Optimizing Suitable Antibiotics for Bacterium Control in Micropropagation of Cherry Rootstock Using a Modified Leaf Disk Diffusion Method and E Test

Abstract

Bacterial contamination is a major and constant threat to the establishment and subculture of in vitro plant culture. In this study, we used a slightly modified qualitative disk diffusion method to screen optimal antibiotics to control the growth of bacterial contaminants isolated from explants of cherry rootstock ‘Gisela 6’. Bacterial susceptibility to eight different antibiotics was tested. The results showed that tetracycline was the most effective antibiotic for controlling bacterial growth; cefotaxime, carbenicillin, kanamycin, and streptomycin were less effective, whereas ampicillin, penicillin, and cefazolin did not inhibit growth of the isolated bacteria. Using the quantitative E test, the minimal inhibitory concentration (MIC) of tetracycline was determined to be 1.0 µg mL−1. We also measured the Fv/Fm values, chlorophyll content, and enzymatic activity of superoxide dismutase and peroxidase to explore the effect of different tetracycline concentrations, 0, 0.064, 0.5, 1.0, 16, and 256 µg mL−1, on the growth of bacteria and explants over 30 days. Results indicated that 1.0 µg mL−1 tetracycline was effective in restricting bacterial growth, with non-significant negative effects on explants at low concentrations, but were enhanced negative effects at high concentrations. The application of the disk diffusion method and E test enabled the identification of an antibiotic and its MIC value effective for eliminating bacterial contaminants while causing minimal damage to explants, indicating a high potential of these methods to control bacterial contaminants in in vitro plant culture.

Cellular, Molecular, and Physiological Aspects of In Vitro Plant Regeneration

“…The medium supplemented with antibiotics such as β-lactam antibiotics and tetracycline has been reported to result in higher regeneration capacity as compared with untreated explants [ 189 , 190 ]. Despite the successful application of antibiotics to suppress bacterial growth in vitro, excessive and regular use of them is detrimental to the regeneration process and results in a reduced regeneration ability of tissue cultures [ 191 ].…”

Effect of Beta-Lactam Antibiotics on Microspore Embryogenesis in Brassica Species

“…When microspores were cultivated in a medium without added antibiotics, developmental scenarios observed in the Petri dishes were as follows: (a) active bacterial growth accompanied by destruction of microspores (Figure 1b), the medium appeared turbid at five days ( Figure 2b); (b) weak bacterial growth seemingly due to weak phytotoxic effect, microspore embryo formation was still observed, although inhibited and reduced in amount ( Figure 3a); and (c) normal microspore embryo development in large amounts (Figure 3b). Multiple studies in different cultures demonstrated that antibiotics can both inhibit the growth and development of explants and cells and induce their regeneration [14][15][16][17][18][19][20]. It is assumed the stimulating effect is due to the auxin-like structure of β-lactam antibiotics that enables them to imitate the effect of plant hormones [21,22].…”

“…Multiple studies in different cultures demonstrated that antibiotics can both inhibit the growth and development of explants and cells and induce their regeneration [14][15][16][17][18][19][20]. It is assumed the stimulating Plants 2020, 9, 489 2 of 10 effect is due to the auxin-like structure of β-lactam antibiotics that enables them to imitate the effect of plant hormones [21,22].…”

Bacteriostatic antibiotic testing in the Allium test

“…Следует напомнить, что тетрациклинбактериостатический антибиотик из группы тетрациклинов; включен в список важнейших лекарственных средств, составляемый ВОЗ (WHO…, 2013). Широко он используется в культуре клеток и тканей растений (Концевая, 2011;Liang et al, 2019). Мы полагаем, что имеет смысл более тщательно рассмотреть роль и значение тетрациклина в антибактериальной терапии, применяемой в биотехнологии растений.…”

“…Введение Исследования по влиянию антибиотиков на клеточные культуры различных сельскохозяйственных и лесных растений довольно многочисленны (Концевая, 2011;Дунаева, 2015;Ray, Ali, 2017;Liang et al, 2019). Выявлен как стимулирующий эффект антибиотиков, так и представлены сведения о негативном их влиянии на рост культуры клеток и тканей растений.…”