Abstract:Camellia oleifera Abel. is an important woody oil species; however, the shortage of rapid and industrialized seedling culture is a large constraint on the development of the tea oil industry. Somatic embryogenesis (SE) is one of the main powerful biotechnological tools for plant mass regeneration, but the largely unknown SE in C. oleifera limits the scale production of clonal plants. In this study, we described a high-efficiency SE system via direct and indirect pathways in C. oleifera and investigated the eff… Show more
“…Protocols for the isolation and purification of protoplasts from C. oleifera suspension have been reported [ 43 ]. In recent years, preliminary progress has been made in the application of biological techniques such as C. oleifera somatic embryogenesis [ 44 ]. However, only a few C. oleifera cultivars induced callus suitable for protoplast isolation, which limited the application of callus and suspension cell lines protoplast isolation system to other C. oleifera cultivars.…”
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.
“…Protocols for the isolation and purification of protoplasts from C. oleifera suspension have been reported [ 43 ]. In recent years, preliminary progress has been made in the application of biological techniques such as C. oleifera somatic embryogenesis [ 44 ]. However, only a few C. oleifera cultivars induced callus suitable for protoplast isolation, which limited the application of callus and suspension cell lines protoplast isolation system to other C. oleifera cultivars.…”
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.
“…Another explanation for the reduced tissue culture period in this protocol is that the QuickCorn media used in this work induce direct somatic embryogenesis. In the direct somatic embryogenesis process, somatic embryos can form from the explant without the formation of an intermediate callus phase ( Raghavan, 1986 ; Zhang et al, 2021 ). It is likely that this improved protocol avoids the conventional callus induction and proliferation steps required by the previous B104 transformation protocol ( Raji et al, 2018 ).…”
For maize genome-editing and bioengineering, genetic transformation of inbred genotypes is most desired due to the uniformity of genetic background in their progenies. However, most maize inbred lines are recalcitrant to tissue culture and transformation. A public, transformable maize inbred B104 has been widely used for genome editing in recent years. This is primarily due to its high degree of genetic similarity shared with B73, an inbred of the reference genome and parent of many breeding populations. Conventional B104 maize transformation protocol requires 16–22 weeks to produce rooted transgenic plants with an average of 4% transformation frequency (number of T0 plants per 100 infected embryos). In this Method paper, we describe an advanced B104 transformation protocol that requires only 7–10 weeks to generate transgenic plants with an average of 6.4% transformation frequency. Over 66% of transgenic plants carried CRISPR/Cas9-induced indel mutations on the target gene, demonstrating that this protocol can be used for genome editing applications. Following the detailed and stepwise procedure described here, this quick and simplified method using the Agrobacterium ternary vector system consisting of a T-DNA binary vector and a compatible helper plasmid can be readily transferable to interested researchers.
“…Direct and indirect somatic embryogenesis pathways can occur in the same explant, but the periods of obtaining regenerated plants differ ( Zhang et al, 2021 ). Compared with the direct somatic embryogenesis pathway, the indirect pathway has a longer period to regenerate plants due to the callus-induction process.…”
Section: Pathways Of Plant Regeneration In Tissue Culturementioning
Plant regeneration occurs when plants repair or replace damaged structures based on the totipotency and pluripotency of their cells. Tissue culture is one of the most widely used regenerative technologies. Recently, a series of breakthroughs were made in the study of plant regeneration. This review summarizes two regenerative pathways in tissue culture: somatic embryogenesis and de novo organogenesis. Furthermore, we review the environmental factors influencing plant regeneration from explant sources, basal culture medium, plant growth regulators, and light/dark treatment. Additionally, we analyse the molecular mechanisms underlying two pathways. This knowledge will promote an understanding of the fundamental principles of plant regeneration from precursor cells and lay a solid foundation for applying plant micropropagation and genetic modification.
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