Culture and selection of somatic hybrids using an auxotrophic cell line

Protoplast fusions between Nicotiana tabacum and N. paniculata and between N. tabacum and N. sylvestris were obtained by polyethylene glycol and Ca(NO3)2 treatment. The protoplasts of one parent originated from cell suspensions, while the protoplasts of the other originated from leaf mesophyll. The heterokaryons were detectable by their intermediate phenotype, namely the green chloroplasts from mesophyll and the dense cytoplasm from suspension cells. They were isolated with micropipettes immediately after fusion using a micromanipulator and were transferred into a protoplast suspension of an auxotrophic cell line serving as a nursery. This mutant is not able to utilize nitrate and had to be supplemented with amino acids. The somatic hybrids were selected by a stepwise reduction of the supplements, which caused the death of the mutant cell colonies, while the autotrophic somatic hybrids continued to grow. The hybrid character of the selected colonies was confirmed by isoenzyme investigations.