Physiological and oxidative stress response of carrot (Daucus carota L.) to jumping plant-louse Bactericera trigonica Hodkinson (Hemiptera: Psylloidea) infestation.

BACKGROUND: Carrot is an important vegetable crop grown worldwide. The major economic problem in carrot cultivation is yellow disease caused by Bactericera trigonica, which induces biotic stress and has the greatest impact on crop productivity. Comprehensive studies on the mechanism of carrot defense response to biotic stress caused by B. trigonica infestation have yet to be conducted. METHODS: The changes in photosynthetic pigments, proline, TPC, H2O2 and MDA content, DPPH radical scavenging ability, and antioxidant enzyme activity of SOD, CAT, and POX in carrot leaves in response to insect sex (female and male), rapid response (during the first six hours), and long-term response to B. trigonica infestation were evaluated. RESULTS: The results of our study strongly suggest that B. trigonica infestation causes significant changes in primary and secondary metabolism and oxidative status of carrot leaves. Photosynthetic pigment content, TPC, and DPPH and CAT activities were significantly reduced in carrot leaves in response to insect infestation. On the other hand, proline, H2O2 content, and the activity of the antioxidant enzymes superoxide dismutase and peroxidase were increased in carrot leaves after B. trigonica infestation. The results indicate that B. trigonica attenuates and delays the oxidative stress responses of carrot, allowing long-term feeding without visible changes in the plant. Carrot responded to long-term B. trigonica infestation with an increase in SOD and POX activity, suggesting that these enzymes may play a key role in plant defense mechanisms. CONCLUSIONS: This is the first comprehensive study strongly suggesting that B. trigonica infestation causes significant changes in primary and secondary metabolism and an attenuated ROS defense response in carrot leaves that enables long-term insect feeding. The information provides new insights into the mechanisms of carrot protection against B. trigonica infestation.

Physiological Response, Oxidative Stress Assessment and Aquaporin Genes Expression of Cherry Tomato (Solanum lycopersicum L.) Exposed to Hyper-Harmonized Fullerene Water Complex.

The rapid production and numerous applications of nanomaterials warrant the necessity and importance of examining nanoparticles in terms to their environmental and biological effects and implications. In this study, the effects of a water-soluble hyper-harmonized hydroxyl-modified fullerene (3HFWC) on cherry tomato seed germination, seedlings growth, physiological response and fruiting was evaluated. Changes in the photosynthetic pigments content, oxidative stress assessment, and aquaporin genes expression in cherry tomato plants were studied after during short- and long-term continuous exposure to 3HFWC nanosubstance (200 mg/L). Increased levels of photosynthetic pigments in leaves, lycopene in fruits, decreased levels of hydrogen peroxide content, activation of cellular antioxidant enzymes such as superoxide dismutase, catalase and peroxidase and increased aquaporin gene expression (PIP1;3, PIP1;5 and PIP2;4) were observed in 3HFWC nanosubstance-exposed plants in comparison to control, untreated cherry tomato plants. The 3HFWC nanosubstance showed positive effects on cherry tomato seed germination, plantlet growth and lycopene content in fruits and may be considered as a promising nanofertilizer.

Treatment of Chrysanthemum Synthetic Seeds by Air SDBD Plasma.

Herein, we present the effect of surface dielectric barrier discharge (SDBD) air cold plasma on regrowth of chrysanthemum synthetic seeds (synseeds) and subsequent plantlet development. The plasma system used in this study operates in air at the frequency of 50 Hz. The detailed electrical characterization of SDBD was shown, as well as air plasma emission spectra obtained by optical emission spectroscopy. The chrysanthemum synseeds (encapsulated shoot tips) were treated in air plasma for different treatment times (0, 5 or 10 min). Plasma treatment significantly improved the regrowth and whole plantlet development of chrysanthemum synseeds under aseptic (in vitro) and non-aseptic (ex vitro) conditions. We evaluated the effect of SDBD plasma on synseed germination of four chrysanthemum cultivars after direct sowing in soil. Germination of synseeds directly sowed in soil was cultivar-dependent and 1.6-3.7 fold higher after plasma treatment in comparison with untreated synseeds. The study showed a highly effective novel strategy for direct conversion of simple monolayer alginate chrysanthemum synseeds into entire plantlets by plasma pre-conversion treatment. This treatment reduced contamination and displayed a considerable ex vitro ability to convert clonally identical chrysanthemum plants.

Phytohormone profiles in non-transformed and AtCKX transgenic centaury (Centaurium erythraea Rafn) shoots and roots in response to salinity stress in vitro.

Plant hormones regulate numerous developmental and physiological processes. Abiotic stresses considerably affect production and distribution of phytohormones as the stress signal triggers. The homeostasis of plant hormones is controlled by their de novo synthesis and catabolism. The aim of this work was to analyse the contents of total and individual groups of endogenous cytokinins (CKs) as well as indole-3-acetic acid (IAA) in AtCKX overexpressing centaury plants grown in vitro on graded NaCl concentrations (0, 50, 100, 150, 200 mM). The levels of endogenous stress hormones including abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) were also detected. The elevated contents of total CKs were found in all analysed centaury shoots. Furthermore, increased amounts of all five CK groups, as well as enhanced total CKs were revealed on graded NaCl concentrations in non-transformed and AtCKX roots. All analysed AtCKX centaury lines exhibited decreased amounts of endogenous IAA in shoots and roots. Consequently, the IAA/bioactive CK forms ratios showed a significant variation in the shoots and roots of all AtCKX lines. In shoots and roots of both non-transformed and AtCKX transgenic centaury plants, salinity was associated with an increase of ABA and JA and a decrease of SA content.

Bulb Dormancy In Vitro-Fritillaria meleagris: Initiation, Release and Physiological Parameters.

In ornamental geophytes, conventional vegetative propagation is not economically feasible due to very slow development and ineffective methods. It can take several years until a new plant is formed and commercial profitability is achieved. Therefore, micropropagation techniques have been developed to increase the multiplication rate and thus shorten the multiplication and regeneration period. The majority of these techniques rely on the formation of new bulbs and their sprouting. Dormancy is one of the main limiting factors to speed up multiplication in vitro. Bulbous species have a period of bulb dormancy which enables them to survive unfavorable natural conditions. Bulbs grown in vitro also exhibit dormancy, which has to be overcome in order to allow sprouting of bulbs in the next vegetation period. During the period of dormancy, numerous physiological processes occur, many of which have not been elucidated yet. Understanding the process of dormancy will allow us to speed up and improve breeding of geophytes and thereby achieve economic profitability, which is very important for horticulture. This review focuses on recent findings in the area of bulb dormancy initiation and release in fritillaries, with particular emphasis on the effect of plant growth regulators and low-temperature pretreatment on dormancy release in relation to induction of antioxidative enzymes' activity in vitro.

Breaking the Dormancy of Snake's Head Fritillary (Fritillaria meleagris L.) In Vitro Bulbs-Part 2: Effect of GA3 Soaking and Chilling on Sugar Status in Sprouted Bulbs.

The bulb is the main propagation organ of snake's head fritillary (Fritillaria meleagris L.), a horticulturally attractive and rare geophyte plant species. In this study, we investigated the effect of soaking bulbs in GA3 solution (1, 2, and 3 mg L-1) combined with low-temperature treatment (7 °C) on breaking the dormancy of in vitro bulbs. Sugar status (total soluble sugars, glucose, and fructose content) was analyzed in different parts of the sprouted bulbs. The results showed that the soluble sugar concentration was highest in bulbs soaked in GA3. The main sugar in fritillary bulbs was glucose, while fructose content was much lower. Glucose concentration dramatically increased after bulb chilling (7 °C), and its accumulation was predominantly detected in the lower sprout portion during the first weeks of sprouting. Sugar concentration was significantly lower in nonchilled bulbs, which indicates the importance of low temperature in bulb development and sprouting.

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.

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.

Esterase and peroxidase isoforms in different stages of morphogenesis in Fritillaria meleagris L. in bulb-scale culture.

Morphogenesis in vitro is a complex and still poorly defined process. We investigated esterase and peroxidase isoforms detected in bulb scale, during Fritillaria meleagris morphogenesis. Bulbs were grown either at 4 °C or on a medium with an increased concentration of sucrose (4.5%) for 30 days. After these pre-treatments, the bulb scales were further grown on nutrient media that contained different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KIN) or thidiazuron (TDZ). Regeneration of somatic embryos and bulblets occurred at the same explant. The highest numbers of somatic embryos and bulblets were regenerated on the medium containing 2,4-D and KIN (1mg/L each), while morphogenesis was most successful at a TDZ concentration between 0.5 and 1mg/L. Monitoring of esterases and peroxidases was performed by growing bulb scales on a medium enriched with 2,4-D and KIN or TDZ (1mg/L), and the number and activity of isoforms were followed every 7 days for 4 weeks. In control explants, six isoforms of esterase were observed. Three isoforms of peroxidase were not detected in the control bulb scale, which has not begun its morphogenesis process.

Alteration of flower color in Iris germanica L. 'Fire Bride' through ectopic expression of phytoene synthase gene (crtB) from Pantoea agglomerans.

KEY MESSAGE: Genetic modulation of the carotenogenesis in I. germanica 'Fire Bride' by ectopic expression of a crtB gene causes several flower parts to develop novel orange and pink colors. Flower color in tall bearded irises (Iris germanica L.) is determined by two distinct biochemical pathways; the carotenoid pathway, which imparts yellow, orange and pink hues and the anthocyanin pathway, which produces blue, violet and maroon flowers. Red-flowered I. germanica do not exist in nature and conventional breeding methods have thus far failed to produce them. With a goal of developing iris cultivars with red flowers, we transformed a pink iris I. germanica, 'Fire Bride', with a bacterial phytoene synthase gene (crtB) from Pantoea agglomerans under the control of the promoter region of a gene for capsanthin-capsorubin synthase from Lilium lancifolium (Llccs). This approach aimed to increase the flux of metabolites into the carotenoid biosynthetic pathway and lead to elevated levels of lycopene and darker pink or red flowers. Iris callus tissue ectopically expressing the crtB gene exhibited a color change from yellow to pink-orange and red, due to accumulation of lycopene. Transgenic iris plants, regenerated from the crtB-transgenic calli, showed prominent color changes in the ovaries (green to orange), flower stalk (green to orange), and anthers (white to pink), while the standards and falls showed no significant differences in color when compared to control plants. HPLC and UHPLC analysis confirmed that the color changes were primarily due to the accumulation of lycopene. In this study, we showed that ectopic expression of a crtB can be used to successfully alter the color of certain flower parts in I. germanica 'Fire Bride' and produce new flower traits.

Micropropagation of Iris sp.

Irises are perennial plants widely used as ornamental garden plants or cut flowers. Some species accumulate secondary metabolites, making them highly valuable to the pharmaceutical and perfume industries. Micropropagation of irises has successfully been accomplished by culturing zygotic embryos, different flower parts, and leaf base tissues as starting explants. Plantlets are regenerated via somatic embryogenesis, organogenesis, or both processes at the same time depending on media composition and plant species. A large number of uniform plants are produced by somatic embryogenesis, however, some species have decreased morphogenetic potential overtime. Shoot cultures obtained by organogenesis can be multiplied for many years. Somatic embryogenic tissue can be reestablished from leaf bases of in vitro-grown shoots. The highest number of plants can be obtained by cell suspension cultures. This chapter describes effective in vitro plant regeneration protocols for Iris species from different types of explants by somatic embryogenesis and/or organogenesis suitable for the mass propagation of ornamental and pharmaceutical irises.

Cloning and functional characterization of a gene for capsanthin-capsorubin synthase from tiger lily (Lilium lancifolium Thunb. 'Splendens').

The orange color of tiger lily (Lolium lancifolium 'Splendens') flowers is due, primarily, to the accumulation of two κ-xanthophylls, capsanthin and capsorubin. An enzyme, known as capsanthin-capsorubin synthase (CCS), catalyzes the conversion of antheraxanthin and violaxanthin into capsanthin and capsorubin, respectively. We cloned the gene for capsanthin-capsorubin synthase (Llccs) from flower tepals of L. lancifolium by the rapid amplification of cDNA ends (RACE) with a heterologous non-degenerate primer that was based on the sequence of a gene for lycopene ß-cyclase (lcyB). The full-length cDNA of Llccs was 1,785 bp long and contained an open reading frame of 1,425 bp that encoded a polypeptide of 474 amino acids with a predicted N-terminal plastid-targeting sequence. Analysis by reverse transcription-PCR (RT-PCR) revealed that expression of Llccs was spatially and temporally regulated, with expression in flower buds and flowers of L. lancifolium but not in vegetative tissues. Stable overexpression of the Llccs gene in callus tissue of Iris germanica, which accumulates several xanthophylls including violaxanthin, the precursor of capsorubin, resulted in transgenic callus whose color had changed from its normal yellow to red-orange. This novel red-orange coloration was due to the accumulation of two non-native κ-xanthophylls, capsanthin and capsorubin, as confirmed by HPLC and ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis with authentic standards. Cloning of the Llccs gene should advance our understanding of the molecular and genetic mechanisms of the biosynthesis of κ-carotenoids in general and in the genus Lilium in particular, and will facilitate transgenic alterations of the colors of flowers and fruits of many plant species.

Spontaneous plant regeneration and production of secondary metabolites from hairy root cultures of Centaurium erythraea Rafn.

We have established an efficient protocol for plant regeneration and production of secondary metabolites in hairy root culture of Centaurium erythraea Rafn. Because the hairy roots and regenerated plants produce bitter secoiridoid glucosides and xanthones similar to the plants in nature, the use of in vitro cultures as an alternative source of their production is feasible. This chapter describes a protocol for the induction of adventitious shoots and transgenic plants from hairy root cultures of C. erythraea and their phytochemical analysis.