Abstract
Versatile protoplast platforms greatly facilitate the development of modern botany. However, efficient protoplast-based systems are still challenging for numerous horticultural plants and crops. Orchids are globally cultivated ornamental and medicinal monocot plants, but few efficient protoplast isolation and transient expression systems have been developed. In this study, we established a highly efficient orchid protoplast isolation protocol by selecting suitable source materials and optimizing the enzymatic conditions, which required optimal D-mannitol concentrations (0.4–0.6 M) combined with optimal 1.2% cellulose and 0.6% macerozyme, 5 μM of 2-mercaptoethanol and 6 h digestion. Tissue- and organ-specific protoplasts were successfully isolated from young leaves [∼3.22 × 106/g fresh weight (FW)], flower pedicels (∼5.26 × 106/g FW), and young root tips (∼7.66 × 105/g FW) of Cymbidium orchids. This protocol recommends the leaf base tissues (the tender part of young leaves attached to the stem) as better source materials. High yielding viable protoplasts were isolated from the leaf base of Cymbidium (∼2.50 × 107/g FW), Phalaenopsis (1.83 × 107/g FW), Paphiopedilum (1.10 × 107/g FW), Dendrobium (8.21 × 106/g FW), Arundina (3.78 × 106/g FW) orchids, and other economically important monocot crops including maize (Zea mays) (3.25 × 107/g FW) and rice (Oryza sativa) (4.31 × 107/g FW), which showed marked advantages over previous mesophyll protoplast isolation protocols. Leaf base protoplasts of Cymbidium orchids were used for polyethylene glycol (PEG)-mediated transfection, and a transfection efficiency of more than 80% was achieved. This leaf base protoplast system was applied successfully to analyze the CsDELLA-mediated gibberellin signaling in Cymbidium orchids. We investigated the subcellular localization of the CsDELLA-green fluorescent protein fusion and analyzed the role of CsDELLA in the regulation of gibberellin to flowering-related genes via efficient transient overexpression and gene silencing of CsDELLA in Cymbidium protoplasts. This protoplast isolation and transient expression system is the most efficient based on the documented results to date. It can be widely used for cellular and molecular studies in orchids and other economically important monocot crops, especially for those lacking an efficient genetic transformation system in vivo.
Highlights
Protoplasts are plant cells from which the cell walls have been enzymatically removed (Eeckhaut et al, 2013)
Leaf tissues are the most commonly used source materials, and high yielding mesophyll protoplasts can be isolated from tobacco (Nagata and Takebe, 1971), Arabidopsis (Menczel, 1980), maize (Z. mays L.) (Kanai and Edwards, 1973), rice (Oryza sativa L.) (Toriyama and Hinata, 1985), and many other non-model plant species, such as Medicago sativa (Song et al, 1990), Panicum virgatum (Mazarei et al, 2008), Elaeis guineensis (Masani et al, 2014), Hevea brasiliensis (Zhang et al, 2016b), Phaseolus vulgaris (Nanjareddy et al, 2016), and Magnolia (Shen et al, 2017)
We found that the D-mannitol concentration significantly affected the efficiency of protoplast isolation
Summary
Protoplasts are plant cells from which the cell walls have been enzymatically removed (Eeckhaut et al, 2013) Protoplast-based platforms allow creation of new plant species via protoplast fusion and regeneration (Melchers et al, 1978; Grosser and Gmitter, 1990). They explore the signal transduction and metabolic pathways transiently respond to hormones and stress factors (Sheen, 2001; Hirata et al, 2012), answer specific questions related to cell types (Petersson et al, 2015; Denyer et al, 2019), and determine the subcellular localization, transport, and interactions of tagged proteins (Goodman et al, 2004; Zhang et al, 2011). Flowers withered within a short period, and petals are not sufficient to supply continuously for protoplast isolation
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