Evelyn Klocke

Somatic hybrids of vegetable brassicas as source for new resistances to fungal and virus diseases

Somatic hybrids were produced by PEG-induced symmetric and asymmetric protoplast fusions in order to transfer resistance to Alternaria brassicicola, A. brassicae, Phomalingam, Plasmodiophora brassicae and Turnip mosaic virus (TuMV) into Brassica oleracea var. capitata (cv. ‘Toskama’) and botrytis (cv. ‘Korso’). As resistance donors, ten species belonging to several genera of the family Brassicaceae including wild relatives were used. Of 2,189 plants (somatic hybrids, partially in vitro cloned) tested, 1,616 (73.8%) were resistant against at least one of the pathogens, indicating that, mostly, a successful resistance transfer has taken place. Five hundred and twenty-two hybrids showed multiple resistances to two, three and, in a single case, to four pathogens. Irrespective of the donor parents used in the fusion, a broad variability in symptom manifestation ranging from 0 (without symptoms) to 9 (highly susceptible) could be observed. With regard to the Alternaria pathogens, Sinapis alba, B. nigra and B. juncea were the most effective resistance donors, whereas fusions with Raphanus sativus resulted in the most hybrids with resistance to clubroot and TuMV. As could be shown especially in asymmetric fusions with S.alba, Barbarea vulgaris and Hesperismatronalis, transferred resistance to a pathogen may not correspond with resistance exhibited by the donor parent. Some combinations in which both parents were highly susceptible, e.g. R. sativus (+) B. oleracea var. capitata, yielded hybrids that exhibited strong resistance, e.g. to A. brassicicola, revealing that a new type of resistance might be occurring. With regard to the Alternaria pathogens, resistance expression was very unstable. Many hybrids into which (also variable) resistance of some donors, such as B. vulgaris, S. alba and B. carinata, was transferred became as highly susceptible as those of which the fusion parents did not show any resistance reaction (e.g. R. sativus). For reliable characterization of the resistance response, hybrids should be subjected to several resistance tests during growth period of the host, at least until flowering.

Monitoring the expression of green fluorescent protein in carrot

Green fluorescent protein (GFP) was successfully used as a visual reporter at various stages of carrot (Daucus carota L.) transformation. GFP-fluorescence was non-invasively observed in protoplasts, callus and plants after the delivery of mgfp5-er gene using two transformation methods: direct DNA transfer into polyethylene glycol (PEG) -treated protoplasts and inoculation of root discs with Agrobacterium rhizogenes. Transient GFP-expression was detected in the treated protoplasts and monitored during the first week of the cell culture until the stable level of expression was observed. It was useful for the comparison of protoplast susceptibility to DNA uptake and the transgene expression as the fluorescence declined with various rates depending on the used carrot genotype and PEG-concentration. GFP-monitoring in callus enabled the selection of stably expressing lines. It also allowed verification of the homogeneous tissue composition with regard to the expression of the transgene. In plants, GFP-performance depended on the assayed tissue and organ despite of the constitutive 35S promoter. The expression was visually detected in both vegetative and generative parts, but particularly strong fluorescence was observed in leaf marginal meristems, petioles, stems, and styles. Those tissues can be convenient for examination of the transgenic plants during their growth. The results encourage that GFP is a valuable reporter and can be routinely used for optimization of transformation protocol, selection of transformants and monitoring transgenic carrot.

Regeneration of protoplasts after somatic hybridisation of Hydrangea

Somatic hybridisation of Hydrangea is a promising tool to obtain new basic material for breeding. Viable mesophyll protoplasts were isolated from 21 cultivars and accessions of H. macrophylla, H. paniculata, H. arborescens, H. quercifolia and H. febrifuga. Induction of cell divisions was observed after electromanipulation and fusion by polyethylene glycol. Multi-cellular structures developed in liquid media. Plated microcalli grew on different solid regeneration media. Several phytohormones, their concentration and combination influenced the development of callus and roots. The regular appearance of endophytes challenged successful plant regeneration. As a result of endophytes, the development of microcolonies stopped and they died in liquid protoplast media. Plated microcalli or growing calli turned brown on regeneration medium. Antibiotic Timentin® inhibited expansion of endophytes for a short time. The addition of ascorbic and citric acid to the regeneration media had inhibiting effect on endophyte growth. Calli showed more vitality and grew faster. The supplement of the regeneration media with karrikinolide, a recently discovered new plant growth regulator, brought contradictory results. After addition of karrikinolide microcolonies looked healthier by their shining green colour in liquid media followed by a severe browning of callus soon afterwards. In the further course, karrikinolide promoted the development of endophytes. Shoot induction and plant regeneration succeeded only once from callus that was a result from H. macrophylla ‘Schneeball’ and H. macrophylla ‘Nachtigall’ fusion.

Verbesserung der Krankheitsresistenz von Kohlgemüse: 1. Turnip mosaic virus(TuMV)

Zusammenfassung:Das Turnip mosaic virus (TuMV) ist weltweit verbreitet und gehört zur Gruppe der Potyviren. Es hat einen außerordentlich großen Wirtspflanzenkreis von über 300 Arten und wird nicht persistent durch 89 Aphidenarten übertragen. Bisher sind beim TuMV zwölf Pathotypen bekannt. Im Kohlgemüse verursacht das TuMV die so genannte Kohlschwarzringfleckigkeit. Dabei handelt es sich um eine wirtschaftlich wichtige Virose, die beim Weißkohl bis zu 25 % Ertragsausfall und Gewebenekrotisierungen, insbesondere während der Kühllagerung, bewirken kann. Zur Sicherung eines hohen Ertrags- und Qualitätsniveaus bei Kohlgemüse wurde begonnen, die Resistenz gegen diese Virose zu verbessern. Mit aktuellen, weitgehend charakterisierten Erregerherkünften des TuMV wurden Resistenzprüfmethoden weiterentwickelt und neue Resistenzquellen in der Familie der Brassicaceae erschlossen. Resistenzen gegen mehrere TuMV-Pathotypen ließen sich in B. oleracea-Primitivformen, in Chinakohl (B. rapa ssp. pekinensis), in Meerrettich (Armoracia ru-sticana), in Radies bzw. Rettich (Raphanus sativus) sowie in sexuell erzeugten Raphanobrassica-Hybriden nachweisen. Die evaluierten TuMV-Resistenzen wurden entsprechend der phylogenetischen Distanz zum Kohl durch Kreuzung bzw. durch Protoplastenfusion in Kulturformen des Kohls übertragen.Summary:Turnip mosaic virus (TuMV) is an important pathogen in vegetable Brassica crops and belongs to the Potyvirus group. The virus occurs worldwide and has a very wide host range of more than 300 plant species including different Brassica crops. In white cabbage (Brassica oleracea var. capitata) TuMV infection causes up to 25 % yield reductions as well as necrosis during cold storage of the heads. An effective way of plant protection is the establishment of resistance to different TuMV pathotypes in cabbage.TuMV isolates were characterized and resistance screening methods utilizable under greenhouse and field conditions were developed. The search for resistance to TuMV in more than 100 accessions of the Brassicaceae family revealed some new resistance donors. Resistance to different TuMV pathotypes was found in B. oleracea primitive forms, Chinese cabbage (B. rapa ssp. pekinensis), horse-radish (Armoracia rusticana) and radish (Raphanus sativus). A high level of resistance to three TuMV pathotypes was also detected in sexually developed Raphanobrassica hybrids. In a B. oleracea landrace five inbred lines (I6) were selected under field conditions receiving a high level of resistance (infection rate 0 %) after inoculation with the high virulent TuMV isolate 2. From two B. oleracea primitive forms seven homozygous lines (I5) were developed by successive selection and self-pollination. After inoculation with five TuMV isolates representing three pathotypes (Mix 1), resistant plants were symptom-less as well as DAS-ELISA negative. Resistance to TuMV in B. oleracea primitive forms could be transferred into white cabbage by crosses followed by successive screening and selection procedures. Somatic hybrids between R. sativus and B. oleracea resistant to TuMV were generated by protoplast fusion.

Use of CACTA Mobile Genetic Elements for Revision of Phylogenetic Relationships in Brassica rapa L. Species

To supplement our previous studies of genetic diversity in Brassica rapa L. by SSR analysis, for the first time, we used Class II transposable elements to clarify the phylogenetic relationships. The core collection of 96 accessions stored at the Vavilov Institute of Plant Industry is used in the study. Experiments with 12 S-SAP primer pairs gave rise to 123 polymorphic markers. Using TE markers, we divide B. rapa crops into two major clusters: East Asian vegetables and Indo-European-Asian oilseed and turnip. The first cluster is divided into the subclusters of pak-choi and bok choi according to botanical classification and the results of SSR analysis. The earlier classification is improved by subclassification of pak-choi into groups, including identification of the separate group of headed cabbages, revision of the positions of the Hiroshimana and Mizuna forms, and subcategorization of oilseed by geographical origin. However, more accurate positions of accessions in the system of the species are obtained by construction of a SAHN dendrogram with 149 SSR and 123 S-SAP markers. The diagram shows that only a few accessions of European turnip are dispersed among the accessions of the other cluster but some accessions form a separate group in the Nepal-Indian subcluster. They are assumed to be the oldest forms closely related to the first domesticated Central-Asian B. rapa form.

Verbesserung der Krankheitsresistenz von Kohlgemüse: 2. Kohlhernie, Alternaria- und Phoma-Blattfleckenkrankheit

Zusammenfassung:Kohlhernie (Plasmodiophora brassicae), Schwarzfleckigkeit (Alternaria brassicicola, A. brassicae) sowie die durch Phoma lingam verursachte Blattfleckigkeit und Fußvermorschung sind wirtschaftlich bedeutsame Erkrankungen des Kohlgemüses. Für ihre Begrenzung bietet sich als Alternative zum Einsatz von Fungiziden die Nutzung der erblichen biotischen Resistenz im Wirt an. Bei Darlegung eigener Untersuchungsergebnisse werden Informationen zum Stand der Evaluierung von Resistenzträgern und dem Resis- tenztransfer in Kulturformen von Brassica oleracea durch konventionell züchterische sowie moderne In-vitro-Verfahren gegeben. Bei der Kohlhernie bestehen gute Aussichten, stabile Resistenzen insbesondere durch Nutzung quantitativ-genetischer Erbfaktoren (vornehmlich rezessive Anlagen, additive und Domi-nanzeffekte) zu entwickeln. Dagegen stellt sich die Situation bei den Alternarien und Phoma lingam als wesentlich komplizierter dar, da effektive Resistenzen nur in mehr/weniger systematisch weiter von B. oleracea entfernten Taxonen präformiert sind. Diese müssen zunächst durch moderne biotechnologische Verfahren in die Kulturformen übertragen, in Bezug auf den Vererbungsmodus charakterisiert und später in fertilen Genotypen durch genetisch-cytologische und züchterische Maßnahmen etabliert und stabilisiert werden. Erste Erfolge zur Übertragung von Phoma-Resistenz in Kohl liegen vor.Summary:Clubroot (Plasmodiophora brassicae), black leaf spot (Alternaria brassicicola, A. brassicae) as well as leaf spot and blackleg caused by Phoma lingam are economically important diseases in Brassica vegetables. For their control, the utilisation of inheritable biotic resistance in the host is an alternative to the application of fungicides. Results of resistance evaluation and transfer of resistance into culture forms of Brassica oleracea by conventional breeding and modern in vitro techniques are presented. Concerning clubroot there are good chances for establishing durable resistance particularly by using quantitative inheritance factors. With regard to the Alternarias and Phoma lingam the situation is more complicated because effective resistance traits only occur in taxa more/less distantly related to B. oleracea. These traits must, first of all, be transfered into the culture forms by modern biotechnological techniques (protoplast fusion, gene transfer), characterized with regard to the mode of resistance in heritance and, thereupon, established and stabilised by genetic, cytogenetic and breeding arrangements in fertile genotypes. First successful attempts in transferring Phoma-resistance into cabbage have been performed.

Morphogeographic and molecular genetic analysis of the VIR white cabbage collection

It is shown that an analysis of microsatellites allows dividing the white cabbage collection into concultivars and geographic groups corresponding to its morphogeographic classification. Despite a different breeding history, cultivars remain belonging to the concultivar.