1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Induction of early floral meristem in saffron (Crocus sativus L.)(Department of Molecular Biology and Biotechnology, College of Agriculture,Vellayani, 2024-02-05) Aparna, S V.; Smitha BhasiThe study entitled "Induction of early floral meristem in saffron (Crocus sativus L.)" was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani during 2023-2024. The objective of the study was to assess the effect of various inducers in inducing early floral meristem transition, morphological analysis of different stages of bud development and differential expression of key genes involved in floral meristem transition in saffron. The experimental design followed was CRD with twenty treatments and three replications each. The explants (corms) were collected from farmer's fields in Pampore, Kashmir and subjected to a rigorous three-step surface sterilization process using 0.02% bavistin (20min), 0.04% mancozeb (20min), 1% sodium hypochlorite bleach (30min) and a final dip in 1.5% mercuric chloride solution. Surface sterilised corms were placed on Murashige and Skoog medium supplemented with various concentration of different inducers viz., GA3 (T1-T3), IAA (T4-T6), Zeatin (T7-T9), Glucose (T10-T11), Fructose (T12-T13), Sucrose (T14-T15), KNO3 (T16-T17) and Paclobutrazol (T18-T19) along with control (T20-Basal MS medium). The cultures were maintained under 24ºC temperature, 16/8hrs photoperiod with 60% humidity. 100% sprouting was noticed within 14 days of treatment using 40 mg L-1 GA3 followed by 30 mg L-1 GA3 (19 days), 10% sucrose (35 days), 5% sucrose (38 days), 10% glucose (37 days) and 5% glucose (37 days) whereas the control plants took around 45-50 days for sprouting. The bud transition from stage 1 to stage 3 was prominent in treatment using 40 mg L-1 GA3 followed by 30 mg L-1 GA3 compared to other treatments. The present study showed 40 mg L-1 GA3 as the best treatment for inducing early floral meristem in saffron. Morphological analysis focussed on the three development stages of bud based on its length viz, stage 1 (length of bud ≤ 1mm), stage 2 (length of bud between 1.5-2.0 mm) and stage 3 (length of bud ≥ 3mm) was carried out. Stage 1 was further divided into stage 1 (a) and stage 1 (b) based on morphological differences noticed around the basal region of the bud. Histological sections revealed undifferentiated floral primordium in stage 1(a) representing the vegetative phase, whereas stage 1(b) showed floral primordia initiation, representing the transition to the floral bud stage. Histological sections of stage 2 revealed prominent floral bud differentiation and initiation of perianth primordia which expanded and elongated in stage 3 accompanied by pistil primordia initiation. Molecular analysis investigated the differential expression of key genes involved in floral meristem transition, such as SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), FLOWERING LOCUS T (FT), SEPALLATA 3 (SEP3) and CHROMATIN REMODELING 4 (CHR4). SEP3 is reported to form complexes with other floral homeotic genes to initiate differentiation of all floral organs viz sepals, petals, stamen and carpel. The differential expression analysis revealed a significant (10-fold) upregulation of SEP3 during stage 2, which correlates with the prominent floral meristem differentiation observed in stage 3, where perianth and pistil primordia became more prominent. CHROMATIN REMODELING 4 (CHR4) is a positive regulator of floral meristem transition and is also associated with the epigenetic silencing of FLC (FLOWERING LOCUS C), a crucial MADS-box transcription factor (TF) that negatively regulates floral transition. A 1.5-fold upregulation of CHR4 during the transition from stage 2 to stage 3 could be correlated with the active floral differentiation. The present investigation reveals that GA3 and sugars can be used for inducing early floral meristem in saffron. Morphological analysis confirmed that stage 1(b) marks the initiation of floral transition. Additionally, upregulation of floral meristem identity genes was observed during the floral meristem transition phase.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) 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 RNA mediated resistance to Yellow vein mosaic virus in okra(Centre for Plant Biotechnology and Molecular Biology, College of Agriculture, Vellanikkara, 2021) Kelkar Vipul Ganesh; Deepu MathewOkra (Abelmoschus esculentus L. Moench, Malvaceae) is one of the leading vegetable crops in hot and humid tropics. Unfortunately, this climate is conducive for many of the pests and diseases. Okra is susceptible to viruses such as Yellow vein mosaic virus (YVMV) and Enation leaf curl virus (ELCV), belonging to the genus Begomovirus (family Geminiviridae). Because of the favourable conditions prevailing in the coastal region, the losses in Kerala state are 60-100%, depending upon the stage of plant growth and the severity of infection. RNAi is one of the promising molecular biology approach against the viral diseases. Keeping the above facts in view, the present study “RNA mediated resistance to Yellow vein mosaic virus in okra” was taken up at the Centre for Plant Biotechnology and Molecular Biology, CoA, Thrissur from September 2017 to May 2021. The high yielding and YVMV susceptible popular okra cv. Salkeerthi was selected for the development of resistance using RNAi mechanism. Total DNA was isolated from the YVMV infected plant and part of the βC1 gene (187 bp) of the virus was amplified using primers VβC1F and VβC1R. Sequence information of PCR product has revealed that the gene is 90-95% identical with the Indian isolates. The βC1 gene sequence was analysed using IDT software and 10 siRNAs were found at three different position (19-44, 34-59, 99-124 bp). Through Restriction Mapper, it was confirmed that the sequence selected for the preparation of sense and antisense strand, do not possess recognition sites for SmaI, HindIII and MauBI restriction enzymes which are present in the pRNAiLIC vector. The output of VSupPred revealed that the fragment does not contain any Viral Suppressor Regions (VSRs), with a high prediction score (0.625). The hairpin RNAi construct harbouring the region of βC1 gene of β satellite of Begomovirus of okra was generated using pRNAi-LIC (CD3-1285) vector. The SmaI digested plasmid produced three fragments, vector backbone (9842 bp), Pdk intron (1641 bp) and ccdB gene (614 bp) and the digested plasmid was treated with dTTP. Product-1 was PCR amplified (215 bp) with VLIC1 and VLIC2 primers, using the DNA from YVMV infected plant as template. Product-2 was PCR amplified (243 bp) with VLIC3 and VLIC4 primers using product-1 as template. Product-1 and product-2 were eluted from the gel and treated with dATP. The dATP treated PCR products and dTTP treated SmaI digested plasmid were mixed together and ligated by incubation at 65ºC for 5 min. followed by 22ºC for 15 min. Ligated product was successfully transformed in competent cells of E. coli (DH5α) and incubated on LB medium containing Kanamycin and Chloramphenicol. Colony PCR was performed, the transformation efficiency was found to be 80%. Plasmid was isolated from the positive DH5α colony and sequenced using the primers VLIC5 and VLIC6. The sequence data had shown that both sense and antisense strands are at right position and direction. Plasmid containing ihpRNA-βC1 cassette was successfully transformed into the competent cells of Agrobacterium (GV3101) and incubated on LB medium containing Kanamycin, Chloramphenicol and Rifampicin. Colony PCR was performed, the transformation efficiency was found to be 100%. Plasmid was isolated from the positive GV3101 colony and sequenced using the primers VLIC5 and VLIC6. Sequence data has further confirmed that both sense and antisense strands are at right position and direction. The ihpRNA-βC1 cassette was successfully transformed into okra cv. Salkeerthi using in planta method of Agrobacterium mediated transformation. The transformation efficiency observed was 11.42% and the transformation was confirmed by the amplification of sense strand using the primers VLIC1 and VLIC5. cDNA was prepared from the total RNA isolated from transformed and control plants. siRNA synthesis was confirmed using the primers VLIC1 and VLIC5 (400bp) and Ubiquitin gene was confirmed using the primer UBQ7 (187 bp). Silencing potential of the RNA interference of βC1 gene and the development of resistance was evaluated by keeping the 15-day old transformed and control plants along with YVMV infected plants inside containment facility, with whiteflies released into insect cage for infection. All the control plants and one transgenic plant have shown the YVMV symptoms after 10 days. Three transgenic plants were healthy with no symptoms. The present investigation was successful in the development of YVMV resistant okra plants carrying ihpRNA-βC1 using pRNAi-LIC (CD3-1285) plasmid vector. The further evaluation is needed in the coming generations for the identification of stable transgenic lines.Item Development of an in vitro regeneration system and validation of genetic stability in phalaenopsis hybrid winter spot with molecular marker(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Asha Amal Raj; Lissamma JosephItem Molecular characterization of candidate gene for pungency in Capsicum spp.(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture,Vellanikkara, 2016) Anju Viswanath; Deepu MathewChilli, also known as “Wonder spice”, has been cultivated since 3000 BC. Out of the 21 identified species of chilli, C. annuum, C. chinense, C. frutescens, C. baccatum and C. pubescens are the domesticated species. It is a major vegetable cum spice crop which can impart pungency, colour and aroma to the human foods. Pungency is one of the most important and peculiar character of all the species belonging to the genus Capsicum. Capsaicinoids are the alkaloid compounds which are responsible for pungency in chilli. Because of the nutraceutical properties possessed by these capsaicinoids, it has much importance in manufacturing several drugs. Though so many studies are conducted to understand the genetic mechanisms behind pungency, the gene action responsible for its production is still an enigma. This experiment was undertaken with the objective to assess the molecular mechanisms behind different levels of pungency in different species of Capsicum. The investigations were carried out in ten chilli genotypes namely, Ujwala, Anugraha, Byadagi Dabbi, Byadagi Kaddi, paprika Kt-Pl-19 and bell peppers Arka Gaurav and Arka Mohini (C. annuum), Vellayani Thejus (C. chinense) and White Khandari, Vellayani Samrudhi (C. frutescence). Among the genotypes Anugraha, Ujwala, Vellayani Thejus, Vellayani Samrudhi and White Kandari are pungent lines and Kt-Pl-19, Byadagi Dabbi, Byadagi Kaddi, Arka Mohini and Arka Gaurav are non-pungent lines. Good quality genomic DNA has been extracted from all the genotypes with an absorbance ratio ranging from 1.79 - 1.85 and concentration more than 1000 ng/μl. The DNA was screened with five pungency specific SCAR (Sequence Characterized Amplified Region) primers. Among the five SCAR primers used, three were specific for Pun1 locus (MAP1F/R, Pun1 1 fwd1/rev, Pun1 3 fwd/rev1) and two were specific for CS (Capsaicinoid synthetase) gene (CSF1/R2, BF7/R9). Pun1 and CS are the loci responsible for the synthesis of putative acyl- transferase and capsaicin synthase enzymes leading to the synthesis of capsaicinoids. The results revealed that MAP1F/R is the most significant primer which gave distinct amplifications in both pungent lines and non-pungent lines. A 15 bp deletion was clearly identified in the non-pungent lines compared to the pungent lines. This resultii revealed that the 15 bp deletion in the non-pungent lines is the reason for the absence of pungeny in them. The other two primers Pun1 1 fwd1/rev, Pun1 3 fwd/rev1 gave amplification only for pungent lines in C. annuum and C. frutescence respectively since Pun1 1 and Pun1 3 are the mutant alleles of Pun1 locus present in the respective species. The capsaicin, which is a capsaicinoid compound contributing about 69 per cent of pungency, is produced with the help of the capsaicin synthase enzyme produced from the CS gene. The primers specific for the CS gene have amplified only in the pungent lines. This result revealed that the nucleotide change in the primer binding region is the reason for the absence of pungency in them. The amplicon sequences of CS gene was subjected to insilico analysis such as BLASTn and Clustal Omega, which identified that the CS gene whose location was not yet confirmed also resides within the Pun1 locus. The insilico analysis has also proven that the 15 bp deletion identified in the non-pungent lines were located at the ORF3 in the Pun1 locus. This deletion in the coding region significantly affects the capsaicinoid formation for the pungency. Irrespective of the species, the deletions occurring in the coding regions of the Pun1 locus and CS gene, are the reasons for the variation of pungency levels in chillies. All the five primers attempted were promising and can be utilized to distinguish the pungent and non-pungent lines even in the seedling stage and hence in marker assisted selection (MAS). Identification of the location of CS gene inside the Pun1 locus is the most striking finding of this study. From this it can be infered that Pun1 locus, which is in the chromosome 2 of chilli is the major deciding locus for the production of capsaicinoids.