Enhancing In Vitro Multiplication of some Olive Cultivars Using Silver, Selenium And Chitosan Nanoparticles

Olive trees are commercially propagated by cuttings or grafting on clonal or seedling rootstocks; recently, olive micropropagation has emerged as a powerful technique for mass production of true to type and pathogen free plants. The current study was carried out to evaluate the effect of silver, chitosan and selenium nanoparticles as microbial decontamination agents, as well as evaluate its possible effects on in vitro shoot growth and multiplication of three olive cultivars namely Koroneiki, Picual and Manzanillo. Validation and characterization of the biosynthesized nanoparticles was carried out by Transmission Electron Microscopy. The produced nanoparticles were added to the olive culture medium, and the growth parameters were determined. The tested nanoparticles showed varied degree of antimicrobial activity, silver nanoparticles were highly effective to inhibit in vitro microbial contamination. The effect of genotypes on shoot growth and multiplication was signicant; Koroneiki and Picual cultivars showed better growth measurements compared with Manzanillo. The addition of nanoparticles to the culture medium had a signicant outcome on growth and multiplication rate of in vitro growing olive shoots. Silver nanoparticles produced higher values of number of shoots, shoots length, leaf number and multiplication rate compared with the other treatments. In conclusion; the current results showed that nanoparticles were ecient as in vitro disinfectant agent and the nanoparticles addition to culture medium especially silver NPs had a positive effect on growth and multiplication rate of in vitro growing olive shoots.


Introduction
Olive (Olea europaea) is one of the traditionally cultivated fruit crops in the Mediterranean region and one of the most important fruit crops in Egypt (Baldoni and Belaj 2009). Olive trees are generally adapted to the semi-arid environment and traditionally grown under arid desert conditions (Dag et al. 2008; Guerfel et al. 2009). Olive fruits, oil and leaves are rich sources of valuable nutrients and bio-active pharmaceutical materials (Ghanbari et al. 2012). The bene ts of olive products to human health have been widely recognized (Visioli et al. 2002). Olive trees are usually propagated by leafy stem cuttings under mist conditions; the hard to root cultivars are propagated by grafting onto seedlings or clonal rootstocks (Fabbri et al. 2004). Olive micropropagation has been reported as a powerful technique for mass production of pathogen-free and true to type cultivars, and offers a valuable tool for genetic improvement and germplasm conservation (Zuccherelli and Zuccherelli 2002;Zacchini and De Agazio 2004). In vitro propagation of olive is affected by several factors including plant genotype, growth medium, cytokinin type and concentration (Grigoriadou et al. 2002;Zuccherelli and Zuccherelli 2002;Peixe et al. 2007).
Generally, microbial contamination, oxidation of phenolic compounds and slow shoot growth are the major problems of olive micropropagation (Lambardi et al. 2012). Moreover, olive shoots are characterized by a strong apical dominance and the formation of axillary shoots is very limited (Fabbri et al. 2004). Microbial contamination is a serious problem of in vitro propagation; eliminate the microbial contamination is one of the basic requirements for successful initiation, growth and development of cultured plant tissues (Abdi et al. 2008; Msogoya et al. 2012). To eliminate microbial contamination, plant materials must be surface sterilized; disinfection procedures usually involve using of sodium hypochlorite, ethyl alcohol, hydrogen peroxide, mercury chloride and antibiotics; nevertheless, these substances are frequently toxic to plant tissues and have shown inhibitory effects on explant growth and development (Teixeira da Silva et al. 2003; Abdi et al. 2008). Therefore, there is a need for more studies to improve olive micropropagation e ciency with inhibition of pathogens growth. Recently, nanotechnology have received much attention, it expected to support a wide range of applications in agriculture elds, e.g. enhancing seed germination, plant growth, yield, nutritional value and production of secondary metabolites, moreover nanotechnology aims to develop a new delivery system of mineral nutrients and pesticide, thereby, reducing environmental pollution (Tarafdar et
Preparation of silver, selenium and chitosan nanoparticles Silver nanoparticles (AgNPs) were prepared using the supernatant free cells of Fusarium oxysporum (Elshahawy et al. 2018). A 250 ml asks containing 100 ml sterilized potato dextrose broth (PDB) medium was inoculated with F. oxysporum and incubated in a rotary shaker at 28˚C for 3 days, then the cell free supernatant was obtained by ltration (Whatman No.1). Ten ml of supernatant was added to 10 ml of 1M silver nitrate solution in bottles and incubated under static conditions at room temperature for 24 h. The prepared AgNPs were obtained by centrifugation (10000 rpm for 10 min) and washed three times by deionized water and then washed by absolute ethanol and oven dried at 50 °C (Marrez et al. 2019). In case of selenium nanoparticles preparation, stock solutions of 100 mM sodium selenite and 50 mM ascorbic acid were prepared in deionized water. The ascorbic acid was mixed drop wise with the sodium selenite solution under magnetic stirring (600 rpm) at room temperature till achieve a nal ratio of 1:4 sodium selenite to ascorbic acid. The mixtures were allowed to react till the color changed from colorless to light orange (Hussein et al. 2019a). Chitosan nanoparticles were prepared by ionic gelation technique using tri-sodium polyphosphate (TPP) ). One gram of medium molecular weight chitosan (deacetylation degree> 80%), were dissolved in 100 ml of 1% (v/v) acetic acid solution by stirring at room temperature overnight, the nal pH of chitosan solutions was adjusted to 5.9 with 1M of NaOH (Tsai and Su 1999). Chitosan nanoparticles were obtained by drop wise addition of 14 ml of 0.1 %TPP solution to 35 ml of chitosan solution under continuous stirring (550 rpm) at room temperature. Chitosan nanoparticles were precipitated by centrifugation at 10000 rpm for 10 min. The pellet was washed with distilled water followed by absolute ethanol then air dried.

Characterization of the synthesized nanoparticles
Dimension and shape of the manufactured nanoparticles have been analyzed using transmission electron microscopy (JEOL, JEM 1400, USA) at an accelerating voltage of 80 kv. Samples for TEM analysis have been prepared by drop-coating nanoparticles solution onto carbon-coated copper grids. The lms on the TEM grids have been allowed to stand for 2 min, then extra solution was removed and the grid was allowed to dry prior to measurement.

Effect of synthesized nanoparticles on in vitro microbial contamination
In order to validate the e ciency of the studied nanoparticles on microbial contamination, silver at 5 and 10 mg l -1 , selenium at 2.5 and 5 mg l -1 and chitosan at 40 and 60 mg l -1 , were added to the non-sterilized Rugini olive medium (Rugini 1984) supplemented with 30g l -1 mannitol and 6 g l -1 agar; the experiment including comparative negative control treatment (non-sterilized free nanoparticles medium) and positive control treatment (autoclave sterilized free nanoparticles medium). The prepared media were dispensed in glass petri dishes and incubated at 25°C with 16 h photoperiod for 48h; fungus or bacteria colony formation was monitored by visual examining of culture plates, and the visible colonies were counted and the contamination percentage was calculated in relation to the negative control treatment.

Plant materials and explants preparation
Active growing shoots of 30 to 40 cm were collected during June and July from mature own rooted olive trees of 'Koroneiki', 'Picual' and 'Manzanillo' cultivars, grown at experimental olive orchard (experimental orchard location is situated at 031°12'65"E longitude, 30°00'48"N latitude, Giza, Egypt). The shoots were collected from each cultivar and immediately transferred to the laboratory; shoots were stripped of leaves, divided into nodal cuttings and washed several times with running tap water. The nodal segments were surface sterilized by 20% commercial bleach (5.5% NaOCl) for 10 min followed by 1000 mg l -1 mercuric chloride (HgCl 2 ) for 5min and then rinsed 3 times with distilled and sterilized water for 5 min.
Effect of the synthesized nanoparticles on in vitro performance of olive explants Sterilized nodal segments of the selected cultivars were cultured on Rugini olive medium (Rugini 1984), supplemented with 2.5 mg l -1 zeatin, 30 g l -1 mannitol and 6 g l -1 agar. The pH was adjusted to 5.8 and the media were sterilized by autoclaving at 121°C for 15 min. The cultures were incubated into growth chamber at 25±2ºC under 4000 lux light intensity for 16h/8h light/dark photoperiods supplied by white cool uorescent lamp. Four weeks later, the sprouted axillary shoots were transferred to fresh Rugini olive medium supplemented with one type of the above mentioned nanoparticles; silver NPs at 5 and 10mg l -1 , selenium NPs at 2.5 and 5mg l -1 and chitosan NPs at 40 and 60mg l -1 , in addition to the control free nanoparticles medium. The nanoparticles were added to the proliferation medium then the media were autoclaved at 121°C for 15 min. Four explants were transferred to 200ml jar containing 50ml semi-solid medium (each treatment consisted of 20) and incubated at 25±2ºC under 4000 lux light intensity for 16h/8h light/dark photoperiods supplied by white cool uorescent lamp, the sub-culture was performed every four weeks. At the 3 rd subculture, number of shoot per explant, shoots length, multiplication rate and number of leaves per shoot was determined. Multiplication rate calculated as the total number of shoots per explant multiplied by number of potentially nodal cuttings per shoot (Lambardi et al. 2012).

Experimental design and statistical analysis
The experiment was carried out in a completely randomized design, the assumptions of normality were tested using Shapiro-Wilk's test (Shapiro and Wilk 1965). Normally distributed data was subjected to two-way analysis of variance to investigate the effect of olive genotype, nanoparticles treatments and their interaction (Snedecor and Cochran 1967). Analysis of variance was performed using the SAS software (version 9.0; SAS Institute, Cary, NC, USA). The mean and standard error (SE) were calculated from three replicates per treatment and the signi cant differences within and between treatments were assessed by means of multiple Duncan range test at signi cance level of 0.01 (Duncan 1955).

Results And Discussion
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   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 signi cantly 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-signi cant differences between ChNPs and control treatment. 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 signi cant 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. Values of interaction (treatments x cultivars) followed by different lowercase letters are signi cantly different (p < 0.01). Mean values of treatment or cultivar followed by different uppercase letters are signi cantly 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   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

Conclusion
According to the obtained results, the tested nanoparticles showed varied degree of antimicrobial activity; AgNPs were highly effective to inhibit in vitro microbial contamination, ChNPs and SeNPs showed low anti-microbial activity. The addition of nanoparticles to the culture medium had a signi cant effect on growth and multiplication rate of in vitro growing olive shoots. AgNPs had a positive effect on growth and multiplication rate of in vitro growing olive shoots while the higher concentration of chitosan and selenium nanoparticles had a negative effect on shoot growth under in vitro conditions.

Declarations
Funding: This research was funded and supported by the Internal Projects Funding of National Research Centre (Project No. 11030115 and 11090339).
Con ict of interest: Authors declare no con ict of interest.
Availability of data and material: It is available when request Ethics approval: Not applicable Consent to participate: All authors con rm to participation in this work Consent for publication: All authors con rm for publication this work in the journal of Plant Cell, Tissue and Organ Culture (PCTOC).