Physiological and biochemical aspects of regeneration snake's head fritillary (Fritillaria meleargis L.) in vitro

Proučavana je in vitro regeneracijia košutice (Fritillaria meleagris), višegodišnje lukovičaste geofite. Indukcija morfogeneze in vitro košutice postignuta je u kulturi zrelih zigotskih embriona, segmenata lukovica kao i bazalnih delova listova in vitro formiranih lukovica. U kulturi zrelih zigotskih embriona regeneracija biljaka je postignuta procesom somatske embriogeneze i organogeneze u isto vreme i na istom eksplalntatu na hranljivoj podlozi bez regulatora rastenja ili hranljivoj podlozi obogaćenoj sa TDZ. U kulturi segmenata in vitro formiranih lukovica indukcija morfogeneze postignuta je na hranljivoj podlozi sa 2,4-D ili TDZ na svetlosti ili u mraku. Regeneracija biljaka je bila postignuta na obe hranljive podloge putem somatske embriogeneze i organogeneze, s tim da je regeneracija putem organogeneze bila uspešnija na hranljivoj podlozi obogaćenoj sa TDZ. U kulturi bazalnih delova listova indukovana je somatska embriogeneza na hranljivim podlogama sa 2,4-D, KIN ili 2,4-D i KIN. Anatomska istraživanja su pokazala da je somatska embriogeneza direktna, a somatski embrioni imaju višećelijsko poreklo i nastaju od epidermalnih i subepidermalnih slojeva ćelija bazalnih delova lista. Ispitan je uticaj niskih temperatura (15 i 4 °C), povećane koncentracije saharoze u hranljivoj podlozi kao i predtretman sa GA3 na rastenje, razviće i prevazilaženje dormancije in vitro formiranih lukovica košutice. Pokazano je da predhodno gajenje lukovica na sniženim temperaturama i hranljivoj podlozi sa 4,5 % saharoze pozitivno utiče na rastenje i umnožavanje lukovica. Pored toga, predtretman rastvorom GA3 pre izlaganja lukovica niskoj temperaturi (4 °C) dovodi do stimulacije umnožavanja i klijanja lukovica. Izlaganjem in vitro formiranih lukovica košutice niskoj temperaturi dolazi do promena u sadržaju šećera (saharoze, glukoze i fruktoze), fotosintetičkih pigmenata i poliola. Ispitana je aktivnost antioksidativnh enzima (SOD, CAT, GR i POX) tokom prevazilaženja dormancije lukovica gajenjem na niskoj temperaturi. Analize su pokazale da enzimi antioksidativnog stresa aktivno učestvuju u procesima prekidanja dormancije in vitro formiranih lukovica košutice, kao i aklimatizacije lukovica na ex vitro uslove. Enzimi antioksidativne zaštite su aktivni i tokom indukcije morfogeneze košutice u kulturi segmenata lukovica, a njihova aktivnost zavisi od sastava hranljive podloge i predtretmana kome su lukovice prethodno izložene. Zimogramskom detekcijom esteraza tokom indukcije morfogeneze in vitro u kulturi segmenata lukovica utvrđeno je prisustvo 6 izoformi i njihova aktivnost kao i zastupljenost pojedinih izoformi zavise takođe od predtretmana na kojima su lukovice predhodno gajene kao i od regulatora rastenja u hranljivoj podlozi. Praćena je promena sadržaja arabinogalaktanskih proteina (AGP) u eksplantatima tokom indukcije morfogeneze in vitro u kulturi bazalnih delova listova i segmenata lukovica na dve hranljive podloge obogaćene sa 2,4-D i KIN ili TDZ. Koncentracija AGP u eksplantatima se povećava već posle 7 dana gajenja bazalnih delova listova odnosno posle 21 dana gajenja segmenata lukovica na hranljivim podlogama sa regulatorima rastenja. Koncentracija AGP je veća u bazalnim segmentima lista gajenim na hranljivoj podlozi sa 2,4-D i KIN nego na hranljivoj podlozi sa TDZ. Analizom profila AGP dobijenog ukrštenom elektroforezom pokazano je prisustvo samo jednog tipa AGP tokom indukcije morfogeneze in vitro.We have investigated in vitro regeneration of snake’head fritillary (Fritillaria meleagris L.), a perennial bulbous geophyte. The induction of morphogenesis in vitro of snake’head fritillary was achieved in mature zygotic embryo culture, scale segment and leaf base culture of in vitro formed bulbs. Plant regeneration via somatic embryogenesis and organogenesis was obtained in mature zygotic embryo culture on a growth regulator-free medium or on medium supplemented with TDZ. The induction of morphogenesis in vitro was achieved in scale segment culture of the in vitro formed bulbs on media supplemented with 2,4-D or TDZ, grown either on light or in darkness, with more efficient regeneration on media supplementned with TDZ. Somatic embryogenesis was induced in leaf base culture of the in vitro formed plants on media with 2,4-D, KIN or 2,4-D and KIN. Anatomical studies revealed that the somatic embryogenesis was direct, with somatic embryos of multicellular origin formed from epidermal and subepidermal leaf base cells . The effect of low temperature (4 and 15 °C), higher concentration of sucrose in the nutritional media and GA3 pretreatment on growth, differentiation and dormancy breaking of the in vitro formed bulbs was investigated. It was shown that pre-cultivation of the in vitro regenerated bulbs at lower temperatures and higher concentration of sucrose in the nutrition media (4,5 %) have stimulatory effect on growth and multiplication of the bulbs. Also, GA3 pretretment followed by cultivation at low temperature (4 °C) had a stimulatory effect on multiplication and germination of the bulbs. Cultivation at low temperature breaks dormancy of the bulbs and causees changes in the sugar content (sucrose, glucose and fructose), photosinthetic pigments and poliols. Activity of antioxidative enzymes (SOD, CAT, GR i POX) during dormancy breaking was investigated. It was shown that these enyzmes are actively involved in dormancy breaking of the in vitro formed bulbs, and in the process of acclimatization of the bulbs to ex vitro conditions. Antioxidative enzymes were active during the induction of morphogenesis in vitro in bulb segment culture and their activity depended on the nutritional media and the pretreatment to which the bulbs were exposed. During morphogenesis in vitro in the scale segment culture of snake’s head fritillary, up to 6 esterase isoforms have been detected, depending on the pretreatment and media composition. The content of arabinogalactan proteins (AGPs) in explants during the induction of morphogenesis in vitro in leaf base and scale segment cultures at media supplemented either with 2,4-D and KIN or with TDZ was determined. Concentration of AGPs increased after seven days of cultivation of explants on media with growth regulators in the leaf base culture and 21 days in the scale segment culture. In the leaf base culture, concentration of AGPs in explants was higher on a medium with 2,4-D and KIN than on a medium with TDZ. The AGP profile obtained by crossed electroforesis reveiled the presence of one AGP type during induction of morphogenesis in vitro of F. meleagris

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.Serbian Ministry of Science and Technological Development [143026B

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.Serbian Ministry of Science and Technological Development [143026B

The effect of low temperature and GA(3) treatments on dormancy breaking and activity of antioxidant enzymes in Fritillaria meleagris bulblets cultured in vitro

We investigated the effect of low temperature and gibberellic acid (GA(3)) treatment on dormancy in Fritillaria meleagris L. bulbs. Also, we studied the effect of dormancy breaking on the antioxidant enzymes activity. To overcome dormancy, bulbs require a period (4-8 weeks) of exposure to low temperature. Bulbs regenerated in vitro were grown in the dark on medium without growth regulators at the standard (24 A degrees C) or at low temperatures (4 and 15 A degrees C) for 4, 6, 8 and 10 weeks. Bulbs were collected after 3, 4 and 5 weeks of cooling at 4 A degrees C. To investigate the influence of GA(3) on dormancy, bulbs were treated for 24 h with GA(3) solutions with 1, 2 and 3 mg l(-1) concentrations. During the period of growth of bulbs at 4 A degrees C, regeneration of bulbs was very weak, while at 15 A degrees C the number of regenerated bulbs increased significantly. Improved bulb sprouting was achieved by a short treatment with gibberellin. Low temperature also represents a kind of oxidative stress for the plant. The activity of superoxide dismutase, catalase (CAT) and peroxidase (POX) in bulbs of F. meleagris L. grown in vitro and ex vitro increased with decreasing temperature in contrast to glutathione reductase. POX showed generally lower activity than CAT which indicates that major role in the breaking dormancy and preparing bulbs for sprouting have catalases.Serbian Ministry of Education, Science and Technological Development [ON173015, TR31019

Genetic Transformation of Centaurium erythraea with AtCKX1 and AtCKX2 Genes Using Agrobacterium tumefaciens

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Genetic Transformation of Centaurium erythraea with AtCKX1 and AtCKX2 Genes Using Agrobacterium tumefaciens

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Cyclisation oxydative d’hexadiène-1,5.

Cytokinin oxidase/dehydrogenase (CKX) is the only known enzyme involved in cytokinin catabolism. Genes coding for two Arabidopsis CKX isoforms, AtCKX1 and AtCKX2, were introduced separately into a binary cloning vector, immobilized into Agrobacterium tumefaciens strain GV3101, and introduced into root explants of centaury (Centaurium erythraea Rafn.). The integration of each transgene was confirmed by genomic PCR. Of the total transformed explants, 30 and 28.2 % of the transformants carried AtCKX1 and AtCKX2 transgenes, respectively. Of these transformants, 50 % exhibited expression of the AtCKX1 transgene, while 64 % of transformants exhibited expression of the AtCKX2 transgene. For all analysed AtCKX transgenic centaury lines, as well as for untransformed control plants, CKX activity was higher in roots than in shoots. Expression of AtCKX in most transgenic lines contributed to enhanced levels of CKX activity in root tissues; whereas, only a few lines demonstrated increased CKX activity in shoot tissues compared to those of control plants. Moreover, overexpression of AtCKX resulted in reduced morphogenetic potential in transgenic plants, but did not significantly affect biomass production in comparison to untransformed control plants.Ministry of Education, Science and Technological Development of the Republic of Serbia [ON173015]; Czech Science Foundation [P506/11/0774

Practical management plan for invasive mosquito species in Europe: I. Asian tiger mosquito (Aedes albopictus)

Aedes albopictus, also known as the “Asian Tiger Mosquito”, is an invasive mosquito species to Europe causing high concern in public health due to its severe nuisance and its vectorial capacity for pathogens such as dengue, chikungunya, yellow fever and Zika. Consequently, the responsible authorities implement management activities to reduce its population density, possibly to below noxious and epidemiological thresholds. In urban areas, these aims are difficult to achieve because of the species’ ability to develop in a wide range of artificial breeding sites, mainly private properties. This document (Management Plan) has been structured to serve as a comprehensive practical and technical guide for stakeholders in organizing the vector control activities in the best possible way. The current plan includes coordinated actions such as standardized control measures and quality control activities, monitoring protocols, activities for stakeholders and local communities, and an emergency vector control plan to reduce the risk of an epidemic