**6.1 Cell integrity**

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

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 plants.

Efficacy and Utilization of Fungicides and Other Antibiotics for Aseptic Plant Cultures 253

water drainage system after rinsing the stock plants. Disinfectants such as HgCl2 are known to be corrosive, and hence can damage the water drainage system. Proper disposal of such disinfectants must be taken into consideration. For instance, it may be advisable to dig a pit where HgCl2 solutions must be disposed off and buried. This is because heavy metals are

The effects of most fungicides and many other disinfectants on different stock plants and explants remain unanswered, especially for the strong fungicides or disinfectants used in decontaminating stock plants in the plant culture laboratories. To our knowledge there has been limited research in this area. Therefore, there is the need for more research studies in this area. For instance, the effects of the fungicide and disinfectant residuals on the growth and survival of plantlets in *in vivo* have not been thoroughly researched. Our experience has shown that the use of HgCl2 may require several rinsing of stock plants. This is to get rid of the residues and we hypothesize that the residues could have deleterious effects on explants

This chapter has outlined the significant roles that fungicides and other disinfectants play in plant culture laboratories in achieving aseptic *in vitro* plant cultures. This is a vital process for the success of *in vitro* plant culture protocols. However, selection of an appropriate fungicide or disinfectant is important since endogenous or endophytic pathogens require the use of systemic and strong fungicides to effectively disinfect stock plants. Proper handling of fungicides and disinfectants and also proper disposal of the used disinfectants in the plant culture laboratory are equally important as they may become corrosive to the water drainage systems, and hence dangerous to the environment. Many research studies are needed in order to increase our understanding with respect to various disinfectants used

We would like to acknowledge the financial support from the World Agroforestry Centre (ICRAF), southern Africa region without which this chapter could have not been published.

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Danso, K.E., Azu, E., Elegba, W., Asumeng, A., Amoatey, H.M. & Klu, G.Y.P. (2011).

Darworth, C.E. & Callan, B.E. (1996). Manipulation of endophytic fungi to promote their

*Micropropagation: Technology and Application*, P.C. Derbergh & R.H. Zimmerman

Effective decontamination and subsequent plantlet regeneration of sugarcane (*Sacchrum officinarum* L.) *in vitro*. *International Journal of Integrative Biology,* Vol. 11

utility as vegetation biocontrol agents. In: *Endophytic Fungi in Grasses and Woody* 

also not friendly to the environment, and hence proper disposal is needed.

**8. Future research areas on fungicides** 

and later on the plantlets.

in the plant culture laboratories.

(Eds), 31– 44, Kluwer, Dordrecht.

No.2, pp 90 – 96 (May 2011)

**10. Acknowledgement** 

**11. References** 

**9. Conclusion** 

#### **6.2 Preconditioning mother plants**

Application of fungicides to the mother plants in the field before collection of explants (preconditioning of the ortet plants) greatly contributes to aseptic cultures. This could reduce the need to use stronger disinfectants such as HgCl2. Furthermore, it may also improve decontamination efficacy. For instance, the use of HgCl2 alone was less effective on field collected *U. kirkiana* (Mng'omba et al., 2007). Thus, preconditioning of grafted *U. kirkiana* stock plants using Benomyl contributed to achieving culture asepsis. Therefore, preconditioning reduces the fungal loads on the stock plants.
