Anther and microspore culture in tobacco

Abstract

Serving as the oldest model species, several variants of tobacco anther and microspore culture have been developed over the years, depending on the timing of the inductive stress treatment and microspore isolation (Heberle-Bors et al., 1996). A convenient, one-step procedure of anther culture in Nicotiana tabacum consists of a cold-treatment of excised flower buds, followed by anther excision and anther culture on a charcoal-containing medium. For microspore culture, sucrose and nitrogen starvation of isolated microspores in combination with a mild heat shock induces the formation of embryogenic pollen which, after transfer to a simple, sucrose and nitrogen-containing liquid medium, divide repeatedly and produce large numbers of embryos (sporophytic pathway, Touraev et al., 1996). Under non-stress conditions in a rich medium isolated microspores develop into mature, fertile pollen (gametophytic pathway, Benito Moreno et al., 1988; Touraev and Heberle-Bors, 1999). The usually haploid microspore embryos are transferred to a low-sucrose agar medium for germination and treated with colchicine for diploidization.