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Experiments were carried out to study the consequences of inoculating Rhododendron ‘Nova Zembla’ and ‘Alfred’ microcuttings and young plants with Serendipita indica (formerly Piriformospora indica), an endophytic fungus. The inoculation at the in vitro rooting stage decreased the in vitro root and shoot quality, but after planting microplants in a greenhouse in a non-fertilised peat substrate, the plants grew 33% taller, with 10% more leaves and with more abundant roots in comparison with the non-inoculated microplants. The young plants rooted in vitro, acclimated in a greenhouse in a peat substrate and inoculated with S. indica at the time of the transplantation to the pots displayed, after 15 months, an increase in the shoot lengths by 35% and 13%, depending on the cultivar, in the leaf number by 47% and in the chlorophyll content by 31% when compared with the non-inoculated plants. The beneficial effect of S. indica on the young Rhododendron plants indicates the possibility of its practical use in the nursery production of this plant.

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.

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.

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.

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.

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).