Exposure to Light of the Abaxial versus Adaxial Side of Detached Kalanchoë blossfeldiana Leaves Affects Anthocyanin Content and Composition Differently.

The accumulation and composition of anthocyanins in leaves of Kalanchoë blossfeldiana, detached and kept for five days under natural light conditions, were investigated. The presence of fifteen derivatives of cyanidin, petunidin, and delphinidin was found. Changes in the content of each anthocyanin in the leaves before and after exposure to light on the abaxial (naturally upper) and adaxial (naturally lower) sides of the leaves were compared. When the adaxial side was exposed to light, the anthocyanin contents of the leaves did not change. In contrast, when the abaxial side of detached leaves was exposed to light, there was enhanced accumulation of delphinidin-rhamnoside-glucoside, cyanidin-rhamnoside-glucoside, cyanidin-glucoside-glucoside, and two unknown derivatives of petunidin and delphinidin. Application of methyl jasmonate (JA-Me) on the abaxial side exposed to light inhibited the accumulation of these anthocyanins. This effect could probably be due to the presence of these anthocyanins in the epidermal cells of K. blossfeldiana leaves and was visible in the microscopic view of its cross-section. These anthocyanins were directly exposed to JA-Me, leading to inhibition of their formation and/or accumulation. The lack of significant effects of JA-Me on anthocyanin mono- and tri-glycosides may indicate that they are mainly present in the mesophyll tissue of the leaf.

A Possible Mode of Action of Methyl Jasmonate to Induce the Secondary Abscission Zone in Stems of Bryophyllum calycinum: Relevance to Plant Hormone Dynamics.

Plants can react to environmental stresses through the abscission of infected, damaged, or senescent organs. A possible mode of action of methyl jasmonate (JA-Me) to induce the formation of the secondary abscission zone (SAZ) in the stems of Bryophyllum calycinum was investigated concerning plant hormone dynamics. Internode segments were prepared mainly from the second or third internode from the top of plants with active elongation. JA-Me applied to the middle of internode segments induced the SAZ formation above and below the treatment after 5-7 days. At 6 to 7 days after JA-Me treatment, the above and below internode pieces adjacent to the SAZ were excised and subjected to comprehensive analyses of plant hormones. The endogenous levels of auxin-related compounds between both sides adjacent to the SAZ were quite different. No differences were observed in the level of jasmonic acid (JA), but the contents of 12-oxo-phytodienoic acid (OPDA), a precursor of JA, and N-jasmonyl-leucine (JA-Leu) substantially decreased on the JA-Me side. Almost no effects of JA-Me on the dynamics of other plant hormones (cytokinins, abscisic acid, and gibberellins) were observed. Similar JA-Me effects on plant hormones and morphology were observed in the last internode of the decapitated growing plants. These suggest that the application of JA-Me induces the SAZ in the internode of B. calycinum by affecting endogenous levels of auxin- and jasmonate-related compounds.

Accumulation of Anthocyanins in Detached Leaves of Kalanchoë blossfeldiana: Relevance to the Effect of Methyl Jasmonate on This Process.

Accumulation of anthocyanins in detached leaves and in excised stems of Kalanchoë blossfeldiana kept under natural light conditions in the presence or absence of methyl jasmonate (JA-Me) was investigated. When the abaxial surface of detached leaves was held lower than the adaxial surface (the normal or natural position) under natural light conditions, anthocyanins were not accumulated on the abaxial side of the leaves. In contrast, when the adaxial surface of detached leaves was held lower than the abaxial surface (inverted position), anthocyanins were highly accumulated on the abaxial side of the leaves. These phenomena were independent of the growth stage of K. blossfeldiana as well as photoperiod. Application of JA-Me in lanolin paste significantly inhibited anthocyanin accumulation induced on the abaxial side of detached leaves held in an inverted position in a dose-dependent manner. Anthocyanin accumulation in the excised stem in response to natural light was also significantly inhibited by JA-Me in lanolin paste. Possible mechanisms of anthocyanin accumulation on the abaxial side of detached K. blossfeldiana leaves held in an inverted position under natural light conditions and the inhibitory effect of JA-Me on this process are described. The accompanying changes in the content of primary metabolites and histological analyses were also described.

Anatomy and Ultrastructure of Galls Induced by Neuroterus quercusbaccarum (Hymenoptera: Cynipidae) on Oak Leaves (Quercus robur).

The structure and ultrastructure of two developmental stages of the spangle gall induced by Neuroterus quercusbaccarum (Hymenoptera, Cynipidae) were investigated using light microscopy (LM), fluorescence microscopy (FM), and transmission (TEM) and scanning (SEM) electron microscopy. The general design of the gall structure was typical of Cynipidae, but some structural features distinguished the spangle gall. Previously undescribed, characteristic multicellular epidermal protuberances with large openings were observed in autumn on the surface of galls. These may facilitate the gas exchange between the atmosphere and the inside of the gall, thus assisting larval respiration. The larval chamber is surrounded by both a sclerenchymatous capsule and numerous cells containing calcium oxalate crystals that may both serve as protective barriers. In young galls, the nutritive tissue is a wall-less protoplasmic mass, potentially easily accessible to young larvae with delicate mandibles. Cell walls only develop at a later stage. The nutritive tissue was found to be rich in proteins and lipids, but starch grains were not observed. Cellular topology suggests that spangle galls grow by anticlinal division of marginal epidermal cells and periclinal division of subepidermal cells. Cellular proliferation (hyperplasia) also occurs in the leaf tissue near the connection with the gall peduncle, which eventually lignifies.

Mode of Action of 1-Naphthylphthalamic Acid in Conspicuous Local Stem Swelling of Succulent Plant, Bryophyllum calycinum: Relevance to the Aspects of Its Histological Observation and Comprehensive Analyses of Plant Hormones.

The mode of action of 1-naphthylphthalamic acid (NPA) to induce conspicuous local stem swelling in the area of its application to the growing internode in intact Bryophyllum calycinum was studied based on the aspects of histological observation and comprehensive analyses of plant hormones. Histological analyses revealed that NPA induced an increase in cell size and numerous cell divisions in the cortex and pith, respectively, compared to untreated stem. In the area of NPA application, vascular tissues had significantly wider cambial zones consisting of 5-6 cell layers, whereas phloem and xylem seemed not to be affected. This indicates that stem swelling in the area of NPA application is caused by stimulation of cell division and cell enlargement mainly in the cambial zone, cortex, and pith. Comprehensive analyses of plant hormones revealed that NPA substantially increased endogenous levels of indole-3-acetic acid (IAA) in the swelling area. NPA also increased endogenous levels of cytokinins, jasmonic acid, and its precursor, 12-oxo-phytodienoic acid, but did not increase abscisic acid and gibberellin levels. It was shown, using radiolabeled 14C-IAA, that NPA applied to the middle of internode segments had little effect on polar auxin transport, while 2,3,5-triiodobenzoic acid substantially inhibited it. These results strongly suggest that NPA induces changes in endogenous levels of plant hormones, such as IAA, cytokinins, and jasmonic acid, and their hormonal crosstalk results in a conspicuous local stem swelling. The possible different mode of action of NPA from other polar auxin transport inhibitors in succulent plants is extensively discussed.

Apple Autotetraploids with Enhanced Resistance to Apple Scab (Venturia inaequalis) Due to Genome Duplication-Phenotypic and Genetic Evaluation.

Among the fungal diseases of apple trees, serious yield losses are due to an apple scab caused by Venturia inaequalis. Protection against this disease is based mainly on chemical treatments, which are currently very limited. Therefore, it is extremely important to introduce cultivars with reduced susceptibility to this pathogen. One of the important sources of variability for breeding is the process of polyploidization. Newly obtained polyploids may acquire new features, including increased resistance to diseases. In our earlier studies, numerous tetraploids have been obtained for several apple cultivars with 'Free Redstar' tetraploids manifesting enhanced resistance to apple scab. In the present study, tetraploids of 'Free Redstar' were assessed in terms of phenotype and genotype with particular emphasis on the genetic background of their increased resistance to apple scab. Compared to diploid plants, tetraploids (own-rooted plants) were characterized with poor growth, especially during first growing season. They had considerably shorter shoots, fewer branches, smaller stem diameter, and reshaped leaves. In contrast to own-rooted plants, in M9-grafted three-year old trees, no significant differences between diplo- and tetraploids were observed, either in morphological or physiological parameters, with the exceptions of the increased leaf thickness and chlorophyll content recorded in tetraploids. Significant differences between sibling tetraploid clones were recorded, particularly in leaf shape and some physiological parameters. The amplified fragment length polymorphism (AFLP) analysis confirmed genetic polymorphism of tetraploid clones. Methylation-sensitive amplification polymorphism (MSAP) analysis showed that the level of DNA methylation was twice as high in young tetraploid plants as in a diploid donor tree, which may explain the weaker vigour of neotetraploids in the early period of their growth in the juvenile phase. Molecular analysis showed that 'Free Redstar' cultivar and their tetraploids bear six Rvi genes (Rvi5, Rvi6, Rvi8, Rvi11, Rvi14 and Rvi17). Transcriptome analysis confirmed enhanced resistance to apple scab of 'Free Redstar' tetraploids since the expression levels of genes related to resistance were strongly enhanced in tetraploids compared to their diploid counterparts.

Formation of the Secondary Abscission Zone Induced by the Interaction of Methyl Jasmonate and Auxin in Bryophyllum calycinum: Relevance to Auxin Status and Histology.

The interaction of methyl jasmonate (JA-Me) and indole-3-acetic acid (IAA) to induce the formation of the secondary abscission zone in the middle of internode segments of Bryophyllum calycinum was investigated in relation to auxin status and histology. When IAA at 0.1% (w/w, in lanolin) was applied to the segments, the formation of the secondary abscission zone at a few mm above the treatment in the apical direction was observed. On the contrary, IAA at 0.5% (w/w, in lanolin) did not induce the formation of the secondary abscission zone. JA-Me at 0.5% (w/w, in lanolin) applied to the middle of internode segments kept in the normal (natural) or inverted positions also induced the formation of the secondary abscission zone below and above parts of the treatment. IAA at 0.5% applied to the cut surface of the upper part of the segments completely prevented the formation of the secondary abscission zone induced by JA-Me. Simultaneous application of IAA 0.5% with JA-Me 0.5% in the middle part of the internode segments induced the formation of the secondary abscission zone at 10 mm to 12 mm above the treatment. Histological analyses indicated that the formation of the secondary abscission zone was characterized by the presence of newly synthesized cell plates that resulted from periclinal cell division within one layer of mother cells in stems. The effects of IAA (0.1%) and JA-Me (0.5%) on the formation of the secondary abscission zone were histologically similar. Comprehensive analyses of plant hormones revealed that the balance of the endogenous levels of IAA in both sides adjacent to the abscission zone was significantly disturbed when the secondary abscission formation was induced by the application of IAA. These results strongly suggest that an auxin gradient is important in the formation of the secondary abscission zone in the internode segments of B. calycinum, and IAA gradient results from polar IAA transport from the application site. IAA is important in the regulation of formation of the secondary abscission zone induced by JA-Me. Further possible mechanisms of the formation of the secondary abscission zone in the internode segments of B. calycinum are also discussed in the interaction of JA-Me and IAA.

Breeding of lilies and tulips-Interspecific hybridization and genetic background.

Lilies and tulips (Liliaceae family) are economically very important ornamental bulbous plants. Here, we summarize major breeding goals, the role of an integrated method of cut-style pollination and fertilization followed by embryo rescue and mitotic and meiotic polyploidization involved in new assortment development. Both crops have been subjected to extensive interspecific hybridization followed by selection. Additionally, spontaneous polyploidization has played a role in their evolution. In lilies, there is a tendency to replace diploids with polyploid cultivars, whereas in tulip a majority of the cultivars that exist today are still diploid except for triploid Darwin hybrid tulips. The introduction of molecular cytogenetic techniques such as genomic in situ hybridization (GISH) permitted the detailed studies of genome composition in lily and tulip interspecific hybrids and to follow the chromosome inheritance in interspecific crosses. In addition, this review presents the latest information on phylogenetic relationship in lily and tulip and recent developments in molecular mapping using different DNA molecular techniques.

An ultra-dense integrated linkage map for hexaploid chrysanthemum enables multi-allelic QTL analysis.

KEY MESSAGE: We constructed the first integrated genetic linkage map in a polysomic hexaploid. This enabled us to estimate inheritance of parental haplotypes in the offspring and detect multi-allelic QTL. Construction and use of linkage maps are challenging in hexaploids with polysomic inheritance. Full map integration requires calculations of recombination frequency between markers with complex segregation types. In addition, detection of QTL in hexaploids requires information on all six alleles at one locus for each individual. We describe a method that we used to construct a fully integrated linkage map for chrysanthemum (Chrysanthemum × morifolium, 2n = 6x = 54). A bi-parental F1 population of 406 individuals was genotyped with an 183,000 SNP genotyping array. The resulting linkage map consisted of 30,312 segregating SNP markers of all possible marker dosage types, representing nine chromosomal linkage groups and 107 out of 108 expected homologues. Synteny with lettuce (Lactuca sativa) showed local colinearity. Overall, it was high enough to number the chrysanthemum chromosomal linkage groups according to those in lettuce. We used the integrated and phased linkage map to reconstruct inheritance of parental haplotypes in the F1 population. Estimated probabilities for the parental haplotypes were used for multi-allelic QTL analyses on four traits with different underlying genetic architectures. This resulted in the identification of major QTL that were affected by multiple alleles having a differential effect on the phenotype. The presented linkage map sets a standard for future genetic mapping analyses in chrysanthemum and closely related species. Moreover, the described methods are a major step forward for linkage mapping and QTL analysis in hexaploids.

An assessment of chromosomal rearrangements in neopolyploids of Lilium hybrids.

Two types of newly induced polyploids (neopolyploids) of Lilium hybrids were monitored for the occurrence of chromosomal rearrangements through genomic in situ hybridization (GISH) technique. One of the populations was obtained through crossing an allotriploid Longiflorum x Oriental hybrid (LLO) with an allotetraploid Longiflorum x Trumpet hybrid (LLTT), both of which were derived from somatic chromosome doubling. The other type of allopolyploid population was derived from meiotic chromosome doubling in which numerically unreduced (2n) gametes from two different interspecific hybrids, namely, Longiflorum x Asiatic (LA) and Oriental x Asiatic (OA), were used to get backcross progeny with the Asiatic parents. GISH clearly discriminated the three constituent genomes (L, T, and O) in the complements of the progeny obtained from mitotic chromosome doubling. A total of 26 individuals were analyzed from this population and there was no evidence of chromosomal rearrangements. However, in the case of meiotically doubled allopolyploid progeny, considerable frequencies of chromosomal rearrangements were observed through GISH. The so-called chromosomal rearrangements in meiotic polyploids are the result of homoeologous recombination rather than translocations. Furthermore, evidence for the occurrence of meiotic recombination in the LA hybrids has been confirmed with GISH on meiotic chromosomes. Thus, there was evidence that neopolyploids of Lilium hybrids did not possess any noticeable chromosome rearrangements.