Sampling cambial region and mature xylem for non structural carbohydrates and starch analyses

Abstract

This paper describes a procedure to analyze non-structural carbohydrates (NSC) and starch in xylem and cambial region extracted by the scraping method. An application on woody logs of 2-year-old Populus × canadensis Mönch ‘I-214’ during tree-ring formation is reported. Samples are freeze-dried in liquid nitrogen and, successively, the bark is removed. After drying, the breaks between bark and xylem take place in correspondence of differentiating xylem cells. The cambial region with the differentiating phloem and xylem is gently scraped with a razor blade from the inner side of the bark and the outermost side of the stem, respectively. Additionally the mature xylem is milled until obtaining a powder. The amount of the cambial region collected by scraping changes with respect to the width of the differentiating zone (differentiating phloem and xylem), with higher amount of dry matter obtained during the maximum rate of xylem formation and high stem water content. After extraction of the powder in an ethanol solution and centrifugation, the supernatant and the resulting pellet are used for assessing the contents of non structural carbohydrates (NSC) and starch, respectively. NSC are determined using the High-Performance Liquid Chromatography (HPLC) analysis and starch by colorimetric method. In both cambial region and xylem, the major NSC are glucose and fructose which represented together more than 80% of the total NSC during wood formation. However, the total NSC are higher in cambial region compared to xylem, especially when the xylem and phloem are differentiating. The scraping technique provides the possibility to sample cambial region during the year, allowing the investigation of metabolic changes during tree-ring formation.