In order to produce human lactoferrin (Lf) in alfalfa (Medicago sativa L.), a construct containing human Lf cDNA under the control of cauliflower mosaic virus 35S promoter was engineered. As a selectable marker bar gene whose expression in plant cells confers tolerance to L-phosphinothricin (ppt) was used. Plants from a highly embryogenic alfalfa clone from the Bulgarian cultivar Obnova 10 were transformed using Agrobacterium tumefaciens mediated leaf disc method. Transgenic alfalfa plants were established from ppt-resistant calli via indirect somatic embryogenesis. The presence of human Lf cDNA in the genome of the selected regenerants was confirmed by polymerase chain reaction (PCR). Reverse transcriptase (RT)-PCR and Western blot showed expression of human Lf in leaf tissue. Studies on antibacterial effect of the recombinant glycoprotein were conducted and resistance of the transgenic alfalfa plants to two phytopathogens, Pseudomonas syringae pv. syringae and Clavibacter michiganensis, was demonstrated. The obtained results suggest that the expression of human Lf in alfalfa could be beneficial not only for producing recombinant protein for clinical application but also for crop quality improvement.

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A new reconstructed barley karyotype, PK88, which is a quadruple homozygote for three unequal translocations, 1-2, 3-4, 5-7, and one pericentric inversion in chromosome 6, was studied. As a result of these chromosome rearrangements, a complete cytological marking of the complement has been achieved. Due to the specific intra or interchromosomal transfer of particular bands, Giemsa staining of somatic chromosomes provided clear-cut indications about the localization of translocation and inversion breakpoints. It was established that the long arms of chromosomes 1, 2, 4, 5 and 7 and the short arm of chromosome 3 have been involved in interchanges 1-2, 3-4, and 5-7. The breakpoints of pericentric inversion proved to be located proximally to the short (satellite) arm and distally in the long arm of chromosome 6. PK-88 offers an essential gain in resolution power and extension of the areas of application in cytogenetics over other reconstructed karyotypes produced so far in barley.

A total of 52 reciprocal translocations and 9 pericentric inversions were induced and identified in both standard and cytologically marked barley karyotypes using gamma-rays as the clastogenic agent. An analysis based upon Giemsa N-banding patterns and arm length measurements of the reconstructed chromosomes enabled a rather precise cytological localization of intra- and interchange breakpoints. This analysis was significantly facilitated and improved, especially for the identification of pericentric inversions, when the reconstructed karyotype T-1586 was used as starting material. The majority, if not all, of the aberration breakpoints proved to be localized in interband regions or in medial and terminal parts of the chromosomes, i.e., in regions which are deficient in constitutive heterochromatin. A great number of the structural mutations produced in this study contain specific cytological markers covering nearly all of the chromosomes of barley karyotype. This material might be of considerable interest in solving various problems of barley cytogenetics and chromosome engineering and especially in constructing a physical map of barley genome.

The potential of cytologically reconstructed barley line D-2946 to cope with the major lesions that hamper genome integrity, namely DNA single- and double-strand breaks was investigated. Strand breaks induced by γ-rays and Li ions were assessed by neutral and alkaline comet assay. Repair capacity after bleomycin treatment was evaluated by agarose gel electrophoresis under neutral and alkaline conditions. Frequencies of radiation-induced chromosome aberrations were also determined. Results indicate that radiation-mediated constitutive rearrangement of the chromosome complement has led to a substantial modulation of the sensitivity of barley genome towards DNA strand breaks, produced by ionising radiation, Li ion implantation and bleomycin in an agent-specific manner, as well as of the clastogenic response to γ-rays. Based on these findings, reconstructed barley karyotype D-2946 can be considered a candidate radio-sensitive line with reduced ability to maintain genome integrity with respect to both DNA and chromosomal damage.