Effect of zinc oxide nanoparticles on growth and development of dendrobium officinale kimura et migo in in vitro culture medium

Zinc-oxide nanoparticles (ZnO-NPs) has positive effects on the growth and development of plants at appropriate concentrations. This study aimed to test the effect of ZnO nanoparticles with concentrations from 0 mg/L to 5 mg/L individually or in combination with ZnSO4 on growth and development of in vitro Dendrobium officinal at 3 stages of shoot multiplication, elongation and rooting.

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EFFECT OF ZINC OXIDE NANOPARTICLES ON GROWTH AND DEVELOPMENT OF Dendrobium officinale KIMURA ET MIGO

IN IN VITRO CULTURE MEDIUM

Dao Thi Sen* and Le Thi Thuy Faculty of Biology, Hanoi National University of Education

Abstract Zinc-oxide nanoparticles (ZnO-NPs) has positive effects on the growth and development of plants at appropriate concentrations This study aimed to test the effect of ZnO nanoparticles with concentrations from 0 mg/L to 5 mg/L individually or in combination with ZnSO 4 on growth and development of in vitro Dendrobium officinal at 3 stages of shoot multiplication, elongation and rooting Results showed that zinc oxide nanoparticles had the effect on stimulating shoot formation of in vitro Dendrobium officinal

at 1 mg/L to 5 mg/L In shoot extension stage, zinc oxide nanoparticles stimulated length of shoots The medium formula with the addition of 1 mg/L zinc oxide nanoparticles and combination with ZnSO 4 obtained the best results after 6 weeks of culture with 2.9 shoots/sample, average length of 2.74 cm/sample, total chlorophyll content of 545.8 µg/g ZnO nanoparticles was used at concentration of 1 mg/L, the root count was the most, reaching 6.7 roots/sample with large and long roots The basic MS control medium (containing full zinc salts) produced lower or similar all tested mediumin some indicators (2.5 shoots/sample after 6 weeks, shoot length of 2.58 cm/sample, total chlorophyll content

of 396.27 µg/g, large roots, reaching 3.5 roots/shoot)

Keywords: Dendrobium officinale Kimura et Migo, ZnO nanoparticles, growth and development, chlorophyll

1 Introduction

Nanotechnology is one of the important areas for investigation in sciences of modern materials based on nanoparticle (NP) properties that are specific such as size, shape, and distribution Nanoparticles (NPs) have diverse properties when compared to respective chemicals due to their structure, surface to volume ratio, morphology, and reactivity In plant tissue culture, there are several research based on the use of NPs in seed germination, plant growth improvement, plant genetic modification, plant protection, improve bioactive compound production and some others [1, 2] The effectiveness of NPs on growth and development of plant in plant tissue cultures depends on their dimensions, size, distribution, and type Among the metal nanoparticles, zinc oxide (ZnO-NPs) is the third most popular metal nanoparticle Zinc plays a vital role in physiological and anatomical responses so ZnO NPs are mostly used in agricultural applications Some of recent studies have evaluated the addition and replacement of nano zinc oxide for commonly used zinc salts that indicate positive inputs Chamani et al reported the accumulation of specific bioactive compounds in Lilium ledebourii and its dependency

Received April 13, 2020 Revised May 4, 2020 Accepted May 11, 2020

Contact Dao Thi Sen, e-mail address: sen.hnue@gmail.com

on the concentration of ZnO NPs in the MS medium The highest content of flavonoids, phenolics and anthocyanins was obtained on MS medium supplemented with 25, 75 and 100 mg L−1 ZnO NPs, respectively The accumulation of steviol glycosides in shoot cultures of S rebaudiana was significantly enhanced on MS medium fortified with 1 mg L−1 ZnO [3] In addition, the total flavonoid and phenolic content also increased with ZnO treatment [4] However, higher concentrations of ZnO led to decreased secondary metabolite production due to the phytotoxic effects of ZnO Treatment of licorice seedlings with copper oxide (CuO) and zinc oxide (ZnO) increased the content of anthocyanins, flavonoids, glycyrrhizin, phenolic compounds and tannins [5] In general, the effects of nanoparticles depend on the type of nanoparticle, the plant species, the duration of exposure and the concentration of nanoparticles used

Micropropagation is one of the major applications of plant tissue culture Flowering plants, medicinal plants are often the subjects of choice Dendrobium officinale Kimura et Migo spreads in several countries over the world This is a valuable medicinal plant that is interested in in vitro cultivation Few studies have been carried out to determine the effects of ZnONPs on growth and development of some plant species However, there has not been any research to determine the effect of ZnO-NPs on in vitroDendrobium officinale growth and development

2 Content

2.1 Materials and methods

2.1.1 Materials

ZnO-NPs with size of 20 - 40 nm, purity of 97% are provided by the Institute of Environmental Technology - Vietnam Academy of Science and Technology Concentrations of ZnO-NPs: 0; 0.1; 0.5; 1; 5 (mg/L)

Shoots of 1.0 cm in length from in vitro cultured Dendrobium officinale at Gennetics - Biochemistry Department, Hanoi National University of Education were used in this study

Culture media:

Shoot multiplication media: MS basal medium (Murashige and Skoog) or MS modified medium (NZn: remove zinc salt completely and N1/2Zn: keep half of the zinc salt) supplemented with 0.3 mg/L BAP + 0.3 mg/L Kinetin + 0.1 mg/L NAA + 100 ml/L coconut water + 100 g/L potato homogenate + 30 g/L sucrose + 7 g/L agar, pH 5,7 - 5,8 + ZnO-NPs (0; 0.1; 0.5; 1; 5 mg/L) Root formation media: MS basal medium (Murashige and Skoog) or MS modified medium (NZn: remove zinc salt completely and N1/2Zn: keep half of the zinc salt) supplemented with 1.0 mg/L IBA + 100 ml/L coconut water + 100 g/L potato homogenate + 20 g/L sucrose + 7 g/L agar + 0.1% activated carbon, pH 5,7 - 5,8 + ZnO-NPs (0; 0.1; 0.5; 1; 5 mg/L)

2.1.2 Methods

Effect of ZnO-NPs on shoot multiplication

Shoot of in vitro Dendrobium officinale (1cm) was used to culture into the above shoot multiplication medium The study was carried out to evaluate the effect of different concentrations of ZnO-NPs on shoot multiplication and development of in vitro Dendrobium officinale

After 3 weeks and 6 weeks of culture, effects of ZnO-NPs to Shoot of in vitro Dendrobium officinale on shoot multiplication were evaluated by the number of shoots/explant, shoot length (cm), shoot morphology, % dry weight / fresh weight, and total chlorophyll content (mg/g) Effect of ZnO-NPs concentrations on root formation

After 3 weeks and 6 weeks of culture, shoots were separated and subcultured on rooting medium The effect of various concentrations of ZnO-NPs on root formation and development

of in vitro Dendrobium officinale was evaluated by number of roots/plantlet, root morphology Experimental arrangement

The experiments were repeated three times, each formula consisting of three flasks, five samples/flask

Statistical analysis

The results were indicated as means ± standard error of the mean (SEM) Comparisons of variables were carried out by using Student’s t test or analysis of variation (ANOVA) with Duncan’s multiple-range examination Differences were considered to be significant when P < 0.05 2.2 Results and discussion

2.2.1 Effect of ZnO-NPs on multiple shoot formation

The results of in vitroDendrobium officinale shoots after 3 weeks of culture on shoot multiplication showed in Table 1

Table 1 Effect of ZnONPs on multiple shoot formation after 3 weeks

Note The same letters in lowercase in the same row mean no significant difference at P ≤ 0.05

There was a significant difference in the average number of shoots/explant in experimental formulas and control formula When ZnO-NPs was added into the cuture medium, it made the positive respone on shoot initiation, number and growth rate of shoot As the concentration of ZnO-NPs added to the medium increased, the rate of shoot formation of the samples increased significantly in both types of NZn and N½Zn media In N½Zn medium, the formula N½Zn5 (5 mg/L ZnO-NPs) was the average number of shoots tends to decrease However, there was no significant difference between the sample and the control

We further evaluated these criteria of shoot culture multiple shoot formation after 6 weeks Shoot of in vitro Dendrobium officinale were evaluated by the number of shoots/explant, shoot length (cm), shoot morphology, % dry weight / fresh weight, and total chlorophyll content (mg/g) The results were shown in Figure 1 and Table 2

Treatme

shoots/

explant

Shoot length (cm)

Shoot morphology Treatment shoots/ No of

explant

Shoot length (cm)

Shoot morphology

NZn0 1.40 a ±0.14 1.36 a ±0.08 small, short,

a ±0.14 1.41 a ±0.05 small, short,

green NZn0.1 1.40 a ±0.28 1.42 a ±0.04 small, short,

green

N½Zn0.1 1.55 a ±0.07 1.71 ab ±0.08 big, long,

green NZn0.5 1.50 ab ±0.14 1.56 ab ±0.05 big, long,

green N½Zn0.5 1.75

ab ±0.21 1.82 ab ±0.19 big, long,

green NZn1 1.70 ab ±0.14 1.67 ab ±0.11 big, long,

green

N½Zn1 2.25 b ±0.21 1.94 b ±0.12 big, long,

green NZn5 2.20 b ±0.28 1.82 b ±0.19 big, long,

green

N½Zn5 1.65 a ±0.07 1.73 ab ±0.11 big, long,

green

MS 1.55 ab ±0.21 1.62 ab ±0.04 big, long,

green

MS 1.55 a ±0.21 1.62 ab ±0.04 big, long

green

When ZnO-NPs addition to the medium increased (except N1/2Zn5), not only on No of shoots/explant, shoot length increased but total chlorophyll content and % dry weight/fresh weight also increased) The explants on N1/2Zn medium with 1mg/L ZnO-NPs gained the maximum number of shoots (2.90), highest shoot length (2.74 cm) These parameters were about 10% higher than those of explants cultured on MS control medium, especially, total chlorophyll content was 13.88% higher (545.80/396.27 µg/g) However, the data recorded about

% dry weight/fresh weight treatment was no significantly different between experiments (p

<0.05) In brief, the medium formula N1/2Zn 1 (removes 1/2 zinc salt and adds 1mg/L ZnO-NPs) was found to be the optimum concentration required to induce maximum shoot growth Table 2 Effect of ZnONPs on chlorophyll content and% Dry weight/Fresh weight after 6 weeks Treatment Chlorophyll a

content (µg/g)

Chlorophyll b content (µg/g)

Total chlorophyll content (µg/g)

% Dry weight/Fresh weight NZn0 91.60a±11.93 134.03a±13.32 225.64a±24.50 10.42%a±1.30 NZn 0.1 110.67ab±4.17 164.11ab±18.61 274.80ab±32.79 10.32%a±0.66 NZn 0.5 132.51ab±13.61 197.74ab±20.02 330.25ab± 3.63 10.26%a±1.07 NZn 1 144.43ab±24.33 213.84ab±34.09 358.28ab±58.42 9.86%a ±1.35 NZn 5 164.70b±22.86 243.13b±33.66 407.84b±56.51 9.51%a ±0.43

ĐC 159.42b±5.30 236.84b±5.77 396.27b±11.08 9.97%a ±0.66 N½Zn0 92.89a±17.21 194.02a±34.28 286.91a ± 51.49 10.70%a±0.70 N½Zn0.1 113.62ab±20.02 240.96ab±39.20 354.58ab±59.24 9.85%a±1.34 N½Zn0.5 153.92bc±17.14 324.46bc±43.64 478.38bc±60.78 9.70%a±0.84 N½Zn1 176.40c±5.72 369.29c±10.83 545.80c±16.56 9.68%a±0.59 N½Zn5 156.97bc±16.58 326.61bc±37.01 483.59bc±53.60 9.70%a±0.70

ĐC 159.42bc±5.30 236.84ab±5.77 396.27ab±11.08 9.78%a±1.24

Note: The same letters in lowercase in the same row mean no significant difference at P ≤ 0.05

Figure 1 Effect of ZnONPs on multiple shoot formation after 6weeks

The obtained results were consistent with the role of zinc and also showed the effects of Zn-NPs outstanding compared to zinc salts The primary sources of zinc micronutrients for fertilizer fortification or types of plant tissue culture media are zinc oxides (ZnO) and zinc sulfates (ZnSO4 H2O or ZnSO4.7H2O) Zinc is contained in most enzymes, necessary for hormones and chlorophyll regulation, and for carbohydrate metabolism In the nanoparticles form, ZnO can be absorbed, metabolized and accumulated in plant systems Zinc is a micronutrient and therefore needed in small quantity; higher concentrations of ZnO NPs can

harm the development of plants, leading to inherent aberration in seed germination, root growth and seedling biomass [6] In this study, when the Zn-NPs concentration increased to 5 mg/L, shoot formation, shoot extension and total chlorophyll content decreased Javed et al (2017) reported that the highest frequency of shoot formation (89.6%) was obtained when nodal explants of Stevia rebaudiana were cultured on MS medium amended with 1 mg/L ZnO-NPs [7] This result was similar to the result we obtained on Dendrobium officinale

2.2.2 Effect of ZnO-NPs on root formation

The results of in vitro Dendrobium officinale shoots after 6 weeks of culture on root formation showed in Table 3 and Figure 2

Table 3 Effect of ZnO-NPs on root formation after 6 weeks Treatment No of roots/ explant morphology Shoot Treatment No of roots/ explant morphology Shoot NZn0 3.75a±0.21 small, short N½ Zn0 3.85ab±0.07 small, short NZn0.1 4.20ab±0.28 big, long N½Zn0.1 4.40ab±0.28 big, long NZn0.5 5.10b±0.14 big, long N½Zn0.5 4.75b±0.21 big, long NZn1 6.70c±0.42 big, long N½Zn1 3.95ab±0.07 big, long NZn5 4.20ab±0.14 big, long N½Zn5 3.50a±0.42 big, long

Note: The same letters in lowercase in the same row mean no significant difference at P ≤ 0.05

ZnO-NPs not only induced shoot multiplication but also influenced root formation of explant More roots were produced in explants inoculated on medium containing ZnO-NPs solution in concentrations from 0.1 to 1 mg/L It was also observed that in vitro shoot cultures

on NZn1 formula containing 1 mg/L ZnO-NPs produced maximum number of roots (6.70) per explant with big, long roots Number of roots/ explant and root morphology were the lowest and worst in Zinc-free culture media (NZn0) and control medium (MS)

Figure 2 Effect of ZnO-NPs on root formation after 6 weeks Zinc is essential for the normal, healthy growth of plants Plant usually requires small amounts of Zn to allow the normal function Many kinds of enzyme require Zn so Zn affects to the metabolism of proteins, carbohydrates and auxin as well as reproductive processes [8] So Zn-deficient plants may be affect the process of root formation and development as well as susceptibility to root diseases

3 Conclusions

Zinc oxide nanoparticles had the effect on stimulating shoot formation of in vitro Dendrobium officinal at 1 mg/L to 5 mg/L.In shoot extension stage, zinc oxide nanoparticles stimulated length of shoots The medium formula with the addition of 1 mg/L zinc oxide

nanoparticles and combination with ZnSO4 obtained the best results after 6 weeks of culture with 2.9 shoots/sample, average length of 2.74 cm/sample, total chlorophyll content of 545.8 µg/g.ZnO nanoparticles was used at concentration of 1 mg/L, the root count was the most, reaching 6.7 roots/sample with large and long roots The basic MS control medium (containing full zinc salts) produced lower or similar all tested mediumin some indicators (2.5 shoots/sample after 6 weeks, shoot length of 2.58 cm/sample, total chlorophyll content of 396.27 µg/g, large roots, reaching 3.5 roots/shoot)

Acknowledgments The study was conducted with the financial support from SPHN 18-02

TD project

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