Mutation Induction for Development of Olive Breeding Material and its Evaluation for Drought Tolerance

Abstract

Olive (Olea europaea L.) is a medium sized evergreen tree that is naturally grown in an arid and semi-arid environment associated with infertile/marginal lands, dry and warm summers, mild winters, soil water deficiency and high rate of irradiance. Olive tree has the tendency to tolerate high drought/salt stress and also can be successfully grown on calcareous soils unlike other fruit crops. Therefore, present study was designed in two experiments to evaluate its stress response against drought and salt stress by using different mutation and genetic transformation techniques. The purpose of first experiment was to develop mutants through x-rays irradiations and to screen the variants for drought tolerance. Thus, in vitro cultured plantlets of CV. Frantoio (4x) and Moraiolo (2x) were exposed to physical mutagenic treatment of X-rays (0, 3 and 5 Gy) at 37 oC with an aim to recover diploid and tetraploid plants from same transition zone. Protoplasts were isolated from root tips in order to study DNA damage through Single Cell Gel Electrophoresis (comet assay). Frantoio (4x) showed less DNA damage when exposed to x-rays, confirming that olive tetraploid plants have four set of genes and it has greater tendency for DNA repair under stress conditions. The DNA analysis with ISSR markers was performed for the identification of mutant plants and Frantoio (4x) produced more stable mutants as compared to Moraiolo (2x) at 3 gy dose of X-rays. Furthermore, these mutant plants were evaluated for in vitro induced drought stress through different concentrations (0, 2 and 4%) of polyethylene glycol (PEG, 8000) and antioxidant activities drought stress conditions were observed. Frantoio mutants showed increased activity of antioxidants and lower quantity of malondialdehyde (MDA) contents. Whereas, the PEG treatments produced higher proline accumulation and oxidative stress in Moraiolo mutants, confirming that net photosynthetic rate was decreased in Moraiolo mutants under drought stress conditions. In second experiment, the salt tolerance of olive cultivars Canino and Sirole was compared with two transgenic lines Canino At17-1 and Canino At17-2, expressing tobacco osmotin, obtained by Agrobacterium-mediated transformation, under the control of constitutive promoter 35S. Shoot cultures were in vitro exposed to salt stress by adding sodium chloride (NaCl) in growth medium at different concentrations: 0, 50, 100, and 200 mM. xix Most of the shoots of wt plants showed stunted growth and ultimate leaf drop by exposure to salt-enriched media, contrary to transgenic lines, that did not showed injuries and exhibited a normal growth. It is considered that S assimilatory pathway could be involved in alleviating the adverse effects of salt stress that has been evaluated in this study. For this purpose, thiols levels as well as extractable activities of ATPS and OASTL, the first and the last enzyme of S assimilation pathway, respectively, have been evaluated. The results have clearly depicted that both transgenic lines overexpressing osmotin gene expressed highest activities of ATPS and OASTL enzymes even at maximum level of NaCl and resulted significant resistant to in vitro induced salt stress as compared to wt cultivars. Malondialdehyde (MDA) content was also measured as an indicator of the level of lipid peroxidation. It can be concluded from this study that Sulphur assimilation pathway along with ATPS and OASTL enzymatic activities, plays a key role in adaptive response of olive plants under salt stress conditions.