In Vitro Mutagenesis For Genetic Improvement In Mat Rush (Juncus Effusus L.)

Mat rush(Juncus effusus L.) is a cosmopolitan,emergent macrophyte inhabits fresh to brackish marshes,wetlands and meadows.It is an economical as well as ecologically important plant grown for reclamations of land,mitigation and construction of wetlands,while its stem is used as raw material for production of various products.Keeping in view the importance of mat rush plant a series of experiments was conducted to induce genetic variations and to improve the quality of plant using in vitro mutagenesis techniques.An efficient tissue culture protocol was developed in the first step as a prerequsite of in vitro mutagenesis experiment.Plants were grown on MS(Murashige and Skoog,1962) medium and effects of hormonal combinations and medium innovation on tissue culture system were investigated.Positive effects of additional benzylaminopurine(BA) in the medium were observed on callus proliferation,plant regeneration and its multiplication.0.5 mg L^-1 concentration of BA in combination with 4 mg L^-1 of 2,4-dichlorophenoxyacetic acid(2,4-D) in the medium resulted in the highest callus induction(90.48%).The effects of various growth regulators and their combinations with successive subculturing(medium replacement) were found to be essential to develop embryogenic calli and to regenerate shoots.Among different combinations of growth regulators tested in the study,the combination of 0.1 mg L^-1 BA and 2 mg L^-1 2,4-D in MS medium was determined to be the best for callus proliferation.Medium innovation was also found necessary for successful plant regeneration.Our results suggested the importance of the ratio of auxin(IAA) to cytokinin(BA and KT) in the manipulation of shoot regeneration in this plant.The maximum plant survival frequency and multiplication rate(90.97%) were recorded in the presence of 0.5 mg L^-1 BA in the 1/2 MS multiplication medium for the plant.An in vitro mutagenesis study was carried out using the tissue culture protocol developed in the first study to induce genetic variability in mat rush and to unveil the mutagenic effects of ethyhnethane sulfonate(EMS at 0.1-1.0%for 12 and 24 h) and ultraviolet radiations(UV for 15,30 and 45 min) on physiological characteristics and ultra-structural responses of the plant.The basal culms of plants were treated with EMS and UV radiations and then transferred to 1/2 MS media to grow and multiply.Physiological and ultra-structural alterations of leaf were observed in the leaves of treated plants.A large number of mutants with changes in leaf colour,length,size and growth rates were found.With the increase in the level of mutagens,the activities of antioxidant enzymes increased invariably,however,at their higher level these activities showed downward trend.Plant growth and biomass significantly reduced at the higher doses of both mutagens.Further study of the selected mutants showed greater reduction in chlorophyll contents as well as noticeable changes at the ultra-cellular levels.Over all reduction in the size of cell and chloroplast,appearance of large number of oil bodies in chloroplasts and disruption of the thylakoid integrity were some of the obvious changes in resultant mutants.Present study suggested that both EMS and UV mutagens caused mutation at functional and ultra-structural levels that can be effectively used to create genetic Variations in mat rush(Juncus effusus L.).