عنوان مقاله [English]
Apple is one of the most important trees in temperate and cold regions, which has attracted many consumers due to its pleasant flavor and high economic value. Traditional apple micropropagation by methods such as cuttings and seed cultivation have disadvantages such as dispersal of traits, hard rooting, spending more time growing, over-consumption of chemical fertilizers, and contamination with a variety of pests and diseases. Glass has partially solved these problems and has created breeding plants, free from diseases and pests and high and strong growth rate. The apple tree (Malus x domestica L.) is one of the most important seed plants of the Rosaceae family in cold and temperate regions of the world. A number of apple varieties are used as a base, one of the most common of which is the MM106 rootstock due to its good compatibility with different apple cultivars. The study of apple characteristics in in vitro conditions has been considered by many researchers due to the precise control of environmental conditions. The use of cytokinins is generally accepted as one of the essential strategies for branch proliferation in plant tissue culture.
The plant materials used in this study were prepared from the young and growing branches of three-year-old apple trees MM106 in Tabriz, Khalatpooshan Research Center of Tabriz University. The prepared branches were transferred to the laboratory with a length of 15 to 20 cm and were divided into small pieces of 1 to 1.5 cm, in which there was at least one bud in each cut piece. After disinfection, the specimens were branched under the hood with a binocular loop. Murashig Skog (MS) basal culture medium was used in this study. Explants are the most important source of infection in plant tissue culture. The disinfection step is performed to remove most bacterial and fungal infections. After splitting into smaller pieces, the young shoots were washed with running water for 5 minutes and then disinfected with 2% sodium hypochlorite for 20 minutes and 70% ethanol for 5 minutes and then sterilized 3 times with distilled water. Laminar hoods were washed. After disinfection under the hood and using binocular loops, the explants were cultured at the tip of the branch and the buds were transferred to the culture medium containing MS and growth regulators after scaling. The number of shoots per experimental unit indicating fertility was obtained by counting emerging lateral shoots.
Bradfor method was used to determine the concentration of total soluble proteins. In this method, first 0.5 g of each leaf sample was crushed and crushed by liquid nitrogen in a Chinese mortar and then 50 mg of polyvinyl pyrrolidone (PVP) was added to each sample. Then 1.5 ml of potassium phosphate buffer (pH = 7) containing sodium metabisulfide (0.019 g per 100 ml of buffer) was added and the contents of the mortar were transferred to 2 ml microtubes and placed in a refrigerated centrifuge at 15000 rpm. Minutes at 4 ° C for 20 minutes. The samples were then slowly removed from the refrigerator and 500 μl of the top phase of the extract was mixed with 175 μl of 50% glycerol and the resulting solution was transferred to 2000 μl microtubes and stored in a freezer at -80 ° C. To read the protein adsorption reading, 20 μl of the extract was mixed with 500 μl of Bradford solution and 2 minutes after adding the extract to the Bradford solution were read using a spectrophotometer based on spectroscopy at 595 nm. Factorial experiment was performed based on a completely randomized design. The number of roots per experimental unit, which indicates the percentage of rooting, was obtained by counting emerging roots. The diameter of the branches was measured using a digital caliper and the measurements were expressed in millimeters. The length of the branches was measured using a ruler and the sizes were expressed in centimeters and then recorded using data analysis software. The fresh weight of the samples was measured on a scale of three zeros after being taken out of the culture medium, then to determine the dry weight, it was transferred to a dryer containing infrared light (to preserve the material and color of the sample) and measured using a scale. Calculations were performed by SPSS statistical software at a probability level of 5% and graphs were drawn by Excel software.
This study studied the effect of BAP and TDZ cytokines on proliferation and creating a suitable environment for MM106 apple tissue culture in a completely randomized design with three replications. Each experimental replicate consisted of five planted branch tips. After collecting the samples from the apple orchards of Khalatpooshan Research Station and disinfecting them with 70% ethanol, the microsamples were transferred to MS medium containing two concentrations of 0.5 and 0.6 mg BAP and 0.2 and 0.4 mg TDZ. The results showed that the suitable culture medium for Plastochron MM106 index was 0.4 mg TDZ and 0.6 mg BAP (number of leaves with average leaf 1.87). The effect of TDZ and BAP treatment on MM106 apples was significant at 1 and 5% probability levels, respectively. The highest fertility index was 0.4 mg / l TDZ and 0.3 mg / l BAP and the lowest fertility index belonged to the control (Figure 2). In this experiment, the leaves formed per shoot were counted in seven-day time sequences for 2 months. Plastocron index or time required for the emergence of each leaf, which indicates the degree of fertility and organ production. The results showed that the effect of TDZ and BAP treatment on MM106 apple was significant at 5% probability level. The highest amount of plastocron index was 0.4 mg / l TDZ and 0.6 mg / l BAP and the lowest selectivity index belonged to the control (Figure 3). The results of determination of soluble proteins showed that TDZ and BAP treatment on apple rootstock was significant at 5% probability level. The amount of total soluble protein in the treatment of 0.2 mg / l TDZ and 0.6 mg / l BAP on MM106 apple was significantly higher compared to other treatments. The lowest ratio of total soluble protein content was related to the 0.3 mg / L BAP treatment (Figure 4).