Nevena Mitić

Agrobacterium-Mediated Transformation and Plant Regeneration of Triticum aestivum L.

The use of two Agrobacterium tumefaciens strains for transformation of Triticum aestivum L. cv. Vesna was studied. Immature embryos, isolated 15 d after pollination, were co-cultivated with the super-binary LBA4404/pTOK233 and the binary AGL1/pDM805 vectors. While the transient GUS-intron expression was high (69.9 and 80.0 %), the number of plants regenerated on selective media containing hygromycin or phosphinotricin did not exceed 0.4 and 0.13 %, respectively. Nevertheless, the regenerated plants were fertile and produced seeds. The T0 plants, as well as the T1 seedlings, displayed the activity in the β-glucuronidase histochemical assay and a positive signal in PCR analysis for the presence of uidA gene sequences in their genomes. The data suggest that the transformation of wheat cv. Vesna with both Agrobacterium strains is feasible.

Efficient genetic transformation of Lotus corniculatus L. and growth of transformed plants in field

An efficient protocol for shoot regeneration and genetic transformation was applied to root segments of a new Lotus corniculatus L. cultivar Bokor. The shoots, that regenerated on root segments, were inoculated with Agrobacterium rhizogenes A4M70GUS, and produced hairy roots, which on media with 0.2 mg dm−3 benzylaminopurine, regenerated shoots. After rooting and acclimation, the transformed plants were planted in the experimental field. Their morphological traits were compared to controls. No signs of the rol genes phenotype were present. The transformants were significantly taller than controls, while there were no significant differences in the leaf area. The glucuronidase activity and the presence of uidA gene was demonstrated in transformed plants of T0 and in seedlings of T1 generations. It is concluded that A. rhizogenes could be a vector of choice for the transfer of desirable genes into the birds foot trefoil genome.

Hairy root exudates of allelopathic weed Chenopodium murale L. induce oxidative stress and down-regulate core cell cycle genes in Arabidopsis and wheat seedlings

The effects of Chenopodium murale root exudates, applied as phytotoxic medias (PMs), were tested on Arabidopsis thaliana and Triticum aestivum. The effects of PMs, where wild-type roots (K), hairy roots derived from roots (R clones) or from cotyledons (C clones) were cultured, were different. K medium suppressed Arabidopsis germination, while other PMs reduced root and leaf elongation and the number of rosette leaves. R media were more phytotoxic than C media. Treatment of Arabidopsis with R8 down-regulated expression of core cell cycle genes: cyclin-dependent kinase (CDK) A1;1, four B-class CDKs, and cyclins CYCA3;1, CYCB2;4, CYCD4;2 and CYCH1 in root and shoot tips. Only CYCD2;1 transcript was elevated in treated shoots, but down-regulated in roots. Wheat Ta-CDC2 and Ta-CYCD2 genes showed the same expression profiles as their Arabidopsis counterparts, CDKA1;1 and CYCD2;1. PMs also caused increase of antioxidative enzyme activities in both plants. Exposure of Arabidopsis to PMs induced one catalase isoform, but repressed another, resulting in no net change of catalase activity. Wheat seedlings treated with PMs had catalase activity significantly elevated in all treatments, particularly in shoots. In both plants, PMs induced the activity of different peroxidase isozymes and total peroxidase activity. Both plants responded to phytotoxic treatments by induction of CuZn-superoxide dismutase. Thus, the phytotoxicity of C. murale root exudates is, at least partially, based on down-regulation of the cell cycle regulators and on generation of oxidative stress in the affected plants. We propose that C. murale root exudates should be considered as means of biological weed control.

Herbicide Phosphinothricin Causes Direct Stimulation Hormesis

Herbicide phosphinothricin (PPT) inhibits glutamine synthetase (GS), a key enzyme in nitrogen assimilation, thus causing ammonia accumulation, glutamine depletion and eventually plant death. However, the growth response of Lotus corniculatus L. plants immersed in solutions with a broad range of PPT concentrations is biphasic, with pronounced stimulating effect on biomass production at concentrations ≤ 50 μM and growth inhibition at higher concentrations. The growth stimulation at low PPT concentrations is a result of activation of chloroplastic isoform GS2, while the growth suppression is caused by inhibition of both cytosolic GS1 and GS2 at higher PPT concentrations. Since the results are obtained in cell-free system (e.g. protein extracts), to which the principles of homeostasis are not applicable, this PPT effect is an unambiguous example of direct stimulation hormesis. A detailed molecular mechanism of concentration-dependent interaction of both PPT and a related GS inhibitor, methionine sulfoximine, with GS holoenzymes is proposed. The mechanism is in concurrence with all experimental and literature data.

Tissue culture and agronomic traits relationship in wheat

There are only a few reports about the relationship between tissue culture and agronomic traits in wheat. In an attempt to enlarge the knowledge in this area, 96 wheat genotypes of worldwide origin were evaluated for their tissue culture response as well as the agronomic performance in the three-year field trials. Plant regeneration was achieved from immature embryos collected 12–15 days after anthesis. Associations between tissue culture and agronomic traits were surveyed by the correlation and path coefficient analyses. A trait-association by year biplot was applied to understand environmental effects on these relationships. All significant trait-associations were of a moderate magnitude and were responsive to the environment. The path coefficient analysis revealed that a grain yield had the highest positive direct effect on a callus formation while the kernel number per spike had the highest positive direct effect on regenerative calli and plant number per embryo. Nevertheless, only productive tillering had significant (positive) direct effect on all studied tissue culture traits.

Evaluation of agronomic traits in tissue culture – derived progeny of bird's-foot trefoil

Two newly selected Lotus corniculatus cultivars (Bokor and Zora), introduced recently as pasture legumes, were studied with respect to their in vitro regeneration capacity and field performance of R1 regenerates. Multiple buds were induced in callus tissues derived from apical buds, petioles, leaf and cotyledon explants. The rooted regenerates, acclimated in a greenhouse, were fertile. Seeds from the R1 generation were sown in the field and the plants found to be superior to controls in stem height and green mass production.

Use of Chenopodium murale L. transgenic hairy root in vitro culture system as a new tool for allelopathic assays

We investigated Chenopodium murale transgenic hairy root in vitro culture system as a new tool for allelopathic assays. Transgenic hairy roots were induced by Agrobacterium rhizogenes A4M70GUS from roots, cotyledons, leaves, and internodes of C. murale seedlings. Roots were found to be the best target explants, providing transformation efficiency of up to 11.1%. Established hairy root clones differed in their morphology and growth potential. Molecular characterization of these clones was carried out by PCR, RT-PCR and histochemical GUS analyses. No differences in rol gene expression were observed. Liquid culture system of characterized hairy root clones was maintained for over 2 years. Six hairy root clones were selected for assaying the allelopathic effect of their growth medium against germination and seedling elongation of wheat and lettuce test plants. The inhibitory potential varied depending on the hairy root clone. Some transgenic clones showed significantly higher inhibition compared to wild-type roots. These results revealed that hairy roots as an independent system synthesize some bioactive substances with allelopathic activity and exude them into the growth medium. Concentrations of caffeic, ferulic and p-coumaric acids (0.07-2.85 μmol/L) identified by HPLC analysis in the growth media were at least 1000 times lower than the inhibitory active concentration (5 mmol/L) of pure grade phenolic acids, suggesting that they have a limited role in the allelopathic phenomena of C. murale. The presented hairy root system appears to be a suitable tool for further investigation of the potential and nature of root-mediated allelopathic interference of C. murale.

Hairy roots formation in recalcitrant-to-transform plant Chenopodium rubrum

Susceptibility of C. rubrum to Agrobacterium-mediated transformation was demonstrated by inoculating the petioles of in vitro grown plants with A. rhizogenes strain A4M70GUS. Hairy roots were produced in 8 % of explants. They were isolated and maintained on plant growth regulator-free solid or liquid half-strength Murashige and Skoog medium for two years. Hairy root fresh mass increased 30 — 90 folds when grown in liquid medium, which was superior to solid medium, where most of the hairy roots produced calli. When these calli were grown on medium supplemented with 0.5 mg dm-3 thidiazuron, embryo-like structures were obtained. Transgenic status of long-term callus and hairy root cultures was confirmed by histochemical GUS assay, by PCR specific to the uidA, rolA&B and ags genes and by Southern hybridization.

Variability in shoot cultures regenerated from hairy roots of Gentiana punctata

Differences among three clones of Gentiana punctata L. hairy root shoot regenerants were investigated in relation to their growth patterns, production of secondary metabolites and 2D protein profiles. Prominent differences in growth parameters were stable thus qualifying regenerant clones as true somaclones. Marked differences in protein spots were registered among the regenerant clones but not in comparison with the non-transformed control. Southern blot hybridization of regenerants showed the absence of rolA, B and C genes, initially present in the main hairy root lines. Orf13 and rolD were present and orf8 was missing in all three regenerant clones whereas orf3 was missing only in clone 2. Although lacking the three major rol genes, plants of regenerant clones retained characteristics of the hairy root phenotype.

Somatic embryogenesis and in vitro shoot propagation of Gentiana utriculosa

Abstract Study describes protocols for in vitro propagation of Gentiana utriculosa L. via axillary shoot multiplication and indirect somatic embryogenesis. Shoot cultures were established from seedling epicotyl explants cultured on MS medium supplemented with 0.25 mg L−1 BA and 0.1 mg L−1 IAA. Medium containing 2% sucrose and 0.2 mg L−1 BA improved multiple shoot production, providing 2.3 shoots per explant. The highest rooting (29.6%) was obtained on medium with 1/2 MS mineral salts and 0.5 mg L−1 NAA. Somatic embryogenesis was induced using different explants, including immature seeds as well as leaves and roots from shoot cultures. Following auxin treatment with either 1.0 mg L−1 2,4-D (immature seeds and leaves) or 0.1 mg L−1 NAA (roots), explants produced embryogenic calli which upon transfer to plant growth regulator-free medium allowed embryo conversion into plantlets. The best embryogenic response (82%) was obtained in calli derived from leaves cultured with their abaxial surface in contact with medium, whereas the highest embryo conversion rate (68%) was recorded for calli induced on immature seed explants. Histological analysis in all explant types revealed development of proembryogenic cell complexes at callus periphery, giving rise to somatic embryos. The presence of embryos at various stages of development indicated asynchronous somatic embryogenesis in G. utriculosa. Derooted embryo-derived plantlets placed on medium with 0.2 mg L−1 BA multiplied further as shoot cultures.