The MM106 rootstock is a semi-shortening apple rootstock that is widely accepted due to its desirable characteristics. Propagation of this rootstock through tissue culture and optimization of the culture medium can increase the micropropagation efficiency of this plant. Therefore, in order to investigate the effect of iron source on multiplication, a factorial experiment was conducted in a completely randomized design. The factors studied included different concentrations of citrate-coated iron oxide nanoparticles (0, 25, 50 and 75 mg/L) along with the iron concentration of the culture medium (without iron, one-quarter, one-half and complete iron MS or control). Single nodes (as explant) of Malling Merton rootstocks were cultured on these media and growth indices such as fresh weight, dry weight, number of leaves, shoot length and root length were investigated. The results of analysis of variance showed that the growth traits of seedlings and leaf iron content were significantly affected by the interaction effect of the iron source of the MS culture medium with iron nanoparticles. The use of high concentrations of iron nanoparticles (75 mg/L) and complete MS iron source improved the growth and physiological parameters of seedlings. However, rooting and shoot growth were observed at low concentrations of iron nanoparticles (25 mg/L) and low iron concentrations in the culture medium. In general, a concentration of 75 mg/L of iron nanoparticles and complete or half MS iron source can be introduced to improve both growth indices and iron uptake for this rootstock.
EXTENDED ABSTRACT


Introduction
MM106 rootstock is a semi-shortening apple rootstock which is currently being used due to its characteristics of shortening, early fruiting, low suckering, resistance to diseases and wax aphids (Mohseni Azar et al., 2009; Mahdavian et al., 2010). Nanoparticles (NPs), due to their unique physicochemical properties, can be used as a stimulus in plant tissue culture (Jadczak et al., 2019). Fe3O4 iron nanoparticles show outstanding properties such as chemical stability, electromagnetic behavior, anti-pollution properties and low toxicity. It also opens water channels in root cells, which leads to greater absorption of water and minerals (Ren et al., 2011). Considering that iron nanoparticles have been used in in vitro culture of plants and their positive effects have been proven, and also the in vitro technique is of high economic importance in increasing of the propagation efficiency of the MM106 rootstock.
Materials and methods
Single-node explants from in vitro seedlings of apple cultivar MM106 were used. Iron oxide nanoparticles were prepared by co-precipitation with 2- and 3-valent iron chloride (with a molar ratio of 2:1) in an alkaline solution. (Lyon et al., 2004). Explants were cultured on MS culture medium containing different concentrations of citrate-coated iron oxide nanoparticles (0, 25, 50, and 75 mg/L) along with different concentrations of medium iron (FeSO4 + Na2 EDTA) (0, 1.2, 1.4, MS iron concentration, and control). The cultures were placed in a growth chamber at 25±2°C and a photoperiod 16/8 light and darkness.
Results and discussion
The results of the analysis of variance showed that the type of iron composition in the culture medium had a significant effect on the vegetative traits of plantlets obtained from the growth of explants in MS medium, and the interaction effect of different concentrations of MS iron source and iron nanoparticles was significant. At 25, 50 and 75 mg/L of iron nanoparticles and in complete MS iron, the root length reached a maximum. The highest rooting percentage was observed at a 25 mg/L of iron nanoparticles in combination with different concentrations of iron source. With increasing iron nanoparticles concentration and iron content in the culture medium, the number of leaves increased, such that the highest number of leaves was observed at a 75 mg/L of iron nanoparticles and complete iron. The maximum leaf area was observed at 75% of MS iron and in the absence of iron nanoparticles. The results showed that the longest and shortest shoot lengths were associated with 75 mg/L of iron nanoparticles and 25% MS iron as well as with iron or iron nanoparticles free medium, respectively. complete iron and 50% MS iron along with 50 and 75 mg/L of iron nanoparticles are probably the optimal concentrations for increasing total iron.
Conclusion:
In general, the effects of citrate-coated iron oxide nanoparticles on the characteristics of MM106 seedlings were investigated and the results showed that the use of nanoparticles at high concentrations and complete MS iron source improved the growth and physiological parameters in MM106 seedlings. The concentration of 75 mg/L of iron nanoparticles and complete MS iron source can be introduced to improve both growth and biochemical indices with value for this base. The best treatment to increase the iron content of MM106 seedlings is complete MS iron source without iron nanoparticles, and in the absence of complete MS iron, the use of iron nanoparticles at a concentration of 75 mg/L or higher concentrations is effective. This study is not only important in terms of the beneficial use of nanoparticles in agricultural sciences, but also provides new insights into the understanding of the mechanism between nanomaterials and plants regarding the increase in growth and biochemical parameters.
EXTENDED ABSTRACT


Introduction
MM106 rootstock is a semi-shortening apple rootstock which is currently being used due to its characteristics of shortening, early fruiting, low suckering, resistance to diseases and wax aphids (Mohseni Azar et al., 2009; Mahdavian et al., 2010). Nanoparticles (NPs), due to their unique physicochemical properties, can be used as a stimulus in plant tissue culture (Jadczak et al., 2019). Fe3O4 iron nanoparticles show outstanding properties such as chemical stability, electromagnetic behavior, anti-pollution properties and low toxicity. It also opens water channels in root cells, which leads to greater absorption of water and minerals (Ren et al., 2011). Considering that iron nanoparticles have been used in in vitro culture of plants and their positive effects have been proven, and also the in vitro technique is of high economic importance in increasing of the propagation efficiency of the MM106 rootstock.
Materials and methods
Single-node explants from in vitro seedlings of apple cultivar MM106 were used. Iron oxide nanoparticles were prepared by co-precipitation with 2- and 3-valent iron chloride (with a molar ratio of 2:1) in an alkaline solution. (Lyon et al., 2004). Explants were cultured on MS culture medium containing different concentrations of citrate-coated iron oxide nanoparticles (0, 25, 50, and 75 mg/L) along with different concentrations of medium iron (FeSO4 + Na2 EDTA) (0, 1.2, 1.4, MS iron concentration, and control). The cultures were placed in a growth chamber at 25±2°C and a photoperiod 16/8 light and darkness.
Results and discussion
The results of the analysis of variance showed that the type of iron composition in the culture medium had a significant effect on the vegetative traits of plantlets obtained from the growth of explants in MS medium, and the interaction effect of different concentrations of MS iron source and iron nanoparticles was significant. At 25, 50 and 75 mg/L of iron nanoparticles and in complete MS iron, the root length reached a maximum. The highest rooting percentage was observed at a 25 mg/L of iron nanoparticles in combination with different concentrations of iron source. With increasing iron nanoparticles concentration and iron content in the culture medium, the number of leaves increased, such that the highest number of leaves was observed at a 75 mg/L of iron nanoparticles and complete iron. The maximum leaf area was observed at 75% of MS iron and in the absence of iron nanoparticles. The results showed that the longest and shortest shoot lengths were associated with 75 mg/L of iron nanoparticles and 25% MS iron as well as with iron or iron nanoparticles free medium, respectively. complete iron and 50% MS iron along with 50 and 75 mg/L of iron nanoparticles are probably the optimal concentrations for increasing total iron.
Conclusion:
In general, the effects of citrate-coated iron oxide nanoparticles on the characteristics of MM106 seedlings were investigated and the results showed that the use of nanoparticles at high concentrations and complete MS iron source improved the growth and physiological parameters in MM106 seedlings. The concentration of 75 mg/L of iron nanoparticles and complete MS iron source can be introduced to improve both growth and biochemical indices with value for this base. The best treatment to increase the iron content of MM106 seedlings is complete MS iron source without iron nanoparticles, and in the absence of complete MS iron, the use of iron nanoparticles at a concentration of 75 mg/L or higher concentrations is effective. This study is not only important in terms of the beneficial use of nanoparticles in agricultural sciences, but also provides new insights into the understanding of the mechanism between nanomaterials and plants regarding the increase in growth and biochemical parameters.