Biochemical and transcriptomic analyses of the symbiotic interaction between Cremastra appendiculata and the mycorrhizal fungus Coprinellus disseminatus

Background Cremastra appendiculata is a rare terrestrial orchid with a high market value as an ornamental and medicinal plant. However, the species depends entirely on fungi for seed germination under natural conditions. In a previous study, we have successfully isolated and identified the mycorrhizal fungus Coprinellus disseminatus which was able to induce the germination of C. appendiculata seeds. We then speculated that C. disseminatus may do so by breaking the testa imposed dormancy of the seeds. In this study, biochemical and transcriptomic analyses were used to characterize the germination of C. appendiculata seeds, collected at different stages of germination, as affected by C. disseminatus. Results The lignocellulose in the seeds coat of C. appendiculata was degraded by the mycorrhizal fungus resulting in facilitated absorption of water. The rate of decline in lignin content was 67 and 73% at 6 and 12 days after sowing, respectively. The water content increased from 13 to 90% during symbiosis. A total of 15,382 genes showing significantly different levels of expression (log2 FPKM≥2.0, Qvalue≤0.05) were successfully identified among all libraries, where the highest number of DEGs was shared between 6 days versus 0 day after symbiotic germination. Gene annotation results suggested that 15 key genes related water-status, such as DHN gene family and Xero 1 were down-regulated. The genes zeaxanthin epoxidase ZEP, 9-cis-epoxycarotenoid dioxygenase NCED3 and β-carotene hydroxylase involved in the biosynthesis of abscisic acid (ABA) were significantly down-regulated in 6 days as compared to 0 day after symbiotic germination. Conclusions This work demonstrates that mycorrhizal fungus C. disseminatus can stimulate C. appendiculata seeds germination through a mechanism of breaking the testa imposed dormancy and inducing water absorption of the embryo.

Chemical Components and Hypoglycemic Activity of Large Scale Micropropagated Cremastra Appendiculata (D.Don) Makino

Cremastra appendiculata (D. Don) Makino (Orchidaceae) is a valuable species which is in ornamental field and pharmaceutical industry, inefficient propagations limit the utilization of C. appendiculata. Here, we reported an improved and efficient method for large-scale micropropagation including seed germination, initiation and proliferation of protocorm like-bodies (PLBs), “shoot cluster” induction, rooting and acclimatization. Factually, the highest shoot multiplication coefficient 3.16 was achieved on 1/4 MS medium supplemented with 1.5 mg·L -1 thidiazuron (TDZ), along with 0.4 mg·L -1 indole-3-acetic acid (IAA), 3% sucrose and additives. Likewise, the best medium for protocorm proliferation was 1.0 mg·L -1 indole-3-butyric acid (IBA), 1.0 mg·L -1 6-benzyladenine (6-BA) and 0.2 mg·L -1 adenylate cyclase (AC) with proliferation of 170.07%, followed by root induction on 1/4MS with 0.5 mg·L -1 α naphthaleneacetic acid (NAA), 0.5 mg·L -1 AC and 3% sucrose. Besides, the achieved micropropagation plants grew vigorously and were steady with a survival rate of 85.3% in vermiculite: humus (1: 2). Assessment of the chemical compositions by high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) displayed that the composition of dried pseudobulbs from micropropagation plantlets and naturally grown plants were similar, containing colchicine, quercetin, rutin, adenosine, oleanolic acid, myristate, rhein and emodin. Furthermore, the hypoglycemic activity in vitro indicated micropropagation plantlets were superior to naturally grown plants. Consequently, the described protocol could be used for the large-scale propagation and conservation of this endangered plant, which might be natural resource for the diabetes patients.

One new species and one new record of Zasmidium in China

Two hyphomycetous species were collected from leaves of Smilax china (Liliales, Smilacaceae) and Cremastra appendiculata (Asparagales, Orchidaceae). ITS barcoding indicated that they belong to the genus Zasmidium. Morphological data in combination with molecular phylogenetic analyses based on ITS, LSU and rpb2 confirmed that our Chinese strains represented a new species, Zasmidium liboense and a new record of Z. citri-griseum.

Links between important values of (Cremastra appendiculata) and elevations by long-time investigation and qualitative analysis and quantitative statistics of "Big data"

(Cremastra appendiculata) of treating lumbago and arthritis not only is a vital medicinal material plant, but also it is a widely distributed wide plant species. This plant species is widely distributed elevation from 500m to 3100m in Mei County of China. However, understanding dynamics of Important Value of this species is very difficult along elevation. This research explained that links between Important Value of this species and elevation is the significant positive correlation from 500m to 1500m (P<0.01) as well as the links between Important Value of this species and elevation are the significant negative correlation from 1500m to 3100m (P<0.01). This study provides six natural landscape types and a series of areas ecological adaptation for finding new medical species along different elevation. Thus, this research has vital theoretical and practical significance for medicinal plant protection at spatial-temporal-environmental-disturbance scales (STEDS).

Links between Leafstalk Biomass of (Cremastra appendiculata) and Elevation by Big Data of Long-time Wild Investigation in Mei-County

(Cremastra appendiculata) of treating lumbago and arthritis not only is a vital medicinal material plant, but also it is a widely distributed wide plant species. This plant species is widely distributed elevation from 500m to 3100m in forest landscapes and vegetation ecosystems in Mei County of China. However, understanding dynamics of biomass of leafstalk of this species is difficult along elevation. This research explained that the links between biomass of leafstalk of this species and elevation is the significant positive correlation from 500m to 1500m (P<0.01) as well as the links between biomass of leafstalk of this species and elevation are the significant negative correlation from 1500m to 3100m(P<0.01). This study provides six ecosystem types and a series of areas ecological adaptation for finding new medicinal species. Therefore, this study has vital theoretical and practical significance for medicinal plant protection along elevation and environments. Keywords: biomass of leafstalk; elevation; correlation; areas ecological adaptation; medicinal species.