A Protoplast Transient Expression System to Enable Molecular, Cellular, and Functional Studies in Phalaenopsis orchids

Abstract

The enigmatic nature of the specialized developmental programs of orchids has fascinated plant biologists for centuries. The recent releases of orchid genomes indicate that orchids possess new gene families and family expansions and contractions to regulate a diverse suite of developmental processes. However, the extremely long orchid life cycle and lack of molecular toolkit have hampered the advancement of orchid biology research. To overcome the technical difficulties and establish a platform for rapid gene regulation studies, in this study, we developed an efficient protoplast isolation and transient expression system for Phalaenopsis aphrodite. This protocol was successfully applied to protein subcellular localization and protein–protein interaction studies. Moreover, it was confirmed to be useful in delineating the PaE2F/PaDP-dependent cell cycle pathway and studying auxin response. In summary, the established orchid protoplast transient expression system provides a means to functionally characterize orchid genes at the molecular level allowing assessment of transcriptome responses to transgene expression and widening the scope of molecular studies in orchids.

Highly Efficient Protoplast Isolation and Transient Expression System for Functional Characterization of Flowering Related Genes in Cymbidium Orchids

“…In addition to viability and quantity of protoplasts, DNA or RNA amount, final concentration of PEG and incubation time are factors affecting transfection efficiency [41,56]. High level transfection efficiency (>80%) has been achieved using Phalaenopsis petal protoplasts [34]. This is equivalent to or better than that of previously reported transfection efficiencies [14,42].…”

“…Previous attempts to isolate higher yield and viability of protoplasts from different tissues of numerous species have been made separately under different conditions [14,34,42]. Though vacuum infiltration or treatment with high osmotic solution of plasmolyzed tissue prior to the enzymatic digestion has been recommended [14], high yielding viable protoplasts were obtained from flower petals of Cymbidium orchids without those treatments.…”

“…Nevertheless, callus induction is time consuming and impractical for protoplast isolation. Hence, flower petals have been selected as an alternative for protoplast isolation for ornamental plants such as Rosa rugose [32], Dendrobium [33] and Phalaenopsis [34]. In addition to species types and source materials, enzyme combination and the concentration of osmotic pressure regulating substance D-mannitol also affect the effective release of protoplasts [22,35].…”

“…For family Orchidaceae, protoplasts are first successful isolated from leaves of Cymbidium [36] and various tissues of Renantanda, Dendrobium and Paphiopedilum [37,38]. Leaf tissues of in vitro grown seedlings of Dendrobium (3.97 × 10 5 /g fresh weight (FW)) [39] and Cymbidium (maximum 1.1 × 10 7 /g FW) [40], and flower petals of Phalaenopsis (1.1-5.9 × 10 6 /g FW) [34,41] have been used for successfully protoplast isolation. However, the protoplast isolation efficiency is lower than that of Arabidopsis (3.0 × 10 7 ) [42], maize (1-5 × 10 6 /g FW) [43], rice (1.0 × 10 7 ) [44] and cassava (4.4 × 10 7 /g FW) [45].…”

“…Protoplast-based transient expression system (PTES) has been proven to be a powerful experimental tool in molecular biology. It is widely used for protein localization, protein-protein interactions, identification of the gene function and gene regulation [14,34,44,[46][47][48][49]. Transient protoplast transfection has been used due to its advantages in high screening efficiency with gene silencing and genome editing technologies, prior to the development of transgenic plants [43,[50][51][52].…”