Angelina Subotić

Morpho-histological study of direct somatic embryogenesis in endangered species Frittilaria meleagris

Direct somatic embryogenesis of Frittilaria meleagris L. was induced using leaf base explants excised from in vitro grown shoots. Somatic embryos occurred at the basal part of leaf explants 4 weeks after culture on a Murashige and Skoog (MS) medium supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or kinetin (KIN). The highest number of somatic embryos (SEs) were formed (9.74) from leaf explant on MS medium supplemented with 0.1 mg dm−3 2,4-D after 4 weeks of culture initiation. An initial exposure to a low concentration of KIN in the medium also enhanced SEs induction. Our observations by light and scanning electron microscopy revealed that SEs originate directly from the epidermal and subepidermal layers of leaf explant. The developmental stages of somatic embryogenesis from the first unequal cell division through the meristematic clusters, multi-cellular globular somatic embryos to the fully formed cotyledonary embryos were determined. After 4 weeks on MS medium without plant growth regulators, SEs developed into bulblets.

Plant regeneration in leaf culture of Centaurium erythraea Rafn. Part 1: The role of antioxidant enzymes

Centaurium erythraea Rafn. is a medicinal plant rich in secoiridoids and xanthones and used for gastrointestinal disorders, fever, anemia and many other conditions. C. erythraea is characterized with extraordinary developmental plasticity and manageability in vitro; thus we propose it as an excellent experimental model system for studies in developmental biology. Hereby we describe regeneration of centaury from leaf explants that can proceed via somatic embryogenesis or organogenesis on inductive media containing 2,4-D and CPPU. In the absence of growth regulators, shoots and roots appeared without callusing, on light or in darkness, respectively. Indirect somatic embryogenesis was induced in the presence of growth regulators occurring both on light and in darkness. Light was obligatory for indirect shoot development, where adventitious buds formed simultaneously with somatic embryos. Dynamic changes of antioxidative activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX) in response to morphogenetic changes were followed during developmental pathways in vitro. Wounding of centaury leaves immediately induced all SOD and CAT isoforms but caused a decrease in POX activity. In control leaves and leaf explants, three Cu/Zn-SOD activities were detected, which gradually decreased on inductive treatments on light, but remained unchanged during growth in darkness. Morphogenetic paths on all hormonal and light treatments where characterized with dynamic changes of CAT activity (comprised of three major CAT isoforms), but generally CAT was reduced during morphogenesis induction. POX activity was strongly induced during morphogenesis in all treatments.

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.

Endogenous Phytohormones in Spontaneously Regenerated Centaurium erythraea Rafn. Plants Grown In Vitro

Phytohormones are important regulators of numerous developmental and physiological processes in plants. Spontaneous morphogenesis of the common centaury (Centaurium erythraea Rafn.) is possible on nutrition medium without addition of any plant growth regulator depending solely on endogenous phytohormone levels. Thus, this plant species represents a very good model system for the investigation of numerous physiological processes under phytohormonal control in vitro. We analysed the total amount of endogenous cytokinins (CKs) including the contents of their individual groups in shoots and roots of C. erythraea plants grown in vitro. The total amount of endogenous CKs was 1.4 times higher in shoots than in roots. Inactive or weakly active N-glucosides found to predominate in both organs of centaury plants, whereas free bases and O-glucosides represented only a small portion of the total CK pool. Consequently, centaury roots showed higher IAA content as well as IAA/free CK base ratios compared to shoots. Centaury tissues also showed increased levels of “stress hormones”. In contrast to SA, considerably higher levels of ABA were found in centaury shoots than in roots. Our results could serve as a basis for understanding and elucidating spontaneous de novo shoot organogenesis and further plant regeneration of C. erythraea in vitro.

Changes in cytokinin content and altered cytokinin homeostasis in AtCKX1 and AtCKX2-overexpressing centaury (Centaurium erythraea Rafn.) plants grown in vitro

The plant hormones cytokinins (CKs) regulate a number of physiological processes. Their homeostasis is controlled by the rate of de novo synthesis and the rate of catabolism. The aim of this work was to analyze the content of total as well as individual groups of endogenous CKs in AtCKX1 and AtCKX2-overexpressing centaury (Centaurium erythraea Rafn.) plants grown in vitro. Transgenic CKX plants represent a suitable model system for studying physiological and morphological processes controlled by CKs. In this work we clearly demonstrate a significant effect of AtCKX transgenes on CK metabolism in transgenic centaury plants. However, shoots and roots of only one AtCKX1 line and three AtCKX2 lines with a significant reduction of bioactive CKs were obtained. We also show that changes in the CKs metabolism considerably affected endogenous indole-3-acetic acid (IAA) levels in plant tissues. All analyzed transgenic AtCKX centaury lines exhibited decreased amount of endogenous IAA in shoots as well as in roots. Consequently, the IAA/bioactive CK forms ratios showed a significant variation in the shoots and roots of all analyzed AtCKX centaury transformants.

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 messageGenetic modulation of the carotenogenesis inI. germanica‘Fire Bride’ by ectopic expression of acrtBgene causes several flower parts to develop novel orange and pink colors.AbstractFlower 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.

The Role of Arabinogalactan Proteins in Morphogenesis of Centaurium erythraea Rafn In Vitro

Histochemical localization using the β-Glc Yariv reagent and immunolocalization with arabinogalactan protein (AGP) reactive antibodies (LM2, JIM13, JIM15, JIM16, MAC207) were performed during morphogenic induction in root cultures of Centaurium erythraea Rafn cultured on half-strength MS medium without plant growth regulators. The observations revealed that β-Glc Yariv reagent specifically bound to AGPs in cells of the root epidermis and central cylinder. Monoclonal antibodies recognizing AGPs were localized in epidermal cells and cells of the central cylinder (LM2 , JIM16), vascular tissue (JIM15), globular somatic embryos (LM2, MAC207), and de novo-formed meristematic centers in the root cortex (JIM16). The effect of β-Glc Yariv reagent was investigated after supplementation (0–75 μM) in the culture medium. The morphogenetic potential was increased at lower concentrations (15–25 μM) of treatment with β-Glc Yariv reagent but inhibited (40 %) at the highest concentration. These results implicate that AGPs play a significant role during the development of somatic embryos and adventitious shoots in root cultures of C. erythraea.

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.

Esterase and peroxidase isoforms during initial stages of somatic embryogenesis in Fritillaria meleagris L. leaf base

The aim of this study was to determine the enzymatic profile of esterases and peroxidases during early stages of somatic embryogenesis of Fritillaria meleagris L. Somatic embryogenesis was induced using the leaf base as explant on a medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). Zymography showed the presence of different moieties, six isoforms of esterases and peroxidases, during morphogenesis as compared to control explants. One isoform of esterases was detected only during the process of somatic embryogenesis, and one isoform was detected in control explants. Analysis of esterases with 1-naphthyl butyrate proved that esterases, which participate in somatic embryogenesis of F. meleagris , belong to the family of aryl esterases. For the first time it was proved that five isoforms of esterases, which are involved in morphogenesis of F. meleagris , belong to the family of aryl esterases, while two isoforms are carboxyl esterases. One isoform of carboxyl esterases was visible in control explants. This is also the first description of peroxidases during the morphogenetic process, and of the difference between aryl and carboxyl esterases. More isoforms of esterases during morphogenesis as compared to control explants are probably responsible for some early physiological process during somatic embryogenesis of F. meleagris . https://doi.org/10.2298/ABS161130007P Received: November 30, 2016; Revised: January 10, 2017; Accepted: March 3, 2017; Published online: March 10, 2017 How to cite this article: Petric M, Subotic A, Jevremovic S, Trifunovic-Momcilov M, Tadic V, Grujic M, Vujcic Z. Esterase and peroxidase isoforms during initial stages of somatic embryogenesis in Fritillaria meleagris L. leaf base. Arch Biol Sci. 2017;69(4):619-25.