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Agrobacterium tumefaciens was used to transform fiber flax with the pBITUBA8 plasmid carrying the mutant alpha-tubulin gene imparting resistance to dinitroaniline herbicides and the nptII selective marker gene imparting resistance to canamycin. The transformants were selected in parallel on media containing canamycin and trifluralin (a dinitroaniline herbicide). The transgenic nature of the resultant regenerants resistant to dinitroaniline herbicides was confirmed by means of Southern blotting and polymerase chain reaction (PCR) analysis using specific probes for the ntpll gene and the gene of alpha-tubulin.

Barley (Hordeum vulgare) grains synthesize starch as the main storage compound. However, some starch is degraded already during caryopsis development. We studied temporal and spatial expression patterns of genes coding for enzymes of starch synthesis and degradation. These profiles coupled with measurements of selected enzyme activities and metabolites have allowed us to propose a role for starch degradation in maternal and filial tissues of developing grains. Early maternal pericarp functions as a major short-term starch storage tissue, possibly ensuring sink strength of the young caryopsis. Gene expression patterns and enzyme activities suggest two different pathways for starch degradation in maternal tissues. One pathway possibly occurs via alpha-amylases 1 and 4 and beta-amylase 1 in pericarp, nucellus, and nucellar projection, tissues that undergo programmed cell death. Another pathway is deducted for living pericarp and chlorenchyma cells, where transient starch breakdown correlates with expression of chloroplast-localized beta-amylases 5, 6, and 7, glucan, water dikinase 1, phosphoglucan, water dikinase, isoamylase 3, and disproportionating enzyme. The suite of genes involved in starch synthesis in filial starchy endosperm is much more complex than in pericarp and involves several endosperm-specific genes. Transient starch turnover occurs in transfer cells, ensuring the maintenance of sink strength in filial tissues and the reallocation of sugars into more proximal regions of the starchy endosperm. Starch is temporally accumulated also in aleurone cells, where it is degraded during the seed filling period, to be replaced by storage proteins and lipids.

Biolistic transformation of soybean by construction carrying a mutant alpha-tubulin gene as selective marker gene conferring resistance to dinitroaniline herbicides, and by additional construction pAHTUBI carrying a full-length barley beta-tubulin gene for correct co-expression of exogeneous tubulin in cells of transgenic soybean lines has been realized. It was established that 10 microM trifluralin is the most optimal selective concentration to pick up transformed soybean lines. Transgenic nature of offained regenerants was confirmed by Southern blotting hybridization using specific probe to selective alpha-tubulin gene.

Tubulins, as major components involved in the organization of microtubules, play an important role in plant development. We describe here the expression profiles of all known alpha-tubulin (TUA), beta-tubulin (TUB) and gamma-tubulin (TUG) genes of barley (Hordeum vulgare), involving eight newly identified TUB sequences, five established TUA genes and one TUG gene. Macroarray and Northern blot-based expression patterns in the pericarp, endosperm and embryo were obtained over the course of the development of the grain between anthesis and maturation. These revealed that the various tubulin genes differed in their levels of expression, and to some extent were tissue specific. Two expression peaks were detected in the developing endosperm. The first and more prominent peak, at 2 days after flowering, included expression of almost all the tubulin genes. These tubulins are thought to be involved in mitoses during the formation of the syncytial endosperm. The second, less pronounced but more extended, peak included only some of the tubulin genes (HvTUA3, HvTUB1 and HvTUG) and might be associated with the cell wall organization in aleurone and starchy endosperm. The HvTUA5 gene is expressed only in embryo of the developing grain and may be associated with shoot establishment. The expression profiles of the tubulin folding cofactors HvTFC A and HvTFC B as well as small G-protein HvArl2 genes were almost perfectly correlated with the global levels of tubulin mRNA, implying that they have a role in the control of the polymerization of alpha/beta-tubulin heterodimers.

Barley endosperm development can be subdivided into the pre-storage, intermediate, storage and desiccation phase. Nothing is known about DNA methylation events involved in different endosperm-specific developmental programmes. A complete set of methylation cycle enzyme genes was identified and investigated by mRNA expression analysis. During the pre-storage phase, methionine synthase and S-adenosylmethionine (AdoMet) synthase genes are expressed at high levels, mainly to produce AdoMet, which might be used for methylation processes as indicated by high expression of methyltransferases HvMET1, HvCMT1 and HvDnmt3-1 as well as AdoHcy hydrolase genes. The methyltransferases, core histones and DNA-unwinding ATPases are co-expressed at the mRNA level. On the contrary, storage protein (prolamin) gene expression is repressed due to CpG methylation. Expression of genes responsible for starch biosynthesis is also developmentally regulated but not methylation-dependent. Thus, during pre-storage phase, activity of HvMET1 and HvCMT1 possibly maintains DNA replication and suppresses specific pathways of maturation. Besides, HvDnmt3-1 might be responsible for differentiation-specific de novo methylation. Expression of methyltransferases HvDnmt3-2 and HvCMT2 peaks during the onset of massive starch accumulation. The enzymes are likely responsible for DNA methylation involved in determining plastid division and amyloplast differentiation as concluded from the patterns of co-expressed genes. Levels of AdoMet decarboxylase mRNA, but not methyltransferase- and AdoHcy mRNA, increase at the beginning of desiccation together with methionine synthase and AdoMet synthase levels. This increase may be indicative for utilization of AdoMet in polyamine production protecting aleuron and embryo cell membranes during desiccation.

Plants of the Brassicaceae family are important oil, vegetable and feed crops. The review is devoted to the latest achievements in genetic engineering of plants from this family. Results concerning development of effective methods both of Agrobacteium-mediated transformation and of direct gene uptake are considered. Particularly, possibilities of plant genetic modification with the aim to improve agronomically and commercially important traits are stressed. Problems of biologically safe introduction of transgenic plants into agricultural production are discussed.

Mesophyll protoplasts of Brassica oleracea var. botrytis were successfully transformed using polyethylene glycol (PEG). The success of plant transformation depended on both gene transfer and plant regeneration. Parameters, such as PEG and vector concentrations and heat shock conditions were tested in experiments on transient expression of the beta-glucuronidase (EC 3.2.1.31) gene and the most suitable conditions for DNA uptake were determined. Two antibiotic resistance marker genes for neomycin phosphotransferase (EC 2.7.1.95) and hygromycin phosphotransferase (EC 2.7.1.104), and three vector plasmids with different lengths were used to obtain stable transformants.

The procedure for genetic transformation of two spring and one winter rapeseed cultivars was developed. No-paline strains of Agrobacterium tumefaciens GV3101 and EHA105 were shown to be preferable for gene transfer, as compared to the octopine strain GV2260. With two types of plant explants, the segments of hypocotyls and cotyledons, transformation was successful; however, its efficiency was somewhat higher with the fragments of hypocotyls. Analysis of regenerated plants by PCR and Southern blotting confirmed the presence of the nptII and nisA genes in transformants. RNA analysis by Northern blotting showed expression of the nisA gene in transformed shoots. The transgenes were inherited in T2 as Mendelian traits. The effect of biotic and abiotic factors on the efficiency of genetic transformation in rapeseed is discussed.