Dataset Information

Highly Efficient Leaf Base Protoplast Isolation and Transient Expression Systems for Orchids and Other Important Monocot Crops.

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 ?M 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.

SUBMITTER: Ren R

PROVIDER: S-EPMC7917215 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Highly Efficient Leaf Base Protoplast Isolation and Transient Expression Systems for Orchids and Other Important Monocot Crops.

Ren Rui R Gao Jie J Yin Dongmei D Li Kai K Lu Chuqiao C Ahmad Sagheer S Wei Yonglu Y Jin Jianpeng J Zhu Genfa G Yang Fengxi F

Frontiers in plant science 20210215

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 ...[more]

PMID: 33659015

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Project description:Protoplasts are single cells isolated from tissues or organs and are considered a suitable system for cell studies in plants. Embryogenic cells are totipotent stem cells, but their regeneration ability decreases or becomes lost altogether with extension of the culture period. In this study, we isolated and cultured EC-derived protoplasts (EC-pts) from carrots and compared them with non-EC-derived protoplasts (NEC-pts) with respect to their totipotency. The protoplast isolation conditions were optimized, and the EC-pts and NEC-pts were characterized by their cell size and types. Both types of protoplasts were then embedded using the alginate layer (TAL) method, and the resulting EC-pt-TALs and NEC-pt-TALs were cultured for further regeneration. The expression of the EC-specific genes SERK1, WUS, BBM, LEC1, and DRN was analyzed to confirm whether EC identity was maintained after protoplast isolation. The protoplast isolation efficiency for EC-pts was 2.4-fold higher than for NEC-pts (3.5 × 106 protoplasts·g-1 FW). In the EC-pt group, protoplasts < 20 µm accounted for 58% of the total protoplasts, whereas in the NEC-pt group, small protoplasts accounted for only 26%. In protoplast culture, the number of protoplasts that divided was 2.6-fold higher for EC-pts than for NEC-pts (7.7 × 104 protoplasts·g-1 FW), with a high number of plants regenerated for EC-pt-TALs, whereas no plants were induced by NEC-pt-TAL. Five times more plants were regenerated from EC-pts than from ECs. Regarding the expression of EC-specific genes, WUS and SERK1 expression increased 12-fold, and LEC1 and BBM expression increased 3.6-6.4-fold in isolated protoplasts compared with ECs prior to protoplast isolation (control). These results reveal that the protoplast isolation process did not affect the embryogenic cell identity; rather, it increased the plant regeneration rate, confirming that EC-derived protoplast culture may be an efficient system for increasing the regeneration ability of old EC cultures through the elimination of old and inactivate cells. EC-derived protoplasts may also represent an efficient single-cell system for application in new breeding technologies such as genome editing.

Dataset Information

Highly Efficient Leaf Base Protoplast Isolation and Transient Expression Systems for Orchids and Other Important Monocot Crops.

Publications

Highly Efficient Leaf Base Protoplast Isolation and Transient Expression Systems for Orchids and Other Important Monocot Crops.

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