New insights into the genetic manipulation of the R2R3-MYB and CHI gene families on anthocyanin pigmentation in Petunia hybrida.

Several R2R3-MYB genes control anthocyanin pigmentation in petunia, and ANTHOCYANIN-2 (AN2) is treated as the main player in petal limbs. However, the actual roles of R2R3-MYBs in the coloration of different floral tissues in the so called "darkly-veined" petunias are still not clear. The genetic background and expression of AN2 paralogs from various petunias with different color patterns were identified. All "darkly-veined" genotypes have the identical mutation in the AN2 gene, but express a different functional paralog - ANTHOCYANIN-4 (AN4) - abundantly in flowers. Constitutive overexpression of PhAN4 in this petunia resulted not only in a fully colored flower but also in a clearly visible pigmentation in the green tissue and roots, which can be rapidly increased by stress conditions. Suppression of AN4 gene resulted in discolored petals and whitish anthers. Interestingly, when a similar white flower phenotype was achieved by knockout of an essential structural gene of anthocyanin biosynthesis - CHALCONE ISOMERASE-A (CHI-A) - the plant responded directly by upregulating of another paralogs - DEEP PURPLE (DPL) and PURPLE HAZE (PHZ). Moreover, we also found that CHI-B can partially substitute for CHI-A in anthers, but not in vegetative tissues. Further, no significant effects on the longevity of white or enhanced colored flowers were observed compared with the wild type. We concluded that endogenous up-regulation of AN4 leads to the restoration of petal color in the "darkly-veined" phenotypes as a result of the breeding process under human selection, and CHI-B is a backup for CHI-A acitvity in some floral tissues.

Mechanical Stimulation Decreases Auxin and Gibberellic Acid Synthesis but Does Not Affect Auxin Transport in Axillary Buds; It Also Stimulates Peroxidase Activity in Petunia × atkinsiana.

Thigmomorphogenesis (or mechanical stimulation-MS) is a term created by Jaffe and means plant response to natural stimuli such as the blow of the wind, strong rain, or touch, resulting in a decrease in length and an increase of branching as well as an increase in the activity of axillary buds. MS is very well known in plant morphology, but physiological processes controlling plant growth are not well discovered yet. In the current study, we tried to find an answer to the question if MS truly may affect auxin synthesis or transport in the early stage of plant growth, and which physiological factors may be responsible for growth arrest in petunia. According to the results of current research, we noticed that MS affects plant growth but does not block auxin transport from the apical bud. MS arrests IAA and GA3 synthesis in MS-treated plants over the longer term. The main factor responsible for the thickening of cell walls and the same strengthening of vascular tissues and growth arrestment, in this case, is peroxidase (POX) activity, but special attention should be also paid to AGPs as signaling molecules which also are directly involved in growth regulation as well as in cell wall modifications.

Physiological changes besides the enhancement of pigmentation in Petunia hybrida caused by overexpression of PhAN2, an R2R3-MYB transcription factor.

KEY MESSAGE: Ectopic expression of PhAN2 in vegetative tissue can improve regeneration and adventitious rooting but inhibit axillary bud outgrowth of petunia, while overexpression specifically in flowers could shorten longevity. Anthocyanin 2 has been only treated as a critical positive regulation factor of anthocyanin biosynthesis in petunia flowers. To determine if this gene had other functions in plant growth, we overexpressed this gene in an an2 mutant petunia cultivar driven by promoters with different strengths or tissue specificity. Various physiological processes of transformants in different growth stages and environments were analyzed. Besides the expected pigmentation improvement in different tissues, the results also showed that ectopic expression of AN2 could improve the regeneration skill but inhibit the axillary bud germination of in vitro plants. Moreover, the rooting ability of shoot tips of transformants was significantly improved, while some transgenic lines' flower longevity was shortened. Gene expression analysis showed that the transcripts level of AN2, partner genes anthocyanin 1 (AN1), anthocyanin 11 (AN11), and target gene dihydroflavonol 4-reductase (DFR) was altered in the different transgenic lines. In addition, ethylene biosynthesis-related genes 1-aminocyclopropane-1-carboxylic acid synthase (ACS1) and ACC oxidase (ACO1) were upregulated in rooting and flower senescence processes but at different time points. Overall, our data demonstrate that the critical role of this AN2 gene in plant growth physiology may extend beyond that of a single activator of anthocyanin biosynthesis.

Blue Light Improves Vase Life of Carnation Cut Flowers Through Its Effect on the Antioxidant Defense System.

Improving marketability and extension of vase life of cut flowers has practical significance for the development of the cut flower industry. Although considerable efforts have been made over many years to improve the vase life of cut flowers through controlling the immediate environment and through post-harvest use of floral preservatives, the impact of lighting environment on vase life has been largely overlooked. In the current study, the effect of three LED light spectra [white (400-730 nm), blue (peak at 460 nm), and red (peak at 660 nm)] at 150 µmol m-2 s-1 on vase life and on physiological and biochemical characteristics of carnation cut flowers was investigated. Exposure to blue light (BL) considerably delayed senescence and improved vase life over that of flowers exposed to red light (RL) and white light (WL). H2O2 and malondialdehyde (MDA) contents in petals gradually increased during vase life; the increase was lowest in BL-exposed flowers. As a consequence, BL-exposed flowers maintained a higher membrane stability index (MSI) compared to RL- and WL-exposed flowers. A higher activity of antioxidant enzymes [superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)] was detected in petals of BL-exposed flowers, compared to their activities in RL- and WL-exposed flowers. In BL-exposed flowers, the decline in petal carotenoid contents was delayed in comparison to RL- and WL-exposed flowers. Maximum quantum efficiency of photosystem II (Fv/Fm) and a higher percentage of open stomata were observed in leaves of BL-exposed flowers. Sucrose and glucose contents accumulated in petals during vase life; sugar concentrations were higher in BL-exposed flowers than in RL- and WL-exposed flowers. It is concluded that BL exposure improves the vase life of carnation cut flowers through its effect on the antioxidant defense system in petals and on photosynthetic performance in the leaves.

Blue light postpones senescence of carnation flowers through regulation of ethylene and abscisic acid pathway-related genes.

Endogenous signals in response to exogenous factors determine the senescence of flowers. Interactions among phytohormones especially abscisic acid (ABA) and ethylene are the major determinant of the senescence. In the present study, complex expression patterns of the genes related to ABA and ethylene as endogenous signals were investigated on cut carnations (Dianthus caryophyllus L.) that were exposed to different light spectra. Expression of ethylene biosynthetic (DcACS and DcACO), and signaling (DcETR and DcEin2) genes and also genes involved in ABA biosynthesis (DcZEP1 and DcNCED1), transport (DcABCG25 and DcABCG40) and catabolism (DcCYP707A1) were evaluated in petals of carnations exposed to three light spectra [white, blue and red]. Lowest relative membrane permeability (RMP) was detected in flowers that exposed to Blue light (BLFs), as a consequence, the longest vase life was found in BLFs. The Red and White lights markedly accelerated flower senescence and increased expression of DcACS and DcACO on day 6 and 10 of vase life assessment respectively; while Blue light inhibited the expression of ethylene biosynthetic genes. Expression of the genes involved in the production and transport of ABA and in signal transduction of ethylene was elevated during vase life of flowers irrespective of exposure to different light spectra. In conclusion, Blue light can be an effective environmental factor to extend the vase life of carnation flowers by delaying the petal senescence through down-regulation of ethylene biosynthetic genes and up-regulation of ABA biosynthetic genes.

Manipulation of MKS1 gene expression affects Kalanchoë blossfeldiana and Petunia hybrida phenotypes.

SUMMARY: The establishment of alternative methods to chemical treatments for growth retardation and pathogen protection in ornamental plant production has become a major goal in recent breeding programmes. This study evaluates the effect of manipulating MAP kinase 4 nuclear substrate 1 (MKS1) expression in Kalanchoë blossfeldiana and Petunia hybrida. The Arabidopsis thaliana MKS1 gene was overexpressed in both species via Agrobacterium-mediated transformation, resulting in dwarfed phenotypes and delayed flowering in both species and increased tolerance to Pseudomonas syringae pv. tomato in transgenic Petunia plants. The lengths of the stems and internodes were decreased, while the number of nodes in the transgenic plants was similar to that of the control plants in both species. The transgenic Kalanchoë flowers had an increased anthocyanin concentration, and the length of the inflorescence stem was decreased. The morphology of transgenic Petunia flowers was not altered. The results of the Pseudomonas syringae tolerance test showed that Petunia plants with one copy of the transgene reacted similarly to the nontransgenic control plants; however, plants with four copies of the transgene exhibited considerably higher tolerance to bacterial attack. Transgene integration and expression was determined by Southern blot hybridization and RT-PCR analyses. MKS1 in wild-type Petunia plants was down-regulated through a virus-induced gene silencing (VIGS) method using tobacco rattle virus vectors. There were no significant phenotypic differences between the plants with silenced MKS1 genes and the controls. The relative concentration of the MKS1 transcript in VIGS-treated plants was estimated by quantitative RT-PCR.

Transformation of miniature potted rose (Rosa hybrida cv. Linda) with P( SAG12 )-ipt gene delays leaf senescence and enhances resistance to exogenous ethylene.

KEY MESSAGE : The P ( SAG12 ) -ipt gene was transferred to miniature rose, as the first woody species, resulting in increased ethylene resistance due to specific up-regulation of the ipt gene under senescence promoting conditions. Transgenic plants of Rosa hybrida 'Linda' were obtained via transformation with Agrobacterium tumefaciens strain harboring the binary vector pSG529(+) containing the P( SAG12 )-ipt construct. A. tumefaciens strains AGL1, GV3850 and LBA4404 (containing P(35S)-INTGUS gene) were used for transformation of embryogenic callus, but transgenic shoots were obtained only when AGL1 was applied. The highest transformation frequency was 10 % and it was achieved when half MS medium was used for the dilution of overnight culture of Agrobacterium. Southern blot confirmed integration of 1-6 copies of the nptII gene into the rose genome in the tested lines. Four transgenic lines were obtained which were morphologically true-to-type and indistinguishable from Wt shoots while they were in in vitro cultures. Adventitious root induction was more difficult in transgenic shoots compared to the Wt shoots, however, one of the transgenic lines (line 6) was rooted and subsequently analyzed phenotypically. The ipt expression levels were determined in this line after exposure to exogenous ethylene (3.5 µl l(-1)) and/or darkness. Darkness resulted in twofold up-regulation of ipt expression, whereas darkness combined with ethylene caused eightfold up-regulation in line 6 compared to Wt plants. The transgenic line had significantly higher content of chlorophyll at the end of the treatment period compared to Wt plants.

Localisation of abundant and organ-specific genes expressed in Rosa hybrida leaves and flower buds by direct in situ RT-PCR.

In situ PCR is a technique that allows specific nucleic acid sequences to be detected in individual cells and tissues. In situ PCR and IS-RT-PCR are elegant techniques that can increase both sensitivity and throughput, but they are, at best, only semiquantitative; therefore, it is desirable first to ascertain the expression pattern by conventional means to establish the suitable conditions for each probe. In plants, in situ RT-PCR is widely used in the expression localisation of specific genes, including MADS-box and other function-specific genes or housekeeping genes in floral buds and other organs. This method is especially useful in small organs or during early developmental stages when the separation of particular parts is impossible. In this paper, we compared three different labelling and immunodetection methods by using in situ RT-PCR in Rosa hybrida flower buds and leaves. As target genes, we used the abundant ß-actin and RhFUL gene, which is expressed only in the leaves and petals/sepals of flower buds. We used digoxygenin-11-dUTP, biotin-11-dUTP, and fluorescein-12-dUTP-labelled nucleotides and antidig-AP/ streptavidin-fluorescein-labelled antibodies. All of the used methods gave strong, specific signal and all of them may be used in localization of gene expression on tissue level in rose organs.

Interspecific somatic hybrids between Cyclamen persicum and C. coum, two sexually incompatible species.

By applying polyethylene glycol (PEG)-mediated protoplast fusion, the first somatic hybrids were obtained between Cyclamen persicum (2n = 2x = 48) and C. coum (2n = 2x = 30)-two species that cannot be combined by cross breeding. Heterofusion was detected by double fluorescent staining with fluorescein diacetate and scopoletin. The highest heterofusion frequencies (of about 5%) resulted from a protocol using a protoplast density of 1 × 10(6)/mL and 40% PEG. The DNA content of C. coum was estimated for the first time by propidium iodide staining to be 14.7 pg/2C and was 4.6 times higher than that of C. persicum. Among 200 in vitro plantlets regenerated from fusion experiments, most resembled the C. coum parent, whereas only 5 plants showed typical C. persicum phenotypes and 46 had a deviating morphology. By flow cytometry, six putative somatic hybrids were identified. A species-specific DNA marker was developed based on the sequence of the 5.8S gene in the ribosomal nuclear DNA and its flanking internal transcribed spacers ITS1 and ITS2. The hybrid status of only one plant could be verified by the species-specific DNA marker as well as sequencing of the amplification product. RAPD markers turned out to be less informative and applicable for hybrid identification, as no clear additivity of the parental marker bands was observed. Chromosome counting in root tips of four hybrids revealed the presence of the 30 C. coum chromosomes and 2-41 additional ones indicating elimination of C. persicum chromosomes.

Regeneration and transformation in adult plants of Campanula species.

Adult plants are known for recalcitrance when it comes to adventitious organ formation and regeneration. Methods used for regeneration in explants from seedlings of Campanula carpatica failed to work for explants from adult plants of the same species. The present investigation generated efficient regeneration methods for mature specimens of four species of Campanula, C. carpatica, C. haylodgensis, C. portenschlagiana and C. poscharskyana. Petiole explants from dark-grown in vitro shoot cultures grown from nodal cuttings of adult plants regenerated successfully (95%), while explants from light-grown in vitro shoot cultures and greenhouse-grown plants regenerated at 12% and zero percentage, respectively. Dark-treatment, along with media manipulation with plant growth regulators, further enhanced regenerative capacity of the explants. A MS-based medium containing 10mg l (-1) TDZ and 0.25 mg l(-1) NAA was the most efficient regeneration medium. Transgenic shoots from C. carpatica (3%) and C. haylodgensis (1%) and transgenic callus from all species were produced using Agrobacterium tumefaciens, and transformation was confirmed by histochemical and Southern blot analyses. Protocols developed in this study may be useful for achieving efficient regeneration and transformation of recalcitrant adult plants.

Transformation of Kalanchoe blossfeldiana with rol-genes is useful in molecular breeding towards compact growth.

Dwarf genotypes of the economically important flowering potted plant Kalanchoe blossfeldiana were developed by molecular breeding. Root inducing (Ri)-lines were regenerated by applying CPPU to the hairy roots, which were produced by inoculating leaf explants with a wild-type Agrobacterium rhizogenes strain ATCC15834. Amplification by polymerase chain reaction (PCR) and Southern blot analysis confirmed the presence of T-DNA in the Ri-lines. Six Ri-lines were characterised in a greenhouse trial revealing that several morphological traits changed with respect to ornamental value such as plant height, number of lateral shoots, leaf size, leaf number, flower size and number of flowers. The Ri-lines differed in their degree of Ri-phenotype, and the internodes of the Ri-lines were clearly shorter, giving a compact growth habit compared to control plants. Time to anthesis was the same in Ri-line 331 as in control plants and delayed by only 3 days in Ri-line 306 as compared to control plants. A compact plant without delayed flowering can be assumed to be valuable for further breeding.

Kalanchoe blossfeldiana plants expressing the Arabidopsis etr1-1 allele show reduced ethylene sensitivity.

Transgenic Kalanchoe blossfeldiana Poelln. with reduced ethylene sensitivity in flowers was obtained by Agrobacterium tumefaciens-mediated transformation using the plasmid pBEO210 containing the mutant ethylene receptor gene etr1-1 from Arabidopsis thaliana under the control of the flower-specific fbp1-promoter from Petunia. Three ethylene-resistent T0 lines, 300, 324 and 331, were selected and analyzed for postharvest-performance and morphological characteristics. Line 324 was found to be infertile and only slightly less ethylene-sensitive than control-plants, but lines 300 and 331 had significantly increased ethylene-resistance and were fertile. These two lines were analyzed for copy-number of the etr1-1 gene by Southern blotting and were crossed with the ethylene-sensitive cultivar 'Celine' to create T1 progeny. Line 300 contains two T-DNA copies per nucleus, one of which is rearranged, and these are unlinked according to segregation data from the crossing to 'Celine' and PCR-analysis of progeny plants. For control plants all flowers were closed after 2 days at 2 microl l(-1 )ethylene, but for line 300 only 33% were closed after 10 days. Line 331 contains three T-DNA copies per nucleus and is more sensitive to ethylene than line 300. In the line 300 the etr1-1 gene was found by RT-PCR to be expressed in petals and stamens but not in carpels and sepals. Both lines 300 and 331, and their progeny, appear morphologically and physiologically identical to control plants except for the higher ethylene resistance. Line 300 and its progeny with only one T-DNA copy have very low ethylene sensitivity and may be useful in future breeding.

Transgenic Campanula carpatica plants with reduced ethylene sensitivity.

Fertile transgenic Campanula carpatica Jacq. plants with flowers, which had reduced sensitivity to ethylene were obtained by Agrobacterium tumefaciens that mediated transformation. The construct used for transformation contained the etr1-1 gene from Arabidopsis thaliana under control of the flower specific fbp1-promoter from petunia. More than 100 flowering T0 lines were tested for their ethylene sensitivity using 2 microl l(-1) ethylene. The tolerance level to ethylene varied among the lines. While control plants stopped flowering within 3 days of exposure to ethylene, one of the transformed lines flowered for up to 27 days. The presence and the expression pattern of the transgene in various tissues were studied by polymerase chain reaction (PCR) and reverse transcription (RT)-PCR techniques. The expression of etr1-1 was significant in flowers and buds. Transgenic lines did not differ morphologically from control plants. The selected transgenic T0 lines, which were re-established from in vitro cultures showed the same degree of tolerance to exogenous ethylene, confirming the stability of the transgene in in vitro cultures. The rooting ability of the transgenic plants was not affected by the presence of etr1-1. T1 progeny were produced by crossing the transgenic line, which showed the most significant reduction in ethylene sensitivity with a control plant, and the analysis of the T1 plants showed 1:1 segregation in terms of ethylene sensitivity and the presence of the transgene.

Proteomic analyses of somatic and zygotic embryos of Cyclamen persicum Mill. reveal new insights into seed and germination physiology.

In the horticulturally important ornamental species Cyclamen persicum Mill., somatic embryogenesis is an efficient vegetative propagation method and the development of artificial seeds is an ultimate aim. This study aims at a systematic comparison of the proteomes of zygotic embryos, somatic embryos grown in liquid medium containing 30 or 60 g l(-1) sucrose, germinating embryos of both types and endosperm in order to obtain novel insights into seed and germination physiology. Using high resolution two-dimensional isoelectric focussing/sodium dodecylsulfate polyacrylamide gel electrophoresis (2D IEF/SDS PAGE), 74% of the proteins expressed in zygotic embryos were found in similar abundance in somatic embryos grown in 60 g l(-1) sucrose. Somatic embryos grown in 30 g l(-1) sucrose accumulated fewer protein species than those grown in 60 g l(-1). Selected proteins were identified following mass spectrometry (nano-LC-MS/MS). Four enzymes involved in glycolysis (UDP-glucose pyrophosphorylase, fructose bisphosphate aldolase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase GAPDH) were specifically induced in somatic embryos. 11S globulin proteins identified by MS were present in high levels in somatic embryos, zygotic embryos and endosperm, whereas 7S globulins were detected mainly in endosperm and zygotic embryos. These are the first storage proteins identified in C. persicum. Xyloglucans are known to be another group of seed storage compounds in C. persicum. Interestingly, xyloglucan endotransglycosylases were found to be highly expressed in endosperm tissue. We discuss the physiological implications of these observations.

Cryopreservation of embryogenic suspension cultures of Cyclamen persicum mill.

We have developed a simple protocol for the cryopreservation of embryogenic suspension cultures of Cyclamen persicum. Embryogenic suspension cultures in the linear growth phase 7-10 days after subculture were used for cryopreservation. Of the different cryoprotectants tested during a 2-day pre-culture, 0.6 M sucrose resulted in the highest re-growth rates of 75%. An additional pre-treatment with 0.6 M sucrose and 10% DMSO (dimethylsulfoxide) for 1 h also positively affected re-growth. Microscopic studies on viability revealed that only few small embryogenic cells survived cryopreservation, while vacuolated single cells died. Experiments in which the duration of the pre-culture period--i.e. the length of time the embryogenic suspension cells were exposed to 0.6 M sucrose--was varied showed that 2-4 days was the most optimal exposure time to 0.6 M sucrose. Callus re-grown after cryopreservation showed growth rates similar to that of unfrozen callus and regenerated even higher numbers of somatic embryos than unfrozen callus.

Agrobacterium tumefaciens-mediated transformation of Campanula carpatica: factors affecting transformation and regeneration of transgenic shoots.

An efficient transformation system for Campanula carpatica was developed using Agrobacterium tumefaciens strains LBA4404 (harbouring the plasmid pBI121), and AGL0 (harbouring the plasmid pBEO210). This is the first report on the transformation of C. carpatica. Various factors affecting the transformation efficiency and subsequent regeneration were identified. The age of seedlings from which the explants for transformation studies were taken, and the growth conditions under which the seedlings were grown had a significant influence on the production of transformed shoots. Hypocotyls taken from 12-day-old seedlings grown in the dark were the most productive, with up to 25% of hypocotyls producing transformed shoots. Explants taken from 5-week-old seedlings produced only transformed callus. The medium used for co-cultivation and incubation also had a significant influence on transformation frequency and shoot regeneration. The cultivar "Blue Uniform" was more responsive than "White Uniform". Both bacterial strains and plasmids were equally effective in producing transformed tissue. Transformed shoots were selected on kanamycin medium, and the presence of the uidA and nptII genes in those selected shoots was confirmed by beta-glucuronidase and ELISA analyses, respectively.