A phenotyping protocol for detailed evaluation of apple root resistance responses utilizing tissue culture micropropagated apple plants

Authors: Zhu, Yanmin; Saltzgiver, Melody; ZHAO, JINGXIAN - Hebei Academy Of Agriculture & Forestry

Submitted to: American Journal of Plant Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/25/2018
Publication Date: 10/19/2018
Citation: Zhu, Y., Saltzgiver, M.J., Zhao, J. 2018. A phenotyping protocol for detailed evaluation of apple root resistance responses utilizing tissue culture micropropagated apple plants. American Journal of Plant Sciences. 9(11):2183-2204. https://doi.org/10.4236/ajps.2018.911158.
DOI: https://doi.org/10.4236/ajps.2018.911158

Interpretive Summary: For better exploiting the innate resistance to combat soilborne diseases, it is critical to elucidate the genetic controls and molecular mechanisms in root tissue as they are challenged with soilborne pathogens. Along this line, the high-quality phenotypes of root resistance responses are essential for unravelling the underlying resistance mechanisms. However, phenotyping root resistance responses is a more challenging task compared to that for plant aerial parts, because of the hidden nature and small stature of fine roots where infection occurs. For rosaceae tree crops such as apple, an extra long-standing barrier is that apple seed germination will not produce genetically identical apple plants. This is due to the self-incompatible (or outcross) of apple reproduction and the high-level heterozygosity of apple genome. The lack of clean and healthy root tissues for the specific genotypes hinders the progress of phenotyping apple root resistance traits. Here we report an established phenotyping protocol which includes a streamlined tissue culture procedure for in vitro propagating uniform apple plants, standardized inoculation procedure using Pythium ultimum, and multilayered evaluating methods on apple root resistance traits. The consistent availability of these uniform apple plants with defined genetic background, equivalent age and non-contaminated root system set the foundation for systematic and detailed phenotypic characterization of apple root resistance traits. Multiple resistance traits such as overall plant survival rates, shoot and root biomass reduction, maximum root lengths, leaf number and cumulative leaf areas can be reliably quantified by repeated infection assays. With the assistance of a dissecting microscope and using a glass-box pot, the genotype-specific resistance responses along the infected apple roots were captured and analyzed for the first time. This reported phenotyping protocol provided unprecedent accessibility of apple root for continuous and non-disruptive evaluation on the necrosis progression patterns, and with minimized invasiveness. This phenotyping protocol represents the significant contribution to methodology in investigating root resistance traits of perennial tree fruit crop.

Technical Abstract: In the post-genomics era, reliable phenotypes are considered the bottleneck for unraveling the genetic control over a biology of interest. Phenotyping resistance responses 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. 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. Genotype-specific plant survival rates, shoot and root biomass reduction, maximum root lengths, leaf number and cumulative leaf areas can be consistently quantified by repeated infection assays. 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.