EFFECT OF METAL NANOPARTICLES ON THE

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EFFECT OF METAL NANOPARTICLES ON THE GROWTH OF NGOC LINH GINSENG (PANAX VIETNAMENSIS) LATERAL ROOTS CULTURED IN VITRO

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Tay Nguyen Institute for Scientific Research, Vietnam Academy of Science and Technology 2

Hue University College of Sciences *Email: [email protected]

ABSTRACT Panax vietnamensis (Ngoc Linh ginseng) plays critical roles in pharmaceutical industry because triterpenoid saponins from its roots produce medicine for improving health and treating many diseases. Metal nanoparticles reveal completely new or improved properties based on specific characteristics such as size, distribution and morphology compare to metal ion or salt; and their potential for in vitro plant cultures. Present study investigated the effects of metal nanoparticles including nZnO (0.5-2.5 mg/l), nAg (1-3 mg/l), and nCu (1-3 mg/l) supplemented in freehormone-MS medium to in vitro Panax vietnamensis lateral root growth. Our results showed that metal nanoparticles have the positive effect on the growth of in vitro Panax vietnamensis lateral roots with nAg, nCu, and nZnO. At different concentrations, in vitro Panax vietnamensis lateral root growth also has various effects on the growth of lateral roots. In supplemented metal nanoparticle treatments, nCu is the most optimum for in vitro Panax vietnamensis lateral root growth; the highest increase was obtained at 1.5 mg/l nCu treatment (99.3% lateral root formation and all root growth indexes are the highest). Besides, 2.5 mg/l nAg is also significantly noticed in ginseng root growth. However, the negative impact on the growth of the in vitro Panax vietnamensis lateral roots showed when culture medium contained the highest concentration; such as the root growing inhibition of nCu and nAg above 2.5 mg/l. Especially, this decrease was higher with the application of nZnO 0.5-2.5 mg/l (decrease the lateral root number) and 2.5 mg/l (decrease percent of lateral root formation). Keywords: in vitro, lateral root culture, metal nanoparticle, MS medium, Panax vietnamensis

INTRODUCTION Nanotechnology is an emerging field that is widely used in many medical, pharmaceutical, cosmetic, biosensors and agriculture and agricultural products [12]. The metal nanoparticles (NPs) are microscopic particles (1-100 nm), which are separated from molecules or atoms [26]. It is because of the surface effect and the critical size of the nanoparticles that make the nanoparticles have unique physical and chemical properties compared to conventional mass materials such as quick reaction and catalysis [26]. Therefore, NPs have a better interact with plants. NPs can create physical or chemical effects on the organs of plants to improve plant growth [12]. Usually, NPs enter the tree through the lateral root system and through the cortex and the lining to the xylem system of main roots [6]. In particular, some NPs of small size and surface properties that are suitable for the size of the holes in the cell membrane will easily enter

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the cell and participate in metabolic reactions within the cell [26]. After entering the cell, the NPs will mix with the metal ions and react with sulfhydryl groups, carboxyl groups and ultimately alter the activity of plant proteins. These effects may be beneficial for the growth and development of plants such as enhanced antimicrobial activity and the ability of plants to grow roots and shoots [12]. Nanoparticles of silver (Ag), copper (Cu), zinc (Zn), and zinc oxide (ZnO) have found applications in agriculture. Although rooting and mineral nutrition are intimately related, few studies have attempted to characterize the effects of specific minerals on each of the three phases of the rooting process, especially, metal nanoparticle effects. To enhance root growth, copper (Cu) is an essential transition metal that is involved in many physiological processes in plant rooting, because it can exist in multiple oxidation states in vitro [4]. Cu was added to the medium in the form of nitrates with low concentrations to improve root growth [4]. Copper nanoparticles (nCu) enhance root growth and mode of application for yield maximization of plant [4]. Silver nanoparticles (nAg) has not only strongly antibacterial activity, but also improves plant rooting such as increasing the root growth of chrysanthemum in microponic system [10]. Root number of in vitro culture was affected by zinc (Zn) [12]. Addition of Zn to culture media has been shown to improve rooting with masses of roots [13][14]. Nano zincs oxide (nZnO) also increase the development of lateral root system on field [5][15]. Panax vietnamensis (Ngoc Linh ginseng), a precious medicinal plant of Vietnam, contains many compounds that are highly medicinal in the treatment of blood pressure, cardiovascular, blood sugar, nerves, liver and kidney or to produce functional products to nourish and enhance health [9]. In order to harvest large amounts of biomass in a short time, many studies have shown that root culture is optimal for ginseng root growth and accumulation of saponins [9][11][25]. However, in order for the Ngoc Linh ginseng to grow more and more, the evaluation of the effect of nanomaterials is very necessary. Understanding the benefits and harms of the nanoparticles can find a new and safe pathway to improve the growth of lateral roots in the in vitro culture of the Ngoc Linh ginseng roots. Therefore, in this study we evaluated the stimulation of copper, zinc, and silver nanoparticles on the growth of Ngoc Linh ginseng roots. MATERIALS AND METHODS Materials Plant material Lateral root explants (length of 1 cm, weight about 10 mg) were separated from adventitious roots culturing MS medium with 5 mg/l IBA after 56 day-old culture the Department of Molecular Biology and Plant Breeding in Tay Nguyen Institute for Scientific Research. Materials characterization As previously stated, silver nanoparticles (nAg), zincs oxide nanoparticles (nZnO), and copper nanoparticles (nCu) were tested for potential rooting on in vitro chrysanthemum shoots, and

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tomato, soybean and oat breeding [12][13][19]. The synthesis procedure for nAg, nCu, and nZnO is presented in the supplementary information from Institute of Environmental Technology in Hanoi. The particle size, morphology and crystallinity were investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Infrared (IR) spectroscopy. nAg was size smaller than 20 nm which had been manufactured using the rate: [AgNO 3] = 750 – 1000 ppm, [β-chitosan] = 250 – 300 ppm, [NaBH4] = 200 ppm, mole rate [NaBH4]/[AgNO3] = ¼, NaBH4 drip speed: 10 – 12 droplet/min [10]. nCu (the average diameter) were synthesized in the presence of chitosan via reduction of Cu(NO3)2 with sodium borohydride with the ratio of 4 ml 2.0 mg/ml chitosan : 2ml Cu(NO 3)2 10 mM:1 ml NaBH4 0.1 M in aqueous solution. Chitosan molecules absorbing on the surface of prepared copper nanoparticles formed the corresponding copper nanoparticle-chitosan [7]. The synthesis of nZnO by the sol–gel method showed wurtzite construction, purity about 99.9%, and lattice parameters (50 nm in average diameter and   100-200 nm in length). Frist, 0.5 M zinc acetate solution was transferred into TEA solution and stirred for 20 min at temperature of 120 °C. Then, 6 mL of NH4OH (25%) was slowly dropped into the solution with aluminum foil to form nZnO after 30 min [1][23]. Methods Effect of silver nanoparticles on the growth of P. vietnamensis lateral root Lateral root explants (length of 1 cm) were cultured on hormone-free MS basal medium, containing 30 g/l sucrose and 8 g/l agar. To evaluate the effect of silver nanoparticles (nAg) on the growth of lateral root, nAg was added into culture medium at concentrations of 0; 0.5, 1, 1.5, 2, 2.5 mg/l nAg. Effect of zinc oxide nanoparticles on the growth of P. vietnamensis lateral root Lateral root explants (length of 1 cm) were cultured on hormone-free MS basal medium, containing 30 g/l sucrose and 8 g/l agar. To evaluate the effect of zinc oxide nanoparticles (nZnO) on the growth of lateral root, nZnO were added into culture medium at concentrations of 0; 0.5, 1, 1.5, 2, 2.5 mg/l nZnO. Effect of copper nanoparticles on the growth of P. vietnamensis lateral root Lateral root explants (length of 1 cm) were cultured on hormone-free MS basal medium, containing 30 g/l sucrose and 8 g/l agar. To evaluate the effect of copper nanoparticles (nCu) on the growth of lateral root, nCu was added into culture medium at concentrations of 0; 0.5, 1, 1.5, 2, 2.5 mg/l nCu. Statistical Analysis Each treatment was repeated three times and the explants in experiments were arranged in a randomized complete bottle design with five root explants per treatment and ten bottles. Data of in vitro cultures was recorded at the 60 days of P. vietnamensis lateral root cultures.

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The expressed data (Percent of formed lateral roots, fresh weight (mg), dry weight (mg), and means of number roots, and root length (cm) were analyzed using the one-way ANOVA. Lateral root anatomical morphology was prepared similar to method of Moore and MacClelen (1983) [17] and observed by Nikon SMZ 800 (Nikon, Japan) in 10x of magnify rate. When significant differences occurred (P