**4. Surface sterilants and disinfectants**

Surface sterilants can be described as chemicals rendering plants free from any pathogens including fungal spores. Generally, surface sterilants act on the outside of the explants. There are several chemicals which have been used to free plants from pathogens before culture initiation onto the media (chemotherapy). They deter growth and proliferation of pathogens (fungi, bacteria and other types of microbes). In many cases, a combination of several surface sterilants, disinfectants and/or fungicides is used to improve the efficiency of subsequent fungicides in decontaminating stock plants. For instance, a few drops of Teepol (0.05%) are often applied to the water and this increases wettability of the plant surfaces. Also, penetration of fungicides into the outer plant cells might be enhanced by rinsing the stock plants in the water. Generally, Teepol (soapy water or detergent) has been one of those disinfectants or surface sterilants commonly used in many plant culture laboratories to enhance the removal of pathogens and/or fungal spores from the stock plants.

Another common disinfectant in plant culture laboratories has been ethanol (an alcohol derivative). Ethanol has been one of the commonest disinfectants used in plant tissue culture to eliminate pathogens and spores. It is used from the preparation room up to the laminar

Efficacy and Utilization of Fungicides and Other Antibiotics for Aseptic Plant Cultures 251

stronger disinfectants or fungicides have been used to remove culture contaminants, especially those which are hard to decontaminate. For instance, mercuric chloride (HgCl2) has been effective in decontaminating pre-conditioned mature *U. kirkiana* stock plants (Mng'omba et al., 2007) where sodium hypochlorite (NaOCl) and Calcium hypochlorite (Ca (OCl2)2) have not been effective in decontaminating the above stock plants. There have been no aseptic cultures obtained regardless of the type and concentration of such disinfectants

Efficacy of fungicides depends on many factors including their active ingredient (a.i.), concentration (dosage), the type of stock plants (mature vs. new shoots), type of fungi (exogenous vs. endogenous), and the exposure time. Generally, rigorous decontamination is required for stock plants derived from mature ortet, especially when weaker disinfectants are used. This will also require longer exposure time. Generally, the use of concentrated (stronger) fungicides or disinfectants to eliminate contaminants from the stock plants

Mercuric chloride as a culture disinfectant is stronger than sodium or calcium hypochlorite solutions for disinfecting stock plants and this could be the reason for its efficacy in decontaminating endogenous fungal contaminants. Danso et al. (2011) reported successful *in vitro* decontamination of sugarcane explants with mercuric chloride where NaOCl and Ca (OCl2)2 were less effective. Therefore, HgCl2 is ideal for the control of endophytic fungal

The use of stronger disinfectants and fungicides could weaken cell membrane and cell wall of the plants. This can lead to the discharge of cell sap (nutrients) which stimulates an outgrowth of endogenous fungi once placed onto the culture media. These endogenous fungi might become pathogenic to the explants under *in vitro* conditions (Darworth & Callan, 1996). For example, HgCl2 might be strong to some plants and hence can easily damage their cells, especially after a long exposure (Mng'omba et al., 2007). Danso et al. (2011) reported a low survival of sugarcane plantlets when decontaminated with HgCl2. Therefore, the application of fungicides should consider the right concentration (dosage)

The mortality of plant cultures could be high due to cell damage by the use of concentrated (strong) fungicides or disinfectants. Where possible, it is important to maintain cell integrity of the stock plants to avoid any undesirable effects on the plant cultures. However, this may be difficult with heavily fungal loaded stock plants. For instance, the use of disinfectants or surface sterilants such as NaOCl, Ca(OCl2)2 and many others for a heavy fungal loaded stock plants could result in the resurgence of endogenous contaminants at any stage after the culture initiation. Therefore, the utilization of strong disinfectants or surface sterilants is warranted for the highly fungal loaded stock plants such as those derived from the old

and exposure time in order to reduce injury to the cells of the explants.

used on *U. kirkiana* stock plants. These fungi were suspected to be endogenous.

**5.2 Efficacy of fungicides** 

requires a shorter exposure time.

**6. Effects of fungicides on plant cultures** 

culture contaminants.

**6.1 Cell integrity** 

plants.

air floor to kill pathogens and spores. This alcohol derivative has often been used to reduce culture contaminants as it kills fungal pathogens and spores on the stock plants.

The concentrations of sterilants and disinfectants widely used vary depending upon the nature of stock plants (soft vs. lignified plant surfaces) to be sterilized. A wide range of ethanol concentrations (20 – 100%) has been used, but high concentrations of ethanol (100% concentration) are rarely used, especially on soft stock plants because they can easily damage or injure the plant tissues. Generally, many epithytic contaminants (fungi) are eliminated, but not endophytes, and hence high concentrations of disinfectants are required to destroy these endophytic culture contaminants. Apart from the concentration of the disinfectants, exposure time must also be considered. Generally, the exposure time should be short (5 - 20 seconds), especially with the tender stock plants which are highly infested. Low concentrations of ethanol (≤50%) have often been used for tender and soft stock plants to avoid injuring the plant cells or tissues.

#### **4.1 Improving decontamination process**

For adequate plant sterilization (decontamination), stock plants are stirred in a beaker to ensure sufficient surface contact between stock plant and the fungicide. In this case, the exposure time, disinfectant concentration, and active ingredient (a.i.) are important factors determining the efficacy of the entire process of *in vitro* decontamination of stock plants. Rinsing the stock plants in the water assists in stopping the reaction between the chemicals (fungicide) and stock plants. The disinfection by fungicides and other surface sterilants in a sealed beaker (with aluminum foils) is followed by rinsing stock plants under running tap water for some time to remove the contaminants and also stop any reaction between the stock plants and the disinfectants.
