Study of the amount of oxidative damage to mitochondrial and chloroplast DNA in clones of white poplar (Populus alba L.) during long-term in vitro cultivation for 26years.

Abstract

Measurement of the amount of damage in non-nuclear DNA can be a convenient method for assessing the senescing of clones in vitro during long-term cultivation. In vitro conservation technology is used to preserve the valuable gene pool of forest woody plants. It is important to maintain genetic stability of the collection material. However, in the course of long-term subculturing, genetic variability of cells and tissues can accumulate, and morphological signs of culture aging can appear. It is likely that this is due to the use of various hormones in nutrient media. We have been supporting in vitro cultures of diploid and triploid clones of white poplar (Populus alba L.) in a hormone-free media for 26 years. We show that in 26-year-old clones, in comparison with 5-year-old clones, the amount of oxidative damage to mitochondrial DNA (mtDNA) and chloroplast DNA (chDNA) increases. In addition, there is an age-related decrease in the number of chDNA copies. Despite this, 26-year-old poplar clones retain regenerative activity, the ability of microshoot rooting, and normal growth, and there were no visible markers of ontogenetic aging. The clones also retained the ploidy typical for the mother tree. We hypothesize that the absence of phenotypic manifestations of mtDNA and chDNA damage is associated with optimal cultivation conditions, while stressful conditions could result in these damages. Thus, we show that the measurement of the amount of damage in non-nuclear DNA, using long-range PCR, can be a convenient method for assessing the senescing of clones in vitro during long-term cultivation; this approach is more sensitive and more accurate than morphological and cytogenetic methods.