The distribution of potato foliage rot and the usual time of its emergence have been analysed. The causes of the increased intensity of the disease have been pointed out. The resistance of potatoes to the disease has been studied, the most resistant varieties are named. The selection work of new resistant hybrids at Priekuli Plant Breeding Station is discussed. The necessity to test and evaluate the new sorts before recommending them to growers is emphasized

The use of ploidy manipulations in potato breeding has been successfully applied by breeders at the International Potato Center (CIP) as a tool for germplasm enhancement and for the introduction of desirable genes of wild species into the cultivated gene pool. This methodology consists in reducing the ploidy level using haploids (2n = 2x = 24) and increasing it through the utilization of 2n gametes. It takes advantage of working at the 2x level to develop genotypes which combine multiple pest/disease resistance and a high frequency of 2n gametes. The specific resistances are obtained mainly from wild species. Then, desirable attributes are transferred to the 4x level by unilateral sexual polyploidization (4x x 2x crosses) using first division restitution (FOR) 2n pollen. CIP's work demonstrates that this approach has been very efficient for the transmission of resistance to cyst and root-knot nematodes, bacterial wilt, early blight and potato tuber moth as well as producing high yielding 4x genotypes which also have yield stability over environments. This recently developed germplasm is now available for potato breeders in developing countries who can produce high yielding and resistant 4x clones adapted to the growing conditions of their countries.

Solanum brevidens synteny groups were examined with 47 widely-distributed RFLP markers in 17 BC2 progeny from six fertile BC1 plants. The BC1 plants were derived from a single S. brevidens x Solanum tuberosum somatic hybrid backcrossed with S. tuberosum (potato). Probes which were linked in potato and tomato were also found to be syntenic along each of the 12 S. brevidens chromosomes. More than half of the S. brevidens synteny groups had lost one or more S. brevidens-specific RFLPs in the BC2, suggesting that recombination had occurred. For eight of the 12 S. brevidens RFLP synteny groups, the frequency of recombinant chromosomes exceeded that of intact parental chromosomes. Using the RFLP data, 161 RAPD markers were tentatively located throughout the S. brevidens genome. Further analyses with 39 of these 161 RAPD markers generally showed that RAPD and RFLP results were comparable, but some inconsistencies were noted with 14 of the 39 RAPD markers. The extent of marker loss and the high frequency of synteny groups which were marked by a single S. brevidens-specific RFLP marker suggest that the S. brevidens chromosomes have some pairing affinity with potato chromosomes. This interaction should facilitate the transfer of novel disease-resistance traits into potato breeding lines. One plant was recovered with the chromosome number of S. tuberosum (2n=48) that carried a single S. brevidens RFLP marker, suggesting transfer of this S. brevidens marker into the genome of S. tuberosum.

Solanum brevidens synteny groups were examined with 47 widely-distributed RFLP markers in 17 BC2 progeny from six fertile BC1 plants. The BC1 plants were derived from a single S. brevidens + S. tuberosum somatic hybrid backcrossed with S. tuberosum (potato). Probes which were linked in potato and tomato were also found to be syntenic along each of the 12 S. brevidens chromosomes. More than half of the S. brevidens synteny groups had lost one or more S. brevidens-specific RFLPs in the BC2, suggesting that recombination had occurred. For 8 of the 12 S. brevidens RFLP synteny groups, the frequency of recombinant chromosomes exceeded that of intact parental chromosomes. Using the RFLP data, 161 RAPD markers were tentatively located throughout the S. brevidens genome. Further analyses with 39 of these 161 RAPD markers generally showed that RAPD and RFLP results were comparable, but some inconsistencies were noted with 14 of the 39 RAPD markers. The extent of marker loss and the high frequency of synteny groups which were marked by a single S. brevidens-specific RFLP marker suggest that the S. brevidens chromosomes have some pairing affinity with potato chromosomes. This interaction should facilitate the transfer of novel disease-resistance traits into potato breeding lines. One plant was recovered with the chromosome number of S. tuberosum (2n = 48) that carried a single S. brevidens RFLP marker, suggesting transfer of this S. brevidens marker into the genome of S. tuberosum.

Rice blast (Pyricularia grisea) is considered the most important disease of rice worldwide. Development of resistant cultivars has been the most effective method to control it; the pedigree method (PM) has been used in this regard but recently, the production of double-haploids through anther culture (AC) has been proposed as an effective, and economic breeding tool. The objectives of this study were to generate rice lines by PM and AC and to compare their efficiency in relation to blast resistance. Crosses were made between the rice cultivar Fanny (highly susceptible to blast), and 11 cultivars with different degrees of resistance/susceptibility to blast. Using PM, the F2, F3, F4, F5, and F6 generations were evaluated and selected for blast resistance through subsequent semesters. Via AC, AC-R2 (from F1 plants), and AC-F2 (from F2-blast susceptible plants) lines were obtained using a liquid potato-medium for callus induction and an MS medium for plant regeneration. Three sets of lines (F6, AC-R2 and AC-F2) were planted together and evaluated for blast resistance under upland conditions in a blast prone environment. Rice blast reactions were evaluated on a 0-9 scale and analyzed as a discrete variable following the multinomial distribution. Three response variables, leaf and neck blast, and general reaction to blast (maximum score between the first two variables), were considered. Stratified analysis using the Cockran-Mantel-Haenszel statistic was performed to test the association between methods and blast resistance, and resistance stability across crosses; all data processing and analysis was performed through SAS version 6.07. The percentage of resistant lines as well as stability of resistance was significantly (P=0.01) greater in PM-derived lines, with a lower mean blast score, suggesting that PM is more efficient than AC in relation to selection for blast resistance. Nevertheless, in Fanny/Ceysvonni, and Fanny/LAC 23 crosses the percentage of stable-resistant lines was greater in the case of AC. Crosses between susceptible cultivars (Fanny/CICA4, and Fanny/Colombia 1) yielded resistant lines by either method (PM or AC). This excludes somaclonal variation as a possible mechanism and indicates that recombination of possibly minor genes took place and was fixed through either method. However, the stability of the resistance was greater in PM-derived lines

Bacterial diseases can cause a drastic decrease of yield in certain crops. Breeding for bacterial disease resistance therefore is of utmost necessity. Up to now, traditional plant breeding was the only method to reach this goal. Recent developments in genetic engineering technology however provide novel ways for the production of disease resistant plants. This thesis describes the results of two research projects that have been undertaken to investigate the potential of such a novel way, namely the introduction and expression of genes coding for antibacterial proteins in plants. In the first project, the potential of the hordothionins from barley (Hordeum vulgare) endosperm, has been investigated, and in the second, the potential of cecropin B from the giant silkmoth (Hyalophora cecropia).In the first part of chapter 1, the literature available on plant thionins is presented. General information on the different thionin types, homology, occurrence in nature, molecular structure and toxicity for microorganisms and cultured cells is listed. Since their role in nature has not yet been established, although a putative role in plant defense has been proposed by several groups, special emphasis is put on the numerous divergent activities displayed by the different thionins in conjunction with possible modes of action and biological roles. Data collected from literature indicate that thionins might expose their toxic activity in vitro by several mechanisms: by acting as a thiol intermediate in reducing and oxidizing proteins or by direct binding to DNA and/or RNA or by interaction with the phospholipid membrane, acting most likely on Ca 2+-channels or -pumps, and/or Ca 2+-ATPases. In the second part of chapter 1, an overview of the literature on insect cecropins is presented. General information on molecular structure, toxicity and known mode of action of these proteins is presented. Special emphasis is put on one of them, cecropin B, which was under investigation in the second research project.Our first choice was to investigate the feasibility of using thionin encoding sequences for engineering bacterial disease resistance into solanaceous crops. The hordothionins originating from barley endosperm were chosen because of the availability of nucleotide and amino acid sequences. To establish the potential of hordothionins, the toxicity for plant pathogenic bacteria had to be determined first. To this end the thionins from wheat and barley endosperm were isolated (chapter 2). The thionins were purified in few steps from flour by petroleum-ether extraction and hydrochloric acid treatment of the resulting lipoprotein, followed by ion-exchange chromatography. The hordothionins were separated into two forms, HTH-1 and HTH-2, probably reflecting the two forms described in literature, namely α- and β-hordothionin. In vitro experiments indicated that purothionin, hordothionin and HTH-1 and HTH-2 were equally toxic to Clavibacter michiganensis subsp. michiganen sis, the causal agent of bacterial canker on tomato, C.michiganensis subsp. sepedonicus, the causal agent of ring rot on potato and Xanthomonas campestris pv. vesicatoria, the causal agent of a foliage and fruit spot disease on tomato and pepper. Erwinia spp. and Pseudomonas spp. were insensitive.Since hordothionins appeared to be toxic for a number of bacteria that affect tomato and potato, several hordothionin encoding constructs were made (chapter 3). Analysis of published hordothionin cDNA clones indicated that the 5 kD mature hordothionin was made as a much larger precursor protein. This precursor consisted of an amino-terminal signalpeptide, followed by the mature hordothionin exhibiting antibacterial activity, and a carboxy-terminal so-called acidic peptide of unknown function. Since no such cDNA or genomic hordothionin clones were available to us, we decided to chemically synthesize the genes making use of the many advantages of synthetic genes. In chapter 3, the design and construction of seven different α- and β-hordothionin encoding constructs is described. Gene constructs were made to study expression in cytosol and secretion into the apoplast. Genes were designed for optimal expression in solanaceous crops by adapting the codon usage and optimizing the translation initiation region, and for convenience in subsequent cloning steps.The seven hordothionin gene constructs made were cloned in a plant expression vector under the control of the constitutive cauliflower mosaic virus 35S promoter and introduced in tobacco to study their expression, processing of precursors, sorting and biological activity (chapter 4). Analysis of a large number of transgenic plants indicated that the signalpeptide was essential for expression, whereas the acidic peptide facilitated transport of mature hordothionin and increased accumulation at least tenfold compared to plants harboring constructs without this acidic peptide coding sequence. Fractionation of protoplasts prepared from transgenic plants indicated that hordothionin accumulated in the microsomes and membranes and was not secreted into the medium. In addition, hordothionin was not secreted into the apoplast in intact leaves. The hordothionin partially purified from leaves of transgenic tobacco plants exhibited in vitro toxicity for C.michiganensis subsp. michiganen sis, at comparable doses as the hordothionin from barley endosperm. The tobacco phytopathogen P.syringae pv. tabaci was however not affected in growth in transgenic tobacco plants exhibiting high hordothionin expression levels (chapter 4).In vitro growth inhibition experiments indicated clearly that hordothionin was toxic for a few bacteria that were pathogenic on tomato and potato. For this reason, the best performing construct was introduced in tomato (chapter 5). Transgenic plants were selected exhibiting high hordothionin expression levels, selfed to obtain plants homozygous for the transgene and evaluated for resistance. No differences were observed between control plants and transgenic tomato plants exhibiting high hordothionin expression levels upon infection with C.michiganensis subsp. michganensis. In addition, no growth inhibition of X.campestris pv. vesicatoria was observed in leaves of these transgenic plants upon infiltration of a bacterial suspension. However, less symptoms were visible on these transgenic plants upon spray-inoculation with a suspension of the latter.Since hordothionins were not toxic to Erwinia spp. and Pseudomonas spp. in vitro (chapter 2), a second antibacterial protein was chosen exhibiting toxicity to these bacteria. Three different cecropin B gene constructs were made and introduced in tobacco. Analysis of transgenic plants indicated that the genes were transcribed into mRNA, however the protein could not be detected (chapter 6). By mixing a synthetic cecropin B peptide with different tobacco cell extracts, it appeared that the peptide was rapidly degraded, whereas boiling of the extracts prior to mixing did not result in cecropin B degradation, suggesting protease degradation. This was confirmed by experiments which indicated that the protease inhibitors chymostastin and to a lesser extent PMSF, also inhibited degradation. Transgenic plants exhibiting high cecropin B-mRNA levels were nevertheless evaluated for resistance to two pathogens, P. solanacearum and P. syringae pv. tabaci. Results from these experiments clearly indicated that transgenic plants were not resistant.Finally, in chapter 7 a general discussion on the topic described in this thesis is presented. Also, other potential approaches to obtain bacterial disease resistant plants are discussed and other applications of hordothionin encoding sequences.