The present study examined the seed morphometric and viability, asymbiotic seed germination, protocorm formation, and plantlet growth in Orchis simia Lam., an at-risk Euro-Mediterranean terrestrial orchid species. Furthermore, tissue histology pattern in vitro grown plantlets and field-established was compared. After one month, shoot and root primordia started to grow on developing protocorms and small plantlets became visible after 23 subculturing. Asymbiotic in vitro seed germination and plantlet growth attributes were significantly (P<0.05) impacted by both type of main organic constituent and nitrogen sources. The seeds sown on media supplemented with pineapple juice (PJ) and casein hydrolysate (CH) had the highest germination percentage. In terms of required days to see the first signs of germination, CW+AV treatment brings about the fastest germination (6.8 § 0.20 days). Protocorms grown on the media comprising PJ in combination with Aminoven (AV) or casein hydrolysate (CH) or Pep showed higher dimensions and therefore higher weight (% 450 increase) compared to protocorms grown on other media. Altogether, treatments with higher germination percentage, faster germination, and bigger protocorms finally resulted in better plantlet growth. Acclimatization of in vitro grown plantlets occurred by converting scattered vascular tubes into fully developed xylem and phloem vessels facilitating sap flow. Taking into account the fragmentation of O. simia populations in the Middle East and the EuroMediterranean basin mainly due to tuber overharvesting and climate change, the present procedure could be used for large-scale multiplication of in vitro raised seedlings to re-introduce into endangered regions.
