1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Identification of molecular marker linked with bacterial wilt resistance in marigold (Tagetes erecta L.)(Centre for Plant Biotechnology and Molecular Biology, College of Agriculture , Vellanikkara, 2022-04-08) Sreekutty ,S S; Deepu MathewThe annual flower crop marigold has gained popularity due to its easiness of cultivation and wide adaptability. It is grown for loose flowers for garland making, wreaths and religious offerings and is ideal for garden display. It is a rich source of some value added compounds - essential oils, carotenoid pigments etc. Bacterial wilt is a major reason for low productivity, especially in Kerala, causing a yield loss up to 65 - 70 per cent under conducive climatic conditions. Moreover, the pathogen being soil borne, management of this disease is very difficult and development of resistant varieties is the most promising strategy. Bulk sergeant analysis (BSA) is a quick strategy to find molecular markers associated with a trait. In this, plants from segregating population are grouped according to their phenotypic response to the target trait and the marker pattern will be associated with the expression. Usually, F2 population has been used for BSA because it provides the best recombination and segregation in the population so that extreme phenotypes can be easily distinguished upon artificial inoculation (Michelmore et al., 1991). The study entitled “Identification of molecular marker linked with bacterial wilt resistance in marigold (Tagetes erecta L.)” was carried out at the Centre for Plant Biotechnology and Molecular Biology and Department of Floriculture and Landscape Architecture, College of Agriculture, Thrissur during 2019 to 2021. The objective of this research programme was to identify inter simple sequence repeat (ISSR) marker for resistance to bacterial wilt disease in marigold (Tagetes erecta L.). To develop the mapping population, pollen from the resistant line M1 was used to pollinate the susceptible cv. Double Yellow. F1 seeds were collected and the hybrids were found to have moderate resistance. Flowers of F1 plants were selfed by bagging and the seeds obtained were sown to raise the F2 population. Two hundred and four F2 plants, resistant and susceptible parents were artificially screened (using fresh bacterial wilt inoculum having an OD value of 0.9 at 600 nm) for bacterial wilt resistance and the most susceptible and most resistant plants in F2 were identified. Wilting in plants was checked daily and infection was confirmed by ooze test. DNA was isolated from the parents and 10 plants each from most susceptible and most resistant F2 plants and S- and R-bulks were prepared. Fifty ISSR primers were initially screened and 21 primers yielding amplification were selected for BSA. BSA was carried out using parental DNA and Sand R- DNA bulks. A total of 179 amplicons were produced from 21 primers in ISSR marker analysis. From these 11 primers, yielded 23 polymorphic bands and four molecular markers were able to produce eight polymorphic bands segregating with bacterial wilt resistance. The markers ISSR 12 (750 bp), ISSR 16 (370 bp), ISSR 30 (150 and 800 bp) and UBC 866 (400 and 350 bp) were found segregating with the expression of resistance. Of these, markers ISSR 12 and ISSR 30 were associated with the susceptibility and the rest were associated with the resistance. Six markers identified in this study through BSA can be used in marker assisted selection for bacterial wilt resistance in marigold.Item Targeting silencing of Root architecture associated 1 gene in rice using CRISPR/Cas9 system(Department of Molecular Biology and Biotechnology, Center for Plant Biotechnology and Molecular Biology, Vellanikkara, 2024-12-12) Arpitha Acharya; Abida, P.SRice (Oryza sativa L.) is the second most cultivated crop, feeding around two-thirds of the population after wheat worldwide (Pirdashti et al., 2009). The semi-aquatic nature of rice makes it more susceptible to water stress (Lafitte et al., 2006). Drought stress is the major obstacle in rice production, affecting around 45% of agricultural areas worldwide (Ambavaram et al., 2014; Todaka et al., 2015; Heinemann et al., 2015). Climate change has resulted in unpredictable, more frequent, and distressing weather patterns, which are likely to continue with increasing global warming (IPCC, 2023). The root structure of a plant plays a significant role in scavenging limited resources and coping with stressed conditions, remains unexplored. Researchers have identified genotypes with deep rooting habits that had a better edge towards growth and survival in stressful conditions before the outbreak of stress period exhibit better productivity (Venuprasad et al., 2002). The ROOT ARCHITECTURE ASSOCIATED 1 (OsRAA1) gene belongs to a new small protein family having GTP binding activity that negatively regulates a wide range of cellular processes and can be controlled by phytohormone signaling, particularly auxin (Ge et al., 2004; Xu et al., 2010). According to Ge et al. (2004), constitutive overexpression of the gene led to an increased number of adventitious roots, reduced gravitropic response, and reduced growth of primary roots in rice. The presence of two auxin response elements (AuRE) suggests that the OsRAA1 gene is probably regulated by auxin (Ge et al., 2004). Xu et al. (2010) found that degradation of RAA1 protein is associated with Anaphase Promoting Complex (APC), ascertaining a functional association between RAA1 and APC/C complex. RAA1 was established as a cell cycle candidate and an APC/C substrate for proteolysis. Degradation of RAA1 by the ubiquitin-proteosome structure is necessary for the transition of the cell cycle to anaphase during root growth in rice (Xu et al., 2010). Although a lot of research conveys the biochemical and regulatory role of OsRAA1, several gaps exist in comprehending the function of the OsRAA1 gene under various stress conditions. Thus, the main goal of this study is to knock out the OsRAA1 gene using CRISPR/Cas9 system to produce mutant rice lines with deeper rooting traits. In the current study, Oryza sativa ssp. japonica cv. Nipponbare was used as plant material. The sequence of OsRAA1 was retrieved from the Rice Annotation Project Database (RAP-DB) and Rice Genome Annotation Project (RGAP). The Locus ID of OsRAA1 was identified from RAP DB. The spacer sequence or guide sequences for the sgRNA that target potential protospacers were designed using the CRISPR-P v2.0 portal. Based on the GC content (60-80%), on-score value, off-target sites, and location in the genome, two 20 bp length gRNAs were selected. The gRNAs located towards the 5’ end of the coding sequence of the gene with fewer off target sites and located mostly on the first or initial exons, were preferred. The secondary structures of the gRNAs were also validated using RNA secondary structure prediction tool. In this study, guide RNA constructs were developed using the pRGEB32 vector. The gRNA scaffold is the vector flanked by BsaI restriction sites, enabling easy insertion of designed spacer sequences. The CRISPR/Cas9 construct for cloning was developed by annealing and ligating the gRNAs to the pRGEB32 vector followed by cloning in E. coli strain DH5α. The putative positive clones were identified by colony and plasmid PCR and further confirmed by Sanger sequencing with M13 reverse primer. The sequences of the clones were confirmed by analyzing the results using BioEdit v7.2 software. The multiple sequence alignment in the software confirmed the presence of both the gRNAs (OsRAA1#R1 and OsRAA1#R2). The vector-gRNA constructs, pRGEB32:OsRAA1#R1 and pRGEB32:OsRAA1#R2 were confirmed positive for cloning after sequence analysis and were further mobilized into Agrobacterium strain EHA105 by freeze-thaw method (Holsters et al., 1978). Randomly selected colonies were screened for inserts by colony PCR with gRNA specific and M13 reverse primer. Expected bands of size ~450 bp were observed on 1% agarose gel. Positive colonies of OsRAA1#R1(4) and OsRAA1#R2(6) constructs in EHA105 were then used for rice genetic transformation. The calli induced from the seeds of rice cv. Nipponbare were inoculated into MS medium supplemented with 2,4-D (2.5 mg/L) for callus induction. After 2 weeks of inoculation, seeds and shoots were removed and only the callus cultures were transferred to fresh callus induction media. The established calli were transformed by co-cultivation with positive EHA colonies. To remove excess Agrobacterium load, calli were washed with a solution of sterile water containing cefotaxime and timentin. The selection of transformed calli was carried out in three steps through selection media I, II, and III for hygromycin resistance. From selection media III, only the micro-calli of the proliferating calli were transferred to regeneration media. In the future, regenerated shoots will be analyzed for mutation and the expected mutant rice lines may confer drought tolerance.Item Marker assisted backcross breeding in rice variety jyothi for drought tolerance(Centre for Plant Biotechnology and Molecular Biology, College of Agriculture, Vellanikkara, 2024-01-03) Shankar, C.; Abida, P SDrought is one of the important abiotic stressors that can substantially reduce rice production by 35 - 40 per cent. Water stress will significantly impact the growth and development of rice crops, but drought at the reproductive stage will directly affect the grain yield. In 2018, the State Disaster Management Authority, Kerala declared major rice growing districts including Palakkad and Alappuzha as drought affected. Jyothi is a high-yielding popular rice variety, widely cultivated variety across Kerala. Despite its widespread popularity, it exhibits poor spikelet fertility per cent under water deficit conditions, indicating that it is susceptible to drought. Considering the negative impact of drought stress on rice production and the losses incurred by farmers, efforts were initiated at the College of Agriculture, Vellanikara to pyramid QTLs (qDTY4.1, qDTY6.1, and qDTY9.1) for yield under drought into the rice variety Jyothi (recurrent parent) from the drought tolerant variety Chuvannamodan (donor parent). The present work was conducted during 2021-2023 with the objective of introgressing the target QTLs of interest into variety Jyothi through marker assisted backcross breeding. Genomic DNA was isolated from both parents and subjected to PCR amplification for parental polymorphism using a set of 208 microsatellites. Out of which, 85 markers were found to be polymorphic between the parents. Parental varieties were hybridized and resultant F1 hybrids were confirmed using foreground markers (RM518, RM587 and RM566). A total of 27 F1 hybrids showed heterozygous banding pattern for all foreground markers. The F1 hybrids were backcrossed with recurrent parent for the production of the BC1F1 population. Also, F1 hybrids were allowed to selfing for the production of F2 and subsequently F3 generation. Gene annotation and enrichment analysis were done using Simple Sequence Repeat (SSR) markers associated with the target QTLs of interest. It was found that 23 genes were associated with drought tolerance, spikelet fertility and root related traits at three different QTL regions. Foreground selection in 200 BC1F1 progenies along with the parents was done to identify the plants with the target QTLs. SSR markers RM518, RM587 and RM566 closely linked with qDTY4.1, qDTY6.1 and qDTY9.1, respectively were used as foreground markers. Screening of BC1F1 population revealed that 60 progenies possessed heterozygous alleles for at least one QTL. A total of 27 BC1F1 progenies including 10 with qDTY4.1 + qDTY6.1, 9 with qDTY6.1 + qDTY9.1 and 8 with qDTY9.1 + qDTY4.1 were identified to possess two QTLs pyramided. Background selection of 27 BC1F1 foreground selected progenies using 76 SSR markers, polymorphic between the parents, revealed the presence of wide variability among these progenies with respect to recurrent parent genome recovery (RPGR). The results of graphical genotyping revealed a maximum RPGR of 57.7 per cent in the BC1F1 progeny 24.165, followed by 56 per cent in progeny 1.2. Drought tolerance of 15 BC1F1 with two QTL combination along with the parental varieties was characterized under reproductive stage drought stress using thirteen traits as per standard evaluation system (IRRI, 2002), including water mining traits. All the backcrossed progenies exhibited better performance under water deficit condition than the susceptible parent, Jyothi. It was found that progeny 20.118 (qDTY4.1 + qDTY6.1) had a higher number of filled grains (FG) per panicle (41) and spikelet sterility (SS) per cent (49.38 %) compared to Jyothi with 1 FG and 99.29 % SS under drought. The progeny 1.10 (qDTY9.1 + qDTY4.1) possessed higher root length (97 cm), root volume (100 cc), root to shoot ratio (1.70), root dry weight (25.9 g) than Jyothi (82.2 cm, 76 cc, 1.4, 14.64 g). Also, progenies 23.153 (qDTY4.1 + qDTY6.1) and 3.30 (qDTY6.1 + qDTY9.1) exhibited delay of 8 days to show the leaf rolling symptoms than Jyothi with 4.2 days. The BC1F1 progenies positive in foreground selection was backcrossed with the recurrent parent and 87 BC2F1 seeds were harvested. The resultant BC2F1, BC1F2 and F3 lines with potential drought tolerant QTLs can serve as the base population for enhancing the drought tolerance in Jyothi. Further, genotypic and phenotypic evaluation of the QTL pyramided progeny in subsequent generations will lead to the development of drought tolerant cultivar in the background of popular rice variety Jyothi.Item Candidate gene analysis on self incompatibility in cocoa (Theobroma cacao L.)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2020) Sharath Prabhakaran; Minimol, J SCocoa is a perennial tree with typical plant habit and specific fruit characteristics. It is highly influenced by climate changes and growing environment, which makes long term and dynamic breeding programme necessary (Malhotra and Hubali, 2016). Physiological and genetic investigations have unveiled that the yield potential of cocoa is not yet fully exploited (Bertus, 2004). Demand for chocolate is increasing at a rate of 15-20 per cent every year. To meet this demand, more area has to be brought under cocoa cultivation using improved genetic stock. Development of superior hybrids have significantly contributed to improve the cocoa productivity in many countries (Kennedy et al., 1987; Dias et al., 2003). Cocoa hybrids showed wide adaptability, low environmental interaction and improved yield, when compared to traditional cultivars (Dias et al., 2003). Self-incompatibility is a pollination control mechanism which prevents self-fertilization. Hence, this can be exploited in hybrid production by avoiding emasculation, which is a cumbersome process (Minimol and Amma, 2013). Moreover, emasculation will damage the flowers leading to reduced success rate. Conventionally, the self-incompatibility is measured by selfing 100 flowers per tree. If no fruit set is observed, then the plant is classified as self-incompatible (Mallika et al., 2006). This is a tedious process which will reduce the pace of breeding programme. In various other crops, many candidate genes have been reported for self-incompatibility (McCormick, 1998). However, the actual sequence variations in candidate genes are yet to be studied in cocoa. Identification of appropriate genes involved in self-incompatibility will help to identify its mechanism at an early stage and quicken the breeding programme. In this study, 10 candidate genes viz. Serine Receptor Kinase (SRK), S Locus Glycoprotein (SLG), Barely Any Meristem 1 (BAM1), Barely Any Meristem 2 (BAM2), COMPASS-like H3K4 histone methylase component (WDR5a), Voltage-dependent L-type calcium channel subunit (alpha-1F), Gamete Expressed Protein (GEX1), Zinc finger AN1 domain-containing stress-associated protein 12 (PMZ), ARM repeat-containing protein (ARC1) and Hapless 2 (McCormic, 1998; Lanaud et al., 2017) were characterized. These genes were reported to have involved in self-incompatibility in other crops. Genomic nucleotide sequences from reported host plant species were retrieved from the NCBI GenBank database. Using this information, homologous gene sequences of the candidate genes in cocoa were retrieved. Primer sets targeting major exonic regions for each of the candidate genes were designed. Genomic DNA was isolated from self-compatible genotype (GVI-167 x GIV-18.5) and self-incompatible genotype (IMC20) and the candidate genes were PCR amplified. Amplified products were sequenced and the variations in the sequences between the self-incompatible and self-compatible genotypes were analyzed, in comparison with a self-compatible reference genome (Argout et al., 2010). Between the self-incompatible and self-compatible genotypes, a total of 31 different SNPs were discovered among the genes studied. All of them were found to be heterozygous at the locus either in self-compatible or self-incompatible genotype. The maximum number of SNPs, a total of 12, were found in GEX1 gene. Four SNPs each were found in genes SRK, BAM2, WDR5a and Alpha1F whereas three SNPs were found in PMZ. No variation was seen in BAM1 and ARC1. SNP locus homozygous in self-compatible and heterozygous in self-incompatible, with the corresponding locus of self-compatible reference genome can be used as potential candidate for developing markers to distinguish them. Such SNPs are identified and recommended for further validation.Item Commercial production of ginger (Zingiber officinale ROSC) microrhizomes using temporary immersion bioreactor system(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2020) Rasha Fathima, A A; Shylaja, M RGinger (Zingiber officinale Rosc.), is an important commercial spice crop grown in India from very ancient times. High seed rate of ginger (1500 kg/ha) and desiccation of seed rhizomes during storage are the problems faced by farmers in ginger cultivation. The tissue culture plants are not commercially distributed in ginger as they require an additional one more season for rhizome formation. Microrhizomes of ginger induced in vitro if used as planting materials, rhizomes can be harvested in the same season as conventional seed rhizomes and year round availability of seed material can be ensured. Hence, in vitro induced microrhizomes are included in the seed chain of ginger and there is high demand for microrhizomes for clean ginger production. The study entitled “Commercial production of ginger (Zingiber officinale Rosc.) microrhizomes using Temporary Immersion Bioreactor (TIB) system” was conducted at Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara during 2018 to 2020. The objective of the study was to develop an efficient commercial production protocol for ginger microrhizomes using a TIB system. The study was conducted in the ginger variety Athira using Plantform TIB purchased from Sweden. The multiple shoot cultures in the 5th subculture stage received from the commercial micropropagation unit, of CPBMB were used for the study. The protocol for in vitro induction of microrhizomes reported by Shylaja et al. (2016) was optimised for bioreactor production and at each stage it was compared with the conventional microrhizome production. The number of clumps/ 500ml of medium to initiate multiple shoot production, media for shoot multiplication and microrhizome induction were optimised for TIB and compared with conventional microrhizome production system. The microrhizome, root and shoot characters in microrhizome plantlets and growth of microrhizome plants after hardening were evaluated in the two culture systems. The clonal fidelity analyses of microrhizome plants derived from 8th subculture cycle were done using the specific ISSR marker as reported by Gavande, (2013). The clump size of 15 clumps/ 500 ml of medium showed higher shoot multiplication in both TIB and conventional system. The shoot proliferation in bioreactor (7.71 shoots/clump) was significantly higher than the conventional microrhizome production system (5.24 shoots/clump). Early induction of microrhizomes was observed in TIB system. In both the culture systems, microrhizome induction was faster in MS medium with 90 gL-1 sucrose. The number of microrhizomes produced in the medium in TIB varied from 87.75 to 96.75/ 500ml medium and in conventional system it varied from 84 to 88/ 500 ml medium. The microrhizome plantlets produced in TIB recorded significantly higher weight of microrhizomes, better shoot and root growth and more number of leaves compared to the microrhizome plantlets produced in the conventional system. The weight of microrhizomes, number of roots and root length were higher in microrhizome plantlets in MS medium with 90 gL-1 sucrose. The mean weight of microrhizome in the medium was 0.29 g in bioreactor and 0.18 g in conventional system. The survival of microrhizome plants after primary hardening was 94.34 per cent and after secondary hardening was 99.29 per cent in TIB. Microrhizome plants from bioreactor recorded significantly higher plant height (18.60 cm) compared to conventional system (14.80 cm). The number of leaves recorded in microrhizome plants of both the culture systems were on par and ranged from 6.97 to 7.18. Clonal fidelity analyses using specific ISSR marker revealed that there were no polymorphism in the ISSR amplification profiles in microrhizome plants produced after the 8th subculture cycle with the source mother plant and hence plants produced from both the culture systems are true to type. The protocol developed in the present study can be further modified by early bulking of the clumps, employing TIB for multiple shoot production in early culture phase and reducing the number of culture cycles so that the entire protocol period can be reduced. Evaluation of TIB microrhizome plants in high tech poly house or field and comparison of yield and quality with microrhizome plants from conventional production system also can be focused for further research.Item Commercial micropropagation of banana (Musa spp.) using a temporary immersion biroeactor system(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2020) Waghmare Vaibhav Gautam; Shylaja, M RItem DNA fingerprinting of selected cocoa (Theobroma cacao L.) varities of Kerala agricultural university(Department of Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2020) Megha Totaganti; Minimol, J SCocoa (Theobroma cacao L.), is an important tropical tree-crop belonging to the family Malvaceae. It has originated in South America (Wood and Lass, 1985) and was introduced to India, in 1798 at Courtallam in Tirunelveli district of the old Madras state (Ratnam, 1961). Now, cocoa is extensively cultivated in South Indian states and its cultivation is slowly extending to North Eastern states. Central sub-committee on crop standards has made the DNA fingerprinting data mandatory for the national release and notification of varieties. DNA markers, act as active tool to categorize and distinguish accessions and cultivars which have similar morphological characteristics. DNA fingerprinting is effectively used for cultivar identification, assessing seed purity of hybrids and to compare genetic similarities between the cultivars. DNA fingerprints act as a powerful tool to protect Plant Breeder’s Rights (PBR). In the present study, seven cocoa varieties, CCRP 3 (selection), CCRP 10, CCRP 11, CCRP 12, CCRP 13, CCRP 14 and CCRP 15 (hybrids) were fingerprinted. Molecular characterization was performed with genomic DNA isolated using modified Delloporta method (Ileana, 2005). Thirty five ISSR (inter simple sequence repeats) primers and 30 SSR (simple sequence repeats) primer combinations were screened for marker polymorphism, of which 23 ISSR and 17 SSR primer combinations were selected for further study. ISSR and SSR amplification patterns differed among the varieties and thus the DNA fingerprints from each primer combination were developed. ISSR primer UBC810 was found to distinguish CCRP 3 from other genotypes. Whereas, UBC835 and UBC857 produced unique amplicons in variety CCRP 10. Primer UBC810, UBC826, UBC841 and UBC854 generated unique amplicons and formed specific DNA fingerprints of the hybrid CCRP 11. Primer ISSR3, UBC815, UBC827, UBC854 and Oligo 05 gave highest (6) unique bands in CCRP12, whereas UBC 854 produced specific band in CCRP 13. Hybrid CCRP 14 generated unique amplicons with primers UBC855 and Oligo 07 which formed specific fingerprint of the hybrid. Similarly, CCRP15 generated three unique fingerprints with the primer UBC844 and Oligo05. In SSR marker analysis, all the seven genotypes have at least one unique band. mTcCIR40 generated unique amplicons at 250bp length in CCRP 3 and SHRSTc53 at 230bp in CCRP 10. mTcCIR10, mTcCIR8, mTcCIR11 and mTcCIR121 generated distinct bands in CCRP 11. In CCRP 12 genotype, primer mTcCIR18 (345bp), mTcCIR40 (200bp) and mTcCIR42 (210bp) produced specific band. Unique bands were generated by primers mTcCIR10(340bp), mTcCIR11 (310bp), mTcCIR12(270bp), mTcCIR22(200bp), mTcCIR24 (150bp), mTcCIR33 (300bp) and SHRSTc53 for CCRP12. DNA fingerprint generated for CCRP 14 revealed that unique bands were developed at 250bp (mTcCIR37) and 200bp (SHRSTc64). Similarly, CCRP15 developed specific fingerprint at 355 bp (mTcCIR18). ISSR amplicons shared by maximum of three varieties and SSR shared in four varieties were considered for developing final DNA fingerprint profile. The present study had facilitated to characterize the selected cocoa varieties of KAU and the data generated will be useful for varietal notification and in case of any third party litigations.Item DNA barcoding of spider mites (Prostigmata:Tetranychidae) associated with ornamental plants(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2019) Jayalakshmi Prakash; Haseena BhaskarTotal area under floriculture in India is estimated to be 0.31 million hectares with a production of 2.08 million tonnes loose flowers and 0.82 million tonnes cut flowers in 2018-19. In Kerala, Thrissur district is considered as the centre of floriculture nursery business. Majority of nurseries in the area do not maintain their own sources of mother plants and hence import planting materials from other countries. Trade of commercial ornamentals has been recognized worldwide as an important invasion pathway for non-native pests, especially insects and mites. Though mites are reported as major pests of commercial ornamental plants from different parts of India, no systematic study has been conducted so far to document the diversity of mites associated with ornamental plants in Kerala. Considering this, the present study, ‘DNA barcoding of spider mites (Prostigmata: Tetranychidae) associated with ornamental plants’ was undertaken with the objectives to generate DNA barcodes for different species of spider mites infesting commercial ornamental plants of central Kerala and to find the genetic variability among them. The study included collection and culturing of spider mites, morphology based identification and molecular characterization of selected accessions. Purposive sampling surveys were carried out in commercial ornamental nurseries and homestead gardens of Thrissur and Ernakulam districts, covering 12 ornamental plants. Mite infested leaf samples were collected, brought to the laboratory and maintained separately as isoline cultures by assigning unique accession numbers. Morphological characterization of 26 isoline cultures revealed the occurrence of six species of spider mites under two genera viz., Tetranychus and Oligonychus. The genus Tetranychus was more diverse with five species viz., Tetranychus truncatus, T. urticae, T. okinawanus, T. neocaledonicus and T. marianae. The genus Oligonychus was represented by only one species, Oligonychus biharensis Hirst. Rose recorded the highest diversity of spider mites with five species. The mite species, T. okinawanus recorded wider host range with eight host plants viz., Adenium, rose, Gerbera, chrysanthemum, orchid, cairo morning glory, marigold and balsam. All the host plants except Adenium are new host records of T. okinawanus from India. The study recorded three new host plants for T. truncatus from India viz., rose, cock’s comb and marigold. In this study, T. marianae was recorded for the first time from Kerala. For molecular characterization, DNA was isolated and COI locus of 868 bp length was amplified using universal primer, specific to COI. Polymerase chain reaction (PCR) products of 12 accessions representing five species were sequenced and in-silico analysis was carried out. Homology analysis of sequences of 11 accessions showed 90-99 per cent similarity with sequences in NCBI database, which were in consensus with morphological identification. The sequence of the accession, Ros1vk (T. marianae) did not show significant similarity with any of the sequences in the NCBI database. In this study, COI sequence of T. marianae was submitted for the first time in GenBank. Barcode gaps among the species were examined by aligning the COI sequences using Clustal Omega tool and species-specific barcodes were identified at different nucleotide positions. Pairwise distance analysis of the sequences showed intraspecific divergence ranging from 0.00 to 0.036 and interspecific divergence ranging from 0.070-0.217. Phylogenetic analysis revealed the monophyly of T. truncatus and T. urticae and the close relationship of T. marianae with T. okinawanus. The study has shown that rose harbours many species of spider mites, indicating the need for imposing strict quarantine regulations for movement of planting materials of rose to avoid entry and invasion of mites into newer areas. The potential of T. okinawanus and T. truncatus to turn invasive in Kerala’s ecosystems is also brought out. The study establishes the reliability of COI locus as a marker for species discrimination in spider mites.Item Inheritance of molecular markers linked to vascular streak dieback disease resistance in hybrid progenies of cocoa (theobroma cacao L.)(Department of Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2019) Midhuna, M R; Minimol, J STheobroma cacao L. (also known as the chocolate tree) is a major cash crop and the costliest beverage crop. Andhra Pradesh is the leading cocoa producing state in India but Tamil Nadu ranks first with an area of 26,969 ha. Vascular Streak Dieback (VSD) caused by the fungus Ceratobasidium theobromae is a serious disease in cocoa. Since it is a vascular pathogen, chemicals have little effect on disease control. The most tenable and economic technique to tackle this disease is by evolving resistant materials. Kerala Agricultural University had initiated VSD resistant breeding since 1995. Seedlings from hybridization, exhibiting high levels of resistance were selected and field established. Nineteen hybrids, exhibiting resistance to VSD (after screening for a period of thirteen years), were selected for the present study. The progeny obtained from these hybrids by crossing it among themselves were used as plant materials for the study. Two thousand two hundred and thirty seven flowers were pollinated and seven pods were obtained. About two hundred and sixty nine seedlings were grown from the seven hybridized pods in which nursery screening for disease resistance was done. Inoculum was dispensed by keeping already infected seedlings around the experimental materials. High humidity was ensured by providing over head sprinkler system. Visual screening recorded one hundred and eighty seedlings as disease resistant, fourteen seedlings as partially resistant and seventy five seedlings as disease susceptible. Three ISSR markers (UBC 811, UBC 815 and UBC 857) and one SSR marker (mTcCIR42) linked with VSD resistance gene, identified and validated from the previous studies were used for screening of the one hundred and twenty seedlings out of which one hundred and six were resistant and fourteen were partially resistant. The polymorphic band of 950 bp, which was found to be linked with the gene conferring VSD resistance was recorded in seventy seven resistant seedlings and three partially resistant seedlings, when screened with the primer UBC 811. When screened with the primer UBC 815 (750 bp) and UBC 857 (450 bp), the polymorphic marker band which was found to be linked with VSD resistant gene from the previous studies, was present in only twenty five resistant and one partially resistant seedling and twenty one resistant and one partially resistant seedling respectively. When screened with SSR marker, the 200 bp marker band, which was tagged with the VSD resistant gene was detected in fourty six resistant and six partially resistant seedlings. The ISSR marker UBC 811 and SSR marker mTcCIR42 were found to be having comparatively good percentage of inheritance among the segregating progeny screened with a mean inheritance percentage of 71.70 per cent and 48.78 per cent respectively. Flanking sequences of the ISSR markers UBC 811, UBC 857 and SSR marker mTcCIR42 were extracted from the whole genome database of cocoa. The ORFs from the flanking sequences of UBC 811 were identified to be uncharacterized proteins by using BLASTp tool. One ORF from the upstream sequence of the UBC 857 had shown identity with beta tubulin chain. Analyzing the distance between the marker and the flanking region, it was deduced that UBC 857 is a part of beta tubulin gene. Two ORFs were identified from both the upstream and downstream flanking sequences of the SSR marker. Using BLASTp tool, it was analyzed that both the ORFs showed more than 97 per cent identity to beta tubulin gene. Analysing the spacing between the marker and the flanking sequences, it was deduced that both the ORFs are part of the same gene and the SSR marker mTcCIR42 lies within the beta tubulin gene. Tubulin beta chain belongs to the microtubular component of cytoskeletal elements which provides resistance by not allowing the fungi to penetrate the outer epidermal wall of the plants, hence protecting the plants from infection. The ISSR marker UBC 857 and the SSR marker mTcCIR42 are linked to the beta tubulin gene, which provides VSD resistance by giving resistance against penetration of the plant cell by the fungus. Nineteen seedlings were identified to be having three or more markers expressed. They can be planted in the field and can be further evaluated for yield and yield contributing characters. Sequence of the beta tubulin gene can be used for primer designing, which can be used for confirmation by screening in resistant genotypes of cocoa.Item Management of biodegradable plant tissue culture lab wastes through biomethanogenesis(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2008) Abdulla Fayas, T; Rajendran, P CGenerating renewable source of energy from tissue culture laboratory waste by the process of biomethanogenesis is the focal theme of present investigation. Unlike developed countries, the developing countries are hesitant to establish more number of biotechnology/ tissue culture laboratories due to financial constraints. Easy and regular availability of biogas from TC wastes will be a boon to establish self-sustainable TC laboratory in view of present energy The biogas experimental units required for the study was designed and various treatments were employed for the biodegradation of tissue culture waste, using the methanogenic bacteria Methanobacterium ruminatium, Methanobacterium formicicum; Methanosarcina barkeri, Bactereoides ruminicola, Selenomonas ruminatium, Eubacterium tortuosum and Clostridium butyricum. Treatment involving TC waste and cow dung was also conducted for biomethanation in the present study. Quantity of gas production and its combustibility was noticed for various treatments. In bacterial treatments the quantity of gas generation was highest for Clostridium butyricum. Only treatments involving cow dung produced combustible gas. Molecular characterization, of methanogenic bacterial cultures was also done for finding the genetic similarity between them. RAPD followed by scoring . of the bands by UPGA routine showed maximum similarity between bacterial cultures of Methanobacterium ruminatium and Methanobacterium jormicicum with Methanosarcina barkeri. Physio-chemical characters like CIN ratio of the TC wastes, pH and temperature of medium and Hydraulic retention time was also observed for the various treatments. The CIN ratio of the TC wastes was found to be very low and nowhere near the optimum CIN ratio of 20-30 required for gas production. Other parameters like pH of the treatments and Hydraulic retention time was also. • noticed. The pH of the treatments involving bacterial cultures was very low, considering the normal pH of 6.8 to 7.5 required in biogas generation. The main constraints in the biogas generation were found out to be the low CIN ratio of the TC waste and the low pH of the medium. The present study indicated the possibility ofbio-gas generation from TC waste through fortification using various supplements like coconut water and coir pith which have higher CIN ratio.