Anther and pollen culture of Nicotiana tabacum.

Pollen of Burley tobacco gave rise to plantlets by anther culture. Not all anthers responded and in those that did the number of plants produced was different. Response varied with different batches of anthers, but this was not a result of the season. Chilling of anthers before culture was not beneficial. Androgenesis was influenced by the stage of pollen development tetrad stages failed completely, and the mitotic-early bicellular stage was optimal. Mature pollen gave rise to plants which were almost all haploid. A dimorphism existed in maturing pollen removed from the plant. At the time starch deposition occurred in normal grains, a small proportion of grains failed to lay down starch or continue increasing in diameter and lost their cytoplasmic staining reaction with aceto-carmine. The size of these S-grains was constant, but their number was different in different anthers, even from the same bud. During culture embryos arose only from S-grains. It is suggested that S-grain formation may be determined during meiosis. The anther wall contributes to the androgenic response. Although ethylene is produced by cultured anthers, neither this gas nor any other appears to be required at levels above those present in air, either in the culture vessels or within the closed anthers themselves. Using an extraction technique involving homogenisation of anthers, few plantlets were obtained from 4 day or 14 day cultured pollen, even using a nurse system involving high density embryogenic carrot cells. Damage to an anther resulted in rapid death of its pollen. The dehiscence lines of anthers could be artificially opened with no effect on androgenesis. Pollen removed by the dehiscence lines gave rise to plantlets on a simple medium with almost the same frequency as intact anthers, but only if a period of 14 days of intact culture was allowed before isolation.