Somatic hybridization between Nicotiana rustica and N. tabacum. I. Isolation and culture of protoplasts and regeneration of plants from cell cultures of wild-type and chlorophyll-deficient strains

Abstract

Preliminary to somatic hybridization between chlorophyll-deficient mutants of Nicotiana rustica and N. tabacum conditions for cell culture, protoplast production and culture and induction of morphogenesis were established. Callus and suspension cultures from wild-type and chlorophyll-deficient strains of both species were established and maintained on Murashige and Skoog medium containing either 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA). The chlorophyll-deficient phenotype was evident in cultures derived from mutants of both species. Protoplasts were readily obtained from cell cultures with N. rustica outyielding N. tabacum and mutant cells of the latter substantially outyielding the wild-type cells. In Nagata and Takebe medium protoplasts regenerated cells walls and 60–80% divided and formed cell colonies. Protoplasts from cultures on 2,4-D medium were healthier but slower to divide than those from cultures grown on an NAA-supplemented medium. Protoplasts derived from 2,4-D-grown cells also gave rise to proembryo-like structures. Shoot and root organogenesis could be induced in cell colonies obtained from both suspension cultures and protoplasts of mutant and wild-type N. rustica. With albino N. tabacum only cell colonies from cell suspension showed morphogenic potential. Maximum frequencies of colonies forming shoots were 77% for N. rustica cv. chlorotica and 100% for albino N. tabacum. The morphogenic capacity of colonies from 2,4-D-grown cells was greater than from cells grown on NAA. Morphogenic capacity of both species declined with increasing age of the suspension cultures. Roots were induced on shoots of the wild type in both species as well as in the chlorophyll-deficient N. rustica mutant.