The thesis is devoted to the development of the new approach in genetic transformation of plants through the use of carbon nanotubes (CNTs) for gene transfer. Having a range of unique properties CNTs are under precise investigation now as potential DNA carriers for the transformation of living cells. The main issues to be solved in this field are overcoming the intrinsic hydrophobicity of pristine CNTs via proper functionalization, the design of procedure for the efficient reversible binding of genes and their delivery into cells, as well as the establishment of the main factors governing the efficiency of DNA transportation process. The thesis presents results of the selection of an effective protocol for noncovalent functionalization of CNTs with enabling subsequent use in plant biotechnology. Noncovalent functionalization of CNTs using a range of molecules of biological origin such as double-stranded DNA, deoxyribonucleoside triphosphates (dNTPs), adenosine triphosphate sodium salt (ATP), bovine serum albumin (BSA), compounds of vitreous humor extract (VHE) and sodium humate is reported. Ultrasound-mediated formation of polydisperse aqueous colloidal systems by functionalized CNTs is shown. Using Raman spectroscopy, atomic force and transmission electron microscopy properties of CNTs conjugates with biomolecules are described and the mechanisms of their formation are discussed. Changes in structure and properties of CNTs due to functionalization process that are considered favorable for their exploitation for the development of nanoscale delivery systems are established. Namely, shortening and appearance of highly reactive loci in walls of CNTs, formation of "open ends", good dispersing ability of functionalized CNTs in water environments. Aqueous colloidal systems of functionalized CNTs were stable for 10 (functionalization with BSA), 6 (functionalization with DNA dNTPs, ATP, VHE) and 3 (functionalization with sodium humate) months. Biologically functionalized CNTs were used for non-covalent attachment of plasmid DNA: the vector pGreen 0029 containing the gene sequence of membrane protein of protein-storage vacuole ?-tip (tonoplast intrinsic protein), fused to the reporter eyfp gene of enhanced yellow fluorescent protein as well as the kanamycin resistance selective marker gene nptII under control of 35S promoter and nos terminator. The procedure for reversible temperature-mediated binding of target DNA molecules to functionalized CNTs was developed. The preservation of the functional activity of the target DNA molecules within conjugates with CNTs was shown in bacterial cells E. coli strain DH5? transformation studies. The effect of functionalized CNTs on plant model objects was investigated. All used types of functionalization coverings of CNTs were found to be equally biocompatible. The decrease of protoplasts survival when co-incubated with higher concentrations of CNTs was established. While lower concentrations of CNTs were associated with good protoplasts survival levels. Multi-walled CNTs (MWCNTs) demonstrated higher damaging effect for protoplasts in comparison with single-walled CNTs (SWCNTs). SWCNTs in concentrations of 1, 15, 30, 45, 60 and 75 ?g/ml corresponded to protoplasts survival levels of 90, 84, 60, 29 and 3 %, respectively. For MWCNTs in the same range of concentration survival of protoplasts was 91, 74, 49 and 21 %, respectively. All protoplasts were damaged when MWCNTs were used in concentrations 60 and 75 ?g/ml. For the transformation of protoplasts appropriate concentrations of SWCNTs and MWCNTs were considered 20 ?g/ml and 15 ?g/ml, for transformation of both callus and leaf explants - 40 and 30 ?g/ml. The method for the delivery of exogenous genetic material into protoplasts, callus and leaf explants of tobacco Nicotiana tabaccum L. using functionalized CNTs was developed. The frequency of transient eyfp reporter gene expression in protoplasts transformed using SWCNTs has been found to reach 16 % and using MWCNTs - 13 %. Absence of difference in frequency of genetic material delivery by CNTs functionalized with DNA dNTPs, ATP, BSA and VHE was observed. While sodium humate-functionalized CNTs were less efficient. For SWCNTs and MWCNTs the values of transformation frequencies were 11 and 7 %, respectively. Genetic transformation of tobacco callus using SWCNTs was found to demonstrate 2.5 times higher frequency of stable transformation in comparison with MWCNTs, with corresponding values of 8 and 3 %. Frequency of stable transformation of leaf explants using SWCNTs and MWCNTs was 6 % and 3 %, respectively. Thus obtained results demonstrate the ability of CNTs to deliver genes into plant protoplasts and walled plant cells. Single-walled non-covalently functionalized CNTs are established to be more suitable for the development of novel gene delivery techniques in comparison with multiwalled CNTs.
