Analyzing the Impact of Protein Overexpression on Ca2+ Dynamics and Development in Tobacco Pollen Tubes.

Overexpression of RFP-tagged proteins in growing tobacco pollen tubes together with the genetically encoded Ca2+ sensor YC3.6 allows to analyze localization and dynamics of the protein of interest, as well as the impact of its overexpression on Ca2+ dynamics and pollen tube growth. Here, we describe a step-by-step instruction for transient transformation of N. tabacum pollen and subsequent in vitro germination and Ca2+ imaging.