A Phenotyping Protocol for Detailed Evaluation of Apple Root Resistance Responses Utilizing Tissue Culture Micropropagated Apple Plants

摘要

In the post-genomics era, reliable phenotypes are considered the bottleneck for unraveling the genetic control over the biology of interest. Phenotyping resistance response of roots to infection by soilborne pathogen is more challenging compared to that of plant aerial parts. In additional to the hidden nature and small stature of fine roots where infection occurs, extra obstacles exist for rosaceae tree crops such as apple. Due to self-incompatible reproduction and high-level heterozygosity of apple genome, genetically identical apple plants cannot be produced through apple seed germination. Here we report an established phenotyping protocol which includes a streamlined tissue culture procedure for micropropagation of uniform apple plants, standardized inoculation procedure using Pythium ultimum, and multilayered evaluating methods on apple root resistance traits. Because of the implementation of tissue culture based micropropagation procedure, constant availability of the uniform plants with defined genetic background, equivalent age and non-contaminated roots overcame a longstanding barrier of systematic and detailed phenotypic characterization of apple root resistance traits. Repeated infection assays by root-dipping inoculation demonstrated the reproducible and wide-range plant survival rates, from single-digit to over 90% survived plants for a given genotype. Genotype-specific values due to P. ultimum inoculation on shoot and root biomass reduction, maximum root lengths, leaf number and cumulative leaf areas were quantified between mock-inoculated and P. ultimum infected plants. Use of a glass-box container offered enhanced accessibility and minimized invasiveness for continuous and non-disruptive observation on the necrosis progression patterns along inoculated roots. With the assistance of a dissecting microscope, the genotype-specific resistance responses along the infected apple roots were captured and analyzed in detail. This reported phenotyping protocol represents a major development and should be easily adopted for other rosacea tree fruit crops with minor modifications.