We genotyped a Hydrangea macrophylla collection including 120 plants. These plants represented 43 cultivars that were developed between 1909 and 1987. These plants were genotyped with 12 SSR markers and 2 to 7 alleles were detected per marker. Surprisingly, we identified 62 instead of 43 distinct fingerprints, because 2 to 4 different genotypes were determined in 25 out of 43 cultivars. Frequently, these genotypes resembled each other very much in phenotype, suggesting a mix-up of genotypes under the label of one cultivar. However, also plants with lacecap and mophead inflorescences as well as diploid and triploid plants were wrongly grouped together. In order to determine cultivar-specific fingerprints, we reconstructed the "Wadenswil" pedigree at molecular level. Based on this pedigree, which encompasses 32 cultivars, we predicted cultivar-specific fingerprints and identified at least 20 "Wadenswil" cultivars by fingerprint (Hempel et al., 2018). Based on the 2C DNA content of these plants, we detected 4 putative interploid crosses between diploid and triploid plants. These interploid crosses resulted in diploid or/and triploid offspring, confirming that crosses with triploids were successfully performed in the "Wadenswil" breeding program. Here we present exemplarily the possibilities and limitations to predict cultivar-specific fingerprints.

The soilborne pathogen Verticillium dahliae invades its host via the root, and spreads systemically throughout the plant. Although a functional root system of appropriate size is essential for water and nutrient uptake, to date, effects of pathogens on root morphology have not been frequently investigated. Therefore, this study aims to improve knowledge of how V. dahliae infection impairs root morphological characteristics of tomato, considering plant growth and physiological responses, particularly those involved in defence in roots and leaves over a growing period of up to 28 days post‐inoculation. Verticillium dahliae infection suppressed the growth of both shoot and root. Diseased plants developed a smaller leaf area, and exhibited a reduction in the rate of photosynthesis and stomatal conductance. An early response to pathogen invasion in the host root was the up‐regulation of several defence‐related genes, such as proteinase inhibitor II (Pin2), β‐1,3‐glucanase A (GluA) and two pathogenesis‐related genes (PR‐1a, PR‐1b). However, this response did not prevent colonization of the roots by the pathogen. Although a high variability in pathogen density was found within the root system, a significant increase of both the specific root length and surface area was observed in response to pathogen invasion; these traits correlated with water use efficiency. Morphological changes of the root may represent an adaptive response evolved to sustain the supply of both water and nutrients in the presence of the pathogen.

The so-called "bud bloomers" are economically very important cultivars of Calluna vulgaris (Ericaceae), a major ornamental crop of the European horticultural sector. Within the bud bloomer group, the diplocalyx and the polystyla type can be distinguished. Both bud bloomer types have an extended flower longevity. Due to a dysfunction in flower opening, they display a duplication of calyx organs and a loss of petals. However, they differ with regard to flower whorl 3: whereas stamens are missing in the diplocalyx type, stamens show a carpelloid character in the polystyla type. The genetic basis of the bud blooming flower type is still unclear. A series of crosses was performed to evaluate the number of loci and alleles involved in the genetics of the trait 'flower type' in C. vulgaris. From the segregation ratios within the resulting populations, a monogenic inheritance involving three alleles was deduced, assuming that the polystyla genotypes are heterozygous. This hypothesis was verified in additional crossing experiments.

Heather (Calluna vulgaris), an important bedding plant in Northern Europe, is a monotypic genus with rather limited phenotypic variability. Hence, it is aimed at starting a breeding program of polyploids that might constitute a new phenotypic class of varieties with attractive strong habitus and larger flowers. As a basis, data on fertility of genotypes of different ploidy levels, fertility of interploid crosses, and ploidy levels of the offspring were generated by setting up a complete interploid crossing experiment in C. vulgaris including diploid (2x), triploid (3x), and tetraploid (4x) genotypes.Determination of pollen viability by staining, in vitro germination and evaluation of pollen tube growth in vivo generated contradicting results and none of the methods proved to be suitable to explain fertilization success as determined by seed set. Seed yield was significantly reduced in any cross direction compared to 2x x 2x crosses. However, the germination rate did not correlate with seed set, hence for determining crossing success, the number of germinating seedlings has to be considered. The rate of germinating seedlings per pollinated flower was 2.95 in the 2x x 2x crosses and on average only 0.025 in the interploid crosses. 2x x 4x and 4x x 2x crosses differed significantly with regard to seed yield, germination rate and ploidy of the offspring. 3x genotypes were generated only by 4x x 2x crosses. Aneuploids resulted from 2x x 3x, 3x x 4x, 4x x 2x and 4x x 3x crosses. (C) 2014 Elsevier B.V. All rights reserved.

In Calluna vulgaris, a common bedding plant during autumn in the northern hemisphere, the bud-blooming mutation of flower morphology is of high economic importance. Breeding of new bud-blooming cultivars suffers from poor seed set in some of the desirable bud-flowering crossing partners. In the current study, fertilisation and seed development in genotypes with good or poor seed set were monitored in detail in order to examine pre- and post-zygotic cross breeding incompatibilities. Whereas no distinct differences were detected in seed development, pollen tube growth was impeded in the pistils of genotypes characterised by poor seed set. Detailed microscopic analysis revealed malformations of the gynoecia due to imperfect fusion of carpels. Hence, a pre-zygotic mechanism hindering pollen tube growth due to malformation of gynoecia was deduced. An interaction of putative candidate genes involved in malformation of gynoecia with floral organ identity genes controlling the flower architecture is discussed.

The ornamental crop Calluna vulgaris is of increasing importance to the horticultural industry due to a flower organ mutation, the so-called 'bud-flowering' phenotype, in which buds remain closed throughout the total flowering period and thereby maintain more colourful flowers for a longer period of time than the wildtype. Moreover, stamens are missing. In order to clarify the genetics of this mutation a comparative study of the wild-type and the 'bud-flowering' flower type of C. vulgaris was initiated. Scanning electron microscopic analyses of flower organs as well as gene expression studies of an AP3-like and a SEP1-like gene in the different flower whorls of both flower types allowed identification of bracts, sepals and petals. However, loss of stamens in the 'bud-flowering' phenotype could not be explained by modified spatial expression of known organ identity genes. Therefore, new approaches (map-based cloning and differential transcriptome analysis) are now envisioned in order to elucidate the genetics of the 'bud-flowering' phenotype.

Prerequisite for the development of F-1-hybrids in Helleborus orientalis Lam. is the evaluation of potential parents' inbreeding levels. As no codominant marker system is available, a method to estimate the inbreeding coefficient based on AFLP-markers has been applied and evaluated based on the progeny of a presumable selfing experiment. The effect of marker numbers used in the analysis and the composition of an out-group for this calculation were analysed. Practical problems of the method are discussed.

Calluna vulgaris (Ericaceae) is an economically important crop for the European horticultural sector. Within the species the so-called 'bud-flowering' phenotypes represent the most significant subgroup. Since several other flower phenotypes exist and classification of these is considered as an important matter for future breeding, a study of flower organ identity based on molecular and morphological aspects was started. We discuss our results within the context of earlier classifications of specific cultivars into botanical subgroups.As a consequence, we propose some re-classifications of relevant cultivars. Furthermore, our results proved perianth organ identity as well as the identity of bracts in the wild-type phenotype and the 'bud-flowering' group.

The establishment of a marker-assisted selection system for the economically important 'bud-flowering' phenotype in the ornamental crop Calluna vulgaris is of great interest to practical breeding companies, as it would allow selection at the juvenile stage. Segregation analyzes revealed a monogenic recessive inheritance of the bud flowering trait. Since in C. vulgaris only sparse molecular data are available, the search for molecular markers in a segregating backcross progeny was accomplished using PCR techniques based on random primers. Two candidate RAPD markers in coupling of the trait of interest were identified. Results on their applicability in different populations and independent varieties are presented. Their transformation capability to sequence characterized amplified region and single strand conformation polymorphism markers are described and discussed in the context of marker-assisted selection strategies in breeding of ornamental crops.

Embryo development and germination of Cyclamen persicum have been comparatively characterized for zygotic and somatic embryos with regard to mitotic activity and morphology in order to identify developmental abnormalities in somatic embryogenesis. Zygotic embryo development proved to be highly synchronous with distinct periods of cell division, cell elongation and embryo maturation within a total period of 17 weeks of seed development. Somatic embryo development was accomplished within only 3 weeks, resulting in a mixture of morphologically highly variable embryos. No distinct developmental periods could be identified and no reduction of the mitotic activity was discovered for non-desiccated somatic embryos. Controlled desiccation of somatic embryos severely reduced their germination rate, demonstrating resemblance of somatic embryos to recalcitrant seeds, whereas zygotic Cyclamen seeds could be characterized as typically orthodox.

The first bryophyte tissue culture techniques were established almost a century ago. All of the techniques that have been developed for tissue culture of seed plants have also been adapted for bryophytes, and these range from mere axenic culture to molecular farming. However, specific characteristics of bryophyte biology for example, a unique regeneration capacity - have also resulted in the development of methodologies and techniques different than those used for seed plants. In this review we provide an overview of the application of in vitro techniques to bryophytes, emphasising the differences as well as the similarities between bryophytes and seed plants. These are discussed within the framework of physiological and developmental processes as well as with respect to potential applications in plant biotechnology.

Summary As a first step in the development of synthetic seed of Cyclamen persicum Mill. various procedures for induction of desiccation-tolerance in somatic embryos of cyclamen have been tested. Most important proved to be the careful selection of mature embryos by morphological characteristics. Torpedo-shaped somatic embryos with a size between 700 and 1000 µm exhibited the highest germination rate after desiccation to only 0.2 g H2O g–1 DW. Addition of 75 g l–1 polyethylene glycol (PEG 4000) and 10 mg l–1 abscisic acid to the maturation medium increased the germination rate of somatic embryos after desiccation to 28% compared to 4% in the control without these supplements.

Gene targeting in the moss Physcomitrella patens has created a new platform for plant functional genomics. We produced a mutant collection of 73329 Physcomitrella plants and evaluated the phenotype of each transformant in comparison to wild type Physcomitrella. Production parameters and morphological changes in 16 categories, such as plant structure, colour, coverage with gametophores, cell shape, etc., were listed and all data were compiled in a database (mossDB). Our mutant collection consists of at least 1804 auxotrophic mutants which showed growth defects on minimal Knop medium but were rescued on supplemented medium. 8129 haploid and 11068 polyploid transformants had morphological alterations. 9% of the haploid transformants had deviations in the leaf shape, 7% developed less gametophores or had a different leaf cell shape. Other morphological deviations in plant structure, colour, and uniformity of leaves on a moss colony were less frequently observed. Preculture conditions of the plant material and the cDNA library (representing genes from either protonema, gametophore or sporophyte tissue) used to transform Physcomitrella had an effect on the number of transformants per transformation. We found correlations between ploidy level and plant morphology and growth rate on Knop medium. In haploid transformants correlations between the percentage of plants with specific phenotypes and the cDNA library used for transformation were detected. The number of different cDNAs present during transformation had no effect on the number of transformants per transformation, but it had an effect on the overall percentage of plants with phenotypic deviations. We conclude that by linking incoming molecular, proteome, and metabolome data of the transformants in the future, the database mossDB will be a valuable biological resource for systems biology.

Abstract Due to its high rate of homologous recombination, the moss Physcomitrella patens (Hedw.) B.S.G. is used as a novel system to facilitate gene/function-analyses. Loss-of-function mutations are easy to identify in the moss, as the dominant phase is the haploid gametophyte. Regenerating protoplasts were used to establish different moss lines with ploidy levels of 1C, 2C, and 4C. Flowcytometric analysis of three haploid, three diploid, and two tetraploid Physcomitrella lines revealed that haploid and diploid lines were cytologically stable, whereas nuclei of tetraploid lines exhibited varying DNA-contents. The effect of polyploidization on the phenotype, growth, and differentiation of Physcomitrella was investigated in vitro. The growth of three haploid, three diploid, and two tetraploid genotypes was evaluated after four weeks of axenic culture. The effect of the genotype on the growth rate of plants cultured on minimal medium was statistically significant; in contrast the ploidy level had no effect. On full medium the effect of the line, as well as the ploidy level, were statistically significant. The dry weight per petri-dish after a culture period of four weeks, was 103–106 mg in haploid lines, 64–78 mg in diploid, and 19–32 mg in tetraploid ones. The effect of the moss line and the ploidy level on differentiation of buds and gametophores was highly significant. Tetraploid moss rarely developed buds and/or gametophores. Sporophyte induction was observed in haploid and diploid genotypes, however not in tetraploid moss. Twelve percent of the germinated spores increased the ploidy level in comparison with the parent plant. A reduction of the ploidy level from 2C to 1C was observed in 7%. Evaluation of older plants (11 weeks after protoplast isolation) showed that polyploidization often resulted in a reduced number of gametophores and a reduced colony diameter on minimal medium compared to haploid plants. More than 70% of the diploid plants had gametophores with phenotypic alterations. Only 1.4% of the diploid plants were indistinguishable from wildtype. Changes in leaf shape and multiple phenotypic deviations from wildtype are most likely indications of polyploidization; however, the unequivocal identification of diploid plants is only possible using flowcytometric analysis, because a multitude of different phenotypic changes were observed in polyploid plants.

The moss Physcomitrella patens (Hedw.) B.S.G. is a novel tool in plant functional genomics as it has an inimitable high gene targeting efficiency facilitating the establishment of gene/function relationships. Here we report, based on flow cytrometric (FCM) data, that the basic nuclear DNA content per cell of Physcomitrella is 0.53 pg, equating to a genome size of 1C = 511 Mbp. Furthermore, we describe a unique tissue-specific cell cycle change in this plant. Young plants consisting of only one cell type (chloronema) displayed one single peak of fluorescence in FCM analyses. As soon as the second cell type (caulonema) developed from chloronema, a second peak of fluorescence at half the intensity of the previous one became detectable, indicating that caulonema cells were predominantly at the G1/S transition, whereas chloronema cells were mainly accumulating at the G2/M transition. This conclusion was validated by further evidence: i) The addition of ammonium tartrate arrested physcomitrella in the chloronema state and in G2/M. ii) Two different developmental mutants, known to be arrested in the chloronema/caulonema transition, remained in G2/M, regardless of age and treatment. iii) The addition of auxin or cytokinin induced the formation of caulonema, as well as decreasing the amount of cells in G2/M phase. Additionally, plant growth regulators promoted endopolyploidisation. Thus, cell cycle and cell differentiation are closely linked in Physcomitrelia and effects of plant hormones and environmental factors on both processes can be analysed in a straight forward way. We speculate that this unique tissue-specific cell cycle arrest may be the reason for the uniquely high rate of homologous recombination found in the Physcomitrelia nuclear DNA.

Attempts for establishing an efficient gene targeting (GT) system in seed plants have hitherto not been successful. In contrast, GT based on homologous recombination is highly efficient in Physcomitrella, making this moss a novel tool in reverse genetics. However, why homologous and illegitimate recombination are differently regulated between Physcomitrella and seed plants is still enigmatic. Here we update the state of the art of GT in Physcomitrella and discuss approaches to unravel this enigma. Identification of molecular factors significantly enhancing GT and their subsequent transfer to crop plants will have a great impact on plant biotechnology by enabling precise genetic engineering. Physcomitrella appears to be the most useful model system in this context.

The effect of temperature and light conditions on sexual reproduction (sporophyte formation) of in vitro cultures of the moss Physcomitrelia patens was analysed. All parameters tested, i.e., temperature, light intensity and day length had a strong impact on the number of sporophytes formed. The highest number of sporophytes, 559 g fresh weight, developed at 15degreesC, 8 h light/day with an intensity of 20 mumol/m(2)/s. in contrast, at 25degreesC, as well as with a day length of 16 h per day, the number of sporophytes was drastically reduced. Vegetative growth, determined as fresh weight per petri dish, was impeded under conditions favouring sporophyte formation, probably due to nutrient transfer to the sporophytes. Microscopic documentation of the developing sporophytes revealed that, although archegonia were arranged in bundles at the gametophore apices, usually only one archegonium per gametophore apex developed into a mature sporophyte. From an EST database six novel MADS-box genes were identified which, in phylogenetic analyses, did not cluster with the known groups of higher plant MADS-box genes. One of these genes was represented only as a singleton in a cDNA library specifically derived from gametophore apices and developing sporophytes, and, therefore, designated PpMADS-S. RNA amounts of PpMADS-S were two to three times higher under conditions that stimulate sporophyte development (15degreesC, 8 h light per day) when compared to conditions favouring vegetative growth (25degreesC, 16 h light per day), indicating a possible function in sexual reproduction of this moss. Thus, an efficient experimental system was established to study sex organ formation, fertilization and embryo development in Physcomitrella.

One prerequisite for plant functional genomic projects is the development of a high-throughput transformation platform. As the moss Physcomitrella patens can be transformed via PEG-mediated DNA-uptake into protoplasts, a semi-continuous bioreactor culture of this plant was optimised regarding protoplast isolation efficiencies. Under standard conditions protoplast yields were 2.8×104/mg dry weight. This yield was increased sixfold by supplementation of the medium with 460 mg/l ammonium tartrate. The same effect was achieved by controlling the pH-value in the bioreactor culture with a setpoint of 4.5. In contrast, pH control with a setpoint of 7.5 reduced the protoplast yield compared to a culture without pH control to 11%. A semi-continuous culture of Physcomitrella in a 5-l bioreactor grown at pH 4.5 yielded sufficient cell material for more than 100 transformations per day.

Based on the relative ease of performing targeted nuclear gene knockout, the moss Physcomitrella patens has recently been developed as a model system for plant functional genomics. To address the need for new promoters that could drive expression of transgenes in this moss, we tested two fragments of the promoter region of the gene for the sugar beet (Beta vulgaris) V-type H+-ATPase subunit isoform c. Four gene knockout constructs were tested in which the neomycin phosphotransferase II selection marker gene was put under the control of two distinct V-type H+-ATPase promoter fragments, the NOS promoter, or the CaMV 35S promoter. In each case the selection cassettes were flanked by moss FtsZ1 cDNA sequences to facilitate chromosomal targeting. From a total of more than 440 transformed plants, the number of plants generated per construct was monitored and found to be in the range of 5 to 11 stable transgenics per transformation. Both V-type H+-ATPase promoter fragments lead to NPTII expression levels that were sufficiently high to generate large numbers of stable transgenic plants. The numbers of plants obtained with the two V-type H+-ATPase promoter fragments were comparable to those with constructs containing the standard NOS and 35S promoters. We propose that the higher plant V-type H+-ATPase promoter can be used for the expression of transgenes in the bryophyte P patens.

The use of the moss Physcomitrella patens as a production system for heterologous proteins requires highly standardised culture conditions. For this purpose a semi-continuous photoautotrophic bioreactor culture of Physcomitrella was established. This culture grew stably for 7 weeks in a 5-l bioreactor with a dilution rate of 0.22/day. Enrichment of the air for aeration in a batch bioreactor culture with 2% (v/v) CO2 resulted in an increase in the specific growth rate to 0.57/day. Changes in the pH of the semi-continuous bioreactor culture medium between pH 4.5 and pH 7.0 influenced protonema differentiation; however it did not negatively affect the growth rate compared to uncontrolled pH. The advantages of Physcomitrella as a system for the production of heterologous proteins in plants are discussed.