TRANSIENT GUS GENE-EXPRESSION IN BRASSICA-NAPUS ELECTROPORATED MICROSPORES

As a prerequisite to the transformation of rapeseed through androgenesis, we assessed the feasability of expressing foreign DNA in the male gametophyte of Brassica napus L. var. oleifera cv. Tapidor microspores by electroporation, using a square-wave-pulse discharge apparatus. Nuclease activities were observed only in young pollen grains and in the tapetum and disappeared after successive washings, The parameters of the electrical pulses used for the introduction of a fluorescent probe (calcein) into microspores without affecting microspore viability were determined in relation to different electroporation media. The optimal field conditions applied to microspores suspended in a BK-modified medium supplemented with 2% PEG were found to be 250 V/cm and 750 V/cm with respective pulse durations of 10 ms and 0.5 ms. Transient gene expression experiments using plasmids bearing the GUS reporter gene under the control of CamV 35S or Zmg 13 promoters were used to test DNA transfer into microspores and pollen. Expression was significantly greater in electroporated microspores than in non-treated microspores. This is the first step towards transgenic plants obtained by the haploid way.