The effects of silver nanoparticles on growth of chrysanthemum morifolium ramat. cv. "jimba" in different cultural systems

Vietnam Journal of Science and Technology 55 (4) (2017) 503-514 DOI: 10.15625/2525-2518/55/4/9322 THE EFFECTS OF SILVER NANOPARTICLES ON GROWTH OF Chrysanthemum morifolium Ramat. cv. "JIMBA" IN DIFFERENT CULTURAL SYSTEMS Luong Thien Nghia1, Hoang Thanh Tung1,2, Nguyen Phuc Huy1, Vu Quoc Luan1, Duong Tan Nhut1, * 1Tay Nguyen Institute for Scientific Research, VAST, 116 Xo Viet Nghe Tinh, Da Lat, Lam Dong 2Hue University of Sciences, Hue University, 77 Nguyen Hue, Hue, Thua Thien – Hue *Email: duongtannhut@gmail.com Received: 14 March 2017; Accepted for publication: 31 July 2017 ABSTRACT Silver nanoparticles (SNPs) are one of metallic nanoparticles widely applied in many fields. Research of SNPs application in plant tissue culture has been gaining attention in recent years. Moreover, novel plant tissue culture systems have been researched and developed for improving SNPs uptake capability in medium. In this study, we investigated effects of SNPs on Chrysanthemum morifolium ramat. cv. "JIMBA" growth and its ability in 3 culture systems: in vitro solid medium system, in vitro liquid medium system and microponic (combined of micropropagation and hydroponic). The 3 cm Chrysanthemum shoots and silver nanoparticles of diameter smaller than 20 nm were used in the experiments. After 4 weeks, the results showed that the SNPs concentration was suitable for growth of Chrysanthemum in vitro solid medium system, in vitro liquid medium system and microponic was 1.5, 1.5 and 5 ppm, respectively. Microponic system not only improved plant growth but also reduced the succulent phenomenon. SNP uptake was likely dependent on concentrations and culture systems. At low concentrations (1, 5, and 10 ppm), SNPs were completely absorbed after 4 weeks of culture in all systems, but not at high concentrations (20 ppm). The amount of absorbed silver nanoparticles was directly proportional to the culture period and inversely proportional to the concentration of SNPs supplemented to the medium. In all of our investigated systems, the hydroponic system showed the highest capability of SNPs absorption. Keywords: absorption, Chrysanthemum sp., growth, microponic, silver nanoparticles. 1. INTRODUCTION The rapid growth of nanotechnology make it become one of the greatest impetuses to technological and industrial development in the 21st century. Among the different type of nanomaterials, silver nanoparticles (SNPs) are extensively used in many fields of science and technology. In plant tissue culture technology, SNPs were applied as a factor which may resist some inhibitors in micro-propagation, such as in vitro contamination (e.g., fungal and bacterial infections) [1, 2, 3, 4, 5, 6] and ethylene [7, 8, 9], hence, improve in vitro plant growth. In recent Luong Thien Nghia, et al. 504 years, nanoparticles absorption mechanisms have gained first attentions, experiments demonstrated SNPs can be absorbed via leafs or roots [10] by penetrated or diffused in symplast and apoplast [11]. Otherwise, cultural conditions are the most important factors which directly impact to nanoparticle absorption. Kumari et al. proved that in liquid medium metal nanoparticles are absorbed more effectively than in solid or semi-solid medium [12]. On the other hand, ventilation condition between inside and outside system is an advantage factor for uptake nutrient from medium due to enhancing transpiration capability. Nevertheless, studying about conditions effect on SNPs absorption is still quite limited. Therefore, the targets in this experiment aim to investigate effect of SNPs on Chrysanthemum sp. growth in various cultural systems and find out which system is appropriate to SNPs absorption. 2. MATERIALS AND METHODS 2.1. Sample source and materials Chrysanthemum morifolium shoots with 3 cm in length were used as explant source. These shoots were obtained from a mass of shoots cultured in vitro on Murashige and Skoog (MS) medium [13] with 8 g/L agar and 30 g/l sucrose after 40-45 days of culture. The used silver nanoparticles (SNPs) were of size smaller than 20 nm which had been manufactured using the rate: [AgNO3] = 750 – 1000 ppm, [β-chitosan] = 250 – 300 ppm, [NaBH4] = 200 ppm, mole rate [NaBH4]/[AgNO3] = ¼, NaBH4 drip speed: 10 – 12 droplet/min. Substrates in microponic and in vitro liquid medium systems are tubes of nylon films with 2 cm in height and 1.5 cm in diameter. Substrates were put into the cultural vessels (bottles or plastic box). 2.2. Cultural systems 2.2.1. In vitro with solid or liquid medium systems Glass bottles with 250 ml in volume, each of bottles contain 40 ml of MS medium, with 30 g/l sucrose (and supplied 8 g/l agar in solid medium system). After that, we supplied SNPs of various concentrations and sterilized by autoclaving at 121 oC, 1 atm in 30 minutes. 2.2.2. Microponic system Microponic system was circular plastic containers with 12 cm of diameter at top, 9 cm of diameter at bottom, and 8.5 cm of height. 40 ml of half-strength sugar-free liquid MS medium was added to system with 15 tubes of nylon film. Top of the system was equipped with one Millipore filter by MillisealTM, of pore size of 0.2 µm (Nihon Millipore Ltd., Tokyo, Japan). All experiments were incubated at 25
2 oC with humidity of 55 – 60 % and photoperiod of 12 hours/day under fluorescent light with 45 µmol.m-2.s-1 of intensity. 2.3. Methods 2.3.1. Evaluating effect of SNPs to Chrysanthemum growth on in vitro solid medium system Shoots were cultured in in vitro solid medium system within different SNPs concentrations (0; 0,5; 1; 1,5; 2; 3; 5; 7; 10 ppm) and sterilized in autoclave to investigate effects of SNPs on Chrysanthemum growth. The effects of silver nanoparticles on growth of Chrysanthemum morifolium Ramat. cv. "JIMBA" 505 2.3.2. Evaluating effect of SNPs to Chrysanthemum growth on in vitro liquid medium system Shoots were cultured in in vitro liquid medium system within different SNPs concentrations (0; 0.5; 1; 1.5; 2; 3; 5; 7; 10 ppm) and sterilized in autoclave to investigate effects of SNPs on Chrysanthemum growth. 2.3.3. Evaluating effect of SNPs to Chrysanthemum growth on microponic system Shoots were cultured in microponic system within different SNPs concentrations (0; 5; 10; 15; 20 ppm) without sterilization to investigate effects of SNPs on Chrysanthemum growth. 2.3.4. Evaluating absorption capability of different systems Shoots were cultured in different culture systems (in vitro solid medium, in vitro liquid medium and microponic system) with various SNPs concentrations (1; 5; 10; 20 ppm). After 1, 2, 3, 4 weeks, medium remained was collected and SNPs content in culture medium was examined to determine absorbed-SNPs contents. 2.4. Collecting and analysing data Data were scored in 4 weeks of culturing and analysis of variance was performed. Investigated growth characteristic include: Plant height (mm); number of shoots; number of leaves; number of roots, root length (mm); SPAD - total chlorophyll content (µg/g), fresh weight (mg); dry weight (mg); net weight rate (%), absorbed-SNPs rate (%). Total chlorophyll contents in leaves were evaluated by SPAD-502 (Minolta Co., Ltd., Osaka, Japan). SNPs contents in medium were evaluated by UV–vis spectroscopic at 480 nm [14] of wavelength (Shimadzu, UV-2450, Japan). Root morphology were observed by Nikon SMZ 800 (Nikon, Japan) in 20x of magnify rate. Net weight rates were calculated by the following formula: Net weight rate %  Dry weight mg     100 % Absorbed-SNP rates were calculated by the following formula: AgH %  Ag0 " AgT Ag0  100 % where: AgH is absorbed-SNPs rates after (1, 2, 3, 4 weeks) (%), Ag0 is total SNPs content in culture medium at the beginning (mg), AgT is total SNPs content in culture medium at 1, 2, 3, 4 weeks (mg). All treatments were in triplicates and each replicate with 10 culture vessel. The means were compared using Duncan's multiple range Test using SPSS (Version 16.0) at α = 0.05 [15]. 3. RESULTS AND DISCUSSION 3.1. Effects of SNP on Chrysanthemum growth in in vitro solid medium system Luong Thien Nghia, et al. 506 The effects of exposure to different concentrations of SNPs on Chrysanthemum plantlets after four weeks of incubation are presented in Table 1 and Figure 1. Table 1. Effects of SNP on chrysanthemum growth in in vitro solid medium system. SNP (ppm) No. of shoots Plant height (mm) No. of leaves No. of roots Root length (mm) SPAD (mg/g) Fresh weight (mg) Dry weight (mg) Net weight rate(%) 0 6.3bcd 39.7bc 10.3a 8.7c 49.7bcd 31.9b 300.3b 14.0e 4.66a 0.5 6.3bcd 39.0bc 8.3bcd 8.0c 45.0d 32.7b 328.0b 19.0de 5.79a 1 7.3b 44.7ab 8.7abc 14.0abc 45.3d 31.7b 531.0a 31.0c 5.84a 1.5 9.0a 48.3a 10.0ab 17.3a 65.0a 37.6a 673.7a 58.7a 8.71a 2 6.7bc 42.0ab 8.0bcd 16.7ab 62.0abc 34.0ab 657.3a 46.0b 7.00a 3 6.7bc 34.7cd 9.7abc 13.7abc 62.2abc 32.5b 354.3b 27.3cd 7.71a 5 5.3cd 32.0d 6.3e 10.3bc 53.7bcd 31.7b 188.7b 15.3e 8.13a 7 5.7bcd 42.7ab 7.7cde 11.7abc 64.3ab 34.9ab 342.7b 22.0cde 6.42a 10 4.7d 31.7d 7.0de 10.7bc 48.3d 32.3b 228.3b 14.3e 6.28a Different letters within a column indicate significant differences at α = 0.05 by Duncan's multiple range tests. Figure 1. Effects of SNPs on Chrysanthemum morphology in in vitro solid medium system. Most of values observed such as number of shoots, plant height, number of leaves, number of roots, root length, SPAD (Soil-Plant Analysis Development), fresh weight and dry weight showed a significant change (Supplementary Table 1; Figure 1). The mean net weight rate of the plantlets showed no significant difference in various SNPs concentrations. Plants cultured on The effects of silver nanoparticles on growth of Chrysanthemum morifolium Ramat. cv. "JIMBA" 507 medium with 1.5 ppm SNPs grew well with almost investigated characteristics reached best as compared to remain SNPs concentrations. When adding higher SNPs concentration - 1.5 ppm in medium, plant growth was slightly lower, severely at 10 ppm, number of shoots, plant height, number of leaves were decreased on the contrary SNPs free. Therefore, in in vitro with solid medium system, SNPs with concentration 1.5 ppm is appropriate to Chrysanthemum plant growth. Some reports confirmed the positive response of SNPs on many plant species. For instance, in vitro Chrysanthemum growth improved at 10 ml/l of SNPs dose [16], Araucaria excelsa explants grown in MS medium supplemented with SNPs demonstrated that explants grown on media supplemented with SNPs were fresher, had a suitable growth, and maintained their green color on the contrary to SNPs free-MS medium. The authors hypothesized that SNPs indirectly affect plant growth but its inhibitory effects of plant phytohormone ethylene - an inhibitor caused senescence, malformation or some phenomenon as hyperhydicity [8]. According to what reported by Sarmast, findings in Tecomella undulata (Roxb.) Seem. micropropagation demonstrated that the ethylene present in culture vessels during its micropropagation caused shedding of leaves, decreased in chlorophyll content and finally would result in the demise of explants. Providing SNPs in MS medium of T. undulata improved survival percentage of explants and increased mean number of shoot and length of explants [17]. Ethylene resistance mechanism of SNPs due to blocking 1-Aminocyclopropane-1-carboxylic acid (ACC) gene - ethylene precursor synthesized gene, subsequently, inhibit ethylene synthesis [17]. On the other hand, in higher concentrations of SNPs negative effects, including seed germination, shoots and roots growth, late flowering and low yield, were observed [18, 19, 20]. Especifically, at 20 ppm of SNPs concentration, yield and antioxidant gene expressions were decreased in Arabidopsis [21], or inhibition of early development at 73.4 ppm [22]. 3.2. Effects of SNPs on Chrysanthemum growth in in vitro liquid medium system After 4 weeks of treatment with different concentrations of SNPs in in vitro liquid medium, the results showed that mean of plant height, number of roots, root length, fresh weight, dry weight were statistically different in various concentrations of SNPs (Table 2, Fig. 2). Among them, 1.5 ppm SNPs reached best effects to Chrysanthemum growth which manifested in the best of plant height, number of shoots, number of roots, root length, fresh weight, dry weight and net weight rate as compared to control (1.12; 1.45; 3.3; 1.3; 1.3; 1.75 times, respectively). Table 2. Effects of SNPs on Chrysanthemum growth in in vitro liquid medium system. SNP (ppm) No. of shoots Plant height (mm) No. of leaves No. of roots Root length (mm) SPAD (mg/g) Fresh weight (mg) Dry weight (mg) Net weight rate (%) 0 10.0ab 71.7b 13.0a 16.0b 16.0d 39.2ab 1221.0b 32.0cd 2.62b 0.5 8.0cd 72.7b 8.3c 16.0b 17.7d 41.3a 790.7c 23.7d 2.99b 1 8.3c 78.3ab 9.7bc 16.7b 57.0a 34.8de 1022.0b 34.7abc 3.39b 1.5 8.7bc 81.0a 9.3bc 23.3a 53.0ab 37.7bc 1590.7a 74.0a 4.65a 2 11.0a 83.0a 13.0a 17.7b 42.3bc 35.9cd 1470.3ab 46.3b 3.15b 3 6.7de 77.0ab 9.0bc 17.8b 63.0a 35.7cd 1308.3ab 38.3bc 2.93b 5 6.3e 52.7c 8.7c 17.0b 53.3ab 37.9bc 1202.0b 30.0cd 2.50b 7 6.0e 41.3d 11.0ab 15.3b 39.3c 33.6e 836.3c 27.0cd 3.23b Luong Thien Nghia, et al. 508 10 6.3e 31.7e 8.0c 15.0b 36.0c 34.2de 1005.3bc 29.3cd 2.92b Different letters within a column indicate significant differences at α = 0.05 by Duncan's multiple range tests. Figure 2. Effects of SNP on Chrysanthemum morphology in in vitro liquid medium system. When supplying 1 ppm SNPs into medium, although root length reached highest value, others still lower as compared with those cultured on medium supplemented with 1.5 ppm SNPs. When SNPs concentration over 1.5 ppm, growth index was suppressed. Particularly, supplied 10 ppm SNPs gained inhibited response in plant. Plant height, number of leaves, number of roots, root length, SPAD, fresh weight, dry weight decreased. In this experiment, hyperhydricity phenomenon on in vitro plantlet in closure vessel was appeared and expressed in lower net weight rate. Exposed SNPs Chrysanthemum at different concentrations were resisted hyperhydricity phenomenon. Especially, 1.5 ppm SNPs help Chrysanthemum gained best as compared to others concentrations and control. 3.3. Effects of SNPs on Chrysanthemum growth in microponic system The effects of silver nanoparticles on growth of Chrysanthemum morifolium Ramat. cv. "JIMBA" 509 Table 3. Effects of SNP on Chrysanthemum growth in microponic system. SNP (ppm) No. of shoots Plant height( mm) No. of leaves No. of roots Root length (mm) SPAD (mg/g) Fresh weight (mg) Dry weight (mg) Net weight rate (%) 0 10.0ab 49.0b 8.7a 21.0a 11.3ab 31.0c 619.0b 37.3b 6.03b 5 11.0a 56.0a 8.0a 14.2ab 13.3a 39.3a 708.3a 45.3a 6.40a 10 8.3c 47.0bc 10.0a 12.0b 6.7bc 35.2b 654.7ab 44.0ab 6.72a 15 8.7bc 45.0bc 8.3a 8.7c 4.7c 34.1b 569.3bc 37.0b 6.50b 20 7.3c 41.7d 9.0a 8.3c 4.0c 29.8c 472.7c 31.3c 6.63ab Different letters within a column indicate significant differences at α = 0.05 by Duncan's multiple range tests. The effects of exposure to different concentrations of SNPs on Chrysanthemum growth are shown in Table 3 and Fig. 3. The overall results indicated that plants which were exposed to different concentrations of SNPs showed significant change in growth characteristics. 5 ppm of SNPs-exposed plant growth is best as compared to the other concentrations. Like in aforementioned in vitro systems, at high concentrations of SNPs, plant growth quite be inhibited. Particularly, 10 ppm of SNPs-exposed plant showed as plants of lowest quality. This result is appropriate with Tung et al. report in 2006, in this study, the authors determined suitable concentration of SNPs in microponic culture on Chrysanthemum is 7.5 ppm, if SNPs concentrations were higher than this value, plant growth would become suppressed [23]. Figure 3. Effects of SNP on Chrysanthemum morphology in microponic system. Luong Thien Nghia, et al. 510 When comparing growth and morphology of Chrysanthemum which were cultured in 3 different systems (in vitro solid medium, in vitro liquid medium and microponic system). The results showed that in vitro liquid medium seem was the best medium for in vitro plant growth due to some of characteristics reach highest as compared to other systems. Due to in aqueous condition, medium can dilute and translocate nutrients easier than in hard condition. Moreover, supported nutrient content (mineral, carbon source) in in vitro liquid medium system was higher in microponic system, consequently, plant growth in in vitro liquid medium system was better. Nevertheless, in vitro liquid medium system with closure vessel condition causes high humidity and hyperhidricity phenomenon expressed in net weight rate lower than in vitro solid medium and microponic system (3.7 and 13.5 times, respectively). Figure 4. SNPs-exposed Chrysanthemum growth in different cultural systems. Figure 5. SNPs-exposed root morphology in different cultural system (left to right: in vitro solid medium, in vitro liquid medium and microponic system, respectively). The effects of silver nanoparticles on growth of Chrysanthemum morifolium Ramat. cv. "JIMBA" 511 Beside of growth values, the alteration in plant morphology under SNP-exposed in different cultural conditions were recognized (Fig. 4). When observed with microscope, root morphologies in different systems have significant change. In liquid medium conditions (in vitro liquid medium and microponic), root-hair developed denser than in solid medium (Fig. 5). Moreover, it was observed that exposure to different concentrations of SNPs has resulted in root tip browning in microponic system. Similar phenomenon was realized in Tung et al. research, they hypothesized that this phenomenon be intensified when SNPs concentration increased and cause root necrosis [23]. Prakash and Ill Min Chung also confirmed root browning by culturing Arabidopsis thaliana in a medium supplemented with SNPs, which occurs due to the accumulation of silver nanoparticles at the root tips [24]. 3.4. SNPs absorption in different cultural systems In the first two weeks of culture, the SNPs absorbed capability of Chrysanthemum were very low in all cultured systems. The SNPs uptake was improved then in subsequent culture weeks. Under in vitro solid medium, the ability to absorb SNPs at 20 ppm was only 15.8 % in the first two weeks, but within the next two weeks absorbed-SNPs increased up to 71 % (Fig. 6). This can be explained at the beginning, Chrysanthemum root system still not formed which makes the absorbed-SNPs capacity be limited and only gradually improved in the next stages. Graphs of absorbed-SNPs rate in in vitro the solid medium system also showed that silver were not completely absorbed at all concentrations over a 4-week period. From 71.7 to 95.9 % and inversely proportional to the SNPs concentration added to the culture medium (Fig. 6). In in vitro liquid medium systems, at low concentrations (1, 5, 10 ppm), silver nanoparticles are completely absorbed after 4 weeks. Especially, 1 ppm SNPs were completely absorbed only for 3 weeks. In this system, absorbed-SNPs capability is also directly proportional to the culture times and inversely proportional to the SNP concentrations. Due to the liquid medium is capable of absorbing growth regulators, dissolving nutrients is better in the solid medium [25]. The study by Suthar et al. also showed that cultured in vitro Boswellia serrata Roxb shoots in liquid medium gave better performance than in solid medium [26]. The authors explained this based on the solubility and flexibility of the environment, which makes the nutrient in the medium easier to be absorbed. In this study, the image was observed under a microscope showing that in a liquid medium, root hairs grow stronger in a solid medium, consequently increases the uptake of nutrients (Fig. 5). In the microponic system after 4 weeks of culture, except the treatment with 20 ppm SNPs- supplementation (only 80.9 % SNPs absorbed), silver nanoparticles were completely absorbed in other treatments. Remaining in all treatments, silver nanoparticles were completely absorbed, in treatments 1 and 5 ppm silver nanoparticles were completely absorbed only after 2; 3 weeks. Silver nanoparticle absorption in this system was directly proportional to the culture time and inversely proportional to the initial silver nanoparticles added to the culture medium, too. As compared to in vitro liquid medium systems, it was found that in microponic system (aeration filter equipped), SNPs absorbed capability were better at all concentrations. Ventilation is also a condition that enhances the uptake of nutrients from the medium, Ventilation condition enhances air, moisture exchange, promotes water and nutrients uptake from the medium. As compared with other systems, the micro hydroponics system included both growth helpful conditions: liquid medium and ventilated, furthermore saved materials (no sugar added, less than 1/2) and energy (medium not through sterilize by autoclaving) and still guaranteed the utility of silver nanoparticles. Luong Thien Nghia, et al. 512 Figure 6. Absorbed-SNPs rate in different culture systems. 4. CONCLUSION Silver nanoparticles are suitable for in vitro Chrysanthemum growth in various culture systems: in vitro solid medium, in vitro liquid medium and microponic system are 1.5; 1.5; 5 ppm, respectively. Different culture systems have different SNPs absorbability which directly proportional to the culture time and inversely proportional to the SNPs concentration in the medium. Microponic culture system with liquid medium, ventilated conditions, reduced mineral content and no sugar gained the best results on SNPs absorption, chrysanthemum growth and cost savings. 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