4.4. DNA Extraction and Southern Blot Analysis of Telomeric Repeats

Immediately after collection, the 300–500-mg samples of the ETs, mature somatic embryos or immature ZE were frozen in plastic bags in liquid nitrogen and stored at −80 °C. Total genomic DNA was isolated from the samples by the modified method of Lodhi et al. [43], as described [44,45]. DNA analysis was performed using Southern blot hybridization, as described by Kilian et al. [14], with the modifications described by Aronen and Ryynänen [9,10]. For the positive control, a synthetic telomere sequence was generated by PCR according to Cox et al. [46], using oligomer primers T1 (5′-TTTAGGG-3′) and T2 (5′-CCCTAAA-3′). Chemiluminescence detection of the hybridization products was performed according to the manufacturer’s (Roche Diagnostics GmbH) instructions. The output was then scanned with the AlphaImager Imaging System (Alpha Innotech Co./ProteinSimple, San Jose, CA, USA), and the size of the signals was analyzed using AlphaEase^®FC software and digoxigenin labeled marker for molecular weight (MW). Only high molecular weight signals representing true telomeres at the end of the chromosomes were analyzed, i.e., clearly separate low molecular weight signals originating from centromeric or interstitial repeats observed in conifers [9] were not measured.