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.
Highlights
The tea oil plant (Camellia oleifera Abel.), an evergreen shrub or dungarunga, is a woody oil species worldwide
For 210-day-old zygotic embryonic axis (ZE), 85 embryogenic callus clumps were induced with an induction rate of 16.7%, while 108 embryogenic callus clumps were obtained from 220-day-old ZE with a maximum induction rate of 40.9%
The results showed that embryogenic calli were induced from the cotyledons of “HuaXin,” “Huashuo,” and “Huajin” in E1-6 medium supplemented with 1.5 mg/L 2,4-D (Supplementary Figures 2A,B) and the embryogenic callus induction rates showed no significant differences between different varieties (Supplementary Table 1)
Summary
The tea oil plant (Camellia oleifera Abel.), an evergreen shrub or dungarunga, is a woody oil species worldwide. The large demand for C. oleifera seedlings is expanding with the rapid development of the tea oil industry and has far outstripped the supply of quality seedlings. In China, approximately 73% of tea oil trees are planted with seed-germinated seedlings. Grafting techniques have been widely applied for low-yield tree transformation and seedling propagation (Ruan and Mopper, 2017). Since heterozygous seeds are primarily employed as rootstocks in the process of grafted C. oleifera seedling propagation, the poor growth consistency of the seedling is one of the major limiting factors for the supply of higher quality seedlings. Owing to the great consumption of seed rootstock and low efficiency, it is relatively difficult to provide a mass of sustainable seedlings for the C. oleifera industry by grafting propagation alone. It is essential and urgent to establish a more efficient and industrialized propagation technique system to raise C. oleifera seedlings
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