A micropropagation protocol for an endangered slipper orchid species, Cypripedium formosanum Hayata, through axillary buds from adult plants has been developed. The season of explant collection is crucial for the initial success of an aseptic culture. Explants collected in the middle of January gave the highest percentage of explant survival (54.2%) and shoot-forming percentage (41.7%). Of the two cytokinins tested, N6-benzyladenine (BA) was found to be superior to thidiazuron for normal shoot formation. The optimum result was obtained in quarter-strength Murashige and Skoog medium containing 22.2 or 44.4 μM BA in which the cultures produced 6.3 and 7.1 shoots per explant with 10.6 to 11.7 mm average length after 90 d of culture. Regenerated shoots rooted for 60 d in the basal medium with 1 g·L−1 activated charcoal and 20 g·L−1 potato homogenate were ready for growth in pots. This is the first report on shoot multiplication in vitro from mature plants of Cypripedium that provides a reliable method for propagating the selected elites.
Rebecca C.-C. Hsu and Yung-I Lee
The histological and histochemical changes in developing seeds of Cypripedium debile Rchb. f., a native slipper orchid species with horticultural potential, were investigated. The effects of timing for seed collection, culture media, and cultural conditions were also examined. The optimum germination percentage occurred when mature seeds were collected and sowed on 1/4 Murashige and Skoog basal medium. Besides, the liquid culture promoted germination of mature seeds. This finding is contrary to most other Cypripedium species, which are relatively easy to germinate with immature seeds. Moreover, two notable cytological changes of C. debile were observed. First, the suspensor cell protruded beyond the micropyle opening of the inner seedcoat, making the inner seedcoat not substantial. Second, Nile red staining indicated that the deposition of cuticular material on the seedcoat was fragmentary. It is proposed that the less hydrophobic nature of the seedcoat makes mature seeds of C. debile easier to obtain water and nutrients for germination.
Cheng-Jung Hu, Nean Lee and Yung-I Lee
The dancing-lady orchid, Oncidesa Gower Ramsey, is an important cultivar for cut-flower production, but it has low pollen fertility in breeding programs. In this study, we compared the pollen germination in vitro, sporad type, and pollinia development of Oncsa. Gower Ramsey and a diploid species, Oncidium sphacelatum Lindl (one of its grandparents). In Oncsa. Gower Ramsey, the pollen germination in vitro was lower as compared with those in Onc. sphacelatum. In addition, the frequency of abnormal sporads in Oncsa. Gower Ramsey was higher than those in Onc. sphacelatum. In Oncsa. Gower Ramsey, the middle layer and the tapetum were disorganized before meiosis, and subsequently they degenerated at the early tetrad stage. In contrast, the middle layer and the tapetum of Onc. sphacelatum began to degenerate at the early tetrad stage and fully disappeared at the bicellular pollen stage. These results suggested that the abnormal meiosis caused by unbalanced genomes and the premature degeneration of the middle layer and the tapetum could probably result in the abnormal pollen development and the low fertility of Oncsa. Gower Ramsey.
Vi Nguyen Tuong Do, Shan-Te Hsu and Yung-I Lee
The aim of this study was to develop an efficient protocol for shoot tip culture from adult plants of Paphiopedilum Pfitzer. A considerable seasonal effect on explant collection was observed in the aseptic cultures established from adult plants, including the survival and microbial contamination of explants. The shoot tip explants excised from adult plants in February and May showed higher survival and had less contamination than those explants excised in August and November. Moreover, the season of explant collection also affected the subsequent shoot forming capacity and multiplication of axillary buds. In Paphiopedilum ‘In-Charm Silver Bell’, higher shoot forming capacity was observed in February and May, whereas higher shoot multiplication was observed only in February. In Paphiopedilum ‘Hsinying Maudiae Leopard’, both February and May were optimal timing for shoot forming capacity and multiplication. We also demonstrated the effectiveness of transcinnamic acid (tCA), an antiauxin chemical in diminishing the apical dominance of shoot tip explant and thus improving the axillary bud outgrowth. In P. ‘In-Charm Silver Bell’, the addition of 100 μM tCA plus 13.3 μM 6-benzylaminopurine (BA) for 1 month promoted axillary shoot bud formation from shoot tip explants as compared with the control.
Yung-I. Lee, Chia-Fu Lu, Mei-Chu Chung, Edward C. Yeung and Nean Lee
Changes in endogenous abscisic acid (ABA) concentrations were investigated in developing seeds and the pretreated seeds of Calanthe tricarinata, a hard-to-germinate terrestrial orchid. ABA concentration was as low as 2.16 to 2.26 ng·mg−1 fresh weight at the proembryo stage [60 to 90 days after pollination (DAP)] and then continuously increased to 11.6 ng·mg−1 fresh weight at 210 DAP. Seed maturation was accompanied by a dramatic decrease in water content and a prominent accumulation of protein and lipid bodies within the embryo proper. The optimum time for asymbiotic seed germination was obtained from immature seeds at 150 DAP. At this stage, the embryo proper reached its maximum size, and the seedcoat became dehydrated and gradually shrunk into a thin layer. By 180 DAP, seed germination declined sharply as seed approached maturity. Mature seeds pretreated with ultrasound (45 min), 1% NaOCl (45 to 60 min), or 1N NaOH (45 min) were effective in improving the germination percentage and lowering seed ABA concentrations. Our results suggest that high concentrations of endogenous ABA in orchid seeds may play a critical role in arresting embryo growth and in preventing seed germination.
Yung-I Lee, Nean Lee, Edward C. Yeung and Mei-Chu Chung
This investigation documents the key anatomical features in embryo development of Cypripedium formosanum Hayata, in association with the ability of embryos to germinate in vitro, and examines the effects of culture media and seed pretreatments on seed germination. A better understanding of zygotic embryogenesis for the Cypripedium L. species would provide insights into subsequent germination events and aid in the in vitro propagation of these endangered species. In seeds collected at 60 days after pollination (DAP), soon after fertilization, no germination was recorded. The best overall germination was found at 90 DAP (≈70%), at which time early globular to globular embryos with a single-celled suspensors can be observed. After 135 DAP, the seeds germinated poorly. At this time the inner integument shrinks and forms a tight layer, which encloses the embryo, the so-called “carapace.” Using Nile red stain, a cuticular substance was detected in the carapace, which may play a role in the impermeability of the mature seed and may help the seeds survive in the stringent environment. At maturity (after 210 DAP), the embryo proper has an average size of eight cells along its length and six cells across the width. Lipids and proteins are the main storage products within the embryo. To improve seed germination, experiments were conducted to test the suitability of various media and pretreatments of seeds. When different media were used, except for the Harvais medium at 120 DAP, there was no significant difference in seed germination at three different developmental stages tested. Soaking mature seeds in 1% NaOCl or treating them with ultrasound may slightly increase the germination percentage. For seed germination, our results indicate that the timing of seed collection outweighs the composition of medium and the seed pretreatments.