Genetic Transformation Of Herbicide Resistant Genes In Upland Cotton And Exploring The Genetic Potential Of Transgenic Cotton Against Cadmium Stress

Cotton,Gossypium spp.,is an economically important crop that is grown throughout theworld.To improve the genetic basis of cotton,two approaches have been adopted,namely,traditional and non-traditional.The non-traditional approach born with theadvent of Recombinant DNA Technology and is mainly based on transformation ofdesirable traits into the host genome.Thus it is very important in crop production toincrease the production efficiency and reduce both the monetary and environmental costof production.In this paper,we compared and utilized the genotype independentapproaches for the Bar gene transformation in cotton as well as to study the inheritanceand segregation of the exogenous gene in the resultant transgenic cotton cultivar(BR001).We also developed bentazon(a herbicide) resistant germplasm throughAgrobacterium-mediated genetic transformation using embryogenic callus as explant.Inaddition,we explore the other genetic potentials of the resultant transgenic cottongermplasm(BR001) apart from the herbicide and insect resistance in comparison withthe extending insect resistant cultivar(GK30) and its wild relative cotton genotype(Coker 312) regarding the cadmium tolerance by peeping into the possible cellularmechanism being involved against cadmium stress.The main experimental results arepresented as follows:1.In order to develop gluphosinate resistance in the cotton genome,we exploitedthe genetic transformation technique using two genotype independent approaches.We also further studied segregation and inheritance of the Bar gene as well asvarious agronomic and fiber quality traits of the resultant transgenic cotton cultivar(BR001) and its parent line(Coker 312).PCR and Southern blot analyses showedsuccessful integration of the foreign gene in case of both approaches.Although thetransformation efficiency of shoot apex culture method was higher than PollenTube Pathway via ovarian injection method,the number of plants developed in thesoils was almost same.Field evaluation revealed that the agronomic traits werelittle bit superior of the parent line as compared to BR001,however,the fiber quality traits like fiber strength,lint percentage of BR001 were almost equal toCoker 312.Moreover,the Mendelian inheritance and segregation studiesconfirmed that a single nuclear dominant gene governed the herbicide resistancecharacters in BR001.Our present study suggests that both genotype independentapproaches can be utilized.2.We report the Agrobacterium-mediated transformation of bentazon resistantgene(named as Cyp81A6) in two Coker cotton genotypes namely,Coker 312 andCoker 201 using embryogenic calli(EC).The EC of both genotypes were firstinoculated with the A.tumefaciens strain LBA4404 harboring binary vectorp35S81A6,which carries the rice herbicide-resistant gene(Cyp81A6) and nptⅡgene driven by CaMV35S promoter(P35S).The infected calli were co-culturedwith A.tumefaciens for 48 hours and then transferred to the selection mediumhaving kanamycin(100 mg/L) and 500 mg/L cefatoxim for 4 weeks.Thekanamycin selection process was repeated for three times and the kanamycinresistant highly proliferating EC were put on normal medium.A large number ofwell-developed somatic embryos were obtained after 2-3 rounds of subculturing onnormal medium.The cotyledon embryos were transferred to 100 ml Erlenmeyerflasks for germination and regeneration.The kanamycin and bentazon resistant testconfirmed the transformation event in both genotypes and the PCR reactionrevealed that the EC were putatively transgenic.The developing plantlets werestepwise transferred to the greenhouse and finally to the field.3.The toxic effect of cadmium(Cd) at increasing concentrations was studied withspecial attention being given to the root morphological and ultrastructural changesin herbicide resistant transgenic cotton germplasm BR001,using extending insectresistant transgenic cotton cultivar(GK30) and its wild relative line(Coker 312) aschecks.In comparison to their respective controls,low concentration(10 and 100μM) of Cd greatly stimulated seed germination,while it was inhibited by highestconcentration of Cd(1000μM) in case of two transgenic cultivars.However,inCoker 312 the seed germination percentage progressively decreased over thecontrol at all Cd levels.Various physiological and morphological parameters of theroot and whole plant in both transgenic cotton cultivars and their relative wild cotton genotype respond differently towards the Cd toxicity.Bioavailability of Cdwas concentration-dependent where seedling roots captured more Cd as comparedto shoot.BR001 accumulated more Cd followed by GK30,while Coker 312 wasless Cd accumulator.The ultrastructural modifications in the root tip cells of boththe transgenic cotton cultivars and their wild relative were also dose-dependent.With the increase in Cd levels,the fine structures of their root cells also invariablychanged.Increase in plasmolysis of the plasma membrane,greater number ofnucleoli and vacuoles and enlarged vacuoles could be observed in both transgeniccotton cultivars.In comparison to them,Coker 312 showed relatively well-developed ultrastructures of the root tips except enlarged vacuoles and greaternumber of mitochondria.Moreover,the accumulation of Cd in the form of electrondense granules and crystals both in vacuoles and attached to cell walls were visiblein both transgenic cotton cultivars and their wild relative.These results suggestthat both transgenic cotton cultivars and their wild relative cotton genotyperesponded positively towards Cd stress at seedling stage,the internal Cd-detoxification might be through apoplastic and symplastic binding.Moreover,as awhole BR001 proved to be sensitive whereasGK30 and Coker 312 were found astolerant.4.The ultramorphological changes in leaves as well as physiological alterations atthe whole plant level in herbicide resistant transgenic cotton germplasm(BR001),extending insect resistant transgenic cotton cultivar(GK30) and their wild relativecotton genotype(Coker 312) were investigated under toxic concentrations ofCadmium(Cd).At all Cd levels(0,10,100,1000μM),the mean lengths of root,stem and leaf and leaf width as well as their fresh and dry biomasses linearlydecreased.Moreover,root,stem and leaf water absorption capacities progressivelystimulated,which were high in leaf followed by root and stem.BR001accumulated more cadmium followed by GK30 and Coker 312.Root and shootcadmium uptakes were significantly and directly correlated with each other andalso with leaf,stem and root water absorption capacities.The ultrastructuralmodifications in leaf mesophyll cells became prominent with elevated levels of Cd,which were more obvious in BR001 followed by GK30 and Coker 312.Change in morphology of chloroplast,increase in number and size of the starch grains as wellas increase in the number of plastoglobuli reveal the toxic effect of Cd onphotosynthetic organ.Cd in the form of electron dense granules could be seeninside the vacuoles and attached to the cell walls in all these cultivars.Theseresults suggest that both apoplastic and symplastic bindings are involved in Cddetoxification in these cultivars.Absence of tonoplast invagination reveals that Cdtoxic levels did not cause water stress in any cultivar.Also,these cultivars possessdifferential capabilities towards Cd accumulation and its sequestration.5.Cadmium-induced oxidative metabolism was studied in herbicide resistanttransgenic cotton germplasm(BR001),extending insect resistant transgenic cottoncultivar(GK30) and their wild relative cotton genotype(Coker 312) under theincreasing concentrations of Cd.The results revealed that under different Cd levels,the antioxidant enzymes and lipid peroxidation differentially regulated.Bothtransgenic cotton cultivars and the wild relative invariably responded towards Cdstress at enzymatic levels.Overall greater enzymatic levels could be found inleaves as compared to other parts of the seedlings in these cultivars.Moreover,there could be found no direct relationship between the antioxidant enzymesactivities and the Cd tolerance in either of these cultivars.Differential regulation ofthese enzymes in our present experimental materials suggests that their mightinvolved different tolerance mechanism at both cellular and molecular levels,which needs to be confirmed.