Production of synthetic tetraploids as a tool for polyploid research

Polyploidization has been traditionally considered a phenomenon that mediates ecological differentiation; however, the adaptive value of genome duplications has been seldom explored. Natural or synthetic polyploids offer unique opportunities to experimentally quantify the immediate consequences of genome duplications in plant traits that may be involved with ecological differentiation. Jasione maritima is a diploid-tetraploid complex with cytotypes distributed allopatrically in an environmental gradient ideal for exploring the role of genome duplications in the success of polyploid lineages. However, like in numerous other polyploid complexes, neotetraploids of J. maritima have not been found in nature. In this study we developed a methodology to obtain synthetic tetraploids from seeds collected in wild diploid plants of J. maritima. We tested the effect of different colchicine concentrations and seedling ages in survival and polyploidization success of J. maritima seedlings, using flow cytometry to assess ploidy level and considering the population of origin. A methodology to synchronize seed germination was also explored. Synchronization of seed germination was best achieved using a cold treatment of 2 weeks before the transference to a growth chamber. An overall survival rate of 11.5±0.7 % and further tetraploid conversion of 35.6±2.9 % was obtained using 3-day-old seedlings of J. maritima. Survival rates were variable depending on colchicine treatment (the highest the most lethal) and seedling origin (i.e. population), while conversion rate was similar across these factors and high rates of tetraploid conversion were obtained. Considering that the main constraint was survival to the colchicine treatment, we suggest the use of the lowest colchicine concentration tested, i.e. 0.1 % colchicine. The use of older seedlings increased survival rates but, in many cases, compromised a complete tetraploid conversion, generating many diploid-tetraploid mixoploid plants.