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2006 - 20073
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Subject: Plant Physiology × Author: KAU × End date: 2009 × Start date: 2006 × Type: Thesis ×
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    ThesisItemOpen Access
    Influence of host plants and soil moisture stress on the water relations in sandal
    (Department of Tree Physiology and Breeding, College of Forestry,Vell, 2006) Dhaniklal, G; KAU; Asokan, P K
    The influence of host plants and soil moisture stress on water relations in sandal was investigated in a pot culture experiment at College of Forestry, Kerala Agricultural University, Vellanikkara. Five host plants, Divi divi (Caesalpinia coriaria Jacq.), Casurina (Casuarina equisetifolia J.R & H.G. Forst), Pongamia (Pongamia pinnata (L.) Pierre), Lantana (Lantana camara L.) and Erythrina (Erythrina indica Lamk.) were selected for this study. The results showed that Sandal seedlings with and without host showed similar height increment, except when erythrina was the host. The host plant had no influence on most of the growth parameters of sandal seedlings. The collar diameter of sandal seedlings with and without host was on par during the early seedling stage and by the end of the experimental period it showed a significant difference. There was no significant difference in the number of leaves, leaf area and root length of sandal with different hosts. Haustorial connections were not recorded even after the experimental period. Sandal seedlings with Erythrina indica as host decreased the pre dawn water potential. Water stress decreased the seedling height, collar diameter, number of leaves, leaf area, shoot dry weight and total dry weight of sandal seedlings. Fully irrigating the pots once in three days resulted in better growth of sandal seedlings as compared to irrigation once in six days. Water stress increased the root length and root dry weight. Root length was found to be more for sandal seedlings which were irrigated once in six days compared to that, irrigated once in three days. Water stress decreased leaf area of sandal seedlings. Transpiration rate at 14:00 hrs was found to be more than that of transpiration at 8:00 hrs. Transpiration rate decreased with increase in soil moisture stress. Pre dawn water potential was lower for sandal seedlings irrigated once in six days compared to that irrigated once in three days. As the haustorial connections were not observed even after 270 days after planting it can be concluded that the difference in the seedling growth parameters of sandal with some of the hosts is due to the above and below ground competition for sun light, water and nutrients.
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    ThesisItemOpen Access
    Effect of abiotic stress factors on growth and secondary plant metabolism in Withania somnifera (L) Dunal
    (Department of Plant Physiology, College of Agriculture, Vellayani, 2006) Lini Jacob; KAU; Manju, R V
    An experiment was conducted in the Department of Plant Physiology, College of Agriculture, Vellayani, to study the effect of abiotic stress factors on growth and secondary plant metabolism in Withania somnifera (L.) Dunal. The study analyzed the effect of abiotic stresses on growth, physiological, biochemical parameters and also withanolide content-the secondary metabolite of this medicinal plant. The abiotic stresses were three levels of light stress at 25%, 50% and 75% shade and three levels of water stress at 25%, 50%, and 75%FC along with control under optimum conditions. The treatments were imposed for a period of 30 days on 5 month old plants. Growth parameters such as plant height, Dry matter production, length of tap root and physiological parameters such as specific leaf area and relative water content were recorded highest in 75% shaded plants. Stomatal conductance was higher in 25% shade condition. Stomatal frequency and stomatal index were higher in control plants. The total chlorophyll, chlorophyll a and chlorophyll b increased under different field capacity levels and recorded the highest value under 25%FC. Stable isotopes discrimination studies showed significant variation among the treatments. 50% shade showed highest value in stable isotopes. Biochemical parameters total soluble protein, starch content, total phenols, ascorbic acid, free amino acids and reducing sugars were recorded from the leaves of the stress imposed plants. The total soluble protein and ascorbic acid were highest in control condition whereas starch recorded maximum under 25 per cent shade condition. Reducing sugar and phenol content showed highest under 75 per cent shade condition. Amino acid was highest under 25 per cent of field capacity. The secondary metabolite were recorded from the stress imposed, sun dried roots of the plants. The secondary metabolite withanolide was estimated spectophotometrically. The withanolide content was high in 75% shade (0.64%) condition after imposing the stress for a period of 30 days. The activity of free radical scavenging enzymes namely peroxidase and polyphenol oxidase were also estimated in the above treatments and was found to be highly active in 75% field capacity level. The water stress at different field capacity levels reduced the growth of the plants while the activities of free radical scavenging enzymes were shown an increase among the treatments. There was a positive relation with the antioxidant enzymes under 75% FC and withanolide content which was reported to have an antioxidant activity. The gene sequence analysis revealed that hmgr in Withania somnifera is having a size of 262 base pairs. Similarity search using BLAST showed that high percent similarity exist in hmgr across many crop plants and important medicinal plants. Expression levels of HMG Reductase were assessed by relative quantitative RT-PCR and there was a maximum expression of hmgr under 75% shade condition followed by 25% shade. The present investigation has shown that exposure to abiotic stress factors invariably leads to better accumulation of secondary metabolites. The differential expression of hmgr can be a crucial factor in deciding the levels of biosynthesis of secondary metabolites in Withania somnifera when exposed to abiotic stress situation. An understanding of this role of abiotic stress factors is of great importance in phytomedicinal enrichment programme.
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    ThesisItemOpen Access
    Physiology and secondary metabolite production in genetically transformed brahmi (Bacopa monnieri L. wettst.) with cytokinin synthesizing isopentenyl transferase (ipt) gene
    (Department of Plant Physiology, College of Agriculture, Vellayani, 2007) Vighnesha; KAU; Roy Stephen
    An experiment was conducted in the Department of Plant Physiology, College of Agriculture, Vellayani, to overexpress cytokinin synthesizing ipt gene in Bacopa monnieri through Agrobacterium tumefaciens mediated transformation and to regenerate the transformed plants through tissue culture for analyzing the influence of overexpression of ipt gene on growth, physiology and secondary metabolite production. The transformation and molecular works were done in Rajiv Gandhi Center for Biotechnology, Trivandrum. Escherichia coli strain JM 109 was transformed independently with pBI B33 ipt and pBI SAG12 ipt. Triparental mating was done using Agrobacterium tumefaciens strain EHA 105, pRK 2013 and recombinant E.coli. Plasmids were isolated from recombinant E.coli and recombinant Agrobacterium cells to confirm the successful transformation of constructs. Both have showed the insertion release when double digested with restriction enzymes EcoRI and HindIII. Pre incubated leaf explants of Bacopa monnieri were co-cultivated with the recombinant Agrobacterium for two days and transferred to regeneration medium containing MS supplemented with 2mgl-1 BA, 15mgl-1 kanamycin and 300mgl-1 cefotaxime. Putative transformants were regenerated from co-cultivated explants when placed on the selection medium containing 15mg/l kanamycin and 300mg/l cefotaxime. Uninfected explants failed to regenerate in presence of kanamycin. Rooting was not found in the MS medium devoid of growth regulators. Sub culturing of shootlets was done in MS medium supplemented with 1ppm GA and 1ppm IAA. Hardening was done to the fully rooted plants and were kept in five replications for further analysis. DNA was isolated from both wild type and transformants. PCR amplification for nptII and ipt gene specific primers showed presence of gene in transformants but not in the wild type. From the selected transformants, RNA was isolated and RT-PCR was done. RT-PCR analysis confirmed the expression of ipt and nptII gene in all the transformants, while there was no expression in the wild type. Expression of constitutively expressed plant gene –actin was used as loading control. Southern hybridization of PCR amplified products gave the evidence for the presence of ipt gene only in transformants but not in wild type. Physiological and biometric observations were performed on both transformants and wild type which served as control over the transformants. Plant height was more in transformants compared to the wild. Both root length and relative water content was more in wild compared to the transformants. Other parameters like number of branches and number of leaves were higher in the transformants than in the wild. Total chlorophyll, chlorophyll a and chlorophyll b were found to increase for first five weeks in all treatments, after that there was a decrease in the total chlorophyll, chlorophyll a and b in wild type but transformants were able to retain higher contents throughout the period of study. Total soluble protein content was higher in the transformants than the wild type. Stomatal frequency showed a significant difference between the treatments. Higher number of stomata was observed in the transgenics compared to the wild type. The distribution of stomata also differed significantly. In wild type the distribution was equal in both upper and lower surface of leaf but in transformants a higher number of stomata were observed at the lower surface than the upper surface. Cytokinin content was estimated using ELISA. There was a significant variation in cytokinin, iPA concentration between wild type and transformants. Transformants had higher cytokinin content than the wild type. The transformant with B33 promoter had more cytokinin content than transformant with SAG promoter. Bacoside, the major secondary metabolite of the plant was estimated by HPLC and its content between the wild type and transformants were found to be on par. In this experiment, the overexpression of ipt gene in bacopa resulted a higher amount of cytokinin in transgenics and hence had higher growth rate, protein and pigment content. Overexpression of ipt may not increase the bacoside content in the bacopa.