Radomirka Nikolić

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.

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.

Morphogenetic responses of embryo culture of wheat related to environment culture conditions of the explant donor plant

Availability of immature embryos as explants to establish wheat (Triticum aestivum L.) by tissue culture can be limited by climatic factors and the lack of high quality embryos frequently hampers experimentation. This study evaluates the effects of rainfall, various temperature-based variables and sunshine duration on tissue culture response (TCR) traits including callus formation (CF), regenerating calli (RC), and number of plants per embryo (PPE) for 96 wheat genotypes of worldwide origin. The objectives of this study were to evaluate the significance of a particular climatic factor on TCR traits and to determine the period of wheat growth during which these factors were the most effective. The genotypes were grown in an experimental field during three seasons differing in meteorological conditions. The relationships between TCR traits and climatic factors within three time periods of wheat growth: 2, 6 and 10 weeks prior to embryo sampling were analysed by biplot analysis. The tissue culture traits were influenced at very different degrees by climatic factors: from 16.8% (RC) to 69.8% (CF). Donor plant environment with high temperatures and low rainfalls reduced (p < 0.05) the tissue culture performance of wheat genotypes. Callus formation was most sensitive to the temperature based factors. The environmental conditions between flowering and the medium milk stage were the most important for CF, while RC and PPE were not particularly related to any period.