Microbial-Based Biopesticides

Steven Reid, Leslie C. L. Chan, Leila Matindoost, Charlotte Pushparajan, Gabriel Visnovsky, Reid, Steven, Chan, Leslie C. L., Matindoost, Leila, Pushparajan, Charlotte, Visnovsky, Gabriel

Travis R. Glare, Maria E. Moran-Diez

While large-scale culture of insect cells will need to be conducted using bioreactors up to 10,000 l scale, many of the main challenges for cell culture-based production of insecticidal viruses can be studied using small-scale (20-500 ml) shaker/spinner flasks, either in free suspension or using microcarrier-based systems. These challenges still relate to the development of appropriate cell lines, stability of virus strains in culture, enhancing virus yields per cell, and the development of serum-free media and feeds for the desired production systems. Hence this chapter presents mainly the methods required to work with and analyze effectively insect cell systems using small-scale cultures. Outlined are procedures for quantifying cells and virus and for establishing frozen cells and virus stocks. The approach for maintaining cell cultures and the multiplicity of infection (MOI) and time of infection (TOI) parameters that should be considered for conducting infections are discussed.The methods described relate, in particular, to the suspension culture of Helicoverpa zea and Spodoptera frugiperda cell lines to produce the baculoviruses Helicoverpa armigera nucleopolyhedrovirus, HearNPV, and Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, AgMNPV, respectively, and the production of the nonoccluded Oryctes nudivirus, OrNV, using an adherent coleopteran cell line.