Agrobacterium Rhizogenes-mediated Transformation For Characterizing Al Tolerance Genes

Legumes(pulse crops)are cultivated to provide a protein source in various locations in Asian countries where acidity(i.e.proton and A1 rhizotoxicity)is common problem in soils.I developed an efficient transformation system of hairy roots that can be used for characterizing functional genes regulating Al tolerance in pulse crops.Firstly,we tried to improve efficiency of transformation in tobacco(Nicotiana tabacum)to generate hairy roots system by Agrobacterium rhizogenes-mediated transformation.The procedures enabled with high rate of transformation of the target genes locating in the co-existing Ti-plasmid vector when the hairy roots were formed by the transformation of T-DNA of internal Ri-plasmid.Rate of co-transformation in the hairy roots attained over 80%,and it could enable us to transform the target genes without any selection marker genes such as resistance to processes by antibiotics marker if the target genes could be recognized by visible marker such as GFP(Green Fluorescent Protein).Using this system,we could analyze the function of MATE(Multidrug And Toxic compound Extrusion)-citrate transporter from Cajanus cajan(L.)Millsp.GFP-fusion proteins of CcMATE1 localized in the plasma membrane,and overexpression in the tobacco NtSTOPl-KD enhanced Al-activated citrate excretion.These observations could fit the expected function of CcMATE01,which is upregulated by Al,and being concomitant with Al-activated citrate excretion.In addition,high efficiency of co-transformation rates of hairy root systems enabled us to characterize the GFP-tagged MATE gene in the transgenic tobacco of NtSTOP1-KD.The NtSTOP1-KD carried multiple antibiotics resistance due to original vectors used for introducing RNAi construct.Without any selection processes by antibiotics,we obtained the transgenic hairy roots efficiently.Most of the transgenic hairy roots expressed CcMATE1 genes,and they showed clear response of Al-activated citrate excretion due to loss of function of citrate regulators NtSTOPl genes in the NtSTOP1-KD.