Protocol on Mutation Induction in Coffee Using In Vitro Tissue Cultures

Abstract

Pathogens are the major limiting factors in coffee production. Approximately 26% of the global annual coffee production is lost to diseases, threatening the income of approx. 125 million people worldwide. Therefore, reducing coffee yield losses by improving coffee resistance to diseases and insect attacks through breeding can make a major contribution to agricultural sustainability. Mutation breeding in vegetatively propagated and perennial crops is hampered in large part due to bottlenecks in the induction of variation (lack of recombination) and challenges in screening. Tissue culture approaches using alternative types of material were developed. This offers a clear advantage of providing the required sample size for mutation induction and subsequent screening within a reasonable time frame. The protocols developed compare different tissue culture systems for mutation induction involving unicellular and multicellular explants requiring different numbers of subsequent subcultures to reduce the impact of chimerism: (a) axillary shoot culture for the provision of donor material for mutation induction and regeneration; (b) leaf disc cultures for the induction of calli; (c) direct and indirect somatic embryogenesis for the production of somatic embryos; (d) the irradiation of somatic embryos at the globular and cotyledonary stage. Mutagenesis was induced by irradiation with a Cobalt-60 Gamma-source at the FAO/IAEA Laboratories in Seibersdorf, Austria. A comparison of the time required for the regeneration of high numbers (hundreds) of plantlets from irradiated in vitro shoots versus irradiated embryogenic calli is clearly in favor of embryogenic calli, since the plantlets regenerate from individual cells and can be used for genotypic and phenotypic analyses directly. This chapter describes the general methods for mutation induction using gamma irradiation and the procedures that can be used to generate large numbers of induced mutants in different tissues of coffee under in vitro conditions.