In vitro insect-feeding bioassay to determine the resistance of transgenic rice plants transformed with insect resistance genes against striped stem borer (Chilo suppressalis)

Summary To determine the degree of insect resistance in transgenic plants, different bioassays are used which typically use either whole plant or small pieces of leaves or stems of transgenic plants, following culture under greenhouse conditions. An in vitro insect-feeding bioassay is presented which permits the infestation of transgenic plantlets with newly hatched larvae from the striped stem borer. The bioassay consists of the germination of rice seeds in vitro using Murashige and Skoog medium in test tubes, and then infestation of each 3–4 cm long seedling with one neonate larva obtained from surfacesterilized eggs of Chilo suppressalis. The infested in vitro plantlets are kept in culture rooms at 25°C for several days and then the seedling damage and the growth of the larvae are analyzed. Senia (japonica variety) homozygous transgenic rice plants were used for these experiments. The plants were transformed with either the cry1B or the maize proteinase inhibitor (mpi) genes. Both genes confer resistance to Chilo suppressalis. With non-transformed plants the larvae grew and developed normally, feeding on the small rice plantlets. In contrast, with cry1B plants, the neonate larvae died during the first days of the infestation. These plantlets recovered completely and developed similarly to the non-infested control plants. With transgenic plants transformed with the mpi gene, the neonate larvae did not die but grew more slowly compared with the controls. Thus, this in vitro insect-feeding bioassay is a rapid and easy method to detect the resistance of cry and mpi transgenic plants to stem borers such as Chilo suppressalis.