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Seeds of Cardiocrinum giganteum var. yunnanense, which is native to China, has underdeveloped embryos when dispersed from parent plants that did not grow until the second autumn and winter after exposure to summer temperatures. Radicles and cotyledons emerged in late winter and spring. Thus, a 15–16 month period was required from dispersal to seed germination. Under laboratory conditions, this period could be shortened to 5–6 months in a 25°C/15°C (60 days) → 15°C/5°C (60 days) → 5°C (60 days) temperature sequence. Based on dormancy-breaking requirements, the seeds have deep simple morphophysiological dormancy (MPD). This is practical knowledge for propagation of the species from seeds.

Ethnopharmacological relevance Seeds of Cardiocrinum giganteum var. yunnanense (Leichtlin ex Elwes) Stearn (Liliaceae), also known as Doulingzi, have been used as a folk substitute for conventional antitussive herb “Madouling” (Aristolochia species) to treat chronic bronchitis and pertussis. The active antitussive phytochemicals in C. giganteum seeds are not known. Aim of the study The present work aims at isolating the active phytochemicals in C. giganteum seeds and confirming their antitussive effects. Materials and methods Active chemicals were isolated from C. giganteum seeds ethanol extract and identified their structures. Antitussive effects were evaluated with the cough frequency of guinea pigs exposed to citric acid. Electrical stimulation of the superior laryngeal nerve in guinea pigs was performed to differentiate the acting site of potential antitussives. Results Two racemic biflavonoids (CGY-1 and CGY-2) were isolated from C. giganteum seeds. CGY-1 was identified as (S)-2″R,3″R- and (R)-2″S,3″S-dihydro-3″-hydroxyamentoflavone-7- methyl ether, which are new compounds and firstly isolated from C. giganteum seeds. Racemic CGY-2 was identified as (S)-2″R,3″R- and (R)-2″S,3″S-dihydro-3″-hydroxyamentoflavone. Both CGY-1 and CGY-2 could significantly inhibit coughs induced by inhalation of citric acid. Further, they acted on the peripheral reflex pathway to inhibit cough after electrical stimulation of the superior laryngeal nerve in guinea pigs. Conclusions These chemicals isolated from C. giganteum seeds showed good antitussive effects. The data provide scientific evidence to support the traditional use of C. giganteum seeds as an antitussive herbal medicine.

Certain biflavonoids have been proven to protect against cognitive dysfunction. A new biflavonoid, CGY-1, isolated from Cardiocrinum giganteum seeds, has not yet been reported to have any neuroprotective effect. In this study, a scopolamine-induced memory deficit model was used to explore the neuroprotective effect of CGY-1. Behavioral experiments, such as tests using the Morris water maze, the Y-maze and the fear conditioning test, were conducted. The results revealed that oral administration of CGY-1 (20 and 40 mg/kg) and donepezil shortened the escape latency, improved the percentage of spontaneous alternation, and increased the freezing times, respectively. CGY-1 decreased the levels of reactive oxygen species and malondialdehyde and increased the activities of superoxide dismutase and glutathione peroxidase in the hippocampus. In addition, CGY-1 decreased the activity of acetylcholinesterase and increased the activities of choline acetyltransferase and acetylcholine in the hippocampus. Furthermore, qPCR and western blot results revealed that the expressions of neurotrophic factors, brain-derived neurotrophic factor and nerve growth factor were upregulated in the hippocampus after CGY-1 treatment. In conclusion, CGY-1 could be a promising candidate for the treatment of cognitive dysfunction.

Giant lily (Cardiocrinum giganteum) is a perennial bulbiferous herb of Liliaceae and an endemic species in the eastern Himalayan region, which has become one of the new flower crops because of its high ornamental value. The floral biology and breeding characteristics of C. giganteum were studied by monitoring floral characteristics, duration of flowering, phenology, flowering behavior, outcrossing index (OCI), pollen-ovule ratio (P/O), pollen viability, stigma receptivity and fruit sets under a series pollination treatment. The result showed that flowering occurred from early May to early July and the mean duration of flowering was 16 days per inflorescence containing 6 to 19 flowers, and every flower has a life span of 7-11 days. The flowers were bisexual, with dichogamy and spatial separation. However, some anthers of a small number of flowers were longer than or equal to the stigma, which were suitable for self-pollination. The flowers were protandrous; the pistils matured in the day of floral opening. The pollen viability peaked around 75% after the pollen dispersed in 2 days, and its stigma had peroxidase activity from the third day after the anther loosing pollen until the end of flowering. The fruit setting rate of natural pollination was as high as 95%, that of artificial self-pollination 57.5%, and that of artificial xenogamy was as high as 100%, suggesting that C. giganteum is self-compatible. According to the artificial pollination experiments, OCI and P/O tests, the breeding system of C. giganteum was outcross and self-compatible, which needed pollinators.

In this article, we studied the pollen morphology and wall development, microsporogenesis, male gametophyte development, and anther wall structure changes during pollen development of Cardiocrinum giganteum (Wall.) Makina from the genus Cardiocrinum (Endl.) Lindl. (Liliaceae) using paraffin sections, scanning and transmission electron microscopy, and fluorescence microscopy. The results showed that C. giganteum has oval-shaped pollen with a single sulcus and reticulate ornamentation. The exine is of the semi-tectum type and can be divided into the tectum layer, columellate layer and basal layer. Meiosis in the microsporocyte is accompanied by successive cytokinesis. The mature pollen is three-celled. The anther wall prior to maturity is built by one layer of epidermis, 1–2 layers of endothecium cells, 4–5 middle layers and 2 layers of tapetum, while upon maturity it is only built by one layer of epidermis, one layer of endothecium cells and one middle layer. The tapetal cells are secretory, with two or more nuclei. Ubisch bodies originate from rough endoplasmic reticulum except a few from mitochondria.

Understanding the potential for ornamental plant species to become naturalized in a nonnative habitat requires information on seed germination in order to help predict responses of the species to the natural environ- mental conditions of its new habitat. Cardiocrinum gi- ganteum var. giganteum, which is native to the Himalayas, has been introduced as an ornamental plant in temperate regions of the world, and was categorized recently as invasive in New Zealand. Seed germination requirements of the species were determined under natural conditions in Hokkaido, Japan, to assess its potential to become natu- ralized in this region of Japan. Mature seeds were collected from its native range in the Indian Himalayas. At maturity in autumn, seeds had underdeveloped embryos, which grew in the second autumn and winter after exposure to summer temperatures. Radicles and cotyledons emerged in late winter and spring. Thus, an 18-19 month period was required from dispersal to seed germination. Under laboratory conditions, this period could be shortened to 10-11 months in a 25/15 � C (120 days) fi 15/5 � C (90 days) fi 0 � C (90 days) fi 15/5 � C (60 days) temperature sequence. GA3 did not substitute for the above temperature requirements. These temperature requirements for seed germination of C. giganteum var. giganteum are very similar to those of its native Japanese congener C. cordatum var. glehnii. Seeds of both taxa have deep simple morphophysiological dormancy. The close similarity in the requirements for regeneration from seeds of the two taxa suggests that the seed stage of the life cycle is not an impediment to the naturalization of the giant Himalayan lily in northern Japan.

Twenty-one microsatellite markers from the genome of Cardiocrinum giganteum var. yunnanense, an important economic plant in China, were developed with a fast isolation protocol by amplified fragment length polymorphism of sequences containing repeats (FIASCO). Polymorphism within each locus was assessed in 24 wild individuals from Gaoligong Mountains in western Yunnan Province, China. The number of alleles per locus ranged from 2 to 4 with a mean of 2.9. The expected and observed levels of heterozygosity ranged from 0.042 to 0.726 and from 0.000 to 1.000, with averages of 0.44 and 0.31, respectively. These polymorphic microsatellite markers should prove useful in population genetics studies and assessments of genetic variation to develop conservation and management strategies for this species.

Phylogenetic relationships among 55 species of Lilium, Cardiocrinum giganteum, and Nomocharis saluenensis were inferred from nucleotide sequence variations in the internal transcribed spacer (ITS) regions of 18S-25S nuclear ribosomal DNA. The phylogeny derived from ITS sequences estimated using maximum-likelihood methods indicated that (1) most of the species construct their own clade according to the classification based on morphological features at the section level; (2) section Daurolirion is not independent of Sinomartagon, and it is appropriate to integrate two sections as Sinomartagon; (3) it is appropriate that L. henryi and L. bulbiferum are classified into subsection 6a and Sinomartagon-Daurolirion, respectively; (4) subsection 6b is much closer to Sinomartagon than subsection 6a and Archelirion, and it arose directly from Sinomartagon; and (5) Lilium is much closer to Nomocharis than Cardiocrinum. Phylogenetic estimation using sequences of the ITS region is suitable at the levels of genus, section, and most of subsection.

Aim of the study The antitussive Chinese herb Madouling derived from Aristolochia species is banned due to aristolochic acid-induced nephropathy. A substitute is found dispensed as Madouling in Taiwan. This study aims to determine the source plant and verify the antitussive properties of the Madouling substitute used in Taiwan. Materials and methods Forensically informative nucleotide sequencing (FINS) approach based on the trn L- trn F and psb A- trn H regions was applied to facilitate identification of the genuine species and substitute. The antitussive effect of both genuine Madouling and the substitute were evaluated in guinea pigs. Results FINS approach based on the trn L- trn F and psb A- trn H regions readily identified the sample of Madouling in Taiwan to the seeds of Cardiocrinum giganteum var. yunnanense . Ethanol extracts of the substitute showed significant antitussive properties in guinea pigs. Conclusion Cardiocrinum seeds may have potential as a replacement of Aristolochia fruits.

Pollen grain germination and pollen tube growth of Chinese giant lily (Cardiocrinum giganteum) have been investigated as follows: scanning electron microscopy (SEM) morphology of pollen germination in vivo, and calcium distribution in germinating pollen grains and growing pollen tubes in vitro with energy-dispersive X-ray analysis (EDAX) and chlorotetracycline (CTC) fluorescence. Research materials were mainly collected from the plants wild-grown at Mount Emei in Sichuan. Self- and cross-pollination was made by hand in the open, and pollen grains collected were germinated in vitro as discribed commonly. Samples were withdrawn at the required intervals and prepared for SEM, EDAX and CTC fluorescence. SEM observation revealed that pollen grains start to germinate on stigmas 30 min after pollination, and there are linked secretions between germinating grains and between pollen and stigma as well as secretion granules on the exine surfaces and at the germinal furrows. For EDAX, measured along the pollen tube axis from the tip to the base, the tubes show a maximum of calcium concentrition at the tip region. Behind the region of about 5-10 um from the apex, the calcium concentritions fall remarkably and remain relative constant in the rest of the tube, The germinating grains show higher calcium amounts at the exine surfaces, especially at the germinal furrow region having a lot of calcium- rich bodies.