Direct and Indirect Somatic Embryogenesis Induction in Camellia oleifera Abel

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 effect of genotype, explant age and phytohormones on SE. In the direct pathway, somatic embryos were highly induced from immature seeds 220 days after full blossom, and the development of embryoids was achieved with a combination of 0.19 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg/L thidiazuron (TDZ). In the indirect pathway, embryogenic calli were induced from the same explants in medium containing 1.5 mg/L 2,4-D, while 0.75 mg/L 2,4-D treatment led to high proliferation rates for embryogenic calli. The addition of 0.19 mg/L 2,4-D alone stimulated the production of globular embryos while causing a 75% loss of the induction rate in the heart embryo stage. Upon transfer of the globular embryos to phytohormone-free medium, an optimal induction rate of 62.37% from globular embryos to cotyledonary embryos was obtained. These data suggest that the subsequent differentiation process after the globular embryo stage in ISE is more similar to an endogenous phytohormones-driven process. Mature embryos germinated to produce intact plantlets on half-strength MS basal medium with a regeneration rate of 63.67%. Histological analysis confirmed the vascular bundle isolation of embryoids from the mother tissue. We further studied the different varieties and found that there were no significant genotype differences for SE induction efficiency in C. oleifera. Thus, we established a high-efficiency induction system for direct and indirect somatic embryogenesis (ISE) in C. oleifera and regenerated intact plantlets via SE, not organogenesis. ISE has a more complicated induction and regulatory mechanism than direct somatic embryogenesis. The improved protocol of SE would benefit mass propagation and genetic manipulation in C. oleifera.

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

“…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.…”

An Improved Agrobacterium-Mediated Transformation and Genome-Editing Method for Maize Inbred B104 Using a Ternary Vector System and Immature Embryos

“…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 ).…”

New Insights Into Tissue Culture Plant-Regeneration Mechanisms

“…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.…”