Cut and paste - Efficient cotyledon micrografting for Arabidopsis thaliana seedlings

Cotyledon micrografting represents a very useful tool for studying the central role of cotyledons during early plant development, especially their interplay with other plant organs with regard to long distance transport. While hypocotyl grafting methods are established, cotyledon grafting is still inefficient. By optimizing cotyledon micrografting, we aim for higher success rates and increased throughput in the model species Arabidopsis thaliana. We established a cut and paste cotyledon surgery procedure on a flat solid but moist surface which improved handling of small plant seedlings. Applying a specific cutting and joining pattern throughput was increased up to 40 seedlings per hour. The combination of short day conditions and low light intensities for germination and long day plus high light intensities and vertical plate positioning after grafting significantly increased ligation efficiency. Together, we achieved up to 46 % grafting success in A. thaliana. Reconnection of vasculature was shown by successful transport of a vasculature-specific dye across the grafting site. On a whole plant level, plants with grafted cotyledons match plants with intact cotyledons in biomass production and rosette development. This cut and paste cotyledon-to-petiole grafting protocol simplifies the handling of plant seedlings in surgery, increases the number of grafted plants per hour and produces higher success rates for A. thaliana seedlings. The developed cotyledon micrografting method is also suitable for other plant species of comparable size.