The production of female-sterile flowers in Arabidopsis thaliana (L.) Heynh. and Brassica napus L
Pylatuik, Jeffrey Dennis
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The production and viability of pollen produced by a female-sterile Arabidopsis mutant, flo10-1, was examined. Although the additional stamens of flo10-1, produced significantly less pollen of lower viability, the pollen still effectively fertilized ovules, producing normal, viable seed. To create a floral phenotype similar to that seen in flo10-1, it was decided to alter the floral morphology using homeotic genes involved in the ABC model. Expression by the regulatory elements of the Arabidopsis class C homeotic gene AGAMOUS (AG) was tested in B. napus. Spatially and temporally, the expression pattern of AG in B. napus was very similar to that in Arabidopsis, being expressed primarily in the reproductive organs A cDNA isolated and cloned from Brassica napus, BnAP3, was found to be homologous to the B class homeotic gene APETALA3 of 'Arabidopsis'. In order to convert a wild-type gynoecium to stamens, BnAP3 was targeted to the reproductive organs of Arabidopsis and B. napus flowers under the control of the AG regulatory elements. In both species the gynoecium of transformed plants was replaced with four organs that were partially converted to stamens. The conversion was only partial because L2 and L3 derived tissues of these organs were converted to those of stamens while L1 derived tissues retained their carpeloid properties. Affected flowers were however, female-sterile. Finally, a gene orthologous to SHATTERPROOF1' SHP1') of Arabidopsis, BnSHP1' was isolated and characterized from B. napus. 'SHP1' is required for the proper development of dehiscence zones in the gynoecium of Arabidopsis. Sequence analysis of the large first intron of BnSHP1, as well as the regulatory regions of both 'SHP1' and 'SHP2' of Arabidopsis, identified a conserved LEAFY binding site within these genes, suggesting that they may be regulated, in part, by the LEAFY protein. These studies were successful in that they demonstrated a viable approach to generating female sterility in flowers of these species. Furthermore, homologous genes controlling flower development were identified in B. napus and aspects of the ABC model of flower development were tested and verified in B. napus.