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A critical step during  in vitro  sexual propagation of terrestrial orchids is the treatment of the microscopic seeds with a disinfecting solution that kills bacteria and fungi attached to the seeds. This treatment is necessary to prevent infection of the culture vessels. At the same time, the treatment serves to scarify the seeds, a process that disrupts seed dormancy and initiates germination. The literature is inconclusive with respect to the proper combination of disinfecting solution strength and treatment duration. Both factors should be adapted to each species to guarantee minimal infection rate without damaging the embryo. This research aims to compare three disinfection/scarification methods for seeds of  Anacamptis laxiflora  (Lam.): (i) soaking in 0.5% NaClO, (ii) soaking in 0.5% NaClO, then centrifugation, and (iii) presoaking the seeds in sucrose solution, then soaking in 0.5% NaClO. The seeds were soaked in the disinfecting solution for 5 to 85 min. Following scarification, the seeds were sown in modified Malmgren nutrient medium. Infected and germinated vessels were counted at 41 and 189 d after sowing. We found that the longer the chemical treatment, the lower the infection rate, and the higher the germination rate. There was no significant difference in germination rate between the NaClO and the NaClO-plus-centrifugation method; in fact, the slight savings in disinfection time effected by centrifugation were more than offset by the added complexity of the method. Moreover, we found that centrifugation significantly delays germination. The sucrose presoak-plus-NaClO method was superior to plain NaClO, as the sucrose stimulates the germination of microbial spores on the surface of the seeds, making them easier to kill. Perhaps seeds with thicker testa as well as whole immature capsules could benefit even more from the pretreatment in sucrose solution.

Influence of scarification method on seed germination of the terrestrial orchid Anacamptis laxiflora (Lam.)