Optimization Tissue Culture of Almond 2-22 Genotype

Background and aim: the almond is one of the most important nut crops in many countries, including iran. on the other hand, due to rooting, it is challenging to propagate this species by conventional methods, mainly cuttings. therefore, this study was aimed at optimizing in vitro culture conditions for the promising 2-22 genotype as a high-yield and late-blooming cultivar. to this end, after collecting apical and lateral buds of 2-22 genotype, surface sterilization and establishment treatments were applied to explants. the second-order equation is selected for the central compound design (ccd) with two variables (hgcl2 and naclo) to obtain a good fit in the sterilization treatment. the results showed that with 81.25% and 100% of healthy seedlings, 1.8% naclo for 12 min + 0.1 % hgcl2 for 3 min and 1.5 % naclo for 8 min respectively were the best treatment in contamination frequency and explant viability. the experimental establishment was conducted as a factorial experiment using a completely randomized design (crd) with four replications. the establishment treatments indicated that the wpm medium was more effective than the ms medium and knop medium. the hormonal composition of 1 mgl-1 ba + 0.05 mgl-1 iba had the best results in the percentage of the establishment (72.25%), number of foliage (7.24), and shoot length (13.77 mm).methods: this study was conducted at the plant tissue culture laboratory of the faculty of agriculture, tarbiat modares university, iran on june and august 2019. explants of almond 2-22 genotype were taken from plants grown in horticultural department of seed and plant improvement institute, karaj, iran, in june 2019. one-year-old shoots were cut into 1–1.5 cm long pieces, each including one bud. the factors that dust and waste of insects with negative effects on sterilization application existing on the shoots were cleaned out with running water for 30 minutes. then, the explants were kept in 70% alcohol for 30 seconds as a pretreatment procedure before the application of the chemical agents. later, they were rinsed with distilled water three times to avoid the caustic effect of the alcohol. then, explants were kept in sterilization solutions prepared at different concentrations, for different periods in such a way that immersion in 2%, 1.8%, and 1.5% (w/v) naclo respectively for 15, 12, and 8 min depending on the status of explant tissues and rinsed three times with sterile distilled water. then, explants were dipped in 0.1% hgcl2 for 5, 3, and 0 minutes. finally, they were washed four times with sterile distilled water. the second-order equation presented for the central compound design with four replications and four explants in each replicate is an equation as following: y: model output (healthy and dead explant percentage, bacterial and fungal contamination), k: number of input variables (concentration of sterilization agents and immersion time) β0: constant coefficient, βi: linear coefficient on variable, βij : the coefficient of interaction effect on the variables, βii: the coefficient of the second-order on the variable, and ε: includes the residual value of the model. in addition, analysis of tests was performed by minitab version 19 software. in the culture establishment stage, culture media ms (murashige & skoog, 1962), wpm (lloyd and mccown, 1980) and knop (knop w, 1865), and containing different concentrations of ba (0. 0.5 and 1 mgl-1) and (0, 0.05 and 0.1 mgl-1 iba) were evaluated on the percentage of active buds and bud growing quality index. the ph of the culture medium was 5.6, and the culture medium was autoclaved at 121°c for 20 minutes. growth chamber conditions were 16 hours of light at 25°c and 8 hours of dark at 23°c. leaf number, shoot length, establishment percentage were measured after six weeks. this study was conducted as a factorial experiment using a crd. each treatment had four replicates and four explants in each replicate in a 125 ml culture glass jar containing 35 ml of medium. statistical analysis of the data was carried out through using (ibm spss statistics version 22) software, and difference among treatment means were compared by duncan's multiple range test (dmrt) at 1% and 5% levels.results: according to the obtained results, t2 and t3 with 81.25% and 100% of healthy seedlings both treatments were the best treatment in production of healthy and viable plants without contamination, respectively. alternatively, t1 resulted in 25% of healthy seedlings and 75% of dead explants were the least effective in the sterilization process. .results of analysis of variance revealed that the effect of concentration and immersion duration of sodium hypochlorite on explants viability both were significant at 0.05 probability level. the use of a higher concentration of sodium hypochlorite and the longer immersion time has been associated with an increase in the percentage of burn or dead explants. in the following, mercury chloride treatment had a significant effect on the viability of explants and was significant at 0.05 probability level. furthermore, the immersion duration of mercury chloride effect on the survival of the explants was significant at the level of 0.01. application of higher concentrations and longer immersion time in mercury chloride solution led to an increase in the percentage of dead explants. moreover, all three treatments were very effective in controlling bacterial and fungal infections. almost no infection was observed in the explants of the three treatments. from this table of analysis of variances the difference between the treatments in terms of infection control was not significant. these results indicate the high efficiency of the applied treatments in terms of contamination control. results of analysis of variance show that different media and different concentrations of ba, iba, as well as the interaction of medium with ba and iba growth regulators on leaf number, shoot length, and establishment percentage at 1% level were significant. based on the results of the mean comparison, the highest leaf number (7.24), shoot length (13.77 mm), and establishment percentage (72.25%) in the wpm medium with 1 mgl-1 ba and 0.05 mgl-1 of iba and lowest leaf number (1.71), shoot length (4.32 mm), and establishment percentage (13.97%) were obtained in hormone-free knop’s medium. wpm medium had the highest number of leaves (5.46), shoot length (12.56 mm), and establishment percentage (58.19%). knop’s medium had the lowest leaf number (3.18), shoot length (5.73 mm) and establishment percentage (27.43%) and ms medium with leaf numbers (4.27), shoot length (8.16 mm), and establishment percentage (39.38%) were placed between wpm and knop’s medium. this study showed that with increasing ba concentration to a certain extent, the number of leaves and establishment rate increased, indicating a positive relationship between the increase in concentration and establishment rate that the percentage of establishment peaked was at a concentration of 1 mgl-1 ba. the establishment treatments indicated that the wpm medium was more effective than the ms medium and knop’s medium. besides, the hormonal composition of 1 mgl-1 ba plus 0.05 mgl-1 iba had the best results in the percentage of establishment and number of foliage. conclusion: the results of sterilization treatment showed that t2 and t3 with 81.25% and 100% of healthy seedlings were selected as the best treatment in contamination frequency and explant viability, respectively and the application of concentrations and immersion time of explants in sodium hypochlorite and mercury chloride were highly effective in the explants viability. the establishment treatments indicated that the wpm medium was more effective than the ms medium and knop’s medium. furthermore, the hormonal composition of 1 mgl-1 ba plus 0.05 mgl-1 iba had the best results in the percentage of establishment, number of foliage and, shoot length and these amounts have been 72.25%, 7.24 and 13.77mm, respectively. furthermore, we can safely say that a certain amount of ba is required to obtain the best effect. thus, in this experiment, the effect of different concentrations of the hormone ba was very significant, and the results reveal that for sleeping buds, the hormone ba is essential, while the hormone iba has not had much effect.