In Vitro Elucidation Of The Impacts Of Plant Growth Regulators On Physio-morphic,biochemical And Genetic Attributes Of Different Olive(Olea Europaea L.)Cultivars

In vitro propagation of olive（Olea europaea L.）always remained a challenging task due to its woody nature and oxidation of culture.As a first of objective,The current study intended to optimize shoot induction and proliferation protocol for different genotypes（‘Leccino’,‘Gemlik’,‘Moraiolo’and‘Arbosana’）of olive on olive media（OM）provided with different concentrations（0,0.5,1.5 and 2.5 mg L-1）of 6-benzylaminopurine（BAP）by pre-exposing their explants（nodal segments）with different regimes（0,24 and 48h）of cooling.The impacts of treatments were evaluated on morphological（shoot induction percentage,primary shoot length,number of leaves shoot-1 and number of shoots per explant-1）,physiological（total chlorophyll,carotenoids,CO2 assimilation and proline）,biochemical（primary and secondary metabolites）attributes of cultivars after 50 to 60 days of culture.The experiment was conducted in three factorial arrangements with pre-cooling treatments as first,varieties as second and BAP concentrations as third factor.Data recorded were subjected to statistical analysis using computer based software statistix8.1 and R-Packages.All traits depicted significant increase in all genotypes with increasing pre-cooling treatments and increasing supplementations of 6-benzylaminopurine（BAP）;however this increase was the highest for the interaction of 48h pre-cooling and 2.5mg L^-1BAP concentration.Moreover,correlation analysis of all traits revealed significant paired association among them in positive direction,while principal component analysis（PCA）revealed extent of association varied with types of treatments and nature of genotypes.Among cultivars,‘Arbosana’depicted more dramatic change in morphological traits,physiological attributes and biochemical contents due to varying interaction of pre-cooling and BAP treatments as compared to‘Moraiolo’,‘Gemlik‘and‘Leccino’within in vitro system.The addition of BAP within OM increases shoots induction and proliferation through regulation of different physiological,metabolic and molecular processes.As a second part of objective the current study was conducted to elucidate the impacts of different supplementations of BAP within OM medium on physiological traits（chlorophyll,CO₂assimilation）,antioxidant enzymes（superoxide dismutase,peroxidase,catalase）and metabolic contents（sucrose,starch,flavonoids）in connection with the expression of corresponding genes（Oe Rbcl,Oe POD10,Oe SOD10,Oe CAT7,Oe SS4,Oe Su SY7,Oe F3GT,Oe Chl H）olive cultivars（Leccino,Gemlik,Moraiolo,Arbosana）under in vitro conditions.The second experiment was conducted in two factorial arrangements and collected data were evaluated statistically using computer based program statistix8.1.All physiological and biochemical traits illustrated significant increase under in vitro conditions due to increasing concentrations of BAP,however this increase was more dramatic at 2.5 mg L^-1while least dramatic at 0.5 mg L^-1.Besides,significant increase in the expression of corresponding traits associated genes was noticed in all genotypes due to varying concentrations of BAP.All genes depicted variation in their expression complementary to the variation of their corresponding physiological and biochemical traits.Although all cultivars depicted significant responsiveness to in vitro varying concentrations of BAP,however the response of Arbosana cultivar was statistically more distinct.In conclusion,current study established a new trend in context of protocol optimization by comprehensively evaluating the impacts of plant biostimulants on physiological,biochemical and genetic traits in integrated way.The third part of Current study was conducted with objective to evaluate the effect of different supplementations of indole-3-butryic acid（IBA）on root morphological（length of primary root,percentage induced shoot,number of roots per shoot,days of root initiation）,biochemical（antioxidant enzymes,starch,sucrose and flavonids）and genetic parameters（Oe Rbcl,Oe POD10,Oe SOD10,Oe CAT7,Oe SS4,Oe Su SY7,Oe F3GT）of olive cultivars（Leccino,Gemlik,Moraiolo,Arbosana）within olive medium（OM）under in vitro conditions.The experiment was conducted in two factorial arrangements with cultivars as one factor and IBA concentrations as other factor.All morphological parameters except days of root initiation recorded significant increase with increasing concentrations of IBA within OM.Likewise,biochemical parameters showed significant improvement in their expression due to increasing concentrations of IBA.Interestingly,metabolites and their associated genes showed complementary variation in their expression with varying concentrations of IBA,that confirmed strong extent of association between biochemical and genetic traits.Although all cultivars responded in parallel way to the varying concentrations of IBA in OM,however the response of‘Arbosana’was statistically more distinct.In conclusion this was first consolidated study that in depth elucidated the impacts of IBA on rooting process by integrating morphological,biochemical and genetic processes.The fourth part of Current study was conducted with objective to acclimatize the in vitro grown olive cultivars（Leccino,Gemlik,Moraiolo,Arbosana）within green house under control conditions by estimating their photosynthesis associated parameters.The data for chlorophyll（chl）,photosynthesis（Pn）,stomatal conductance（Gs）,transpiration rate（Tr）and stomatal conductance were taken at 20,40,100 and180 days of acclimatization period.Chlorophyll content and photosynthesis（Pn）showed consistent increase with increasing days of acclimatization period,while transpiration rate（Tr）,stomatal conductance（Gs）and stomatal density recorded consistent decrease with increasing days of acclimatization period.In conclusion,the changes reported in the leaves of in vitro-grown olive cultivars during long acclimatization period indicated that the plantlets are well acclimated due to proper functioning of photosynthetic apparatus and plant assumes full autotrophic capacity.From obtained results we can further conclude that monitoring photosynthesis（Pn）and chlorophyll content on one hand and the densities of stomata on the other hand can facilitate to predict the necessary duration of the acclimatization.Overall current study proved that the plant growth regulators demonstrate their impact by changing the multiple traits that collectively determine plant growth and development processes.Although in past different studies were conducted for elucidation of the impacts of hormones on different traits but they did not provide the knowledge about their relation with plant physiology,biochemistry and genetics.This is the first ever study that unanimously taken into consideration the plant physiology,biochemistry and genetics for optimization of the impacts of growth regulators on micro propagation of olive cultivars.