In vitro pollen germination and transient transformation of Zea mays and other plant species

To study pollen-specific gene expression, fast and convenient methods involving in vitro pollen germination and bombardment with promoter deletion constructs are needed. Unfortunately, because of variation of pollen germability and tube growth, conducting these experiments is often unsatisfying for many plant species, including maize, especially when pollen is collected at different times of the day or season. We have overcome these problems by defining a novel medium (PGM) that guarantees germination efficiencies of more than 90% for maize pollen from at least 7 genotypes (A188, AC 3572 C, B73. H99. Hi-II, Q2. Tx232). This medium is also suitable to germinate pollen of other monocot species, such as Pennisetum americanum and Tradescantia species, and dicot species, such as Arabidopsis thaliana, Arachis hypogaea, Columnea oesterdiana, Nicotiana tabacum, Phaseolus vulgaris, Pisum sativum, Solanum lycopersicum, Solanum tuberosum, and Vicia faba. On average, reproducible germination rates ranging from 50-100% were observed with all plant species tested. In addition, we report a transient transformation assay using the luciferase (Luc) reporter gene. Biolistic parameters were defined to obtain reproducible Luc activity measurements after bombarding thick-walled pollen, such as maize pollen. For comparison, samples of germinated maize and tobacco pollen were bombarded with the reporter gene under control of the constitutive ubiquitin- and pollen-specific ZmMADS2 maize promoters. The important parameters necessary to apply both in vitro pollen germination and transient transformation for a large range of plant species are discussed.