ORIGIN OF TETRAPLOIDIZATION IN PROTOPLAST CULTURES OF PETUNIA (PETUNIA-HYBRIDA)

We have previously studied chromosomal and morphological variation in protoplast cultures of diploid petunia (Petunia hybrida) plants. We fo und that 85% of the plants regenerated were tetraploid (2n = 4x = 28). To understand the origin of cytogenetic variation in protoplast cultu res of petunia, nuclear behavior during culture of mesophyll protoplas ts isolated from diploid and tetraploid petunias was investigated by s taining the nuclei with 4,6-diamidino-2-phenylindole (DAPI). Freshly i solated protoplasts were cultured in modified Murashige and Skoog medi um supplemented with 16 combinations of auxins (2,4-D, NAA, IAA) and B AP. During culture of diploid mesophyll protoplasts, varying numbers o f multinucleate cells were observed to be formed prior to initiation o f the first cell division. At the optimum concentration of plant growt h regulators for the induction of cell division, the formation of mult inucleate cells was the highest (up to 53.7%). Although the majority o f the protoplasts subsequently entered into morphologically normal cel l division, DAPI staining demonstrated that the cells were multinuclea te. In contrast with diploid protoplasts, the frequencies of multinucl eate cells in protoplast cultures of tetraploid petunia were only 0.1% to 2.3%, and the majority of protoplasts in such cultures showed norm al cell divisions when stained with DAPI. These results indicate that the formation of multinucleate cells during the initiation of the firs t cell divisions in diploid mesophyll protoplast cultures was primaril y influenced by exogeneous plant growth regulators. Thus, such multinu cleate cells appear to be the origin of tetraploidization. The implica tion of these results in relation to polyploidization in diploid petun ia protoplast cultures is discussed.