Plant regeneration in vitro and genetic transformation of Viola cornuta (Violaceae) using capsanthin-capsorubin-synthase gene

Razvijen je protokol za regeneraciju biljaka Viola cornuta L. cv. ’Lutea Splendens’, sitnocvetne ljubičice sa cvetovima žute boje, i genetičku transformaciju genom za kapsantin-kapsorubin-sintazu u cilju promene boje cveta metaboličkom modifikacijom biosinteze karotenoida. Najuspešnija indukcija adventivnih izdanaka dobijena je od eksplantata hipokotila gajenih na ½MS hranljivoj podlozi sa 0,1 mg/l 2,4-D i 2 mg/l BAP u uslovima dugog dana. Indukcija adventivnih izdanaka odvijala se procesom indirektne i direktne organogeneze. Transformisani izdanci dobijeni su genetičkom transformacijom pomoću ”praznog” (Llccs-) i pWBVec10a/CaMV 35S::Llccs::TNos (35S-Llccs) vektora, dok je korišćenjem pWBVec10a/PchsA-Llccs::TNos (PchsA-Llccs) vektora dobijen transformisani kalus. Prisustvo Llccs, hpt i uidA gena u genomu V. cornuta potvrđeno je PCR analizom, dok je qPCR analiza pokazala neujednačen nivo ekspresije Llccs gena u kruničnim listićima cvetova i listovima 35S-Llccs transformisanih linija. Boja kruničnih listića cvetova i žiga tučka promenjena je iz žute, odnosno zelene u nijanse narandžaste boje, usled akumulacije kapsantina, novosintetisanog crvenog pigmenta u hromoplastima 35S-Llccs transformisanih biljaka. Fenotip transformisanih biljaka se značajno razlikovao od fenotipa netransformisanih biljaka. Razvijen je protokol za dugotrajno čuvanje (krioprezervaciju) netransformisanih i transformisanih linija korišćenjem metode vitrifikacije sa PVS3 rastvorom. Oporavak vrhova izdanaka i regeneracija biljaka transformisanih linija (Llccs- i 35S-Llccs) posle krioprezervacije bili su značajno manji u odnosu na oporavak vrhova netransformisanih izdanaka. Nivo ploidnosti biljaka V. cornuta regenerisanih u kulturi in vitro, transformisanih linija kao i biljaka regenerisanih posle krioprezervacije bio je identičan.A protocol for the plant regeneration of Viola cornuta L. cv. ’Lutea Splendens’, horned pansy with yellow flowers and the genetic transformation with the capsanthin-capsorubin-synthase gene in order to change flower color by metabolic modification of carotenoid biosynthesis were developed. The most efficient induction of adventitious shoots was obtained from hypocotyl explants grown on ½MS medium with 0,1 mg/l 2,4-D and 2 mg/l BAP under 16h light/8h dark conditions. The induction of adventitious shoots was achieved through a process of indirect and direct organogenesis.Transformed shoots were obtained by genetic transformation using ”empty” (Llccs-) and pWBVec10a/CaMV 35S::Llccs::TNos (35S-Llccs) vectors, while using pWBVec10a/PchsA-Llccs::TNos (PchsA-Llccs) vectors transformed calli were obtained. The presence of the Llccs, hpt and uidA genes in the V. cornuta transformed plants were confirmed by PCR analysis, while qPCR analysis showed an unequal level of Llccs gene expression in the petals and leaves of 35S-Llccs transformed lines. The color of the flower petals and the stigma changed from yellow or green to shades of orange, due to the accumulation of capsanthin, a newly synthesized red pigment in the chromoplasts of 35S-Llccs transformed plants. The phenotype of transformed plants differed significantly from the phenotype of untransformed plants. A protocol for the long-term storage (cryopreservation) of untransformed and transformed lines using the vitrification method with PVS3 solution has been developed. The recovery of shoot tips and regeneration of transformed lines (Llccs- and 35S-Llccs) after cryopreservation was significantly lower compared to the recovery of the shoot tips of the untransformed shoots. The ploidy levels of V. cornuta plants regenerated in culture in vitro, transformed lines and plants recovered after cryopreservation were similar

Recent advances in flower color alteration by metabolic manipulation of carotenoid biosynthesis

U hortikulturi je prisutna stalna potreba za ukrasnim biljkama sa novim karakteristikama, gde boja cveta predstavlja jednu od najvažnijih osobina koja određuje njihovu komercijalnu vrednost. Sa razvojem metoda genetičkog inženjeringa otvorena je mogućnost kreiranja biljaka sa željenom bojom cvetova koja se ne može postići klasičnim ukrštanjem ili mutagenezom. Boja cvetova kod biljaka određena je sadržajem tri biljna pigmenta: antocijanina, karotenoida i betalaina. Do sada, najveći napredak postignut je genetičkom modulacijom biosinteze antocijanina. Na ovaj način postignute su nove boje cvetova kod najmanje 50 ukrasnih vrsta, a neki od tih modifikovanih varijeteta su već dugi niz godina u slobodnoj prodaji. Međutim, promena boje cveta manipulacijom biosintetskog puta karotenoida je dokumentovana kod svega nekoliko ukrasnih vrsta i poslednjih godina intenzivirana su istraživanja u tom pravcu. U ovom radu je razmatran potencijal ovog pristupa, sa posebnim osvrtom na rezultate postignute na promeni boje cvetova kod kultivara ljubičice uvodjenjem gena za kapsantin-kapsorubin sintazu.In horticulture, there is a constant need for ornamental plants with new characteristics, where the flower color is one of the most important features that determines their commercial value. With the development of genetic engineering methods, it has been possible to create plants with the desired flower color which cannot be achieved by classical breeding or mutagenesis. The flower color in plants is determined by the content of three plant pigments: anthocyanins, carotenoids and betalains. Up to date, the greatest progress has been made by genetic modulation of anthocyanin biosynthesis. In this way, the new flower colors have been achieved in at least 50 ornamental species, and some of these modified varieties have been on market for many years. However, the alteration of flower color by manipulating the carotenoid biosynthetic pathway has been documented in only a few ornamental species, and the research has been significantly increased last few years. In this paper, the potential of this approach is considered, with special reference to the results achieved on flower color alteration of pansy cultivars by introducing the gene for capsanthin-capsorubin synthase

Bringing nature indoors: Design and development of indoor living spaces in harmony with nature for active and healthy ageing in urban environments

The focus of this research is improving the well-being and happiness of the elderly in terms of the design and development of their indoor living spaces in harmony with nature. Multiple scientific studies have pointed out the benefits and importance of nature for people, especially for children and older populations. These benefits (increased participation in physical activities, improved mental health and cognitive function and an increase in social interaction) found through access to nature are key ingredients to well-being during ageing. As our population ages rapidly, and at the same time, the majority of people live in cities since urbanisation is continuing worldwide, it is important to provide and foster reconnection with nature for the senior members of our communities. Today’s cities suffer from many health and environmental problems. In recent years, the growing awareness of sustainability and climate change issues has made it even more apparent that bringing nature into our homes is essential. Bringing nature indoors causes people to feel happier, healthier, calmer, and, at the same time more energetic and optimistic about their lives. Natural environments also help to improve sleeping patterns, reduce pain, speed up recovery and even increase longevity. This research deals with different design strategies, principles, scales, concepts and patterns of biophilic design for elderly people, as well as their different benefits for health and well-being, with the aim of improving the design of dwelling environments in urban areas. The research also stimulates further discussions about the question of how bringing nature indoors through design (of interior/ architectural spaces, furniture) can improve the well-being of the elderly and how these can enable them to undertake activities that contribute to their well-being. It means creating a sensitive and responsive design that highlights a) a visual connection to nature, b) pulpability and soundness of nature and c) nurturing a sense of place, a community in which the role of aesthetics is crucial for behavioural change

Supplementary data for article:Trmčić, M.; Matović, R. V.; Tovilović, G.; Ristic, B. Z.; Trajković, V. S.; Ferjančić, Z.; Saičić, R. A Novel C,D-Spirolactone Analogue of Paclitaxel: Autophagy Instead of Apoptosis as a Previously Unknown Mechanism of Cytotoxic Action for Taxoids. Organic and Biomolecular Chemistry 2012, 10 (25), 4933–4942. https://doi.org/10.1039/c2ob25514f

Supplementary material for: [https://doi.org/10.1039/c2ob25514f]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1304

Krioprezervacija vrhova izdanaka Impatiens hawkeri W. Bull

Impatiens hawkeri (Balsaminaceae) je ukrasna biljna vrsta poreklom sa Nove Gvineje. Krioprezervacija predstavlja metod za dugotrajno čuvanje biljnog materijala na -196C u tečnom azotu koji se koristi i za eliminaciju patogena kod biljaka.1 Vrste roda Impatiens su izrazito osetljive na tretmane dehidratacije koji prethode zamrzavanju.2 Takođe, koncentracija saharoze u hranljivim podlogama utiče na morfogenetski potencijal tokom gajenja u kulturi in vitro.3 U ovom radu smo ispitivali uticaj prekulture vrhova izdanaka na povišenoj koncentraciji saharoze (6%), kao i vitamina C na preživljavanje i regeneraciju biljaka posle hemijske dehidratacije vrhova izdanaka sa rastvorima za vitrifikaciju (PVS2 i PVS3). Vrhovi izdanaka I. hawkeri su izrazito osetljivi na tretman sa 100% PVS2 rastvorom. Najveći morfogenetski potencijal posle izlaganja PVS2 rastvoru su pokazali vrhovi tretirani sa 50% PVS2 rastvorom kada je dolazilo samo do kalusiranja vrhova izdanaka. Potpuna regeneracija biljaka posle PVS2 tretmana postignuta je uz dodatak vitamina C u hranljivoj podlozi u koncentraciji od 100 mg/l. Najbolji rezultati preživljavanja i regeneracije biljaka posle odmrzavanja postignuti su korišćenjem metode vitrifikacije sa 100% PVS3 rastvorom u trajanju od 45 min. Izdanci gajeni na hranljivoj podlozi sa 6% saharoze su pokazali veće preživljavanje (49,4%) posle izlaganja ultra niskim temperaturama u odnosu na izdanke gajene na podlogama sa 3% saharoze (26,5 %)

Cryopreservation of Viola cornuta shoot tips using vitrification procedure

Cryopreservation represents a suitable method for long term storage of different plant genetic resources. The aim of this study was to develop protocol for cryopreservation of Viola cornuta shoot tips using one step freezing method with chemical dehydration of tissue with modified Plant Vitrification Solutions (PVS2 or PVS3). Shoot tips (1-2 mm) of two-week cold acclimated shoots were cultured on ½MS medium with 0.3 M sucrose for one day before treatment with loading solution (2 M glycerol, 0.4 M sucrose) for 30 min. Osmotic dehydration with PVS2 solution (30% glycerol, 15% ethylene glycol and 15% DMSO in liquid ½MS medium with 0.4 M sucrose) were tested at 0 °C or 24 °C. Osmotic dehydration with PVS3 (50% sucrose, 50% glycerol in liquid ½MS medium) were tested at 24 °C for 45 min. After the treatment the explants were directly immersed in liquid nitrogen (LN) for at least one day. Re-warming was performed at 42 °C in water bath for 2 min. After re-warming, the PVS solutions were replaced with unloading solution containing 1.2 M sucrose for 20 min. Re-warmed shoot tips were cultured on ½MS medium with 0.1 mg L-1 BAP. We observed that PVS2 solution is cytotoxic for V. cornuta shoot tips and cannot be used for cryopreservation. However, cryopreservation with PVS3 solution was successful, where 71.9-100% shoot tips survived treatment before immersion to LN and 31-40% survived after re-warming from LN. Regrowth of cryopreserved shoot tips with new well-formed leaves was obtained after four weeks of culture

Alteration of Flower Color in Viola cornuta cv. “Lutea Splendens” through Metabolic Engineering of Capsanthin/Capsorubin Synthesis

Flower color is an important characteristic that determines the commercial value of ornamental plants. The development of modern biotechnology methods such as genetic engineering enables the creation of new flower colors that cannot be achieved with classical methods of hybridization or mutational breeding. This is the first report on the successful Agrobacterium-mediated genetic transformation of Viola cornuta L. The hypocotyl explants of cv. “Lutea Splendens” variety with yellow flowers were transformed with A. tumefaciens carrying empty pWBVec10a vector (Llccs−) or pWBVec10a/CaMV 35S::Llccs::TNos vector (Llccs+) for capsanthin/capsorubin synthase gene (Llccs) from tiger lily (Lilium lancifolium). A comparative study of shoot multiplication, rooting ability during culture in vitro, as well as phenotypic characteristics of untransformed (control) and transgenic Llccs− and Llccs+ plants during ex vitro growth and flowering is presented. Successful integration of Llccs transgene allows the synthesis of red pigment capsanthin in petal cells that gives flowers different shades of an orange/reddish color. We demonstrate that the ectopic expression of Llccs gene in ornamental plants, such as V. cornuta “Lutea Splendens” could successfully be used to change flower color from yellow to different shades of orange

Interview with architect Philippe Rahm: Imagination without scientific knowledge is weak

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STEPEN BELINE PAMUČNE TKANINE DOBIJENE IZBELJIVANJEM POMOĆU FLUORESCENTNOG SREDSTVA UVITEKS BHT I H2O2

In this paper, the influence of fluorescent bleaching agent or UVITEX BHT optical bleach, on the degree of whiteness of cotton fabric was examined. In the first phase of the test, the bleaching of cotton with the bleaching agent hydrogen peroxide was done, and in the second phase, the effect of fluorescent bleaching agents on the whiteness of cotton wool was tested, which was bleached in the first phase by chemical agents.The color strength (K/S) and CIE L*a*b* was examined under Coloreye - 300 spectrophotometer, too. The whiteness degree achieved with bleaching agent UVITEX BHT is almost twice larger than with chemical bleaching compounds, while the largest WI cotton fabrics whitened with hydrogen peroxide using the single-bone stress method was obtained for the concentration of the fluorescent UVITEX BHT bleach of 0.1%.U ovom radu analiziran je uticaj fluorescentnog sredstva za beljenje, UVITEKS BHT optičkog belila, na stepen beline pamučne tkanine. U prvoj fazi izvršeno je beljenje pamuka vodonik-peroksidom kao sredstvom za izbeljivanje, a u drugoj fazi praćen je uticaj fluorescentnih sredstava za beljenje na belinu pamučne tkanine, koja je u prvoj fazi beljena hemijskim sredstvom. Snaga boje (K/S) i CIE L* a* b* parametri određeni su pomoću spektrofotometra Coloreye – 3000. Stepen beline postignut sredstvom za izbeljivanje UVITEKS BHT gotovo je dvostruko veći nego stepen beline ostvaren hemijskim jedinjenjima za izbeljivanje. Najveći stepen beline (WI) pamučne ostvaren je pri koncentraciji fluorescentnog izbeljivača UVITEKS BHT od 0,1%