Phenolic Exudation Control and Indirect Somatic Embryogenesis of Garlic-Fruit Tree (Malania oleifera Chun & S.K. Lee)-An Endangered Woody Tree Species of Southeastern Yunnan Province, China

Abstract

Malania oleifera Chun & S.K. Lee, an endemic monotypic species that belongs to the family Olacaceae, is under continuous pressure of decline owing to several ecological and physiological factors. The present study aimed to establish an efficient in vitro protocol for callus-mediated indirect somatic embryogenesis in M. oleifera by alleviating tissue browning. Internodes and leaves obtained from seedlings were used as explants. Antioxidant pre-treatment (ascorbic acid, AA) followed by different carbon sources (sucrose, maltose, glucose, and fructose) and plant growth regulators in various concentrations and combinations were employed in Woody Plant Medium (WPM) to alleviate explant browning and induce callus formation from the explants. AA pre-treatment and subsequent culture on maltose at a concentration of 116.8 mM were optimal for controlling phenolic exudation on >90% of both explants. The highest responses of 53.77% and 57.43% for embryogenic calli were induced from internode and leaf explants, respectively. The highest responses, 85.22% and 93.80%, were observed for somatic embryos that matured into the globular, heart-shaped and torpedo stages at different percentages on NAA 2.5 mg/L in combination with BA 1.0 mg/L for both explants. The matured somatic embryos were finally germinated at a maximum concentration of GA₃, 2.0 mg/L. All plantlets were successfully hardened and acclimatized under culture room conditions and then transferred to the greenhouse. The current study suggests an efficient protocol for indirect somatic embryogenesis by alleviating phenolic exudation from the explants of M. oleifera. This first successful report of in vitro culture establishment in M. oleifera may offer an effective alternative measure to conserve this species and provide a system for analyzing bioactive chemicals and for use in the oil industry.

Keywords: Malania oleifera; carbon source; endemic tree; nervonic acid; phenolic exudation; reintroduction; somatic embryogenesis.

Figure 1

Impact of various carbon sources on phenolic exudation from internode explants on WPM supplemented with different carbon sources, after 4 weeks of culture. Values represent means ± S.E.

Figure 2

Effect of different carbon sources on phenolic exudation from leaf explants on WPM supplemented with different carbon sources, after 4 weeks of culture. Values represent means ± S.E.

Figure 3

Embryogenic callus induction and somatic embryo formation in M. oleifera. (a) Induction of embryogenic callus, (b) proliferation of embryogenic callus, (c,d) somatic embryo induction and maturation (globular and heart-shaped embryos), (e) torpedo stage somatic embryo with bipolar structure, (f–i) somatic embryo germination into plantlets, (j,k) acclimatized plantlets. Scale bars: (a–d,h) = 5 mm, (e–g,i) = 1 mm, (j) = 1 cm, (k) = 2 cm.

Figure 4

Different developmental stages of somatic embryos and histological analysis of M. oleifera. (a–c) Different developmental stages of somatic embryos from internode explant derived callus, (d–f) different developmental stages of somatic embryos from leaf explant derived callus, (g) histology of globular embryo, (h) histology of heart-shaped embryo, (i) histology of cotyledonary stage embryos showing bipolar structures derived from torpedo-shaped embryo. Scale bars: (a–f) = 1 mm, (g–i) = 500 μm.