Dragoljub Grubišić

The effects of potassium nitrate and NO-donors on phytochrome A- and phytochrome B-specific induced germination of Arabidopsis thaliana seeds

Nitrogenous compounds, such as potassium nitrate, potentiate germination of different species of light-requiring seeds. Using light-induced Arabidopsis thaliana seed germination as a model system, our data suggested that only phytochrome A (phyA)-specific induced germination was affected after the exogenous application of nitrates, different nitric oxide (NO)-donors (such as organic nitrates) or sodium nitroprusside. The stimulative effect was very pronounced. Treated seed samples reached maximal germination after very short periods of red-light irradiation. To a far lesser extent, these substances affected phytochrome B (phyB)-specific induced germination. In phyB-specific induced germination, potassium nitrate was most effective, but germination percentages never exceeded 50%. The least effective was sodium nitroprusside, which practically did not affect phyB-specific induced germination. These results were confirmed using corresponding phytochrome mutants.

Effect of nitric oxide - releasing compounds on phytochrome - controlled germination of Empress tree seeds

Using different nitric oxide releasing compounds and appropriate controls we have obtained data strongly suggesting the involvement of nitric oxide in the phytochrome controlled germination of Paulownia tomentosa seeds. Direct detection of nitric oxide, under various experimental conditions, was performed by a spin-trapping technique combined with electron paramagnetic resonance (EPR) spectroscopy. The addition of methylene blue prevented light-induced and NO donors-potentiated germination of P. tomentosa seeds. This inhibition could be completely overcome by addition of gibberellin. The promotive effect of nitrite was pH dependent, maximally pronounced at the pH range where nitrite undergoes dismutation and liberates nitric oxide. Under these conditions, nitrite exerted its efficacy at the same concentrations at which nitric oxide releasing compounds such as sodium nitroprusside (SNP), S-nitroso acetylpenicillamine (SNAP), and 3-morpholinosydnonimine (SIN-1), were the most effective. Likewise, the potentiation of P. tomentosa seed germination could be achieved by chemical reduction of nitrite with Na2S2O4 during which liberation of nitric oxide could be detected.

Agrobacterium rhizogenes-mediated transformation and plant regeneration of four Gentiana species

Shoots of micropropagated Gentiana acaulis, G. cruciata, G. lutea, and G. purpurea were inoculated with suspensions of Agrobacterium rhizogenes cells, strains ATCC 15834 or A4M70GUS. Adventitious roots appeared at the sites of inoculation in all 4 species. Root tips were excised and cultured on growth regulator-free media for 2-6 years. They exhibited very high branching and plagiotropism. Spontaneous bud initiation occurred in roots of G. cruciata. Roots of G. lutea, G. acaulis and G. purpurea were cultured on media with high kinetin concentration, which induced the formation of friable callus tissues. Only in G. purpurea were these calluses organogenic. Regenerated shoots of G. cruciata and G. purpurea gave rise to plants, that displayed the typical phenotypes of A. rhizogenes-transformed plants: short internodes and rolled leaves. In the roots of G. acaulis and G. cruciata, transformed with A. rhizogenes A4M70GUS, a positive reaction with X-gluc indicated the activity of β-glucuronidase. The DNA extracted from hairy roots and from the roots of transgenic plants hybridized with the appropriate genomic probes in Southern blotting. This is taken as evidence of the stable genetic transformation in the 4 Gentiana species.

Direct Regeneration of Shoots from Hairy Root Cultures of Centaurium erythraea Inoculated with Agrobacterium rhizogenes

Stable transformation and expression of transgenes was achieved in Centaurium erythraea Gillib. using Agrobacterium rhizogenes-mediated system. Five hairy root clones exhibited the transformed phenotype. Shoot regeneration, with green organized structures, was apparent in 4 clones, after the first subculture on Murashige and Skoog (MS) half strength medium. The integration of Ri-plasmid T-DNA was confirmed by polymerase chain reaction (PCR) analyses.

Micropropagation of four Gentiana species (G. lutea, G. cruciata, G. purpurea and G. acaulis)

The growth of axillary shoots was initiated on nodal stem segments, excised from aseptically grown seedlings of Gentiana acaulis L., G. cruciata L., G. lutea L. and G. purpurea L. In later subcultures, a basal callus tissue developed on the shoots, giving rise to de novo formed buds. Optimum benzyladenine and indoleacetic acid combinations for shoot development were established. They were slightly different in the four species. From 35-70% of shoots rooted spontaneously, except in G. lutea, in which adventitious roots were induced by applying naphthaleneacetic acid. It was conduded that the four Gentiana species were amenable to propagation in vitro.

Nitrogen oxides as environmental sensors for seeds

Nitrates have long been known to promote seed germination in many species, and various proposals have been made regarding the role of nitrates in this process. One hypothesis suggests a possible ecological role for nitrate, providing the seeds with a report of soil nitrogen status and the proximity of already established plants. However, nitrogen oxides (NO, NO 2 ), which are present in the soil, also may be information carriers that indicate not only soil nitrate content, but also microbial activity and, therefore, soil quality. Because of annual variation of soil trace gas fluxes, seeds could be provided with information on seasonal and climate changes in their surroundings. Thus, nitrogen oxides would be the outer information carriers providing the seeds with integral data about many important factors required for successful germination and seedling establishment.

Nepetalactone content in shoot cultures of three endemic Nepeta species and the evaluation of their antimicrobial activity.

Rapid micropropagation of Nepeta rtanjensis, N. sibirica and N. nervosa was performed. Qualitative and quantitative nepetalactone content in methanol extracts of in vitro grown plants was analysed by reverse-phase HPLC coupled with UV and MS detection. Only trans,cis-nepetalactone was detected in shoots of N. rtanjensis, while cis,trans-nepetalactone stereoisomer was present in N. sibirica. No nepetalactone was observed in shoots of N. nervosa. The antimicrobial activity of methanol extracts, against eight bacterial and eight fungal species, was evaluated. All the tested extracts showed significant antibacterial and strong antifungal activity. However, N. rtanjensis extract exhibited the best antimicrobial potential.

Micropropagation of Salvia brachyodon through nodal explants

A protocol for in vitro propagation of Balkan endemic plant Salvia brachyodon Vandas from nodal segments was developed. 6-benzylaminopurine was more effective in axillary buds promotion when compared to thidiazuron. The rooting of regenerated shoots was induced by transferring them to the media supplemented with auxins. All tested auxins (indole-3-acetic acid, indole-3-butyric acid, and α-naphthaleneacetic acid) stimulated the rooting of S. brachyodon shoots. The acclimatization of in vitro rooted shoots was successful.

In vitro regeneration and transformation of Blackstonia perfoliata

In vitro root culture of yellow wort (Blackstonia perfoliata (L.) Huds.) was initiated on Murashige and Skoog (MS) medium. In the presence of benzylaminopurine (BAP) numerous adventitious buds formed, which developed into shoots. Presence of indole-3-butyric acid (IBA) in media significantly decreased number of buds, but increased development of lateral roots. On hormone-free medium shoots successfully rooted and developed flowers and viable seeds that formed another generation. Shoot cultures of B. perfoliata inoculated with suspension of Agrobacterium rhizogenes strain A4M70GUS developed hairy roots at 3 weeks and they were cultured on hormone-free MS medium. Spontaneous shoot regeneration occurred in 3 clones.

Susceptibility of pathogenic bacteria and fungi to essential oils of wild Daucus carota

The compositions of the essential oils of wild Daucus carota L. (Apiaceae) from the ripe fruits, unripe fruits, flowers, root, leaves, and stem, collected in Serbia, were analyzed by GC and GC/MS. The oils from all samples contained α-pinene (7.05–51.23%) and sabinene (2.68–36.69%) as major constituents. The other dominant compound in ripe and unripe fruits essential oil was α-muurolene (8.23–10.97%). Antibacterial and antifungal properties of these oils, against eight bacterial and eight fungal strains, were tested by a microdilution technique. The most prominent biological activity was achieved by isolated essential oils of ripe and unripe fruits of D. carota.