Establishment Of Regeneration And Transformation Systems And Germplasm Innovation Of Mat Rush (Juncus Effusus L.) Through In Vitro Mutagenesis

Wetland species mat rush (Juncus effusus L.) is an important economic plant,butno information is available regarding the callus proliferation and plant regenerationfrom seed-derived calluses.Keeping in view the importance of mat rush plant a seriesof experiments was conducted to induce genetic variations and to improve the qualityof plant using genetic transformation and in vitro mutagenesis techniques.An efficienttissue culture protocol was developed in the first step as prerequsite of genetictransformation and in vitro mutagenesis.The present study investigates the effects of growth regulator combinations andmedium innovation on tissue culture system of five varieties of mat rush.Addition ofN6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D) in Murashige andSkoog medium (MS) showed significantly positive effect on callus proliferation,plantregeneration and its multiplication as compared to the medium devoid of BA.Thehighest callus induction (80.95,90.48,75.40,70.83 and 83.33 %) was observed in MSmedium containing 0.5 mg L^-1 BA in Yinlin-1,Nonglin-4,Gangshan,Taicao and Taiwangreen,respectively.Various growth regulators combination with successive subculturing(medium replacement) were found to be essential to develop embryogenic calluses andto regenerate shoots.The combination of 0.1 mg L^-1 BA and 2 mg L^-1 2,4-D in MSmedium was determined to be the best for callus proliferation for all the varieties undertrial.The plant regeneration required two steps involving successive mediumreplacements as well as optimal hormonal balances.Successful plant regeneration (over70%) was observed only by transferring the embryogenic callus from regenerationmedium-â… (MS medium containing 0.5 mg L^-1 BA and 1.0 mg L^-1 KT) to theregeneration medium-â…¡(MS medium containing 0.5 mg L^-1 BA,1.0 mg L^-1 KT and 3.0mg L^-1 IAA.Our results confirmed the importance of the ratio of auxin (IAA) tocytokinin (BA and KT) in the manipulation of shoot regeneration in J.effusus L.Themaximum plant survival frequency and multiplication rate (90.97 % and 5.40,and 94.23% and 8.25) were recorded in the presence of 0.5 mg L^-1 BA in the 1/2 MSmultiplication medium for the varieties of Nonglin-4 and Taicao,respectively.Theefficient plant regeneration system developed here has now been used for genetictransformation and mutagenesis breeding in J.effusus L.Agrobacterium-mediated genetic transformation was explored for inducing insectresistance in mat rush (Juncus effusus L.).Firstly,the callus regeneration system wasestablished,and secondly the Agrobacterium tumefaciens strain EHA105 with plasmid pKUB carrying cryIA (b),hph and gus genes,coding for hygromycin phosphotransferaseandβ-glucuronidase,respectively,was used for transformation.Results indicated that150μM acetosyringone in both of the induction and co-cultivation media increased thefrequency of GUS⁺ calli,and OD₆₀₀ value 0.4 of Agrobacterium enhanced the rate ofGUS⁺ calli,significantly.The infection duration of 45-60 min,co-cultivation duration of4 days,and infection in the 12^th or 16^th day were the best combination for transformation.Agrobacterium-treated plants achieved a transformation frequency of 9.56 % to thehighest.Transient GUS expression and hygromycin-resistance tests were practicable andreliable for selecting the positive transformants.This transformation system was foundsuitable for manipulating the desired traits though transgenic techniques,as thetransgenic Juncus effusus L.plants were produced from seeds to embryogenic calli,thento plantlets.The Agrobacterium-mediated transformation of Bt gene into mat rush withsome optimizations is highly successful and reproducible as experienced from theestablished protocol.Keeping in view the ecological and economic importance of mat rush (Juncuseffusus L.),an in vitro mutagenesis study was carried out to induce genetic variability ina commercial variety Yinlin-1 and to unveil the mutagenic effects of ethylmethanesulfonate (EMS,at 0.1-1.0 % for 6,12 and 24 h) and ultraviolet-C (UV-C,for 15,30,45and 60 min) on physiological characteristics and ultrastructural responses of the plant.The basal culms segments of plants were used for in vitro mutagenesis treatment.Afterthe treatment with EMS and UV radiations,these segments were first transferred to 1/2Murashige and Skoog media and then shifted to soil-perlite mixture to grow andmultiply.Plant growth and biomass were significantly reduced with the increasing dosesof both of the mutagens.With the increase in the level of mutagens,the activities ofantioxidant enzymes increased in the treated plant,however,at their higher level theseactivities showed downward trend.Physiological and ultrastructural alterations wereobserved in the leaves of selected mutants.Further study of these mutants showedgreater reduction in chlorophyll contents as well as noticeable changes at theultracellular levels.Over all reduction in the size of cell and chloroplast,appearance oflarge number of plastoglobuli in chloroplasts and disruption of the thylakoid integritywere some of the obvious changes in resultant mutants.Both EMS and UV radiationscaused mutation at functional and ultrastructural levels that could be effectively used tocreate genetic variations in this crop.Some useful mat rush mutants with novelmorphological and physiological characteristics have been identified and are beingutilized in the breeding programmes as well as for further genetic studies. An experiment of in vitro mutagenesis was conducted to induce tetraploidy in alocal leading cultivar Nonglin-4 of mat rush (Juncus effusus L.) through exposure toantimitotic chemical colchicine.The basal culm segments of plants were treated in vitrowith colchicine (0,50,100 and 500 mg L^-1) for 6,12 and 24 hours,and then transferredto 1/2 Murashige and Skoog media to grow and multiply.Flow cytometric analysis wasused to confirm the ploidy level.Tetraploid plants appeared to be superior to the diploidcontrol plants with the thicker and stronger leaves and more biomass on themorphological basis.The effects of colchicine on cellular and subcellular levels werestudied in the resultant tetraploid plants.While anatomical and ultrastructural analysesdepicted variations at cellular and subcellular levels in tetraploid compared with diploidcontrol plants,study of leaf epidermis revealed larger stomatal size but low stomataldensity in these tetraploid plants.In addition,mesophyll cells in tetraploidy appearedmore compact and showed less intercellular spaces along with increased size of vascularbundles.Interestingly,on an overall basis significant reduction of chlorophyll contentwas observed in tetraploid plants that might be the result of thylakoid disintegration.Amicrographic study of cellular organelles showed that higher doses of colchicine proveddestructive to the chloroplast causing disruption of thylakoid membrane system.Thesetetraploid plants developed during the study have been utilized in the breedingprogrammes as well as for further genetic studies of mat rush.In the present study,DNA damage caused by EMS was evaluated in mat rush(Juncus effusus L.),by the alkaline single-cell gel electrophoresis (SCGE).SCGE alsocalled commet assay is a method which can detect DNA rupture under level of singlecell.The results showed that comet percentage,the head DNA percentage,tail DNApercentage and tail length of the tested groups were significantly different from thecontrolled group (P＜0.01).In addition,the significant concentration or treatingtime-dependent reduce in percentage of survival rate was found in mat rush segmentsexposed to EMS and UV.To our knowledge,this was the first report describing thegenotoxicity assessment of mutagen using mat rush plant by the alkaline SCGE.