Enhancement of abiotic stress tolerance In Canola (Brassica napus) through genetic engineering approach

© 2016 Dr. Aqsa TabasumAgricultural production encounters multifaceted environmental challenges such as high salinity, water deficiency and extreme temperatures. Crop plants are most often influenced by multiple abiotic stresses that are subject to complex interactions therefore designing strategies to contest abiotic stress is a challenge. Expression of genes regulating stress adaptive mechanisms has prime importance in improving stress tolerance. Helicases member of DEAD-box protein family are capable of transiently catalysing the unwinding of energetically stable duplex DNA or RNA molecules. They play vital roles in basic cellular processes and are involved in protein turnover and protection by removing the RNA secondary structures to empower the tolerance to multiple abiotic stresses. PDH45, a DNA helicase isolated from pea that exhibits striking homology with the eukaryotic translation initiation factor eIF-4A was reported to be up-regulated in pea plant in response to high salinity, cold stress, abscisic acid (ABA), dehydration and early wounding. In present study, genetic engineering approach was employed to generate transgenic lines of canola (Brassica napus) harbouring PDH45 DNA helicase gene. Transformation was successfully achieved with 26.30% transformation frequency. Fifty three transgene PCR positive independent transgenic lines were obtained that were confirmed for gene and protein expressions. Genetic and molecular analyses confirmed the integration and inheritance of the transgene. Transgenic canola plants expressing PDH45 helicase displayed dramatically enhanced salt tolerance and moderately improved drought and heat stress tolerance. Transgenic lines manifested vegetative and reproductive stage salt stress tolerance that was found to be functionally stable for studied traits. Improved physiological seedling growth particularly seedling biomass was evident in transgenic lines under hydroponic salt stress (150 mM NaCl). Seedling growth decline in term of plant fresh and dry biomass was several times less in transgenic lines than wild type. During the reproductive stage stress, transgenic lines manifested better morpho-physiological growth under salt stress (100 mM NaCl) in particular through increased plant height, larger leaf area, number of leaves, enhanced photosynthesis yield and chlorophyll contents with lower membrane damage than wild type plants. Furthermore, transgenic plants were able to grow under continuous salt treatment (100 mM NaCl) and produced fertile flowers, pods and finally set seeds. Improvement of reproductive performance was evident by considerably high pollen fertility and less reductions in yield and yield components in transgenic lines. Under salinity stress, the transgenics produced significantly greater stem dry biomass, pod weight, number of pods per plant, pod length, number of seeds per pod leading to significantly higher seed yields than wild type plants which demonstrated drastic reductions. PDH45-transgenic plants maintained relatively high K+ ion and lower Na+ levels in leaves than WT plants under salinity stress. Moreover, PDH45 expressing transgenic plants showed osmotic stress tolerance to in-vitro PEG induced osmotic stress having dramatically higher chlorophyll contents in leaf disc under osmotic stress than wild type. Transgenics exhibited better recoveries after short period (10 days) of water deficiency stress by displaying enhanced photosynthesis yield, leaf number, leaf area, fresh plant biomass and shoot weight. In this study, non-significant differences were recorded for dry shoot weight; shoot length and relative water contents (RWC). Relative membrane permeability an indicator of membrane damage was significantly high (61.22%) in wild type plants while PDH45 transformed events maintained their membrane integrity under water stress condition. In this study, after monitoring the effect of high temperature (maximum 35oC) stress during flowering it was observed that pod and seed development was significantly improved in transgenic lines than wild type plants under heat stress. Under high temperature stress (HTS) during flowering, transgenic lines indicated significantly increased plant height and number of branches than wild type plants (WT). Under HTS, transgenic lines exhibited significantly less pod sterility and produced significantly greater pod weight, pod length and 100-seed weight. While comparing with non-stressed controls, highest reductions were observed in wild type plants for pod weight, seed number per pod and 100-seed weight under HTS. By contrast, transgenic lines exhibited relatively less reductions in pod weight and seed weight while no reduction was observed for seeds per pod. HTS resulted in remarkable increase (40.31%) in pod sterility in wild type than transgenic lines (12.87 to 15.83%). In our study, HTS caused highest yield decline in WT (82.15%) whereas the reductions in different transgenic lines were significantly lower (37% to 60%). Furthermore, T3-transgenic seeds indicated higher germination percentage and less seed electrolyte leakage than wild type seeds. Collectively, increase seed number per pods, increased seed weight and reduced pod sterility contributed to enhance seed yield in PDH45 transformed lines of canola under heat stress. This study provided the evidence that members of DEAD-box helicase family are potentially useful genes for engineering crops for enhanced tolerance to multiple abiotic stresses. PDH45-transgenic plants indicated the ability to minimize the toxic effects of sodium by restricting the entry of Na+ and maintaining the nutrient balance by accumulating more K+ ion. Mainly the differences in Na+ accumulation, photosynthesis, chlorophylls and cell membrane stability in WT and PDH45 overexpressing transgenic plants were responsible for their differential tolerance towards studied stresses therefore; PDH45 is proposed to regulate these processes as adopted mechanisms for stress tolerance. This study along with the further detailed mechanism (s) associated with PDH45 for multiple stress tolerance will contribute to the knowledge of crop improvement strategies and findings of this project can be applied to other oilseed crops