Analysis of ancient DNA from in vitro grown tissues of 1600-year-old seeds revealed the species as Anagyris foetida

Abstract

Seven ancient seeds, about 1600 years old, were found during an archaeological excavation in Asar Island which is located in south-western Turkey. These seeds were subjected to germination, in vitro callus induction and molecular characterization experiments to test the viability and plant origin of the seeds. Six of the seven seeds had viable seed components (such as cotyledons) and produced callus tissue in Murashige and Skoog medium supplemented with 2mgl(-1) 6-benzylamino purine (BAP), 0.2 mgl(-1) 1-naphthaleneacetic acid (NAA), 20 gl(-1) sucrose, 2mgl(-1) glycine and 7 gl(-1) agar, but the calli from these seeds failed to yield adventitious shoots. DNA samples from callus tissues produced by ancient seeds in vitro were of good quality. The internal transcribed spacer (ITS) region in nuclear DNA (nDNA) of ancient seeds was amplified successfully. The sequences from amplified ITS DNA products of six ancient seeds indicated that their ITS sequences matched those of Anagyris foetida after subjecting them to BLAST searches in international sequence databases (NCBI). A. foetida is a relict species endemic to the Mediterranean region and used as a herbal medicine. We believe that seed characteristics such as the very hard, extremely smooth and shiny testa, toxic anagyrine alkaloid content and their storage in a pot further improved the longevity of these ancient seeds.