TY - JOUR
T1 - Isolation of MTX-resistant cell line NP-19 of Nicotiana plumbaginifolia
T2 - Phenotypic, genetic and biochemical study
AU - Shabtai, Sara
AU - Ziv, Meira
AU - Barg, Rivka
PY - 1992/4
Y1 - 1992/4
N2 - Several cell lines of Nicotiana plumbaginifolia resistant to methotrexate (MTX) were isolated upon gradual elevation of the MTX concentration in the growth medium. One of the MTX-resistant cell lines, NP-19, acquired resistance to 10 mmol m-3 MTX, that is, at least 50-fold higher than the lethal dose for the wild-type cell suspension. Its resistance was stably maintained upon prolonged withdrawal of the drug. The acquisition of resistance was accompanied by severe deterioration of regeneration potential. Regenerated shoots still manifested resistance to 5·0 mmol m-3. MTX-resistant colonies and tiny shoots were recovered following asymmetric fusion between γ-irradiated NP-19 protoplasts and N. plumbaginifolia leaf mesophyll-derived protoplasts on a medium containing 1·0 mmol m-3 MTX. The development of the shoots derived both from NP-19 calli and following somatic fusion was arrested at the stage of 6-8 leaves. No difference was found in uptake of 3H-MTX between cell suspension of NP-19 and the parental cell line. A 30-fold increase in binding of 3H-MTX to protein extract was found in cell line NP-19, suggesting differential capacity of MTX binding as a mechanism involved in the MTX resistance of this cell line. Since differentiated organs seem more sensitive to MTX than undifferentiated tissues, this cell line is a promising source for a gene(s) conferring enhanced MTX-tolerance both in non-differentiated and differentiated tissues.
AB - Several cell lines of Nicotiana plumbaginifolia resistant to methotrexate (MTX) were isolated upon gradual elevation of the MTX concentration in the growth medium. One of the MTX-resistant cell lines, NP-19, acquired resistance to 10 mmol m-3 MTX, that is, at least 50-fold higher than the lethal dose for the wild-type cell suspension. Its resistance was stably maintained upon prolonged withdrawal of the drug. The acquisition of resistance was accompanied by severe deterioration of regeneration potential. Regenerated shoots still manifested resistance to 5·0 mmol m-3. MTX-resistant colonies and tiny shoots were recovered following asymmetric fusion between γ-irradiated NP-19 protoplasts and N. plumbaginifolia leaf mesophyll-derived protoplasts on a medium containing 1·0 mmol m-3 MTX. The development of the shoots derived both from NP-19 calli and following somatic fusion was arrested at the stage of 6-8 leaves. No difference was found in uptake of 3H-MTX between cell suspension of NP-19 and the parental cell line. A 30-fold increase in binding of 3H-MTX to protein extract was found in cell line NP-19, suggesting differential capacity of MTX binding as a mechanism involved in the MTX resistance of this cell line. Since differentiated organs seem more sensitive to MTX than undifferentiated tissues, this cell line is a promising source for a gene(s) conferring enhanced MTX-tolerance both in non-differentiated and differentiated tissues.
KW - MTX uptake
KW - MTX-binding capacity
KW - Methotrexate resistance
KW - Tobacco cell culture
UR - http://www.scopus.com/inward/record.url?scp=0343900751&partnerID=8YFLogxK
U2 - 10.1093/jxb/43.4.471
DO - 10.1093/jxb/43.4.471
M3 - ???researchoutput.researchoutputtypes.contributiontojournal.article???
AN - SCOPUS:0343900751
SN - 0022-0957
VL - 43
SP - 471
EP - 478
JO - Journal of Experimental Botany
JF - Journal of Experimental Botany
IS - 4
ER -