Optimization of Tissue Culture Medium and the Combination of Growth Regulators to Increase Plantlet Production Efficiency in Walnut Micropropagation

Introduction In our country, walnut tree propagation is traditionally done through seed cultivation, often resulting in seed rot and death due to fungal, bacterial, and viral contamination (MC Granahan et al., 1986; Driver & Kenyuki, 1984; Saadat & Henry, 2002). The traditional method, in addition to low multiplication rates, leads to high variation in resulting seedlings, potential loss of seedlings due to contamination, and reduced efficiency in subsequent stages (Unit, 2012; Kaur et al., 2006). Previous research has mainly utilized concentrations of one milligram per liter of benzyl adenine along with small amounts of indole butyric acid for Iranian walnut growth and enrichment (Rodrigues, 1982; Revilla et al., 1989; Penuela, 1988; Mejzadeh et al., 2010, 1997; Amiri & Qaraati, 2012; Riosleal et al., 2012). This research aims to build upon and optimize previous work, evaluating the effectiveness of different concentrations of two growth regulators, benzyl aminopurine and adenine sulfate, on walnut plantlet regeneration and growth traits in tissue culture.   Materials and Methods This study was conducted to optimize the tissue culture protocol for the "Chandler" cultivar walnut and determine the most suitable culture medium and hormonal composition for micropropagation. Lateral and terminal buds from the current season's branches were sterilized and cultured in DKW medium containing 2 mg/liter of benzyl adenine hormone and 100 mg/liter of indole butyric acid hormone, with polyvinyl pyrrolidine at one g/liter and activated charcoal at 2 g. Two-factorial experiments were used to process and multiply the plant after the establishment phase. The first factor was DKW culture medium containing five levels of adenine sulfate (0, 20, 40, 60, and 80 mg/liter), and the second factor was benzylaminopurine plant growth regulator with five hormonal levels containing 0, 0.5, 1, 1.5, and 2 mg/liter in combination with 0.01 mg/liter of indole butyric acid hormone. DKW base culture medium without any plant growth regulating substances was considered as control. After two months, growth traits including plantlet weight, stem length, number of leaves, number of buds, and number of leaflets per plantlet were measured in different culture media. The resulting data were statistically analyzed using SAS 9.1 software, and means were compared using Duncan's multiple range test with a five percent probability level.   Results and Discussion The analysis of variance showed that both plant growth regulators, benzyl aminopurine and adenine sulfate, had a very significant effect at 1% probability level on plantlet weight, stem length, number of leaves, number of buds, and number of leaflets. The interaction effect of benzyl aminopurine with adenine sulfate treatment on plantlet weight and stem length was significant at the 1% probability level. However, the interaction effect of benzyl aminopurine with adenine sulfate treatment on the number of leaves, number of buds, and number of leaflets was not significant. The results indicated that an increase in the levels of growth regulators benzyl aminopurine and adenine sulfate led to an increase in plantlet weight. The positive effects of increasing the levels of growth regulating substances in increasing plantlet weight are likely due to their direct effect on nutrient absorption, utilization, and the photosynthesis process. These results align with the research of Hatemzadeh et al. (2017) and Saadat and Henrati (2002). The positive effects of higher concentrations of both growth regulators on the increase in the number of sprouts and the lack of significant difference between the two high concentrations confirm that the use of high levels does not exceed the economic threshold. It can be justified that in excessive and unconventional concentrations, positive effectiveness is not achieved, but it can also impose more costs on the walnut tissue culture program. The appropriate concentration of BAP and adenine sulfate increases the leaf surface through the effect on cell divisions, resulting in receiving more light radiation and increasing the rate of photosynthesis. It seems that the two growth regulating substances in the appropriate concentration intensified each other's effect, affecting the rate of absorption and utilization of materials from photosynthesis, leading to an increase in the fresh and dry weight of the seedling. This, in turn, leads to a decrease in the length of the reproduction period in the resulting seedlings and an increase in the efficiency of the seedling production in walnut tissue culture.   Conclusion The use of both studied growth regulators significantly increased plantlet weight, stem length, number of leaves, number of buds, and number of leaflets compared to the control treatment. Plantlet growth was achieved with the use of plant growth regulators, whereas no growth was observed in their absence. All assessed traits increased significantly with the addition of plant growth regulators, with the highest trait values obtained through the simultaneous use of benzylaminopurine and adenine sulfate.