Somatic Hybridization by Fusion of Protoplasts : 1. The combination of Nicotiana tabacum and Nicotiana rustica

Abstract

Mesophyll protoplasts of Nicotiana tabacum. cv. Bright Yellow mutant Aurea and Burley 21, and of N. rustica. cv. Rustica were prepared from leaf tissue by enzymatic digestion and were fused with the aid of polyethylene glycol. When the fused protoplasts were plated on an agar medium or in a liquid medium for cell culture, new cell walls were regenerated from them and the first cell division could be seen beginning on the 12th day of culture, and cell division process took place on and after the 21th day (Fig. 1). After one month culture, almost all of the surviving fused protoplasts grew to cell colonies of 1-2 mm in diameter. The cell colonies were subcultured on an agar medium for callus culture. On the medium, they grew vigorouly and formed calli of 2 cm in diameter after 2 months culture. At this stage, the hybrid calli showing greenish white with a compact appearance were selected in contrast to parental type calli tinged with white or green color (Fig. 2). Ten somatic hybrid calli of the combination of Aurea and Rustica and 3 calli of the combination of Burley 21 and Rustica were obtained. Many plantlets were differentiated from these calli when transfered to a medium enriching zeatin (Fig. 3). All of the mature flowering plants showed to be perfectly hybrid, and the shape of the leaves and flowers indicated an intermediate between N. tabacum and N. rustica (Fig. 5, 6). The plant height of the normal hybrid morphologically was higher than that of the parent plant and all of the hybrid plants were highly resistant to the tobacco mosaic virus (Fig. 4, Table 1, 2). The chromosome numbers of the somatic hybrids showed to be the aneuploid type having from 60 to 91 chromosomes (Table 1, 2). The normal hybrid plants morphologically, however, have a pollen fertility, and seeds and progenies were obtained from 4 of the Aurea-Rustica hybrids and 3 of the Burley 21-Rustica hybrids (Table 1, 2).