Efficient genetic transformation of Lotus corniculatus L. using a direct shoot regeneration protocol, stepwise hygromycin B selection, and a super-binary Agrobacterium tumefaciens vector

Cotyledons from 6-day-old Lotus corniculatus cv. Bokor seedlings, transversally cut into two halves, were capa­ble of regenerating buds without intervening callus formation. The explants were co-cultivated with the Agrobacterium tumefaciens LBA4404/pTOK233 superbinary vector carrying the uidA-intron gene and the genes hpt and nptII. They were cultured for 14 days on a regeneration medium, then subjected to a stepwise hygromycin B selection procedure consisting of gradually increasing antibiotic concentrations (5-15 mg L-1) over 21 weeks. Transformed shoots were obtained within 5 months after co-cultivation. Out of 124 initially co-cultivated explants, 52 (42%) plants survived hygromycin B selection. The presence of transgenes in regenerated plants was verified by β-glucuronidase histochemical assays and PCR analysis for the presence of uidA gene sequences. Hygromycin B-resistant and PCR-positive T0 plants were cultured in the greenhouse to produce flowers and seeds. The obtained data demonstrate that the reported transformation protocol could be useful for introducing agriculturally important genes into the new L. corniculatus cultivar Bokor

Apple dihydrochalcones phloridzin and phloretin as novel allelochemicals

Sekundarni metaboliti jabuke (Malus × domestica Borkh.) florizin i floretin su dihidrohalkoni visoko specifični za vrste roda Malus. Autoalelopatski potencijal ovih fenolnih jedinjenja se ogleda u suzbijanju rasta klijanca i sadnica zasađenih na mestu prvobitnih voćnjaka jabuke. U osnovi njihove fitotoksičnosti su ultrastrukturne i molekularne promene koje se ogledaju u povećanju sadržaja malondialdehida, prolina i aktivnosti antioksidativnih enzima: superoksid-dismutaze, peroksidaza i katalaza, kao i nivoa ekspresije proteina uključenih u odbrambene mehanizme ćelije. Najnovija istraživanja ukazuju na fitotoksičnost ovih dihidrohalkona i prema drugim biljnim vrstama, kao npr. korovskoj model biljci Arabidopsis thaliana. Tretman floretinom izaziva značajnu inhibiciju rastenja i razvića klijanaca A. thaliana, poremećaj gravitropskog odgovora i pojavu morfoloških i ultrastrukturnih malformacija u čijoj osnovi leži promena ekspresije CDKA1;1, CDKB2;1, CYCA3;1 i CYCB2;4 gena ćelijskog ciklusa, kao i gena uključenih u održanje homeostaze auksina (PINs, AUX1, LAX3, ABCBs, TAA1 i YUCs) i biosinteze giberelina (GA20ox2 i GA3ox1). Ovo otvara mogućnost za dalja istraživanja njihovog alelopatskog delovanja ali i potencijalnu primenu kao ekološki bezbednih bioherbicida.Phloridzin and phloretin are dihydrochalcones highly specific to domestic apple (Malus × domestica Borkh.) and other species from genus Malus. Auto-allelopathic potential of these phenolics was recognized in ‘Apple Replant Disease’ (ARD) characterized by stunted growth, reduced root system and low yields in apple trees replanted on lands that previously supported apple orchards. Physiological basis of phloridzin and phloretin autotoxicity are ultrastructural and molecular alterations that include increased catalase, peroxidases and superoxide dismutase activities, raised malondialdehyde and proline contents, and high expression of genes involved in cell defence system. The latest research on model plant Arabidopsis thaliana indicates phytotoxicity of these dihydrochalcones towards other plant species. Phloretin inhibits A. thaliana seedlings growth and development inducing agravitropic phenotype and morphological and ultrastructural malformations in treated seedlings. Altered expression of CDKA1;1, CDKB2;1, CYCA3;1 and CYCB2;4 cell cycle genes and genes involved in auxin homeostasis (PINs, AUX1, LAX3, ABCBs, TAA1 and YUCs) and gibberellin biosynthesis (GA20ox2 and GA3ox1) are in the physiological bases of phloretin phytotoxicity. This mechanism makes phloretin a prospective candidate for an eco-friendly bioherbicide and paves the way for further research of phloretin role in ARD

Efficient genetic transformation of Impatiens hawkerii Bull. (Balsamiaceae) using agrobacterium rhizogenes

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-2301

Effect of nitrogen salts on the growth of Ceratonia siliqua L. Shoot cultures

Effects of reduced nitrogen salt nutriton on the growth, lenticel hypertrophy and anthocyanin accumulation of carob (Ceratonia siliqua L.) shoot cultures were investigated in conditions of light and darkness. Growth of shoot cultures was not significantly affected until nitrogen salts were reduced to less than ¼ of full-strength MS (Murashige and Skoog, 1962) values. Cultures in darkness were less affected and their main shoots even increased in length. Appearance of hypertrophied lenticels in light decreased, while in darkness they were absent in all treatments. Reduced nitrogen salt nutrition strongly affected anthocyanin accumulation of shoots and leaves, which greatly increased in both light and darkness. .Kod kultura izdanaka rogača ispitivani su efekti smanjenja koncentracije azotnih soli u podlozi MS mineralnog rastvora na multiplikaciju i izduživanje izdanaka, hipertrofiju lenticela i sintezu i akumulaciju antocijanina na svetlosti i u mraku. Smanjenje koncentracije N u podlozi bitno menja parametre rastenja izdanaka (multiplikaciju i izduživanje) tek na ¼ (na svetlosti) tj. 1/10 (u mraku). Hipertrofirane lenticele se ne razvijaju na etioliranim izdancima gajenim u mraku dok su kod izdanaka gajenim na svetlosti brojne. Smanjenjem koncentracije N u podlozi broj HL se smanjuje kao i njihovo akropetalno prostiranje. Sinteza i akumulacija antocijanina u izdancima i listovima rogača bila je direktno zavisna od koncentracije N u podlozi i na svetlosti i u mraku. .nul

Efekti salicilne kiseline na morfološke osobine i formiranje krtola kod netransformisanog i AtCKX2-transformisanog krompira (Solanum tuberosum L. cv Désirée) gajenog in vitro

Salicilna kiselina (SA) je biljni regulator rastenja čije su glavne fiziološke funkcije odbrana od biotičkog stresa i termogeneza tokom cvetanja kod određenih biljnih vrsta. U novije vreme sve je više podataka o ulozi SA u odbrani biljaka od abiotičkog stresa. SA se često označava kao biljni hormon, mada su podaci o njenim specifičnim efektima na rastenje i razviće kod biljaka sporadični i ograničeni na mali broj vrsta. U literaturi nema podataka o efektima SA na rastenje i razviće krompira, kao ni o interakciji SA sa biljnim hormonima koji regulišu ekonomski značajan proces tuberizacije kod ove vrste. Cilj sprovedenog istraživanja je bio da se utvrdi da li egzogeno primenjena SA ima efekte na morfološke osobine i tuberizaciju kod krompira gajenog in vitro. Ispitivan je efekat SA kod netransformisanog krompira (K) i jedne linije AtCKX2-transformisanog krompira (AtCKX2-51), koju karakteriše veoma nizak sadržaj bioaktivnih citokinina (CK).1 SA je primenjivana u niskim koncentracijama (0,01–10 μM), dodavanjem u podloge za gajenje izdanaka krompira in vitro, a morfološki parametri, uključujući i parametre tuberizacije, određivani su u kulturama starim 30 dana. Rezultati su pokazali da SA (u najvećoj primenjenoj koncentraciji) ima efekat samo na pojedine procese rastenja i razvića, a da ne remeti tuberizaciju. Uočeni efekti se razlikuju kod K i AtCKX2-51 krompira. Prikazani rezultati sprovedenog istraživanja ukazuju na moguću interakciju između SA i CK u regulaciji rastenja i razvića krompira in vitro

The effects of β-lactam antibiotics and hygromycin B on de novo shoot organogenesis in apple cv. Golden Delicious

Since the genetic transformation of the apple is strongly genotype-dependent and generally inefficient, the evaluation of factors affecting shoot regeneration are crucial for the establishment of a successful transformation process. In this report, we evaluated the effects of the β-lactam antibiotics meropenem and timentin on in vitro regeneration via de novo shoot organogenesis from leaf explants of apple cv. Golden Delicious, as well as on the growth of the Agrobacterium tumefaciens strain EHA 105, and compared them with the commonly used β-lactam cefotaxime. Also, we report for the first time the effect of hygromycin B as a selective agent in the domesticated apple, as regards shoot regeneration and shoot multiplication efficiency. We observed that cefotaxime and timentin at concentrations higher than 100 mg L-1 were sufficient to prevent Agrobacterium growth during a two-week period, while meropenem exhibited an inhibitory effect on bacterial growth at all tested concentrations (25-150 mg L-1). Cefotaxime at a concentration of 300 mg L-1 increased the number of regenerated shoots per explant (9.39) in comparison with the control (7.67). In contrast to cefotaxime, meropenem and timentin caused a decrease in shoot regeneration efficiency, but larger and more developed shoots were obtained on meropenem (25-125 mg L-1) after the same period of cultivation. Hygromycin B at a concentration of 5 mg L-1 or higher completely inhibited shoot regeneration and induced explant tissue necrosis. Therefore, the selection procedure with a final concentration of 4 mg L-1 throughout organogenesis and 10 mg L-1 for further shoot growth and multiplication is recommended for an efficient transformation process in apple cv. Golden Delicious

Efficient genetic transformation of Impatiens hawkerii Bull. (Balsamiaceae) using agrobacterium rhizogenes

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-2301

Effect of nitrogen salts on the growth of Ceratonia siliqua L. Shoot cultures

Effects of reduced nitrogen salt nutriton on the growth, lenticel hypertrophy and anthocyanin accumulation of carob (Ceratonia siliqua L.) shoot cultures were investigated in conditions of light and darkness. Growth of shoot cultures was not significantly affected until nitrogen salts were reduced to less than ¼ of full-strength MS (Murashige and Skoog, 1962) values. Cultures in darkness were less affected and their main shoots even increased in length. Appearance of hypertrophied lenticels in light decreased, while in darkness they were absent in all treatments. Reduced nitrogen salt nutrition strongly affected anthocyanin accumulation of shoots and leaves, which greatly increased in both light and darkness. .Kod kultura izdanaka rogača ispitivani su efekti smanjenja koncentracije azotnih soli u podlozi MS mineralnog rastvora na multiplikaciju i izduživanje izdanaka, hipertrofiju lenticela i sintezu i akumulaciju antocijanina na svetlosti i u mraku. Smanjenje koncentracije N u podlozi bitno menja parametre rastenja izdanaka (multiplikaciju i izduživanje) tek na ¼ (na svetlosti) tj. 1/10 (u mraku). Hipertrofirane lenticele se ne razvijaju na etioliranim izdancima gajenim u mraku dok su kod izdanaka gajenim na svetlosti brojne. Smanjenjem koncentracije N u podlozi broj HL se smanjuje kao i njihovo akropetalno prostiranje. Sinteza i akumulacija antocijanina u izdancima i listovima rogača bila je direktno zavisna od koncentracije N u podlozi i na svetlosti i u mraku. .nul

In vitro multiplication of oryzacystatin II transformed Alfalfa on GA3-containing medium

Projekat ministarstva br. 14302

Growth habit and photosynthetic activity of shoot cultures of Medicago sativa L. transformed with the oryzacystatin II gene

Abstract Background and Purpose: Introduction of a foreign gene into plant genome may induce morphological and physiological alterations in transgenic plants. The purpose of this study was to assess the impact of gene introduction on morphology and photosynthetic activity of two transgenic alfalfa shoot cultures. Material and Methods: In vitro maintained shoot cultures of alfalfa (Medicago sativa L. cv. Zaječarska 83) that were transformed with the oryzacystatin II (OC-II) gene and propagated on growth regulator-free medium were subjected to analysis of morphological characteristics and photosynthetic activity. For analysis of phological characteristics, the length of the main axis, the number of axillary and adventitious shoots, total number of leaves and the number of senesced leaves as well as plant dry mass were determined. Net photosynthetic rate was determined as CO2 influx using a LI-6200 closed photosynthesis system. Photosynthetic function was assessed as the rate of basic chlorophyll fluorescence and determined with a Plant Stress Meter by method of induced fluorometry. Chlorophyll content in leaf samples was determined spectrophotometrically. Results: The most striking feature of transformed cultures was reduced apical dominance and the absence of adventitious roots. In comparisonwith control, main axis length was also reduced. In addition, a general decrease in photosynthesis in transgenic shoots was also observed. Conclusion: Both morphology and the key processes in photosynthesis were modified in transgenic shoots.However, the fact that transgenic plants carry a gene that confers pest resistance gives potential agronomic value to at least some of the clones whose morphological characteristics and photosynthetic acivity were minimally disturbed