Outbreeding and asymbiotic germination in the conservation of the endangered Italian endemic orchidOphrys benacensis

The Insubric bee orchid, Ophrys benacensis (Reisigl) O.&E. Danesch & F. Ehrend, occurs in fragmented populations only in northern Italy, and suffers from inbreeding depression. We found that pollen from a depressed population at Monte Barro, Lecco, did not fertilise plants of the same population, nor of a larger population at nearby Valmadrera. Fertilisation was successful at both sites when pollen from Valmadrera was used, although the proportion of seeds containing embryos was almost six times greater at Valmadrera. Embryos produced by both populations were equally likely to develop in vitro Sowing seed on medium enriched with 50 mL L -1 coconut milk more than doubled the germination rate with respect to a non-enriched control (i.e. from 14.5% to 39.8%; p = 0.024, Student's t-test), whereas other complex organic media inhibited germination. We conclude that both pollen and ovules have inherent developmental problems that can be partially overcome by outbreeding with larger populations. Once seed is produced propagation is relatively easy: sufficient plantlets were produced to enlarge the Monte Barro population to 195 times its current size.