Integrating the Roles for Cytokinin and Auxin in De Novo Shoot Organogenesis: From Hormone Uptake to Signaling Outputs.

De novo shoot organogenesis (DNSO) is a procedure commonly used for the in vitro regeneration of shoots from a variety of plant tissues. Shoot regeneration occurs on nutrient media supplemented with the plant hormones cytokinin (CK) and auxin, which play essential roles in this process, and genes involved in their signaling cascades act as master regulators of the different phases of shoot regeneration. In the last 20 years, the genetic regulation of DNSO has been characterized in detail. However, as of today, the CK and auxin signaling events associated with shoot regeneration are often interpreted as a consequence of these hormones simply being present in the regeneration media, whereas the roles for their prior uptake and transport into the cultivated plant tissues are generally overlooked. Additionally, sucrose, commonly added to the regeneration media as a carbon source, plays a signaling role and has been recently shown to interact with CK and auxin and to affect the efficiency of shoot regeneration. In this review, we provide an integrative interpretation of the roles for CK and auxin in the process of DNSO, adding emphasis on their uptake from the regeneration media and their interaction with sucrose present in the media to their complex signaling outputs that mediate shoot regeneration.

Extraordinary Adaptive Plasticity of Colorado Potato Beetle: "Ten-Striped Spearman" in the Era of Biotechnological Warfare.

Expanding from remote areas of Mexico to a worldwide scale, the ten-striped insect, the Colorado potato beetle (CPB, Leptinotarsa decemlineata Say), has risen from being an innocuous beetle to a prominent global pest. A diverse life cycle, phenotypic plasticity, adaptation to adverse conditions, and capability to detoxify or tolerate toxins make this insect appear to be virtually "indestructible". With increasing advances in molecular biology, tools of biotechnological warfare were deployed to combat CPB. In the last three decades, genetically modified potato has created a new challenge for the beetle. After reviewing hundreds of scientific papers dealing with CPB control, it became clear that even biotechnological means of control, if used alone, would not defeat the Colorado potato beetle. This control measure once again appears to be provoking the potato beetle to exhibit its remarkable adaptability. Nonetheless, the potential for adaptation to these techniques has increased our knowledge of this pest and thus opened possibilities for devising more sustainable CPB management programs.

Growth and development of Colorado potato beetle larvae, Leptinotarsa decemlineata, on potato plants expressing the oryzacystatin II proteinase inhibitor.

Plant proteinase inhibitors (PIs) are attractive tools for crop improvement and their heterologous expression can enhance insect resistance in transgenic plants. PI oryzacystatin II (OCII), isolated from rice, showed potential in controlling pests that utilize cysteine proteinases for protein digestion. To evaluate the applicability of the OCII gene in enhancing plant defence, OCII-transformed potatoes were bioassayed for resistance to Colorado potato beetle (Leptinotarsa decemlineata Say). Feeding on transformed leaves of potato cultivars Desiree and Jelica significantly affected larval growth and development, but did not change mortality rates. During the L2 and L3 developmental stages larvae consumed the OCII-transformed foliage faster as compared to the nontransformed control. Also these larvae reached the prepupal stage (end of L4 stage) 2 days earlier than those fed on control leaves. However, the total amounts of consumed OCII-transformed leaves were up to 23% lower than of control, and the maximal weights of prepupal larvae were reduced by up to 18% as compared to larvae fed on nontransformed leaves. The reduction in insect fitness reported in this study in combination with other control measures, could lead to improved CPB resistance management in potato.

Cytokinin deficiency confers enhanced tolerance to mild, but decreased tolerance to severe salinity stress in in vitro grown potato.

Cytokinin (CK) is a plant hormone that plays crucial roles in regulating plant growth and development. CK-deficient plants are widely used as model systems for investigating the numerous physiological roles of CK. Since it was previously shown that transgenic or mutant CK-deficient Arabidopsis and Centaurium plants show superior tolerance to salinity, we examined the tolerance of three CK-deficient potato lines overexpressing the Arabidopsis thaliana CYTOKININ OXIDASE/DEHYDROGENASE2 (AtCKX2) gene to 50 mM, 100 mM, 150 mM, and 200 mM NaCl applied in vitro. Quantification of visible salinity injury, rooting and acclimatization efficiency, shoot growth, water saturation deficit, and chlorophyll content confirmed that the CK-deficient potato plants were more tolerant to low (50 mM) and moderate (100 mM) NaCl concentrations, but exhibited increased sensitivity to severe salinity stress (150 and 200 mM NaCl) compared to non-transformed control plants. These findings were corroborated by the data distribution patterns according to principal component analysis. Quantification of the activity of superoxide dismutases, peroxidases, and catalases revealed an impaired ability of AtCKX2-transgenic lines to upregulate the activity of antioxidant enzymes in response to salinity, which might contribute to the enhanced sensitivity of these potato lines to severe salt stress. Our results add complexity to the existing knowledge on the regulation of salinity tolerance by CK, as we show for the first time that CK-deficient plants can exhibit reduced rather than increased tolerance to severe salt stress.

UHPLC-OrbiTrap MS Characterization of Phenolic Profiles in French Marigold Extracts and Analysis of Their Antifeedant Activity against Colorado Potato Beetle.

French marigold is an aromatic plant rich in polyphenolic secondary metabolites, which pesticidal potential was examined in this study. Ultra-high-performance liquid chromatography (UHPLC) connected with OrbiTrap mass spectrometer (MS) identified 113 phenolics and revealed the most detailed phytochemistry of French marigold published so far. Depending on plant material (flowers or leaves) and solvents used for extraction (water, methanol, dichloromethane), the phenolic composition varied. Methanol extract of flowers, with 89 identified phenolics and high antioxidant activity statistically comparable with positive control Trolox, was chosen for testing of antifeedant potential against the 3rd and 4th instars of Colorado potato beetle (CPB). A significant reduction in final body mass of 4th larval stage fed with potato leaves coated with methanol extract of flowers in the concentration of 10 mg/mL was observed (157.67 mg vs. 182.26 mg of controls fed with non-treated leaves). This caused delayed molting since treated larvae reached the maximal mass a day after controls and this delay persisted during the entire larval development. Continuous feeding caused a 25% decline in digestive proteolytic activity of the 4th instar in comparison to controls. The results suggest that French marigold methanol extract of flowers could be proposed as a promising antifeedant for CPB management, with an impact on the reduction in the environmental footprint associated with synthetic pesticide application.

Transcriptome Profiling of the Potato Exposed to French Marigold Essential Oil with a Special Emphasis on Leaf Starch Metabolism and Defense against Colorado Potato Beetle.

Flower strips of French Marigold are commonly used pest repellents in potato fields. However, the effect of French Marigold volatiles on potato metabolism, physiology and induced defense is unknown. Thus, a microarray transcriptome analysis was performed to study the effects of French Marigold essential oil (EO) on laboratory-grown potato. After 8 h of exposure to EO, with gas chromatography/mass spectrometry (GC/MS)-detected terpinolene and limonene as dominant compounds, 2796 transcripts were differentially expressed with fold change >2 compared to expression in controls. A slightly higher number of transcripts had suppressed expression (1493 down- vs. 1303 up-regulated). Since transcripts, annotated to different photosynthesis-related processes, were mostly down-regulated, we selected a set of 10 genes involved in the leaf starch metabolism pathway, and validated microarray patterns using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Except for decreased synthesis and induced decomposition of starch granule in leaves, 8 h long EO exposure slightly elevated the accumulation of sucrose compared to glucose and fructose in subjected potato plants. An in vitro feeding bioassay with Colorado potato beetle showed that EO-induced alternations on transcriptional level and in the sugars' metabolism caused the enhancement of feeding behavior and overall development of the tested larvae. Results of comprehensive analysis of transcriptional responses in potato exposed to French Marigold EO provide a basis for further elucidation of molecular mechanisms underlying eco-physiological interactions in companion planting cropping systems.

Molecular Characterization and Expression of Four Aquaporin Genes in Impatiens walleriana During Drought Stress and Recovery.

Aquaporins comprise a large group of transmembrane proteins responsible for water transport, which is crucial for plant survival under stress conditions. Despite the vital role of aquaporins, nothing is known about this protein family in Impatiens walleriana, a commercially important horticultural plant, which is sensitive to drought stress. In the present study, attention is given to the molecular characterization of aquaporins in I. walleriana and their expression during drought stress and recovery. We identified four I. walleriana aquaporins: IwPIP1;4, IwPIP2;2, IwPIP2;7 and IwTIP4;1. All of them had conserved NPA motifs (Asparagine-Proline-Alanine), transmembrane helices (TMh), pore characteristics, stereochemical properties and tetrameric structure of holoprotein. Drought stress and recovery treatment affected the aquaporins expression in I. walleriana leaves, which was up- or downregulated depending on stress intensity. Expression of IwPIP2;7 was the most affected of all analyzed I. walleriana aquaporins. At 15% and 5% soil moisture and recovery from 15% and 5% soil moisture, IwPIP2;7 expression significantly decreased and increased, respectively. Aquaporins IwPIP1;4 and IwTIP4;1 had lower expression in comparison to IwPIP2;7, with moderate expression changes in response to drought and recovery, while IwPIP2;2 expression was of significance only in recovered plants. Insight into the molecular structure of I. walleriana aquaporins expanded knowledge about plant aquaporins, while its expression during drought and recovery contributed to I. walleriana drought tolerance mechanisms and re-acclimation.

Morpho-Physiological and Molecular Evaluation of Drought and Recovery in Impatiens walleriana Grown Ex Vitro.

This study was carried out to examine the drought effect on development, physiological, biochemical and molecular parameters in Impatiens walleriana grown ex vitro. Experiment design included three treatments: Control plants-grown under optimal watering (35%-37% of soil moisture content), drought-stressed plants-non-irrigated to reach 15% and 5% of soil moisture content and recovery plants-rehydrated for four days to reach optimal soil moisture content. Drought reduced fresh weight, total leaf area, as well as dry weight of I. walleriana shoots. Drought up-regulated expression of abscisic acid (ABA) biosynthesis genes 9-cis-epoxycarotenoid dioxygenase 4 (NCED4) and abscisic aldehyde oxidase 2 (AAO2) and catabolic gene ABA 8'-hydroxylase 3 (ABA8ox3) which was followed by increased ABA content in the leaves. Decrement in water potential of shoots during the drought was not accompanied with increased amino acid proline content. We detected an increase in chlorophyll, carotenoid, total polyphenols and flavonols content under drought conditions, as well as malondialdehyde, hydrogen peroxide and DPPH (1,1'-diphenyl-2-picrylhydrazyl) activity. Increased antioxidant enzyme activities (superoxide dismutase, peroxidase and catalase) throughout drought were also determined. Recovery treatment was significant for neutralizing drought effect on growth parameters, shoot water potential, proline content and genes expression.

Breaking the Dormancy of Snake's Head Fritillary (Fritillaria meleagris L.) In Vitro Bulbs-Part 1: Effect of GA3, GA Inhibitors and Temperature on Fresh Weight, Sprouting and Sugar Content.

Bulbs are the main vegetative reproductive organs of Fritillaria meleagris L. In nature, as well as in vitro, they become dormant and require low temperatures for further growth during the next vegetative period. In the present study, using 10 µM of gibberellic acid (GA3), or gibberellin biosynthesis (GA) inhibitors-ancymidol (A) and paclobutrazol (P)-the dynamic changes in soluble sugars, fructose and glucose content, fresh weight and sprouting capacity were investigated. F. meleagris bulbs were cultured on medium with GA3 and GA inhibitors for 1, 2 and 5 weeks at two different temperatures (24 and 7 °C). GA3 improved bulb fresh weight, as well as sprouting percentage at both tested temperatures, compared to the control. The highest fresh weight increase (57.7%) and sprouting rate (29.02%) were achieved when bulbs were grown at 24 °C for 5 weeks. In addition, soluble sugar content was the highest in bulbs grown for 5 weeks on medium supplemented with GA3. The main sugar in fritillary bulbs was glucose, while fructose content was lower. The sensitivity of bulbs to GA inhibitors differed and significantly affected sugar content in bulbs. To our knowledge, this is the first study of the sugar composition in F. meleagris bulbs during breaking of the bulb's dormancy and its sprouting.

Endogenous levels of cytokinins, indole-3-acetic acid and abscisic acid in in vitro grown potato: A contribution to potato hormonomics.

A number of scientific reports published to date contain data on endogenous levels of various phytohormones in potato (Solanum tuberosum L.) but a complete cytokinin profile of potato tissues, that would include data on all particular molecular forms of cytokinin, has still been missing. In this work, endogenous levels of all analytically detectable isoprenoid cytokinins, as well as the auxin indole-3-acetic acid (IAA), and abscisic acid (ABA) have been determined in shoots and roots of 30 day old in vitro grown potato (cv. Désirée). The results presented here are generally similar to other data reported for in vitro grown potato plants, whereas greenhouse-grown plants typically contain lower levels of ABA, possibly indicating that in vitro grown potato is exposed to chronic stress. Cytokinin N-glucosides, particularly N7-glucosides, are the dominant cytokinin forms in both shoots and roots of potato, whereas nucleobases, as the bioactive forms of cytokinins, comprise a low proportion of cytokinin levels in tissues of potato. Differences in phytohormone composition between shoots and roots of potato suggest specific patterns of transport and/or differences in tissue-specific metabolism of plant hormones. These results represent a contribution to understanding the hormonomics of potato, a crop species of extraordinary economic importance.

Advancement in protocol for in vitro seed germination, plant regeneration and cryopreservation of Viola cornuta.

The aim of this study was to develop a fast, reliable and true-to-type protocol for in vitro plant regeneration and long-term storage of horned pansy (Viola cornuta L). Seed germination over 60% was recorded after 12 weeks of growth at 10 °C or 4 °C. Calli formation and shoot induction were obtained in petiole and hypocotyl culture on half-strength MS mineral salts with full concentration of Na-FeEDTA and vitamins (½MS medium) with 2,4-dichlorophenoxyacetic acid (2,4-D, 0.1 mg/L) and 6-benzylaminopurine (BAP, 2.0 mg/L) and leaf culture on ½MS medium with thidiazuron (TDZ,1.0 mg/L). The highest frequency of adventitious shoot induction (50%) with six shoots/explant was achieved in hypocotyl culture from top hypocotyl segments, close to epicotyl which was grown 8 weeks at 16 h light/8 h dark photoperiod. Subsequent shoot multiplication was achieved on ½MS medium with α-naphthaleneacetic acid (NAA, 0.1 or 0.5 mg/L) and BAP (1.0 mg/L). Rooting of shoots was obtained on ½MS medium with low concentration (0.1 mg/L) of auxins: indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or NAA, or without growth regulators. In vitro-derived plantlets were acclimatized under greenhouse conditions. All plants developed normally, bloomed and set seeds. Shoot tips were cryopreserved succssefully using modified plant vitrification 3 (PVS3-based vitrification procedure). Cold acclimation for 2 weeks significantly improved shoot regrowth (64%) after thawing in comparison to non-acclimated shoots (39%). Clonal fidelity of regenerated plantlets at ploidy level was confirmed by chromosome counting. The presented protocol can be useful for mass propagation, genetic transformation studies and long-term storage of valuable Viola spp.

Expression profiles of organogenesis-related genes over the time course of one-step de novo shoot organogenesis from intact seedlings of kohlrabi.

Kohlrabi (Brassica oleracea var. gongylodes) is an important vegetable crop that is able to undergo shoot regeneration in culture from intact seedlings in a single-step regeneration process, using cytokinin as the only plant growth regulator. In this work, we present the expression profiles of seven organogenesis-related genes over the time course of shoot regeneration from intact seedlings of kohlrabi cv. Vienna Purple on shoot regeneration media containing trans-zeatin, cis-zeatin, benzyl adenine or thidiazuron. Two auxin transporter genes - PIN3 and PIN4, a cytokinin response regulator - ARR5, two shoot apical meristem-related transcription factors - CUC1 and RGD3, and two cell cycle-related genes - CDKB2;1 and CYCB2;4 - displayed bimodal expression patterns on most cytokinin-containing media when their expression levels were normalized against control plants grown on hormone-free media. The first expression peak corresponded to direct upregulation by cytokinin from the growth media, and the second one reflected transcriptional events related to callus formation and/or acquisition of organogenic competence, corresponding to the shoot regeneration phases that have already been characterized in Arabidopsis thaliana. We demonstrate that the genes involved in the two-step shoot regeneration of Arabidopsis display their expected expression profiles during the single-step shoot regeneration of its close phylogenetic relative kohlrabi confirming the universality of their roles in the distinct phases of the regeneration process in Brassicaceae. The results presented here represent a first step towards genetic characterization of the morphogenetic processes in this important crop species.

Beneficial implications of sugar beet proteinase inhibitor BvSTI on plant architecture and salt stress tolerance in Lotus corniculatus L.

Food demands of increasing human population dictate intensification of livestock production, however, environmental stresses could jeopardize producers' efforts. Forage legumes suffer from yield losses and poor nutritional status due to salinity increase of agricultural soils. As tools aimed to reduce negative impacts of biotic or abiotic stresses, proteinase inhibitors (PIs) have been promoted for biotechnological improvements. In order to increase tolerance of Lotus corniculatus L. to salt stress, serine PI, BvSTI, was introduced into this legume using Agrobacterium rhizogenes, with final transformation efficiency of 4.57%. PCR, DNA gel-blot, RT-PCR and in-gel protein activity assays confirmed the presence and activity of BvSTI products in transformed lines. Plants from three selected transgenic lines (21, 73 and 109) showed significant alterations in overall phenotypic appearance, corresponding to differences in BvSTI accumulation. Lines 73 and 109 showed up to 7.3-fold higher number of tillers and massive, up to 5.8-fold heavier roots than in nontransformed controls (NTC). Line 21 was phenotypically similar to NTC, accumulated less BvSTI transcripts and did not exhibit an additional band of recombinant trypsin inhibitor as seen in lines 73 and 109. Exposure of the transgenic lines to NaCl revealed different levels of salt stress susceptibility. The NaCl sensitivity index, based on morphological appearance and chlorophyll concentrations showed that lines 73 and 109 were significantly less affected by salinity than NTC or line 21. High level of BvSTI altered morphology and delayed salt stress related senescence, implicating BvSTI gene as a promising tool for salinity tolerance improvement trials in L. corniculatus.

Co-expression of the proteinase inhibitors oryzacystatin I and oryzacystatin II in transgenic potato alters Colorado potato beetle larval development.

Colorado potato beetle (CPB; Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae) has shown a remarkable adaptability to a variety of control measures. Although oryzacystatin I and II (OCI and OCII) have potential in controlling pests that use cysteine proteinases for food digestion, expression of a single OC gene in potato exhibited a minimal or no effect on CPB fitness traits. The aim of this study was to examine the effect of coexpressed OCI and OCII in potato (Solanum tuberosum L.) cultivars Desiree, Dragacevka and Jelica on CPB larvae. Growth parameters, consumption rates and food utilization, as well as activity of proteases of CPB larvae were assayed. Second and third instar larvae fed on transformed leaves molted earlier and had higher relative growth and consumption rates than larvae fed on nontransformed leaves, while efficiency of food utilization was unaffected. In contrast, fourth instar maximum weight gain and amount of leaves consumed were about 20% lower for the larvae fed on transgenic potato. Analysis of total protease activity of third instar larvae revealed reduction in overall proteolytic activity measured by azocasein hydrolysis, accompanied with inhibition of cysteine proteinase activity 24 h after ingestion of potato leaves expressing OCI and OCII. However, after long-term feeding on transformed leaves proteolytic activities of larvae became similar to the controls. Although feeding on OCI/OCII leaves did not affect larval survival, coexpression of OC genes reduced the development time and thus significantly decreased plant damage caused by CPB larvae.