Sex Plant Reprod (2004) 17:49–53 DOI 10.1007/s00497-004-0203-2 SHORT COMMUNICATION Laura C. Hudson · C. Neal Stewart Effects of pollen-synthesized green fluorescent protein on pollen grain fitness Received: 22 September 2003 / Accepted: 6 January 2004 / Published online: 7 February 2004  Springer-Verlag 2004 Abstract Genetically engineered pollen with a visible marker gene could be useful to monitor the movement of transgenic pollen provided there are no negative physi- ological or fitness effects of expressing such a gene. In this study, we measured the fitness of Nicotiana tabacum cv. Xanthi pollen expressing the marker gene green fluorescent protein (GFP). Average pollen tube germina- tion frequencies and pollen tube growth rates in vitro were measured in three different types of plants: (1) plants producing GFP in pollen cells only (LAT59-GFP), (2) plants synthesizing GFP under the control of a constitutive promoter (CaMV 35S) in which no GFP was produced in pollen, and (3) non-transgenic plants. Pollen synthesizing the GFP protein did not differ significantly in average pollen germination frequencies from pollen without GFP (P=0.65). Average pollen tube growth rates over a 5-h period did not differ significantly between transgenic and non-transgenic types (R 2 =0.89, 0.98, and 0.95, respectively, for GFP-tagged, 35S-GFP, and wild type). Overall, GFP expression in pollen grains of tobacco was not found to have an effect on pollen fitness under the controlled experimental conditions of this study. Keywords Nicotiana tabacum · GFP · LAT59 promoter · Pollen tube · Pollen fitness Introduction The male gametophyte of a flowering plant is a highly specialized tissue that is essential for reproductive suc- cess. A mature pollen grain is responsible for the recognition of a compatible stigma and delivery of sperm nuclei to the plant ovule (Cheung 1996). Tobacco pollen is essentially a bicellular structure, independent of the sporophyte, originating from two meiotic and one mitotic division. A tobacco pollen grain consists of only two cells—a vegetative nucleus and a generative cell—sur- rounded by a relatively thick exine cell wall (McCormick 1993). The rise of the haploid microspore after the first two meiotic divisions is considered to be the start of plant gametophyte gene expression and development, which makes the male gametophyte an attractive experimental system for various issues in plant molecular biology. Being bicellular, tobacco pollen is an ideal model in the study of pollen tubes. Techniques for in vitro and semi vivo germination of pollen are well established in tobacco, and elongated tubes can be easily obtained (Mulcahy and Mulcahy 1985). Arabidopsis thaliana and some Brassica species are members of the 30% of angiosperm plants species that have tricellular pollen and do not germinate well in vitro (Preuss et al. 1993). In vitro growth of most tricellular pollen types subsides in less than 1 h; however, bicellular pollen tubes routinely elongate for over 5 h (Hoekstra 1979). Although in vitro germination provides a controlled experimental environment, tube growth in vitro does not completely mimic growth in vivo. Under optimum culture conditions, in vitro pollen tubes reach approximately 30– 40% of in vivo lengths (Read et al. 1993). Although pollen-pistil interactions are not duplicated in vitro, in many species, including tobacco, pollen germination and tube growth are robust under experimentally defined conditions, rendering in vitro-based studies relevant to the in vivo situation (Taylor and Hepler 1997). As a molecular marker, green fluorescent protein (GFP) has many applications that could not have been imagined 10 years ago. GFP has been used as a molecular L. C. Hudson Department of Plant Pathology, North Carolina State University, 840 Method Road, Rm. 213, Unit 4 Bldg., Campus Box 7903, Raleigh, NC 27607, USA C. N. Stewart Jr. ( ) ) Department of Plant Sciences, Ellington Plant Sciences, University of Tennessee, 2431 Joe Johnson Drive Rm 252, Knoxville, TN 37996–4561, USA e-mail: nealstewart@utk.edu Fax: +1-865-941989