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Item Glucosinolate profiling and molecular analysis of MYB28 gene for metabolome editing in Moringa oleifera Lam(Department of Plant Biotechnology, College of Agriculture ,Vellanikkara, 2024-02-29) Muhammed Ameer; Rehna AugustineMoringa (Moringa oleifera Lam.), an indigenous herb native to South Asia, recognized for its health benefits, is often referred to as "the miracle tree." Thriving in the foothills of the Himalayas, moringa belongs to the Moringaceae genus, comprising 14 known species. Each part of the plant possesses medicinal properties, contributing to antimicrobial, anti-inflammatory, detoxifying, and anticancer activities. Glucosinolates (GSLs) are stable secondary metabolites derived from sulfur and nitrogen-rich amino acids. Myrosinase, the sole known β-thioglucosidase, is responsible for driving their breakdown. The resulting product, isothiocyanates (ITCs), possesses fungicidal, bacteriocidal, nematocidal, and herbivore-deterrent qualities. The R2R3-MYB class of transcription factors significantly influences glucosinolate (GSL) biosynthesis. Studies manipulating the MYB28 gene, a key transcription factor, have been reported to regulate GSL levels in plants. GSLs are suggested to be produced in green tissues and then transported to developing reproductive tissues through the phloem. While extensive research explores the control of the GSL pathway in Brassica and Arabidopsis, in members of Brassicales like moringa, the regulatory mechanism of GSLs is yet unclear. It is reported that the GSLs or ITCs in M. oleifera are probably responsible for many of the therapeutic benefits that have long been associated with them in traditional medicine. M. oleifera is home to several unusual GSLs with unique properties. Glucomoringin, also known as 4-(α-L-rhamnopyranosiloxy) benzyl glucosinolate (4RBGS) is the most prevalent GSL in all of M. oleifera's components, especially the pulp seed, followed by 3-hydroxy- 4-(α-L-rhamnopyranosyloxy) benzyl glucosinolate (4-OHBGS). Due to the presence of a second saccharide residue in the aglycon side chain, this chemical could show biological effects that are very different when compared to those of other GSLs because of its unusual structure. Thus, one of the main goals of M. oleifera breeding is to improve nutritional and medicinal characteristics by creating high GSL lines. The objective of the current research is glucosinolate profiling, isolation and expression analysis of MYB28 gene from M. oleifera variety PKM-1 and development of CRISPR/Cas construct for functional analysis of MYB28 gene. Desulphoglucosinolate profiling using High Performance Liquid Chromatography showed a major peak of 4RBGS and a minor peak of 4-OHBGS in aerial tissues, suggesting them as the predominant GSLs. However, in roots, in addition to the benzyl GSLs, indole GSL (indol-3-ylmethyl GSL, I3M) was also detected. The highest concentration of total GSLs was found in moringa seeds (399.48 µmoles/g dry weight). This was followed by stem (63.95 µmoles/g dry weight) and flowers (30.61 µmoles/g dry weight) had the least amount of GSL accumulation. Isolation and sequence analysis of MYB28 gene in moringa proved that the sequence is diverse from that of its related species coming under the order Brassicales. Homology search using NCBI BLASTn showed 86% identity with the predicted mRNA sequence of Abelmoschus esculentus transcription factor MYB28. Bioinformatic analysis of the genomic and CDS sequences were performed using softwares like NCBI Splign, Molbiotools, ORFfinder and InterPro to characterize the MYB28 gene isolated from moringa. Expression profiling of the MYB28 gene was performed in different tissues of moringa using Real-time qRT-PCR. Highest level of MYB28 expression was found in the stem followed by immature pod tissue, flower and leaf tissue. Lowest level of gene expression was found in seeds, where no GSL synthesis occur, which act as only sink for GSLs. In order to elucidate the role of the putative MYB28 in GSL biosynthesis, a gene knock-out cassette based on CRISPR/Cas9 system was generated in the study. The genomic sequence of MYB28 obtained in the study was used to design gRNA using Cas-designer of ‘CRISPR RGEN Tools’ software. The MYB28 CRISPR/Cas9 construct was ligated to CRISPR/Cas9 binary vector pKSE401 and cloned in to E. coli strain DH5α. The positive clones were confirmed by Sanger sequencing of the plasmid DNA. The construct was further mobilized to A. tumefaciens strain GV3101. Positive clones were identified by colony PCR using vector and gRNA specific primers. The construct will be used for moringa genetic transformation. Callus and cell suspension cultures of moringa was established for moringa genetic transformation in future.Item Premeiotic ovular culture and candidate gene expression studies for in vitro gamete production in rice(Department of Plant Biotechnology, College of Agriculture , Vellanikkara, 2024-03-20) Abhishek Gangadharan; Gibence, H Rose WinnieDeveloping genetic variation and recombining the traits of interest are critical factors affecting genetic gain in genetic engineering and crop breeding programmes. The essential steps in a breeding programme, namely, hybridisation and the repeated backcrossing and selection in the field, consume considerable time. Thus, the length of breeding cycles and the steps in breeding programmes delay the timely release of crop varieties. In vitro, gamete production can reduce the time required for this and overcome seasonal impacts and space limitations. The accumulating information on the well-mapped genetic markers and cross-over information, combined with the advanced techniques of in vitro gamete development and in vitro fertilisation, can help crop breeders recombine the desired allelic combinations in a shorter period. Meiosis involves DNA replication and two nuclear division events in germ cells. During meiotic division, DNA double-strand breaks (DSBs) are generated and are repaired via a homology-dependent DNA repair pathway (HDR), resulting in cross-over mediated genetic recombination (Mercier et al., 2015; Osman et al., 2011). During meiotic progression, the somatic cells get committed to the meiotic pathway in the S or G2 phases (Ito and Takegami, 1982). Under appropriate regulation of relevant genes, a somatic cell can behave like a germ cell to undergo meiosis and produce haploid cells, which has been achieved in the animal system. High auxin, temperature, and starvation are suggested to induce meiotic progression like changes in the cells cultured in vitro. Genes responsible for the transition from mitosis to meiosis have been elucidated in several plant species, namely, Arabidopsis, Maize, Rice, etc. OSH1, OsMADS13, OsSPL, OsAM1, OsMEL2, Os Cyclin D2, OsYUC4, OsPAIR1, and OsPAIR2 are some of the genes reported to be involved or regulated during this transition. Six genes among these viz OsMADS13, OsSPL, OsMEL2, OsCyclin D2, OsPAIR1 and OsPAIR2 were selected for analysis in the current study. Except for the OsCyclin D2, a mitosis-related gene, all others are critical to meiosis. When the expression pattern of these genes in the developing panicles of rice is analysed, one can get to know whether the panicle is undergoing premeiotic, meiotic, or postmeiotic development. Itoh and co-workers have reported that the premeiotic, meiotic, and postmeiotic panicles can be identified and isolated by following the length of the panicles (Itoh et al., 2004). In the present study, twenty-two different growth regulator combinations in ½ MS media were tested to support the growth of the post-meiotic ovules of Rice (Oryza sativa L. ssp japonica cv. Nipponbare), where seven among them were found to support the growth and development of the ovules. The seven media were with the following plant hormone combinations - IBA (1 mg/L), IBA + BAP (2+1 mg/L), 2,4-D (2 mg/L), BAP (4 mg/L), 2,4-D + IBA (2+1 mg/L), 2,4-D + BAP (2+1 mg/L) and 2,4-D (4 mg/L). One of the objectives of the current study was to support the premeiotic florets of rice to complete the meiotic progression in vitro and to monitor the expression of the six candidate genes. The candidate gene expression analysis in the premeiotic florets grown in vitro showed an expected gene expression pattern in the seven different media except for the gene OsSPL. Even though the expressions of the other genes varied between the different media conditions, OsMADS13, OsMEL2, OsPAIR1 and OsPAIR2 increased many folds in expression, as expected. The expression of OsCyclin D2 was either downregulated or remained constant under culture conditions. Further, callus culture was established from the scutellum of Oryza sativa L. ssp japonica cv. Nipponbare was subjected to the same culture conditions to test for the changes occurring at the expression levels of the candidate genes. Being cell division/meiosis-related genes, the candidate gene expression changes indicate the influence of culture conditions on the cell division mode. All the candidate genes were expressed in the callus tissues. The expression pattern of meiosis-related genes observed in ½ MS media with IBA + BAP (2+1 mg/L) was similar to the post-meiotic ovule, a sign of probable meiotic induction. However, the OsMADS13 expression level, even though upregulated, was low. All other media have shown an anomalous expression pattern for the OsSPL, OsMADS13, OsMEL2 and OsPAIR1 genes. The study shows that changing the culture conditions could induce meiosis-like gene expression patterns. When a suitable culture condition is given, it may be possible to cause and complete the meiotic transition in the somatic cells of rice. However, more studies are warranted to confirm the induction of meiosis and rule out any anomalous cell behaviour in the culture. Genes responsible for the transition from mitosis to meiosis have been elucidated in several plant species, namely, Arabidopsis, Maize, Rice, etc. OSH1, OsMADS13, OsSPL, OsAM1, OsMEL2, Os Cyclin D2, OsYUC4, OsPAIR1, and OsPAIR2 are some of the genes reported to be involved or regulated during this transition. Six genes among these viz OsMADS13, OsSPL, OsMEL2, OsCyclin D2, OsPAIR1 and OsPAIR2 were selected for analysis in the current study. Except for the OsCyclin D2, a mitosis-related gene, all others are critical to meiosis. When the expression pattern of these genes in the developing panicles of rice is analysed, one can get to know whether the panicle is undergoing premeiotic, meiotic, or postmeiotic development. Itoh and co-workers have reported that the premeiotic, meiotic, and postmeiotic panicles can be identified and isolated by following the length of the panicles (Itoh et al., 2004). In the present study, twenty-two different growth regulator combinations in ½ MS media were tested to support the growth of the post-meiotic ovules of Rice (Oryza sativa L. ssp japonica cv. Nipponbare), where seven among them were found to support the growth and development of the ovules. The seven media were with the following plant hormone combinations - IBA (1 mg/L), IBA + BAP (2+1 mg/L), 2,4-D (2 mg/L), BAP (4 mg/L), 2,4-D + IBA (2+1 mg/L), 2,4-D + BAP (2+1 mg/L) and 2,4-D (4 mg/L). One of the objectives of the current study was to support the premeiotic florets of rice to complete the meiotic progression in vitro and to monitor the expression of the six candidate genes. The candidate gene expression analysis in the premeiotic florets grown in vitro showed an expected gene expression pattern in the seven different media except for the gene OsSPL. Even though the expressions of the other genes varied between the different media conditions, OsMADS13, OsMEL2, OsPAIR1 and OsPAIR2 increased many folds in expression, as expected. The expression of OsCyclin D2 was either downregulated or remained constant under culture conditions. Further, callus culture was established from the scutellum of Oryza sativa L. ssp japonica cv. Nipponbare was subjected to the same culture conditions to test for the changes occurring at the expression levels of the candidate genes. Being cell division/meiosis-related genes, the candidate gene expression changes indicate the influence of culture conditions on the cell division mode. All the candidate genes were expressed in the callus tissues. The expression pattern of meiosis-related genes observed in ½ MS media with IBA + BAP (2+1 mg/L) was similar to the post-meiotic ovule, a sign of probable meiotic induction. However, the OsMADS13 expression level, even though upregulated, was low. All other media have shown an anomalous expression pattern for the OsSPL, OsMADS13, OsMEL2 and OsPAIR1 genes. The study shows that changing the culture conditions could induce meiosis-like gene expression patterns. When a suitable culture condition is given, it may be possible to cause and complete the meiotic transition in the somatic cells of rice. However, more studies are warranted to confirm the induction of meiosis and rule out any anomalous cell behaviour in the culture.Item Elicitation of phenylpropanoid glycosides biosynthesis and expression profiling of key acteoside biosynthetic genes in Artanema sesamoides Benth (Vathomvaretti)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2022-02-21) Anusha, R; Soni , K BThe study entitled “Elicitation of phenylpropanoid glycoside biosynthesis and expression profiling of key acteoside biosynthetic genes in Artanema sesamoides Benth (vathomvaretti)” was carried out during 2019-2021, at the Department of Plant Biotechnology, College of Agriculture, Vellayani. The objective was to enhance the phenylpropanoid glycosides synthesis in the callus culture of Artanema sesamoides using elicitors like salicylic acid, yeast extract and pectin and to study their effect on the expression of acteoside biosynthetic genes PAL (phenylalanine ammonia-lyase) and HCT (Hydroxycinnamoyl-CoA Shikimate). In vitro raised seedlings of A. sesamoides were used for the establishment of callus culture in MS medium supplemented with 0.5 mgL-1 BA and 0.5 mgL-1 NAA. Two-week old callus cultures were transferred to liquid MS medium with 0.5 mgL-1 NAA and 0.5 mgL-1 BA and stabilized for 10 days. Elicitors viz., salicylic acid (SA 40 and 100μM), yeast extract (YE 1 and 2 gL-1) and pectin (100 and 200 mgL-1) were added to the suspension culture and incubatedin an orbital shaker (120 rpm) at 24°C for 3 weeks. After elicitation, callus was harvested, dried and finely powdered for analysis. The spent medium was also analysed for the PPGs. Phenylpropanoid glycosides (PPGs) were extracted using n-butanol and the extracts were evaporated to get the crude residues. The total yield of the crude residue of phenylpropanoid glycosides was maximum (44.88 mgg-1) in the suspension culture treated with pectin (200 mgL-1) and this treatment showed maximum residue (41.6 mgg-1) in the callus also. While the additionof pectin increased the accumulation of PPGs in the callus, the addition of SA and YE increased their exudation in the medium. Six important PPGs such as acteosides, artanemoside, isoacteoside, leucosceptoside, martynoside and plantainoside were identified in the butanol extracts using HPLC. Phenylpropanoid glycosides showed maximum absorbance at 330 nm and the peaks appeared within the retention time of 40 to 75 min. All the elicitors increased the synthesis of plantinoside. Treatment with SA (40µM) enhanced the content of martynoside (3.032%) and plantainoside (45.444%) in the callus by 10 and 50 folds respectively. In this study, the acteoside content was found to be more in the medium than the callus. Treatment with (SA 40µM) and YE (2 gL-1) was found to increase the acteoside content to a maximum of 10 to 17 folds in the medium. Up to 34.4% increase in the artanemoside content was observed in the medium with the elicitation by Pectin (100 mgL-1). Isoacteoside content was increased to 17.3% with elicitation by SA (100 μM). Real-time PCR was performed to find the effect of elicitors on the expression of acteoside biosynthesis genes such as PAL and HCT in the callus after 24h and 48h of elicitation. The quality of cDNA was confirmed by PCR using β-ACTIN gene-specific primers. The Cq values obtained in RT-qPCR for each gene was further analysed and relative expression values were obtained using 2-ΔΔCq (Livak) method with ACTIN as the reference gene. After 24h of elicitation, 83-fold increase in the expression of PAL gene was observed in the callus treated with SA (40 μM). Elicitation with YE (1 gL-1) and Pectin ((100 mgL-1) showed 8 and 2.3-fold increase in the expression of PAL gene after 24h. The expression of both PAL and HCT genes was increased up to 2-folds in the callus treated with SA (100 μM), pectin (100 mgL-1) and YE (1 gL-1). After 48h of elicitation, the expression of PAL and HCT genes was decreased drastically in most of the treatments. The study shows a possible use of yeast extract, salicylic acid and pectin for the in vitro production of phenylpropanoid glycosides from Artanema sesamoides.Item Anther culture of Capsicum L. for doubled haploid production(Department of Plant Biotechnology, College of Agriculture,Vellayani, 2022-04-13) Ninitha Vijayan; Swapna AlexThe study entitled “Anther culture of Capsicum annuum L. for doubled haploid production” was carried out at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2019 - 2021. The objective of the study was to develop haploids of Capsicum annuum var. Arka Meghana by anther culture. Standardisation of the suitable stage of bud of Capsicum annuum var. Arka Meghana for anther culture was carried out by staining the anthers of different bud stages with 1% aceto-orcein and calculating the percentage of microspores at late uninucleate and early binucleate stages. Buds collected at six days after bud initiation had 57% of microspores in the late uninucleate stage and 34% in the early binucleate stage. Buds collected at nine days after bud initiation had 21% of microspores in the late uninucleate stage and 49% in the early binucleate stage. Hence the buds collected at six and nine days after bud initiation were found to be most suitable for anther culture. The optimum surface sterilization condition for buds was standardised by treating the buds with 70% of ethanol and 4% of sodium hypochlorite for different time intervals. Surface sterilization with 70% ethanol for 30 seconds followed by 4% sodium hypochlorite for 15 minutes was found to be most effective. Anther culture was carried out in nine media compositions for optimization. Anthers were inoculated onto full strength Murashige and Skoog (1962) medium (MS) and Dumas de Vaulx (1981) medium (CP) with different concentrations of plant growth regulators Kinetin, BA, NAA, IAA and 2,4-D with AgNO3 and activated charcoal as additives. The anthers were incubated at 25°C for two and eight days in darkness. The treatment T5 - MS + 4.00 mg/L NAA + 0.10 mg/L BA + 0.25% activated charcoal + 10 mg/L AgNO3 incubated at 25°C for two days in darkness showed callogenesis (3.17%) at six weeks after inoculation. All the treatments incubated at 25°C for eight days in darkness showed response at sixth week of inoculation with callogenesis varying from 3.75% to 15.38%. Among the nine treatments, highest callogenesis of 15.38% was observed in the treatment T5 at sixth week of inoculation. Globular embryo initiation (1.25%) was observed in the treatment T4 - MS + 4.00 mg/L NAA + 1.00 mg/L BA at sixth week of inoculation. The six treatments that showed good response at 25°C incubation temperature and eight days darkness were incubated at 35°C in two days darkness. All the treatments showed response varying from 4% to 15.54% of callogenesis at second week of inoculation. Callogenesis varying from 10.66% to 34.48% and embryonic calli induction varying from 3.17% to 17.24% was observed in the fourth week of inoculation. At sixth week of inoculation callogenesis varied from 11.11% to 37.93% and embryonic callus induction varied from 3.17% to 19.54%. Among the six treatments, the maximum callogenesis of 37.93% and embryonic calli induction of 19.54% was observed in the treatment T5 at fourth week of inoculation. Embryogenesis was observed in the treatment T7 – MS + 4.00 mg/L NAA + 0.50 mg/L BA+ 0.25% activated charcoal + 15 mg/L AgNO3 (1.82%) and treatment T6 - MS + 4.00 mg/L NAA + 0.10 mg/L BA + 0.25% activated charcoal + 15mg/L AgNO3 (1.33%) at fourth week of inoculation. At sixth week of inoculation, embryo induction (1.15%) was observed in the treatment T5 – MS + 4.00 mg/L NAA + 0.10 mg/L BA + 0.25% activated charcoal + 10mg/L AgNO3. Highest embryogenesis of 2.72% was observed in the treatment T7 – MS + 4.00 mg/L NAA + 0.50 mg/L BA+ 0.25% activated charcoal + 15 mg/L AgNO3. To conclude, among the treatments tested in Capsicum annuum var. Arka Meghana the best treatment for indirect embryogenesis and direct embryogenesis was MS + 4.00 mg/L NAA + 0.10 mg/L BA + 0.25% activated charcoal + 10 mg/L AgNO3 and MS + 4.00 mg/L NAA + 0.50 mg/L BA + 0.25% activated charcoal + 15 mg/L AgNO3 respectively at culture conditions of 35°C initial incubation temperature and two days darkness.Item Expression profiling of laccase and β-Glucan synthase genes in Pleurotus ostreatus during different developmental stages(Department of Plant Biotechnology, College of Agriculture , Vellayani, 2022-10-21) Nesma, A S; Susha S TharaThe study entitled “Expression profiling of laccase and β-glucan synthase genes in Pleurotus ostreatus during different developmental stages” was conducted at College of Agriculture, Vellayani. The objective of this study was to analyze the differential expression of laccase and β-glucan synthase genes in two strains of P. ostreatus during different developmental stages using quantitative Real-Time PCR (qRT- PCR). Two strains of P. ostreatus viz., DMR P115 collected from the Directorate of Mushroom Research (DMR), Solan, Himachal Pradesh and HUC collected from the Department of Plant Pathology, College of Agriculture, Vellayani were used for the study. Samples were collected from four different developmental stages viz., mycelium, primordium, young fruiting body and mature fruiting body of P. ostreatus strains. Potato Dextrose Agar (PDA) and Potato Dextrose Broth (PDB) were used for maintaining the pure cultures of both strains. The cultural and morphological studies of mycelia revealed that DMR P115 strain formed maximum radial growth on the 3rd, 5th, 7th and 9th day. However, white, thick and fluffy growth with radiating margins were observed in both strains. To obtain fruiting bodies, the spawn of both strains was prepared on paddy grains and inoculated on sterilized rubber sawdust substrate. The morphological studies of the mushroom fruiting body revealed that DMR P115 took minimum days for spawn run, days to produce primordia, days to produce young fruiting body and days for maturity. Sporophore characters like stipe length, stipe diameter, pileus length, pileus diameter and average weight of fruiting body were higher in DMR P115 strain than in HUC strain. Maximum yield (747.429 g/kg of the substrate) and the number of sporophores (84.429) were observed in DMR P115 strain. The expression of laccase (POXA3) and β-glucan synthase (FKS) genes that are important in fruiting body development and cell wall formation respectively was analysed at different developmental stages of two strains by performing qRT-PCR. There was a significant difference in the expression of POXA3 and FKS genes in different developmental stages of the two strains. The expression of POXA3 was higher in the mycelial stage of DMR P115 (5.58 fold) and HUC (2.03 fold) strains. FKS gene 81 was upregulated in mycelium (29.24 fold) and mature fruiting body (58.48 fold) of DMR P115 strain whereas in HUC strain, there was a significant upregulation only in the mycelial stage (17.15 fold). It can be concluded that the growth and gene expression pattern in DMR P115 and HUC strain varies. The expression of POXA3 and FKS genes differ significantly in different developmental stages of the two strains. POXA3 gene, which is essential for its fruiting body development and degradation of the substrate was upregulated in the mycelial stage of both strains. FKS gene was upregulated in the mycelium and mature fruiting body of DMR P115 strain and the mycelial stage of HUC strain which indicates the antioxidant and immunostimulatory properties of that strain. The findings obtained from this study can be used as a foundation for future lines of research related to strain improvement of P. ostreatus.Item Physiological and molecular studies in mitigating drought stress of Tomato (Solanum lycopersicum) by biochar with organic amendments(Department of Plant Biotechnology, College of Agriculture , Vellayani, 2022-11-09) Anjusha, P; Viji, M MThe study entitled “Physiological and molecular studies in mitigating drought stress of tomato (Solanum lycopersicum) by biochar with organic amendments” was conducted at College of Agriculture, Vellayani, Thiruvananthapuram, during 2021-2022. The primary objective of this study was to determine the physiological and molecular attributes of tomato grown under water stress as influenced by biochar with organic amendments. The experiment on tomato variety ‘Vellayani Vijay’ was conducted in a completely randomized design in pot culture with five replications. In order to assess the effect of biochar (BC) (0.5% w/w) and organic amendments such as vermicompost (VC) and farm yard manure (FYM) in mitigating the consequences of water stress, individual treatments viz. (Biochar (0.5% w/w), VC (50g/10kgsoil) and FYM (50g/10kg soil)) as well as combined treatments viz. ((Biochar+VC) and (Biochar+FYM)) were carried out along with T1 control (normal irrigated condition) and T7 control (water stress). The study consisted of 12 treatments, in which T1 to T6 were irrigated normally whereas treatments designated as T7 to T12 were subjected to 25% water stress. The present study was carried out using biochar derived from rice husk that was subjected to maximum pyrolysis temperature of 550°C in a cylindrical iron tank. Water stress of 25% was imposed by gravimetric method for a period of 80 days (after three weeks of sowing). The seedlings were maintained in protected condition for the first three weeks. Various morpho-physiological and yield parameters were evaluated at different critical stages of the crop, such as seedling stage, vegetative stage, flowering stage, fruiting stage and harvesting stage. Molecular studies were carried out at 50% flowering stage. Water stress had a negative impact on morphological, physiological and yield attributes of S. lycopersicum except total soluble solids, when compared to plants under normal irrigated condition. Biochar treatment was found to effectively mitigate the deleterious impacts of water stress. Plants treated with biochar outperformed the plants grown in both VC and FYM with regard to all the physiological and morphological parameters studied. However the best yield performance (yield and yield parameters) was obtained in the treatment with 124 biochar in combination with vermicompost. According to the results obtained it was clear that the combined application of biochar and vermicompost increased the relative water content, membrane stability index, chlorophyll stability index, photosynthetic rate, total protein content, stomatal conductance, transpiration rate, plant height and primary branches per plant under both water stress and normally irrigated conditions when compared to the non-treated controls. This indicates the ability of biochar to retain water and thereby increasing soil moisture content. Furthermore, under water stress, there was a significant improvement in the morphological traits such as, root weight (57.07%), shoot weight (33.33%), root: shoot ratio (17.91%), fruit number (43.54%), fruit size (69.93%), fruit weight (13.10%), total soluble solids (10.32%) and yield (62.42%). SlWRKY8 gene has an important role under drought stress conditions and is reported to have role in the regulation of ROS pathways and mitigating the detrimental effects in plants grown under water stress condition. In the present study, quantitative real time PCR amplification of SlWRKY8 gene in tomato leaves (taken at 50% flowering stage) showed an increase in the expression level of this gene in the treatments with combined application of biochar and vermicompost, both under water stress condition (T11) (3.24 fold) and normal irrigated condition (T5) (1.51 fold) compared to their respective controls (T7 and T1). This indicates that BC and VC combinations can be applied to tomato plants grown under water stress for the upregulation of SlWRKY8 genes which regulate the ABA signaling and helps in ABA-mediated stomatal closure in water stress condition. Taken together, the study demonstrate that, the combined application of vermicompost and biochar not only boost crop production, but also mitigate the destructive impacts of water stress by influencing physiological, morphological and molecular parameters of tomato.Item Effect of stingless bee propolis on proliferation of human stem cells(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2022-01-17) Drishya Prakashan; Shanas, SThe present research work entitled “Effect of Stingless bee propolis on the proliferation of human stem cells” was carried out in the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram and Department of Genomic Science, Central University of Kerala during the year 2020 2021, with the objective to study the effect of propolis collected from two different genera of Stingless bees, viz., Lisotrigona and Tetragonula on the proliferation of human induced pluripotent stem cells (hPSCs). Crude propolis samples were collected from the nest of three species of stingless bees Lisotrigona sp. (Kollam), Tetragonula calophyllae (Thiruvananthapuram) and Tetragonula travancorica (Thiruvananthapuram). Propolis samples were macerated at room temperature and extracted with 95% ethanol. MTT assay was performed and IC-50 values were calculated in-order to determine the influence of propolis on the proliferation of hPSCs. Five different concentrations of propolis, viz., 150µg/ml, 300µg/ml, 450µg/ml, 600µg/ml and 900µg/ml respectively, were added to D14/C2 cells for a period of 24 hrs. Cells treated with the propolis extracted from Lisotrigona sp., T. calophyllae and T. travancorica obtained IC50 values of 410.904 ug/ml, 480.097 ug/ml and 215.157 ug/ml respectively. The cells treated with the three different propolis displayed significant proliferation rate after 24 hrs treatment in relative to the control however, higher concentration were observed to be cytotoxic to cells. When the right concentration of propolis was used, marked difference in the morphology of cells was observed. The cells lost its border integrity, uniformity and started to migrate. RNA of the control and propolis treated cell was isolated by QIAzol reagent method. Quality and quantity of the samples were analyzed through nanodrop, which gave an absorbance value of 1.8 to 2.0 for all the samples. The concentration of the samples observed was between 30-200 ngµl-1. The RNA samples were then subjected to 99 cDNA conversion (iScriptTM cDNA Synthesis Kit). The cDNA of control and treated samples were then subjected qRT- PCR. The real-time quantitative PCR (qRT-PCR) was performed with the Applied Biosystems PCR system in a total volume of 10 µl and expression of different genes was studied. Gene expression analysis revealed pluripotency markers (OCT4 and NANOG), important for preserving pluripotency were down regulated upon treatment with propolis, which in turn confirmed early differentiation of hPSCs. Further investigation on the gene expression of early differentiation markers revealed that propolis supported the cells to differentiate into mesendoderm and endoderm lineage, which is a novel finding. To conclude, the result of the study proved that the propolis obtained from stingless bees Tetragonula spp. probably has more therapeutic value in terms of its effect on human pluripotent stem cells compared to the propolis obtained from Lisotrigona sp.Item Expression profiling of water stress responsive miroRNAs in banana (Musa spp.)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2022-03-08) Amal Mohamud Naushad; Soni, K BThe study entitled “Expression profiling of water stress responsive microRNAs in Banana (Musa spp)” was conducted during 2019-2021, in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The study envisaged expression profiling of selected computationally predicted miRNAs in banana cultivar Nendran (Musa AAB) under water stress conditions. Fifty-two novel miRNAs and their targets have been computationally predicted in banana genome A, using NOVOMIR (Mathew, 2018) in a previous study conducted in the Department of Plant Biotechnology. Among five miRNAs were selected in this study for their validation and identifying their association with water stress response. The microRNAs selected are miR-3900-5p (target: Heat shock protein, HSP gene), miR-2172-5p (target: Putative Ethylene Responsive Transcription factor 1 (ERT) gene), miR-971-5p (target: Argonaute, (ARG) gene, miR-6928-5p (target: FAD dependent oxidoreductase gene), and miR-2172-5p (target: Diacylglycerol, DAG gene). Six months old in-vitro raised banana plants of cv. Nendran (Musa AAB) were used in this study. Water stress was induced in the potted plants by withholding irrigation for seven consecutive days. After 7 days, the relative water content in the leaf samples was reduced from 93.84 to 59.32, indicating water stress. The expression profile of the miRNAs and their target genes in banana plants under water stress conditions was analysed by performing Real-time quantitative PCR (RT-qPCR). Total RNA was extracted from leaf samples using the modified Rodrigues Garcia method and reverse transcribed to cDNA using the miRNA specific stem-loop primers designed. Selected miRNAs were amplified using miRNA specific forward and universal reverse primers. The target genes were also amplified by designing primers. The specificity of the primers was determined using a melt curve analysis. All the reactions are conducted with three biological and two technical replicates. Out of the four, three miRNAs (miR-2172-5p, miR-6928-5p and miR-971 5p) and their target genes showed amplification in all the samples. All the water stressed 70 plants showed upregulation of miR-2712-5p (4 to 11fold), and downregulation of miR971-5p (0.1 to 0.4fold) and miR-6928-5p (0.2 to 0.7fold). The two targets of miR2712-5p showed differential expression under the water stressed conditions, DAG gene was downregulated (0.1 to 0.2folds) and ERT gene was upregulated (2 to 3folds). Expression of FAD dependant oxidorductase (The target of miR-6928-5p) and ARG (the target of miR971-5p) was increased up to 3 to 4folds respectively in water stress conditions. Relative gene expression analysis in the water-stressed plants indicated an inverse correlation between all the three miRNAs and their targets, suggesting their strong miRNA: target relation. The study showed that miR-2172-5p, miR-971-5p, miR-6928-5p are water stress-responsive in banana cv. Nendran. Since the products of all the target genes are related to stress responses in plants, an in-depth study of these miRNAs may help develop strategies for water stress management in bananas.Item Cloning and characterization of coat protein gene of Tomato leaf curl virus infecting tomato and its phylogenetic relationship with other members of geminiviridae(Department of Plant Biotechnology, College of Agriculture , Vellayani, 2022-10-27) Athira ,S M; Ayisha ,RThe study entitled ‘Cloning and characterization of coat protein gene of Tomato leaf curl virus infecting tomato and its phylogenetic relationship with other members of Geminiviridae’ was carried out at College of Agriculture, Vellayani during the year 2021- 2022. The objective of the study was molecular characterization and cloning of coat protein gene of Tomato leaf curl virus (ToLCV) infecting tomato and its phylogenetic analysis with other members of Geminiviridae. Symptomatology of virus infected tomato plants was studied. Infected plants were collected from different regions of Vellayani campus, Kerala Agricultural University and the virus was maintained by graft inoculation. The virus was serologically characterized using DAS-ELISA (Double Antibody Sandwich-ELISA) and DIBA (Dot Immuno Binding Assay) using ToLCNDV (Tomato leaf curl New Delhi virus) antisera and higher viral titer was shown by plants with severe reduction in leaf size (8-fold absorbance value). Genomic DNA was extracted from the infected samples, and coat protein (CP) gene-specific primers were used for molecular detection. PCR yielded expected amplicon size of 500bp and 600bp and were sequenced. The BLAST analysis of the sequence revealed similarities with Tomato leaf curl Palampur Virus (ToLCPMV) and ToLCNDV of 95% and 94%, respectively. Both bipartite virus and monopartite virus with a satellite DNA were detected by rolling circle amplification (RCA), which was followed by Restriction Fragment Length Polymorphism (RFLP). PCR was done using RCA fragments as template with DNA A specific primers and the amplicons obtained were cloned. Sequencing of cloned genes followed by BLAST analysis showed 98.61 per cent similarities with ToLCPMV isolates. Phylogenetic analysis of partial DNA A gene of Vellayani isolate with other strains of ToLCV showed close relation to ToLCPMV infecting cucurbits. Comparitive analysis of partial DNA A sequence with other viruses in genera Begomovirus showed closest relation with Melon leaf curl virus and Cotton leaf curl virus from Pakistan. Comparison of partial amino acid sequence of CP of ToLCV Vellayani isolates with other mono and bipartite begomoviruses revealed a maximum of 99.11 per cent similarity with pre coat protein genes of ToLCPMV. According to the current investigation, the Begomovirus that infects tomatoes in the Vellayani region is bipartite as well as monopartite with a satellite DNA. The CP and DNA A genome phylogenetic analyses revealed a strong relationship between the Vellayani isolate and the ToLCPMV isolates infecting cucurbitsItem Targeted editing of rice micro RNA osa-miR396b through CRISPR/Cas9 system(Department of Plant Biotechnology, Centre for Plant Biotechnology and Molecular Biology, College of Agriculture , Vellanikkara, 2022-10-15) Sanjay Sathian; Rehna AugustineRice (Oryza sativa L.) is one of the most produced and consumed food crops in the world. There is an urgent need to increase rice production to feed the increasing population. Rice yield is determined by several components like grain size, grain weight, number and architecture of panicles, number of spikelets per panicle and grain filling. The microRNA family osa-miR396 is known to suppress the expression of rice growth regulating factors (OsGRFs) resulting in reduced growth and yield. The miRNA osa-miR396b is reported to be a negative regulator of spikelet number and inflorescence development. CRISPR/Cas9 mediated knockout of osa-miR396b gene can thus possibly result in an enhanced yield in rice. Hence, the current study ‘Targeted editing of rice microRNA, osa-miR396b through CRISPR/Cas9 system’ was conducted during the period from 2019 to 2022 at the Department of Plant Biotechnology, College of Agriculture, Kerala Agricultural University, Vellanikkara, Thrissur. Oryza sativa ssp. japonica cultivar Nipponbare was selected for the study due to well established transformation protocols and higher transformation efficiency. Initially, the sequence information of the rice microRNA gene osa-miR396b was retrieved from ‘miRbase’. The stem loop sequence obtained was used to design guide RNAs (gRNAs) using the software CRISPR-P v2.0 and CRISPR-PLANT v2. The gRNAs were selected for further studies mainly based on GC content and number of off-target sites. The target osa-miR396b gene sequence was confirmed by amplifying the genomic region flanking the target using gene specific primers followed by sequencing and the sequence analysis using Clustal Omega and BLASTn showed 100% similarity with reported sequences. The osa-miR396b G1 CRISPR/Cas9 construct was generated by restriction digestion of CRISPR/Cas9 binary vector pRGEB32 using BsaI restriction enzyme followed by ligation with annealed and phosphorylated gRNA. The osa-miR396b G1 construct was cloned to E. coli strain DH5α. The positive clones were confirmed by Sanger sequencing of the plasmid DNA isolated from the colonies and sequence analysis using Clustal Omega. Three (osa-miR396b G1 #2, osa-miR396b G1 #3 and osa-miR396b G1 #4) out of four plasmids sequenced were having gRNA insertion. The osa-miR396b G1 #4 CRISPR/Cas9 construct was mobilized to A. tumefaciens strain EHA105. Positive clones were confirmed by PCR amplification of hygromycin resistance gene (hptII) using specific primers. Colony #1 of A. tumefaciens with osamiR396b G1 CRISPR/Cas9 construct out of the two positive colonies was used for rice transformation. Genetic transformation of rice was achieved through Agrobacterium-mediated transformation. The first step was induction of calli from dehusked and sterilized Nipponbare seeds. Five-day-old calli were infected with Agrobacterium harbouring osa-miR396b G1 CRISPR/Cas9 construct for 1.5-2 min. and co-cultivated for 48 hours. An empty vector was also transformed as control. After washing off excess Agrobacterium growth with sterile distilled water containing Augmentin (300 mgL-1 ) or carbenicillin (250 mgL-1 ), the calli were kept for selection in selection medium supplemented with hygromycin (50 mgL-1 ) and Augmentin (300 mgL-1 ) or carbenicillin (400 mgL-1 ). The calli showing proliferation of microcalli were transferred to regeneration medium supplemented with NAA (0.02 mgL-1 ) and kinetin (2.0 mgL-1 ) for inducing somatic embryogenesis. The somatic embryos were allowed to develop into small plantlets which were transferred to rooting medium for root development. The rooted plantlets were initially maintained in sterile distilled water and then hardened in sterile soil-cocopeat mixture in paper cups and transferred to pots with soilsand-cow dung mixture. A total of 94 putative transformed plants for osa-miR396b G1 CRISPR/Cas9 construct and four vector control plants were obtained. For confirming successful transformation, PCR amplification of hptII gene using hygromycin gene specific primers was done. DNA extracted from 35 plants and two vector control were used as PCR template. A total of 16 out of 35 transformed and two vector control plants were hygromycin positive, indicating successful transformation. The osa-mir396b partial gene sequence was amplified using gene specific primers and sequenced by Sanger sequencing for detecting mutation. Detection of mutation was carried out using ‘Inference of CRISPR Edits (ICE)’ software by Synthego. Analysis using ‘ICE’ detected indel mutations in seven plants. Five plants (71.42%) had deletions and two (28.57%) had insertions around the cut site. Four plants (57.14%) had heterozygous mutations (mutation in one allele) and three (42.86%) had chimeric (more than two) mutations. The mutation efficiency was calculated to be 43.75%. The mutations obtained could lead to a non-functional osa-miR396b gene in these plants. The study successfully demonstrated application of CRISPR/Cas9 system to mutate rice microRNA gene. The knockout of osa-miR396b gene will likely promote the expression of rice GRF genes improving the grain yield. Further studies should be conducted to study inheritance pattern of mutations in the subsequent generations. Genotypic and phenotypic analyses is to be done to study effect of mutated osamiR396b gene on its target genes and on yield and other agronomically important traits.