Antisense Oligodeoxynucleotide-Mediated Gene Knockdown in Pollen Tubes - Plant Cell Morphogenesis: Methods and Protocols - page 221

Chapter 19

Antisense Oligodeoxynucleotide-Mediated Gene
Knockdown in Pollen Tubes

Radek Bezvoda , Roman Pleskot , Viktor Žárský , and Martin Potocký

Abstract

Specifi c gene knockdown mediated by the antisense oligodeoxynucleotides (AODNs) strategy recently
emerged as a rapid and effective tool for probing gene role in plant cells, particularly tip-growing pollen
tubes. Here, we describe the protocol for the successful employment of AODN technique in growing
tobacco pollen tubes, covering AODN design, application, and analysis of the results. We also discuss the
advantages and drawbacks of this method.

Key words Antisense oligodeoxynucleotide, AODN, Pollen tube, Gene knockdown, Tip growth

1 Introduction

Angiosperm fertilization requires delivery of sperm cells to embryo
sac. This is accomplished by the polarized growth of the male
gametophyte, the pollen tube, throughout the pistil tissues into
the ovary. Along with root hairs, pollen tube serves as an example
and useful model for highly polarized cell expansion with a very
high growth rate (reviewed in ref. [ 1 ]). This process is regulated by
small GTPases, ions, reactive oxygen species, phospholipids, and
cytoskeleton remodeling ( see , e.g., refs. [ 1 - 4 ]). Given the fact that
pollen of many species can be easily germinated and cultivated in a
liquid media in vitro, it can be utilized as a tool to investigate regu-
lation of polar secretion sustaining polarized cell growth.
Arabidopsis, tobacco, and lily pollen are typically used in the
research, where Arabidopsis serves as model of tricellular pollen
and tobacco and lily as model of bicellular pollen ( see Note 1 ).
In modern biology, various gene-specifi c knockdown
approaches have become invaluable tools for elucidating the func-
tional role of candidate genes. One particular method uses short
(18-22 nucleotides long) antisense oligodeoxynucleotides
(AODNs) that are complementary to target gene mRNA and thus
suppress gene expression. AODNs are widely used in biomedical