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Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 956-963 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 11 (2017) pp. 956-963 Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2017.611.112 Antibiotics Efficacy on In Vitro Growth Parameters of Important Potato Cultivars Tanuja Buckseth1*, R. K. Singh1, Sumita Sharma1, Ashwani K. Sharma2, Vaishali Moudgil1 and Aastha Saraswati1 1 ICAR-Central Potato Research Institute, Shimla- 171001 (HP), India 2 ICAR-Central Potato Research Station, Kufri-171012 (HP), India *Corresponding author ABSTRACT Keywords Microplants, Antibiotics, Amphotericin-b, Antimycotic, Varieties, Potato. Article Info Accepted: 10 September 2017 Available Online: 10 November 2017 The use of plant tissue culture as a routine method of potato seed production would be costly but these techniques can be used to eliminate the pathogens, produce required initial material and then, use another efficient and cheaper system to rapidly produce high quality seed tubers for commercial production. Considering this, the liquid formulation of antimycotic 100x, and amphotericin-B 250 µg/ml of PAA make was used. These test antibiotics were added aseptically at various concentrations (0.5%, 1%, 1.5%, and 2%) in the sterilized MS medium culture tubes to find out contamination free, vigorous in-vitro growth of the microplants. It was observed that amphotericin-b at lower conc. i.e., 0.5% and 1% could be incorporated in the micro propagation medium of potato to minimize fungal contaminations as well as for improving the microplants vigour. However, all the tested antimycotic concentrations had negative effects on almost all the morphological characters studied, which need further investigation with lower concentrations. Introduction most of the potato growing nations can be overcome through micro-propagation techniques on account of faster rate of multiplication (Singh et al., 2016). Besides, rapid multiplication, disease freedom on account of multiplication of disease free mother stocks under controlled conditions followed by reduced number of field exposures as compared to conventional multiplication system is an added advantage of seed potato production through tissue culture techniques (Venkataslam et al., 2017). Due to these numerous advantages, the new system of seed potato production involving micro-propagation is finding favour among Seed constitutes a major and important input in potato (Solanum tuberosum L.) cultivation. On account of vegetative propagation, the requirement of seed potatoes (tubers) is voluminous and accounts for 40- 50% of the total production (Buckseth et al., 2016). Potato productivity in India is low in comparison to developed countries due to the non-availability of quality seed in required amounts. Seed potato production involving micro-propagation (tissue culture) techniques can overcome many of the problems associated with the conventional multiplication system (Buckseth et al., 2017). The everlasting shortage of seed potatoes in 956 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 956-963 the seed potato entrepreneurs. Tissue culture based hi-tech seed potato production is spreading very fast in the country. Micropropagation at faster rate and free from cultural contamination is an integral component for the success of in-vitro mass multiplication of potato. Plant tissue cultures can be contaminated by different microorganisms, which include bacteria and fungi. They can reduce the productivity and can completely prevent their cultivation (Sen et al., 2013). Fungal and bacterial contamination can also reduce the growth rate, retard rooting, and can even cause plant death (Leifert and Waites, 1992). Tissue cultures can become contaminated at any stage of the tissue culturing process (Leifert, 2000). The most difficult to control are endogenous bacteria, which do not cause any visible symptoms in the contaminated culture. Successful tissue culture protocols start with effective sterilization process. Though, surface sterilization eliminates the exophytic microbes (George, 1993) but, endophytic contamination can only be eradicated by antibiotic therapy (Jung, 2003). Antifungal agents like amphotericin B and antimycotic are one of the polyene antibiotics and is widely used for the treatment of systemic fungal infections. In plants grown in vitro, the presence of microorganisms with the culture medium often is associated to contamination, thus their unwanted presence, since one of the objectives of this technique is precisely to obtain axenic plants, or entirely free microorganisms (Leifert and Cassells, 2001; Harshal et al., 2014). Materials and Methods The research was carried out at ICAR-Central Potato Research Institute, Shimla with four important varieties of potato viz., Kufri Jyoti, Kufri Sadabahar, Kufri Lauvkar and Kufri Sindhuri. The broad spectrum antibiotics in liquid commercial formulation of PAA (Phenyl acetic acid) make was used in the experiment. The liquid formulation of antimycotic 100x, and amphotericin-B 250 µg/ml of PAA make was used. These test antibiotics were added aseptically at various concentrations (0.5%, 1%, 1.5%, and 2%) in the sterilized MS medium culture tubes (Murashige and Skoog, 1962). Three single node pieces of explants were vertically inserted in MS culture medium per tubes at equilateral distance (Naik et al., 2007). The culture tubes were kept for 21days in culture room with 16 hours light (irradiance of 60 µmol/m2/s) and 8 hours dark photoperiodism at 22±1oC temperature. Various growth parameters viz. microplant height, root length, no. of nodes, leaves and roots, fresh weight as well as dry weight was recorded after 21 days of incubation (Buckseth et al., 2017). In case of roots, as there was secondary branching, only primary roots were counted. Root length was recorded for the longest root in each plant. Fresh and dry weight was taken for all the three plantlets along with the root. Micro-plants from each test tube were dried at 80˚C for 48 h in the hot air oven and dry weight was recorded after bringing it to room temperature (Venkataslam et al., 2013). The experiment was conducted in a factorial (2x4x5) completely randomized design. Each treatment comprised four replicates, each replicate consist of four test tubes having three plantlets. The three-way analysis of variance was done using the software AGRES and means were separated according to the least significant differences at 0.05 level of probability. Therefore, a study was conducted to investigate the effect of these antifungal agents (Amphotericin-B and Antimycotic) for different concentrations for four varieties (Kufri Jyoti, Kufri Sadabahar, Kufri Lauvkar and Kufri Sindhuri) to find out contamination free, vigorous in-vitro growth of the microplant in MS media. 957 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 956-963 Sadabahar (Table 2). However, Antimycotic at all tested concentrations had negative effect on no. of roots and root length in all the tested varieties. Results and Discussion Amphotericin-b at 0.5% and 1.0% significantly increased the microplants height as compared to control in Kufri Jyoti, K. Sindhuri, K. Lauvkar and Kufri Sadabahar respectively. Rest of the concentrations were at par with control (Table 1). However, Antimycotic at all concentrations retarded microplants height compared to control. In all the tested varieties i.e., K. Jyoti, K. Sindhuri, K. Lauvkar and K. Sadabahar, the no. of leaves were recorded more in each applied conc. of amphotericin-b as compared to control but the effect of different conc. was found to be statistically at par with each other. However, antimycotic at all concentrations decreased the no. of leaves in all tested varieties at all concentrations as compared to control (Table 1). Zhang et al., (1999) reported that at higher concentrations of antibiotic products so formed degrade the polyribosome that can inhibit protein synthesis and disrupt the membrane permeability. In Kufri Sadabahar, amphotericin-b at all concentrations significantly increased the fresh weight and dry weight as compared to control. The effects of all the tested concentrations were significant with each other (Table 3). In Kufri Jyoti, 0.5% amphotericin-b had positive significant effect on fresh weight and dry weight as compared to control. All the tested concentrations of amphotericin-b were at par with control in Kufri Sindhuri and Kufri Lauvkar. Antimycotic irrespective of concentrations reduced the fresh weight and dry weight respectively. Plant growth promoting effect of amphotericin-b upto certain concentrations may be responsible for its significant positive response on morphological characters of different varieties. Borrelli et al., (1992), Holford and Newbury (1992), Nakona and Mii (1993), Teng and Nicholson (1997), Venkataslam et al., (2013) have reported that in different crops morphogenetic response were promoted through the breakdown of the products formed by the metabolic activities of the cells during incubation period which mimic plant growth regulators. Negative effect of the tested concentrations of antimycotic on almost all the morphological characters of all the varieties as observed in the present investigation may be due to toxic effects of this antibiotic and such effects due to different antimicrobial agents have also been reported in Barley by Mathias and Mukasa (1987). The present study explains that the genotype is sole responsible to confer the microplants sensitivity as well as its tolerance limit to different antibiotic agents (Venkataslam et al., 2017; Buckseth et al., 2016). In Kufri Lauvkar, amphotericin-b at 0.5% conc. and 1.5% conc. in Kufri Sadabahar showed significant increase in no. of nodes/plantlet whereas; the effect of all the tested conc. was at par or slightly better than control. Antimycotic at all tested concentrations had negative effect on all the tested varieties for no. of nodes/plantlet. In Kufri Sadabahar, all the tested concentrations of amphotericin-b significantly increased the root no. compared to control whereas, in Kufri Sindhuri, higher conc. of amphotericinb had positive effect on root number (Table 2). In Kufri Jyoti and Kufri Sindhuri, the root length was more in control. No positive effect of amphotericin-b was recorded. Amphotericin-b upto 1.5% conc. in Kufri Lauvkar showed increased in root length compared to control whereas, at all conc. it had positive effect on root length in Kufri 958 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 956-963 Table.1 Effect of antibiotics and their concentrations on microplant height and number of leaves Variety Antibiotics Microplant height C1 K. Jyoti Amphotericin C2 C3 C4 C5 4.70 3.86 3.74 3.98 3.60 Number of leaves Mean C1 C2 2.12 2.46 C3 C4 C5 2.48 2.40 2.64 3.92 Mean 2.42 Antimycotic 3.60 2.74 1.62 1.12 1.12 2.04 2.12 2.28 1.46 1.12 1.40 1.68 Mean 3.60 3.72 2.59 2.43 2.55 2.98 2.12 2.37 1.97 1.76 2.02 2.05 Amphotericin 7.04 7.70 8.36 7.96 8.22 7.86 4.11 5.14 5.54 5.54 5.60 5.19 Antimycotic 7.04 6.94 5.06 5.36 4.84 5.85 4.11 4.98 4.40 4.00 4.08 4.31 Mean 7.04 7.32 6.71 6.66 6.53 6.85 4.11 5.06 4.97 4.77 4.84 4.75 Amphotericin 6.60 7.64 7.84 7.40 5.80 7.06 4.80 5.40 4.46 4.74 3.68 4.62 Antimycotic 6.60 4.78 3.02 2.82 2.40 3.92 4.80 3.34 2.40 2.02 2.26 2.96 Mean 6.60 6.21 5.43 5.11 4.10 5.49 4.80 4.37 3.43 3.38 2.97 3.79 Amphotericin 3.80 4.90 4.92 4.72 3.90 4.45 2.80 3.12 3.12 3.56 3.00 3.12 Antimycotic 3.80 3.06 2.20 1.50 0.84 2.28 2.80 2.26 2.02 1.74 1.26 2.02 Mean 3.80 3.98 3.56 3.11 2.37 3.36 2.80 2.69 2.57 2.65 2.13 2.57 Grand Mean 5.26 5.31 4.57 4.33 3.89 3.46 3.62 3.23 3.14 2.99 V A C VA AC VC VAC V A C VA AC VC VAC 0.2 0.1 0.2 0.2 0.3 0.4 0.6 0.1 0.09 0.1 0.1 0.2 0.2 0.4 CD (0.05) 0.3 0.2 0.4 0.5 0.6 NS NS 0.2 0.1 0.2 0.3 0.4 0.5 NS K. Sindhuri K. Lauvkar K. Sadabahar SEd V: Variety; A: Antibiotics; C: Concentrations; Amphotericin (C1-Control, C2-0.5%, C3-1.0%, C4-1.5% and C5-2.0%); Antimycotic (C1-Control, C2-0.5%, C31.0%, C4-1.5% and C5-2.0%) 959 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 956-963 Table.2 Effect of antibiotics and their concentrations on number of nodes/plantlet, number of roots/plantlet and root length Variety Antibiotics Number of nodes/plantlet C1 C2 C3 C4 Number of roots/plantlet C5 Mean C1 C2 C3 C4 Root length C5 Mean C1 C2 C3 C4 C5 Mea n K. Jyoti K. Sindhuri K. Lauvkar K. Sadabahar Amphotericin 2.30 2.56 2.54 2.52 2.74 2.53 8.54 8.86 7.46 5.50 8.68 7.81 3.60 3.14 3.48 2.54 2.88 3.13 Antimycotic 2.30 2.28 1.46 1.18 1.40 1.72 8.54 3.68 1.12 0.01 0.01 2.67 3.60 2.24 0.24 0.01 0.01 1.22 Mean 2.30 2.42 2.00 1.85 2.07 2.13 8.54 6.27 3.79 2.75 4.34 5.14 3.60 2.69 1.86 1.27 1.44 2.17 Amphotericin 5.20 5.22 5.66 5.48 5.86 5.48 6.11 5.60 5.86 7.26 8.80 6.73 8.81 7.82 6.78 7.34 8.54 7.86 Antimycotic 5.20 4.44 3.94 3.78 4.00 4.27 6.11 2.26 0.66 0.32 0.06 1.88 8.81 3.58 1.22 0.50 0.38 2.90 Mean 5.20 4.83 4.80 4.63 4.93 4.88 6.11 3.93 3.26 3.79 4.50 4.32 8.81 5.70 4.00 3.92 4.46 5.38 Amphotericin 4.94 5.46 4.26 4.74 3.66 4.61 7.02 7.60 8.26 7.06 5.00 6.99 6.04 7.06 6.50 6.10 5.40 6.22 Antimycotic 4.94 3.54 1.94 2.00 2.26 2.94 7.02 7.60 0.76 0.88 0.06 3.26 6.04 3.78 2.90 1.18 0.24 2.83 Mean 4.94 4.50 3.10 3.37 2.96 3.77 7.02 4.60 4.51 3.97 2.53 4.53 6.04 5.42 4.70 3.64 2.82 4.52 Amphotericin 2.81 3.20 3.12 4.02 3.08 3.25 6.26 9.76 8.18 9.06 8.54 8.36 4.14 4.84 4.66 4.42 4.44 4.50 Antimycotic 2.81 2.26 2.02 1.74 1.26 2.02 6.26 2.82 0.54 0.52 0.20 2.07 4.14 3.06 0.80 0.84 0.26 1.82 Mean 2.81 2.73 2.57 2.88 2.17 2.63 6.26 6.29 4.36 4.79 4.37 5.21 4.14 3.95 2.73 2.63 2.35 3.16 Grand Mean 3.81 3.62 3.12 3.18 3.03 6.98 5.27 3.98 3.83 3.93 5.65 4.44 3.32 2.87 2.77 C VA AC VC 0.1 0.08 0.1 0.1 0.1 0.3 0.3 V SEd CD (0.05) 0.2 A 0.1 0.2 VAC V A C VA AC VC VAC V A 0.2 0.3 0.2 0.1 0.3 0.3 0.4 0.6 0.8 0.2 0.1 0.5 NS 0.5 0.3 0.5 0.7 0.8 1.2 1.7 0.3 0.2 C VA AC VC VAC 0.2 0.2 0.3 0.4 0.6 0.4 0.5 0.6 0.8 1.2 V: Variety; A: Antibiotics; C: Concentrations; Amphotericin (C1-Control, C2-0.5%, C3-1.0%, C4-1.5% and C5-2.0%); Antimycotic (C1-Control, C2-0.5%, C31.0%, C4-1.5% and C5-2.0%) 960 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 956-963 Table.3 Effect of antibiotics and their concentrations on fresh weight (mg) and dry weight (mg) Variety Fresh weight Dry weight C5 Mean 10.76 13.58 13.26 11.10 10.36 8.80 10.64 13.05 12.96 10.56 11.19 11.95 19.72 15.70 11.42 11.90 17.48 15.02 129.63 19.72 13.44 10.70 8.46 7.94 12.05 164.61 170.86 19.72 14.57 11.06 10.18 12.71 13.65 229.12 161.64 188.56 10.02 10.74 10.12 11.96 12.04 10.98 88.84 116.38 97.36 125.97 10.02 9.94 9.34 7.38 9.24 9.18 162.09 132.34 172.75 129.50 157.26 10.02 10.34 9.73 9.67 10.64 10.08 285.76 386.22 377.08 410.40 378.68 367.63 18.44 21.64 19.88 25.36 23.78 21.82 Antimycotic 285.76 185.58 142.58 121.80 112.96 169.74 18.44 16.42 15.60 15.62 13.06 15.83 Mean 285.76 285.90 259.83 266.10 245.82 268.68 18.44 19.03 17.74 20.49 18.42 18.82 Grand Mean 230.90 207.49 166.19 181.93 174.88 15.04 14.25 12.87 12.73 13.24 V A C VA AC VC VAC V A C VA AC VC VAC 9.2 6.5 10.3 13.1 14.6 20.7 29.3 0.6 0.4 0.7 0.9 1.1 1.5 2.2 12.9 20.4 25.9 28.9 40.9 NS 1.3 0.9 1.5 1.9 NS 3.0 4.3 K. Jyoti K. Sindhuri K. Lauvkar K.Sadabahar SEd Antibiotics CD (0.05) 18.3 C1 C2 C3 C4 C5 Mean C1 C2 C3 C4 Amphotericin 206.78 251.16 188.46 203.0 221.76 214.23 11.98 15.14 14.82 Antimycotic 206.78 142.90 100.02 104.62 97.44 130.35 11.98 10.96 Mean 206.78 197.03 144.24 153.81 159.60 172.29 11.98 Amphotericin 241.40 224.06 161.68 180.90 252.46 212.10 Antimycotic 241.40 145.80 95.00 89.18 76.76 Mean 241.40 184.93 128.34 135.04 Amphotericin 189.64 186.56 175.84 Antimycotic 189.64 137.62 Mean 189.64 Amphotericin V: Variety; A: Antibiotics; C: Concentrations; Amphotericin (C1-Control, C2-0.5%, C3-1.0%, C4-1.5% and C5-2.0%); Antimycotic (C1-Control, C2-0.5%, C31.0%, C4-1.5% and C5-2.0%) 961 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 956-963 Therefore, it is considered of utmost importance that the use of antibiotics is done in a controlled manner, (both choice and concentrations) in order to maintain the endophytes / microplanta balance. Edington, England: 574p. 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