Isolation, purification and PEG-mediated transient expression of mesophyll protoplasts in Camellia oleifera

Abstract

Background Camellia oleifera (C. oleifera) is a woody edible oil crop of great economic importance. Because of the lack of modern biotechnology research, C. oleifera faces huge challenges in both breeding and basic research. The protoplast and transient transformation system plays an important role in biological breeding, plant regeneration and somatic cell fusion. The objective of this present study was to develop a highly efficient protocol for isolating and purifying mesophyll protoplasts and transient transformation of C. oleifera. Several critical factors for mesophyll protoplast isolation from C. oleifera, including starting material (leaf age), pretreatment, enzymatic treatment (type of enzyme, concentration and digestion time), osmotic pressure and purification were optimized. Then the factors affecting the transient transformation rate of mesophyll protoplasts such as PEG molecular weights, PEG4000 concentration, plasmid concentration and incubation time were explored. Results The in vitro grown seedlings of C. oleifera ‘Huashuo’ were treated in the dark for 24 h, then the 1st to 2nd true leaves were picked and vacuumed at − 0.07 MPa for 20 min. The maximum yield (3.5 × 107/g·FW) and viability (90.9%) of protoplast were reached when the 1st to 2nd true leaves were digested in the enzymatic solution containing1.5% (w/v) Cellulase R-10, 0.5% (w/v) Macerozyme R-10 and 0.25% (w/v) Snailase and 0.4 M mannitol for 10 h. Moreover, the protoplast isolation method was also applicable to the other two cultivars, the protoplast yield for ‘TXP14’ and ‘DP47’ was 1.1 × 107/g·FW and 2.6 × 107/g·FW, the protoplast viability for ‘TXP14’ and ‘DP47’ was 90.0% and 88.2%. The purification effect was the best when using W buffer as a cleaning agent by centrifugal precipitation. The maximum transfection efficiency (70.6%) was obtained with the incubation of the protoplasts with 15 µg plasmid and 40% PEG4000 for 20 min. Conclusion In summary, a simple and efficient system for isolation and transient transformation of C. oleifera mesophyll protoplast is proposed, which is of great significance in various aspects of C. oleifera research, including the study of somatic cell fusion, genome editing, protein function, signal transduction, transcriptional regulation and multi-omics analyses.

Cotyledon Peeling Method for Passion Fruit Protoplasts: a versatile cell system for transient gene expression in passion fruit (Passiflora edulis)

“…In our system, the most effective transfection time for passion fruit protoplast is 5 min (Fig. 3C, D), which is consistent with the optimization results for Magnolia (Shen et al, 2017), but different from Camellia oleifera, Chinese cabbage and cassava (Wu et al, 2017;Sivanandhan et al, 2021;Li et al, 2022).…”

“…2A, B), which is consistent with that of grape and camellia protoplast. However, the plasmid DNA required for optimal passion fruit protoplast transfection was about twofold lower (Wang et al, 2015;Zhao et al, 2016;Li et al, 2022). In this study, we found that a passion fruit protoplast density of 1.0 × 10 6 cells/ml resulted in the best transformation efficiency (Fig.…”

Efficient isolation and purification of tissue-specific protoplasts from tea plants (Camellia sinensis (L.) O. Kuntze)

“…Protocols for the isolation and purification of protoplasts from tea leaves have been reported [12,13]. However, the separation efficiency remains relatively low and experimental replicability is poor; moreover, these protocols do not apply to the preparation of protoplasts from the roots and branches of tea plants.…”

“…All purification operations were conducted at 4 °C. Compared the previous results from Liu et al [12] and Peng et al [13], the procedures of protoplast isolation and purification were optimized with vacuum assisted treatment for improving enzymolysis efficiency, and only once rinsing.…”

“…It is acknowledged that the lack of effective and sustainable methods for preparing tea plant protoplasts have seriously limited the development of tea biology. Notably, poor results have been reported for protoplast preparation from tea plants, as indicated by low efficiency, yield, and viability of specific tissues [12,13]. A rapid, effective, and sustainable system for the isolation and purification of protoplasts from tea plants has yet to be established.…”