In Vitro Chromosome Doubling of Prunus laurocerasus 'Otto Luyken'

Prunus laurocersaus (2n=22x=176), common cherrylaurel, is an evergreen shrub often used as a hedge or screen.  This species has become naturalized in parts of Northwestern U.S., which has lead to concern regarding its invasive potential.  Therefore, a sterile form of common cherrylaurel would be useful in curbing its escape from cultivation.  As part of a breeding program to develop sterile common cherrylaurel cultivars we attempted to induce polyploidy using in vitro exposure of ‘Otto Luyken’ shoots to oryzalin.  Shoots that were 1.5 cm long with two to three meristems each were placed on solid media in 30 × 200 mm culture tubes.  Shoots were treated by covering with 10 mL of liquid MS media supplemented with oryzalin.  Each experimental unit was one culture tube containing a single shoot.  The experiment was completely randomized with 20 replications.  Shoots were treated for 1, 2, 14, or 28 days with 0, 6.25, 12.5, 25, 50, 100, or 150 μM oryzalin.  After treatment, the liquid phase was removed and shoots were transferred to fresh media.  Ploidy level of surviving shoots was determined using flow cytometry analysis of DAPI stained nuclei.  The 14 d treatment had reduced survival compared to 1 d and 2 d treatments and there was still greater mortality in the 28 d treatment.  Duration affected mortality more than oryzalin rate.  Percentage of surviving shoots that were 22x was similar among all treatment durations.  As the rate of oryzalin increased, percent 22x decreased up to 100 μM and then increased.  For the 28 d treatment, there were no 22x shoots except at 0 μM.  Percentage of surviving shoots that were mixoploid increased with rate of oryzalin for 1, 2, and 14 d to a maximum at 86, 86.5, and 85 μM, respectively, before decreasing.  The 28 d treatment had 100% mixoploid shoots at each oryzalin rate except 50 μM, at which survival was 0%.  Percentage of surviving shoots that were 44x increased with rate in the 1 and 2 d treatments. Percentage of surviving shoots in the 14 d treatment reached a maximum at 90 μM and no surviving shoots were homogenous 44x.  In addition to analyzing each tube as a whole, individual shoots were collected and the ploidy level of each was determined.  As expected, when each meristem was analyzed individually, there was a reduced number of mixoploids, as more of the separated meristems were 22x and 44x.