Genome Status of Lippia alba Polyploid Complex Long-term in vitro Cultivated

This study is the first report of a genetic stability analysis of a polyploid complex maintained in vitro for a long-time. Twenty-two accessions of Lippia alba, a medicinal species of economic importance, had been maintained under in vitro culture conditions for 7 years through sprouting of axillary buds. Four clones of each accession were analyzed, being three plants from in vitro bank and one cultivated in the field. We investigated the genetic stability of diploid, aneuploid, triploid, tetraploid, and hexaploid accessions. The investigation was carried out using flow cytometry, inter simple sequence repeat (ISSR) and simple sequence repeat (SSR) markers. No significant variation in nuclear DNA content was observed between the in vitro conserved plants and their respective field plant. Out of 23 ISSR primers screened, 8 primers were found to produce clear reproducible bands resulting in a total of 5456 bands. 86.36% of the analyzed plantlets (19 accessions) showed at least one polymorphic band. The polymorphic rate ranged from 1.61 to 33.87%. The SSR markers were used to confirm the absence or low occurrence of variation in accessions that showed no polymorphism or polymorphism for only one ISSR primer. The genetic instability detected in our study at the molecular level may be attributed to the natural instability of L. alba genome combined with the long-time in vitro maintenance.