Plant regeneration via direct somatic embryogenesis from leaf and petiole explants of Epipremnum aureum 'Marble Queen' and characterization of selected variants

Leaf and petiole explants of Epipremnum aureum 'Marble Queen' were cultured on Murashige and Skoog basal medium containing three concentrations of either N-(2-chloro-4-pyridl)-N'-phenylurea (CPPU) or N-phenyl-N'-1, 2, 3-thiadiazol-5-ylurea (TDZ) with 1.07 mu M alpha-naphthalene acetic acid (NAA). Somatic embryos appeared directly from explants after 4-6 weeks of culture. TDZ at 4.54 mu M with 1.07 mu M NAA induced 75% of leaf explants to produce somatic embryos and 62.5% of explants to produce germinated embryos. Both 8.07 mu M CPPU and 9.08 mu M TDZ, respectively, with 1.07 mu M NAA induced 100% of petiole explants to produce somatic embryos and 78.1 and 91.7% of explants to produce germinated embryos. Plantlets with completely green, variegated, and whitish leaves were identified among regenerated individuals. Some variegation patterns differed greatly from the parent 'Marble Queen' within the variegated plants. Flow cytometry analysis of stock plants and selected variants showed that all plants had one identical peak. Analysis of simple sequence repeats amplified from 14 universal chloroplast primer pairs showed no variation between stock plants and selected variants. RT-PCR analysis of EaZIP, a marker gene involved in leaf variegation of E. aureum 'Golden Pothos', suggested little expression difference between green and whitish plants or between green and whitish sectors of a variegated leaf. However, SDS-PAGE analysis of proteins showed differences in band intensity and patterns. These results suggest that mechanisms underlying leaf variegation of 'Marble Queen' differ from the closely related 'Golden Pothos'. The established regeneration system and identified variants could be important materials for further investigation of leaf variegation in E. aureum.