sesquiterpenes were the major class of compounds among the identified volatiles. The prominent ones from this terpene classes include bicyclogermacrene, α-gurjunene, α-pinene, α-phellandrene, E-caryophyllene, terpinolene, δ-3-carene, limonene, α-humulene, germacrene D, β-selinene and germacrene B. While the statistical analysis of the varieties using their phytochemical composition clustered Vellari and Kilichundan as the nearest neighbours, Moovandan and Pulinji emerged as two distinct varieties from others in comparison. SCoT Primers proved to be a fine tool in studying the genetic diversity of M. indica varieties with an average polymorphism of 82% among the ten studied varieties. The varieties Kilichundan, Moovandan, Neelam and Kottoorkonam appeared as the ones with highest genetic similarity. Though the phytochemical and genetic diversities observed among the varieties are not directly linkable, the analyses helped to establish fine distinction between the varieties.Item Development of molecular markers for blight disease resistance in taro using bioinformatics tools(Department of Plant Biotechnology College of Agriculture, Vellayani, 2018) Athul, V S; Sreekumar, JDevelopment of molecular markers using sequential information publicly available in the biological databases has enhanced their credibility over the years. The study entitled “Development of Molecular markers for blight disease resistance in taro using bioinformatics tools” was conducted at the Central Tuber Crop Research Institute (CTCRI) during 2017-2018. The objectives of the study included the development and evaluation of various Single Nucleotide Polymorphism (SNP) and Simple Sequence Repeats (SSR) prediction pipelines, computational prediction and validation of the molecular markers for blight disease resistance in taro. The preliminary data set for the study was obtained from the Sequence Read Archive (SRA) section of NCBI. A total of 6,479,882 sequences obtained initially were reduced to 6,319,834 after pre-processing. The processed sequences were reduced to 79,608 sequences after de novo assembly and were finally assembled to 8547 contigs and 59,242 singlets. The contigs were then processed with various prediction pipelines to predict SSRs and SNPs. The tools, QualitySNP and AutoSNP were employed to detect the SNPs present within the contig sequences. The efficiency of these tools in determining the number of synonymous and non-synonymous SNPs was also analyzed. The tools, MISA and SSRIT were used to detect the SSRs within the sequences. The efficiency in predicting more number and types of reliable repeats were considered. The analysis was done with a wide range of repeats such as mono-, di-, tri-, tetra-, penta-, hexa-, and poly repeats and their numbers. QualitySNP identified 518 synonymous and 44 non-synonymous SNPs from the 8547 contigs. MISA identified 967 mono-, 1484 di-, 558 tri-, 14 tetra-, 2 penta-, 9 hexa-, and 393 compound SSRs. Five SNP and SSR primers were designed and synthesized from the contigs containing SSRs and SNPs. The synthesized SNP and SSR primers were then validated against tolerant and susceptible varieties of taro leaf blight. Among the primers synthesized the SSR primer CeSSR4 and SNP primer CeSNP3 were capable of differentiating leaf blight resistant and susceptible varieties. The markers need to be analyzed further with a large number of samples to develop them as a marker for taro leaf blight. Once analyzed, they could be used in marker-assisted selection and breeding programmes of taro.Item Identification and characterization of viruses in sweet potato (Ipomoea batatas (L.) Lam.)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2015) Jayalekshmi, V S; Makeshkumar, T