Evaluation of somaclonal variation of Arnebia pulchra (Boraginaceae) calli versus seeds using ISSR markers

This is the first report on the analysis ISSR markers for assessing the genetic variation in Arnebia pulchra. As the rate of cell division and propagation in A. pulchra is very low, the achievement of biomass in this valuable plant requires techniques and strategies that can guarantee the survival of the callus. The present study was performed to investigate genetic variation in two in vitro culture samples of the medicinal plant A. pulchra including new (one month old) and old (four years old) calli versus seeds by Inter Simple Sequence Repeat (ISSR) marker. In total, 79 bands were produced by 10 ISSR primers. The total expected heterozygosity (He) was calculated 0.028 and the greatest value was contributed to seeds samples (0.054) followed by old and new calli, respectively. The maximum values of different alleles (Na), effective alleles (Ne) and Shannon’s index were also observed in seeds. Cluster analysis was performed in the form of dendogram to indicate the genetic stability of the samples. UPGMA tree discriminated samples in two major groups and principal component analysis (PCoA) confirmed the clustering result. The first major cluster included seeds and the second involved new and old calli. Although it was thought that the morphological changes of A. pulchra could be due to the somaclonal variation caused by successive subcultures, however, the results indicated that the new and old calli were clustered in the same group. Therefore, it can be concluded that the morphological changes of the old and new calli could be due to environmental parameters or epigenetic changes but not directly due to somaclonal variation.