Acid scarification as a potent treatment for an in vitro germination of mature endozoochorous Vanilla planifolia seeds

Abstract

Background: Vanilla planifolia is the most widely cultivated species of vanilla with high economic importance. However, seed germination under artificial conditions is difficult and yields low germination percentages. The seeds are adapted to endozoochorous dispersal, and we therefore tried to simulate the conditions in the digestive tract by acid scarification of seeds.

Results: Immature seeds lacking dormancy, used as a control, showed the highest germination percentage. Among the treatments tested for mature seeds, the hydrochloric acid treatments were significantly the best in breaking dormancy and inducing germination, irrespective of the acid concentration and the presence of pepsin. Conventional treatment with a hypochlorite solution induced much lower germination percentage. Sulphuric acid at concentration 50% was too strong and caused damage to the seeds. Important factor is also high cultivation temperature 30 °C as there was nearly no germination at 25 °C.

Conclusions: Our protocol significantly improves the efficiency of generative propagation of vanilla and allows for significantly higher germination percentages than previously described. The strongly positive effect of hydrochloric acid may be related to the adaptation of seeds to endozoochorous dispersal.

Keywords: Acid scarification; Calcium hypochlorite; HCl; Hydrochloric acid; In vitro cultivation; Orchid; Seed germination; Sulfuric acid; Vanilla planifolia.

Fig. 1

Germination percentage (A) and protocorm size (B) of Vanilla planifolia after different treatments of seeds measured 5 months after sowing. Immature seeds excerpted from surface sterilized unripe pods served as a positive control as they lack dormancy. Mature seeds treated with chlorinated lime represent conventional treatment published before. The cultures were incubated asymbiotically on medium BM1 at 30 °C in dark. Pepsin was used in concentration 0.5 g/100 ml. Different letters indicate significantly different groups of data according to the result of post-hoc multiple comparison test (α = 0.05). Boxplots compose of whiskers (minimum and maximum), edges of the box (lower and upper quartiles), dividing line (median) and points (outliers). Number of measured objects and other statistics can be found in Additional file 2: Table S1. Only those experimental variants that yielded a reasonable number of protocorms are included in (B)

Fig. 2

Representative protocorms of Vanilla planifolia after different treatments of seeds. Cultures were incubated for 5 months on BM1 medium at 30 °C in dark. Mature seeds were disinfected with solutions of HCl (A–D), solutions of H₂SO₄ (E—Immature seeds excerpted from surface sterilized unripe pods served as a positive control as they lack dormancy (J). The seed treatments were (in the same order as in Fig. 1A): 30 min 0.1 M HCl (A), 30 min 0.1 M HCl + pepsin 0.5 g/100 ml (B), 4 h 0.1 M HCl + pepsin 0.5 g/100 ml (C), 15 min 35–38% HCl (D), 3 min 50% H₂SO₄ (E), washed in 50% H₂SO₄ (F), 5 min 50% H₂SO₄ (G), 5 min 96% H₂SO₄ (H), 30 min Chlorinated lime 66 g/l (I). Scale bars 1 cm