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Possible maintainer lines were selected from CC XXXII and crossed onto cytoplasmically male-sterile plants. Complete male sterility was maintained in both the F₁ and BC₁ generations of 46.4% of the lines. Four cultivars with maintainer genotypes that were in both normal and msm1 cytoplasm were intercrossed using the male-sterile forms as the female parents. All F₁'s were completely male-sterile. Restoration of male fertility by 22 lines selected from CC XXXII was shown in each case to be due to a single dominant gene. In some lines, restoration was influenced by environment and genetic background. Partial restoration was observed in cultivars in the World Collection and lines selected from CC XXXII. Partial restoration appeared to be due to several genes that were subject to environmental influence. Accumulation of some of these genes increased the amount of restoration. There was no evidence that cytoplasmic factors were passed through the pollen. Twenty-two F₁ hybrids were produced by crossing restorer lines onto male-sterile msm1 lines. The 22 hybrids, their 44 parental restorer and maintainer lines and six check cultivars were grown in a four-replication yield trial. Total yield, 1000-seed weight and hectaliter weight were measured for each plot. All the F₁ hybrids outyielded their midparent values and 17 of the hybrids outyielded their high parents. Half of the F₁'s outyielded the high check cultivar, which yielded about 9,130 kg/ha. Twenty-one F₁'s had greater 1000-seed weights than their midparent values while only 11 F₁'s had greater 1000-seed weights than their high parents. The high check cultivar had the greatest 1000-seed weight, 49.0 gm. The hybrids with the greatest 1000-seed weights were not the hybrids with the greatest yields. Eighteen of the F₁'s had greater hectaliter weights than the midparent values, but only seven had greater hectaliter weights than their high parents. The high check cultivar had the greatest hectaliter weight, 75 kg. The hybrids with the greatest hectaliter weights were not the highest yielding hybrids.

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In plants, retrotransposon copy numbers have been shown to vary from several hundreds to several million. The factors that regulate retrotransposon copy number are poorly known. A model by FLAVELL et al. (1997) proposing different effects of selection in genomes with low numbers of copia-like retrotransposons, against genomes with higher copy numbers of this retroelement, was tested. Non-synonymous to synonymous substitutions ratio (d N/dS), together with the frequency of occurrence of stop codons within the reverse transcriptase sequence of copia -like retrotransposons, were analyzed in relationship to copy number estimates of this retrotransposons. Evidence of purifying selection was detected in an enzyme directly involved in transposition. Genomes with less than 10,000 copia-like elements showed significantly smaller dN /dS ratios and fewer stop codons compared with genomes containing more than 10,000 copia-like elements. Genome size also appeared to be correlated with dN/dS ratios and frequencies of stop codons. Heterogeneity of copia-like retrotransposons was not related to dN/dS ratios. Copia-like retrotransposons with different copy numbers inside a single genome showed the same dN/d S ratios. The findings were discussed in relation to different possible selective scenarios in plants with low versus high retrotransposon copy number.

An isoenzyme survey of several taxa within the genus Lotus was undertaken to provide markers for genetic research and to test hypotheses concerning the phylogenetic origin of Lotus corniculatus L. A preliminary study identified seven enzyme systems PGI, TPI, PGM, MDH, IDH, 6-PGDH and ME, that produced consistent clear phenotypes in L. uliginosus Schkuhr. Variation in phenotype with tissue type and stage of development suggested the presence of several isozyme zones in the phenotypes. Enzyme phenotype was constant for shoot tissue of plants older than six weeks. A second study examined recombination and segregation of isoenzyme phenotypes in interspecific hybrids, allo- and autopolyploids, and in L. corniculatus. Duplication and quadruplication of pgi2 loci in hybrids, amphidiploids, and in L. corniculatus was used as evidence that the latter is a segmental allotetraploid. A third study surveyed the occurrence of various isoenzyme alleles in L. alpinus Schleich., L. japonicus (Regel) Larsen, L. tenuis Waldst. & Kit., L. uliginosus and L. corniculatus. Lotus uliginosus had unique distinct alleles for several enzymes that did not occur in the other species. This evidence argues against the involvement of L. uliginosus in the origin of L. corniculatus.

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Towards the aim of producing transgenic rice with enhanced resistance to one of its phloem sap-sucking insect pests, the brown planthopper (BPH), two potential phloem-specific promoters, of the rice sucrose synthase-1 (RSsl) and the cucurbit phloem protein PP2 genes, were isolated and investigated. Using a PGR fragment of the maize sucrose synthase-1 (Shi) gene, genomic clones containing the RSsl and RSs2 (rice sucrose synthase-2) genes were isolated from a genomic library of rice (Oryza sativa L. Japonica) and characterised. A full- length RSsl gene from one of the genomic clones was sequenced, including 1756 bp of 5'-flanking sequence and 710 bp of 3'-flanking sequence. The gene had an identical intron-exon structure (16 exons and 15 introns) to the maize Shi gene. The RSsl 5'- flankmg region contained a number of promoter-like sequences, including putative exacting elements homologous to those found in several endosperm-specific, anaerobiosis-inducible, or phloem-specific promoters. The RSsl promoter region, including 1.9 kb 5'-flanking sequence, the first intron, the first exon, and the translational start codon, was fused with coding sequences for β-glucuronidase (GUS) and snowdrop (Galanthus navalis) lectin (GNA). Tobacco plants were transformed with these chimaeric genes in order to determine the expression pattern directed by the RSsl promoter. Histochemical and immunochemical assays demonstrated that the expression of both GUS and GNA was restricted to phloem cells in various tissues (i.e. stem, root, leaf, petiole) and in different transformants. In addition, GNA was detected by immunological assay in the honeydew excreted by peach potato aphids (Myzus persicae), also a phloem sap- sucking insect, feeding on RSsl-GNA transgenic tobacco plants. This provided direct evidence that GNA was not only expressed in the phloem tissue, but was also present in the phloem sap of transgenic tobacco plants. Since GNA has been shown to have antimetabolic effect against BPH, the RSsl-GNA construct is being used to transform rice plants by collaborating groups elsewhere. In order to isolate the phloem protein PP2 gene promoter, the PP2 polypeptide from 3 months old Cucurbita pepo plants was partially sequenced after in situ cleavage with cyanogen bromide vapour, giving 75 residues of sequence on two cyanogen . bromide fragments. Using an oligonucleotide probe based on this amino acid sequence, cDNA clones encoding PP2 were isolated from a C. pepo cDNA library constructed from mRNA of 3-5 days old seedling hypocotyls. A cDNA clone was used as probe to screen a C. pepo genomic library, and several genomic clones were isolated. Restriction mapping showed that these clones contained different genes, consistent with results from Southern blots of C. pepo genomic DNA probed with PP2 cDNA, in which multiple bands were detected in all restriction endonuclease digests. One of the clones was partially sequenced, and was shown to contain a gene encoding PP2. A 1.2 kb 5'-flanking region of this clone was fused with a GUS reporter gene, and this construct was used to transform tobacco. Initial histochemical analysis showed that this chimaeric gene was not expressed in the putative transgenic plants examined. Possible reasons for this failure are discussed.

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