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Hypericum perforatum, H. umbellatum, H. maculatum, and H. hircinum accessions originating from botanical gardens across Europe were examined by flow cytometry and molecular markers. 2C DNA content of 17 Hypericum perforatum accessions (Hp) and the H. perforatum cultivar Topaz amounted to between 1.56 pg and 1.62 pg. In four Hp accessions some individual plants were found with a DNA content corresponding to 6Cx (2.34 - 2.39 pg). All plants of accession Hp8 showed a DNA content of 6Cx (2.41 pg). In root tips of Hp plants with an average DNA amount of 1.58 pg, 32 chromosomes were detected, corresponding to 2n = 4x. This is the first ploidy and/or DNA content report for H. umbellatum, H. maculatum and H. hircinum. H. umbellatum and H. maculatum, each contained 0.76 pg DNA and 16 chromosomes were counted. The 2C DNA content of H. hircinum was 1.00 pg with the best metaphase plate revealing 32 chromosomes. Additionally, a combined marker analysis, based on inter-simple sequence repeats (ISSR) and sequence related amplified polymorphism (SRAP), was conducted to gain a better understanding of diversity especially within the accessions of H. perforatum. A total of 27 (11 ISSR and 16 SRAP) primer combinations were screened, showing 699 bands, of which 661 were polymorphic. UPGMA clustering revealed that accessions from the same geographic area tended to be more closely related, while H. maculatum was grouped separately from all H. perforatum accessions. Both methods have shown similar sensitivities in detecting the genetic diversity of the analyzed genotypes. Our results may be useful for Hypericum breeding programs and the development of effective conservation strategies.

The centromere is the chromosome region where the microtubules attach during cell division. In contrast to monocentric chromosomes with one centromere location, holocentric species usually distribute hundreds of centromere units along the entire chromatid. We assembled the chromosome-scale reference genome and analyzed the holocentromere and (epi)genome organization of the lilioidChionographis japonica.Remarkably, each of its holocentric chromatids consists of only 7 to 11 evenly-spaced megabase-sized centromere-specific histone H3-positive units. These units contain satellite arrays of 23 and 28 bp-long monomers capable of forming palindromic structures. Like monocentric species,C. japonicaforms distinctly clustered centromeres in chromocenters at interphase. Additionally, the large-scale eu- and heterochromatin arrangement differs betweenC. japonicaand other known holocentric species. Using polymer simulations, we modeled the formation of prometaphase line-like holocentromeres from interphase centromere clusters. Our findings broaden the knowledge about the diversity of centromere organization, showing that holocentricity is not restricted to species with numerous and small centromere units.

Asparagus is a valued vegetable in people's diet in many countries of the world. The production of asparagus increased remarkably in the last few years. Comprehensive evaluations have shown that there exist no cultivars of A. officinalis with resistance against Asparagus virus 1 (AV-1) causing a high loss of yield and quality. Resistance was found in wild species of the genus Asparagus such as in A. amarus. Somatic hybridization could be a tool to overcome crossing barriers between species. The sophisticated technology requires plant species adapted protocols. Here, a method starting with suspension cultures of A. officinalis and A. amarus was presented as source for protoplasts. Fusion was performed using either electromanipulation or polyethylene glycol solution. Fused protoplasts were cultivated in liquid protoplast cultivation medium PPM1 supplemented with 1 g L-1 L-glutamine. First cell divisions were noticed already after eight to ten days. About five weeks after fusion microcolonies were plated on solid Murashige and Skoog medium (MS) + 1 mg L-1 2,4-dichlorophenoxyacetic acid. After several subcultures, developing plants were transferred to MS + 0.2 mg L-1 α-naphthalene acetic acid for further growth. Regenerated plants were acclimatized to greenhouse conditions. By fluorescence flow cytometry the DNA amount was estimated. RAPD analyses revealed regenerated plants with DNA profiles with fragments from only one protoplast donor but also with fragments from both donors as it is expected for somatic hybrids. Resistance to AV-1 was tested by DAS-ELISA.

Caraway (Carum carvi) is a widespread and frequently used spice and medicinal plant with a long history of cultivation. However, due to ongoing climatic changes, the cultivation is becoming increasingly risky. To secure caraway cultivation in future, timely breeding efforts to develop adapted material are necessary. Analysis of genetic diversity can accompany this process, for instance, by revealing untapped gene pools. Here, we analyzed 137 accessions using genotyping by sequencing (GBS). Hence, we can report a broad overview of population structure and genetic diversity of caraway. Population structure was determined using a principal coordinate analysis, a Bayesian clustering analysis, phylogenetic trees and a neighbor network based on 13,155 SNPs. Genotypic data indicate a clear separation of accessions into two subpopulations, which correlates with the flowering type (annualvs. biennial). Four winter-annual accessions were closer related to biennial accessions. In an analysis of molecular variance, genetic variation between the two subpopulations was 7.84%. In addition, we estimated the genome size for 35 accessions by flow cytometry. An average genome size of 4.282 pg/2C (± 0.0096 S.E.) was estimated. Therefore, we suggest a significantly smaller genome size than stated in literature.

Rucola is a leafy vegetable from the Brassicaceae family with continuously increasing economic demand. Under this name rocket salad (Eruca sativa) as well as the perennial wall rocket (Diplotaxis tenuifolia) are commercially distributed. So far, for both species no hybrid system is available. Therefore, breeding lines of both species were used to pollinate cytoplasmic male sterile (CMS) cauliflower (Brassica oleracea) plants. Intergeneric Fi-hybrids were achieved by manual crosses and embryo rescue. Allotetraploid plants obtained after colchicine treatment were recurrently backcrossed with the respective rucola parent. The elimination of Brassica genomic DNA was verified using molecular markers, GISH analysis, flow cytometry and chromosome analysis. All backcross plants from BC2 up to the BC6 generation of the B. oleracea x E. sativa (BE) cross showed male sterility and full seed set compared to the E. sativa parent. No further incompatibilities were noticed between the CMS inducing cytoplasm and the E. sativa genome. Agronomical traits such as plant vigor, biomass production, flowering and seed set were recorded for the different backcross generations. The developed alloplasmic lines are diploid with 2n=2x=22 chromosomes and could be introduced without any problems into the breeding process. Also BC1 plants of the cross B. oleracea x D. tenuifolia (BD) had completely male sterile flowers. The phenotype was similar to that of the D. tenuifolia parent. Unlike the BE combination, for BD combination embryo rescue was necessary to establish BC2 plants. The flowers of all BC2 plants of BD combination showed besides the male sterility a pronounced female sterility, expressed by the abort of ovaries and failed seed set. Hence, this combination seems to be not applicable for the breeding process. Reasons for this strikingly different behaviour are under investigation.

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.

Experiments were carried out on the use of Piriformospora indica, an endomycorrhizal-like fungus, to increase the resistance of two rhododendron cultivars ‘Nova Zembla’ and ‘Alfred’ to the pathogens Phytophthora cinnamomi and P. plurivora. The interaction between P. indica and the pathogens was evaluated in dual cultures on KM and PDA media. The growth of P. indica colonies was restricted by pathogens during the 7–13 days of culture due to their quicker growth. Neither hyphae lysis nor atypical morphology was observed. No inhibition zones were observed between colonies of pathogens and P. indica but they were present in monocultures of P. indica. The ability to release zoospores by Phytophthora was not affected in the cultures with P. indica. In an experiment performed in vitro, P. indica colonised the roots of microplants, but after pathogen inoculation, its protective role was limited only to delaying dieback. In pot greenhouse experiments, all the young plants grown in virgin peat substrate, and in the substrate inoculated with P. indica survived, whereas all the plants grown in the substrate infected with the pathogens died. From 40 to 60% of plants grown in the substrate inoculated with P. indica, to which pathogen infected substrate was added after 21 days, survived one year without symptoms. The rate of success depended on the pathogen species and rhododendron cultivar. Our results show a practical possibility of biological protection of rhododendron plants against Phytophthora with the help of P. indica.

Species with holocentric chromosomes are often characterized by a rapid karyotype evolution. In contrast to species with monocentric chromosomes where acentric fragments are lost during cell division, breakage of holocentric chromosomes creates fragments with normal centromere activity. To decipher the mechanism that allows holocentric species an accelerated karyotype evolution via chromosome breakage, we analyzed the chromosome complements of irradiated Luzula elegans plants. The resulting chromosomal fragments and rearranged chromosomes revealed holocentromere-typical CENH3 and histone H2AThr120ph signals as well as the same mitotic mobility like unfragmented chromosomes. Newly synthesized telomeres at break points become detectable 3 weeks after irradiation. The presence of active telomerase suggests a telomerase-based mechanism of chromosome healing. A successful transmission of holocentric chromosome fragments across different generations was found for most offspring of irradiated plants. Hence, a combination of holokinetic centromere activity and the fast formation of new telomeres at break points enables holocentric species a rapid karyotype evolution involving chromosome fissions and rearrangements.

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.

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.

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.

Microbial endochitinase CHIT36 is one of the lytic enzymes secreted by Trichoderma harzianum that exhibits antifungal activity in vitro. To evaluate its activity when expressed in planta, a plasmid containing chit36 gene under the control of CaMV 35S promoter and the nptII selection gene were introduced into polyethylene glycol (PEG)-treated carrot (Daucus carota L.) protoplasts. The transgenic plants expressing CHIT36 were used in resistance assays to evaluate their susceptibility to fungal pathogens. Laboratory-based assays on detached leaves and petioles showed that the transgenic carrots had less severe disease symptoms. The resistance response depended on the transgenic clone, but all clones had significantly enhanced tolerance to Alternaria radicina and Botrytis cinerea, on average by 50%. Slower disease progress caused by Alternaria dauci was observed for two transgenic clones while the remaining clone was more susceptible than the control. The most resistant transgenic clones were also more tolerant to the pathogens than ‘Bolero’ F1, which is a conventionally bred cultivar tolerant to A. dauci. This is the first report of the use of microbial chitinase to enhance carrot resistance. The results indicate that CHIT36 expressed in planta has the potential to reduce development of fungal diseases.

Hybrid callus was formed from the successful protoplast fusion between pollen protoplasts of Brassica oleracea var. italica and haploid mesophyll protoplasts of Brassica rapa. The pollen protoplast isolation frequency in broccoli was highly related to the ratio of trinucleate pollens in the male gametophyte population. Large quantities of pollen protoplasts with high vigor could be isolated, and the isolation frequency reached up to 90% in 6.0-7.0 mm long flower buds with about 94.7% trinucleate-stage pollens. Pollen protoplasts could be collected and purified by discontinuous gradient centrifugation. In 1% Na-alginate embedding culture, cell divisions were observed but no further development was found. The haploid mesophyll protoplasts were isolated from in vitro haploid plants of B. rapa. Results strongly showed the variability in culturability of mesophyll protoplasts from different haploid lines. Both pollen protoplasts and haploid mesophyll protoplasts retained a stable round shape in the designed prefusion solution with an osmotic pressure of 0.74 osmol/kg. Polyethylene glycol was used for the protoplast fusion, and 40% polyethylene glycol 4000 enabled the highest fusion frequency of about 20%. Some postfusion protoplasts showed cell divisions up to callus proliferation. Calli were screened by random amplified polymorphic DNA analysis for their hybrid character. Results revealed the existence of the hybrid calli. Some of the hybrid calli grew well with green color and shoot primordia. According to our knowledge, this is the first report about a hybrid formation between two haploid protoplasts. Potential comprehensive applications, as well as problems of this technique, are discussed.

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.

Alloplasmic male sterile breeding lines of Eruca sativa were developed by intergeneric hybridization with CMS- Brassica oleracea, followed by recurrent backcrosses and determination of the breeding value.Male sterile breeding lines of rocket salad (Eruca sativa) were developed by intergeneric hybridization with cytoplasmic male sterile (CMS) cauliflower (Brassica oleracea) followed by recurrent backcrosses. Five amphidiploid F1 plants (2n = 2x = 20, CE), achieved by manual crosses and embryo rescue, showed an intermediate habit. The plants were completely male sterile and lacked seed set after pollination with the Eruca parent. Allotetraploid F1-hybrid plants (4n = 4x = 40, CCEE) obtained after colchicine treatment were backcrossed six times with pollen of the Eruca parent to select alloplasmic diploid E. sativa lines. The hybrid status and the nucleo-cytoplasmic constellation were continuously controlled by RAPD and Southern analysis during subsequent backcrosses. The ploidy level was investigated by flow cytometry and chromosome analysis. Premeiotic (sporophytic) and postmeiotic (pollen abortive) defects during the anther development were observed in the alloplasmic E. sativus plants in comparison to the CMS-cauliflower donor. No further incompatibilities were noticed between the CMS-inducing cybrid cytoplasm and the E. sativa nuclear genome. The final alloplasmic E. sativa lines were diploid with 2n = 2x = 22 chromosomes and revealed complete male sterility and restored female fertility. Plant vigor and yield potential of the CMS-E. sativa BC5 lines were comparable to the parental E. sativus line. In conclusion, the employed cybrid-cytoplasm has been proven as a vital source of CMS for E. sativa. The developed lines are directly applicable for hybrid breeding of rocket salad.

Laboratory experiments were conducted to develop an assay for evaluation of carrot (Daucus carota L.) leaf reaction to Botrytis cinerea Pers. ex Pers. The detached carrot leaflets were inoculated with the colonized agar plugs attached to the cut surface of the midrib or by spraying conidial suspension. The estimation of the infected leaflet area expressed as the area under the disease progress curve enabled the discrimination between carrot genotypes differing in their susceptibility to the pathogen. Evaluation based on the measurement of the length of the midrib lesion did not allow such differentiation. The inoculation of leaf surface with suspension of conidia was less reliable and the development of the disease symptoms was not reproducible. The established assay using colonized plugs enables a fast assessment of carrot leaf susceptibility to grey mould and can be particularly useful for a non-destructive, preliminary evaluation of precious and limited source materials. © 2006 The Authors.

Agrobacterium rhizogenes mediated transformation combined with a visual selection for green fluorescent protein (GFP) has been applied effectively in carrot (Daucus carota L.) transformation. Carrot root discs were inoculated with A4, A4T, LBA1334 and LBA9402 strains, all bearing gfp gene in pBIN-m-gfp5-ER. The results indicate that transformed adventitious roots can be visually selected solely based on GFP fluorescence with a very high accuracy. The method requires no selection agents like antibiotics or herbicides and enables a reduction of labour and time necessary for tissue culture. Moreover, individual transformants can be easily excised from the host tissue and cultured separately. All of the 12 used carrot cultivars produced transformed adventitious roots and the frequency of discs producing GFP expressing adventitious roots varied from 13 to 85%. The highest transformation rate was found for A4T and LBA1334 strains possessing chromosomal background of A. tumefaciens C58. The results encourage that visual selection of transformed, fluorescing adventitious roots can be highly effective and applied routinely for the production of carrot transgenic plants.

A total of 243 transgenic T1-plant lines were produced by in planta transformation of Arabidopsis by a mixture of three distinct Agrobacterium tumefaciens strains carrying different plasmid constructs. PCR analysis indicated that about 30% of the transgenic plants were transformed with two different T-DNAs and additional 9.5% of the plants carried all genes from three different T-DNAs. Southern analysis revealed the integration from one to nine gene copies for each T-DNA used. Southern analysis of progenies in six lines displayed that the different transgenes were preferentially integrated into the same locus in a single plant genome. Analysis of fragment lengths, obtained in Southern analysis, allows to suppose the co-integration of several different T-DNAs in the single locus in direct, head-to-head or tail-to-tail orientation. PCR analyses of additional plants from these lines confirmed predominantly linked inheritance of all three transgenes integrated. About a half of T1-plants carrying all three transgenes were investigated by Northern hybridisations showing expressed mRNA at different levels. The expression of one transgene appears to have no effect on expression of the others and there is no correlation between the amount of mRNA and the number of gene copies in a single plant genome. The results reported here may be useful in plant metabolic engineering.

Das Turnip mosaic virus (TuMV) ist weltweit verbreitet und gehort zur Gruppe der Potyviren. Es hat einen auserordentlich grosen Wirtspflanzenkreis von uber 300 Arten und wird nicht persistent durch 89 Aphidenarten ubertragen. Bisher sind beim TuMV zwolf Pathotypen bekannt. Im Kohlgemuse verursacht das TuMV die so genannte Kohlschwarzringfleckigkeit. Dabei handelt es sich um eine wirtschaftlich wichtige Virose, die beim Weiskohl bis zu 25 % Ertragsausfall und Gewebenekrotisierungen, insbesondere wahrend der Kuhllagerung, bewirken kann. Zur Sicherung eines hohen Ertrags- und Qualitatsniveaus bei Kohlgemuse wurde begonnen, die Resistenz gegen diese Virose zu verbessern. Mit aktuellen, weitgehend charakterisierten Erregerherkunften des TuMV wurden Resistenzprufmethoden weiterentwickelt und neue Resistenzquellen in der Familie der Brassicaceae erschlossen. Resistenzen gegen mehrere TuMV-Pathotypen liesen 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 ubertragen.

Kohlhernie (Plasmodiophora brassicae), Schwarzfleckigkeit (Alternaria brassicicola, A. brassicae) sowie die durch Phoma lingam verursachte Blattfleckigkeit und Fusvermorschung sind wirtschaftlich bedeutsame Erkrankungen des Kohlgemuses. Fur 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 Resistenztragern und dem Resis- tenztransfer in Kulturformen von Brassica oleracea durch konventionell zuchterische 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 praformiert sind. Diese mussen zunachst durch moderne biotechnologische Verfahren in die Kulturformen ubertragen, in Bezug auf den Vererbungsmodus charakterisiert und spater in fertilen Genotypen durch genetisch-cytologische und zuchterische Masnahmen etabliert und stabilisiert werden. Erste Erfolge zur Ubertragung von Phoma-Resistenz in Kohl liegen vor.

Introgression of genes from distantly related donors within the family Brassicaceae is useful for augmenting the genetic base for achieving specific crop improvement objectives. Hypocotyl-derived protoplasts of B. oleracea var. capitata cv. 'Toskama' were fused with non-irradiated hypocotyl protoplasts as well as X-ray irradiated mesophyll protoplasts of Lepidium meyenii to produce symmetric versus asymmetric intergeneric hybrids. In addition, hypocotyl-derived protoplasts of B. oleracea cv. botrytis 'Korso' were fused with X-ray irradiated mesophyll protoplasts of L. meyenii. Using polyethylene glycol, 76 plants were regenerated, 37 with symmetric fusion and 39 with asymmetric fusion. Regenerated plants were characterized by RAPDs, AFLPs and Southern hybridization. Due to the small amount of template DNA (20 ng) required, the RAPD markers are suitable for detecting true fusion plants in very early stages of in vitro development. Whereas the large number of markers provided with AFLP was useful for detecting a high degree of genetic polymorphisms in the fusion plants and for quantifying the degree of asymmetric fusion. More plants with the morphology of B. oleracea were obtained with asymmetric fusion.

SUMMARY Breeding research for improvement of the homogeneity of marjoram pollinator lines requires reliable information on the genetic diversity. Three progenies of marjoram pollinator lines were examined by means of RAPD assays. For this reason a suitable protocol for DNA isolation and the conditions for the PCR reactions were worked out. A single plant analysis as well as an analysis with bulked DNA samples were used. The RAPD fingerprints of single plants illustrate the heterogeneity inside the accessions in the first year and the enhancement of the homogeneity in the next progenies causing by selfing. For revealing the genetic distances of the accessions, the pooling strategy is more suitable than the single plant analysis. The simplicity of the RAPDs makes it possible to realize a high number of analyses, but the results should be evaluated with caution, with knowledge about some drawbacks of the method. Nevertheless, there is the conclusion that RAPDs are a useful tool in marjoram breeding programs.

Mesophyll protoplasts of Brassica oleracea var. botrytis were successfully transformed using polyethylene glycol (PEG). The success of plant transformation depended on both gene transfer and plant regeneration. Parameters, such as PEG and vector concentrations and heat shock conditions were tested in experiments on transient expression of the beta-glucuronidase (EC 3.2.1.31) gene and the most suitable conditions for DNA uptake were determined. Two antibiotic resistance marker genes for neomycin phosphotransferase (EC 2.7.1.95) and hygromycin phosphotransferase (EC 2.7.1.104), and three vector plasmids with different lengths were used to obtain stable transformants.

Cabbage (Brassica oleracea var. capitata L.) which is cultivated in many countries of north and south climatic zone on more than 2 million ha, is an important vegetable crop. The yield of cabbage cultivation is limited by diseases and pests. The most important diseases are caused by pathogenic fungi. Clubroot (Plasmodiophora Brassica) is spread all over the world and causes high losses, particularly on humid acid soils. In many other Brassicaceae black leg (Phoma lingam) becomes more important to cabbage cultivation, and also Alternaria (A. brassicae, A. brassicicola) is among the most important diseases reducing yields in cabbage. Heavy destruction in cabbage fields is caused by turnip mosaic potyvirus.

The regeneration of plants in tissue culture is generally possible through two morphological and anatomic different pathways: (1) somatic embryogenesis and (2) shoot/root organogenesis involving adventitious shoot bud development (see Reinert et al. 1977).