Bypassing prefertilization barriers to hybridization in Nicotiana using in vitro pollination and fertilization

In vitro pollination of placenta attached ovules was useful in bypassing unilateral incongruity barriers for several Nicotiana interspecific hybrid combinations (N. tabacum cv. 'Ky 17' X N. amplexicaulis, 'Ky 17' X N. benthamiana, and 'Ky 17' X N. repanda). By measuring the pollen tube growth over time, prefertilization barriers were determined to be the cause of the incongruity. Seedling necrosis was a problem in the development of the N. amplexicaulis hybrid and it prevented maturation of the N. repanda hybrid. Callus produced from cotyledons of the N. amplexicaulis hybrid eventually resulted in plants that survived to maturity. This procedure was not successful for the N. repanda materials. The N. amplexicaulis and N. benthamiana hybrids were sterile but following chromosome doubling by midrib culture, male and female fertile plants were produced.Conventional hybridization, fertilized ovule culture, and in vitro pollination were unsuccessful in obtaining hybrids of 'Ky 17' crossed with N. arentsii or N. bonariensis. Apparently, strong postfertilization barriers prevent the production of viable seed of these hybrids. Each of the N. repanda - N. tabacum reciprocal hybrids could not be rescued using callus culture; this adds support to the existence of strong sexual postfertilization barriers. A recent report, however, showed that it was possible to obtain this hybrid using the technique of somatic hybridization. Thus, it appears that it may also be possible to obtain asexual hybrids of N. arentsii and N. bonariensis with N. tabacum.