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Title: In vitro studies on salt and water stress tolerance in Eucalyptus tereticornis Sm.
Authors: Singh, Davinder
Supervisor: Kumar, Anil
Keywords: Osmotic stress;Peroxide redicle;Membrane peroxidation;Antioxidant enzymes;Sodium transported;Gene expression
Issue Date: 13-Apr-2022
Abstract: The present work focuses on the screening and characterization of selected elite clones (‘CE2’, ‘KE8’, ‘KE2’, ‘Y8’, ‘T’ and ‘T1’) of Eucalyptus tereticornis for salt stress. These commercially important elite clones were selected from the plantation of Ballarpur Industries Ltd. and screened for salt tolerance on Murashige and Skoog’s (MS) medium fortified with different concentrations of NaCl. The seeds and shoot cultures were used as explants for screening. This resulted in the identification of salt-tolerant, moderately salt-tolerant, and salt sensitive clones. The results of the in vitro experiments were further verified under greenhouse and field conditions. Further, the salt tolerance of sensitive clone ‘Y8’ was improved in two different ways: exposure to iron-oxide nanoparticles (IONPs) and overexpressing the osmotin gene of potato. The three different in vitro screening approaches (direct, indirect, and gradual exposure to salt stress) were employed for the identification of salt-tolerant (ST) seedlings. The screening via gradual step-wise exposure to salt stress (0 to 400 mM NaCl) was highly efficient and promoted tolerance level up to 400 mM NaCl. Shoot growth of seedlings demonstrated a 91.93% increase due to physiological acclimatization to salt stress. The indirect screening approach was also effective but only a 12.8% rise in shoot length was recorded when cultured on a medium fortified with 400 mM NaCl. On the other hand, the direct screening approach could only identify seedlings with a tolerance level of 200 mM NaCl. Further, when salt sensitive (SS) and salt-tolerant (ST) seedlings were exposed to salt stress (0, 200, and 400 mM NaCl) for 14 days, a significant increase in chlorophyll, osmolyte accumulation, and antioxidant enzyme activity was only observed in ST seedlings. The tolerance of ST seedlings was also associated with significantly higher transcript levels of genes encoding superoxide dismutase, peroxidase, and catalase. The response of individual shoot cultures of six elite clones of E. tereticornis was evaluated on basal MS medium supplemented with different concentrations of NaCl (0, 100, 200, 300, 400, and 500 mM) for 28 days. The increase in NaCl concentration affected the survival of microshoots along with inhibition in shoot and root growth in all the clones. Better performance of clone ‘KE8’ was evident with higher survival of microshoots (65.18%) in comparison to clones ‘CE2’ (36.29%), and ‘Y8’ (10.37%) on medium containing 500 mM NaCl. The highest salt tolerance index (0.30) was recorded for clone ‘KE8’, followed by 0.24 for clone ‘CE2’ and 0.10 for clone ‘Y8’ on a medium containing 500 mM NaCl. The 50% xx growth inhibition dose of NaCl was also found to be 479 mM for clone ‘KE8’, as compared to 385 mM and 206 mM for clones ‘CE2’ and ‘Y8’, respectively. Various physiological and biochemical parameters were investigated in microshoots at different intervals of the culture period (7, 14, 21, 28 days) under control and stress conditions. The relative water content (RWC) and pigment levels (total chlorophyll and carotenoid content) declined significantly during culture on a medium containing 500 mM NaCl with an increasing culture period. Furthermore, the decline was higher in the case of clone ‘Y8’ than ‘CE2’ followed by ‘KE8’. A significant increase in the activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidase), accumulation of proline, and lower levels of malondialdehyde content was recorded in clone ‘KE8’ as compared to the other clones ‘CE2’ and ‘Y8’. This endorsed the tolerant nature of clone ‘KE8’ to salt stress. Furthermore, the variations in tolerance level and various parameters were investigated in micropropagated plants under greenhouse conditions. The three elite clones (‘KE8’, ‘CE2’ and ‘Y8’) of E. tereticornis with contrasting salt tolerance were exposed to salt-stressed and well-watered conditions for 30 days. Regardless of tolerance level, salinity resulted in significant reductions in plant height, root length, leaf number, shoot and root dry mass, however, higher growth was observed in plants of tolerant clone ‘KE8’. The better performance of salt-stressed plants of ‘KE8’ was also associated with low hydrogen peroxide, malondialdehyde accumulation, and improved osmotic adjustment under salinity. The impact of iron oxide nanoparticles (IONPs) on salt stress improvement of salt sensitive clone ‘Y8’ of E. tereticornis was also studied against an inhibitory salt concentration i.e., 300 mM NaCl. Exposing the microshoots of E. tereticornis with an optimized, 25 ppm IONPs dose resulted in a distinct biochemical change in superoxide dismutase (~3.8-fold rise in activity), malondialdehyde (lowered by ~33%) concentration, total soluble sugars (~1.9-fold rise) and proline (11.8-fold rise) content. The examination of gene expression encoding HKT1, SOS1, and NHX1 using qRT-PCR indicated that IONPs may up-regulate their transcript levels either by the efflux of Na+ ions from cell or their sequestration in vacuole under stress conditions. Systematic analysis of morpho/physiological growth parameters also showed a remarkable increase in shoot length and the chlorophyll content by ~2.5 fold and 128.5%, respectively as compared to the control, under similar test conditions. Even under non-stressed conditions, IONPs acted as nano-supplements for promoting the shoot growth of E. tereticornis by increasing the gene expression of various antioxidant enzymes and synergistically improving the activity of catalase and peroxidase(s) enzymes. xxi Shoot regeneration is one of the crucial steps to undertake the genetic improvement of any plant. An efficient shoot organogenesis protocol was also developed from the leaf explants of E. tereticornis. Out of the different auxins tried, the highest frequency of shoot organogenesis (66.67 %) was seen on MS medium supplemented with 15 M NAA. Random amplified polymorphic DNA (RAPD) examinations demonstrated clonal fidelity of the regenerated plants and these were observed to be consistent with the mother plant. Apart from this, high frequency of leaf explants of clone ‘CE2’ differentiated somatic embryos on basal MS medium enriched with 1.0 µM NAA and 10.0 µM 2, 4-D. The other elite clones were found to be highly responsive to this medium composition for the induction of somatic embryos. The optimization of various factors like pre-culture, bacterial density, co-cultivation period, and acetosyringone concentration affecting Agrobacterium-mediated genetic transformation protocol resulted in a 64.44% increase in transient GUS expression of clone ‘Y8’ with the incorporation of 100 µM acetosyringone to the co-cultivation medium. The osmotin gene was cloned and characterized from the cDNA of Solanum tuberosum L. cultivar ‘Kufri Chipsona 1’. The open reading frame of alleles of the osmotin gene varied from 187 to 192 amino acids. Sequence analysis revealed the presence of 16 phosphorylation sites with no glycosylation sites. This gene was mobilized into binary vector pBI121 under the strong constitutive promoter CaMV35S. The modified plasmid was mobilized into Agrobacterium tumefaciens (EHA105) and used for the genetic transformation of salt-sensitive clone ‘Y8’. A total of seven transgenic lines overexpressing the osmotin gene of potato were regenerated via an optimized Agrobacterium-mediated genetic transformation protocol and exposed to salt stress (300 mM NaCl) for 28 days. Osmotin overexpression led to improved shoot growth under salt stress and facilitated the water uptake from the medium. The transgenic shoots growing under stress conditions displayed a larger accumulation of proline and total soluble sugars and a significant increase in the activity of antioxidant enzymes.
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