In vitro culture conditions for asymbiotic germination and seedling development of Anacamptis pyramidalis (L.) Rich. and Gymnadenia conopsea (L.) R. Br.

Abstract

• Positive influence of MM medium on germination of A. pyramidalis and G. conopsea was determined. • Germination was influenced by full darkness and nutrient medium enriched with organic aditives. • In seedling growth, 2iP initiated the formation of the aboveground part of G. conopsea. • In A. pyramidalis, Kin initiated the leaf formation, while Ba promote root formation. Protocols for asymbiotic seed germination and seedling development of two terrestrial orchid species – Gimnadenia conopsea and Anacamptis pyramidalis are presented in this paper. Detail examination covered nutrient media: Knudson C (KC) and Malmgren (MM); organic supplements: coconut water (CW), pineapple juice (PJ), peptone (PE), casein hydrolysate (CA) and glutamine (A); different lighting conditions – 0/24 Light / Dark (L / D) and 16/8 (L / D) as well as cytokinins: 6-benzyladenine (BA), kinetin (Kin), and 6- (γ, γ-dimethylallylamino) purines (2 -iP) aiming to assess asymbiotic seed germination in controlled in vitro conditions, to define the optimal protocol for their mass propagation. The obtained results indicate that the nutrient medium MM and conditions of complete darkness encourage seed germination in both investigated species, while the organic supplements amendment resulted in a shortened period necessary for seed germination as well as a significantly higher percentage of germination. In Gymnadenia conopsea , the highest percentage of germination (73,79%) accompanied by the highest embryo/protocorm morphometric characteristics (1.40 mm average height, 1.21 mm average width, and 0.30 mm average shoot height) was achieved on MM-CW nutrient medium. A similar result was observed for Anacamptis pyramidalis with 69,88% germinated seeds on MM-CW nutrient medium, while the highest morphometric values were 1.10 mm (on MM-CW) for height, 1.00 mm (on MM-PE) for width and 0.57 mm (on MM-PE) for bud height. On the contrary, the results obtained on nutrient medium KC and under the 16/8 L / D lighting conditions indicate the inhibitory effect of these conditions on the seeds germination in examined species. A experiment with chitokinin treatments indicates that the best result in G. conopsea was achieved on the medium with the addition of 0.3 mg L −1 2iP with 5.33 mm for plant height, 1,13 root numbers and 12.26 mm root lenght, while in A. pyramidalis 0.3 mg L −1 Kin emerged as the most suitable for the development of the upper part of the plant (3.33 mm), while the medium enriched with 0.3 mg L −1 BA had a positive effect on the formation of the root system.