Volume 13, Issue 2 (10-2025)
Ecol Iran For 2025, 13(2): 116-129 |
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Amirian S, Hosseini Nasr S M, Jalilvand H, Ahmadi A. (2025). Contamination Control of Leaf and Petiole Explants of Red Maple (Acer rubrum L.) in Vitro. Ecol Iran For. 13(2), 116-129. doi:10.61882/ifej.2025.528
URL: http://ifej.sanru.ac.ir/article-1-528-en.html
URL: http://ifej.sanru.ac.ir/article-1-528-en.html
1- Faculty of Natural Resources, Sari University of Agricultural Sciences and Natural Resources, Sari, Iran
2- Department of Forestry, Faculty of Natural Resources, Sari University of Agricultural Sciences and Natural Resources, Sari, Iran
3- Department of Natural Resources Research, Golestan Province Agricultural and Natural Resources Research Center, Agricultural Research, Education and Extension Organization, Gorgan, Iran
2- Department of Forestry, Faculty of Natural Resources, Sari University of Agricultural Sciences and Natural Resources, Sari, Iran
3- Department of Natural Resources Research, Golestan Province Agricultural and Natural Resources Research Center, Agricultural Research, Education and Extension Organization, Gorgan, Iran
Abstract: (689 Views)
Extended Abstract
Background: The standing red maple (Acer rubrum) is native to the northeastern United States of America for various reasons, including the beauty of colored leaves, being suitable and widely used for urban green space design, resistance to cold, humidity, and drought tolerance, and ecological characteristics, and a wide ability to be compatible with different types of soil. It is one of the imported species of maple in Iran, which is difficult to reproduce through seeds and cuttings in high production, and has not yielded favorable results at a high scale due to the limitations created in terms of the season and the number of cuttings. Unfortunately, the success of laboratory cultivation methods is always hampered by microbial contamination, while contamination-free cultivation is a prerequisite for the success of laboratory-based plant biotechnology. Therefore, surface disinfection is a vital step in preparing healthy and living explants in tissue culture. Most surface contamination can be removed by disinfecting the surface with a suitable disinfectant. Very few studies have been conducted in the field of preparing different sterile explants in the red maple genus. Therefore, this study aimed to provide an optimal disinfection method for the micropropagation of the commercial species of red maple.
Methods: First, treatments were selected through library studies and a literature review. The required plant resources were obtained from seed organs, leaves, and petioles of one to three-year-old young red maple seedlings from Shahsavar City, Mazandaran Province, and the research procedures were carried out in the tissue culture laboratory of the Golestan Agricultural and Natural Resources Research Center. Then, a culture medium was prepared in six replicates of each treatment. In the next step, the required explants were separated from the mother base, and after removing the damaged explants or those with signs of fungal contamination, they were initially washed using a 500 cc container of Teepol and 0.04% benomyl fungicide, and transferred under a laminar air flow to continue the disinfection process. The treatments were applied separately, the explants were cut into 1-cm pieces, and cultivated in jam glass containers using tweezers and scalpels with at least 20 cc of the intended culture medium. Then, samples were transferred to a growth chamber (12 hours light and 12 hours dark) in optimal environmental conditions with a temperature of 25 °C, and their contamination level was examined after 7 days of cultivation. Statistical analysis was based on a factorial experiment as a completely randomized design with three types of explants, two types of culture media, and 13 treatments. Each treatment was cultivated in six replications, and three explants were cultivated in each replication. Finally, the data were analyzed using SPSS software.
Results: The sources of changes investigated in the experiment were statistically different from each other at the level of 0.01%. In this study, fungal infections were diagnosed by the morphological method and with the naked eye, showing that the fungal diseases were caused by Penicillium sp., Aspergillus sp., Cryptococcus sp., Mucur sp., Rhizopus sp., Fusarium sp., and Pythium sp. Based on the results of average comparisons, the rate of growth and spread of fungi in the PDA culture medium (83.2%) showed more contamination than in the MS culture medium (76.5%). A comparison of the contamination rate of the explants revealed that the petiole explant
Background: The standing red maple (Acer rubrum) is native to the northeastern United States of America for various reasons, including the beauty of colored leaves, being suitable and widely used for urban green space design, resistance to cold, humidity, and drought tolerance, and ecological characteristics, and a wide ability to be compatible with different types of soil. It is one of the imported species of maple in Iran, which is difficult to reproduce through seeds and cuttings in high production, and has not yielded favorable results at a high scale due to the limitations created in terms of the season and the number of cuttings. Unfortunately, the success of laboratory cultivation methods is always hampered by microbial contamination, while contamination-free cultivation is a prerequisite for the success of laboratory-based plant biotechnology. Therefore, surface disinfection is a vital step in preparing healthy and living explants in tissue culture. Most surface contamination can be removed by disinfecting the surface with a suitable disinfectant. Very few studies have been conducted in the field of preparing different sterile explants in the red maple genus. Therefore, this study aimed to provide an optimal disinfection method for the micropropagation of the commercial species of red maple.
Methods: First, treatments were selected through library studies and a literature review. The required plant resources were obtained from seed organs, leaves, and petioles of one to three-year-old young red maple seedlings from Shahsavar City, Mazandaran Province, and the research procedures were carried out in the tissue culture laboratory of the Golestan Agricultural and Natural Resources Research Center. Then, a culture medium was prepared in six replicates of each treatment. In the next step, the required explants were separated from the mother base, and after removing the damaged explants or those with signs of fungal contamination, they were initially washed using a 500 cc container of Teepol and 0.04% benomyl fungicide, and transferred under a laminar air flow to continue the disinfection process. The treatments were applied separately, the explants were cut into 1-cm pieces, and cultivated in jam glass containers using tweezers and scalpels with at least 20 cc of the intended culture medium. Then, samples were transferred to a growth chamber (12 hours light and 12 hours dark) in optimal environmental conditions with a temperature of 25 °C, and their contamination level was examined after 7 days of cultivation. Statistical analysis was based on a factorial experiment as a completely randomized design with three types of explants, two types of culture media, and 13 treatments. Each treatment was cultivated in six replications, and three explants were cultivated in each replication. Finally, the data were analyzed using SPSS software.
Results: The sources of changes investigated in the experiment were statistically different from each other at the level of 0.01%. In this study, fungal infections were diagnosed by the morphological method and with the naked eye, showing that the fungal diseases were caused by Penicillium sp., Aspergillus sp., Cryptococcus sp., Mucur sp., Rhizopus sp., Fusarium sp., and Pythium sp. Based on the results of average comparisons, the rate of growth and spread of fungi in the PDA culture medium (83.2%) showed more contamination than in the MS culture medium (76.5%). A comparison of the contamination rate of the explants revealed that the petiole explant
showed signs of contamination (59.6%) less than those of the leaf (8%) and the seed (96.2%). Among all the applied treatments, one drop of Teepol (two minutes) + benomyl fungicide (four g/liter) (25 min) + 75% alcohol (60 sec) + 25% sodium hypochlorite (15 min) + 0.1% mercury chloride (5 min) was determined as the best treatment for the decontamination of red maple explants for use in red maple micropropagation in Golestan Province, Iran.
Conclusion: Maintaining disinfection conditions is a new, definite prerequisite for the successful reproduction of species in laboratory conditions. In the cultivation of ornamental species, such as red maple, which is aimed at commercial plant propagation, the control of contamination and preparation of sterile explants are of paramount importance due to the high volume of work, because the smallest contamination in the explant quickly reproduces in the environment and causes high financial damage, which is often not possible to compensate. In the present study, the use of disinfectants as a combination of (Tipol (2 min) + benomyl fungicide (4 g/L) (25 min) + 75% alcohol, 60 sec + sodium hypochlorite 25% (15 min) + 0.1% mercury chloride (5 min) had a greater effect on controlling the fungal infection of red maple seedlings. In the present study, using these substances alone or in combination with another substance does not generally have a significant effect on the decontamination of the red maple cultivation environment. The use of these materials is one of the most important controlling methods for bacterial and fungal contamination in red maple glass culture.
Conclusion: Maintaining disinfection conditions is a new, definite prerequisite for the successful reproduction of species in laboratory conditions. In the cultivation of ornamental species, such as red maple, which is aimed at commercial plant propagation, the control of contamination and preparation of sterile explants are of paramount importance due to the high volume of work, because the smallest contamination in the explant quickly reproduces in the environment and causes high financial damage, which is often not possible to compensate. In the present study, the use of disinfectants as a combination of (Tipol (2 min) + benomyl fungicide (4 g/L) (25 min) + 75% alcohol, 60 sec + sodium hypochlorite 25% (15 min) + 0.1% mercury chloride (5 min) had a greater effect on controlling the fungal infection of red maple seedlings. In the present study, using these substances alone or in combination with another substance does not generally have a significant effect on the decontamination of the red maple cultivation environment. The use of these materials is one of the most important controlling methods for bacterial and fungal contamination in red maple glass culture.
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