PG Thesis
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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 Standardisation of dehydration, storage and packaging of drumstick (Moringa oleifera Lam.) leaves(Department of Postharvest Management, College of Agriculture,Vellanikkara, 2025) Fathima Ismath.; Anupama, T VLeafy vegetables are an essential part of a healthy diet, providing an affordable source of vital vitamins, minerals, and antioxidants. Among them, Moringa oleifera Lam., often called the "miracle tree," stands out for its exceptional nutritional and medicinal properties. Its leaves are rich in bioactive compounds with antioxidant, anti-inflammatory, and antimicrobial benefits, contributing to improved nutrition and addressing malnutrition, especially in rural households. However, the high moisture content of fresh Moringa leaves makes them highly perishable, necessitating effective post-harvest management to extend their shelf life. Proper dehydration techniques not only reduce spoilage but also help retain their nutritional value, ensuring year-round availability. Converting Moringa leaves into powder enhances their stability and facilitates their incorporation into value-added products. Additionally, suitable packaging and storage conditions play a crucial role in preserving quality and minimizing post-harvest losses. Despite its significance, research on optimizing postharvest handling of Moringa leaves remains limited in Kerala. Hence with this background the present study entitled “Standardisation of dehydration, storage and packaging of drumstick (Moringa oleifera Lam.) leaves” was undertaken to standardize pretreatment methods, dehydration techniques, and suitable packaging materials and storage conditions to enhance the shelf life and preserve the nutritional integrity of Moringa oleifera Lam. leaves. The study was structured into three experiments. The first experiment was to standardise the pretreatments of Moringa leaves. Fresh Moringa leaves were collected, destalked, washed, and subjected to four treatments: control (no blanching), hot water blanching (80°C for 1 min), steam blanching (1 min in a steam cooker), and microwave blanching (800 W for 30 s). Blanched leaves were rapidly cooled, shadedried, powdered, and analysed for physical and biochemical properties including recovery percentage, moisture content, crude fibre, crude fat, total protein, total ash, total carbohydrate, ascorbic acid, total chlorophyll content, total carotenoids and total phenols. The results revealed that blanching treatments significantly influenced the physical and biochemical parameters of Moringa leaves. Microwave blanching (T4) emerged as the most effective pre-treatment, yielding the highest recovery percentage (22.81%), lowest moisture content (8.48%), and maximum retention of crude fibre (13.50%), total carbohydrates (42.00%) and carotenoids (114.48 mg/100g). Steam blanching (T3) and hot water blanching (T2) also showed significant improvements in nutrient retention compared to the control (T1). The control treatment exhibited the lowest recovery (17.94%) and highest moisture content (11.64%), highlighting the importance of blanching in reducing moisture and enhancing nutrient concentration. Microwave blanching also retained higher levels of total ash (12.38%), total protein (24.23%), ascorbic acid (115.61mg/100g), and total chlorophyll (299.80%) and crude fat (7.53%), making it the best pre-treatment method. Moringa leaves blanched by microwave blanching were subjected to different dehydration methods, including shade drying (23–31°C), cabinet drying (50±5°C), microwave oven drying (60°C), and vacuum drying (35±5°C). After drying, the leaves were powdered and analysed for physical (recovery percentage), biochemical (moisture content, crude fibre, crude fat, total protein, total ash, total carbohydrate, ascorbic acid, total chlorophyll content, total carotenoids and total phenols.), mineral (Fe, Ca and K), and antioxidant properties. The results demonstrated that dehydration methods significantly influenced the physical, biochemical, mineral, and antioxidant properties of Moringa oleifera leaves. Vacuum drying (T4) resulted in the highest recovery percentage (28.23%), total carbohydrate (48.00%), total protein (26.28%), total ash (22.42%), ascorbic acid (139.02 mg/100 g), and total phenols (160.91 mg GAE/100 g), while also exhibiting the highest antioxidant activity (IC₅₀: 3.82 mg/ml). Microwave drying (T3) recorded the highest total carotenoid content (119.43 mg/100 g) and retained notable amounts of crude fat (7.42%) and iron (13.34 mg/100 g). Cabinet drying (T2) yielded the highest crude fibre (9.70%) but the lowest crude fat (5.77%) and protein content (23.33%). Shade drying (T1) retained the highest total chlorophyll (324.41 mg/100 g) and crude fat (8.69%) but had the lowest recovery (24.28%) and total carbohydrate content (42.33%). Vacuum drying emerged as the most effective dehydration method, followed by microwave drying, due to their superior retention of key nutrients and antioxidant properties. The vacuum-dried whole leaf and leaf powder of Moringa were packaged using HDPE (200 gauge), LDPE (200 gauge), and polyethylene-laminated aluminium pouches and stored under ambient and refrigerated (4–6°C) conditions for three months. Biochemical, mineral, microbial, sensory, and antioxidant analyses were conducted at monthly intervals to evaluate storage effects. The results of the third experiment revealed that packaging materials and storage conditions significantly influenced the biochemical, mineral, antioxidant, microbial, and sensory properties of dried Moringa oleifera leaves over a three-month storage period. Leaf powder stored in polythene-laminated aluminium pouches under refrigerated conditions (T12) emerged as the most effective method, maintaining the lowest moisture content (5.95–6.02%), highest retention of total phenols (158.21– 159.63 mg GAE/100g), ascorbic acid (133.28–133.32 mg/100g), and total chlorophyll (357.66–390.53 mg/100g). In contrast, whole leaves stored in LDPE pouches under ambient conditions (T2) resulted in the highest moisture content (7.97–10.21%), significant nutrient degradation, and the lowest overall acceptability (5.72–6.57). Refrigerated storage also minimized microbial load, with T12 recording the lowest microbial count ( aerobic plate count - 0.40–2.00 × 10⁴ cfu/g), while ambient-stored samples (T2) exhibited the highest microbial growth (1.50–10.30 × 10⁴ cfu/g). Sensory evaluation confirmed that leaf powder stored in polythene laminated aluminium pouch under refrigeration (T12) retained superior sensory attributes, achieving the highest overall acceptability score (8.71) by the end of the storage period. Mineral content, including iron, calcium, and potassium, showed a gradual decline over time, with refrigerated storage (T7, T8, T9, T10, T11, T12) preserving higher levels compared to ambient storage. For instance, T7 (whole leaves stored in HDPE under refrigerated conditions) and T10 (leaf powder stored in HDPE under refrigerated conditions) retained the highest iron content (15.26–15.29 mg/100g), while T12 maintained the highest potassium content (0.86–0.92%). Antioxidant activity, measured by IC₅₀ values, also declined over time, with refrigerated samples (T12) exhibiting the lowest IC₅₀ values (4.59–6.03 mg/ml), indicating better retention of antioxidant potential compared to ambient-stored samples (T2) with IC50 value of 7.87–14.88 mg/ml. Overall, refrigerated storage in polythene-laminated aluminium pouches (T12) proved to be the most effective method for preserving the nutritional, sensory, and microbial quality of Moringa leaves, making it the preferred choice for long-term storage. The findings of the study revealed that Moringa leaves can be effectively preserved using microwave blanching, vacuum drying, and refrigerated storage in polythene-laminated aluminium pouches, maintaining their nutritional, sensory, and microbial quality for up to three months. The findings highlight the importance of advanced preservation and packaging techniques in retaining nutrient content and quality. Future research should focus on advanced preservation techniques, ecofriendly packaging, and scaling up production for commercial use. Additionally, exploring value-added products, nutrient bioavailability, and smart packaging technologies can enhance the sustainable utilization of Moringa leaves for global nutrition and food security.Item Arthropod diversity in drumstick Moringa oleifera Lam.(Department of Agricultural Entomology, College of Agriculture, Vellayani, 2023-04-12) Niveditha K P; Anitha, N