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Common centaury (Centaurium erythraea Rafn.) is a plant species that can inhabit saline soils. It is known as a plant with high spontaneous regeneration potential in vitro. In the present work we evaluated shoots and roots salinity tolerance of non-transformed and three AtCKX transgenic centaury lines to graded NaCl concentrations (0, 50, 100, 150, 200 mM) in vitro. Overexpression of AtCKX genes in transgenic centaury plants resulted in an altered cytokinins (CKs) profile leading to a decline of bioactive CK levels and, at the same time, increased contents of storage CK forms, inactive CK forms and/or CK nucleotides. Significant increment of fresh shoot weight was obtained in shoots of non-transformed and AtCKX1 transgenic line only on medium supplemented with 50 mM NaCl. However two analysed AtCKX2 transgenic lines reduced shoot growth at all NaCl concentrations. In general, centaury roots showed higher tolerance to salinity than shoots. Non-transformed and AtCKX1 transgenic lines tolerated up to 100 mM NaCl without change in frequency of regeneration and number of regenerated plants. Roots of two analysed AtCKX2 transgenic lines showed different regeneration potential under salt stress. Regeneration of transgenic AtCKX2-26 shoots even at 200 mM NaCl was recorded. Salinity stress response of centaury shoots and roots was also evaluated at biochemical level. Free proline, malondialdehyde and hydrogen peroxide content as well as antioxidative enzymes activities were investigated in shoots and roots after 1, 2, 4 and 8 weeks. In general, adition of NaCl in culture medium elevated all biochemical parameters in centaury shoots and in roots. Considering that all analysed AtCKX transgenic centaury lines showed altered salt tolerance to graded NaCl concentrations in vitro it can be assumed that CKs might be involved in plant defence to salt stress conditions. This is the peer reviewed version of the following article: Trifunović‐Momčilov M, Paunović D, Milošević S, Marković M, Jevremović S, Dragićević IČ, Subotić A. Salinity stress response of non‐transformed and AtCKX transgenic centaury (Centaurium erythraea Rafn.) shoots and roots grown in vitro. Ann Appl Biol. 2020;aab.12593, which has been published in final form at [http://dx.doi.org/10.1111/aab.12593]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.

Snake's head fritillary (Fritillaria meleagris L.) is an endangered and protected plant species in the family Liliaceae mainly inhabiting moist grasslands. As a result of negative anthropogenic impacts such as ameliorization and urbanization, the habitats rapidly disappear and/or are affected by pollutants such as heavy metals. The aim of the study is to estimate level of sensitivity and/or resistance of snake's head fritillary to nickel (Ni), a metabolically important microelement though also toxic when present at excessive amounts in soil. Plants grown in culture conditions were subjected to increasing Ni concentrations (0-5 mM) for four weeks. Relative growth rate (RGR) and certain indicators of oxidative stress - soluble proteins, malondialdehyde content and antioxidant enzyme activities were measured. A decrease of RGR based on fresh weight and protein content was noted in response to growing Ni concentrations (0-5 mM) whereas malondialdehyde (indicator of lipid peroxidation) content increased. Antioxidant response was observed under Ni stress, with large increases in catalase and peroxidases. The results suggest induction of oxidative stress under Ni toxicity, but also demonstrate capacity of F. meleagris to upregulate its antioxidative defense, thus indicating certain degree of plant's tolerance to Ni exposure.

One of the leading areas of modern biotechnology is to develop plants resistant to pathogens using genetic engineering methods. The genetic transformation of I. hawkerii Bull. with Agrobacterium tumefaciens C58C1pac1 was observe in this study, and the aim was to derive plants carrying the gene for pac1 resistance to tomato spotted wilt virus and other plant RNA viruses. I. hawkerii Bull. transformants on a selective medium rapidly failed, and not a single clone survived the fifth passage. This decline was due to great susceptibility of this type to kanamycin or failure of the transformation. Jedna od vodećih oblasti savremene biotehnologije je dobijanje biljaka otpornih na patogene pomoću metoda genetičkog inženjeringa. U ovom radu je proučavana genetička transformacija Impatiens hawkerii Bull. pomoću Agrobacterium tumefaciens C58C1pac1, a cilj je dobijanje biljaka koje nose pac1 gen za rezistentnost na virus bronzavosti paradajza i druge biljne RNK viruse. Transformanati I. hawkerii Bull. na selektivnoj podlozi su brzo propadali, a ni jedan klon nije preživeo peti pasaž. Ovo propadanje je posledica velike osetljivosti ove vrste na kanamicin ili neuspešnosti transfomacije. Projekat ministarstva br. TR 31019

In order to detect and identify viruses in the plant material or to confirm the expression of a gene after genetic transformation it is necessary to have a fast, simple and reliable method for the extraction of DNA and RNA. In this study, we tested a protocol for DNA and three protocols for RNA extraction from plants of I. walleriana L. RNA obtained by different extraction methods significantly differed in its yield and purity. The optimum method proved to be the one applicable to plant species that contain large amounts of secondary metabolites (polyphenols and polysaccharides). Nucleic acid extracted by the application of these methods showed satisfactory results in PCR and RT-PCR analysis. Za detekciju i identifikaciju virusa u biljnom materijalu ili potvrdu ekspresije nekog gena nakon genetičkih transformacija neophodno je imati brzu, jednostavnu i pouzdanu metodu za ekstrakciju DNK i RNK. U ovom radu smo testirali jedan protokol za izolaciju DNK i tri za ekstrakciju RNK iz biljaka Impatiens walleriana L. RNK dobijena primenom različitih metoda za ekstrakciju znatno se razlikovala po prinosu i čistoći. Kao optimalna metoda se pokazala ona koja se primenjuje za biljne vrste koje sadrže velike količine sekundarnih metabolita (polifenola i polisaharida). Nukleinske kiseline ekstrahovane primenom ovih metoda pokazale su zadovoljavajuće rezultate u PCR i RT-PCR analizama. Projekat ministarstva br. TR 31019

We investigated possibility of ex vitro genetic transformation of Impatiens walleriana using Agrobacterium rhizogenes A4M70GUS and Agrobacterium tumefaciens C58C1pac1 bacterial suspension mixture with aim to obtain composite plants carrying GUS and pac1 genes. Compared to the control, treated plants exhibited increase of 27% in number of roots, while a difference in shoot length and number of leaves was not statistically significant. The PCR analysis did not confirmed integration of transgenes in the plant genome. The reason for this can be inadequate sampling or low transformation efficiency. U ovom radu je proučavana mogućnost ex vitro genetičke transformacije Impatiens walleriana pomoću smeše bakterijskih suspenzija Agrobacterium rhizogenes A4M70GUS i Agrobacterium tumefaciens C58C1 pac1, a cilj je dobijanje kompozitnih biljaka koje nose GUS i pac1 gene. Tretirane biljke su imale 27% veći broj korenova, a razlike u dužini izdanaka i broju listova u odnosu na kontrolu nisu bile statistički značajne. PCR analizom nije potvrđena inkorporacija navedenih gena u biljni genom, što može biti posledica lošeg izbora uzoraka ili malog procenta transformacije. Projekat ministarstva br. TR 31019

The content of anthocyanin was studied in different tissues of Impatiens walleriana L. and Impatiens hawkeri Bull., which are annual herbaceous plants of the family Balsamineaceae, and are significantly applied in horticulture. The anthocyanin content was measured spectrophotometrically in nodes, internodes, leaf petioles, leaves, petals and pedicels. Studies show that the accumulation of anthocyanin in all observed plants was higher in younger parts of the shoot (higher nodes and internodes). In Impatiens hawkeri with orange and pink flowers, the anthocyanin accumulation in the node was significantly higher than in the internode. In both species with orange and pink flowers, petals had more anthocyanin than pedicels. A varying content of anthocyanin in plants of both species with orange and pink flowers in different tissues, was higher and statistically more significant than in plants with white flowers. . U ovom radu proučavan je sadržaj antocijanina u različitim tkivima Impatiens walleriana L. i Impatiens hawkeri Bull. Ove vrste su jednogodišnje biljke iz familije Balsamineaceae, i imaju značajnu primenu u hortikulturi. Spektrofotometrijska očitavanja sadržaja antocijanina su obavljena u nodusima stabla, internodijama, lisnim drškama, listovima, cvetnim drškama i kruničnim listićima ovih biljaka. Istraživanja su pokazala da je akumulacija antocijanina kod svih proučavanih biljaka veća u mlađim delovima stabla (višim nodusima i internodijama). Kod Impatiens hawkeri sa narandžastim i ciklama cvetovima akumulacija antocijanina u nodusima je značajno veća nego u internodijama. Kod obe vrste, sa narandžastim i ciklama cvetovima, antocijanina ima više u kruničnim listićima u odnosu na cvetne drške. Variranje sadržaja antocijanina kod biljaka obe vrste sa narandžastim i ciklama cvetovima, u različitim tkivima, bilo je veće, i statistički značajnije nego kod biljaka sa belom bojom cvetova. . Projekat ministarstva br. TR23010A

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A simple protocol has been developed for plant regeneration by somatic embryogenesis of Southern Adriatic iris (Iris pseudopallida Trinajstić), an endemic species of the Balkan Peninsula. Somatic embryogenesis was induced in zygotic embryo culture on media supplemented with 2,4-dichlorophenoxy acetic acid (2-10 mgL-1) as the sole plant growth regulator, where both embryogenic calli and somatic embryos were induced. Subsequent decrease of 2,4-D in the media promoted formation of somatic embryos. Developed somatic embryos germinated on medium without growth regulators. The regenerated plantlets had diploid chromosome number. Planted plantlets acclimatized very well under greenhouse and garden conditions. Jednostavan protokol za regeneraciju biljaka procesom somatske embriogeneze razvijen je za južno-jadranski iris (Iris pseudopallida Trinajstić), endemit Balkanskog poluostrva. Somatska embriogeneza je indukovana u kulturi 'zrelih' zigotskih embriona na hranljivoj podlozi obogaćenoj 2,4-dihlorofenoksi sirćetnom kiselinom (2,4-D), (kao jedinim regulatorom rastenja), gde su embriogeni kalus i somatski embrioni formirali. Smanjenje koncentracije 2,4-D u hranljivoj podlozi dovodi do povećanja broja formiranih somatskih embriona. Klijanje somatskih embriona je postignuto na podlozi bez regulatora rastenja. Regenerisane biljčice su imale diploidan broj hromozoma. Aklimatizacija biljaka je bila dobra u uslovima staklenika i bašte. Projekat ministarstva br. 142630

Transformation of Impatiens hawkerii Bull. mediated by Agrobacterium rhizogenes strain A4M70GUS was studied. Hairy roots developed 10 days after inoculation were excised from the shoot explants and transferred onto Murashige and Skoog's (MS) basal medium lacking plant growth regulators. More than 20 hairy root clones were established and eight of them were further analyzed. Each clone differed significantly from the others in growth capacity and lateral branching. Clone C2 showed the highest biomass (20.6 g L-1) as well as the highest number of lateral roots (37 ± 2.2). The transgenic nature of the established hairy root clones was confirmed by GUS assay and PCR analysis. In conclusion, hairy roots were developed for the first time in I. hawkerii Bull., and transgenic hairy root clones showed a distinct morphological nature and growth patterns. Proučavana je genetička transformacija Impatiens hawkerii Bull. posredstvom Agrobacterium rhizogenes soja A4M70GUS. Deset dana posle inokulacije formirali su se transgeni korenovi na eksplantatima izdanaka, a zatim gajeni na Murashige and Skoog's (MS) osnovnoj hranljivoj podlozi bez biljnih regulatora rastenja. Uspostavljene su kulture više od 20 klonova, a 8 je dalje analizirano. Klonovi su se međusobno značajno razlikovali u odnosu na kapacitet rastenja i bočnog grananja. Klon C2 je imao najveću biomasu (20.6 g L-1), kao i najveći broj bočnih korenova (37 ± 2.2). Prisustvo stranih gena u klonovima transgenih korenova je potvrđeno GUS eseja i PCR analize. Transgeni korenovi su dobijeni prvi put kod Impatiens hawkerii Bull. i pokazuju značajne razlike u morfologiji i parametrima rastenja. Projekat ministarstva br. TR-23010

To establish an efficient procedure for in vitro plant regeneration of Impatiens walleriana L., the effects of urea-type cytokinins for chlorofenuron (CPPU) and thidiazuron (TDZ) on the auxillary shoot induction are examined The shoot meristem explants produced multiple axillary shoots when cultured on Murashige and Skoog's (MS) medium containing different concentration of CPPU 411761 TDZ (0.01-1.0 mu M). Explant activity varied significant, del)ending on the cytokinin type as well as concentration. We have found that the 0.1 mu M CPPU induced the largest number of axillary shoots (4.59). This cytokinin was more c effective for the induction and development of axillary shoots than IDZ in the same concentration. The seperated shoots are rooted and elongated on MS medium without hormone. Rooted plantlets were successfully acclimatized. Shoot meristem explants card urea-type cytokinins could he useful for improvement of regeneration procedure and genetic engineering of this important oranmental species. Serbian Ministry of Science [TR6833B]

An efficient plant regeneration protocol via somatic embyogenesis by leaf base Culture of in vitro grown Iris pumila shoots was developed. Induction of embryogenic calli was achieved on MS media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (4,5 mu M each) and some additives (L-proline, casein hydrolysate, adenine sulphate and tyrosine), Further differentiation of embryogenic calli was achieved on MS hormone-free media. and on media supplemented with either BAP (4.5 mu M) or BAP + zeatin (4.5 and 0.2 mu M. respectively), which allowed somatic embryos, as well as shoot-like structures to form. Fully developed somatic embryos germinated on an MS hormone-free medium. An anatomical study confirmed that shoot-like structures represented early germinating stages of somatic embryos, Acclimatization of plants derived from somatic embryos was 64% after 1 year and no morphological variation was observed. (c) 2005 Elsevier B.V. All rights reserved. null

Morphological characteristics of flowers of long term regenerated chrysanthemum, cv. "White Spider", after ten years of micropropagation are investigated. Shoot cultures are established and maintained more than ten years by stem segment culture on MS medium supplemented with BAP and NAA (1.0, 0.1 mgL-1, respectively). Rooting of shoots (100 %) has done on MS medium without hormones and it was very successful after ten years, as well as, after two or eight years of micropropagation. Acclimation of rooted chrysanthemum plantlets at greenhouse conditions was excellent and after appropriate photoperiod "in vitro" plants flowered 90.3 % and have the same flower color, shape and size as mother plants. Flower color changes of "in vitro" plants are observed during another flowering cycle one year after acclimatization. Observed variations of chrysanthemum flowers could be attributed to epigenetic factors. Praćene su morfološke karakteristike cvetova regeneranata cv. "White Spider" posle deset godina gajenja u kulturi in vitro. Izdanci su umnožavani u kulturi segmenata stabla preko aksilarnih pupoljaka na MS podlozi sa α-naftilsirćetnom kiselinom i benzilaminopurinom (0,5 odnosno 1,0 mgL-1), . Praćeno je umnožavanje izdanaka u periodu od devetnaest pasaža (tri godine) kada je došlo do sukcesivnog smanjenja indeksa umnožavanja. Oživljavanje izdanaka je praćeno na MS podlozi bez hormona i bilo je podjednako uspešno posle dve, kao i posle osam i deset godina mikropropagacije. Oživljene biljke hrizantema su uspešno aklimatizovane na uslove staklenika (97 %). Nakon odgovarajućeg fotoperioda, 90,3 % biljaka je cvetalo i imale su identičnu boju cveta matičnim biljkama hrizantema. Promene u boji cveta su uočene kod "in vitro" biljaka u sledećem ciklusu cvetanja, tj. godinu dana posle aklimatizacije. Ove promene bi se mogle pripisati epigenetskim faktorima. null