In vitro Propagation of Vanda testacea (Lindl.) Reichb. F, a Medicinally Important Threatened Orchid

The present study was planned to enable in vitro conservation of Vanda testacea, a highly medicinal orchid species through in vitro asymbiotic seed germination technique in Mitra orchid medium supplemented with cytokinins (Kn - 4.65 μM, BAP - 4.44 μM), and auxin (NAA- 5.37 μM). The germination frequency and initiation of germination was higher in NAA augmented medium and seedlings developed in 12.50 ± 0.50 weeks. Coconut water (20%) proved optimum for the multiplication of protocorm like bodies. Activated charcoal successfully checked the release of brownish exudates in the cultures. Plant Tissue Cult. & Biotech. 31(2): 153-160, 2021 (December)

Seed Micromorphology, In Vitro Germination, and Early-Stage Seedling Morphological Traits of Cattleya purpurata (Lindl. & Paxton) Van den Berg

In the context of a symbiotic plant-fungus interaction study concerning Cattleya purpurata, we focused on some aspects of seed morphology and biology, and the early stages of seedling development. Seed morphology was characterized using light and scanning electron microscopy. In vitro seed germination capability was evaluated, comparing symbiotic and asymbiotic methods. The morphology of the seeds was overall comparable to that of other congeneric species, showing classical adaptations related to the aerodynamic properties and to the wettability of seeds, but calcium oxalate druses were identified inside the suspensor cells. Asymbiotic seed germination was successful in all tested media (17.1–46.5%) but was higher on 1/2 Murashige & Skoog. During symbiotic interaction with the fungal strain MUT4178 (Tulasnella calospora), germination rate was significantly lower than that obtained with the best three asymbiotic media, suggesting a low fungal compatibility. Seedling morphology was in line with other taxa from the same genus, showing typical characteristics of epiphytic species. Our observations, in particular, highlighted the presence of stomata with C-shaped guard cells in the leaves, rarely found in Cattleyas (where usually they are reniform), and confirm the presence of tilosomes in the roots. Idioblasts containing raphides were observed in both roots and leaves.

An Improved Protocol for Asymbiotic Seed Germination and Seedling Development of Paphiopedilum tigrinum

Paphiopedilum tigrinum is an endangered orchid with high ornamental value. However, seed germination and seedling regeneration in P. tigrinum is very difficult in vitro. Little is known about why P. tigrinum seedlings are difficult to propagate or how to improve the seed germination and seedling rates of this species. In this study, we investigated the developmental process of P. tigrinum from asymbiotic seed germination to seedling rooting by comparing it with P. appletoniantum, a much easier species for germination and seedling formation. We found that asymbiotic seed germination in P. tigrinum is limited by severe browning of the protocorm at the seed germination stage, and protocorm rooting at the differentiation stage was also proved to be difficult. The optimal medium for seed germination of P. tigrinum was a modified Harvais (mHa) medium supplemented with 0.5 mg·L−1 kinetin (Kin), 0.1 g·L−1 activated charcoal (AC) and 100 mL·L−1 coconut water (CW). At the protocorm differentiation stage, seedlings with 1–2 leaves were obtained on a 1/4 MS medium supplemented with 1.0 mg·L−1 6-benzylaminopurin (BA), 0.3 g·L−1 AC and 50–100 mL·L−1 CW after culturing for 120 day. At the seedling subculture stage, a 1/2 MS medium supplemented with 0.5–1.5 g·L−1 AC and 100 mL·L−1 CW was better for leaf and root growth of P. tigrinum. At the rooting stage, a 1/2 MS medium supplemented with 1.0 g·L−1 AC, 0.5 g·L−1 dolomite flour, 15 g·L−1 potato homogenate and 30 g·L−1 banana homogenate was most suitable for the growth and rooting of seedlings. This study has established an effective protocol for seed germination and seedling regeneration of P. tigrinum.

Asymbiotic seed germination and in vitro seedling development of Orchis militaris, an endangered orchid in Siberia

Background Terrestrial orchids belonging to the Orchis genus are difficult to propagate and are under great pressure in their natural habitats. Studies regarding the influence of photoperiod and temperature regimes on Orchis militaris germination and morphological changes during immature seed development in vitro are scarce. Our aim was to identify photoperiod, temperature, and different nutrient media requirements for optimization of O. militaris seed germination and vigorous seedling production. Results Post-germination morphological changes were recorded with O. militaris seeds collected from 32-day-old fruits, where the percentage of O. militaris seeds without embryo was 38.4%. The highest rate of O. militaris seed germination (82.6%) was obtained on Malmgren modified terrestrial orchid medium (mM), enriched by 5% coconut water, 5% birch sap, and 0.1% AC. Nine percent of seedlings were able to reach the advanced seedling stage (stage 6) after 12 months of maintenance on this medium. In all 3 modified media (Harvais, Knudson С and Malmgren), regeneration was via the production of protocorms and seedlings without callus formation. It was proved that more abundantly vigorous protocorms were formed on the modified Harvais 2 under continuous darkness, while the subculture in Knudson C medium with AC addition could be necessary to stimulate their further development. The regeneration success of the species for in vitro conditions was increased by following its natural seasonal cycle. Conclusion This study demonstrated an efficient micropropagation system for O. militaris using immature seeds and thus widely opened the perspectives for its conservation in nature. The favorable conditions of seed germination periods for in vitro culture, identified as the definite shift of temperatures and photoperiod regimes intrinsic to the species in nature, could improve seedling survival of this medicinally important orchid.

IN VITRO REGENERATION OF ARUNDINA GRAMINIFOLIA (D. DON) HOCHR

We can see Orchids come in a wide variety of shapes, sizes, colours, and textures far beyond the human mind’s imagination. They emerge from seeds in nature, but in the absence of suitable hosts, they do not germinate in sufficient numbers. This problem was solved by using the tissue culture technique for its germination. One of the successful method used for mass propogation of orchid plantlets is in vitro techniques. Therefore, an initial analysis was conducted in order to establish an appropriate procedure for mass multiplication of Arundina graminifolia. MS (Murashige and Skoog) medium was found to be suitable for the asymbiotic seed germination of Arundina graminifolia. Direct protocorm like bodies were induced by using combinations and individual supplement of MS medium with IAA (Indole-3-acetic acid), IBA (Indole-3- butyric acid), BAP (6-Benzylaminopurine) and KIN (Kinetin). Hormone-free MS basal medium was found suitable for the conversion of PLBs (protocorm-like bodies) into complete plantlets

A Review on Asymbiotic Seed Germination in Orchids through Plant Tissue Culture

Orchid seeds are very small, dust like in appearance, fusiform in shape, lacking endosperm and have undifferentiated embryo covered by transparent seed coat. Mycorrhizal association is required for seed germination of naturally growing orchids. In this symbiotic association, orchid species are dependent on mycorrhizal association for supply of mineral nutrients. In in-vitro condition, such demand of minerals may be compensated by external supply of sugar and mineral nutrients that are required for seed germination of orchid. Several orchids are responded by this asymbiotic seed culture and have commercial importance. Therefore, formulation of efficient in-vitro protocol is important for commercially important orchid species as well as endangered orchid species for conservation. This review paper is focused on various aspects of asymbiotic seed germination of orchids and the role of organic additives in successful seed germination.