Isolation, Protoplast Culture, and Plant Regeneration of PFP-Resistant Variants of Nicotiana tabacum Sulsu

Abstract

Summary p-Fluorophenylalanine (PFP) resistant variants of Nicotiana tabacum Sulsu were routinely isolated from rapidly growing suspension cultures by plating in the presence of 0.5 mM PFP. The PFP-resistant phenotypes of about 10% of the variants isolated were stable, and calli have retained resistance to PFP after more than one year in culture. Resistance was retained whether the cells were grown in the presence or absence of PFP. Calli of PFP-resistant cell lines were induced to regenerate shoots on MS medium with 5 µM 6-benzylaminopurine. In some PFP-resistant lines shoots were regenerated in the presence of 0.5 mM PFP. Shoot regeneration from PFP-resistant cell lines was only achieved shortly after their isolation. Although shoots could be rooted, the albino Su mutantion precluded transfer of the plants to soil. Suspension cultures of PFP-resistant cell lines retained the same chromosome number (2n = 48) of intact plants, even after several months in liquid culture. Protoplasts (isolated from suspension cultures established from calli of PFP-resistant cell lines) were cultured in the presence of 0.125 mM PFP, a concentration sufficient to inhibit cell wall regeneration and cell division of normal Sulsu protoplasts. Suitable conditions are therefore defined for the use of PFP resistance as a selective marker in recognition of somatic cell hybrids accomplished via protoplasts fusion.