Facilitate Seed Germination of the Golden Shower Tree ( Cassia fistula ) in vitro Using TiO 2 Nanoparticles and Scarification Treatments

The main propagation method of Cassia fistula is sowing seeds. The seed germination is usually low because of its impermeable hard coat. Therefore, this experiment evaluated the effects of TiO2 nanoparticles and scarification methods on seed germination and seedling growth in vitro condition. The tree seeds were treated with, hot water, H2SO4 (36N), and mechanical scarification and culture on 1⁄4 MS salt mixture. The medium was supplemented with different concentrations of TiO2 nanoparticles. The results showed that the highest percentage and rate of germination was recorded in seeds treated with mechanical scarification. The highest shoot and root dry weight was recorded for seeds treated with mechanical scarification and grown on MS media supplemented with 1.5 mg/ml TiO2 nanoparticles. TiO2 nanoparticles did not show any significant effects on the percentage and rate of germination. Different growing soil mixtures had significant effects on the growth of the ex vitro transferred plantlets. Coco peat and peat moss mixture (1:1) was found to be more effective in increasing the number of leaves and root length of the seedlings.


Introduction
Cassia fistula L. (family Fabaceae), commonly known as the golden shower Indian laburnum, is mainly found in Asian countries such as India, Sri Lanka and China.It is also found in various other countries including Mexico, Mauritius and South Africa (Neelam et al., 2011).The fruits of this plant are cylindrical, brown, septate, 25-50 cm in length, 1.5-3 cm in diameter, and have 25-100 seeds (Danish et al., 2011).In southwest of Iran (Ahwaz and Abadan cities) Cassia is cultivated as an ornamental tree.However, the main issues encountered in propagating Cassia seedlings is poor seed germination caused by the water-impermeable seed coat, which exerts physical dormancy (Rolston, 1978).Thus the seeds should be given pre-germination treatments before planting.
The recent discovery of interesting properties of nanoparticles has led to its wide variety of applications in the chemical industry, medical field, electronics and agriculture as well (Savithramma et al., 2012).Several experiments have been carried out to evaluate the effects of different nanoparticles on seed germination and seedling growth of many plants.Some of these experiments include effect of ZnO NPs on onion seeds (Raskar & Laware, 2014); wheat, rice, and cucumber (Kumar et al., 2015); TiO 2 NPs on parsley and spinach seeds (Hong et al., 2005a;Dehkourdi & Mousavi, 2013); Silver NPs on Pennisetum glaucum seeds (Parveen, 2015); CuO NPs on soybean and chickpea seeds (Adhikari et al., 2012); Fe, Co and Cu NPs on soybean seeds (Ngo et al., 2014); SiO 2 NPs on tomato (Siddiqui & Al-Whaibi, 2014); carbon nanotubes on strawberry seeds (Yosefi et al., 2012); Mo and SiO 2 NPs on rice seeds (Adhikari et al., 2013); and Apatite NPs on soybean (Liu & Lal, 2014).Among the different nanoparticles examined, the TiO 2 NPs showed more effectiveness in increasing plant growth.
Therefore, the aim of this experiment was to investigate the use of TiO 2 nanoparticles along with different scarification methods, including mechanical, acid, and hot water, on the seed germination and growth of C. fistula seedlings in vitro.

Method
This experiment was conducted in the tissue culture laboratory of the Shahid Chamran University of Ahwaz, Iran.

Seed Scarification and TiO 2 NPs Treatments
Murashige and Skoog (MS1/4) medium was prepared and poured into the glass bottles.Each bottle contained 50 ml of MS media.Different concentrations of TiO 2 NPs were added into the medium, which was sterilized in an autoclave (121 °C/15 psi, for 20 min).Mineralogical structure of anatase titanium dioxide nanoparticles with sol gel solution was prepared by the Physics Department of Sharif University in Iran.Scanning microscopy (TEM) nano-sized titanium dioxide particles were about 30 nm and spindle-shaped.
The seeds were sterilized by 70% ethanol for 30 sec, then added to sodium hypochlorite (2.5%) for 5 min and washed 3 times with sterile distilled water.The seeds were treated before sterilization using the following steps:  The seeds were cultured on the medium and incubated at 25 °C with 16 h photoperiod and checked every day.

Germination Parameters
The number of seeds was counted with visible radicle as sprouted seeds (seeds that have sprouted length root of it considered two millimeters) (Miller & Chapman, 1978).Then it was measured as Equation (1).
Where, GP is germination percentage, ƩG is the number of seeds germinated on that particular day, and N is the total number of seeds (Hartmann & Kester, 1983).The germination rate was calculated using Equation (2).
Where, GR is the germination rate, Σn is the number of seeds germinated on the day, and Σdn is the number of days from the start of the experiment (Maguire, 1962).

Measurement of Seedling Growth Parameters
After 30 days, five randomly selected seedlings were taken from each petri dish to measure their shoot and root lengths using a measuring scale.The measurements were expressed in centimeters.The number of leaves and radicles were also measured.For the fresh and dry weights of the shoots and roots of the seedlings, the fresh weight of each tissue was weighated first.Then the tissues were dried in an oven at 70 °C for 48 hours and their dry weight was measured.

Plantlets Ex-Vitro Acclimatization
In vitro grown seedlings were ex vitro transferred to acclimatization in a transparent plastic box.Then the agar was washed and disinfected with the fungicide Mancozeb.
The four tested soil mixtures are: The seedlings were fed with MS1/4 basal salt mixture solution during their growing period.Finally, the shoot and root length, number of leaves, and diagonal shoots were measured.

Research Design
The experiment was carried out in factorial complete randomized design that included five replicates with five seeds.Factor A included (mechanical scarification, sulfuric acid, hot water, and control).Factor B includes TiO 2 NPs at four concentrations (0, 1.5, 4.5, and 13.5 mg/ml).
The data were analyzed using the MSTATC software.The significant levels of difference for all measured traits were calculated, and the means were compared by the multiple ranges Duncan test at 5% level.

Results
The analysis of variance showed that there was a significant difference among all measured indices under different scarification treatments as well as indices affected by TiO 2 NPs except the seed germination percentage, germination rate, and shoot fresh weight in the case of TiO 2 NPs treatments.The interactions between scarification treatments and TiO 2 NPs concentrations also showed the same difference as their individual effects except in the seed germination percentage, germination rate and shoot fresh weight (Table 1).Note. * Significant at 5 % level.
The comparison of the means as showed in Table 2 indicates that the highest shoot length, number root, root fresh weight, root length, leaf number and dry weight of the shoot and roots of seedling were significantly increased when the seeds were treated with mechanical scarification along with 1.5 mg/ml of TiO 2 NPs.However, no significant difference was observed between 0 and 1.5 mg/ml TiO 2 NPs for root length, leaf number, and dry weight of the shoot and roots of the seedling.
The highest percentage and rate of germination were observed for seeds under mechanical scarification treatments, and the lowest percentage and rate of germination were observed for seeds with no treatment (control) (Figures 2a and 2b).The effects of scarification treatments and TiO 2 NPs concentration on shoot fresh weight are individually showed in Figures 2c and 2d, respectively.
The results obtained from a comparison among different soil mixture showed that they had a significant difference (Table 3).The maximum leaf number and length root were observed for seedlings that had been transplanted in coco peat-peat moss media (  Note.Means with different letters at each row have a statistical difference at 5 % level.Note.Means with different letters at each row have a statistical difference at 5 % level.

Discussion
The results obtained in this study showed that the mechanical scarification treatment of C. fistula seeds significantly enhanced seed germination percentage from 20 to 80 percent.Similar to the seeds of many leguminous species, C. fistula seed have impermeable hard coat dormancy, which may be due to impermeability of testa to water.Damaging the coat or breaking dormancy, facilitates germination of the seeds as it helps in supplying adequate water to their inner tissues and starts the germination process.Compared to hot water and sulfuric acid treatments, mechanical scarification effectively increases the germination with fewer side effects on the seed tissues.Pant and Chauhan (2013) reported that soaking of Cassia tora seeds in water for 24 h after mechanical scarification (by rubbing the seeds with sand paper) showed a significant level of improved germination (76.88%) when compared to sulfuric acid scarification, hot water and alcohol treatments.However, Karaboon et al. (2005) found that scarification the Cassia fistula seeds with sulfuric acid for 15-20 min represented the best treatment for enhancing germination, but mechanical scarification (by piercing the seeds at the part of cotyledon) showed slightly lower effects (statistically significant).Recently Wu et al. (2014) reported that TiO 2 nanoparticles effectively electrospray (using single-capillary electrospray) dispersed nanoparticles for breaking the seed coat to enhance seed germination percentage of lettuce seeds.However, this method may be not simple to use due to the requirement of a technical apparatus and absence of adequate information on its efficiency in seeds with hard coat dormancy.
On the other hand, the presence of TiO 2 NPs in the germination medium did not further improve the seed germination percentage and rate but showed significantly enhanced seedling growth indices.To the best of our knowledge, there is no report on the application of TiO 2 NPs to enhance C. fistula seed germination or seedling growth.Feizi et al. (2012) found similar results for wheat seed germination.They showed that the treatment of wheat seeds with bulk and nanosized TiO 2 could not significantly enhance germination percentage and rate compared to control but the nanosized TiO 2 increased shoot and seedling lengths at 2 and 10 ppm concentrations which was higher than those of the untreated control and bulk.Zheng et al. (2005) reported that nanosized TiO 2 helped in water absorption by the spinach seeds and enhanced the germination of the seeds.Hatami et al. set an experiment to determine the effects of different concentrations of the TiO 2 NPs on germination indices and seedling vigor of five medicinal plants, namely, Alyssum homolocarpum, Salvia mirzayanii, Carum copticum, Sinapis alba, and Nigella sativa.They concluded that the nanosized TiO 2 treatments in appropriate concentrations accelerates the germination characteristics of the reference plant seeds and increases their vigor.Maroufi et al. (2011) reported that priming the Vigna radiata seeds with TiO 2 NPs increased germination percentage, seedling dry weight and seedling vigor.Seedling emergence losses in the soil are not generally due to germination failure, but the failure of seedlings to grow and emerge above the soil surface (Halmer & Bewley, 1984).Farooq et al. (2005) demonstrated that priming of the tomato seed increased the rate of cell division in the root tips of seedlings.Primed seeds might have better plasma membrane structure by slow hydration (Jett et al., 1996).
The photosynthesis process starts after C. fistula seedling emerges above the soil surface and its expanded green leaves are exposed to the light.TiO 2 NPs promotes the photosynthesis rate by changing the light energy to electron energy and then to active chemistry energy.Sayama et al. (2002) demonstrated that the mechanism to  This characteristic of nanosized TiO 2 can also provide a low-cost, environmentally friendly solar-hydrogen production (Ni et al., 2007).Qi et al. (2013) reported that the net photosynthetic rate, conductance to H 2 O, and transpiration rate of tomato leaves increased after the application of TiO 2 NPs.Elghniji et al. (2014) suggested that photocatalytic degradation characteristic of the TiO 2 helped in removing the cytotoxicity of 4-CP in irrigation wastewater and enhance seed germination and seedling growth of tomato, turnip and onion when they were exposed to sunlight.
It was reported that the TiO 2 NPs strongly absorbed light not only in the UV region, but also in the visible region from 400 to 800 nm (Zheng et al., 2007).Zheng et al. (2005) reported that TiO 2 NPs treatments markedly promoted seed vigor, chlorophyll biosynthesis, and improvement of growth and development in spinach but bulk TiO 2 treatments had comparatively little effects.Hong et al. (2005a) reported that photosynthesis promoted by TiO 2 NPs might be related to the activation of a photochemical reaction of chloroplasts of spinach.The TiO 2 NPs treatment could protect chloroplasts from aging for long-time illumination by enhancing the activities of the enzymes, such as catalase, peroxidase, and superoxide dismutase, lowering the accumulation of free radicals of the reactive oxygen and the level of malondialdehyde, and maintaining the stability of membrane structure of chloroplast under light (Hong et al., 2005b).Hong et al. (2005c) showed that the TiO 2 NPs could improve spinach growth through increased chlorophyll contents and photosynthetic rate by enhance light absorbance, accelerating the electron transport and efficient transformation of the light energy.Gao et al. (2006) reported that TiO 2 NPs could not only improve the light absorbance and the transformation from light energy to electron energy and active chemical energy but also promoted carbon dioxide assimilation of spinach.In this mechanism the TiO 2 NPs actives Rubisco and promotes Rubsico-carboxylation, which increases the rate of photosynthetic carbon reaction.

Conclusion
The results obtained in this study demonstrated that the scarification mainly increased the germination percentage and germination rate and the TiO 2 NPs treatment markedly enhanced the seedling growth.Therefore, the combination of the scarification with 1.5 mg/ml TiO 2 NPs treatment provided a good strategy for the propagation of C. fistula through seed.


Mechanical scarification with damaging of the seed coat (Figure1);  Hot Water 80 °C for 3 minutes;  H 2 SO 4 (36N) for 30 minutes.The non-scarified seeds were used as control.

Figure 1 .
Figure 1.The Cassia fistula seed (a) mechanical scarification by damaging the hard seed coat (b)

Figure 2 .
Figure 2. Effect of different scarification methods on Cassia fistula seed germination percentage (a) ; germination rate (b) and shoot fresh weight (c); effect of TiO 2 NPs on shoot fresh weight (d); effect of different soil mixture on number leaf (e) and root length (f) H 2 and O 2 by TiO 2 under the visible light irradiation was similar to the Z-scheme reaction in the natural photosynthesis.

Figure 3 .
Figure 3. Ex vitro grown seedlings in different soil mixtures in the acclimatization box (a); seedling of Cassia fistula grown from seed after mechanical scarification and nano-TiO 2 treatment in vitro (b)

Table 1 .
Analysis of variance the indices of Cassia fistula seed germination affected by scarification treatments and TiO 2 NPs dioxide

Table 2 .
Effect of scarification methods and nanoparticles of titanium dioxide on growth indices of Cassia fistula seedling

Table 3 .
Analysis of variance the effects of different soil mixture on growth of Cassia fistula seedling ex vitro

Table 4 .
Effects of the different soil mixture on growth of Cassia fistula seedling