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Yu-Chu Chang, Chou-Tou Shii, and Mei-Chu Chung

Lycoris species of the Amaryllidaceae are important ornamental and medicinal plants in Asia. Karyotypes of Lycoris species have been studied extensively since the time when their chromosome numbers were first counted over 80 years ago. Based on karyotype, Lycoris taxa can be classified into the monomorphic A group, dimorphic MT group, and the sterile dikaryotype MT-A group. Numerous reports dealing with karyotype analysis and phylogenetic relationship in the genus Lycoris have been published. However, there are disputes and controversies regarding karyotype evolution resulting from lacking efficient and reliable markers for chromosome identification in the genus Lycoris. In this study, we applied fluorescent in situ hybridization (FISH) to visualize the 5S and 45S rDNA loci on chromosomes as landmarks for chromosome identification in Lycoris taxa. In total, 12 accessions of three karyotype groups, including nine species and three artificial dikaryotype hybrids, were investigated. A high degree of variation in the number and position of 5S and 45S rDNA loci was detected among Lycoris taxa. There were four to 14 FISH signals of 5S rDNAs and two to 12 FISH signals of 45S rDNAs observed in each investigated Lycoris accession. Lycoris accessions with the same karyotype 2n = 22A may have different numbers of rDNA loci, which distributed at different chromosomal positions. In an interspecific hybrid, the number and chromosomal position of both 5S and 45S rDNA loci were either the combinations of those in their parental species or considerably modified. Overlapping FISH signals of 5S and 45S rDNAs were colocalized with a 4′, 6-diamidino-2-phenylindole-positive band at the end of the p-arm on almost every T-type chromosome (but not the A-type chromosomes). Based on the features of T-type chromosomes, the possibility of centromeric fission in karyotypic evolution of Lycoris is discussed.

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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.

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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.