Quantitative Evaluation of Hybrid Aspen Xylem and Immunolabeling Patterns Using Image Analysis and Multivariate Statistics

Abstract

A new method is presented for quantitative evaluation of hybrid aspen genotype xylem morphology and immunolabeling micro-distribution. This method can be used as an aid in assessing differences in genotypes from classic tree breeding studies, as well as genetically engineered plants. The method is based on image analysis, multivariate statistical evaluation of light, and immunofluorescence microscopy images of wood xylem cross sections. The selected immunolabeling antibodies targeted five different epitopes present in aspen xylem cell walls. Twelve down-regulated hybrid aspen genotypes were included in the method development. The 12 knock-down genotypes were selected based on pre-screening by pyrolysis-IR of global chemical content. The multivariate statistical evaluations successfully identified comparative trends for modifications in the down-regulated genotypes compared to the unmodified control, even when no definitive conclusions could be drawn from individual studied variables alone. Of the 12 genotypes analyzed, three genotypes showed significant trends for modifications in both morphology and immunolabeling. Six genotypes showed significant trends for modifications in either morphology or immunocoverage. The remaining three genotypes did not show any significant trends for modification.

Anatomical and Immunocoverage Observations on SuSy, C4H, and Pectate Lyase Family Protein Down-regulated Aspens Genotypes

“…Kanamycin-resistant lines were clonally propagated in vitro and planted in a greenhouse. (Bjurhager et al 2010); c (Sandquist et al 2015).…”

“…Each tree has one knocked down gene each, abbreviated as KR112 (sucrose synthase, Sjödin et al 2009), KR139 (cinnamate 4-hydroxylase, Gerber et al 2014 and K213 (pectate lyase coding genes, Sandquist et al 2015) in comparison with their reference wild type control (T89).…”

“…This has been achieved using quantitative image analysis of transverse sections (Sandquist et al 2015) and macerated fibers, UV-microscopy and histochemical staining of syringyl-and guaiacyl lignin micro-distributions, and ultrastructural observations on changes in fiber wall structure as determined using TEM.…”

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This study aims to complement previous studies on down-regulation of sucrose synthase and cinnamate 4-hydroxylase in hybrid aspen (Bjurhager et al 2010;Gerber et al 2014) with a quantitative anatomical comparison (Sandquist et al 2015). The main focus was placed on evaluating quantitative modifications to fiber morphology and lignin composition.

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“…Overall there was good agreement between the morphological results in this study compared to the previously published chemical and physical reports, particularly on cinnamate 4-hydroxylase down-regulated aspen. The previously reported statistical correlations between fiber lumen area and plant diameter, and fiber length and tree height (Sandquist et al 2015) have been further refined. Knock-down of genes coding for biologically important proteins such as sucrose synthase and cinnamate 4-hydroxylase had broad spanning morphological impacts.…”