In-Vitro Regeneration And Agrobacterium-Mediated Genetic Transformation Of Catharanthus Roseus And Citrus

Periwinkle(Catharanthus roseus)belongs to family Apocynaceae,which is a perennial plant found in tropical regions commonly recognized by the following names,Vinca roseus or Madagascar periwinkle.C.roseus is also an alternative host of Huanglongbing(HLB)bacteria Candidatus Liberibacter asiaticus(CLas).C.roseus is an ideal experimental plant for the pathogenesis studies of CLas bacterium.In this study,we analyzed the effects of plant growth hormones on callus induction and regeneration of C.roseus using hypocotyls,leaves and shoots explants.Red fluorescent protein(Ds Red2)was firstly successfully used as a visual marker for C.roseus genetic engineering.The main results are as follows:To optimize the conditions of regeneration of C.roseus,we used different combinations of plant growth hormones of 6-Benzylamino purine(BAP),Naphthalene acetic acid(NAA)and 2,4-Dicholophenoxyacetic acid(2,4-D),Indole butyric acid(IBA)with full MS and half medium.The maximum callus growth rate from hypocotyls was 98.8% observed after 30 days(d)of growth on M6 callus medium: [MS + BAP(2.0 mg/L)+ NAA(1.0 mg/L)+ 2,4-D(1.0 mg/L)].For leaves,the maximum callus growth rate was 94.0% observed after 30 d on M5 callus medium: [MS + BAP(2.0 mg/L)+ NAA(0.5 mg/L)+ 2,4-D(0.5 mg/L)].The maximum direct shoot regeneration ratio was 97.0% on MS1 medium: [MS + BAP(1.0 mg/L)+ NAA(0.4 mg/L)].And averagely 7.0 ± 2.0 shoots were produced by single-bud.Whereas,for indirect shoot regeneration,the maximum shoots developed from calli was 94.0% on MS1 [MS + BAP(1.0 mg/L)+ NAA(0.4 mg/L)] and MS5 [MS + BAP(2.0 mg/L)+NAA(0.3 mg/L)].For shoot regeneration per hypocotyl,the maximum average 18.7 ± 3.51 of shoots was observed in MS1 [MS + BAP(1.0 mg/L)+ NAA(0.4 mg/L).Whereas,minimum average of shoots per hypocotyl 15.7 ± 5.13 was observed in MS5 [MS + BAP(2.0 mg/L)+ NAA(0.3 mg/L)].While the maximum rooting rate was 88.0% observed on R3 medium [1/2 MS + IBA(2.5 mg/L)+ NAA(0.5 mg/L)+ sucrose(30 g/L)+ agar(5.5 g/L)] along with best quality roots.Agrobacterium tumefaciens strain EHA105 and binary vector p CAMBIA2300::35S::Ds Red2 containing an enhanced 35 S promoter was used for the optimization of gene transformation of C.roseus with Pacific white(PW),Pacific grapevine red(PGR)and Pacific cherry red(PCR).The optimal transformation condition was obtained using C.roseus hypocotyls as explants: A.tumefaciens was adjusted to OD600=0.5,infection of 25 min and co-cultivated at temperature 19 °C for 48 h in 1/2 MS medium with 100 ?M acetosyringone(AS).With the optimization of the concentration of Kanamycin(Kan)(40,50,70 mg/L),the highest regeneration rate of transgenic plants observed in PGR,PCR and PW was 55.5%,45.5% and 38.0% respectively.Visual observation under auto-fluorescence microscopy revealed that Ds Red2 was not only observed in callus but also in most of the organs(leaves,stems)of the transgenic C.roseus plants.The transgenic plantlets were further confirmed by PCR targeting Ds Red2 transgenic plantlets.A total of 30 different lines were tested,and 21 lines detected positive by PCR,the positive ratio of PGR,PCR and PW was 77.7%,75.0% and 55.5% respectively.Ds Red2 was also used for genetic transformation with two citrus species,sweet orange(Citrus sinensis)and pomelo(Citrus maxima).Epicotyls were used as explants.Various parameters were examined to optimize the genetic transformation in C.sinensis and C.maxima.A.tumefaciens was adjusted to OD600=0.6,infection of 25 min was the best for Agrobacteriummediated transformation in C.sinensis and C.maxima.The best condition for co-culture was 72 h under 19 °C with 100 ?M of AS in MT containing malt extract(0.5 g/L)and L-glutamine(1.5 g/L).We regenerated several Ds Red2 expressing transgenic lines from C.sinensis and C.maxima.The maximum regeneration rate of C.maxima and C.sinensis was 50.0% and 35.5%,respectively.Further,we detected Ds Red2 protein expression under auto-fluorescence microscopy,Ds Red2 was present in most of the tissue(leaves,shoots,stem)of the C.sinensis and C.maxima.In a word,we firstly successfully used Ds Red2 as a visual marker for gene transformation in C.roseus and citrus,which laid a firm foundation for future study on gene editing for molecular breeding against CLas-interaction to host.