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

Abstract

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 How to cite this paper: Zhu, Y.M.,

Laccase Directed Lignification Is One of the Major Processes Associated With the Defense Response Against Pythium ultimum Infection in Apple Roots

“…Six apple rootstock genotypes included in this study were previously identified to have contrasting resistance traits based on repeated root infection assays with P. ultimum , as shown in Figure 1A ( Zhu et al, 2018a , b ). In addition to remarkable differences of plant survivability, microscopic observation revealed necrosis progression patterns and the intensity of pathogen hyphae growth along infected root that were distinguishable between resistant and susceptible apple rootstock genotypes.…”

“…These genotypes originated from an “Ottawa 3” × “Robusta 5” (O3R5) apple rootstock F1 population developed in the mid-1970s. Individual plants for each apple rootstock genotype and mock-inoculation controls were produced using synchronized plant tissue culture or micro-propagation procedures, as described previously ( Zhu et al, 2018a ). Shoot propagation and root induction/elongation required 6 weeks and 4 weeks, respectively.…”

“…In contrast, deficient detoxification, weakened effector-triggered immunity (ETI), or existence of a susceptibility ( S ) gene may contribute to a disrupted defense activation process in the roots of the susceptible genotype. Parallel to these transcriptome analyses, a systematic phenotyping effort has recently identified a panel of apple rootstock germplasm with contrasting and reliable resistance traits against P. ultimum infection ( Shin et al, 2016a ; Zhu et al, 2018a , b ). Besides the expression patterns of the protein coding genes, elucidating post-transcriptional control of genotype-specific defense responses may facilitate the identification of key genes underpinning apple roots resistance traits.…”

Challenges and Progress in Evaluating Apple Root Resistance Responses to <i>Pythium ultimum</i> Infection

“…Various inoculation methods and inoculum levels were tested using two rootstock genotypes, G.935 and B.9 as examples before expanding the evaluation to O3R5 apple rootstock genotypes [30] [31]. The root-dipping method with a pathogen inoculum concentration of 2 × 10 3 oospores was demonstrated to be an effective inoculum dose to distinguish the resistance levels between genotypes [31].…”

“…In contrast, the swift expansion of necrotic tissues accompanied by profuse hyphae growth along the root branches of the susceptible genotypes, such as O3R5-132, clearly demonstrated an inability to suppress pathogen progression [17]. These microscopic features on infected root tissues appeared to align with whole plant resistance levels between resistant and susceptible genotypes [17] [30] [31]. These closeup observations at tissue and cellular levels provided valuable information to eventually connect specific gene and pathways with observed resistance traits in apple root to P. ultimum infection.…”