The Effect of Heat Treatment and Fungicide on Controlling the Infestation of Narcissus tazetta L. In Vitro Culture

Success in tissue culture technique, especially in bulbous plants, depends on the microbial contamination control during in vitro culture. Applying different treatments, such as heat treatment and usage of fungicides, can control the microbial contamination and consequently increase the percentage of explant survival.

This study aimed to investigate the effect of heat treatment and fungicide on reducing the contamination during in vitro culture of narcissus. So, an experiment was done as a factorial experiment in a completely randomized design with two factors, including benomyl concentration in the medium (1 and 2 g/l) and heat treatments (two levels, with and without heat treatment), with 10 replications. In order to sterilizing the plant materials, damaged and infected scales were removed firstly and then bulbs were washed for 30 minutes with running tab water and a few drops of dishwashing liquid. For applying heat treatment, bulbs were divided into two groups. In the first group, heat treatment was not applied and in the second group heat treatment 54 °C was applied for one hour using water bath. After this step, bulbs surface were sterilized by dipping in 70% ethanol for one minute and rinsed with sterile distilled water, followed by immersing in 1.5% sodium hypochlorite solution for 30 min. After sterilization with sodium hypochlorite solution, bulbs were washed three times with sterile distilled water under laminar air flow hood. After sterilization step, bulbs were cut into 32 twin scales explants and cultured in MS medium supplemented with 1 mg/l BA and 0.2 mg/l NAA + benomyl (1 or 2 g/l). After 30 days, the response of explants (number of produced bulbs, percentage of explant survival, percentage of bacterial contamination, percentage of fungal contamination, percentage of browning) was evaluated. Data preparation was done in the Excel program and data analysis was done using JMP-8 software. Mean comparison of the treatments was done by LSD test and finally the charts were drawn using the Excel program.

The results showed that increasing the concentration of benomyl in the medium and applying heat treatment had negative effect on regeneration potential of explants, so that the maximum regeneration mean were observed when heat treatment was not applied for explants and medium contains 1 g/l benomyl. Using the heat treatment and application of 2 g/l benomyl in the medium leads to the lowest regeneration amount. On the other hand, evaluating the browning percentage of explants showed that the effect of treatments was significant in this trait. Applying the heat treatment and using 2 g/l benomyl in the medium had severe effect on the increasing of explant browning and the maximum mean was observed in this treatment. But reducing the benomyl concentration in the medium and none application of heat treatment caused the lowest amount of explant browning. Contamination percentage that includes bacterial and fungal contamination is an important parameter in this study. Explants that cultured in the medium containing 1 g/l benomyl and applying heat treatment showed the highest contamination percentage, which contains 21% fungal and 14% bacterial contamination. The lowest percentage of contamination was observed when heat treatment applied and medium contains 2 g/l benomyl. However, this treatment caused the highest percentage of explant browning that lead to reduction of explants regeneration potential. Researches showed that the use of fungicides can help to control tissue culture contamination and according to previous studies, benomyl is the most effective treatment against fungal infection. As benomyl is considered as a systemic fungicide, so it is useful to eliminate the internal fungus. On the other hand, there are some reports about the positive effect of heat treatment on the control of tissue culture contamination. As regards this investigations were done in 1914 to 1997 and then stopped, so it seems that application of this treatment had no sufficient efficiency for contamination control during in vitro condition.

Therefore, by considering the browning, regeneration and contamination percentage, non-application of heat treatment and usage of 1g/l benomyl fungicide in the medium for in vitro culture of Narcissus twin scales explants is recommended.