Isolation and characterization of molecular markers for Brassica napus microspore embryogenesis.

Brassica napus microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. This switch in developmental pathways has been shown to be accompanied by the induction of high levels of napin seed storage protein gene expression (DeMoor, 1992). Specific members of the napin multigene family that were expressed at this time were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), are members of the highly conserved BnmNAP subfamily of napin genes. DNA gel blot analysis, using a subfamily-specific probe, suggested that this subfamily may consist of up to 5 members. RNA gel blot analysis, also using the subfamily-specific probe, indicated that the BnmNAP subfamily was also expressed during embryo development. BnmNAP mRNA was detected as early as the globular stage of development in microsporic embryos, but not until the late torpedo/early cotyledon stage of development in zygotic embryos. A BngNAP1 promoter-$\beta$-glucuronidase (GUS) gene fusion was introduced into B. napus and Nicotiana tabacum (tobacco) plants in order to examine the spatial and temporal pattern of expression of one member of the BnmNAP subfamily. The BngNAP1-GUS construct was shown to be highly expressed in microspores that had been induced to undergo embryogenesis, but was not expressed in microspores continuing pollen development in culture. Furthermore, BngNAP1-directed GUS activity appeared to be predominantly localized in those microspores that have been shown to have the greatest potential to form embryos in culture. Fluorogenic and histochemical analysis of developing microsporic and zygotic embryos of B. napus indicated that the BngNAP1-GUS fusion was expressed as early as the globular stage of development. GUS activity was first detected in the micropylar region of the future embryonic axis and continued to spread upward during subsequent stages of development. In tobacco, GUS activity was first detected in the endosperm of seeds containing globular stage embryos. GUS activity did not begin to accumulate in tobacco embryos until the early heart stage of development, where it appeared as a band in the middle of the embryo, just under the lobes of the emerging cotyledons. This activity continued to spread outward in both directions as development proceeded. Thus the timing, but not the spatial localization, of BngNAP1-directed GUS expression was maintained in transgenic tobacco