Introduction
Success in tissue culture technique, especially in bulbous plants, depends on the microbial contamination control during in vitro culture. Decontamination is considered as a fundamental challenge in the technique of cell, tissue and plant organ culture. Although there are various methods for this purpose, the development of disinfection methods specific to each species is considered an important factor in the establishment and success of the tissue culture system. Applying different treatments can control the microbial contamination and consequently increase the percentage of explant survival. This plant is among the poisonous plants of pastures and the chemical compounds present in it have important medicinal effects, so that the existence of alkaloid and glycosidic compounds has been reported for it. These chemical compounds are used to treat rheumatic pains, lymphatic system diseases and also as liver cleansers. This ornamental plant also has the ability to produce pots and its export is possible in pots . Currently, in order to overcome the issues of the usual methods of propagation of this ornamental plant and to produce pollution-free plants, the use of in vitro cultivation methods in this plant has become very important. bulbes is one of the most common explants of inverted tulip tissue culture, but it often faces very high internal and external fungal and bacterial contamination. In the in vitro culture of onion plants, the existence of fungal and bacterial contaminations are one of the main problems that can affect the efficiency of this type of propagation methods. Fungal and bacterial contaminations are one of the most important problematic factors in different in vitro culture methods. Internal contamination usually appears several weeks after planting. These contaminations may not be seen externally, but they affect the growth, division and greening of small samples. Mercuric chloride is mainly used as a surface disinfectant along with sodium hypochlorite and it controls the spectrum of bacteria and fungi and increases the disinfection efficiency. The results of the study showed that mercuric chloride relatively controls the bacterial and fungal contamination of the samples, but it reduces the survival percentage of the samples in high concentration. It seems that some plants are sensitive to mercuric chloride and determining the appropriate amount of this substance plays a very important role in preparing a healthy and living specimen. Determining the duration of disinfectant treatment was also considered in various researches, so that sometimes carbendazim 1% fungicide for 20 minutes, 70% alcohol for 60 seconds, sodium hypochlorite 2.5% for 10 minutes. And 0.1% mercury chloride was used for 7 minutes to sterilize aloe vera sample. Considering the importance of plant tissue culture techniques and the need to create new protocols to reduce contamination and increase the number of healthy cultures, the main goal of this study is to create an efficient protocol for the disinfection of Fritillaria spp. onion explants in order to increase the efficiency of in vitro cultures.
Materials and Methods
This study aimed to investigate the effect of time and different concentrations of sodium hypochlorite, carbendazim fungicide and mercuric chloride in inhibiting the contamination of inverted tulip bulbs (Fritillaria spp.) in tissue culture environment. So, an experiment was done as a completely randomized design at four replications in the biotechnology laboratory of Zanjan University during 2023. The experimental treatments consisted of 0.1% fungicide at different times (30, 25, 35 and 40 minutes), 5 levels of sodium hypochlorite (0,1, 1.5, 2, 2.5 and 3%) at different times (7, 9, 10, 12 and 15 minutes), 70% Ethanol at two different times (0,60 and 90 seconds) and mercury chloride (0,0.1 and 0.2%). Bulbs that collected from nature were transferred to the Biotechnology Laboratory of the Faculty of Agriculture, Department of Horticulture, Zanjan University and kept them in the refrigerator at 4°C for two weeks. After this period of time, the bulbs were washed with detergent and then remained in water for 30 minutes. Then they were disinfected by using the above-mentioned treatments: It should be noted that at the end of each step, Fritillaria bulbs were washed with sterile distilled water. Also, after the end of the disinfection treatments, the desired explants were cultured in MS basic culture medium. The statistical analysis of this experiment was carried out using SAS software, version 9.1. The differences between mean values were compared using Duncan’s multiple range test method at the 5% significant level (p < 0.05).
Results and Discussion
The control of fungal and bacterial contamination, especially in relation to bulbous and bulbous plants that are in direct contact with the culture medium, is one of the basic steps in in vitro culture conditions. Applying different treatments including the use of fungicides, alcohol, sodium hypochlorite and mercuric chloride in the cultivation environment can help control fungal and bacterial contamination in the Fritillaria spp. plant. However, the application of such treatments can have a negative effect on the regeneration potential of the explants, so it is important to choose the appropriate treatment in a way that controls the fungal and bacterial contamination and on the other hand preserves the regeneration potential of the explants. This study investigated the effectiveness of various disinfection protocols on Laleh-Avgagun onion explants. While the lowest contamination (0%) was achieved using 0.1% fungicide for 40 minutes, 70% alcohol for 90 seconds, 3% sodium hypochlorite for 15 minutes, and 0.1% mercury chloride, this treatment significantly reduced explant survival and regeneration, causing browning. Conversely, applying 0.1% fungicide for 30 minutes, 70% alcohol for 60 seconds, 2% or 2.5% sodium hypochlorite for 12 or 10 minutes respectively, and 0.1% mercury chloride effectively reduced contamination while maintaining explant survival. This optimized protocol improved the growth and regeneration ability of the explants.Therefore, in the conditions of in vitro cultivation, the use of the mentioned treatments is recommended to control contamination and optimal reproduction of the bulbs of Fritillaria spp. plant. |
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