عنوان مقاله [English]
Micropropagation plays an important role in the production of vigorous and healthy plants in apple tree. Carbon nanoparticles have a significant effect on increasing the growth and germination of different plant species under in vitro condition. In this study, the effect of different kinds of nanoparticles on explant proliferation of two apple genotypes (red fleshed and Malling Merton 106) was investigated. The treatments were multi walled of carbon nanotubes and graphene oxide in 3 concentrations of 0, 50 and 100 mg L-1. In order to investigate the entrance and accumulation of carbon nanoparticles in the plant, a fluorescence microscope with a light spectrum of 530 nm was used. Carbon nanotubes and graphene oxide showed a positive effect on explant proliferation by increasing leaf production and stem length compared to the control. The both treatments improved leaf growth by decreasing plastochron. In this study, the penetration of nanoparticles into leaf tissue was observed. The effect of graphene oxide was most than carbon nanotubes.
The increasing of the world population and the reduction of food resources is the important reason to formation and use beneficial methods for plant production. Various methods are used to propagate apples, such as cuttings and bud grafting. These methods do not ensure the production of healthy and disease-free plants, they are seasonal and require a lot of manpower, time and space consuming. Micropropagation is another important method for propagation, especially in plants such as apples, which are difficult to propagate by traditional methods. In the micropropagation process a large number of plants can be obtained in a small space and at a appropriate time. Micropropagation has been used for commercial production of many rootstocks and grafted cultivars of fruit trees such as cherries, grapes and apples (Sun et al., 2014). In apples, the micropropagation of some cultivars is inefficient. However, the use of in vitro methods will be the only option to the production of transgenic plants, preserve the germplasm and secondary metabolites production (Shi et al., 2017).
Red fleshed apple, which is a valuable genotype of the apples with high anthocyanin content, is under danger of extinction. On the other hand, preliminary studies performed on the red fleshed genotype in the tissue culture laboratory of the Horticulture Department of Tabriz University showed that the reproduction of this genotype had a very slow rate under in vitro condition. Considering that the red color of apple flesh is a valuable feature from the perspective of horticulture and consumer market needs (Zhang et al., 2020). Due to the ability of red fleshed apple to production and introduction of a new cultivar, application of the new methods to improve in vitro proliferation of this genotype is necessary. In this regard, innovation and the use of compounds that increase tissue culture efficiency will be very useful.
The results of researchers have shown that carbon nanotubes have a significant effect on increasing the growth and germination of in vitro plants (Patel et al., 2019; Rao and Srivastava, 2014). Materials with a particle size of less than 100 nm in at least one dimension are generally classified as Nano (Neme et al., 2021). Multilayer carbon nanotubes (MWCNTs) are able to penetrate to cell walls and membranes and enter the cytosol of plant cells (Zhai et al., 2015).
Graphene oxide plates are single-layer carbon atoms. The solubility of graphene oxide in solvents, especially water, is very important in bioengineering applications. The maximum solubility of graphene oxide in a solvent depends on the polarity of the solvent and the amount of surface activity during oxidation. Studies have shown that graphene oxide materials, such as graphene oxide films, are highly environmentally friendly materials that allow human and mammalian cells to reproduce effectively without toxicity (He et al., 2021). In this study, fluorescence was used to accurately track nanoparticles in leaves to investigate the penetration of nanoparticles into plants tissues. Also, the effect of nanoparticles on the proliferation of treated plants was compared with control plants.
Apple plant samples were obtained from Khalatpooshan Agricultural Research Center of Tabriz University. After sterilization, the shoot samples were placed in MS culture medium. In order to eliminate fungal or bacterial contamination, the samples were sterile as follows: 10 minutes dipping in running water, 20 minutes washing with 2% sodium hypochlorite, 3 minutes cleaning with 70% ethanol, washing with sterile distilled water under a laminar hood.
The samples were cultured in MS medium containing gibberellin (1 mg L-1) and benzyl aminopurine (1.5 mg L-1). The pH of the culture medium was adjusted to 5.7. Nanoparticles (multi walled of carbon nanotubes and graphene oxide) were first added to distilled water at a certain concentration in order to be added to the culture medium. Ultrasonic device was used for 30 minutes to completely disperse of the nanoparticles. The solutions at the specified amount (0, 50 and 100 mg L-1) were added to the sterilized culture medium under the sterile condition.
In order to synthesize of carbon nanotubes, first increased the number of carboxylic acid groups on carbon nanotubes using the oxidation reaction and then were placed on carbon nanotubes using chemical amides and fluorescein acid coupling reagents (Sager et al., 2014).
In order to synthesize of graphene oxide, graphene in the presence of nitric acid and sulfuric acid are oxidized by diffusion of graphene layers using ultrasonic waves and singly the graphene oxide plates were dispersed in the solution and activated using fluorescein (Eftekhary et al., 2016).
Samples were cultured in the supplemented MS medium containing three different concentrations of carbon nanoparticles (0, 50, 100 mg L-1) with three replications and three samples per each replicate.
All cultures were sub-cultured for 1 month and every 7 days. Shoot length, leaf number and amount of Plastochron per replication were assayed in the mentioned time periods. Plastochron is the interval between the emergences of two leaf primers. In order to observe the texture of the samples treated with carbon particles to detect the locations and penetration of these materials, after preparing the samples, a fluorescence microscope was used.
By oxidation of the nanomaterial surface using nitric acid solution, CNT-CO2H functionalized nanomaterials were produced. IR-FT spectroscopy was performed to study the presence of active groups during chemical changes of nanomaterials. Plants that treated with nanoparticles were studied by fluorescence microscope. According to the obtained results, nanoparticles have penetrated into the plants leave tissues and have accumulated mostly in vascular tissues and stomata. The presence of graphene oxide inside the leaf tissue is clearly seen in red fleshy apples and Malling Merton apples with purple color. Carbon nanotubes that have penetrated into the leave tissue of both plants are seen as black particles. The positive effects of nanoparticles on the proliferation indices were obvious in this experiment including leaf number, internode length and Plastochron.