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Hairy root (HR) cultures are quickly evolving as a fundamental research tool and as a bio-based production system for secondary metabolites. In this study, an efficient protocol for establishment and elicitation of anthocyanin-producing HR cultures from black carrot was established. Taproot and hypocotyl explants of four carrot cultivars were transformed using wild-type Rhizobium rhizogenes. HR growth performance on plates was monitored to identify three fast-growing HR lines, two originating from root explants (lines NB-R and 43-R) and one from a hypocotyl explant (line 43-H). The HR biomass accumulated 25- to 30-fold in liquid media over a 4 week period. Nine anthocyanins and 24 hydroxycinnamic acid derivatives were identified and monitored using UPLC-PDA-TOF during HR growth. Adding ethephon, an ethylene-releasing compound, to the HR culture substantially increased the anthocyanin content by up to 82% in line 43-R and hydroxycinnamic acid concentrations by >20% in line NB-R. Moreover, the activities of superoxide dismutase and glutathione S-transferase increased in the HRs in response to ethephon, which could be related to the functionality and compartmentalization of anthocyanins. These findings present black carrot HR cultures as a platform for the in vitro production of anthocyanins and antioxidants, and provide new insight into the regulation of secondary metabolism in black carrot.

Theban Desert Road Survey II: The Rock Shrine of Paḥu, Gebel Akhenaton, and Other Rock Inscriptions from the Western Hinterland of Qamûla. By John Coleman Darnell. Yale Egyptological Publications, vol. 1. New Haven: Yale Egyptological Institute, 2013. Pp. xvii + 454, 246 plts. $150. [Distributed by ISD, Bristol, Conn.]

Compact plant growth is an economically important trait for many crops. In practice, compactness is frequently obtained by applying chemical plant growth regulators. In view of sustainable and environmental-friendly plant production, the search for viable alternatives is a priority for breeders. Co-cultivation and natural transformation using rhizogenic agrobacteria result in morphological alterations which together compose the Ri phenotype. This phenotype is known to exhibit a more compact plant habit, besides other features. In this review, we highlight the use of rhizogenic agrobacteria and the Ri phenotype with regard to sustainable plant production and plant breeding. An overview of described Ri lines and current breeding applications is presented. The potential of Ri lines as pre-breeding material is discussed from both a practical and legal point of view.

Anna Müller leidet seit ihrer Kindheit an Knick-Senkfüßen. 2018 kommt für den linken Fuß nur noch eine OP in Frage. Ein Jahr später empfehlen ihr die Ärzte, auch den rechten Fuß operieren zu lassen. Doch die Patientin zögert wegen ihrer vielen Nebenerkrankungen. Sie entscheidet sich für den konservativen Weg und arbeitet mit einer Physiotherapeutin, einer Orthopädietechnikerin und einer Tanzpädagogin.

There is increasing demand for natural food colourants that can substitute synthetic colours due to both legislative restrictions and consumer concerns. Black or purple carrots (Daucus carota ssp. sativus var. atrorubens) have strong bluish-purple colour due to the natural pigment anthocyanin, which is used as natural food colourant due to its high pH, light, and heat stability (labelled E163 in Europe). However, new production and storage strategies of black carrot are required in order to increase pigment yield and, therefore, to be competitive against synthetic colours. In the present study, black carrot ‘Deep Purple’ was used in field experiments conducted in the 2015 growing season in Denmark, to determine if foliar applications of ethephon, an ethylene realising compound, can improve carrot anthocyanin content and yield. Total monomeric anthocyanins were measured spectrophotometrically, and anthocyanin composition was analysed by LC-MS/Q-TOF approach. The results documented substantial increase in anthocyanin concentration upon ethephon treatment, whereas root size was not affected. Five major cyanidin-based anthocyanin forms were detected, being the acylated (i.e., more stable) anthocyanins clearly predominant. In addition, a correlation between enhanced anthocyanin contents and decreased root dry matter and soluble sugar contents was found. Taken together, these traits make elicited ‘Deep Purple’ carrots a suitable basis for obtaining natural colourants.

Indoor potted plants are able to remove volatile organic compounds (VOC) from air, but only few studies have investigated the removal of compounds in mixtures. Here, we present a non-targeted pixel-based fingerprinting analysis documenting the removal of a complex mixture of gasoline VOCs by Hedera helix under dynamic chamber conditions allowing for air exchange and continuous gasoline exposure. For 15 days, the entire potted plant was exposed to gasoline; subsequently, the epigeous plant parts were removed and the soil microcosm (i.e. soil, plant roots and microorganisms) was exposed to gasoline for another eight days. Quantitative analysis was performed for heptane, 3-methylhexane, toluene, ethylbenzene and m,p-xylenes, and the CHEMSIC method (CHEMometric analysis of Selected Ion Chromatograms) was used for non-targeted pixel-based fingerprinting analysis. The quantitative analysis demonstrated that the presence of potted plants or pots without epigeous plant parts led to a reduction of selected VOCs by 16.7-22.6%. The CHEMSIC method confirmed this and revealed that all gasoline VOCs were reduced in concentration when H. helix was present. The estimate for the total VOC removal was in the range of 11-32%. The removal was highest for samples where the epigeous plant parts were absent and compounds known to be hard to degrade by microorganisms such as dimethylcyclopentanes were removed the least compared to compounds more easily degraded by microorganisms such as heptane when epigeous plant parts were removed. All findings support the conclusion that the soil microcosm was the main responsible for the removal of VOCs.

Reduced growth habit is a desirable trait for ornamental potted plants and can successfully be obtained through Rhizobium rhizogenes transformation in a stable and heritable manner. Additionally, it can also be obtained by transformation with Agrobacterium tumefaciens harboring specific genes from R. rhizogenes. The bacterial T-DNA harbors four root oncogenic loci (rol) genes and 14 less known open reading frames (ORFs). The four rol genes, i.e., rolA, rolB, rolC, and rolD, are conceived as the common denominator for the compact phenotype and the other less characterized ORFs seem auxiliary but present a potential breeding target for less aberrant and/or more tailored phenotypes. In this study, Kalanchoë blossfeldiana ‘Molly’ was transformed with individual rol genes and selected ORFs in 35S overexpressing cassettes to comprehensively characterize growth traits, gene copy and expression, and ethylene tolerance of the flowers. An association of reduced growth habit, e.g. height and diameter, was observed for rolB2 and ORF14-2 when a transgene single copy and high gene expression were detected. Chlorophyll content was reduced in overexpressing lines compared to wild type (WT), except for one ΔORF13a (a truncated ORF13a, where SPXX DNA-binding motif is absent). The flower number severely decreased in the overexpressing lines compared to WT. The anthesis timing showed that WT opened the first flower at 68.9 ± 0.9 days and the overexpressing lines showed similar or up to 24 days delay in flowering. In general, a single or low relative gene copy insertion was correlated to higher gene expression, ca. 3 to 5-fold, in rolB and ΔORF13a lines, while in ORF14 such relation was not directly linked. The increased gene expression observed in rolB2 and ΔORF13a-2 contributed to reducing plant growth and a more compact habit. Tolerance of detached flowers to 0.5 μl L−1 ethylene was markedly higher for ORF14 with 66% less flower closure at day 3 compared to WT. The subcellular localization of rolC and ΔORF13a was investigated by transient expression in Nicotiana benthamiana and confocal images showed that rolC and ΔORF13a are soluble and localize in the cytoplasm being able to enter the nucleus.

Plant transformation with root oncogenic loci (rol) genes and open reading frames (ORFs) from Rhizobium rhizogenes have not yet targeted the underground root phenotype of these transformants. Hence, there is a need to develop plants with more efficient root system architecture (RSA). Here, RSA was assessed in naturally transformed (NT) and single rol/ORF Kalanchoë blossfeldiana 'Molly' lines in an aeroponic growth system combined with gene expression analysis. Three NT lines; 306, 324 and 331; exhibited better-developed RSA with longer roots and increased root biomass. In line 306, longest root was 6.3 ± 0.3 cm while WT had 4.8 ± 0.1 cm. However, root length of all overexpressing lines was ca. 30% shorter than WT. Root fresh weight of NT lines was 4.5-fold higher than WT. The expression of rolB, ∆ORF13a and ORF14 in the leaves of overexpressing lines was many folds higher than in NT lines. Increased expression of ∆ORF13a and ORF14 in leaves and roots may contribute more to a stronger compact phenotype than previously assumed. The moderate compact phenotype of NT lines combined with improved RSA compared to the overexpressing lines and WT strongly indicate that the use of R. rhizogenes has great potential to produce Kalanchoë phenotypes with enhanced RSA.

Reduced growth habit is a desirable trait for ornamental potted plants and can successfully be obtained through Rhizobium rhizogenes transformation in a stable and heritable manner. Additionally, it can also be obtained by transformation with Agrobacterium tumefaciens harboring specific genes from R. rhizogenes. The bacterial T-DNA harbors four root oncogenic loci (rol) genes and 14 less known open reading frames (ORFs). The four rol genes, i.e., rolA, rolB, rolC, and rolD, are conceived as the common denominator for the compact phenotype and the other less characterized ORFs seem auxiliary but present a potential breeding target for less aberrant and/or more tailored phenotypes. In this study, Kalanchoë blossfeldiana ‘Molly’ was transformed with individual rol genes and selected ORFs in 35S overexpressing cassettes to comprehensively characterize growth traits, gene copy and expression, and ethylene tolerance of the flowers. An association of reduced growth habit, e.g. height and diameter, was observed for rolB2 and ORF14-2 when a transgene single copy and high gene expression were detected. Chlorophyll content was reduced in overexpressing lines compared to wild type (WT), except for one ΔORF13a (a truncated ORF13a, where SPXX DNA-binding motif is absent). The flower number severely decreased in the overexpressing lines compared to WT. The anthesis timing showed that WT opened the first flower at 68.9 ± 0.9 days and the overexpressing lines showed similar or up to 24 days delay in flowering. In general, a single or low relative gene copy insertion was correlated to higher gene expression, ca. 3 to 5-fold, in rolB and ΔORF13a lines, while in ORF14 such relation was not directly linked. The increased gene expression observed in rolB2 and ΔORF13a-2 contributed to reducing plant growth and a more compact habit. Tolerance of detached flowers to 0.5 μl L−1 ethylene was markedly higher for ORF14 with 66% less flower closure at day 3 compared to WT. The subcellular localization of rolC and ΔORF13a was investigated by transient expression in Nicotiana benthamiana and confocal images showed that rolC and ΔORF13a are soluble and localize in the cytoplasm being able to enter the nucleus.

Rhizobium rhizogenes exploits horizontal gene transfer as part of its mechanism of pathogenesis. In this respect, genetic material, transfer DNA (T-DNA) from the bacterium, is transferred transiently to the host plant genome. However, plant species within Nicotiana, Linaria and Ipomea genera contain genomic traces of ancient bacterial T-DNA. To determine if ancient bacterial T-DNA is present in uncultivated plants of European genera within Scrophulariaceae, seeds of Linaria, Antirrhinum, Digitalis, and Veronica were analysed for the presence of one of the root oncogenic loci genes; rolC of R. rhizogenes. This study discloses remnants of ancient rolC haplotypes in several species of Linaria, Antirrhinum, Digitalis and Veronica. The distribution of plant species harbouring rolC sequences within each genus was not uniform. In total, 7 of the 16 investigated species were found to be positive for at least one of the rolC haplotypes, where 6 of these are new additions to the group of naturally transformed plants. Sequence alignment showed high interspecies homology of rolC. Five unique rolC haplotypes (rolCa-rolCe) were found in several plant species. The most abundant, rolCa, was identified in all the plant species holding ancient bacterial DNA. Transcripts of rolC were not detected in leaves, which indicates that rolC may not have a function in leaves under non-stress conditions. This study adds Antirrhinum, Digitalis and Veronica to the list of plant genera within Scrophulariaceae which have been subjected to ancient transformation events via horizontal gene transfer from bacteria to plants.

Kalanchoë is an economically important genus comprising numerous potted plants and recently is also emerging as cut flowers. However, the lack of information about flower-inducing factors limits the number of species that can be used in commercial production and breeding programs. Therefore, the aim of this study was to investigate the effects of exogenous application of gibberellic acid (GA3) on flower induction and flowering quality of Kalanchoë longiflora and Kalanchoë pinnata. The experiment was conducted under a short day (SD) photoperiod with a day temperature of 22 °C and a night temperature of 15 °C for 8 weeks. The treatments consisted of four applications of either 0.25 or 0.50 μg of GA3 per plant per week, providing a total of 100 μg or 200 μg/plant and 0 μg/plant for the control. The volume of 100 μL of GA3 solution containing 1% agarose was applied to the shoot apex using a pipette. For both species, flowering was enhanced by the GA3 treatments compared with the control plants. Gibberellin-treated plants flowered earlier, produced more inflorescences, and exhibited an increased number of flowers compared with the control plants. Moreover, the GA3 treatments in K. longiflora delayed the appearance of wilted flowers. Plant height increased in plants that received GA3, but the number of nodes did not differ from the control plants. Thus, we conclude that the application of GA3 improves flowering of Kalanchoë species and can be a useful tool for the production of cut flower cultivars.

Dried bodies of the female insect Dactylopius coccus Costa were extracted using various extraction solvents. Whereas the amount of extracted carminic acid was between 4 and 24 % of insect material, 4-aminocarminic acid was detected in the extracts in only in low amounts of about 0.1 % of insect material. Compared to ratios of 4-aminocarminic acid to carminic acid, which had been reported to be present in some food additive and beverage samples, ratios in the insect extracts were very low (between 1:211 and 1:154, w/w). However, mixing carminic acid and ammonium hydroxide resulted in formation of significant amounts of 4-aminocarminic acid. Depending on the molar ratio of carminic acid to ammonia, the ratio of 4-aminocarminic acid to carminic acid was up to 1:1.2 (w/w). Spontaneous formation of 4-aminocarminic acid leading to a false positive result was also observed for the analysis of a ketchup sample when ammonia was used during sample preparation.

Screening of strawberry anthocyanin composition and antioxidant capacities are important in the characterization and development of sources of natural colors. Here, Fragaria vesca (wild strawberry) and three commercial (cultivars ‘Elsanta’, ‘Korona’ and ‘Honeyoe’) and 14 non-commercial cultivars of Fragaria × ananassa Duch. were characterized in terms of anthocyanin content and composition, total phenolic content and antioxidant activities for two consecutive growing seasons. Twelve non-commercial strawberry cultivars had higher anthocyanin contents when compared to previous reports, varying between 1.13 and 1.89 g kg-1 fresh weight (fw) in total, with pelargonidin 3-O-glucoside as the major (50–90%) and cyanidin 3-O-glucoside as the second most abundant (1–47%) anthocyanins. Among the characterized cultivars, S 94 combined high antioxidant activities (4.6 mmol Trolox equivalents g-1 fw and 24.6 mmol Fe2+ g-1 fw on average for both years) and total anthocyanin (1.76 g kg-1 fw) and cyanidin 3-O-glucoside contents. Furthermore, the breeding potential of wild strawberry was highlighted as it had the highest antioxidant activity among tested cultivars and 44% of anthocyanins as cyanidin-3-O-glucoside. This study identified strawberry cultivars with traits desirable for colorants, which might guide future breeding programs for healthier strawberries.

Kalanchoe pinnata from the stonecrop family (Crassulaceae) is a medicinal plant with high content of bioactive compounds. The plant is known for its anti-allergic, antioxidant, anti-inflammatory, antimicrobial and antibacterial activities, which are mainly attributed to flavonoids. Low yields of bioactive compounds in medicinal plants have led to new strategies for enhancing their biosynthetic capacity in order to increase the content of specialized metabolites. Herein, the accumulation of specialized metabolites in plants can be triggered by elicitation methods. In the current study, different elicitation strategies were conducted towards the enhancement of bioactive compounds in K. pinnata leaves. We investigated the effect of natural transformation with the Agrobacterium rhizogenes strain A4 as means of biological elicitation on the total content of flavonoids in the leaves of K. pinnata. Furthermore, the effect of supplemental UV-B radiation, as physical elicitor, was assessed on the total flavonoid content of both wild-type (WT) and rol-transformed plants. The combined effect of the two mentioned elicitation methods was also examined. The data showed that presence of rol genes resulted in an increase of 24% in the total flavonoid content compared to WT plants. The supplemental UV-B radiation increased the total content of flavonoids with 95% and 89% in the WT and the rol+ plants, respectively. Collectively, a synergistic effect was shown as the combination of the two factors dramatically increased (133%) the total flavonoid content in K. pinnata leaves.

A high yield of isolated protoplasts and efficient regeneration protocols are prerequisites for successful development of somatic hybrids. In the present study, protoplast isolation and regeneration were evaluated in 12 Kalanchoe accessions belonging to nine species. The highest protoplast yield was obtained from K. blossfeldiana Charming Red Meadow' with 10.780.51x10(5) protoplasts per gram fresh weight. We observed significant differences of protoplast yield while there was no distinct difference in viability among the accessions. Seven accessions reached the microcolony stage and four developed microcalli in medium supplemented with 1.0mg/l 1-naphthaleneacetic acid (NAA), 0.5mg/l 6-benzylaminopurine (BAP) and 0.5mg/l 2,4-dichlorophenoxy acetic acid (2,4-D). Using five selected accessions we optimized the PGR (plant growth regulators) concentrations using combinations of NAA, BAP and 2,4-D. K. blossfeldiana cultivars Charming Red Meadow' and Paris' produced significantly different numbers of calli depending on the PGR concentrations. For plant regeneration, the medium was supplemented with 1mg/l NAA and 2mg/l BAP or 2mg/l zeatin. Shoots were regenerated on medium supplemented with NAA and BAP for K. blossfeldiana Charming Red Meadow' and K. blossfeldiana Paris'. The plants successfully developed roots on the medium supplemented with IAA. The medium containing zeatin induced root formation directly from callus in K. blossfeldiana Charming Red Meadow'. Our findings have the potential to facilitate the use of Kalanchoe species in somatic hybridization breeding programs. Key Message The study revealed a strong genotype-dependent efficiency of colony and microcallus formation. Plants were regenerated from two Kalanchoe blossfeldiana cultivars on medium supplemented with NAA and BAP.

Plant transformation with the wild type Ri plasmid T-DNA of Agrobacterium rhizogenes is a promising method for breeding of compact plants and has been the subject of numerous studies. However, knowledge concerning the isolated functions of single genes and ORFs from the plasmid is limited. The rolB and ORF13 oncogenes of A. rhizogenes show considerable promise in plant breeding, but have not been comprehensively studied. Detailed information regarding the morphological impact of specific genes of the Ri plasmid will allow for optimized targeted breeding of plants transformed with the wild type Ri plasmid T-DNA. rolB and ORF13 were recombined into the genome of Arabidopsis thaliana using Gateway® cloning and the effect on plant growth was assessed biometrically throughout the plants' life cycle. rolB-lines exhibited dwarfing, early necrosis of rosette leaves, altered leaf and flower morphology, and developed an increased number of inflorescences per rosette area compared to the wild type. ORF13-lines were extremely dwarfed, attaining only ca. 1% of the rosette area of the wild type, leaf and flower size was reduced, and the shape modified. The study documents that the traits inferred by the rolB oncogene yield plants with increased formation of generative shoots, but also result in some degree of premature senescence of vegetative organs. The extreme dwarfism seen in ORF13-lines indicate that this oncogene may be more important in the dwarfing response of plants transformed with the wild type Ri plasmid T-DNA than previously assumed and that transformation with this oncogene induces a very compact phenotype.

Rose mallows belong to the Muenchhusia section of the Hibiscus genus. They represent a small group of cold tolerant North American plants and are popular ornamentals mainly because of their abundant, large and colorful flowers. Due to their geographical origin they are well suited for garden use in temperate regions worldwide. The aim of the study was to investigate hybridization barriers in crosses among cultivars of Hibiscus species from the Muenchhusia section: H. coccineus, H. laevis and H. moscheutos. Crossing barriers were identified as both pre- and post-zygotic. The analysis of pollen tube growth revealed inhibition of pollen tubes and their abnormal growth. In specific crosses the fertilization success was low. The pre-fertilization barriers did not cause a complete reproductive isolation between the hybridization partners. In relation to post-fertilization barriers, the occurrence of hybrid incompatibilities such as unviability, chlorosis, necrosis, stunted growth and albinism were the main drawback in production of hybrids. The appearance of symptoms of hybrid incompatibilities was dependent upon specific parental plants. The obtained progeny had intermediate leaf morphology and flower morphology compared to parental plants. Hybridity state was verified by morphological analysis and RAPD markers. Based on the overall plant morphology, 472 hybrid progenies were obtained.

Plants have been used decoratively indoors for centuries. For the last 25-30 years, their beneficial abilities to reduce the levels of harmful volatile organic compounds (VOCs) from the indoor air have also been investigated. Previous studies have shown that VOCs are removed by the plant itself, but also by microorganisms in the soil. Furthermore, the rate of removal is dependent on the plant species and can be influenced by exogenous factors such as light intensity and VOC concentration. The research within this field is, however, limited by the fact that the knowledge gained from laboratory experiments is not directly transferrable to real life settings. The largest problem is the use of closed chambers where there is no air exchange. This also results in a declining VOC concentration over time. Due to this limitation, we constructed a new experimental system which among others can allow for air exchange and a constant VOC concentration. With the new system it was found that removal rates obtained in chambers with air exchange and constant VOC concentration were significantly higher than removal rates obtained in closed chambers. This means that removal rates obtained in closed chambers may be an underestimation of the plants’ real potential. The next step will be to use the new system to investigate the effects of the exogenous factors temperature, light intensity and CO2 concentration on VOC removal rates.