Optimizing Polyploidization of In Vitro-grown Prunus x cistena

Interspecific crosses generally lead to offspring with reduced fertility and can limit further breeding efforts. Polyploidization of allodiploid hybrids to generate amphidiploids is a popular method to restore balanced chromosome pairing during meiosis and increase fertility. Prunus x cistena (purpleleaf sand cherry; P. pumila var. besseyi x P. cerasifera var. atropurpurea) is a popular, near sterile, allodiploid landscape shrub which is very cold hardy and has attractive dark purple foliage.  Polyploidization agents (e.g., colchicine, oralyzalin, and trifluralin) can cause significant stress to plant tissues and high mortality rates are common. Trehalose is a non-reducing disaccharide suspected to play a role in stress responsive plant activities. The objective of this study was to optimize polyploid recovery of purpleleaf sand cherry exploring different durations of trifluralin (a,a,a,-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) exposure and the effect of the disaccharide trehalose in reducing plant stress and impacting continued growth. Longer exposure times and lower concentrations of the trifluralin were expected to reduce the occurrence of mixaploids. Trifluralin was incorporated into hormone free Murashige and Skoog (MS) media at 0.01% for exposures of 0, 2, 4, 7, and 10 days. Trehalose at 0.005% was either present or absent during treatment or for a period of three weeks post treatment in MS media containing 2 µM benzyladenine (BA) as a factorial arrangement on doubling treatments. Recently propagated shoots at 7 days post subculture were completely submerged in the solid media treatments. Trehalose availability during exposure to doubling treatments had a significant negative effect (P = 0.05) on the shoot proliferation rate while no significant effect was observed when added in the recovery medium.  Samples for flow analysis were taken from two to three well-developed leaves from two to three newly formed axillary shoots developed on a single treatment sample. Flow cytometric analysis of recovered plantlets showed that 2 days of exposure were sufficient for induction of tetraploid plantlets. Treatments between 4 and 7 days of exposure had increased peak tetraploid fluorescence levels meaning fewer diploid tissues were screened during the sampling process. Higher proportions of diploid plant sampling and higher propagation rates arising from treatments void of trehalose during trifluralin exposure suggest higher diploid or mixaploid escapism rates.  The results support narrowing exposure window of in vitro grown P. x cistena shoots between 4 and 7 days with the use of trehalose.