174 The Nature of Biological Diversity 



difference in nuclear genotype. However, in order to leave no possi- 

 bility of reasonable doubt about this conclusion, the breeding analysis 

 was carried further by backcrosses and by crosses between, and autog- 

 amy within, Fl clones. All results agreed. No segregation of singlets 

 from doublets occurred in these generations from parents of either 

 type. All exconjugant clones from singlet parents, regardless of geno- 

 type, were composed of singlet cells; and all exconjugant clones from 

 doublet parents, regardless of genotype, were composed of doublet 

 cells. Clearly, genotypic differences here had nothing to do with the 

 hereditary difference between singlets and doublets. 



The exclusion of genotypic differences suggests a cytoplasmic basis 

 for the hereditary difference between singlets and doublets. A direct 

 test of this possibility involves obtaining and detecting cytoplasmic 

 transfer between conjugants (Sonneborn, 1950, pp. 119, 120, 127). 

 Normally there is no detectable transfer of cytoplasm between mates 

 although it sometimes occurs spontaneously in certain stocks. It is 

 readily inducible, however, by subjecting conjugants for a short time 

 to specific immobilizing antiserum. This establishes a cytoplasmic 

 bridge in the region where gamete nuclei pass across, i.e., just to the 

 right of the mouths. The bridge persists after separation has occurred 

 at all other points, but it may eventually disappear, freeing the mates. 

 The degree of persistence and breadth of the bridge are roughly pro- 

 portional to the amount of cytoplasm transferred. When bridges are 

 broad, flow of cytoplasm between mates can be directly seen. Transfer 

 of cytoplasm can also be detected indirectly by use of the cytoplasmic 

 marker, kappa, the visible cytoplasmic particle that determines the 

 hereditary killer trait. Each killer cell carries hundreds of kappa 

 particles in its cytoplasm. Cytoplasm which flows from a killer to a 

 sensitive conjugant carries kappa with it; consequently, the cells of the 

 clone from the sensitive mate bear kappa and are killers. If cytoplasm 

 bearing kappa is not introduced in this way into the sensitive mate, 

 the cells of the clone it produces lack kappa and are sensitive. 



Free flow of cytoplasm between mates had led to phenotypic identity 

 of the resulting clones with regard to all previously studied hereditary 

 traits that had been shown by breeding analysis not to be due to 

 genotypic differences (Sonneborn, 1943; 1954a; Sonneborn and 

 Lesuer, 1948) . Its effect on the inheritance of the singlet versus doublet 

 difference was therefore tested by using kappa and the killer trait as 

 a cytoplasmic marker in addition to gene markers and by inducing 

 cytoplasmic bridges between mates by the antiserum method. As 

 shown in Figs. 41 and 411, reciprocal crosses were made with respect 

 to which kind of mate bore kappa and was a killer: kappa-bearing 



