Mariana Vlahova

Assessment of polysomaty, embryo formation and regeneration in liquid media for various species of diploid annual Medicago.

To avoid polyploidy in regenerants the source of explant material should be monosomatic. Therefore, the leaf and petiole tissue of five diploid Medicago species (Medicago ciliaris, Medicago murex, Medicago orbicularis, Medicago polymorpha and Medicago truncatula cv. Jemalong, and the ecotype R108-1) was assessed for polysomaty by flow cytometry. For the species studied the frequency of 2C nuclei was about 90% in leaves compared with that in petioles. Embryos were readily formed from tissue of leaves in liquid media containing 1 mg l(-1) or 4 mg l(-1) dichlorophenoxyacetic acid (2,4-D). For embryo development two procedures were tested - prolonged use of induction medium and treatment with polyethylene glycol Mw 6000 (PEG). The highly regenerable genotypes M. truncatula cv. Jemalong and R108-1 showed efficient conversion of embryos after maturation in liquid medium. The regenerated plants were diploid and with normal phenotype.

GENETIC TRANSFORMATION OF MEDICAGO TRUNCATULA USING SYSTEM FOR DIRECT SOMATIC EMBRYOGENESIS PROMOTED BY TDZ

ABSTRACT The efficient procedure for Agrobacterium mediated transformation of Medicago truncatula cv. R 108 1 was developed. Using the advantage of recycling embryo formation of system for direct somatic embryogenesis promoted by TDZ, transgenic plantlets were obtained for a short period of 60 days. Transformation was performed with bacterial strain—LBA 4404, containing binary vector pBI 121 carries two genes—uid A gene exprecing β- glucoronidase (GUS) and npt II gene for resistance to kanamicin. Different parameters of the gene transfer—pre-treatment of the embryo clusters, density of the bacterial suspension, time for inoculation and co-cultivation, type of selection were optimized. For evaluation of the transformation efficiency, histochemical GUS assay was performed and glucoronidase activity was detected in embryos, plantlets and seedlings of T1 progeny. Transgenic nature of plantlets was confirmed by npt II-specific PCR amplification and Southern hybridization. In the progeny transgenes segregated in Mendelian manner.

Somatic Embryogenesis of the Model Legume - Medicago Truncatula and other Diploid Medics

ABSTRACT This review outlines the information of somatic embryogenesis in model legume Medicago truncatula and diploid medics. Following the data considerable attention is paid on the factors affecting the process during the induction, development, maturation and conversion stages- genotype, explant choice and preparation, origin of somatic embryos and hormonal composition of culture media. A brief consideration is paid to phenotype and fertility of obtained regenerants.

High frequency plant regeneration of diploid Medicago coerulea through somatic embryogenesis

ABSTRACT A successful procedure for in vitro regeneration of M.coerulea (McG) via indirect somatic embryogenesis using different donor explants has been designed. Regenerable callus tissue from leaf, petiole, stem and root explant sources, was formed at good efficiency on MS or SH media, supplemented with BAP (0.2 mg/l) and 2,4 D in two concentrations (4 mg/l and 2 mg/l). However the petiole used as an initial explant, showed the best morphogenic response. Further development of the induced somatic embryos was promoted on free of hormones medium or after short term cultivation (2 weeks) on medium containing BAP (lmg/l) and NAA (0.01mg/l). At the applied culture conditions normal growth and conversion to plantlets of a part of primarily induced somatic embryos was blocked and secondary embyogenesis also takes place. This significantly increased the number of the obtained regenerants and allowed maintenance of the morphogenic culture for a long period.

PLANT REGENERATION VIA DIRECT ORGANOGENESIS AND SOMATIC EMBRYOGENESIS OF TWO NEW BULGARIAN SPRAY CARNATION CULTIVARS

ABSTRACT Conditions for plant regeneration of leaf expiant from Bulgarian spray carnation cultivars (Fea and Rossitza) were established. Solid MS medium supplemented with BAP and NAA was used for direct induction of adventitious shoots. The optimal medium for regeneration for both cultivars contained 0.9 mg/l BAP and 0.9 mg/l NAA. The mean number of regenerants per explant varied 7–8 for tested cvs. Regenerated plants possessed normal phenotype. At dark and light conditions induction of embryogenic callus were observed for both cultivars. Vigor somatic embryos were obtained on embryo formation medium supplemented with 0.05 mg/l BAP and 250 mg/l casein hydrolysate. They developed to plants with normal phenotype and rooted easily at in vitro condition.

Cell Cycle Plasticity in Response of Low Temperature in Root Tips of Tetraploid Medicago

ABSTRACT Flow-cytometric analysis was carried out to determine the effect of low temperature on the changes in proportion of 2C:4C complement of DNA in root cells from three different zones (meristem apex, elongation and differentiation) in seedlings of Medicago sativa cv. Sitel. 2C:4C complement of DNA in cells were estimated after the treatments of the roots at control temperature of24°C, elevated temperatures of26°C, 30°C, 37°C and low temperatures—10°C and 5°C (in three different periods of duration 15, 38, 45 days). The observed results showed that at control temperature percentage of nuclei 2C:4C in meristem apex is 65:35, in elongation zone 50:50 and in differentiation zone is 40:60. The elevated temperatures (26°C, 30°C, 37°C) influenced this proportion very slightly while treatments of roots with low temperature especially in a period of 45 days changed 2C:4C complement of DNA drastically. It was found that root treated for 45 days at temperature of 10°C the 2C:4C in meristem apex is 78:22, elongation zone 74:26 and in differentiation zone is 70:30. In the case of temperature of 5°C it was 85:15 in meristem apex, 80:20 in elongation zone and 71:29 in differentiation zone. In order to visualize the impact of low temperature on cell cycle plasticity with decrease of dividing cells, roots of transgenic Medicago falcata plants were exposed at control (24°C) and low (5°C) temperatures for the period of 15 and 45 days. These plants carried gus gene under the promoters (cycl, cyc3a)from cell cycle genes After this temperature treatment roots cells were activated for division with different treatments for 1hour. GUS assay were performed for control and pre-treated root tips. The expression of marker gene was observed in highly divided cells of pre-treated root at control temperature. GUS gene was not expressed in pre-treated roots exposed to low temperature for 45 days.

Plant organic farming research – current status and opportunities for future development

ABSTRACT This paper reviews the recent development of the scientific, legislative, economic and environmental aspects of plant organic farming. The impact of organic farming on biodiversity and soil fertility is discussed in comparison with conventional systems. A significant barrier for wide application and future development of organic farming is the existing diversity of national and international policy instruments in this sector. Special attention is paid to up-to-date research techniques that could help solve a number of the problems typically faced in plant organic farming. It is argued that organic farming is still not productive enough to be considered fully sustainable. This underlines the necessity of strong support for more effective implementation of scientific research innovations and improvement of the networking between all stakeholders – organic producers, scientists and corresponding policy makers at the national and international level.

SHAPE OF PODS OF MEDICAGO TRUNCATULA AS A PROMISING MORPHOLOGICAL MARKER FOR EMBRYOGENIC CAPACITY

ABSTRACT Three different types of pods (N- normal with spines, LS- less spines and WS-without spines) were harvested from Medicago truncatula cv. R 108 1 plants grown in greenhouse. The pods with changed shape LS and WS were developed at the end of period of plant maturity at the upper part of plants. In vitro plants originated from seeds from three types of pods were obtained. Callus tissue produced from leaves and petioles of plants from type N, LS and WS showed different embryogenic potential. Callus tissue of type N formed embryos from both -leaves and petioles, while the callus from type WS was able to form embryos only from petioles. Regardless of the initial explants callus from LS plants did not show any embryogenic ability. In contrast to the regenerants from N type capable to form normal root system, those from WS type possess poor developed root system.

Investigation of the Potential of two Wild Medicago Species - Medicago Orbicularis and Medicago Arabica for in Vitro Callusogenesis and Direct Organogenesis

ABSTRACT Plant regeneration via direct shoots formation for two annual diploid medics—Medicago orbicularis and Medicago arabica was established. On medium supplemented with 0.5 mg/l TDZ, petioles base and nodal stem segments formed cluster of shoots. For Medicago orbicularis the number of regenerants varied from 11–13 per explant, while for Medicago arabica, they were less (5–6). Medicago orbicularis also showed the higher potential for callus formation in all tested explants, media and culture conditions. Medium B5 supplemented with 1 mg/l 2,4-D was determined as the optimal for callusogenesis of Medicago orbicularis at both studied conditions—darkness and light. The positive correlation between small genome size of Medicago orbicularis and better in vitro response was found.

EXPRESSION OF THE HUMAN ACIDIC FIBROBLAST GROWTH FACTOR IN TRANSGENIC TOMATO AND SAFETY ASSESSMENT OF TRANSGENIC LINES

ABSTRACT The establishment of transgenic tomato (Solanum lycopersicum) lines expressing human acidic fibroblast growth factor (haFGF) was reported. A nonantibiotic selection involving the manA gene from E. coli that confers resistance to mannose was applied. The transgenic nature of four selected tomato lines cultivar Bela was proved with PCR analysis. The expression of the gene for haFGF was confirmed with RT-PCR analysis and the protein synthesis was quantified with ELISA. A step of purification of haFGF from plant tissue was also performed and the probes were subjected to immunodetection with anti-haFGF and anti-His-tag antibodies. The portion of the recombinant protein out of the total soluble protein extracted from leaves was 0.0015%. In order to evaluate the risk of unintended negative effects of the ripe transgenic tomato fruits, toxicity safety assessment with laboratory mice was carried out including weight analysis, morphophysiological indices and blood parameters measurements. Our data showed that the transgenic fruits as a novel modified product do not pose a risk of intoxication upon the treated animals. Allergenicity evaluation of both recombinant proteins haFGF and phosphomannose isomerase, the product of the selectable marker gene, via alignment of their sequences with those of known allergens did not reveal any sufficient similarity.