Artemisia tilesii Ledeb hairy roots establishment using Agrobacterium rhizogenes-mediated transformation.

An efficient and rapid protocol for the establishment of Artemisia tilesii "hairy" root culture is reported. Leaf explants of aseptically growing plants were cocultured with Agrobacterium rhizogenes A4 wild strain or A. rhizogenes carrying the plasmids with nptII and ifn-α2b genes. Root formation on the explants started in 5-6 days after their cocultivation with bacterial suspension. Prolongation of explant cultivation time on the medium without cefotaxime led to stimulation of root growth. The effects of sucrose concentration as well as of the levels of synthetic indole-3-butyric acid (IBA) and native growth regulator Emistim on the stimulation of A. tilesii "hairy" root growth were studied. Maximum stimulating effect both for the control and for transgenic roots was observed in case of root cultivation on the media supplemented with IBA-up to 7.95- and 9.1-fold biomass increase, respectively. Cultivation on the medium with 10 µl/L Emistime has also led to the control roots growth stimulation (up to 2.75-fold). Emistime at 5 µl/L concentration led to 5.46-fold mass increase in only one "hairy" root line. Higher sucrose content (40 g/L) stimulated growth of two hairy root lines but had no effect on growth of the control roots.

Screening of plant cell culture collection for efficient host species for Agrobacterium-mediated transient expression.

Agrobacterium-mediated transient expression is an approach for short-time expression of heterologous genes in plant systems. During the last decade transient expression was regarded as a potent protocol for high scale production of foreign proteins in plants including pharmaceutically valuable proteins. In vitro grown plant cell cultures represent a suitable system for accumulation of heterologous proteins under controlled conditions. Since host characteristics may strongly influence transient expression efficiency, we performed screening of undifferentiated cell cultures for transient expression ability using GUS as a reporter. Analysis of 248 plant species belonging to 49 families from the National Germplasm Bank of the World Flora of the Institute of Cell Biology and Genetic Engineering (Kyiv, Ukraine) allowed for selection of about 50 plant species exhibiting detectable beta-glucuronidase activity.

[Methods of biotechnology in the system of efforts for plant biodiversity preservation].

The main methods of conservation of plant biodiversity using in situ and ex situ approaches are analyzed in the review. Potentials and problems of biotechnology (in vitro culture, cryopreservation) for storage of plant germplasm are discussed.

[Effect of lox-sites of the Cre lox recombination system on promoterless bar gene expression in transgenic plants].

We demonstrate that localization of lox site between the right border of T-DNA and promoterless bar gene (RB-lox-bar-) led to its highly efficient expression in transgenic plants of Nicotiana tabacum and N. africana. Plasmid vectors used in gene integration experiments contained neomycin phosphotransferase II (npt II) gene under nos promoter as well. Transgenic plants were selected according to their capacity to grow on the medium with kanamycin and then they were tested on the selective medium containing phosphinothricin. 80% of transgenic plants expressed bar gene at the level similar to that in plants transformed with the bar gene under widely used constitutive promoter. Transformation of plants with the plasmid vector containing only promoterless bar gene near T-DNA right border (RB-bar-) and with the vector containing lox site and promoterless bar gene in the middle of the construction (-lox-bar-) led to obtaining no more than 4.5% of transgenic plants resistant to phosphinothricin. PCR analyses confirmed both the absence of tandem repeats and of plasmid recombination resulting in transference of bar gene under promoter in plasmid vector. Nos-terminator situated between the lox site and the right border of T-DNA did not decrease bar gene expression.

[Using methods of plant biotechnology for conservation and study of the world flora biodiversity].

Ex situ germplasm bank of plant species of world flora has been created at the Institute of Cell Biology and Genetic Engineering of the National Academy of Sciences of Ukraine as a result of investigations in the fields of botany and plant biotechnology. At present the bank contains more than 4000 samples in seed bank and more than 2000 cell lines in in vitro bank. The germplasm bank which has been put on the List of scientific objects of national property of Ukraine is the basis of elaboration ofbiotechnological methods of plant biodiversity preservation and of plant material screening for biologically active substances.

[In vitro regeneration of Nicotiana africana plants from explants of different type and mesophyll protoplasts]. / Regeneratsiia in vitro rastenii Nicotiana africana iz éksplantov raznogo tipa i mezofil'nykh protoplastov.

Methods of in vitro cultivation of an African endemic species Nicotiana africana (Solanaceae) possessing some valuable traits have been elaborated. Influence of different concentrations of auxin (alpha-naphtaleneacetic acid) and cytokinins (6-benzylaminopurine, zeatin, thidiazuron) on morphogenesis and plant regeneration has been analysed using leaves and internodes as explants. The optimum method of regeneration of N. africana shoots in leaf explants is cultivation in the presence of 0.1 mg/l NAA and 1 mg/l BA; in internode explants--cultivation on the medium containing 0.5 mg/l NAA and 1 mg/l zeatin. The use of thidiazuron was the most effective at the concentration of 0.4 mg/l with subsequent transfer of explants to hormone-free medium. Method of isolation and culture of mesophyll protoplasts enabling to regenerate N. africana plants during 3-4 months has been proposed.

[Genetic transformation of Nicotiana africana Merxm. with plasmids containing lox recombination]. / Geneticheskaia transformatsiia rastenii Nicotiana africana Merxm. plazmidami, soderzhashchimi saity recombinatsii lox.

An efficient genetic transformation method for african tobacco Nicotiana africana Merxm. has been established. African tobacco is a valuable source for cytoplasmic male sterility (CMS) and nuclear encoded resistance to potato virus Y (PVY). N. africana transgenic plants have been obtained using both Agrobacterium-mediated and direct transformation of leaf explants with gold particle bombardment using particle inflow gun. Plasmid vectors containing phosphinothricin resistance gene (bar gene) coding region without promoter and independent 35S promoter between lox sites (lox-bar-35S-lox) and nptII gene were used. Transgenic plants were selected according to growth capacity on the selective medium containing 50 mg/l kanamycin. PCR analyses of kanamycin-resistant plants confirmed the presence of nptII and bar genes in their genome. Agrobacterium-mediated transformation of root explants has proved to be the most efficient transformation method for N. africana.