Lab-on-a-Chip for Studying Growing Pollen Tubes - Plant Cell Morphogenesis: Methods and Protocols - page 227

Chapter 20

Lab-on-a-Chip for Studying Growing Pollen Tubes

Carlos G. Agudelo , Muthukumaran Packirisamy , and Anja Geitmann

Abstract

A major limitation in the study of pollen tube growth has been the diffi culty in providing an in vitro testing
microenvironment that physically resembles the in vivo conditions. Here we describe the development of
a lab-on-a-chip (LOC) for the manipulation and experimental testing of individual pollen tubes. The
design was specifi cally tailored to pollen tubes from Camellia japonica , but it can be easily adapted for any
other species. The platform is fabricated from polydimethylsiloxane (PDMS) using a silicon/SU-8 mold
and makes use of microfl uidics to distribute pollen grains to serially arranged microchannels. The tubes are
guided into these channels where they can be tested individually. The microfl uidic platform allows for
specifi c testing of a variety of growth behavioral features as demonstrated with a simple mechanical obsta-
cle test, and it permits the straightforward integration of further single-cell test assays.

Key words Pollen tube, Camellia japonica , Cell culture, Lab-on-a-chip, Microfl uidics, Microstructures,
MEMS, Soft lithography, Tip growth

1 Introduction

In order to reach its target, the ovule, the pollen tube needs to
invade the pistillar tissues of the receptive fl ower and follow guid-
ance cues emitted by the sporophytic tissues and the female game-
tophyte [ 1 - 3 ]. Studying the roles of chemical, proteic, and
mechanical cues that direct pollen tube growth and the mechanism
by which the tube turns has become an important aspect of pollen
tube research [ 4 - 7 ]. Conventionally, experimentation on pollen
tubes is performed on cells germinated in bulk samples and grow-
ing in essentially homogeneous and isotropic growth matrices,
either a liquid medium or an agarose-stiffened substrate. This in
vitro environment is in stark contrast with the in vivo growth con-
ditions which present a microstructured environment consisting of
the various cell types and tissues the pollen tube encounters on its
path through the pistil [ 6 ]. To test the behavior of pollen tubes in
structured microenvironments featuring complex geometrical
challenges or simple or superimposed chemical gradients, we have