Roderick John Scott

The molecular biology of anther differentiation

Abstract This review attempts to summarise recent progress in our understanding of androgenesis in plants. There are two major divisions within this work. The first is a cytomorphological description of anther development and gametogenesis in model hermaphrodite plants. This is intended as a background against which to view the molecular biological information; this aspect constitutes the second main division in this chapter. The approach taken in describing these recent advances is intended to reflect, more or less, how these were made in practice. Gene cloning via cDNA library construction from whole anther organs or microspores represents the main technique, and therefore the merits of this approach, as applied to the developing anther, are discussed. In particular, the influence of cDNA library construction and screening methods on the type or class of genes identified to date are highlighted. The information generated from gene cloning is divided roughly into three sections. The first of these describes how the synthesis of specific mRNAs has provided valuable insights into the physiology and ontogeny of anther development. The physical and functional properties of cloned cDNAs cognate to mRNAs expressed within the anther are the subject of the second section. The third of these sections deals with anther-expressed genes, including analysis in transgenic plants. Finally, the commercial spin-off from this part of plant science, artificial male sterility, is discussed.

Transformation of carrot tissues derived from proembryogenic suspension cells: A useful model system for gene expression studies in plants

A method is described for the high frequency transformation of carrot proembryogenic suspension culture cells by a non-oncogenic Ti-plasmid vector (pGV3850::1103) which carried a chimaeric kanamycin resistance gene (nos-NPT-II). Plants were regenerated efficiently from transformed material by somatic embryogenesis in the presence of kanamycin. Transformed tissues expressed readily detectable levels of both NPT-II and nopaline. NPT-II could be detected in total protein extracts by Western blotting. This analysis indicated that NPT-II was produced as a single, full length polypeptide. The T-DNA copy number in individually selected transformants was analysed by Southern blotting and ranged from 1–8 per diploid genome. The copy number and organization of the T-DNA was retained in plants regenerated from these transformants by somatic embryogenesis. These data suggested a clonal origin for the selected kanamycin resistant colonies. NPT-II expression levels appeared to be directly related to gene dosage.

Genetic transformation of flax (Linum usitatissimum) by Agrobacterium tumefaciens: regeneration of transformed shoots via a callus phase

Genetic transformation of flax (Linum usitatissimum) has been achieved using an A. tumefaciens strain carrying a non-oncogenic Ti plasmid-derived vector containing a chimaeric npt-II gene and a wild type nopaline synthase gene. Fertile, transformed shoots were most easily obtained from Kmr callus developing on hypocotyl sections. The totipotency of the Kmr callus was dependent upon its origin. T-DNA was visualised by Southern blotting in all Kmr tissues. Efficient expression of nopaline synthase and the chimaeric npt-II gene was found in transformed Kmr callus and regenerated shoots.

The restoration of fertility in male sterile tobacco demonstrates that transgene silencing can be mediated by T-DNA that has no DNA homology to the silenced transgene.

Male sterile tobacco plants expressing a pathogenesis-related (PR) β-1,3-glucanase gene driven by the Arabidopsis thaliana A3 or A9 tapetum-specific promoter, were partially restored to fertility by retransformation with a range of pA9-driven sense and antisense PR glucanase fragments. The restored plants exhibited improved seed set. PR glucanase protein was undetectable in the anthers of these plants and there was an associated increase in microsporocyte callose, the structural target of the A3 and A9-driven PR glucanase. This phenotype was not solely dependent on interactions between sense and antisense PR glucanase transcripts since a pA9-driven restorer was also capable of down regulating a pA3-GUS construct in the absence of extensive promoter, coding region, or terminator sequence homology. Since the A3 and A9 promoters have similar temporal and spatial expression patterns, it is possible that trans-acting factors common to both promoters become limiting in the PR glucanase double transformants resulting in improved levels of fertility. An alternative hypothesis is that additional sequences present in both the silencing and target T-DNAs can mediate the silencing of adjacent non-homologous transgenes.