Introgression in interspecific hybrids of lily

In order to introduce new desirable characters into the cultivar assortment of lily a range of interspecific crossing barriers has to be overcome. By using various pollination and embryo rescue techniques pre- and postfertilization barriers were overcome and a range of wide interspecific lily hybrids between species and cultivars from the different sections of the genus Lilium could be made. Important breakthroughs include the development of the LA- (L. longiflorum x Asiatic hybrids), the LO- (L. longiflorum x Oriental hybrids) and the OA- (Oriental x Asiatic hybrids) hybrids. In general wide interspecific lily hybrids show F1-sterility. Using somatic chromosome doubling techniques (mitotic polyploidization) tetraploids with restored fertility can be produced from these diploid hybrids. An alternative method is the use of unreduced (or 2n) gametes (meiotic polyploidization), which are rarely found in some hybrids. Introgression of alien chromosome segments from donor species into recipient cultivar through backcrossing of F1 hybrid was studied using in situ hybridization techniques (GISH). Mitotic polyploidization showed no homoeologous recombinations between the parental genomes whereas meiotic polyploidization detected many. The use of 2n-gametes seems to be the most promising way for the introgression of desirable characters

Genome composition of 'Elatior'-begonias hybrids analyzed by genomic in situ hybridisation

Interspecific hybridization of various tuberous Begonia species hybrids with Begonia socotrana results in so-called 'Elatior'-begonias hybrids (B. x hiemalis Fotsch). In our study, genomic in situ hybridization (GISH) has been employed to assess the genome composition in eleven 'Elatior'-begonias hybrids and their ancestor genotypes. Genomic DNA of tuberous Begonia was sonicated to 1-10-kb fragments, labelled by nick translation with digoxigenin-11-dUTP and used as a probe whereas B. socotrana DNA was autoclaved to 100 bp fragments and used as block. The genome of tuberous Begonia was clearly pronounced in 'Elatior'-begonias when the probe concentration was similar to 3.75 ng/mu l (150 ng/slide), with 30 times the excess of B. socotrana blocking DNA and stringency of post hybridization washings at 73% (0.1x SSC at 42A degrees C). In 'Elatior'-begonias hybrids GISH distinguished two groups comprising short (0.6-1.03 mu m in length) and relatively longer chromosomes (1.87-3.88 mu m) which represent B. socotrana and tuberous Begonia genomes, respectively. The number of chromosomes derived from tuberous Begonia ranged from 14 to 56 and for B. socotrana from 7 to 28 which suggest the presence of different ploidy levels in analyzed 'Elatior'-begonia hybrids. Intergenomic recombination has not been detected through GISH in hybrids analyzed. Genomic in situ hybridization turned out to be useful to identify the genome constitution of 'Elatior'-begonia hybrids and thus gain an insight into the origins of these cultivars. This knowledge on the ploidy level and genome composition is essential for further progress in breeding Begonias

Veredelingsonderzoek naar de ontwikkeling van virusresistente broei tulpen = Application of GISH-techniques in breeding research of virus resistant forcing tulips

Door toepassing van Genomische in situ hybridisatie (GISH), een chromosoomkleuringstechniek bij de veredeling van virusresistente broeitulpen hebben aangetoond dat er een belangrijke doorbraak is bereikt. Het is bekend dat Tulipa fosteriana TBV resistentie bezit en doorgeeft aan de Darwin (GF) hybriden (T. gesneriana x T. fosteriana). Tot nu toe waren deze Darwin hybriden steriel en was introgressie van virus resistentie in het broeisortiment onmogelijk. In een project gericht op gestapelde resistenties bij tulp zijn echter GF hybriden gevonden met fertiliteit. Hieruit zijn diverse terugkruisingspopulaties (GGF) verkregen. Met behulp van GISH is van 99 GF-hybriden de genoomsamenstelling vastgesteld. 92 bleken inderdaad echte GF-hybriden te zijn. In GGF hybriden is aanzienlijke intergenomische recombinatie aangetoond. Dit toont aan dat door introgressie van T. fosteriana chromosoomsegmenten in het T. gesneriana genoom introgressie van virusresistentie mogelijk is. In een tweede terugkruisingsgeneratie van ‘Purissima’, een GF-hybride werd voortgaande introgressie aangetoond. In deze hybriden (G x GGF) bleek nog slechts 10% van het genoom afkomstig te zijn van T. fosteriana. In dit materiaal wordt in voortgaand TTI-onderzoek met behulp van moleculaire merkers de virus resistentie aangetoond

Doorbreking van kruisingsbarrières tussen Oriental- en Aziatische hybriden t.b.v. van introgressie van virus, Fusarium en Botrytisresistentie

Dit project heeft als doel om de laatste hindernissen in het gebruik van OA-hybriden voor de lelieveredeling weg te nemen. Dit betekent: Herstel van fertiliteit d.m.v. (geïnduceerde) mitotische en meiotische polyploïdisatie (chromosoomverdubbeling). Selectie van het meest fertiele materiaal en analyse van nakomelingschappen met streven naar maximale introgressie van de diverse genomen. Onderzoek gericht op een combinatie van ziekteresistenties (Lily Mottle Virus, Fusarium en Botrytis)

Genetics of fungal resistance to systemic fungicides

Since the introduction of the systemic fungicides, fungicide resistance has become a serious problem in plant disease control. This study was carried out in order to contribute to the knowledge about the genetics of fungal resistance to fungicides both from a practical and a fundamental point of view.The potential of a fungus to acquire fungicide resistance, measured as mutation frequency and degree of resistance, was investigated in various fungi. The fungicides employed were: the benzimidazole fungicides benomyl and thiabendazole; carboxin, an oxathiin compound; chloroneb, an aromatic hydrocarbon; imazalil, an imidazole derivative; and the two antibiotics cycloheximide and pimaricin, a glutarimide and a polyene antibiotic, respectively (chapter 3). Induced resistance occurred in all cases, in which it was searched for. The mutation frequencies varied from 10 -7 for pimaricin and benomyl to 2.10 -4 for chloroneb resistance (Table 6). Both mutation frequency and degree of resistance were independent on the fungal species. However, considerable differences between the fungicides were found in the highest level of resistance observed among the resistant strains. From the results it was concluded that imazalil and pimaricin, for which the level of resistance in relation to the wild type increased at most by a factor 10 and 4, respectively, might be used in agriculture without a considerable chance of interference with control. On the other hand the development of resistance to the other fungicides of 50-5000 times, makes their practical use rather questionable.Pathogenicity and in vivo fungitoxicity tests were carried out with benomylresistant strains of five plant pathogens, viz. Cladosporium cucumerinum , Cercosporella herpotrichoides , Fusarium nivale , Penicillium expansum and Phialophora cinerescens (chapter 4). Generally, a close correlation between resistance in vitro and in vivo could be established. With respect to pathogenicity some strains were as pathogenic as the wild type, others were less pathogenic and a few had lost their pathogenicity. However, the results did not allow the conclusion that loss of pathogenicity was due to the mutation to resistance.Characterization of the resistant mutants in vitro was performed especially in Aspergillus nidulans (chapter 5). Dosage response relationships of fungicide resistance in agar growth tests were presented. With the four fungi examined, viz. A. nidulans, Aspergillus niger , P. expansum and Ustilago maydis , crossresistance of the chloroneb-resistant strains was observed to pentachloronitrobenzene (PCNB), a related aromatic hydrocarbon fungicide, and to 3-phenylindole. Usually, resistance to either benomyl or thiabendazole involved resistance to the other compound but in rare cases resistance to benomyl only or more frequently to thiabendazole only occurred, the latter involving increased sensitivity to benomyl. This phenomenon, known as negative crossresistance was found in all five fungi examined, viz. A. nidulans , A. niger , P. expansum , Rhodotorula rubra and U. maydis. A number of pleiotropic effects in the imazalil-resistant mutants of A. nidulans and A. niger was found, viz. resistance and hypersensitivity to various inhibitors. This suggested that the action of imazalil might be sought in changes in cell membrane permeability.It is known that for Saccharomyces cerevisiae and U.maydis carboxin is ten times more toxic on a medium with acetate as sole carbon source than with glucose. This effect was verified in the wild-type strain of A. nidulans. However, in the case of the carboxin-resistant strains this difference was the smaller the more resistant the strains were. This strengthened the observations that reactions in the tricarboxylic acid cycle are involved in the mechanism of action of carboxin, since carboxin has been shown to inhibit mitochondrial succinate oxidation.Using genetically well-defined strains of A.nidulans, it was possible to establish the genetic basis of characters such as patterns of cross-resistance. Genetic analysis with the non-pathogenic A.nidulans was carried out by means of the sexual and parasexual cycles (chapter 6). A single nuclear gene relationship of all mutations to resistance was found. The number of loci involved in the resistance to a fungicide was determined by recombination analysis of different mutants. It appeared that with some fungicides such as chloroneb and oligomycin only one locus was responsible for all the mutations to resistance; on the other hand in the case of resistance to pimaricin and to carboxin two and three genes, respectively, were identified, whereas in the case of cycloheximide and imazalil resistance a multigenic: system was involved. High-level resistance to benomyl and/or thiabendazole appeared to be based on mutations at one locus. Twenty- three genes conferring resistance were assigned to seven different linkage groups (chromosomes), sixteen of which were mapped. Using heterozygous diploids the dominance behaviour of the mutations to resistance was studied. A range of degrees of dominance was found from a practically recessive condition of mutations to benomyl, chloroneb and pimaricin resistance to an almost complete dominance of some genes conferring carboxin and cycloheximide resistance. In the case of imazalil resistance a positive interaction of genes resulting in a high degree of resistance in recombinant strains was observed.In order to verify the results obtained with A.nidulans in plant-pathogenic fungi, genetic analysis was also performed with U.maydis, using the sexual cycle, and with A.niger and C.cucumerinum, by means of the parasexual cycle (chapter 7). As a result of this approach it was demonstrated that in A. niger and U. maydis benomyl and thiabendazole resistance including negative crossresistance was based, as in A.nidulans, on one single gene in which all resistant strains carry a different mutation. Analysis through the parasexual cycle, which involves heterokaryosis, diploid formation and recovery of haploid and diploid segregants, gave similar results in A.niger as known from the literature. In C.cucumerinum , for which a parasexual cycle was not known, diploid strains were isolated, from which recombinant haploids were also recovered, although, surprisingly, without observing a heterokaryotic state. The parasexual cycle in C.cucumerinum provided information on the genetic basis of pathogenicity, because loss of pathogenicity of auxotrophic mutants was fully complemented in the heterozygous diploid strains, which appeared to be as pathogenic as the haploid prototrophic wild-type strain.<p/

Partial resistance to Erwinia carotovora SUBSP. carotovora and plant vigour among F1 hybrids of Zantedeschia cultivars

The potential of breeding Zantedeschia cultivars for resistance to soft rot caused by Erwinia carotovora subsp. carotovora (syn. Pectobacterium carotovorum subsp. carotovorum) was determined. Hybrids of six cultivars (`Back Magic`, Galaxy`, Pink Persuasion`, Sensation`, `Treasure` and `Florex Gold`) were screened using a non-destructive resistance test (leaf disk test) and scored different levels of susceptibility. Some hybrid families were less susceptible than the very susceptible reference `Florex Gold`, while all hybrid families were more susceptible than the partial resistant reference Z. rehmannii. The half-sib family of `Pink Persuasion` was more resistant, but less vigorous than the half-sib family of `Florex Gold`. We concluded that resistance and vigour may be negatively correlated in the deployed cultivars and that resistance should be bred from other sources than the used cultivar

Relevance of unilateral and bilateral sexual polyploidization in relation to intergenomic recombination and introgression in Lilium species hybrids

Sexual polyploids were induced in diploid (2n = 2x = 24) interspecific F1 hybrids of Longiflorum × Asiatic (LA) and Oriental × Asiatic (OA) Lilium hybrids by backcrossing to Asiatic (AA) parents as well as by sib-mating of the F1 LA hybrids. A majority of the BC1 progenies were triploid and the progenies from sib-mating were tetraploid or near tetraploids. Genomic in situ hybridization (GISH) technique was applied to assess the intergenomic recombination in the BC1 populations of LA and OA hybrids obtained after unilateral sexual polyploidization. A total of 63 LA (LA × AA and AA × LA) and 53 OA hybrids were analysed. LA hybrids were originated through the functioning of 2n gametes either as 2n eggs or 2n pollen while those of OA hybrids originated through functional 2n pollen of diploid OA genotype. In both type of crosses, a majority of the progenies had originated through First Division Restitution (FDR) mechanism of functional 2n gamete either with or without a cross over. However, there were nine LA- and four OA-genotypes where Indeterminate Meiotic Restitution (IMR) was the mechanism of 2n gamete formation. Based on GISH, total amount of introgression of Longiflorum and Oriental genome into Asiatic genome was determined. Most of the BC progenies exhibited recombination and the amount of recombination was higher in LA hybrids as compared to OA hybrids. Intergenomic recombination was also determined cytologically in the 16 plants of sib-mated LA hybrids where both parents had contributed 2n gametes. Based on these results the nature of interspecific lily hybrids obtained from uni- and bilateral sexual polyploidization leading to allotriploid and allotetraploid formation is discussed in the context of introgression and intergenomic recombinatio

Erwinia resistentie in Zantedeschia : eindverslag juni 2003

Het gewas Zantedeschia heeft zich de afgelopen jaren razendsnel ontwikkeld als nieuw siergewas. De veredeling van Zantedeschia staat echter nog in de kinderschoenen. De ontwikkeling van kennis, technieken en materiaal op het gebied van Zantedeschia is van belang met het oog op de positie die het gewas in de toekomst kan verwerven in de Nederlandse sierteeltsector. Eén van de belangrijkste problemen m.b.t. Zantedeschia is de gevoeligheid voor de bacterie Erwinia carotovora. Binnen een zeer korte tijd kan een gehele teelt vernietigd worden. Vooral gekleurdbloemige types (botanisch behorend tot de sectie Aestivae) zijn gevoelig. Een sluitende bestrijdingsmethode van Erwinia is echter niet voorhanden, zodat het inbouwen van resistenties grote aandacht verdient. Soortkruisingstechnieken zijn beproefde technieken die voor Zantedeschia verder uitgewerkt dienen te worden. Binnen dit project zal de aandacht uitgaan naar inzicht in genetica van resistentie

Interspecific hybridization and polyploidization as tools in ornamental plant breeding

Interspecific hybridisation and polyploidy are recognized as the most impor-tant sources of evolution and domestication of flowering plants. In ornamental plant breeding these phenomena go hand in hand and can be observed in the breeding his-tory of many ornamental crops (Rosa, Chrysanthemum, Gladiolus, Alstroemeria, Lil-ium, orchids etc). With lily as model crop techniques developed for overcoming pre- and post-fertilisation barriers are reviewed. For overcoming F1-sterility mitotic and meiotic polyploidisation are applied and can result in fertile allopolyploids. The mechanism of viable pollen production of mitotic and meiotic polyploidisation is quite different. Mitotic polyploidisation possess one homologous chromosome set. They undergo normal meiotic division like diploid cells. However, meiotic poly-ploidisation often show irregular chromosome division resulting in two of unreduced chromosome number instead of reduced chromosome number in tetrads. In contrast to mitotic doubling homoeologous recombination can occur. There are two signifi-cant mechanisms, FDR and SDR, for the formation of 2n-gametes. The FDR-gamete increase heterozygosity while SDR-gamete increase homozygosity. Genomic in situ hybridisation (GISH) has been used to discriminate parental chromosomes and to detect homoeologous recombination. Mitotic polyploidisation showed no homoeolo-gous recombinations between the parental genomes whereas in meiotic polyploids it can detected frequently. The use of 2n-gametes is therefore the most promising way for the introgression of desirable characters in the breeding with interspecific hy-brids. Although spontaneous occurring in the domestication of many ornamental crops the systematically detected unreduced gametes proved to be highly efficient tool for introgression of characters