Establishment of In Vitro Propagation System and Induction of Autotetraploidy in Monarda fistulosa and M. punctata (Lamiaceae)

Induced polyploidy offers great potential for increasing stress tolerance, biomass, essential oil production and the ornamental appeal of plants. In our research we have been evaluating native species adapted to more northerly latitudes for potential to survive in environments characterized by low water and nutrients. Monarda fistulosa and M. punctata are two species that have great potential because they may have some level of drought tolerance but they are also valued for their production of essential oils. Polyploidy has the potential to increase both of these characters. The proposed research aims to investigate how polyploidy will affect the overall growth and essential oil production of two native ornamental species, Monarda fistulosa and M. punctata. Firstly, an in vitro propagation system was required for these Monarda species. Leaf, petiole and nodal sections were used as explant donors and placed onto a solid MS media supplemented with a range of seven BA concentrations (0-25µM). Explants were transferred to fresh media every four weeks, and after twelve weeks the total number of shoots produced per explant was examined. The nodal sections produced significantly more shoots per explant compared to leaf and petiole explant donors and 25µM of BA was determined to be the optimal hormonal concentration for shoot regeneration. M. fistulosa (3.4 shoots per explant) was more responsive than M. punctata (1.5 shoots per explant). This media was then used to grow the in vitro material for the induction of polyploidy in the two species. Oryzalin and Trifluralin are both known to interrupt spindle fiber formation resulting in increased chromosome numbers. Oryzalin and Trifuralin were dissolved in DMSO and placed directly into the solidified media in a range of eight concentrations (0, 1, 5, 15, 30, 60, 90, and 120µM). The nodal sections were taken from in vitro cultures and placed directly onto petri plates containing the anti-mitotic media for a duration of 1, 3 or 6 days. The plates were placed in a growth cabinet at 16 hour day length and a day/night temperature of 25°C. After the period of exposure, explants were transferred to MS media without Oryzalin or Trifluralin and allowed to produce plantlets. Phenotypic traits such as leaf and stem thickness, plant vigour and stomatal characteristics will be used to screen for potential polyploid plants. Chromosome number will be verified through a combination of flow cytometry and root tip squashes.