RU2577995C1 - Method of elimination of bacterial infections during plating of cell cultures of plants using silver nanoparticles - Google Patents

Abstract

FIELD: bioengineering.

SUBSTANCE: invention is the method of elimination of bacterial infections of cell cultures of plants, including plating of the suspension cell cultures using the antimicrobial agent, where the water suspension of silver nanoparticles with diameter 20-80 nm is used as the antimicrobial agent, it is added to the suspension of cell cultures until final concentration in the culture medium 50 mcg/ml, wherein the planting is performed 24 hours under standard conditions.

EFFECT: invention ensures sterilisation of the cell cultures of plants, and is accompanied by acceleration of the cultures cleaning of the infection, extended antibacterial action, and absence of the cytotoxic action.

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Description

The invention relates to biotechnology and can be used to eliminate bacterial infections in the cultivation of plant cell cultures in the biotechnological industry.

One of the main conditions that determine the success of working with plant cell cultures is the lack of contamination at all stages of cultivation. In the process, a number of measures are taken to ensure sterility, including autoclaving nutrient media, disinfecting the plant material used, sterilizing work tools, and organizing sterile transplant areas [Leifert C. and Waites W. Dealing with microbial contaminants in plant tissue and cell culture: hazard analysis and critical control points. Physiology, Growth and Development of Plants in Culture. 1994. Pp. 363-378; Reed B. M and Tanprasert P. Detection and control of bacterial contaminants of plant tissue cultures. A review of recent literature. Plant Tissue Culture and Biotechnology. 1995.1: 137-142]. Despite the observance of sterile conditions when working with cell cultures, spontaneous bacterial infections cannot be avoided, which leads to the death of the culture and an increase in financial costs for its restoration. This happens both in research laboratories and in industrial production, when several bacteria that enter the bioreactor, for example 4 tons, are able to completely suppress the growth of plant cells due to their faster reproduction [Leifert C. & Woodward S. Laboratory contamination management: the requirement for microbiological quality assurance. Plant Cell, Tissue and Organ Culture. 1998. 52: 83-88; Cassells A. Pathogen and Biological Contamination Management in Plant Tissue Culture: Phytopathogens, Vitro Pathogens, and Vitro Pests. Plant Cell Culture Protocols: Methods in Molecular Biology. 2012.877: 57-80; Hayward A. Latent infections by bacteria. Annu. Rev. Phytopathol. 1974. 12: 87-97]. In addition, the removal of potentially dangerous bacteria for humans from plant cell cultures (E. coli and others) is also relevant [S. Leifert; A.S. Cassells. Microbial hazards in plant tissue and cell cultures. In Vitro Cellular & Developmental Biology - Plant 2090 Vol: 37 (2): 133-138].

In medicine, silver nanoparticles that inhibit hospital-acquired infections resistant to antibiotics are used as antibacterial agents [Rana S. And Kalaichelvan P. Antibacterial Activities of Metal Nanoparticles. Adv Bio Tech. 2011.11 (02): 21-23; Mohammad J. et al. Antibacterial properties of nanoparticles. Trends in biotechnology. 2012.30 (10): 499-511; Azam A. et al. Antimicrobial activity of metal oxide nanoparticles against Gram-positive and Gram-negative bacteria: a comparative study. Int J Nanomedicine. 2012.7: 6003-9].

In this invention, the authors use the same principle of action of silver nanoparticles, which is used to affect hospital infections, but in another area - plant biotechnology, where it has not been used before. The premise of using silver nanoparticles in biotechnology is the same, that is, the replacement of standard substances with a new class of disinfectants.

The antibacterial effect of nanoparticles is due to their ability to sequentially damage the cell wall and penetrate into the bacteria, which leads to the initiation of protein dephosphorylation processes necessary to ensure vital activity, and this in turn leads to the death of bacteria [Shrivastava S. et al. Characterization of enhanced antibacterial effects of novel silver nanoparticles. Nanotechnology. 2007. 18: 225103]. The efficacy of using silver nanoparticles as a bactericidal agent has been investigated for a wide range of pathogens [Galdiero S. et al. Silver nanoparticles as potential antiviral agents. Molecules 2011.16: 8894-8918].

In recent years, publications have appeared that indicate the possible use of metal nanoparticles as an antibacterial agent for the disinfection of plant explants, i.e. primary material used for in vitro culture [Mahna N., Vahed S. and Khani S. Plant In vitro Culture goes Nano: Nanosilver-Mediated Decontamination of Ex vitro Explants. J Nanomed Nanotechol. 2013.4 (2): 1000161; Abdi G. Evaluation the potential of Nano silver for removal of bacterial contaminants in valerian (Valeriana officinalis) tissue culture. L. J. BIOL. ENVIRON. SCI. 2012.6 (17): 199-205]. However, silver or other metal nanoparticles have not been previously used to disinfect plant cell cultures. An important parameter for the use of metal nanoparticles as antimicrobial agents is their size [Ai J. et al. Nanotoxicology and nanoparticle safety in biomedical designs. Inter. J Nanomedicine 2011.6: 1117-1127]. Large nanoparticles larger than 100 nm are practically not used, since they are not effective. Particles up to 20 nm in size have found wide technical application, however, with a decrease in particle size, their toxicity to living organisms increases. The smaller the particles, the higher their antimicrobial activity. However, silver particles smaller than 15 nm in size can cause an oxidative explosion, which leads to destructive changes in cells [Carlson C. et al. Unique cellular interaction of silver nanoparticles: size-dependent generation of reactive oxygen species. J Phys Chem B. 2008. 112 (43): 13608-19; Liu W. et al. Impact of silver nanoparticles on human cells: effect of particle size. Nanotoxicology. 2010.4 (3): 319-30].

Currently, in plant biotechnology there is no other way to eliminate bacterial infections in living cell cultures, so the search for a new, more effective one is relevant. The present invention is the creation of a new method for the effective elimination of infection, using an agent that provides prolonged antibacterial resistance of the culture and the absence of cytotoxic effects. It was not obvious that silver nanoparticles will be effective in cell cultures of plants, since their effect has not been previously tested. Thus, the interaction with the elements of the culture medium (22 components) and high molecular weight compounds (proteins and polysaccharides secreted into the medium) was not studied, and the stability and activity of nanoparticles in these systems were also not studied. The method of using metal nanoparticles to eliminate infection, described in this application as an example of silver nanoparticles, is today the first example of the application of new scientific knowledge in the biotechnology of plant cell cultures.

The most common way to eliminate emerging bacterial infections during the cultivation of plant cells is the introduction of antibiotics into culture media [Fellner et al. Identification and antibiotic sensitivity of microbial contaminants from callus cultures of garlic Allium sativum L. and Allium longicuspis Regel. Plant Science. 1996. 113: 193-201]. However, with antibiotics, many bacteria become resistant to them [Schmieder R. and Disclosures R. Insights into Antibiotic Resistance Through Metagenomic Approaches. Future Microbiol. 2012.7 (l): 73-89; Dever L. and Dermody T. Mechanisms of bacterial resistance to antibiotics. Arch Intern Med. 1991.151 (5): 886-95; Dessen A. et al. Molecular mechanisms of antibiotic resistance in gram-positive pathogens. Curr. Drug Targets Infect. Dis. 2001. 1: 63-77]. Another disadvantage of antibiotics is the cytotoxic effect on cultured cells, which negatively affects the biomass growth parameters or causes culture death [Catlin D. The effect of antibiotics on the inhibition of callus induction and plant regeneration from cotyledons of sugarbeet (Beta vulgaris L.) . Plant Cell Reports. 1990.9 (5): 285-8; Cassels A. Problems in tissue culture: Culture contamination. Micropropagation: technology and application. 1991. Pp. 31-44]. It is also important to note the low chemical resistance of antibiotics to environmental factors during cultivation. Thus, the decay period of antibiotics is 2-3 days, which is sometimes insufficient to inhibit bacterial growth [Kazemi EM, Jonoubi P, Majd A and Pazhouhandeh M. 2014. Reduction of negative effects of cefotaxime in tomato transformation by using FeEDDHA. IJFAS Journal. Vol 3, p. 538-542; de Oliveira ML, Costa MG, da Silva CV and Otoni WC. 2010. Growth regulators, culture media and antibiotics in the in vitro shoot regeneration from mature tissue of citrus cultivars. Pesq. agropec. bras., Brasilia, v. 45, p. 654-660].

The authors are not aware of other methods of eliminating bacterial infections in cultured plant cells, therefore this method is taken as the closest, as a prototype. A known method for the elimination of bacterial infections in the cultivation of cell cultures of garlic Allium sativum L., Allium longicuspis Regel, where antibiotics erythromycin, gentamicin and imipenem are used as an antibacterial agent of the culture medium [M. Feltner et al. Identification and antibiotic sensitivity of microbial contaminants from callus cultures of garlic Allium sativum L. and Allium longicuspis Regel. Plant Science. 1996. 113: 193-201], however, one of the disadvantages of the method is the inability to quickly and effectively eliminate the infection, to ensure long-term antibacterial resistance of the culture. The reason is that many representatives of bacterial microflora infecting cell cultures have developed mechanisms of protection against the damaging effects of antibiotics, the use of which becomes ineffective or critical due to the cytotoxic effect. In addition, the instability of antibiotics to environmental factors often does not allow reliable destruction of bacterial contamination.

Calli of Allium sativum L. and Allium longicuspis Regel were cultured in the dark at 25 ° C on an agarized nutrient medium [Dunstan and Short. Improved growth of tissue cultures of the onion, Allium sulfur. 1997. Physiologia Plantarum. 41: 70-72]. Upon visual detection of infectious foci, infected calluses were transplanted onto fresh growth media with the addition of antibiotics. The presence of infection was determined by the appearance of light yellow colonies after incubation with antibiotics. Colonies were counted. It was shown that only the simultaneous addition of three antibiotics to the culture medium (erythromycin, gentamicin and imipenem at a concentration of 10, 10 and 5 mg / l, respectively) led to a complete inhibition of bacterial infection in cultures of Allium sativum and Allium longicuspis. However, in a culture of A. longicuspis, latent bacteria were detected.

The problem is solved in that in the proposed method, including the cultivation of suspension cell cultures of plants, according to the invention, the elimination of bacterial infection is carried out by introducing into the culture medium, as an antibacterial agent, an aqueous suspension of silver nanoparticles with a diameter of 20-80 nm in a final concentration of 50 μg / ml and cultured 24 hours under standard conditions.

The technical result provided by the invention is to achieve the effect of disinfection of crops, accompanied by an acceleration of the process of cleaning crops from infection, prolonged antibacterial action, and the absence of cytotoxic effect.

The effectiveness of the method of eliminating bacterial contamination during the cultivation of suspension cell cultures of plants using silver nanoparticles was studied for different types of microorganisms, both gram-positive and gram-negative - Escherichia coli, Bacillus subtilis, Agrobacterium rhizogenes and model plant cultures - madder heart-shaped Rubia cordifolia, grapes vinifera and ginseng present Panax ginseng.

Suspension callus cultures of plants Rubia cordifolia, Vitis vinifera, Panax ginseng are cultivated in the standard way on W _{B / A} nutrient media in the dark at 20-30 ° C, relative humidity 50-80% with a passivity of 14-21 days [Mischenko NP, Fedoreyev SA, Glazunov VP, Chernoded GK, Bulgakov VP, Zhuravlev YN Anthraquinone production by callus cultures of Rubia cordifolia. Fitoterapia. 1999. V. 70. N6. P. 552-557].

Further, the resulting suspension plant cultures artificially infect, simulating contamination in an industrial environment. For this, daily cultures of Escherichia coli, Bacillus subtilis, Agrobacterium rhizogenes are used. Bacteria are grown at a temperature of 28 ° C in a known bacterial medium LB [Feltner M. et al. Identification and antibiotic sensitivity of microbial contaminants from callus cultures of garlic Allium sativum L. and Allium longicuspis Regel. Plant Science. 1996. 113: 193-201]. Infection is carried out by co-cultivation of cell cultures with 20 μl of bacteria at a concentration of 1 × 10 ⁶ CFU / ml, in the dark, at a temperature of 23-26 ° C and constant stirring at a speed of 100 rpm for 30 minutes.

To remove artificially induced bacterial infection, an aqueous suspension of silver nanoparticles with a diameter of 20-80 nm was added to flasks with infected cell cultures to a final concentration of 50 μg / ml and cultured for 24 hours under standard conditions: in the dark, with stirring at a speed of 100 rpm , temperature 23-26 ° C and relative humidity 50-70%. For the elimination of using any commercially available silver nanoparticles of the specified size. After a day, the calluses purified from the infection are used for their intended purpose.

Evaluation of the quality and rate of elimination is carried out using the well-known method of bacteriological research, by sowing a solid lawn aliquots of the culture fluid on the bacterial medium LB. Bacteriological tests are carried out one day after adding a suspension of nanoparticles to the culture and with an interval of 7 and 14 days. The results of the experiments showed that complete elimination of the infection occurs one day after the introduction of an aqueous suspension of silver nanoparticles at a concentration of 50 μg / ml and remains throughout the experiment (table 1).

To confirm the effectiveness of elimination and the duration of the antibacterial effect, the purified suspension cultures are transferred to W _{B / A} growth media containing no silver nanoparticles and grown under standard conditions for several passages. The absence of bacterial infection in cultures indicates the effectiveness of using silver nanoparticles as an antibacterial agent.

It is known that silver nanoparticles are widely used in various industries [Shankar S.S. et al. Biological Synthesis of Triangular Gold Nanoprisms. Nature Materials. 2004.3: 482-488; Mohanpuria P. et al. Biosynthesis of nanoparticles: technological concepts and future applications. J Nanopart Res. 2008. 10: 507-517], but due to the toxicity of silver nanoparticles, their scope for humans and animals is limited [Carlson C. et al. Unique cellular interaction of silver nanoparticles: size-dependent generation of reactive oxygen species. J Phys Chem B. 2008. 112 (43): 13608-19; Liu W. et al. Impact of silver nanoparticles on human cells: effect of particle size. Nanotoxicology. 2010.4 (3): 319-30]. However, the authors found that silver nanoparticles exhibit low toxicity to plant cells. Thus, the experimental results showed that the introduction of silver nanoparticles into the culture medium did not suppress the growth of biomass, which indicated the absence of a cytotoxic effect (table 2).

The method is illustrated by the following example.

Example 1. The method of elimination of contamination is shown using the example of the madder heart culture Rubia cordifolia as an option for using any suspension cell culture. Suspension cell culture R. cordifolia is grown by the standard method in 250 ml Erlenmeyer flasks containing 50 ml of W _{B / A} culture medium of the following composition, mg / l of water:

NH ₄ NO ₃ 400 Kno ₃ 1900 CaCl ₂ * 6H ₂ O 665 MgSO ₄ 370 KN ₂ RO ₄ 170 H ₃ IN ₃ 6.2 MnSO ₄ 16.9 CoC1 ₂ * 6H ₂ O 0.025 CuSO ₄ * 5H ₂ O 0.025 ZnSO ₄ * 7H ₂ O 8.6 NaMoO ₄ * 2H ₂ O 0.25 Ki 0.83 FeSO ₄ * 7H ₂ O 27.8 Na ₂ EDTA * 2H ₂ O 37.3 Meso-inositol one hundred Vitamin B ₁ 0.2 Vitamin B ₆ 0.5 Vitamin PP 0.5 Sucrose 25000 Water 1000

Suspension culture is grown on the specified medium with constant stirring at a speed of 100 rpm, at a temperature of 25 ° C and a relative humidity of 60%, for 14 days. If a bacterial infection is detected, or at the risk of microbial contamination, silver nanoparticles with a diameter of 20-80 nm are added to the suspension cell culture to a final concentration in the culture medium of 50 μg / ml. The suspension culture is then cultured for 24 hours under similar conditions.

Thus, the proposed method allows you to quickly and effectively carry out the elimination of bacterial infections during the cultivation of cell cultures of plants. The use of silver nanoparticles as a new antibacterial agent makes it possible to increase the resistance of the culture to the action of microbial contaminants and to provide a long-term anti-bacterial effect. The absence of the cytotoxic effect of nanoparticles allows the method to be used not only to eliminate an infection that has already occurred, but also to protect calluses from possible contamination during cultivation in laboratory or industrial conditions.

Claims (1)

A method for eliminating a bacterial infection of plant cell cultures, including culturing suspension cell cultures using an antibacterial agent, characterized in that an aqueous suspension of silver nanoparticles with a diameter of 20-80 nm is used as an antibacterial agent, which is introduced into the suspension cell culture to a final concentration in the culture medium 50 μg / ml, while cultivation is carried out 24 hours under standard conditions.