A selection method for the synthesis of triploid hybrids by fusion of microspore protoplasts (n) with somatic cell protoplasts (2n).

Microspore protoplasts (n) isolated at the tetrad stage from plants of Nicotiana tabacum Km(+) (2n=4x=48) were fused with somatic cell protoplasts (2n) of WT N. rustica (2n=4x=48) to produce triploid plants. A total of 21.2×10(6) microspore protoplasts were fused with 11.2×10(6) somatic cell protoplasts using the high pH/Ca(+ +) method. Microspore protoplasts did not divide and WT N. rustica protoplasts stopped dividing when the protoplast-derived colonies were transferred to a selection medium containing kanamycin. A total of 104 actively growing green colonies were recovered on the selection medium. Ninety-six of these colonies were tested for their hybrid nature by PAGE of peroxidases and were found to contain bands characteristic of both parents. Hybrid nature of the plants regenerated from some of the selected colonies was confirmed by IEF of leaf esterases, by NPT II activity assay and by hybridizing total DNAs restricted with EcoR I to a cloned 18s rDNA fragment. Root tip squashes of six of the hybrid plants revealed chromosome numbers ranging from 58-72. From chromosomal and biochemical analysis, it can be concluded that the procedure of fusing microspore protoplasts (n) of species A carrying a dominant selection marker with WT somatic cell protoplasts (2n) of species B can be a convenient selection method for the synthesis of triploid plants. The significance of triploids lies in their subsequent use for transferring alien chromosomes and genes of species A to species B.