Somatic Hybridization by Fusion of Protoplasts : II. The combinations of Nicotiana tababum and N. glutinosa and of N. tabacum and N. alata

Abstract

Mesophyll protoplasts of Nicotiana tabacum. cv. Bright Yellow mutant Aurea and N. glutinosa and of N. tabacum. cv. Burley 21 and N. alata were prepared from leaf tissue by enzymatic digestion and were fused with the aid of polycthylcne glycol. When the fused protoplasts were platcd on an agar medium or in a liquid medium for cell culture, new cell walls were regenerated from them and the cell division could be seen beginning on the 14th day of culture in the combination of N. tabacum and N. glutinosa, whereas in that of N. tabacum and N. alata 10th day of culture. Cell division process took place over again after that. After one month culture, almost all of survived protoplasts grew to cell colonies of 1-2 mm in diameter. The cell colonies were placed on an agar medium containing 3.0 mg/l l-Naphthalenacetic acid and 1.0 mg/l 6-Benzylaminopurine in order to induce callus formation. On the medium, they grew vigorously and formed calli of 2 cm in diametcr after 2 months culture. However, it was too difficult to select the hybrid callus. They were trans-fered to a modified LINSMAIER and SKOOG medium containing 1.0 mg/l kinetine and 1.0 mg/l indoleacetic acid. After one month of culture on this mcdium, the hybrid calli, showing greenish white with a compact appearance werc selectcd in contrast to the parental type calli tinged with a white or green color. Thirty-three somatic hybrid calli of the combination of N. tabacum and N. glutinosa and 2 calli of the combination of N. tabacum and N. alata were obtained. Many plantlets were differentiated from these calli when transfered to enriching zeatin. All of the mature flowering plants showed to be perfectly somatic hybrid, and the shape of the leaves and flowers indicated that many somatic hybrids were of an intermediate characteristic between N. tabacum and N. glutinosa and between N. tabacum and N. alata. A few plants had a strong resemblance to a parent or a scxual hybrid. The plant height of the many somatic hybrids were higher than that of the parental plants, and the male sterility of the greater part of the hybrid plants was visible under investigation, and it was possible to back cross. They were highly resistant to the tobacco mosaic virus. The chromosomc number of the somatic hybrids of the combination of N. tabacum and N. glutinosa was determined: fivc were amphidiploid, twenty-eight showed aneuploid with 50 to 88 chromosomes. On the other hand, thc chromosome number of the somatic hybrids of the combination of N. tabacum and N. alata also was determined : one was amphidiploid and the other aneuploid.