Characterization of the synthesized nanoparticles
The produced silver, chitosan and selenium nanoparticles were characterized by TEM instrument. The diameter and shape of the tested nanoparticles were illustrated in Fig. (1). TEM micrograph demonstrates the formation of spherical shape with a narrow range of particle size distribution, the spherical nanoparticles were produced with size of 5-15 nm (Silver NPs), 15-35 nm (Selenium NPs) and 20-50 nm (Chitosan NPs). Analysis of TEM micrograph demonstrated that, the mean size of the obtained nanoparticles are comparable to the particle size that has been reported in previous studies for silver (Hmmam et al. 2021), selenium (Srivastava and Mukhopadhyay 2015), and chitosan nanoparticles (Asgari-Targhi et al. 2018). Therefore, the prepared nanoparticle in our study represented typical nanoparticle in term of shape and size.
Effect of synthesized nanoparticles on in vitro microbial contamination
Regarding the variation in inhibition potential of the tested nanoparticles on in vitro microbial contamination (Fig. 2), the obtained results showed that, there was a significant difference between the different nanoparticles types and concentrations on microbial contamination. Silver nanoparticles at concentration of 10 mg l-1 recorded the lowest value of microbial contamination in tissue culture medium compared with the negative control plates. Chitosan nanoparticles were ranked in the second order, while selenium nanoparticles recorded the lowest value. The obtained results indicated the feasibility for applying the nanoparticles in the tissue culture medium as antimicrobial agents without sterilization. The inhibition potential of nanoparticles agents in vitro growth of microorganisms confirm our previous studies about the antimicrobial activity of the tested nanoparticles (Darwesh et al. 2018; Elshahawy et al. 2018; El-Shanshoury et al. 2020). Silver NPs has a high potential for eliminates microbial contamination in culture medium; AgNPs recorded the lowest contamination percentage, which is statistically similar to media sterilization by autoclaving (Fig. 2). The high antimicrobial activity of AgNPs may be attributed to the strong toxicity of silver ions to a wide range of microorganism (Abdi et al. 2008; Jo et al. 2009; Sobha et al. 2010; Hwan et al. 2017; Ismail et al. 2017). Moreover, the small particle size (5-15 nm) of the obtained silver nanoparticles are important for interactions and binding of silver ions with cell membrane proteins, which resulting cell death (Sondi and Salopek-Sondi 2004).
Chitosan nanoparticles showed relatively higher anti-microbial activity compared with selenium nanoparticles and control treatment; the anti-microbial activity of chitosan nanoparticles against different plant pathogens was reported previously (Qi et al. 2004; Du et al. 2009; Ali et al. 2011). Chitosan NP affecting cell membrane permeability and inhibiting DNA replication (Chandrasekaran et al. 2020). Our results show that SeNPs have low anti-microbial activity compared with AgNPs and ChNPs; the low toxicity of Se NPs may be due to the negative charge of Se NPs which results in relatively repulsive with bacterial membrane (Nguyen et al. 2017). Also, higher concentrations of SeNPs may be needed to show significant effect on microbial contamination. A previous study showed that, 50 mg l-1 is the minimum dose for inhibition of E. coli or S. aureus (Guisbiers et al. 2016). According to Nguyen et al. (2017), SeNPs exhibited dose-dependent antimicrobial properties; 10 mg l-1 inhibited in vitro growth of S. aureus but have a no effect on E. coli, Salmonella, and L. monocytogenes.
Effect of the synthesized nanoparticles on in vitro performance of olive explants
According to the data illustrated in Tables (1), in vitro growth of olive shoots was significantly affected with both of plant genotype and nanoparticles treatments at the proliferation stage. Picual recorded higher number of shoots/explant compared with Koroneiki and Manzanillo. The highest number of shoots /explant was recorded for AgNPs at 5 and 10 mg l-1 (1.77±0.43 and 2.28±0.56, respectively), while SeNPs treatments had a negative effect on shoot growth and recorded the lowest value of number of shoots/explant, there was a non-significant differences between ChNPs and control treatment.
Table 1 The effect of nanoparticles type and concentration on shoot number of different olive cultivars
Treatments
|
Concentration
(mg L-1)
|
Manzanillo
|
Picual
|
Koroneiki
|
Mean
|
Selenium NP
|
2.5
|
1.00±0.00 g
|
1.50±0.05 e
|
1.30±0.05 f
|
1.27±0.07D
|
5
|
1.00±0.00 g
|
1.00±0.00 g
|
1.00±0.00 g
|
1.00±0.00E
|
Silver NP
|
5
|
1.20±0.05f
|
2.00±0.00bc
|
2.12±0.02 b
|
1.77±0.14B
|
10
|
1.83±0.05 cd
|
2.00±0.11bc
|
3.00±0.05 a
|
2.28±0.18A
|
Chitosan NP
|
40
|
1.00±0.00 g
|
1.80±0.20 d
|
1.30±0.00 f
|
1.36±0.11C
|
60
|
1.00±0.00 g
|
1.00±0.00 g
|
1.00±0.00g
|
1.00±0.00CD
|
Control
|
--------
|
1.00±0.00 g
|
2.00±0.00bc
|
1.00±0.00 g
|
1.33±0.16CD
|
Mean
|
|
1.14±0.06 B
|
1.61± 0.09 A
|
1.53±0.15 A
|
|
Values of interaction (treatments x cultivars) followed by different lowercase letters are significantly different (p < 0.01). Mean values of treatment or cultivar followed by different uppercase letters are significantly different (p < 0.01); each value represent mean of three replicate± standard error (SE).
As shown in Table (2), Koroneiki cv. recorded the highest shoot length compared with Picual and Manzanillo cv. It is evident that the addition of nanoparticles to culture medium affected growth of in vitro cultured olive shoots compared with the control. The shoot length varied between the different nanoparticles treatments, AgNPs at 10 mg L-1 recorded the highest value, while SeNPs at 5 mg L-1 recorded the lowest value. Due to the strong apical dominance of olive the formation of secondary axillary shoots is very limited; olive shoot multiplication is achieved by segmentation of elongated shoots at each subculture (Fabbri et al. 2004; Lambardi et al. 2012). The highest multiplication rate of the cultivated olive cultivars was recorded for Picual cv. (Table 3) compared with the other cultivars. There was a significant difference between the nanoparticles treatments regarding multiplication rate; silver NPs at both concentration improved multiplication rate of the studied olive cultivars compared with the other treatments while selenium NPs at 5 mg L-1 and chitosan NPs at 60 mg L-1 produced the lowest value.
Table 2 The effect of nanoparticles type and concentration on shoot length (cm) of different olive cultivars
Treatments
|
Concentration
(mg L-1)
|
Manzanillo
|
Picual
|
Koroneiki
|
Mean
|
Selenium NP
|
2.5
|
6.0±0.57c
|
6.0±0.57c
|
7.0±0.57bc
|
6.33±0.33B
|
5
|
3.0±0.00d
|
4.0±0.00d
|
3.0±0.00d
|
3.33±0.16D
|
Silver NP
|
5
|
6.0±0.57c
|
6.0±0.00c
|
8.0±0.57b
|
6.67±0.41B
|
10
|
7.0±0.00bc
|
8.0±0.57 b
|
10.0±0.00 a
|
8.33±0.47A
|
Chitosan NP
|
40
|
7.0±0.57bc
|
6.0±0.00 c
|
6.5±0.28bc
|
6.50±0.24 B
|
60
|
4.0±0.00d
|
5.5±0.00c
|
6.0±0.57c
|
5.17±0.34C
|
Control
|
-------
|
7.0±0.00bc
|
6.0±0.00 c
|
7.0±0.57bc
|
6.67±0.24B
|
Mean
|
|
5.71± 0.35 B
|
5.92± 0.26 B
|
6.78± 0.46 A
|
|
Values of interaction (treatments x cultivars) followed by different lowercase letters are significantly different (p < 0.01). Mean values of treatment or cultivar followed by different uppercase letters are significantly different (p < 0.01); each value represent mean of three replicate± standard error (SE).
Table 3 The effect of nanoparticles type and concentration on multiplication rate of different olive cultivars
Treatments
|
Concentration
(mg L-1)
|
Manzanillo
|
Picual
|
Koroneiki
|
Mean
|
Selenium NP
|
2.5
|
3.0±0.12d
|
3.2±0.05d
|
2.2±0.12e
|
2.80±0.16C
|
5
|
1.2±0.00fg
|
1.3±0.00f
|
1.2±0.00fg
|
1.23±0.07E
|
Silver NP
|
5
|
3.2±0.00d
|
4.3±0.05b
|
4.8±0.12 a
|
4.10±0.24B
|
10
|
4.3±0.17b
|
4.0±0.00c
|
5.0±0.00 a
|
4.43±0.16A
|
Chitosan NP
|
40
|
2.0±0.00 e
|
2.0±0.00e
|
1.3±0.12f
|
1.77±0.12D
|
60
|
1.0±0.00 g
|
1.2±0.00fg
|
1.1±0.00fg
|
1.10±0.03E
|
Control
|
--------
|
2.0±0.00e
|
2.0±0.00e
|
1.1±0.00fg
|
1.70±0.15D
|
Mean
|
|
2.38± 0.24 B
|
2.57±0.26 B
|
2.38±0.36 A
|
|
Values of interaction (treatments x cultivars) followed by different lowercase letters are significantly different (p < 0.01). Mean values of treatment or cultivar followed by different uppercase letters are significantly different (p < 0.01); each value represent mean of three replicate± standard error (SE).
Data presented in Table (4) showed that the highest leaf number was recorded for Picual cv. compared with the other cultivars, there was a statistically significant difference between the nanoparticles treatments regarding the leaf number, silver NPs at 10 mg L-1 results in the highest leaf number followed by silver NPs at 5 mg L-1 while selenium NPs at 5 mg L-1 and chitosan NP at 60 mg L-1 showed the lowest leaf number. The nanoparticles application may lead to stimulate or inhibit effects on plant growth and development; the impact of nanoparticles on plant growth depending on particle size, shape, concentrations, plant genotype and age (Khan et al. 2019; Goswami et al. 2019; Kranjc and Drobne 2019). The obtained results indicated the positive effect of nanoparticles, especially AgNPs in improving the efficiency of olive micropropagation. As reported previously, silver ion (Ag+) has a positive effect on plant tissue culture e.g. increased survival and delayed explants senescence (Sarmast et al. 2015), improve somatic embryogenesis (Al-Khayriet al. 2001), organogenesis (Ricci et al. 2020), increase shoot multiplication rate and plant growth (Kotsias and Roussos 2001). Shoot growth and number of shoots per explant were increased in Brassica juncea, Tecomella undulate Roxb. and Vanilla planifolia cultured on medium supplemented with AgNPs (Kumar et al. 2009; Aghdaei et al. 2012; Sharma et al. 2012; Spinoso-Castillo et al. 2017), which was attributed to the effect of Ag+ as an ethylene blockage agent (Songstad et al. 1988). Selenium is not an essential nutrient for higher plants and their effect differs depending on the concentration. Selenium at low concentration can stimulate plant growth, whereas, higher doses had an inhibitory effect (Hawrylak et al. 2015). It was reported that selenium nanoparticles stimulate callus induction and improve morphogenesis of tobacco and artichoke (Domokos-Szabolcsy et al. 2012; Abdalla et al. 2021). Recent studies showed that selenium stimulate shoot growth and increased fresh and dry weight of in vitro growing olive shoots (Regni et al. 2021).
Table 4 The effect of nanoparticles type and concentration on leaf number of different olive cultivars
Treatments
|
Concentration
(mg L-1)
|
Manzanillo
|
Picual
|
Koroneiki
|
Mean
|
Selenium NP
|
2.5
|
9.0±0.57cd
|
10.0±0.57c
|
6.0±0.57fg
|
8.33±0.66C
|
5
|
4.0±0.00hi
|
5.0±0.00gh
|
4.0±0.00 hi
|
4.33±0.17E
|
Silver NP
|
5
|
8.0±0.00de
|
10.0±0.57c
|
12.0±0.57b
|
10.00±0.62B
|
10
|
12.0±0.57b
|
10.0±0.57c
|
14.0±0.57a
|
12.00±0.65A
|
Chitosan NP
|
40
|
6.0±0.00fg
|
7.0±0.00ef
|
6.7±0.33efg
|
6.55±0.18D
|
60
|
3.0±0.00 i
|
6.0±0.00fg
|
6.7±0.66efg
|
5.22±0.60E
|
Control
|
--------
|
7.0±0.57ef
|
7.0±0.00ef
|
6.0±0.00fg
|
6.67±0.24D
|
Mean
|
|
7.00± 0.64 B
|
7.85± 0.45 A
|
7.90± 0.76 A
|
|
Values of interaction (treatments x cultivars) followed by different lowercase letters are significantly different (p < 0.01). Mean values of treatment or cultivar followed by different uppercase letters are significantly different (p < 0.01); each value represent mean of three replicate± standard error (SE).
In contrast, our results showed that SeNPs had a negative effect on growth of olive shoots, which may be due to the higher absorption and mobility of selenium nanoparticles in plant tissues. Also, there are limited studies on the in vitro applications of chitosan NPs on the plant growth, supplementations of culture medium with chitosan NPs promote plant growth, however the higher doses of chitosan NPs dramatically caused reduction of plant growth and development, the toxicity of chitosan NP was higher than the chitosan bulk type, which may be due to the physicochemical properties of chitosan NP (Asgari-Targhi et al. 2018). Despite the fantastic effects of nanoparticles, the application of nanoparticles in culture media should be optimized to avoid the toxic effect of high doses; according to our results, the higher concentration of selenium and chitosan NPs had a negative effect on olive shoot growth. Several studies have shown that, higher concentrations of NPs had adverse effects on cell viability, organogenesis, shoot growth and plant regeneration (Chichiriccò and Poma 2015; Da Costa et al. 2016; Salehi et al. 2018). NPs affect the mitotic activity and change the DNA structure and gene expression in different plant species (Ewais et al. 2015; Tripathi et al. 2017; Hassan et al. 2019). According to Nakasato et al. (2017), high concentration of chitosan NP severely inhibited germination, and negatively affected growth of Zea mays, Brassica rapa and Pisum sativum. Se toxicity may be due to the replacement of sulfur atoms by selenium in sulfur containing amino acids; which result in changes in proteins structure and function, simultaneously selenium can cause oxidative and stresses, cellular damage and disrupts plant metabolism and reduce plant growth (Terry et al. 2000; Hasanuzzaman et al. 2011). Therefore, the effects of different types and concentration of NPs on plant tissue should be optimized in order to determine the optimum dose with minimal phytotoxicity (Kim et al. 2017).