Enzymatic scarification facilitates seed coat lignin degradation, water uptake and germination

Seed integuments of many species contain hydrophobic lignins and in soil the action of ligninase enzymes produced by microorganisms may contribute to release from physical dormancy. Laboratory use of ligninases to stimulate germination is promising because of the specific action on integuments, whereas chemical scarification agents may also corrode the embryo. We hypothesized that exposure of Anacamptis morio (Orchidaceae) seeds to fungal laccase stimulates germination, and that the mechanism involves lignin degradation and increased imbibition. Germination capacity in vitro was quantified with 1U of filter-sterilized laccase added to agar medium following autoclaving, compared to a 10% bleach solution (a standard bleach surface sterilization/scarification method used in orchid seed sowing). Lignin degradation was quantified using an optical method (phloroglucinol staining) combined with image analysis, following experimental pretreatments involving immersion in laccase solution, distilled water (negative control) or bleach (positive control). Water uptake after experimental treatments was quantified as the proportion of seeds exhibiting visible uptake of an aqueous fluorochrome under UV excitation. Laccase stimulated a doubling of germination in vitro with respect to bleach surface sterilization/scarification alone, from 23.7 to 49.8% (p=0.007). Laccase and bleach methods both significantly decreased the optical signal of phloroglucinol (for laccase, to 79.9 ±1.3% of controls; ANOVA: F=10.333, p=0.002). Laccase resulted in a modest but highly significant (p<0.0001) increase in water uptake with respect to the control (11.7%; c.f. 99.4% for bleach). Laccase scarification can stimulate germination of A. morio by a mechanism of targeted seed coat degradation and increased imbibition. The results demonstrate the potential of this relatively non-invasive enzymatic scarification technique.