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

Virus elimination from ornamental plants using in vitro culture techniques

Viruses are responsible for numerous epidemics in different crops in all parts of the world. As a consequence of their presence great economic losses are being incurred. In addition to the development of sensitive techniques for detection, identification and characterization of viruses, substantial attention has also been paid to biotechnological methods for their elimination from plants. In this review article, the following biotechnological in vitro culture techniques for virus elimination from ornamental plants are presented: meristem culture, thermotherapy, chemotherapy, cryotherapy or a combination of these methods. The plant species, as well as the type of virus determine the choice of a most suitable method. The state of the art in investigation of virus elimination from Impatiens sp. in Serbia is summarized.Virusi su odgovorni za brojne epidemije na različitim usevima u svim delovima sveta. Posledica njihovog prisustva su velike ekonomske štete, pa osim razvoju osetljivih tehnika za detekciju, identifikaciju i karakterizaciju virusa, velika pažnja se poklanja i biotehnološkim metodama za njihovu eliminaciju. U ovom preglednom radu predstavljene su tehnike in vitro kulture za eliminaciju virusa iz biljnog materijala: kultura meristema, termoterapija, hemoterapija, krioterapija ili kombinacija ovih metoda. Koja će metoda biti primenjena zavisi od biljne vrste, kao i od vrste virusa. U radu je dat pregled istraživanja na eliminaciji virusa iz Impatiens sp. u Srbiji.Projekat ministarstva br. TR-31019 and III4300

Dimethyl sulfoxide improves sensitivity and specificity of RT-PCR and qRT-PCR amplification of low-expressed transgenes

The expression of transgenes in a host plant may be low for a number of reasons. Both low expression and poor specificity of amplification were encountered during analysis of the expression of the Arabidopsis cytokinin oxidase/dehydrogenase (AtCKX1) gene in transgenic Centaurium erythraea. The optimization of the PCR protocol involved a gradient of annealing temperatures, as well as the application of seven PCR enhancers: formamide, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, trehalose, BSA and Tween-20. The best results for AtCKX1 amplification were obtained at 55.1ºC, with the addition of 5% DMSO. Glycerol and trehalose also improved the sensitivity of amplification, while formamide, ethylene glycol and BSA enhanced only the amplification of control purified targets, but not the transcripts. Tween-20 inhibited PCR. DMSO enhanced AtCKX1 PCR amplification and improved the specificity of qPCR amplification, as well as the assay reproducibility. This work emphasizes the usefulness of additives, which are rarely used for PCR optimization in real-time experiments.Projekat ministarstva br. ON173015 i br. ON17302

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

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

Projekat ministarstva br. 14302

The activity of peroxidases and superoxide dismutases in transgenic phosphinothricin-resistant lotus corniculatus shoots

The aim of this study was to investigate the effect of the non-selective herbicide Basta®, with phosphinothricin (PPT) as active compound, on antioxidative enzymes in transgenic PPT-resistant Lotus corniculatus cv. Bokor shoots grown under in vitro conditions. Analysis of peroxidases (POD) and superoxide dismutases (SOD) showed that the activity of these enzymes was affected by herbicide application more in control PPT-sensitive than in transformed resistant shoots. These results confirmed the capacity of genetically modified resistant shoots to reduce the influence of PPT on the physiological processes and disturbance of oxidative balance in cells.Projekat ministarstva br. 14302

Virus elimination from ornamental plants using in vitro culture techniques

Viruses are responsible for numerous epidemics in different crops in all parts of the world. As a consequence of their presence great economic losses are being incurred. In addition to the development of sensitive techniques for detection, identification and characterization of viruses, substantial attention has also been paid to biotechnological methods for their elimination from plants. In this review article, the following biotechnological in vitro culture techniques for virus elimination from ornamental plants are presented: meristem culture, thermotherapy, chemotherapy, cryotherapy or a combination of these methods. The plant species, as well as the type of virus determine the choice of a most suitable method. The state of the art in investigation of virus elimination from Impatiens sp. in Serbia is summarized.Virusi su odgovorni za brojne epidemije na različitim usevima u svim delovima sveta. Posledica njihovog prisustva su velike ekonomske štete, pa osim razvoju osetljivih tehnika za detekciju, identifikaciju i karakterizaciju virusa, velika pažnja se poklanja i biotehnološkim metodama za njihovu eliminaciju. U ovom preglednom radu predstavljene su tehnike in vitro kulture za eliminaciju virusa iz biljnog materijala: kultura meristema, termoterapija, hemoterapija, krioterapija ili kombinacija ovih metoda. Koja će metoda biti primenjena zavisi od biljne vrste, kao i od vrste virusa. U radu je dat pregled istraživanja na eliminaciji virusa iz Impatiens sp. u Srbiji.Projekat ministarstva br. TR-31019 and III4300

Dimethyl sulfoxide improves sensitivity and specificity of RT-PCR and qRT-PCR amplification of low-expressed transgenes

The expression of transgenes in a host plant may be low for a number of reasons. Both low expression and poor specificity of amplification were encountered during analysis of the expression of the Arabidopsis cytokinin oxidase/dehydrogenase (AtCKX1) gene in transgenic Centaurium erythraea. The optimization of the PCR protocol involved a gradient of annealing temperatures, as well as the application of seven PCR enhancers: formamide, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, trehalose, BSA and Tween-20. The best results for AtCKX1 amplification were obtained at 55.1ºC, with the addition of 5% DMSO. Glycerol and trehalose also improved the sensitivity of amplification, while formamide, ethylene glycol and BSA enhanced only the amplification of control purified targets, but not the transcripts. Tween-20 inhibited PCR. DMSO enhanced AtCKX1 PCR amplification and improved the specificity of qPCR amplification, as well as the assay reproducibility. This work emphasizes the usefulness of additives, which are rarely used for PCR optimization in real-time experiments.Projekat ministarstva br. ON173015 i br. ON17302

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

Projekat ministarstva br. 14302

Agrobacterium-mediated transformation of ornamental species: A review

Integration of desirable traits into commercial ornamentals using genetic engineering techniques is a powerful tool in contemporary biotechnology. However, these techniques have had a limited impact in the domain of ornamental horticulture, particularly floriculture. Modifications of the color, architecture or fragrance of the flowers as well as an improvement of the plant tolerance/resistance against abiotic and biotic stresses using plant transformation techniques, is still in its infancy. This review focuses on the application of Agrobacterium-mediated transformation, a major plant genetic engineering approach to ornamental plant breeding and the impact it has had to date.Serbian Ministry of Education, Science and Technological Research {[}TR 31019