Li Sufang, Zhao Rui, Ye Tianwen, Guan Rui, Xu Linjie, Ma Xiaoling, Zhang Jiaxi, Xiao Shixin, Yuan Deyi
Key Laboratory of Cultivation and Protection for Non-Wood Forest Trees, Ministry of Education, Central South University of Forestry and Technology, Changsha, 410004, Hunan, China.
Key Laboratory of Non-wood Forest Products of State Forestry Administration, Central South University of Forestry and Technology, Changsha, 410004, Hunan, China.
Plant Methods. 2022 Dec 22;18(1):141. doi: 10.1186/s13007-022-00972-1.
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.
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 × 10/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 × 10/g·FW and 2.6 × 10/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.
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.
油茶是一种具有重要经济价值的木本食用油料作物。由于缺乏现代生物技术研究，油茶在育种和基础研究方面都面临着巨大挑战。原生质体和瞬时转化系统在生物育种、植物再生和体细胞融合中发挥着重要作用。本研究的目的是建立一种高效的油茶叶肉原生质体分离、纯化及瞬时转化体系。对油茶叶肉原生质体分离的几个关键因素进行了优化，包括起始材料（叶龄）、预处理、酶处理（酶的种类、浓度和消化时间）、渗透压和纯化等。然后探讨了影响叶肉原生质体瞬时转化率的因素，如聚乙二醇（PEG）分子量、PEG4000浓度、质粒浓度和孵育时间等。
将油茶‘华硕’组培苗暗处理24 h，摘取第1至第2片真叶，在-0.07 MPa下真空处理20 min。用含有1.5%（w/v）纤维素酶R-10、0.5%（w/v）离析酶R-10、0.25%（w/v）蜗牛酶和0.4 M甘露醇的酶解液对第1至第2片真叶酶解10 h时，原生质体产量最高（3.5×10⁶/g·FW），活力达90.9%。此外，该原生质体分离方法也适用于另外两个品种，‘TXP14’和‘DP47’的原生质体产量分别为1.1×10⁶/g·FW和2.6×10⁶/g·FW，‘TXP14’和‘DP47’的原生质体活力分别为90.0%和88.2%。以W缓冲液为清洗剂进行离心沉淀时纯化效果最佳。原生质体与15 μg质粒和40% PEG4000孵育20 min时，转染效率最高（70.6%）。
综上所述，本研究提出了一种简单高效的油茶叶肉原生质体分离和瞬时转化体系，这在油茶体细胞融合、基因组编辑、蛋白质功能、信号转导、转录调控及多组学分析等油茶研究的各个方面都具有重要意义。
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