Storability, post-storage conversion and genetic stability assessment of alginate-encapsulated shoot tips of monopodial orchid hybrid Aranda Wan Chark Kuan ‘Blue’ × Vanda coerulea Grifft. ex. Lindl.

An efficient short-term storage system of synthetic seeds, produced using in vitro shoot tips of the monopodial orchid hybrid Aranda Wan Chark Kuan ‘Blue’ × Vanda coerulea Grifft. ex. Lindl. (AV), was developed. In vitro shoot tips (3–4 mm) were successfully encapsulated, resulting in uniform spherical beads (capsules), using 3 % sodium alginate with 75 mM CaCl₂·2H₂O. Maximum (~100 %) conversion (into plantlets with shoot and root) of capsules (or synthetic seeds) was achieved on quarter-strength Murashige and Skoog regrowth medium, while full-strength MS medium was required for effective conversion of non-encapsulated shoot tips. The capsules showed distinct difference in their response to temperature during storage. The conversion efficiency declined upon storage duration at both 4 and 25 °C, with those stored at 25 °C being more tolerant to storage. Capsules stored at 4 °C had rapid deterioration and faced complete death within 160 days while those stored for 200 days at 25 °C showed relatively high conversion (71.6 %). An inter-simple sequence repeats fingerprinting approach, employed on indiscriminately chosen plantlets from converted capsules (following 4 and 25 °C of storage), ensured the post-storage genetic stability.     

Alginate-encapsulation, short-term storage and plant regeneration from protocorm-like bodies of Aranda Wan Chark Kuan ‘Blue’?×?Vanda coerulea Grifft. ex. Lindl. (Orchidaceae)

This article demonstrates the single bead alginate-encapsulation and conversion (complete plantlet regeneration) from protocorm-like bodies (PLBs) of Aranda Wan Chark Kuan ??Blue???×?Vanda coerulea Grifft. ex. Lindl. (AV) (a monopodial orchid hybrid) for the first time. PLBs, induced from leaf segments of AV were isolated from in vitro proliferating PLB clusters. Individual PLBs (4?±?1?mm diameter) were encapsulated in calcium alginate beads to manage mass propagation, short-term storage and germplasm sharing. The superior gel matrix for encapsulation was obtained using 3?% sodium alginate and 75?mM calcium chloride (CaCl₂·2H₂O). Highest percentage of germination (98.1?%) and conversion (96.2?%) of encapsulated PLBs (capsules) was obtained on plant growth regulator-free half-strength MS (Murashige and Skoog, Physiol Plant 15:473?C497, 1962) medium. Successful storage of capsules, until 180?days, was achieved at 25?°C under zero-irradiance with germination and conversion frequency of 76.9 and 70.2?%, respectively. Plantlets regenerated from capsules were acclimatized successfully with 92?% survival rate.     

Induction of multiple shoots in a monopodial orchid hybrid (Aerides vandarum Reichb.f × Vanda stangeana Reichb.f) using thidiazuron and analysis of their genetic stability

The effect of thidiazuron (TDZ) on direct multiple shoot induction in axenic seedlings of a monopodial orchid hybrid Aerides vandarum × Vanda stangeana, using a dual phase culture medium was studied. The culture system consisted of a basal agar solidified half-strength Murashige and Skoog medium overlaid by a liquid fraction of the same composition. Highest regeneration of multiple shoots (15.8 shoots per seedling) was obtained in the medium containing 2% sucrose (w/v) supplemented with 2 mgl⁻¹ TDZ. Genetic stability of the regenerated shoots was assessed using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), and restriction fragment length polymorphism of the PCR amplified (PCR-RFLP) nrITS region, as well as those of the coding (matK) and non-coding (trnL-F) regions of the cpDNA. Across the randomly selected mother plant and nine of its regenerated shoots, 2,680 bands were generated by 19 RAPD and 12 ISSR primers, exhibiting monomorphic banding profiles. Homogenous PCR-RFLP profiles were generated for nrITS using four restriction enzymes (REs), matK using five REs, and trnL-F using six REs. These molecular analyses showed no genomic alterations in all regenerated shoots obtained on medium containing 2 mgl⁻¹ TDZ.