RUSSELL, SCOTT D.1*, ZHAOJIE ZHANG1, HUILING XU2, PREM BHALLA2, AND MOHAN B. SINGH2. 1Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019; 2Institute of Land and Food Resources, The University of Melbourne, Parkville, VIC., 3052, Australia. - Plumbago zeylanica sperm isolation, collection and cDNA library characterization.
Sperm cells of Plumbago zeylanica express structural dimorphism
in which the sperm cell associated with the vegetative nucleus
(Svn) usually fuses with the central cell. The sperm cell
unassociated with the vegetative nucleus (Sua) fuses with
the egg cell. In order to investigate potential differences in gene
expression between these two cells, paired sperm cells were released
from pollen in a 10 mM MOPS buffer containing 0.8 M mannitol (pH 4.6).
Sperm cell types were identified in paired cells based on size
differences and sperm cells were collected individually with a
microinjector. Viability, tested using FCR, indicates that sperm cells
isolated under these conditions remain viable for at least 2 h. Two
PCR based cDNA libraries were constructed using mRNAs isolated from
separated Sua and Svn sperm cell populations.
Preliminary screening of libraries establishes the feasibility of the
technique and suggests differences in gene expression profiles between
the two sperm cell types.
Key words: cDNA library, male gametes, Plumbaginaceae, Plumbago zeylanica, sperm collection, sperm isolation