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

Hsiang-Yin Lin,Jhun-Chen Chen,Su-Chiung Fang

doi:10.3389/fpls.2018.00843

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.

Highlights

Orchidaceae represent one of the largest angiosperm families comprising more than 25,000 species that are grown in a wide range of habitats including rainforest, grassland, and even mangrove swamp and low arctic tundra
Petal protoplast isolation has been reported in Dendrobium orchid (Hu et al, 1998)
The release and integrity of petal protoplasts was visually inspected in cellulose- and macerozyme-containing enzyme solution adjusted to different osmotic conditions

Summary

Introduction

Orchidaceae represent one of the largest angiosperm families comprising more than 25,000 species that are grown in a wide range of habitats including rainforest, grassland, and even mangrove swamp and low arctic tundra. Orchids have distinct morphological and physiological characteristics such as the co-evolution of pollinators and distinct floral structure (Waterman and Bidartondo, 2008), lack of cotyledon development during embryogenesis (Kull and Arditti, 2002), formation of pollen dispersal units (pollinia) (Pacini and Hesse, 2002), and unique growth and development coupled with mycotrophic strategies (Rasmussen, 2002). These unique developmental programs or strategies have drawn the attention of many evolutionary and plant biologists. Despite the enormous interest in understanding the molecular mechanisms of the specialized developmental or physiological programs in orchids, the lack of a robust molecular toolkit hampers the advancement of orchid biology.

Methods

Results

Conclusion