Protocol: An improved high-throughput method for generating tissue samples in 96-well format for plant genotyping (Ice-Cap 2.0)

BACKGROUND: We previously developed a high-throughput system called 'Ice-Cap' for growing Arabidopsis seedlings in a 96-well format and rapidly collecting tissue for subsequent DNA extraction and genotyping. While the originally described Ice-Cap method is an effective tool for high-throughput genotyping, one shortcoming of the first version of Ice-Cap is that optimal seedling growth is highly dependent on specific environmental conditions. Here we describe several technical improvements to the Ice-Cap method that make it much more robust and provide a detailed protocol for implementing the method. RESULTS: The key innovation underlying Ice-Cap 2.0 is the development of a continuous watering system. The addition of the watering system allows the seedling growth plates to be incubated without a lid for the duration of the growth period, which in turn allows for much more uniform and robust seedling growth than was observed using the original method. We also determined that inserting wooden skewers between the upper and lower plates prior to tissue harvest made it easier to separate the plates following freezing. Seedlings grown using the Ice-Cap 2.0 method remain viable in the Ice-Cap plates twice as long as seedlings grown using the original method. CONCLUSION: The continuous watering system that we have developed provides an effective solution to the problem of sub-optimal seedling growth that can be encountered when using the originally described Ice-Cap system. This novel watering system and several additional modifications to the Ice-Cap procedure have improved the robustness and utility of the method.