The Effects of Titanium Dioxide Nanoparticles on the Growth and Development of Sorghum Bicolor (L.) Moenech

The Effects of Titanium Dioxide Nanoparticles on the Growth and Development of Sorghum Bicolor (L.) Moenech

Adam G Shoemaker, D Alexander Wait^*Department of Biology. Missouri State University. Springfield, MO 65897, USA

Received date: October 6, 2020; Accepted Date: October 15, 2020; Published Date: October 26, 2020;

^*Corresponding author: D Alexander Wait, Department of Biology, Missouri State University, Springfield, USA. Email: alexanderwait@missouristate.edu

Citation: Shoemaker AG and DA Wait (2020) The Effects of Titanium Dioxide Nanoparticles on the Growth and Development of Sorghum Bicolor (L.) Moenech. Adv in Agri, Horti and Ento: AAHE-132.

DOI: 10.37722/AAHAE.202052

Abstract
Engineered nanoparticles (ENPs) have seen a drastic increase in their use over the past decade in various consumer products. ENPs will therefore enter terrestrial ecosystems and soils with increasing frequencies, yet research into the effects of ENPs on living organisms and crops is greatly lacking. Currently, there is only one major study reported on the effects of a single ENP, silver quantum dots, on Sorghum bicolor, the 5^th largest crop in the world. We examined the effects of a commonly used metal oxide nanoparticle, titanium dioxide (TiO₂), on the growth and development of sorghum grown in petri dishes (n=25) with agar media and Murashige and Skoog (MS) media with concentrations of 5, 10, 20, and 40 µg/ml. We measured seedling germination rates, gas exchange rates using a LI-6400 Portable Photosynthesis System, and biomass after 14 days of growth in a growth chamber. There was a significant decrease in instantaneous water use efficiency rates due to increasing TiO₂ concentrations, but all other gas exchange variables, germination rates, and biomass accumulation were not significantly affected. While our research indicates no negative effects of this ENP on the growth or development in sorghum, further research will be necessary to identify if TiO₂ ENPs are taken up and translocated by the plant, as well as possible intergenerational effects of ENP exposure. Future research may also be conducted into the possible application of TiO₂ as an antimicrobial agent for use in pesticides or herbicides since there were no negative effects on germination and early development.

Keywords: Engineered Nanomaterials; Growth; Nanoparticles; Seed Germination; Sorghum bicolor; Titanium Dioxide

Introduction
Engineered nanomaterials and nanoparticles (ENMs and ENPs, respectively herein) have become a common material in many products with billions of dollars invested in their development and use worldwide [1-3]. ENMs are small particles that range from 1 to 100 nm. Naturally occurring nanoparticles are common in the environment, originating mainly from volcanic ash, burning fossil fuels and trash, the breakdown of plastics and dust storms [3, 4-6]. On the other hand, ENMs have only recently been produced for a wide range of uses, including many pharmaceutical, biomedical, cosmetic, computer chip, and environmental applications and products [7-10]. It is estimated that in 2004, there were several thousands of tons of ENMs that were released into the environment through sewage sludge, landfills, and biosolid applications for fertilization, and the amounts released are expected to increase to almost half a million tons by 2020 [11]. Over 58,000 tons of metal oxide ENMs were estimated to be produced yearly between 2011 and 2020 [9, 11, 12].

While research into the development and use of ENMs in various sectors has increased greatly, research on the biological impacts of ENMs is lacking [13]. EMN’s can enter the environment through both intentional and unintentional releases, including waste streams from manufacturers and atmospheric emissions, biosolids from waste treatment facilities, pesticide applications to crops, and accidental spillage of consumer products [2]. This allows the ENMs to encounter organisms through multiple routes [9]. The mechanisms behind the uptake of various ENMs by organisms is not completely understood, and risk assessments and toxicological studies of ENMs on living organisms is essential to understand the potential negative effects [14,15].

Due to the small size of ENMs, they generally have unique properties that vary from similar bulk materials with a larger size, which can change their chemical, mechanical, optical, electric, and magnetic properties [4, 9, 13, 16]. While these differences may make ENMs useful for various industrial processes, they can cause harm to the environment and agricultural systems [9, 17].

Most studies on the biological and ecological effects of ENMs focus on aquatic organisms, and there is a need for understanding how they can affect the growth and development of plants, especially in major crop species [9, 11, 18, 19]. Several studies reported higher ENM concentrations in soil than in water or air [15]. Due to most nanoparticles being reactive in the environment, there is a high potential for them to be taken up by various crop plants and transferred through the roots to the shoot and leaves, especially during the juvenile stage of development [6,19]. Depending on the size of the ENM, it is possible for them to be taken up through the roots of the plant and translocated through various tissues, as demonstrated with iron oxide ENP uptake by pumpkins [6].

It is hypothesized that plant growth inhibition due to ENPs may not be solely due to chemical phytotoxicity but may also be due to the physical interactions of the ENPs with plants, such as ENPs blocking apoplastic trafficking in intercellular spaces of the cell wall [6]. For example, in a study with Zea mays, seedlings exposed to TiO₂ ENPs and bentonite had inhibition of leaf growth and transpiration due to a reduction in hydraulic conductivity, as well as a 3.3 nm decrease in the cell wall pores [20].

The application of ENMs in agriculture has been proposed to improve the yield of various plants for food, fuel, and animal feed; however, that research still leaves large gaps in knowledge about the effects of ENMs on most plants [9, 21]. The response of plants to various ENMs can range from increasing yield and growth rate to being toxic and inducing cell death. Carbon nanotubes (CNTs) are an extensively studied nanoparticle, and a variety of responses have been observed ranging from boosting the germination rate of tomato seedlings and increasing the water uptake, to inhibiting the mutigenerational reproductive capacity and biomass of wheat [9,22]. CNT exposure also increased germination rates and shoot length of sorghum seedlings, and increased biomass production, which could be useful for increasing production of sorghum grown for biofuel production [21]. Nanotechnology is also being used in agriculture to increase the efficiency of pesticides, stop fertilizer from leaching into the ground and water, and to increase the growth and development of plants [19].

Information on the impact of TiO₂ ENPs on plants is currently lacking, but current studies indicate they may generate ROS when interacting with living organisms [23]. TiO₂ ENMs may also have antimicrobial properties, one of which could be an impact on the soil microbiome [15, 24]. It is thought that TiO₂ ENPs can act as a photocatalyst and induce redox reactions, as well as promoting seed vigor, chlorophyll formation, and the stimulation of rubisco, which increases photosynthesis and plant growth [15, 19]. Several other studies report that TiO₂ ENPs may stimulate plant growth at lower doses but can prove toxic at higher concentrations [5]. Various studies report that TiO₂ ENPs may have positive impacts on the growth and development of soybeans [25], canola [26], wheat [27], corn [20], and willow [28]. Overall, the acute toxic effects of TiO₂ENS have been found to be low, with most effects not showing a clear dosage-effect relationship [9, 23], although when combined with other metal ENMs (e.g., zinc ENMs, [29]), there can be toxicity.

Sorghum bicolor (L.) Moenech (Referred to as sorghum herein), is the 5^th largest cereal crop in the world, and is grown on over 42.8 million hectares (Ha) worldwide [30]. Sorghum is an annual C₄grass crop plant that was primarily cultivated in arid and semi-arid regions that are drought-prone, which has helped various sorghum cultivars develop the ability to produce high yields under water stressed conditions [31, 32]. Sorghum is a staple crop for over 500 million people worldwide, has a high nutrient content, and is also gluten-free, which can help provide a staple food source for many people with Celiac’s disease [30]. Sorghum is tolerant to a wide range of stresses including drought and nitrogen stress and can still produce a relatively high yield in low nutrient and water conditions, which also makes it a viable plant for biofuel production [21]. In addition to its use as a food source, sorghum has a wide range of uses including brewing, fodder, feed, forage, and diesel biofuel. In the US and Australia, sorghum is mainly grown as feed for cattle and as a biofuel source, and in India and African countries it is mainly grown for human consumption.

Our research aimed to document the dose response effects that TiO₂ ENPs have on Sorghum bicolor seed germination, early growth and physiology. To our knowledge there have been no studies on the effects of TiO₂ ENPs on sorghum, and we hypothesized that growth would only be affected at extremely high concentrations. We addressed two questions. First, do increasing concentrations of TiO₂ affect seed germination rate? We hypothesized that there would be no change in the germination rate due to ENP applications at low concentrations, but there would be at high concentrations. Second, is growth and gas exchange rates of sorghum grown for 14 days in agar affected by increasing TiO₂ concentrations? We hypothesized there would be an increase in the growth of the juvenile plants at low concentrations associated with protection against fungal pathogens, but high concentrations would be toxic.

Materials and Methods
We germinated and grew sorghum in a growth chamber in agar media for 14 days with increasing concentrations of TiO₂. We carried out four separate trials. We applied concentrations of 0, 5, 10, 20, and 40 µg/ml, where the high concentrations mimic Ag-NP concentrations that have been shown to be toxic to root tips of sorghum [18]; therefore, our results can be compared and contrasted to other metal/transition metal ENPs.

TiO₂ nano powder (anatase, 99.5% purity, 15nm diameter, obtained from US Research Nanomaterials, Inc.) was suspended in distilled water at 1 µg/ml; note that the nanoparticles were handled according to training protocols provided by Jordan Valley Innovation Center. Sorghum seeds were provided by the Donald Danforth Plant Science Center.

Sorghum bicolor seeds were sterilized by placing them in a sterilization chamber in a fume hood. In a beaker, 100ml bleach and 3ml Hydrochloric acid (HCl) were mixed under the sterilization chamber, and seeds were kept in the chamber under the fume hood for three hours while exposed to the chlorine gas.

For each trial, media was prepared for 20 petri dishes. In 10 250ml flasks, agar (1.0g) was added into each of 5 flasks, while distilled water was added to the other 5 flasks. 25ml of distilled water was added to flasks corresponding to control petri dishes, and at: 24.38ml, 23.75, 22.50, and 20.00 ml for the flasks corresponding to the petri dishes of nanoparticle concentrations of 5, 10, 20, and 40 µg/ml, respectively. In a separate beaker, 3-Morpholinopropane-1-sulfonic acid (MOPS) buffer (0.63g), and MS salts (2.71g) were dissolved in 375 ml of distilled water. The pH of the solution was adjusted to 7.0 by adding 100 mM KOH and distilled water with a final volume up to 500 ml. The solution (100ml) was added to each of the flasks containing agar. All 10 flasks were autoclaved at 121 °C for 20 minutes. Agar flasks were placed in a warm water bath set at 55 °C to prevent agar from solidifying. After 2 minutes of sonication, nanoparticle suspension was added to the flasks containing water at volumes of 0.13, 0.25, 0.50, and 1.00 ml, to flasks corresponding to 5, 10, 20, and 40 µg/ml, respectively. Flasks with unsterilized nanoparticles were supplemented for two of the replicate trials with a fungicide (250 µl of Amphotericin B) and a bactericide (25 µl of carbenicillin) to prevent bacterial or fungal contamination. Two additional replicate trials did not contain Amphotericin B and carbenicillin to observe if TiO₂ nanoparticles had additional antibacterial properties. The flasks were sonicated, and the flasks containing agar was poured into the flasks containing the mixed nanoparticles with distilled water and were held in the sonicator for 2 minutes to ensure even distribution. After the sonication, the flask composition was distributed evenly across 5 petri dishes for each of the treatments and left to cool at room temperature.

Four seeds were evenly placed onto each of the 20 petri dishes. The petri dishes were sealed with parafilm and placed in a refrigerator. After 3 days, the petri dishes were taken out of the refrigerator and the parafilm removed. The petri dishes were placed into a growth chamber (Conviron Model Adaptis A1000-AR Chamber) at 29 °C, photosynthetically active radiation of 500 µmol m^-2 s^-1, 15-hours light and 9-hours night cycle at 72% humidity. Petri dishes were rotated randomly each day within the growth chamber to avoid potential position effects. Water was added to the medium daily to prevent desiccation. Gemination rates were recorded daily, and gas exchange data were collected after 14 days of growth in the chambers. The experiment was repeated 4 times, with each repeat treated as trial number for statistical analysis.

Gas exchange was measured using a LI-6400 Portable Photosynthesis System (Licor, Lincoln, NE) equipped with a 6 cm² leaf chamber. The measurements were recorded at saturated photosynthetically active radiation (PAR), which was 1000 µmol m^-2 s^-1. Flow rate in chamber was set to 400 µmol s^-1 and fan speed was set at high. Leaves were set in the chamber and area reading was adjusted for each leaf due to varying sizes. Leaf area and root area was measured visually for two of the replicates by aligning seedlings on a 1 cm² grid and taking photos. Further analysis of leaf area was conducted by superimposing additional grid with 25 boxes for each 1 cm² and visually observing how often the shoots and roots cross each box. For chlorophyll content, random leaves were measured non-destructively using a SPAD Chlorophyll Content Meter (Apogee Instruments, model MC-100). Finally, the fresh weight of the shoots and the roots were measured, followed by the dry weight of the roots and shoots after 3 days of drying. The second replicate had a malfunction in the fan of the leaf drying oven which caused the leaves to get burned, therefore, no dry weights were recorded.

Data were analyzed using statistical software Rstudio version 3.5.1. We used ANOVA to examine treatment effects of TiO₂ nanoparticles on the germination rate, dry weight (of roots and shoots), gas exchange rates (photosynthetic rate, transpiration rates, and instantaneous water use efficiency measured at 1000 µmol m^-2 s^-1), percent moisture, root/shoot ratio, and root area of Sorghum bicolor a priori. Gas exchange rate data were analyzed post priori as repeated measures due to data from final trial being discarded. Trial number, treatment applications, and bactericide were treated as fixed effect factors. The interactions between treatment and trial number were also tested. In trial 4, only biomass accumulation data were analyzed. Data are presented as mean ± standard error of mean. Tukey’s test was performed for pairwise comparisons when main treatment effects in the ANOVA were statistically significant at p<0.05. Any analysis relating to dry weight biomass and water content has discarded data from trial two because of burned plants.

Results
Early development of sorghum was not negatively or positively affected by TiO₂ in the growing media (Table 1). There were no significant differences in mean germination between controls and TiO₂ treatments (Table 1). No patterns in response to different concentration were consistent, which would have led us to not consider adding more trials. Treatments of 5 and 10 µg/ml had lower germination rates by 8.1 and 9.7 percent, respectively, and treatments of 20 and 40 µg/ml had higher germination rates by 4.6 and 9.5 percent, respectively, compared to controls.

Response	Control	5 µg/ml	10 µg/ml	20 µg/ml	40 µg/ml
Germination Rate (%)	77.5 ± 3.67	71.25 ± 6.50	70.0 ± 1.77	81.25 ± 9.16	85.63 ± 5.63
Root Length (cm)	7.83 ± 0.77	6.51 ± 1.44	7.39 ± 1.06	6.27 ± 1.08	4.89 ± 0.13
Root Area (cm²)	9.12 ± 0.72	7.33 ± 0.89	7.24 ± 0.92	6.45 ± 0.86	5.39 ± 0.08

Table 1: Seedling germination rate, plant dry weight biomass, root length, and root area of Sorghum bicolor plants grown for 14 days in agar media. The indicated variables for growth in agar media are not significantly different (p < 0.05) between treatments. Values are Mean± SE (n = 100).

Early stages of growth in sorghum was not negatively or positively affected by TiO₂ in the growing media (Figure 1). There were no significant differences in mean total, shoot, or root dry weight between controls and TiO₂ treatments (Figure 1). However, shoot mass was marginally affected (p = 0.08; Figure 1). Patterns in response to treatments were consistent. Treatments of 5, 10, 20, and 40 µg/ml had lower shoot dry weights by 34.1, 27.8, 27.0, and 16.5 percent respectively, compared to controls. Allocation to roots and shoots was not affected; treatments of 5, 10, 20, and 40 µg/ml had lower, but not significantly different, root to shoot dry weight ratios by 10.1, 16.5, 18.9, and 23.3 percent respectively, compared to controls.

Figure 1: Mean dry weight of full plant, shoots, and roots of S. bicolor in controls and different concentrations of TiO₂ nanoparticle treatments after 14 days of growth in agar media. The error bar is the mean of standard error for each treatment (n = 75).

There were no statistical differences in mean root length and root area values between controls and TiO₂ treatments (Table 1). Patterns in response to different concentration levels were consistent. The 40 µg/ml had the shortest root length (37.5 percent lower than control). The 40 µg/ml had the lowest root area (40.9 percent lower than control). Increasing the number of trials was warranted, but we were simultaneously running a greenhouse experiment in soil (see Discussion, no results shown).

Gas exchange in sorghum was not negatively or positively affected by the presence of TiO₂ in the growing media, indicating that growth beyond the harvest date (14 days) would most likely continue to not be negatively or positively affected (Table 2). There were no significant differences in mean photosynthetic rate (A_max) values between controls and TiO₂ treatments (Table 2). Even if the statistical results (p = 0.12; Table 2) had been significant at p<0.05, the reductions in photosynthesis were greatest at the lowest concentrations. Treatments of 5, 10, 20, and 40 µg/ml had lower photosynthetic rates by 23.8, 31.9, 15.5, and 3.7 percent, respectively, compared to controls. No patterns in toxicity indicated that additional trials would reveal any patterns in toxicity.

Response	Control	5 µg/ml	10 µg/ml	20 µg/ml	40 µg/ml
^aA_max(µmol CO₂ m^-2s^-1)	7.53 ± 0.92	5.74 ± 2.07	5.13 ± 2.04	6.36 ± 1.54	7.25 ± 1.60
^bE_max (mol H₂O m^-2s^-1)	7.91 ± 0.91	12.47 ± 3.08	9.46 ± 0.35	10.41 ± 0.58	8.73 ± 0.72
^cA_max/E (µmol CO₂ mol^-1H₂O)	0.96 ± 0.14a	0.56 ± 0.24a,b	0.55 ± 0.24b	0.60 ± 0.13b	0.86 ± 0.23a,b
^aA_max; photosynthetic rate at PAR=1000 µmol m^-2 s^-1, ^bE_max; transpiration rate at PAR=1000 µmol m^-2 s^-1, ^cA_max/E; instantaneous water use efficiency at PAR=1000 µmol m^-2 s^-1. The letters (a/b) indicate significant differences (p < 0.05) between treatments. Values are Mean± SE (n= 75).

Table 2: Photosynthetic rate, transpiration rate, and water use efficiency of sorghum grown for 14 days in agar media at various concentrations of TiO₂ ENPs.

There were no statistically significant differences in mean transpiration rates (E_max) between controls and TiO₂ treatments (Table 2). There was evidence that the concentrations of TiO₂ nanoparticles affected the instantaneous water use efficiency (A_max/E) of S. bicolor grown in agar media (Table 2). Patterns in response to different concentration levels were consistent. Treatments of 10 and 20 µg/ml had significantly lower (p < 0.024) instantaneous water use efficiency rates by 52.7 and 47.4 percent respectively, compared to controls. These data suggest a dose response effect on water use efficiency.

Discussion
There have been several studies on the effects of TiO₂ ENPs on the growth, development, or gas exchange rates of plants [5, 15, 19, 20, 22-29], but to our knowledge, there have been no studies on the effect of TiO₂ ENPs on S. bicolor. The results of the studies listed above range from TiO₂ ENPs having mildly positive effects on plant growth, to neutral effects on plant growth, to negative effects on plant growth, with no seemingly obvious pattern. The negative effects of silver quantum dots on sorghum have been reported [18], although direct comparisons are not possible due to the significantly larger inherent negative qualities silver has on plants compared to titanium. The methods followed for this experiment mirror those of the research with silver [18].

We found that there was a significant difference in the instantaneous water use efficiency (p = 0.024) for treatments of TiO₂ ENPs of 10 and 20 µg/ml, while all other gas exchange data were not significant. This is similar to results found by Asli and Neumann [20], who reported a reduction in hydraulic conductivity and a decrease in cell wall pore size of Zea mays when TiO₂ ENPs were applied. Other reasons for a significant difference in the water use efficiency may include damage to chlorophyll and thylakoid membranes of S. bicolor due to TiO₂ENPs generating excess ROS. Excess generation of ROS can also cause changes in hormonal responses to stress and inflammatory responses, both of which may affect the development of plants [5]. We found no significant effects of TiO₂ applications on the biomass of S. bicolor grown in agar media for 14 days. This is similar to results found in multiple studies [9, 23, 28, 29], where all reported no statistically significant differences in plant growth due to applications of TiO₂ ENPs. Although not found to be significant, visual observations of juvenile sorghum seedlings seemed to indicate concentrations of 20 and 40 µg/ml TiO₂ resulted in an increase in growth and biomass compared to the control and concentrations of 5 and 10 µg/ml TiO₂. In a separate greenhouse study using concentrations of 100, 200, 500, and 1000 mg/kg TiO₂ we found evidence that higher concentrations of TiO₂ nanoparticles marginally increased shoot biomass of S. bicolor grown in soil (p = 0.113) (see Figure 2 [33]) but without a relative increase in flowering biomass.

Figure 2: Mean dry weight of shoots, roots, and tassel head of S. bicolor in controls and different concentrations of TiO₂ nanoparticle treatments after growing to full maturity in soil. The error bar is the mean of standard error for each treatment (n = 36). (Adopted from [33]).

Studies on the sedimentation and aggregation of TiO₂ in soil will be needed to determine its ability to transform in media. Studies on the mechanisms of ENP uptake and translocation by plants are needed to determine if ENPs can be found in leaf and grain tissue, and if trophic transfer is possible [14]. Molecular assays may help determine S. bicolor responses to stress from excess ROS generation and hormonal response changes due to ENPs. Studies on the intergenerational effects TiO₂ ENPs can have on S. bicolor are needed to determine possible long-term exposure effects. However, our results suggest no plant toxicity effects for TiO₂ ENPs.

Acknowledgments: The authors would like to thank Missouri State University Graduate College and Department of Biology for financial support.

References