Impaginato 43 Adv. Hort. Sci., 2020 34(1): 43­48 DOI: 10.13128/ahsc­8300 Cefixime manages internal bacterial contamination during tissue culture operation A. Ameri 1, G.H. Davarynejad 1 (*), A. Tehranifar 1, N. Moshtaghi 2 1 Department of Horticultural Science and Landscape, Ferdowsi University of Mashhad, Mashhad, Iran. 2 Department of Biotechnology and Plant Breeding, Ferdowsi University of Mashhad, Mashhad, Iran. Key words: antibiotic, in vitro culture, growth, Pyrus communis. Abstract: Large­scale propagation of Pyrus communis, which is a difficult­to­ root species, is dependent on tissue culture technique. However, internal bac­ terial contaminations are an obstacle in tissue culture of fruit tree species. For this purpose, this investigation was conducted with several experiments to manage bacterial contamination. First, gram test for bacterial contamination related to Pyrus shoots proliferating was conducted. Gram test revealed that bacteria contaminating pear shoots were negative gram. Then, we investigated the application of cefixime (0, 100, 300 and 500 mg L­1) or ampicillin (0, 100, 300 and 500 mg L­1) for managing bacterial contaminations. It was found that the contaminated area on medium containing 500 mg L­1 cefixime (63.585 mm2) was lower than other treatments (803.84 mm2). Therefore, cefixime at 500 mg L­1 was selected to control the bacterial contamination. Next, different proce­ dures were used included shaking with (1: sterile distilled water, 2: 500 mg L­1 cefixime and culturing in media with 500 mg L­1 cefixime, 3: 500 mg L­1 cefixime, culturing and subculturing in media with 500 mg L­1 cefixime 4: Disinfection). The third procedure was known the best due to the low bacterial contamina­ tion percentage and rate also the healthy growth of plants. Finally, the effect of gibberellic acid at 0 and 1 mg L­1 was investigated to compensate for shoot growth reducing in the presence of cefixime. 1 mg L­1 gibberellic acid improved the growth indices in the presence of cefixime. 1. Introduction Fire blight, the most devastating disease of pear, leads to the death of the whole pear tree through the systematical infection in all underground and aerial parts of the tree (Vanneste, 2000; Evrenosoğlu et al., 2019). From the horticultural science perspective, the revival of the pear orchards is dependent on large­scale propagation. The majority of culti­ vated pear is Pyrus communis (Morgan et al., 1994) and P. communis cv. Williams is sensitive to fire blight (Abdollahi et al., 2010). However, Pyrus communis is difficult­to­root (Zhu et al., 2003; Sun et al., 2011); therefore, the tissue­culture technique can support large­scale propagation of pear. (*) Corresponding author: davarynej@um.ac.ir Citation: AMERI A., DAVARYNEJAD G.H., TEHRANIFAR A., MOSHTAGHI N., 2020 ­ Cefixime manages inter‐ nal bacterial contamination during tissue culture operation ­ Adv. Hort. Sci., 34(1): 43­48 Copyright: © 2020 Ameri A., Davarynejad G.H., Tehranifar A., Moshtaghi N.. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 20 October 2018 Accepted for publication 6 March 2019 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2020 34(1): 43­48 44 One of the most important factors in tissue culture is t h e c o n t r o l o f m i c r o b i a l c o n t a m i n a ti o n . P l a n t immune system acts against pathogens. There are three steps of plant defense responses included the response in the step of entry, establishment, and spread of pathogens. Overall, plant immune systems can be classified into cell wall reinforcement and pro­ grammed cell death. In pathogen entry condition, cell wall reinforcement is efficient and in pathogen estab­ lishment and spread conditions programmed cell death can be restrictive (Abramovitch and Martin, 2004). When pathogens suppress the plant defenses responses, the plant was invaded by pathogens and contaminated. One of the phytopathogens is bacte­ ria; they cause serious troubles in vitro conditions. Some of the modes of bacteria actions to suppress the host defenses refer to the use of type III effector proteins and toxins (Abramovitch and Martin, 2004) as well as, type IV secretion systems to inject effector proteins into cells (Angot et al., 2007). Tissue culture technique is very sensitive to special pathogenic fac­ tors and all of the microbes in air condition and equipment. In other words, all of the microbes infect cultures with the aim of nutrition (Nadha et al., 2012). Internal infections in plant cultures had often harmful effects for shoot proliferation, shoot rooting and quality of plant growth (Nadha et al., 2012). In tissue culture, the selection of the appropriate antibi­ otic is important. There are several reports about the use of antibiotics to manage the bacterial contamina­ tion (Phillips et al., 1981; Falkiner, 1990; Kneifel and Leonhardt, 1992; Barrett and Cassells, 1994; Falkiner, 1997; Nadha et al., 2012). Cefixime is an antibiotic belonging to cephalosporin class. The mode of action of cephalosporins is related to inhibiting the cell wall biosynthesis so that this class arrests the formation of peptide bonds (Kohanski et al., 2010). Affect sites of various antibiotics are different; therefore the efficiency of different antibiotics is dif­ ferent in the removal of bacteria. There are some observations for different antibiotics affect sites in the previous studies, such as, inhibiting of cell wall synthesis that is related to benzylpenicillin and phos­ phomycin, inhibiting protein synthesis related to chloramphenicol and streptomycin, inhibiting of RNA and DNA synthesis related to rifampicin and nalidixic acid (Phillips et al., 1981). One of the side effects of antibiotic application in plant tissue culture is the reduction of growth. However, in control of bacterial infection in Guadua angustifolia, streptomycin sul­ fate decreased growth shoot, but kanamycin caused intensive growth with high­quality; therefore, the effects of antibiotics are different on plant growth (Nadha et al., 2012). The management of contamina­ tion in tissue culture leads to the prevention of waste of time and energy. In this investigation, we examine the use of antibiotics to manage the bacterial conta­ minations coupled with the use of GA3 to compen­ sate for the poor growth of the plant in the presence of antibiotic in the media. 2. Materials and Methods Plant materials Three months old proliferating micro­shoots of Pyrus communis cv. Williams, as the most common cultivar in the world, exhibited bacterial contamina­ tion. Contaminated micro­shoots were picked to investigate the experiments of bacterial control for the large­scale production of P. communis. These m i c r o ­ s h o o t s w e r e m a i n t a i n e d i n M S m e d i u m (Murashige and Skoog, 1962) supplemented with 1.5 mg L­1 BA, 0.1 mg L­1 NAA and 3% sucrose. Gram test of bacteria Two drops, approximately 50 µL, of a 3% (W/V) solution of potassium hydroxide were placed on a clean glass slide as outlined by Ryu (1940). Bacterial cells were transferred from culture media aseptically with a flat wooden toothpick and placed into the drop of KOH with rapid, circular agitation. After 5­8 sec, the toothpick was alternately raised and lowered just off the slid surface to detect a stringing effect. It was considered gram­negative bacteria if drop vis­ cosity increased within 15 sec (Suslow et al., 1982; Schaad et al., 2001). Antibiotic selection test Contaminated shoots were cultured on MS medi­ um (Murashige and Skoog, 1962) supplemented with either ampicillin 0, 100, 300 and 500 mg L ­1 or cefixime at 0, 100, 300 and 500 mg L­1. Vessel cul­ tures with 32 mm inner diameter were maintained at a constant temperature of 25±1°C and in 16/8 h light/dark photoperiod (45 µmol m­2 s­1) using cool white fluorescent lamps (Sylvania, Germany). After a week, the contaminated area was measured in each treatment. Bacterial contamination removing In the before step, we selected the proper antibi­ otic (cefixime at 500 mg L­1). Then, we used four dif­ ferent procedures using cefixime at 500 mg L­1 to control any eventual bacterial contamination. For each procedure, 3 micro­shoots were cultured in a Ameri et al. ‐ In vitro plant rescue by Cefixime 45 candle jar as a replicate. 1) Shaking of contaminated shoots with sterile dis­ tilled water (control); 2) The first, shaking of contaminated shoots with cefixime at 500 mg L­1 then, cultured in media with cefixime at 500 mg L­1. Finally, sub­culturing in free antibiotic media; 3) The first, shaking of contaminated shoots with cefixime at 500 mg L­1, then, cultured in media with cefixime at 500 mg L­1. Finally, sub­culturing in media with cefixime at 500 mg L­1; 4) Disinfection of contaminated shoots (immersing in 1% hypochlorite sodium for 5 min then rinsed with sterile water three times). MS medium (Murashige and Skoog, 1962) supple­ mented with 1.5 mg L­1 BA, 0.1 mg L­1 NAA and 3% sucrose were used for each procedure. Cultures were maintained at a constant temperature of 25±1°C and in 16/8 h light/dark photoperiod (45 µmol m­2 s­1) u s i n g c o o l w h i t e fl u o r e s c e n t l a m p s ( S y l v a n i a , Germany). After 30 days, several traits were evaluat­ ed: percentage of fungal contamination and bacterial contamination (BC), bacterial contamination rate (BCR) and general health. Based on the following equation (E1) bacterial contamination rate was counted per each micro­ shoot in each candle jar: BCR =∑ Ni Di w h e r e B C R = B a c t e r i a l c o n t a m i n a ti o n r a t e , N i = Number of the contaminated shoot in each day, Di = Day number. Rescued shoot improvement Micro­shoots related to the best procedure were transferred to MS medium (Murashige and Skoog, 1962) supplemented with 1.5 mg L­1 BA, 0.1 mg L­1 NAA, 3% sucrose, 500 mg L­1 cefixime and gibberellic acid (GA3) treatments. The concentrations of GA3 were 0 and 1 mg L­1. pH was adjusted at 5.8 with NaOH prior to autoclaving at 98 kPa and 121°C, and the media were solidified using 0.8% agar. Cefixime antibiotic and GA3 added to the media after autoclav­ ing by filtering. Related traits of this experiment were included: the percentage of new growth, the per­ centage of proliferation, the average number of bud and leaf, as well as, the average shoot length. The evaluation of declined antibiotic dose After the six months using cefixime at 500 mg L­1, proliferated shoots were divided into two groups. Each group of plants was cultured in media with cefixime at either 500 mg L­1 or 250 mg L­1. pH was adjusted at 5.8 with NaOH prior to autoclaving at 98 kPa and 121°C, and the media were solidified using 0.8% agar. Cefixime antibiotic and GA3 were added to the media after autoclaving with the syringe filter (pore size: 0.22 µm). After 10 days, the percentage of bacterial contaminations and bacterial contamination rate were measured per each micro­shoot in each candle jar based on (E1). Rooting micro‐shoots After six months, micro­shoots were transferred to ½­strength QL (Quoirin and Lepoivre, 1977) medi­ um supplemented with 1.5 mg L­1 Naphthaleneacetic acid (NAA) and 500 mg L­1 cefixime. Cultures were maintained a week in dark conditions, then, trans­ ferred to 16/8 h (light/dark) photoperiod and light intensity of approximately 45 µmol m­2 s­1 photosyn­ thetic photon flux density (PPFD) emitted by cool­ white fluorescent tubes in 35% relative humidity. Statistical analysis These experiments were arranged as a completely randomized design with three replications. The data were statistically analyzed using a one way ANOVA test and means were compared with the Duncan test at the 5% level of confidence. All of the statistical tests were performed using SAS (Statistical Analysis System) software v9.1. All of the percentage data were transformed to Arcsin √x. 3. Results and Discussion The result of the gram type detection showed contamination of this investigation is related to gram­negative bacteria. In the antibiotic selection step, we compared ampicillin and cefixime. We used 0, 100, 300 and 500 mg L­1concentrations of each antibiotic. Ampicillin is a common antibiotic in tissue culture, and its activity spectrum is related to gram­ positive and gram­negative bacteria, whereas the cefixime is antibiotic acting against gram­negative bacteria. In antibiotic selection test, results revealed 500 mg L­1cefixime could overcome contamination better than other treatments. The contaminated area on medium containing 500 mg L­1cefixime (63.585 mm2) was lower than other treatments (803.84 mm2). Therefore, we selected 500 mg L­1 cefixime to control the contamination for later experiments. ANOVA revealed that the difference between the four procedures was significant (P≤0.01) for bacterial contamination percentage and the rate of bacterial Adv. Hort. Sci., 2020 34(1): 43­48 46 contamination (Table 1). Four procedures were used for the survival and rescuing of shoot from bacterial contamination. The third procedure (the first, shak­ ing of contaminated shoots with 500 mg L­1 cefixime; then, cultured in media with 500 mg L­1 cefixime. Finally, sub­culturing in media with 500 mg L ­1 cefixime) was known the best due to the low bacteri­ al contamination percentage, rate and finally the healthy and fresh growth of plants. These indices in other procedures were not desirable; as the highest percentage of bacterial contamination was observed in the first procedure (shaking with sterile distilled water). All of the procedures led to necrotic plants except for procedure 3 (Table 2). The presence of antibiotic in media effected on plant growth and weakened their growth; therefore, we used GA3 and its effect evaluated on plants growth. GA3 application in media containing 500 mg L­1 cefixime had a signifi­ cant effect on the percentage of new growth, the average of bud number, the average shoot length and the average of leaf number (P<0.05) (Table 3). Without the application of GA3 were not observed any proliferation and new bud formation; while in media containing GA3, 22.53% proliferation and 1.46 the average number of bud were observed (Table 4). As well as, the results showed the decrease in the antibiotic dose to 250 mg L­1 cefixime increased BCP Table 4 ­ Evaluation of Gibberellic acid in MS medium along with PGRs and 500 mg/L cefixime on secondary growth traits Values are mean ± standard error. Table 1 ­ Analysis of variance traits under study Table 2 ­ Evaluation of different procedure to come over bacterial contamination during the culture NS= no significant; **= significant at the 0.01 level of probability according to Duncan Test. **= significant at the 0.01 level of probability according to Duncan Test. *= significant at the 0.05 level of probability according to Duncan Test. Source of variance DF Means square Bacterial contamination Rate of bacterial contamination Treatment 3 2963.807 ** 45.48649 ** Error 8 7.350.308 0.5436 Procedure Fungal contamination (%) Bacterial contamination (%) Bacterial contamination rate General health after 30 days 1 0 90 ± 0 9.47 ± 0.77 Necrotic leaves 2 0 74.55 ± 2.42 4.93 ± 0.119 Necrotic leaves 3 0 17.01 ± 1.22 0.45 ± 0.053 Green leaves and healthy 4 0 57.85 ± 1.56 7.47 ± 0.323 Necrotic leaves 1= shaking with sterile water. 2= the first, shaking of contaminated shoots with cefixime (500 mg/l) then, culturing in media with antibiotic. Finally, sub­culturing in free antibiotic media. 3= the first, shaking of contaminated shoots with cefixime (500 mg/l), then, culturing in media with antibiotic. Finally, sub­culturing in with antibiotic media. 4= disinfection (immersing in 1% hypochlorite sodium for 5 min then shaking with sterile water for three times. Values are mean ± standard error. Table 3 ­ Analysis of variance of traits under study Source of variance DF Means square Percentage of new growth Percentage of proliferation Average of bud number Average shoot height Average of leaf number Treatment 1 514.20 ** 761.53 ** 3.22 ** 60.16 * 54.0 * Error 4 6.022 4.53 0.0066 7.33 0.33 Concentration (mg/L) New growth (%) Proliferation (%) Average bud number Average shoot height (mm) Average leaf number 1 43.93 ± 1.23 22.53 ± 1.74 1.46 ± 0.07 9.66 ± 2.03 10.33 ± 0.33 0 25.41 ± 1.58 0 ± 0 0 ± 0 3.33 ± 0.88 4.20 ± 0.33 Ameri et al. ‐ In vitro plant rescue by Cefixime 47 Leifert and Cassells (2001) mentioned alternatives for the antibiotic in their review. These alternatives included medium acidification and autotrophic cul­ ture (e.g. culture without carbohydrate) (Leifert and Cassells, 2001). In other literature were noted to acti­ vating of endogenous bacteria as a result of sub­cul­ t u r i n g i n m e d i a w i t h c y t o k i n i n s ( K n e i f e l a n d Leonhardt, 1992). However, plant tissue culture with­ out cytokinins, carbohydrates and with the modifica­ tion in media acidity is impossible. In this regards, this investigation showed with the presence of cefixime in media containing cytokinins, carbohy­ drates could manage the bacterial contamination. Based on the results of this investigation, cefixime at 500 mg L­1 had not any toxicity effect on growth and proliferation. Cefixime is an antibiotic belonging to cephalosporins class. The cephalosporin antibiotics have been introduced as the appropriate antibiotic plant tissue culture since they have low eukaryote toxicity (Mathias and Boyd, 1986) which our results emphasize this point. 4. Conclusions Bacterial contamination incidence is common and unavoidable during the in vitro propagation of fruit tree species. This investigation presented a proce­ dure to manage the bacterial contamination of P. communis cv. Williams during the in vitro culture. Based on the results of this investigation, cefixime at 500 mg L­1 could control the bacterial contamination. The use of antibiotic in a medium is associated with a decrease in the growth of plants. This side effect of antibiotic was managed with the application of GA3 at 1 mg L­1. Therefore, we suggest cefixime at 500 mg L­1 for in vitro propagation of fruit trees. References ABDOLLAHI H., TAHZIBI F., GHAHREMANI Z., 2010 ­ Correlation between fire blight resistance and morpho‐ logical characteristics of pear (Pyrus communis L.). ­ Acta Horticulturae, 896: 339­345 ABRAMOVITCH R.B., MARTIN G.B., 2004 ‐ Strategies used by bacterial pathogens to suppress plant defenses. ­ Curr. Opin. Plant Biol., 7(4): 356­364. 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