Study on the germination of nano-TiO 2 synthetic seeds and the physiology of Dendrobium officinale regenerated plants

Synthetic seed of biological technology has been suggested as an effective strategy to improve breeding. Nano-TiO 2 is a photosemiconductor material widely used in life science and agriculture. Nano-TiO 2 has been recently shown to improve photosynthetic function, promote plant growth and development ， but the physiology mechanism is still unclear. The aim of the current study was to confirm whether or not nano-TiO 2 synthetic seed has an effect on germination and physiology of Dendrobium officinale . Using D. officinale protocorm models, we demonstrate that appropriate nano-TiO 2 leads to growth and development of germination seedlings, and improves the root activity, nitrate reductase activity and antioxidant system activity. Our data also provide a basis for the improvement of the preparation process and practical production of D. officinale synthetic seeds.


Introduction
Synthetic seed is a kind of small granule which can develop into a complete plant under suitable conditions by using the totipotency of cells to wrap the somatic embryo or meristem which can develop into a complete plant in vitro culture into an outer membrane which contains nutrients and has protective function. As a new biological technology, synthetic seed has made great progress after more than 20 years of research, and it has been reported that it can germinate into seedlings or be used in field and greenhouse production. Alginate encapsulated somatic embryos have many kinds of advantages over organogenesis for propagation, including long-term storage, higher scale-up potential and low cost of production [1]. In recent years, the research on synthetic seeds has shifted from the early model plants (such as alfalfa [2] and Sandalwood [3]) to numerous crops (such as conifers [4] and pistachio [5]), aquatic macrophytes [6] and commercial plants (such as Paulownia elongata [7]), but only very few in Orchidaceae species.
The complete synthetic seed includes three basic parts: synthetic embryo, synthetic endosperm and synthetic seed coat. At present, the synthetic seed embryo has developed from embryoid to some embryo analogues, such as adventitious buds and protocorms [8]. synthetic endosperm provides nutrients and growth regulators for the metabolism and development of synthetic embryo. In addition, active carbon, natural organic matter, fungicide, herbicide and other substances can be added to the synthetic endosperm. The synthetic seed coat, as the natural seed coat, is a protective membrane wrapped outside the synthetic embryo and endosperm. It not only prevents the loss of water and nutrition in the seed, but also ensures ventilation and prevents the pressure brought by external mechanical shock. Alginate, gelatin, sodium pectinate, agar, Gel-rite-TM, chitosan and polyethylene glycol are suitable for synthetic seed coat, and sodium alginate is the most widely studied and applied in synthetic seed coat [7].
At present, there are few reports about the synthetic seeds of D. officinale. Sodium alginate embedding system is convenient and has high germination rate, but its water retention is poor, it is easy to air dry quickly in the air and contaminate miscellaneous bacteria, and the growth of germinating seedlings is not consistent, there will be secondary seedlings or secondary protocorms, and some will even be deformed, not rod, rootless or root thick. Therefore, it is necessary to optimize the preparation conditions of D. officinale synthetic seeds, so as to lay the foundation for its practical use and large-scale production.
There are multiple nano materials, each with different functions and applications, among which nano-TiO2 is widely used in life science and agriculture [9][10]. At present, few reports show that the photobiological effects are induced by photosemiconductor material nano-TiO2 in living plants, such as improving photosynthetic function [11], promoting plant growth and development [12][13]. The present route enables the utilization of nanoparticles synthesized under any conditions as the starting seeds for nanomaterial growth inside protein nanocages [14]. In addition, nano-TiO2 has antibacterial and antiviral properties [15][16]. Therefore, nano-TiO2 has a certain hormone effect [17], which can promote the buds regeneration of leaves and the rooting of hypocotyls in tissue culture, and has antibacterial and slow-release properties. In the past, MS + 0.5mg/L 6-BA + 0.5mg/L NAA + 3g/L activated carbon + 30g/L sodium alginate + 3g/L chlorothalonil were used as the basic endosperm in our research group. It was found that 10g/L nano-TiO2 and 10g/L nano-SiO2 in the seed coat could significantly promote the germination and seedling formation of synthetic seeds, while the 90 day seedling formation rate of 10g/L nano-SiO2 was the highest, reaching 68.1%. It can be seen that the application of nano-TiO2 in the production of D. officinale synthetic seeds may promote the air permeability, antibacterial, germination and growth of the synthetic seeds, which is expected to improve the existing problems in the application of synthetic seeds.
The study reported here was carried out to rapid propagation of D. officinale plants via directly protocorms from internodal explants without involving a callus phase. We also demonstrate a method for developing artificial seed that can grow to seedling under in vitro conditions. Moreover, germination of nano-TiO2 synthetic seeds and the physiology of regenerated plants was investigated. These results on artificial seed production can provide a practical means of mass clonal propagation for this precious orchid. In addition, nano-TiO2 encapsulation of D. officinale protocorms will be useful for limited breeding methods that lack the appropriate cultivation for conventional sterile micropropagation.

Preparation of nano synthetic seeds
The calcium alginate bulb was used to immerse the protocorm in the synthetic endosperm in the super clean platform. After 5min, the semi gel state of sodium alginate packaging protocorm was dripped into the 2% CaCl2 solution by suction tube to ensure that each protocorm was wrapped only with a synthetic seed. After 15min ion exchange, the seeds were solidified into granules by self action, washed out with distilled water, and put on the filter paper to absorb the surface moisture. Taking MS + 2.0mg/L 6-BA + 0.5mg/L NAA as the basic synthetic endosperm, 3.0% sodium alginate, 3.0% cassava starch, 1.0% water retaining agent, (0%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%) nano-TiO2 (Hangzhou Wanjing new materials Co. Ltd) as the synthetic seed coat matrix, the material was mixed with it and exchanged with 100mmol/L CaCl2 for 15min.
Using MS + 2.0mg/L 6-BA + 0.5mg/L NAA + 0.1% activated carbon + 2.0% -5.0% sodium alginate + 2.0%-3.0% cassava starch as the basic synthetic endosperm, adding (0, 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5, 25.0) g/L nano TiO2 as the synthetic seed coat matrix. The protocorm was dipped into the synthetic endosperm containing nano-TiO2 in the super clean platform by the drop method. After 5min, synthetic endosperm of the semi gel state containing the protocorms was dripped into 2.0%CaCl2 solution with straw to ensure that each protocorm was wrapped only with a synthetic seed and was automatically solidified by 15min ion exchange. Took it out and washed it with sterilized redistilled water, put it on the filter paper to absorb the surface water. The nano synthetic seeds of D. officinale were obtained.

Germination of nano synthetic seeds
The seeds were seeded in 1/2MS medium. The germination rate of 30 days and the seedling rate of 60 days were observed. The germination standard was to break through the seed coat by 2 mm, and the seedling standard was that the whole seedlings could completely break through the seed coat and survive on the medium.
Each group was treated with 30 synthetic seeds and repeated 3 times. The formula is as follows: Germination rate = germination number / number of seeds Seedling rate = number of seedlings / number of seeds After 6 months of germination of D. officinale synthetic seeds, 10 plants were randomly selected for each treatment, and the plant height, stem node number, internode length, leaf number, leaf length and maximum leaf width were measured.
The leaf length was the distance from leaf base to leaf tip, and the maximum leaf width was the measured value of shoulder width at the bottom of leaf. The measurement of leaf area is obtained by multiplying the product of leaf length and maximum leaf width by a coefficient of 0.77. The coefficient comes from the correlation equation between the actual leaf area and the product of leaf length and maximum leaf width, and its correlation coefficient is r 2 = 0.98.

Determination of root activity of germinating seedlings
Take out the root, absorb the water and grind it with 3-4mL ethyl acetate and a small amount of quartz sand to get TTF. Transfer the red extraction solution into the test tube, wash the residue with a small amount of ethyl acetate for 2-3 times, transfer them all into the test tube, add ethyl acetate to make the total amount of 10ml, compare the color with spectrophotometer at 485nm wavelength, read out the optical density with blank as reference, check the standard curve, and calculate the reduction amount of tetrazolium.
The reduction strength of tetrazolium was calculated by taking the data into the following formula.
Reduction strength of tetrazolium = Reduction amount of tetrazolium( μ g) / Determination of nitrate reductase activity of germinating seedlings Take 1.0mL of the reaction solution and put it into the centrifuge tube, add 2.0mL 1.0% sulfanilic acid solution and 2.0mL 0.2% a-naphthylamine solution, and put it in a water bath at constant 30℃ for 30min. At last, the absorbance value was measured at 520nm, the blank was used as reference, the standard curve was checked, and the NO2concentration was calculated.
Take the data into the formula below to calculate the activity of nitrate reductase.
Definition: the number of micrograms of NO2generated by catalysis per hour per gram of fresh weight material is 1U.

Determination of total superoxide dismutase (T-SOD) of germinating seedlings
The T-SOD test box provided by Nanjing Jiancheng Bioengineering Research Institute was used for determination. After all the reagents were evenly mixed, they were left at room temperature for 10min, and then the absorbance was measured at 550nm wavelength with 1.0cm optical diameter cuvette and 0-redistilled water.
Bring the data into the formula below to find out the total SOD activity.
Total SOD activity (U/g FW) = (ODcontrol-ODdetermination) × Dilution ratio of reaction system / [ODcontrol×50%×Homogenate concentration(g/ml)] Definition: the amount of SOD corresponding to the inhibition rate of 50% of SOD per gram of tissue in 1.0ml of reaction solution is a unit of SOD activity (U).

Determination of peroxidase (POD) of germinating seedlings
The POD test box provided by Nanjing Jiancheng Bioengineering Research Institute was used for determination. After all the reagents were evenly mixed, they were centrifuged at 3500 rpm for 10min, the supernatant was taken, and the absorbance was measured at 420nm wavelength with 1cm optical diameter cuvette and 0-redistilled water.
Bring the data into the formula below to calculate the POD activity.
POD activity(U/g FW) = (ODdetermination-ODcontrol) × Total volume of reaction Definition: at 37 ℃ , the amount of enzyme catalyzing 1.0 μ g substrate per gram of tissue per minute is one enzyme activity unit.

Determination of catalase (CAT) activity of germinating seedlings
The CAT test box provided by Nanjing Jiancheng Bioengineering Research Institute was used for the determination. After all the reagents were well mixed, the absorbance was measured at 405nm wavelength with 0.5cm optical diameter cuvette and 0-redistilled water.
Bring the data into the formula below to calculate the CAT activity.

Determination of total antioxidant capacity (T-AOC) of germinating seedlings
The T-AOC test box provided by Nanjing Jiancheng Bioengineering Research Institute was used for the determination. After all the reagents were evenly mixed, they were placed for 10 minutes. The absorbance was measured at 520nm wavelength with 1cm optical diameter cuvette and 0-redistilled water.
Take the data into the formula below to calculate the T-AOC activity.
T-AOC activity(U/g FW) = (ODdetermination-ODcontrol) × Total amount of reaction liquid(mL) / [0.01×30×Sampling quantity(mL)×Homogenate concentration(g/ml)] Definition: at 37 ℃ , the absorbance (OD) value of the reaction system is one unit of total antioxidant capacity per gram of tissue per minute, with each increase of 0.01.

Nano TiO2 particles
Through X-ray diffraction analysis, the average grain size of TiO2 particles in the nano-TiO2 sol was 2.6nm (see figure 1 and table 1); through particle size analyzer analysis, the average effective grain size of TiO2 particles was 0.27 μ m, which showed that the nano-TiO2 sol would agglomerate, because the nano materials were easy to agglomerate (see figure 2); scanning electron microscopy showed that the size of TiO2 particles had reached the nano level, and its distribution in the sol was uniform (see figure 3).

No significant germination rate and seedling rate rising first
The effects of different concentrations of nano-TiO2 on the germination of synthetic seeds are shown in table 2 and figure 4-5. The results showed that the addition of 0-5.0% nano-TiO2 had no significant effect on the germination rate of synthetic seeds of D. officinale, while the addition of 1.0% nano-TiO2 could significantly improve the seedling rate of synthetic seeds, up to (37.78±1.11)%, but with the increase of nano-TiO2 concentration, the seedling rate gradually decreased, which was significantly lower than that of the control group. significantly improve the plant height and the number of stem nodes, and 5.0% was the best. In addition, it was found that when the concentration of nano-TiO2 was 5.0%, the length of internode, the number of leaves and the area of leaves could be significantly increased.

Root activity rising first and then falling
It can be seen from figure 8 that the root activity of the nano-TiO2 treatment group increases first and then decreases. When the concentration of nano-TiO2 is 15.0g/L, the root activity reaches the maximum value, which is 1.92 times of the control group. Then with the increase of TiO2 concentration, the root activity decreased.

Increasing activity of nitrate reductase
It can be seen from figure 9 that the activity of nitrate reductase in the nano-TiO2 treatment group shows a gradual upward trend, which is higher than that in the control group. When the concentration of nano-TiO2 is 22.5 g/L, the activity of nitrate reductase reaches the maximum value, which is 1.15 times of that in the control group, with a significant difference compared with the control group.

Total SOD activity rising first and then falling
From figure 10, it can be seen that the total SOD activity of the nano-TiO2 treatment group first increases and then decreases. When the concentration of nano-TiO2 is 17.5g/L, the total SOD activity reaches the maximum value, which is 1.36 times of the control group. Then, when the concentration of TiO2 was more than 17.5g/L, the total SOD activity decreased gradually. In general, the total SOD activity of nano-TiO2 treatment group was significantly higher than that of the control group.

Increasing activity of POD
It can be seen from figure 11 that the POD activity of the nano-TiO2 treatment group shows a gradual upward trend, which is larger than that of the control group.
When the concentration of nano-TiO2 is 22.5g/L, the POD activity reaches the maximum value, which is 1.17 times of the control group. There is a significant difference compared with the control group. In general, the POD activity of the nano-TiO2 treatment group is higher than that of the control group. When the concentration of nano-TiO2 is more than 10.0g/L, there are significant difference compared with the control group.

CAT activity rising first and then falling
From figure 12, it can be seen that the CAT activity of the nano TiO2 treatment group shows a gradual upward trend, which is higher than that of the control group.
When the concentrations of nano TiO2 are 10g/L and 12.5g/L, the CAT activity reaches the maximum value, which is 5.66 and 5.78 times of that of the control group, respectively. There are significant differences compared with the control group.
However, when the nano-TiO2 is more than 12.5g/L, the CAT activity decreases gradually. In general, the CAT activity of nano-TiO2 treatment group is higher than that of the control group. When the concentration of nano-TiO2 is more than 2.5g/L, there are significant differences compared with the control group.

Increasing activity of T-AOC
It can be seen from figure 13 that the T-AOC activity of the nano-TiO2 treatment group shows a gradual upward trend, and the T-AOC activity is higher than that of the control group (except for the concentration of 2.5g/L, which may be a problem of error). When the concentration of nano-TiO2 is 22.5g/L, the T-AOC activity reaches the maximum value, which is 2.74 times of the control group, with a significant difference compared with the control group.

Statistical analysis
The experimental data were expressed by mean ± SD. The statistical analysis method was IBM SPSS statistics v19.0 and one way ANOVA (one way analysis of variance). The significance of the differences between groups was tested by Duncan test.

Discussion
Through the analysis of X-ray, particle size analyzer and scanning electron microscope, it can be seen that the size of TiO2 particles in the sol has reached nanometer level, the average grain size is 2.6nm, and it is evenly distributed in the sol, but agglomeration phenomenon appears, which may be that nano materials are prone to agglomeration. The results showed that the addition of 0% -5.0% nano-TiO2 had no significant effect on the germination rate of D. officinale aritificial seeds, but the addition of 1.0% nano-TiO2 could significantly improve the rate of seedling, which may be due to the application of its energy conversion performance, the increase of seed activity, and the increase of enzyme activity in the seeds of D. officinale, so as to promote the seedling of D. officinale seeds. However, with the increase of TiO2 concentration, the germination of D. officinale may be inhibited. The bacteria-existing germination of synthetic seeds is the key to their application. In this experiment, some researches were carried out (sowing on outdoor agar gel system), but synthetic seeds withered with time and failed to germinate, so this needs further study.
The results showed that the plant height, stem node number, internode length, leaf number and leaf area of D. officinale germination seedlings were comprehensively investigated, 5.0% nano-TiO2 had a significant effect on the growth of the germination seedlings of D. officinale. However, with the increase of TiO2 concentration, the number of stem nodes, the length of internode, the number of leaves, the area of leaves and so on were inhibited, and then the growth of D. officinale germination seedlings was inhibited.
Root activity index is an important target to reflect root growth and activity level.
It was reported that the length of Lemna minor roots decreased by 99% when exposure to nano TiO2 [18]. In this study, with the increase of nano-TiO2, the root activity of D. officinale germination seedlings showed an upward trend. When the concentration of nano-TiO2 was 15.0g/L, the root activity reached the maximum.
Nano-TiO2 semiconductor sol can promote the ability of plants to absorb water, which may be related to the increase of stomatal conductance and transpiration rate of leaf cells.
Nitrate reductase is the rate-limiting enzyme in the process of nitrate assimilation in plants, which plays an important role in nitrogen metabolism. The activity of nitrate reductase in plants directly affects the utilization of inorganic nitrogen in soil, thus affecting the quality and yield of plants [19][20]. The results showed that the addition of nano-TiO2 could promote the increase of nitrate and nitrite content in the cells of D. officinale germination seedlings, and the NO content of 22.5g/L nano-TiO2 group is the highest. When the concentration of nano-TiO2 was more than 22.5g/L, the activity of nitrate reductase decreased gradually.
SOD enzyme is an enzyme containing metal cofactors, which is the first to react with O 2-, and plays an important role in the process of disproportionation of O 2to generate H2O2 and O2 [21]. Therefore, the induction of SOD activity indicates that a large amount of O 2is produced in the plant, which makes the plant in an oxidative stress state. When nano materials contact with organisms, cells will generate a lot of reactive oxygen [22]. In this study, it was found that at the concentration of 17.5g/L, POD enzyme can remove the H2O2 produced in plants, reduce the concentration of H2O2 in plants, prevent membrane lipid peroxidation, and protect the body from the damage of active oxygen substances. Therefore, the induction of POD enzyme activity indicates that a large amount of H2O2 is produced in the plant, which makes the plant in an oxidative stress state. In this study, it was found that with the increase of the concentration of nano-TiO2, the activity of POD enzyme increased significantly, and the activity of POD enzyme in the treatment group was higher than that in the control group, which indicated that the activity of POD enzyme in the germination seedlings of D. officinale treated with nano-TiO2 could better resist oxidative damage.
This result is consistent with the Tian's study [25].
CAT enzyme can remove H2O2 produced in plants and prevent membrane lipid peroxidation. Therefore, the induction of CAT enzyme activity indicates that a large amount of H2O2 is produced in plants, which makes plants in an oxidation stress state.
In this study, the activity of CAT enzyme in the treatment group of nano-TiO2 firstly increased and then decreased, which showed that when the concentration of nano-TiO2 was more than 2.5g/L and less than 15.0g/L, the content of H2O2 in the germination seedling of D. officinale increased gradually, which promoted the mass synthesis of CAT enzyme, eliminated the excessive H2O2 and prevented the oxidative damage; while with the concentration of nano-TiO2 more than 12.5g/L, the activity of CAT enzyme in the germination seedling of D. officinale is decreasing. In general, the CAT activity of D. officinale germination seedling treated with nano-TiO2 was higher than that without nano-TiO2. With the increase of the amount of nano-TiO2, Tian et al found that the CAT enzyme activity showed an overall upward trend, which was the difference of this research results due to different plant species and nano-TiO2 phase types [25].
T-AOC is an index to measure the total antioxidant capacity of the body [26]. Its level represents the comprehensive level of antioxidant capacity of body fluid, cell, tissue enzyme system and tissue non-enzyme system [27]. The results showed that the T-AOC activity of germination seedlings of D. officinale was significantly higher than that of the group without nano-TiO2 when nano-TiO2 was more than 2.5g/L, indicating that the antioxidant function of germination seedlings treated with nano-TiO2 was increased, and increased with the increase of nano-TiO2.
To sum up, adding appropriate nano-TiO2 into the synthetic endosperm is beneficial to the growth and development of germination seedlings, and improves the root activity, nitrate reductase activity and antioxidant system activity, which provides a basis for the improvement of the preparation process and the practical production of D. officinale synthetic seeds.