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.

Camellia oleifera is an important woody edible oil crop in China with high ecological and economical values. It is a traditional oilseed crop with high levels of desirable fatty acids. The fruits of C. oleifera were harvested from 18 geographic provenances in southern China. In this paper, we analyzed the key environmental factors of diverse geographic provenances that caused the variation in the fruit morphology and fatty acid composition (FAC). Our study indicated an average coefficient of variation of fruit width (FW) of 18.63%, and 15.81% for fruit length (FL). The most abundant fatty acids (FA) were oleic acid (C18:1; 70.21–85.23%), followed by palmitic acid (C16:0; 6.93–13.89%) and linoleic acid (C18:2; 5.02–14.26%). In addition, the fruit width had a negative correlation with the equivalent latitude (ELAT) and a positive correlation with the annual mean air temperature (MAT). The fruit length-to-width ratio and oleic acid level had a positive correlation with ELAT but a negative correlation with MAT, annual precipitation (AP), and precipitation of wettest quarter (PWQ). A positive correlation was observed between MAT, AP, and PWQ with palmitic acid. Meanwhile, a negative correlation was found between longitude (LON), maximum temperature of warmest month (MTW), and ELAT and palmitic acid. The cluster analysis indicated six groups for the selected 18 populations. Our results showed the most influential environmental factors for variation in fruit morphology and FAC are ELAT and MAT.