The use of ligninase enzymes to target the scarification of the seed coat: application for the propagation of the orchids Himantoglossum adriaticum and Anacamptis morio.

Seed dormancy may be associated with the presence of a rigid and impermeable integument that consists for the most part of lignin. Traditional methods for the scarification of the seed coat use corrosive chemicals, such as sulfuric acid or sodium hypochlorite, that can induce collateral effects on the embryo. In natural ecosystems wood/lignin is broken down by ligninase enzymes produced by fungi, and this is of particular relevance to the germination and symbiotic development of certain groups of plants such as orchids. Here we analyze the hypothesis that ligninases derived from fungi can be used to break the seed coat and stimulate germination, with particular reference to wild orchids of semi-natural grasslands. In an initial experiment the enzyme laccase was administered to seeds of Himantoglossum adriaticum and Anacamptis morio under sterile conditions in vitro, using two methods: 1). incorporation of the enzyme directly into the agar substrate using cool filtration, or 2). "bathing" the seeds after sowing with the addition of a sterile solution of the enzyme. In both cases a concentration of 1 unit of active enzyme per seed batch/Petri dish was used. In a second experiment germination of A. morio was compared on substrates containing one of three different ligninase enzymes (laccase, lignin peroxidase and manganese peroxidase) added to the substrate at a concentration of 0.04 U/Petri (the highest achievable concentration within cost constraints – while laccase is inexpensive, lignin peroxidase and manganese peroxidase cost around two hundred times the price of gold). In the first experiment, after six months the final rate of germination for both species was double that of the controls, only when laccase was added to the substrate (in the case of A. morio from 25 to 50 %, while for H. adriaticum from 2 to 5 %; statistically significant at the P ≤ 0.05 level). In contrast, the “bathing” treatment significantly reduced germination compared to the control and also introduced contamination. In the experiment to compare enzymes, no treatment means showed significant differences from the control. In conclusion, lignin peroxidase and manganese peroxidase are too costly to allow useful amounts of enzyme to be applied. However, as laccase is inexpensive larger amounts can be applied and were found to be effective. The germination rates obtained may appear low, but for species that produce thousands of seeds per fruit such as orchids a doubling in germination can result in a significant increase in terms of the number of plants produced. The use of ligninases, particularly laccase, in the propagation of rare species thus appears to be highly promising because the intervention is targeted at the seed coat without the risk of potential complications during the development of the embryo.