Abstract:
The present invention relates to biotechnology. The invention can be used for stimulation of regeneration of plants from tissues and undifferentiated cells cultivated under artificial conditions. The present invention can also be applied in agriculture for acceleration of germination of seeds, increase in germination of aged, long-stored seeds as well as for increase of resistance of plants to biotic and abiotic stresses.

Description:
FIELD OF THE INVENTION 
     This invention relates to biotechnology. The invention can be used for stimulation of regeneration of plants from tissues and undifferentiated cells cultivated under artificial conditions. The present invention can also be applied in agriculture for acceleration of germination of seeds, increase in germination of aged, long-stored seeds as well as for increase of resistance of plants to biotic and abiotic stresses. 
     BACKGROUND 
     Regeneration of plants is obligatory and often limiting stage of such biotechnologies as cell selection, genetic engineering, somatic hybridization, generation of haploid and diploid plants, microclonal proliferation. 
     Microclonal proliferation is a method of vegetative proliferation of plants by activation of dormant buds, induction of formation of new (adventitious) buds or callus tissues with subsequent generation of plans from them. Apart from proliferation, this method also provides partial resistance of planting material to fungal, bacterial and virus diseases. To date all of elite planting material of potato, berry, vegetable and decorative cultures is produced by using microclonal proliferation. Clonal proliferation of lignosa is of especially great interest [Ahuja, M. R. and W. J. Libby (Eds.). 1993. Clonal Forestry I: Genetics and biotechnology, Clonal Forestry II: Conservation and application. Springer-Verlag. Berlin]. When plans are produced from cultivated tissues, the two basic problems arise: how to increase amount of regenerants produced and how to implant them. To solve the first problem, nutrient medium is supplemented with growth regulators possessing cytokinin activity. However, shoots formed are “overfed” with cytokinin and, therefore, they take roots badly. Offered programs of gradual exclusion of cytokinins, addition of different growth regulators with auxin action demand a lot of time and not always produce a positive result. Long-term cultivation of plant tissues in vitro can cause undesirable mutations. To increase efficiency of clonal proliferation, more sophisticated regulation of plant regeneration is required (Tree Physiol. 2000 August; 20(14):921-8; Curr Opin Biotechnol. 2000 June; 11(3):298-302; Methods Mol Biol. 1999; 111:127-34). 
     Generation of haploid plants is applied to fix heterosis effect in hybrids. Upon usual proliferation by seeds, the optimum combination of parental genomes quickly collapses and, therefore, heterosis is not preserved longer 1-2 generations. To fix hybrid genotypes, haploid plants are produced from pollen of best plants F 2  and then duplication of chromosomes is achieved. Dihaploid plants produced can blossom normally and produce seeds. Since chromosomes in pair are identical, properties of such hybrid remain constant upon proliferation. To produce haploids, anthers or isolated pollen grains at early stages of development are introduced into in vitro culture and somatic embryogenesis or callusogenesis is induced. Probability of haploid plant generation does not exceed 5-10% of a number of planted anthers and significantly depends on a genotype of initial plants. Usually albinism can often be observed among regenerants. Upon production of haploids, the main goal is reprogramming of pollen grains from normal development (germination) to somatic embryogenesis. In this case, exogenetic regulation by phytohormones is ineffective. Some signals changing synthesis or activity of endogenous growth regulators are necessary. 
     Somatic hybridization is a process of fusion of isolated protoplasts separated from plant cells. Somatic hybridization allows to overcome noncrossing barriers upon distant hybridization, enables to generate unique combinations of nuclear and plastomic genomes of parents. Method of somatic hybridization is based on fusion of protoplasts induced by different ways followed by regeneration of cell wall at special nutrient media, hybrid cell division and callus formation. Then callus is used for plant regeneration. The most simple stage of this method is isolation and fusion of protoplasts, it is more difficult to achieve hybrid cell division, and the most difficult stage is assumed to be regeneration of plants from calli formed. 
     Isolation of plant tissues and their cultivation under artificial conditions induce genetic variation, which can also be shown in plants generated from cultivated cells. This phenomenon named somaclonal variation can be used, along with induced mutagenesis, for increase of genetic variation of crops in agriculture. Using selective nutrient media, it is possible to select cells with the given attributes directly in in vitro culture-to produce plants. Cell selection is mainly applied to increase resistance of plants to diseases and pests, and also to such adverse environmental factors as drought, salting, extreme temperatures, flooding etc. The selective systems aimed at increasing both specific and nonspecific resistance have been developed. 
     The combination of traditional and cell selection methods has already allowed to produce improved forms and new varieties of tomatoes, sugar cane, rice, barley, potato, spinach, fodder grasses and some other kinds of plants possessing resistance to biotic and abiotic stresses and high productivity [Karp A. Somaclonal variation as a tool for crop improvement//Euphytica, 1995, v. 85, p. 295-302]. 
     Genetic engineering offers novel opportunities for generation of new varieties. Crops in agriculture tolerant to insects-pests, herbicides, adverse climatic conditions have been produced by introduction of genes derived from bacteria or plants. Plants with increased content of protein and essential amino acids, improved oil quality etc have been generated [Ya. I. Bur&#39;yanov. Advances in Plant Gene Engineering Biotechnology.//Russian journal of plant physiology ( Fiziologiya rastenii )-1999—v. 46—No 6—p. 930-944]. Almost all methods of genetic transformation comprise stages of cultivation of tissues in vitro and regeneration of plants from transgenic cells. 
     Thus efficiency of application of all cell biotechnologies depends on a possibility to produce a plant from cultivated cells (Plant Cell Culture Protocols. Second edition. V. M. Loyola-Vargas and F. Vazquez-Flota (eds). Humana Press, Mexico, 2005, 416 pp.; Plant Tissue Culture. 100 years since Gottlieb Haberlandt. M. Laimer and W. Rucker (eds.). Springer, Wien N.Y., 2003, 260 pp.). 
     The basic obstacle in wide use of cell cultures in selection is low regeneration ability of many lines and varieties. For example, in cotton, variety Coker and its derivatives are highly capable for morphogenesis, whereas a majority of other varieties has lowered or zero regeneration potential (Theor Appl Genet. 2004 August; 109(3):472-9.). Production of regenerants in some leguminous plants including such important plant as soya is a serious problem (Planta. 2004 October; 219(6):1042-9). In grain cereals, only cell cultures produced from embryos are really capable for morphogenesis (Vasil V., Chin Yu. L., Vasil I. K. Histology of somatic embryogenesis in cultured immature embryos of maize ( Zea mays  L.)//Protoplasma, 1985, v. 127, p. 1-8). A possibility of plant regeneration strongly depends on genotype (J Exp Bot. 2005 July; 56(417):1913-22.). The majority of commercial corn hybrids is characterized by low morphogenetic potential (Phillips R. L., Somers D. A., Hiberd K. A. Cell/tissue culture and in vitro manipulation. In: Corn and Corn Improvement—Agronomy Monograph 18. (Sprague G. F., Duddley J. W. eds) Am. Soc. of Agronomy, Madison, Wis., 1988, p. 345-387). 
     It is important that regeneration of plants from undifferentiated tissues and cells is a key stage upon production of any genetically modified plants. 
     Choice of Explant Tissue 
     The abilities of different plant tissues to form morphogenetic callus in in vitro culture are distinctive. This feature is most pronounced in monocotyledons. Meristematic tissues: unripe germs, inflorescences, meristem in nodes of tillering and in bases of leaves are characterized by highest regeneration potential. Differentiated tissues of leaves or roots have low ability to callusogenesis. Therefore, if plants are to be produced from cultivated cells, it is necessary to produce callus from competent tissues (Phillips R. L., Somers D. A., Hiberd K. A. Cell/tissue culture and in vitro manipulation. In: Corn and Corn Improvement—Agronomy Monograph 18. (Sprague G. F., Duddley J. W. eds) Am. Soc. of Agronomy, Madison, Wis., 1988, p. 345-387). 
     Variations of Hormonal Content of Nutrient Medium 
     The basic way of switching cells from unorganized growth to differentiation is change in concentration and ratio of hormones. To regenerate many plant species, it is necessary to increase concentration of cytokinins in a medium (Adv Biochem Eng Biotechnol. 2001; 72:157-82.). Ratio and concentration of phytohormones in a nutrient medium optimum for morphogenesis are species- and even variety-specific; therefore anew morphogenesis conditions should be selected upon introduction of new varieties or species of plants. In some cases a probability of morphogenesis increases upon replacement of traditionally used natural and synthetic phytohormones with compounds of other chemical nature but possessing a hormonal activity. However upon screening of a large amount of varieties, different authors have shown that stimulation is also variety-specific, and in case of success frequency of plant regeneration has increased no more than 20% [Wilkinson, Thompson, 1987], [Ignatova et al., 1993; Dias S., Dolgikh Y. I. Role of physiological factors in increase of efficiency of plant regeneration from cultivated maize tissues. Biotechnology (in Russian), 1997, No 11-12, p. 32-36]. 
     Ethylene clearly inhibits callus regeneration. Addition of ethylene precursors 1-aminocyclopropane-1-carboxylic acid or aminoethoxyvinylglycine to a nutrient medium for callus initiation caused significant reduction of frequency of embryogenic callus formation and decrease of a number of produced regenerants by 68% [Songstad D. D., Duncan D. R., Widholm J. M. Effect of 1-aminocyclopropane-1-carboxylic acid, silver nitrate and norbornadiene on plant regeneration from maize callus cultures//Plant Cell Rep., 1988, v. 7, p. 262-265.] [Vain P., Flament P., Soudain P. Role of ethylene in embryogenic callus initiation and regeneration in  Zea mays  L.//J. Plant Physiol., 1990, v. 135, p. 537-540]. Inclusion of compounds inhibiting physiological effect of ethylene-norbornadiene or silver nitrate, into the medium, on the contrary, stimulated embryogenic callus formation and raised regeneration efficiency in a majority of tested genotypes by 15-20% [Vain P., Flament P., Soudain P. Role of ethylene in embryogenic callus initiation and regeneration in  Zea mays  L.//J. Plant Physiol., 1990, v. 135, p. 537-540]. [Hoisington D. A., Bohorova N. E. Towards the production of transgenic tropical maize germplasm with enhanced insect resistance. In: Current issues in Plant Molecular and Cellular Biology (Terzi M., Cella R., Falavigna A. eds.), Kluwer Acad. Publishers., Netherlands, 1995, p. 327-221]. 
     In some cases, addition of abscisic acid to the medium enhances morphogenesis. In wheat, addition of abscisic acid at micromolar concentration inhibited premature germination of isolated embryos and stimulated embryogenic callus formation [Brown C., Brooks F. J., Pearson D., Mathias R. J. Control of embryogenesis and organogenesis in immature wheat embryo callus using increased medium osmolarity and abscisic acid//J. Plant Physiol., 1989, v. 133, p. 727-733; Carman J. G. Improved somatic embryogenesis in wheat by partial simulaton of the in-ovulo oxygen, growth-regulators and desiccation environments//Plants, 1988, v. 175, p. 417-424; I. F. Shayakhmetov, F. M. Shakirova. Somatic Embryogenesis in Wheat Cell Suspension Cultures in the Presence of Abscisic Acid.//Russian journal of plant physiology ( Fiziologiya rastenii ), 1996, v. 43, p. 101-103]. In tissue culture of wild turnip ( Brassica napus ) and maize, effect of abscisic acid on regeneration of plants was positive for some varieties but negative for others [Raldugina G. N., Sobolkova G. I. Genotypic differences upon abscisic acid action on callus cultures  Brassica napus  L.//Russian journal of plant physiology ( Fiziologiya rastenii ), 1994, v. 41, p. 702-706; Yu. I. Dolgikh, T. N. Pustovoitova, N. E. Zhdanova. Hormonal regulation of somatic embryogenesis on maize. In Phytohormones in Plant Biotechnology and Agriculture, Proceedings of NATO-Russia Internation Workshop, Kluwer Academic Publishers, 2003, p. 243-247]. 
     Rather often, none of the media used provide production of plants-regenerants [Armstrong C. L., Romero-Severson J., Hodges T. K. Improved tissue culture response of an elite maize inbred through backross breeding, and identification of chromosomal regions important for regeneration by RFLP analysis//Theor. Appl. Genet., 1992, v. 84, p. 755-762]. 
     Inclusion of Individual Amino Acids into Medium Content 
     Amplification of morphogenetic potential has been observed in some cultures upon addition of some amino acids to medium. For instance, agrinine-containing media are used for wheat, proline-containing media are used for maize [Salmenkallio M, Sopanen T. Amino Acid and Peptide Uptake in the Scutella of Germinating Grains of Barley, Wheat, Rice, and Maize//Plant Physiol. 1989 v. 89 p. 1285-1291]. It was recommended to use proline at high concentrations for increase of frequency of formation of friable embryogenic callus with prolonged ability of regeneration [Duncan D. R., Williams M. E., Zehr B. E., Widholm J. M. The production of callus capable of plant regeneration from immature embryos of numerous  Zea mays  genotypes//Planta, 1985, v. 165, p. 322-332]. According to Rapela&#39;s data, inclusion of 400 mg/L proline into medium resulted in 2-3-fold increase of frequency of embryogenic callus formation and plant regeneration [Rapela M. A. Organogenesis and somatic embryogenesis in tissue culture of Argentine maize ( Zea mays  L.)//J. Plant Physiol., 1985, v. 121, p. 119-122]. In other work, 20 mM proline caused increase in a number of regenerants per explant from 2.8 up to 7.6 [Kamo K. K., Becwar M. R., Hodges T. K. Regeneration of  Zea mays  L. from embryogenic callus//Bot. Gaz., 1985, v. 146, p. 327-334]. The data on application of asparagine are ambiguous: along with stimulation of somatic embryogenesis in cultivated maize tissues [Lupotto E. In vitro culture of isolated somatic embryos of maize ( Zea mays  L.)//Maydica, 1986, v. 31, p. 193-201; Morocz S., Donn G., Nemeth J., Dudits D. An improved system to obtain fertile regenerants via maize protoplasts isolated from a highly embryogenic suspension culture//Theor. Appl. Genet., 1990, v. 80, p. 721-726] its oppression has also been observed [Kamo K. K., Becwar M. R., Hodges T. K. Regeneration of  Zea mays  L. from embryogenic callus//Bot. Gaz., 1985, v. 146, p. 327-334]. The mechanism of influence of proline or asparagine on ability of morphogenesis has not been described. 
     Influence of Oligosaccharides 
     Oligosaccharides are formed during disintegration of plant cell walls. For instance, xyloglucan pentasaccharide 1.5-2-fold enhanced morphogenesis in wheat tussue culture [Pavlova Z. N., Ash O. A., Vnuchkova V. A., Babakov A. V., Torgov V. I., Nechaev O. A., Usov A. I., Shibaev V. N. Biological Activity of a Synthetic Pentasaccharide Fragment of Xyloglucan//Plant Sci. 1992 V. 85. P. 131-134], and trimerous oligosaccharide promoted somatic embryogenesis in cotton [Yu. I. Dolgikh, E. Yu. Shaikina, A. I. Usov, V. N. Shibaev, V. V. Kuznetsov. The Trisaccharide Fragment of Xyloglucan as a Regulator of Plant Morphogenesis// Doklady Akademii Nauk.  1998. v. 360. p. 417-419]. 
     Electrostimulation of Morphogenesis 
     Passing the weak (1-2 μA) constant electric current through calli was shown to stimulate shoot regeneration. 1 to current resulted in increase of tobacco callus weight by 70% and 5-fold increase of a number of shoots formed [Goldsworthy A. The electric compass of plants//New Sci., 1986, No 1, p. 22-23]. In wheat callus, two-fold amplification of rhizogenesis and shoot formation were shown whereas they were entirely absent in the control [Rathore K. S., Goldsworthy A. Electrical control of shoot regeneration in plant tissue culture//Bio/technology, 1985, v. 3, p. 1107-1109]. Stimulation of somatic embryogenesis in lucerne protoplast culture [Dijak M., Smith D. L., Wilson T. J., Brown D. C. W. Stimulation of direct embryogenesis from mesophyllprotoplasts of  Medicago sativa //Plant Cell Rep., 1986, v. 5, p. 468-470], and activation of shoot formation in cabbage, poplar, maize [Wang X., Wang Q., Song M., Zheng E. Effect stimulation with weak electric currents on in vitro culture of cabbage//Acta Bot. Sinica, 1993, v. 35, Suppl., p. 66-70; Dutta R. Studies on the mechanism of electrically induces growth and differentiation in plants in vitro: the cytomorphological profile. Abstr. VIII Intern. Congr. “Plant Tissue and Cell Culture”, Florence, Italy, 1994, p. 49; Kitlaev G. B., Dolgikh Yu. I., Butenko R. G. Physiological action of electric current on maize cell culture in vitro. Doklady Akademii Nauk, 1994, v. 335, No 3, p. 393-395] were also found out. Electric current-induced stimulation of plant regeneration is independent of plant variety or species though quantitative variations are possible. Application of said method of increase of frequency of plant regeneration is limited by necessity to have rather complex technique and small throughput. 
    
    
     
       BRIEF DESCRIPTION OF FIGURES 
         FIG. 1 . SkQ1 stimulates shoot formation from undifferentiated tissue of sugar cane. 
         FIG. 2 . Development of sugar cane shoots at regeneration medium after 10 nM SkQ1-induced shoot formation. 
         FIG. 3 . A sugar cane plant produced from callus by 10 nM SkQ1-induced stimulation of regeneration. 
         FIG. 4 . SkQ1 stimulates root formation from tobacco undifferentiated tissue. 
         FIG. 5 . Effect of different SkQ1 concentrations on formation of shoots and roots from maize calli. 
         FIG. 6 . Effect of SkQ1 on formation of embryogenic callus in lucerne. 
         FIG. 7 . SkQ1 at concentration of 1 nM dramatically enhances germination of rice seeds under water. 
         FIG. 8 . SkQ1 enhances resistance of sugar cane calli to anaerobic stress. 
     
    
    
     DESCRIPTION 
     The invention is based on the principle that biologically active compounds, being linked with a Skulachev-ion, can be specifically delivered to mitochondria at the expense of energy of electrochemical potential of hydrogen ions. Such an approach has allowed to multifold decrease the amount of biologically active compounds administered and to specifically affect mitochondria which are the key element in the most important intracellular processes. Thus the elaborated approach appeared to allow to dramatically decrease unfavorable side effects of the biologically active compound employed. 
     Thus, one of the aspects of the present invention is the way to affect a plant by biologically active compounds targetedly delivered to mitochondria at the expense of energy of electrochemical potential of hydrogen ions. 
     In general, such a compound can be described by the following structure (I): 
     
       
                 
         
             
             
         
      
     
     including solvates, isomers, agrochemically or physiologically acceptable salts thereof, 
     wherein A is an effector-antioxidant of structure: 
     
       
                 
         
             
             
         
      
     
     and/or reduced form thereof, 
     wherein m is an integer from 1 to 3; each Y is independently selected from the group consisting of: lower alkyl, lower alkoxy; or two adjacent Y groups, together with carbon atoms to which they are attached to form the following structure: 
     
       
                 
         
             
             
         
      
     
     and/or reduced form thereof, 
     wherein R1 and R2 may be the same or different and are each independently lower alkyl or lower alkoxy; 
     L is a linker group, comprising: 
     a) a straight or branched hydrocarbon chain optionally substituted with one or more substituents and optionally containing one or more double or triple bonds; or 
     b) a natural isoprene chain; 
     n is an integer from 1 to 20; 
     B comprises: 
     a) a Skulachev-ion Sk:
 
Sk + Z − 
 
     where Sk is a lipophilic cation, Z is an acceptable anion; and/or 
     b) a charged hydrophobic peptide containing 1-20 amino acid residues; 
     provided that in compound of structure (I) A is neither ubiquinone (e.g. 2-methyl-4,5-dimethoxy-3,6-dioxo-1,4-cyclohexadienyl) nor tocopherol or mimetic of superoxide dismutase or ebselen; when L is neither divalent decyl nor divalent pentyl or divalent propyl radical; and when B is triphenylphosphonium cation. 
     A further aspect of the present invention is the composition for targeted delivery of biologically active compound to mitochondria of a cell used for regeneration, stimulation of growth of plants comprising a compound of structure (I), wherein A—plastoquinone of structure: 
     
       
                 
         
             
             
         
      
         
         
           
             wherein Y is methyl, m=2; 
             L is a linker group, comprising: 
             a) a straight or branched hydrocarbon chain optionally substituted with one or more substituents and optionally containing one or more double or triple bonds; or 
             b) a natural isoprene chain; 
             n is an integer from 1 to 20; 
             B comprises: 
             a) a Skulachev-ion Sk:
 
Sk + Z − 
 
             where Sk is a lipophilic cation, Z is an acceptable anion; and/or 
             b) a charged hydrophobic peptide containing 1-20 amino acid residues; 
             provided that in compound of structure (I) A is neither ubiquinone (e.g. 2-methyl-4,5-dimethoxy-3,6-dioxo-1,4-cyclohexadienyl) nor tocopherol or mimetic of superoxide dismutase or ebselen; when L is neither divalent decyl nor divalent pentyl or divalent propyl radical; and when B is triphenylphosphonium cation; 
             including solvates, isomers, agrochemically or physiologically acceptable salts thereof. 
           
         
       
    
     A further aspect of the present invention is a composition useful for plants to overcome various stress factors comprising a compound of structure (I) -SkQ1: 
     
       
                 
         
             
             
         
      
     
     A further aspect of the present invention is a compound of structure (I) that can be used: 
     to stimulate regeneration of plants from tissues and undifferentiated cells cultivated under artificial conditions; 
     to accelerate germination of seeds; 
     to increase germination of aged, long-stored seeds; 
     to stimulate regeneration of plants from cuttings (rooting); 
     to increase flowering period; 
     to increase lifetime of cut flowers; 
     to increase biomass of plants and fruits; 
     to prevent fruit falling; 
     to enhance resistance of plants to pesticides; 
     to increase resistance of plants to biotic and abiotic stresses (salting, extreme temperatures, changes in light conditions), and also to enhance resistance to phytopathogens. 
     A further aspect of this invention is an agrochemically or physiologically acceptable composition to affect a plant or plant seeds comprising agrochemically or physiologically justified amount of a compound of Structure (I) and at least one agrochemically or physiologically acceptable diluent or filler. Agrochemically or physiologically acceptable diluent or filler may present a solvent, solid carrier or surfactant. In other words, agrochemically or physiologically acceptable composition of the present invention for affecting a plant or plant seeds can be used in the form of sprays, emulsive concentrates, suspended concentrates, concentrate solutions, spreading pastes, diluted emulsions, soluble powders, dispersive powders, moistened powders, dust, granules or as incapsulated in polymeric substance. 
     Examples of acceptable solvents are alcohols such as ethanol, propanol or butanol; glycols and their ethers or esters such as propylene glycol, dipropylene glycol ether, ethylen glycol or ethylen glycol monomethyl ether; ketones such as cyclohexanone, isophorone; water, nonepoxy or epoxy vegetable oils such as nonepoxy or epoxy turnip, castor, coconut or soybean oils. 
     Examples of acceptable solid carriers are natural minerals such as calcite, talc, kaolin, montmorillonite or attapulgite. Other granular inorganic or organic substances, for example, dolomite or grinded plant fragments can also be used. 
     Examples of acceptable surface-active compounds (surfactants) are nonionic, cationic and/or anionic surfactants depending on a type of active ingredient included in a composition. 
     A plant suitable to be affected by active ingredient or a composition of the present invention is any biological object related to plant kingdom according to the commonly accepted classification in biology. 
     A compound of Structure (I) can be applied both in itself and together with addition of natural and synthetic phytohormones (for example, 2,4-dichlorophenoxyacetic acid, cytokinins, abscisic acid, 3,6-dichloro-ortho-anisic acid, tricamba, chloramben etc.), compounds inhibiting physiological effect of ethylene (for example, norbornadiene, silver nitrate etc.), individual amino acids, oligosaccharides, and also with the subsequent electrostimulation or other physical exposure. 
     The following non-limiting Examples illustrate the preparation and use of the compounds of structure I but should not be understood as limiting the invention as modifications in materials and methods will be apparent to the skilled person. The following examples should not be construed as limiting the scope of this disclosure. 
     EXAMPLES 
     Examples of agrochemically acceptable compositions are given below in Tables: 
     1. Examples of Compositions in the Form of Solution for Stimulation of Plant Regeneration 
     
       
         
               
               
               
               
             
           
               
                   
                 TABLE 1.1 
               
               
                   
                   
               
               
                   
                   
                   
                 Approximate 
               
               
                   
                 Component 
                 Role of component 
                 amount 
               
               
                   
                   
               
             
             
               
                   
                 SkQ1 
                 Active compound 
                     1-50 μg 
               
               
                   
                 Water 
                 Diluent 
                 1000 ml 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
               
               
             
           
               
                 TABLE 1.2 
               
               
                   
               
               
                   
                   
                 Approximate 
               
               
                 Component 
                 Role of component 
                 amount 
               
               
                   
               
             
             
               
                 SkQ1 
                 Active compound 
                     1-50 μg 
               
               
                 Water 
                 Diluent 
                 1000 ml 
               
               
                 10 mM sodium phosphate buffer, 
                 Diluent 
               
               
                 (pH 4.5-6.5) 
               
               
                   
               
             
          
         
       
     
     2. Examples of Compositions in the Form of Water-Soluble Granules for Stimulation of Plant Regeneration 
     
       
         
               
               
               
             
               
               
               
             
           
               
                 TABLE 2.1 
               
               
                   
               
               
                   
                   
                 Approximate 
               
               
                 Component 
                 Role of component 
                 amount 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 SkQ1 
                 Active compound 
                 45.6% 
               
               
                 Lactose monohydrate 
                 disaccharide 
                 50.6% 
               
               
                 Polyvinylpyrrolidone 
                 coupling compound 
                 0.8% 
               
               
                 Co-polymers of ethylene oxide and 
                 Surfactant 
                 3.0% 
               
               
                 propylene oxide 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
             
               
               
               
             
           
               
                 TABLE 2.2 
               
               
                   
               
               
                 Component 
                 Role of component 
                 Approximate amount 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 SkQ1 
                 Active compound 
                 45.6% 
               
               
                 Lactose monohydrate 
                 Disaccharide 
                 51.9% 
               
               
                 Polyvinylpyrrolidone 
                 coupling compound 
                 1.0% 
               
               
                 Tween 20 
                 Surfactant 
                 1.5% 
               
               
                   
               
             
          
         
       
     
     3. An Example of a Spray for Stimulation of Plant Regeneration 
     
       
         
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                   
                   
                 Approximate 
               
               
                 Component 
                 Role of component 
                 amount 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 SkQ1 
                 Active compound 
                 0.1-1.0% 
                   
               
               
                 Vegeol SPO ™ 
                 Surfactant 
                 0.01-1.0% 
               
               
                 Tween 20 
                 Surfactant 
                 0.01-0.1% 
               
               
                 Liquid vegetative wax 
                 substance retarding penetration 
                 0.01-0.1% 
               
               
                 jojoba 
                 of a composition into a plant 
               
               
                 Plastinol ™ 
                 substance retarding penetration 
                 0.01-0.1% 
               
               
                   
                 of a composition into a plant 
               
               
                 Guar gum 
                 substance preventing a 
                 0.1-0.3% 
               
               
                   
                 composition from evaporation 
               
               
                 Tixoseal ™ 
                 substance forming a covering 
                 0.01-0.05% 
               
               
                   
                 at particles 
               
               
                 Difluoro- 
                 compressed liquid 
               
               
                 dichloromethane 
               
               
                   
               
             
          
         
       
     
     4. An Example of a Medium for Plant Regeneration 
     
       
         
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                   
                   
                 Approximate 
               
               
                 Component 
                 Role of component 
                 amount 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 SkQ1 
                 Active compound 
                 1-1000 
                 nM 
               
               
                 Agarized Murashige-Skoog 
                 Medium 
               
               
                 medium 
               
               
                 Sucrose 
                 Osmotic 
                 30 
                 mg/L 
               
               
                 2,4-dichlorophenoxyacetic 
                 phytohormone 
                 1 
                 mg/L 
               
               
                 acid 
               
               
                   
               
             
          
         
       
     
     5. 1-50 nM SkQ1 included in media for hydroponics. 
     5. Production of Normal Sugar Cane Plants from Calli Induced by SkQ1 to Regeneration 
     In this work, calli and explants of tobacco, sugar cane, maize, lucerne, potato and tomatoes were used. Calli were incubated at Petri dishes containing agarized Murashige-Skoog medium with 30 mg/L sucrose and 1 mg/L 2,4-dichlorophenoxyacetic acid. The cultivation was carried out at temperature 26° C., illumination intensity of 2 cd and the length of daylight of 16 hours. SkQ1 was added to autoclaved and cooled (up to 45° C.) nutrient medium immediately before callus planting. SkQ1 at concentrations of 0, 1.5, 5, 10, 20, 30 and 50 nM was used. Increase of callus green weight, a fraction of calli with shoots and roots and an average number of organs per a morphogenetic callus were taken into account after one month of cultivation. 
     6. SkQ1 Induces Regeneration from Calli of a Wide Range of Plants 
     Sugar Cane 
     SkQ1 at low concentration (10 nM) was found to induce formation of shoots from calli beginning with 19-th day of incubation ( FIG. 1 ). Control calli did not reveal any features of shoot formation. At higher SkQ1 concentrations, though shoot formation was also observed but it occurred at later time (after 31-st day) and in lesser amount of calli. 
     When calli with shoots formed at 10 nM SkQ1-containing medium were transferred onto agarized regeneration medium, the formed shoots have continued to develop normally at the presence of 10 nM SkQ1 ( FIG. 2 ). At the same time, development of shoots of calli transferred onto regeneration medium from medium without SkQ1 has been at the very initial stage. 
     The presence of higher SkQ1 concentrations in the regeneration medium was accompanied by termination of further development of shoots (100 nM SkQ1), or led to destruction of shoots (1000 nM SkQ1). 
     To produce normal plants, sugar cane shoots formed by 10 nM SkQ1 were transferred onto rootage agarized medium containing the same optimum SkQ1 concentration (10 nM). After root formation, the plants were transferred into water medium and then displanted into the ground. Estimation of the plant state was performed at every stage. It was shown that sugar cane shoots induced by 10 nM SkQ1 take roots properly and give normal viable plants ( FIG. 3 ). 
     It is worthy of note that shoots formed at a medium supplemented with 100 nM or 1000 nM SkQ1 and further cultivated at a medium containing the same high SkQ1 concentrations appeared to be nonviable and died. 
     Tobacco 
     Effect of SkQ1 on callus formation (dedifferentiation) in tobacco leaf explants was investigated. Sterile leaf disks of plants  Nicotiana tabacum  cv Samsun were sliced with approximately 1 cm 2  size. The resulting explants were placed at standard MS medium for callus formation supplemented with phytohormones 0.5 mg/L 6-benzylaminopurine and 1 mg/L naphthylacetic acid. Test Petri plates were also supplemented with 0.6 μM SkQ1. Explants were cultivated in darkness for 5 weeks. During incubation callus was initially formed at wound explant surfaces. Then explants at test Petri dishes supplemented with SkQ1 revealed onset of secondary differentiation of callus tissues with root formation. Control Petri dishes without SkQ1 did not show any root formation ( FIG. 4 ). In the course of a further incubation at test Petri dishes areas of differentiated explant tissues necrotized and died (it did not concern newly formed differentiated root tissues which did well), whereas control Petri dishes showed normal dedifferentiation of explant tissues. 
     Maize 
     Dose-dependent effect of SkQ1 on morphogenetic processes in maize callus was shown. At 1-10 nM SkQ1, increase of inoculum fraction that forms shoots and increase of an average number of shoots per callus were observed. No reliable increase in amount of rhizogenic calli was established but an average number of roots per callus increased significantly ( FIG. 5 ). SkQ1 at 20 nM and higher concentrations inhibited morphogenesis, and the influence on shoot formation was more efficient than influence on rhizogenesis. Said compound did not affect essentially callus accretion. Variations in green weight resulted mainly from amount of formed roots. No reliable growth oppression induced by high SkQ1 concentrations was observed. 
     When callus cultivation was repeated using the same medium composition, the similar effect of SkQ1 was observed, with even more pronounced amplification of rhizogenesis. 
     Lucerne 
     Lucerne calli were cultivated for 1 month in the presence of different SkQ1 concentrations (0-10 nM). Effect of SkQ1 on callus growth (weight change) and regeneration ability (formation of calli with “green areas”) were assessed. It was shown that SkQ1 does not affect significantly callus growth but 5 nM SkQ1 markedly (2.5-3-fold) stimulates regeneration. At higher SkQ1 concentrations, said positive effect eliminates ( FIG. 6 ). 
     Potato and Tomato 
     Effect of SkQ1 on formation shoots and roots from leaf potato explants and stem tomato explants was investigated. Explants were incubated for 10 days in the presence of 10 nM SkQ1 or with no SkQ1. The data allow to conclude that SkQ1 significantly enhances formation of both shoots and roots in potato and tomato. 
     7. SkQ1 and Anaerobiosis of Plants 
     Germination of Rice Seeds Underwater 
     Effect of SkQ1 on germination of rice seeds under water (under anaerobic conditions) was studied. SkQ1 was added to water at concentrations of 0, 1, 3, 10, 30 nM. 1 nM SkQ1 was shown to enhance significantly germination of rice seeds ( FIG. 7 ). SkQ1 at higher concentrations does not reveal such a stimulating effect. 
     Survival of Sugar Cane Calli After Anaerobic Stress 
     Effect of SkQ1 on survival of sugar cane calli following anaerobic stress was assessed. For this purpose, sugar cane calli were incubated for 48 hours under anoxic conditions and then were transferred under aerobic conditions at a nutrient medium containing 10 nM SkQ1or no SkQ1 (control). In the presence of SkQ1, said calli were shown to possess markedly higher capability for survival and, besides, they retain capability for morphogenesis ( FIG. 8 ).