As part of a study of pollen embryogenesis from in vitro anthers, flower buds were induced on inflorescence stem pieces of Nicotiana tabacum cv. Virginica, and also on thin epidermal layers excised from similar stem pieces. The medium used was M & S containing 3% sucrose, IAA and kinetin (10-6M each), solidified with agar. The epidermal layers were more productive: 80 to 90% yielded one or more flower buds, as against only 30% with stem pieces. In vitro induced flower buds contained viable pollen grains. Browning in culture affected about 40-50% of vivo anthers irrespective of hormone treatment; the extent of browning of in vitro anthers, on the other hand, was sensitive to the presence or absence of hormones in the medium. Both anther response and anther productivity, based on green anthers, were drastically depressed by exogenous hormones in anthers of both types. Anther response was somewhat more dependent on anther stage in vitro anthers than in in vivo ones. Conditions for the reliable induction of large numbers of flower buds vitro, and the merits of using anthers derived from them for the study of pollen embryogenesis are discussed. In the second part of the work, suspension cultures were initiated from calli induced on epidermal explants in order to explore the extent to which tobacco cell suspensions from adult tissue-derived callus can exhibit embryogenesis. The latter was done by removing the auxin from the suspension cultures, while retaining some kinetin; removal of kinetin along with the auxin prevented all morphogenesis. NAA at the callus and suspension initiation stage proved more satisfactory as an auxin than 2,UD; the thoroughness of the removal of the auxin was found to be critical. The populations of regenerated structures were classified either as 'foliose structures', or as roots attached to callus aggregates, or as whole plantlets with shoot and root on a common axis. Foliose structures were the most common; they were either single or multiple. Single foliose structures had mostly two, occasionally three leaf-like organs attached to a single cylindrical axis terminating in a single basal end. Multiple foliose structures were composed of several axes with mostly two leaf-like organs each. The axes grew out of a common centre through which, as revealed by subsequent work, they were connected by vascular tissues. The ratio of single to multiple foliose structures depended on the kinetin concentration in the auxin-free 'morphogenetic' medium. High levels of ammonium generally favoured the development of single foliose structures, sometimes at the expense of multiple ones, particularly when the extra ammonium was present either in the auxin containing suspension initiation media, or in the 'morphogenetic' media without auxin, but not in both. When the extra ammonium was present at both stages, morphogenesis was almost completely represssed. Casein hydrolysate has also markedly affected the pattern of morphogenesis. When present in the suspension-initiation media with auxin, its main effect was to induce root development, particularly where the cytokinin concentration was low enough to allow its expression. When present in the 'morphogenetic' medium, the development of foliose structures, particularly that of single ones, was strongly enhanced. When present at both stages, casein hydrolysate tended to support only rhyzogenesis at low kinetin concentrations and only the formation of foliose structures and plantlets at higher concentrations of kinetin. A histological examination of the multiple foliose structures confirmed their identity as shoot clusters. The general appearance of single foliose structures was very reminiscent of zygotic embryos. Histological examination revealed certain features characteristic of such embryos. Consistent with these findings, the single foliose structures were designated as embryo-like structures. Zygotic embryos excised from seeds were also put in suspension cultures and a range of abnormalities were observed in their development.
|Date of Award||1982|