Isolation of MTX-resistant Cell Line NP-19 of Nicotiana plumbaginifolia: Phenotypic, Genetic and Biochemical Study

Several cell lines of Nicotiana plumbaginifolia resistant tomethotrexate (MTX) were isolated upon gradual elevation of theMTX concentration in the growth medium. One of the MTX-resistantcell lines, NP-19, acquired resistance to 10 mmol m^–3MTX, that is, at least 50-fold higher than the lethal dose forthe wild-type cell suspension. Its resistance was stably maintainedupon prolonged withdrawal of the drug. The acquisition of resistancewas accompanied by severe deterioration of regeneration potential.Regenerated shoots still manifested resistance to 50 mmol m^–3.MTX-resistant colonies and tiny shoots were recovered followingasymmetric fusion between -irradiated NP-19 protoplasts andN. plumbaginifolia leaf mesophyll-derived protoplasts on a mediumcontaining 10 mmol m^–3 MTX. The development of the shootsderived both from NP-19 calli and following somatic fusion wasarrested at the stage of 6–8 leaves. No difference wasfound in uptake of ³H-MTX between cell suspension of NP-19 andthe parental cell line. A 30-fold increase in binding of ³H-MTXto protein extract was found in cell line NP-19, suggestingdifferential capacity of MTX binding as a mechanism involvedin the MTX resistance of this cell line. Since differentiatedorgans seem more sensitive to MTX than undifferentiated tissues,this cell line is a promising source for a gene(s) conferringenhanced MTX-tolerance both in non-differentiated and differentiatedtissues. Key words: Methotrexate resistance, MTX-binding capacity, MTX uptake, tobacco cell culture