Callus growth kinetics and accumulation of secondary metabolites of Bletilla striata Rchb.f. using a callus suspension culture.

Bletilla striata is an endangered traditional Chinese medicinal plant with multiple uses and a slow regeneration rate of its germplasm resources. To evaluate the callus growth kinetics and accumulation of secondary metabolites (SMs), a callus suspension culture was proven to be a valuable approach for acquiring high yields of medicinal compounds. An effective callus suspension culture for obtaining B. striata callus growth and its SMs was achieved with the in vitro induction of calluses from B. striata seeds. The callus growth kinetics and accumulation of SMs were analyzed using a mathematical model. The resulting callus growth kinetic model revealed that the growth curves of B. striata suspension-cultured calluses were sigmoidal, indicating changes in the growth of the suspension-cultured calluses. Improved Murashige and Skoog callus growth medium was the most favorable medium for B. striata callus formation, with the highest callus growth occurring during the stationary phase of the cultivation period. Callus growth acceleration started after 7 days and thereafter gradually decreased until day 24 of the cultivation period and reached its highest at day 36 period in both the dry weight and fresh weight analyses. The coelonin concentration peaked during the exponential growth stage and decreased afterward during the stationary stage of the callus suspension culture. The maximum content of coelonin (approximately 0.3323 mg/g callus dry weight) was observed on the 18th day of the cultivation cycle, while dactylorhin A and militarine reached the highest concentrations at day 24, and p-hydroxybenzyl alcohol at day 39. This investigation also laid a foundation for a multimathematical model to better describe the accumulation variation of SMs. The production of SMs showed great specificity during callus growth and development. This research provided a well-organized way to increase the accumulation and production of SMs during the scaled-up biosynthesis of calluses in B. striata callus suspension cultures.

Chemical Constituents, Pharmacologic Properties, and Clinical Applications of Bletilla striata.

Bletilla striata is a plant from the Orchidaceae family that has been employed as a traditional Chinese medicine (TCM) for thousands of years in China. Here, we briefly review the published studies of the last 30 years that were related to chemical constituents, pharmacologic activities, and clinical applications of B. striata. Approximately 158 compounds have been extracted from B. striata tubers with clarified molecular structures that were classified as glucosides, bibenzyls, phenanthrenes, quinones, biphenanthrenes, dihydrophenanthrenes, anthocyanins, steroids, triterpenoids, and phenolic acids. These chemicals support the pharmacological properties of hemostasis and wound healing, and also exhibit anti-oxidation, anti-cancer, anti-viral, and anti-bacterial activities. Additionally, various clinical trials conducted on B. striata have demonstrated its marked activities as an embolizing and mucosa-protective agent, and its application for use in novel biomaterials, quality control, and toxicology. It also has been widely used as a constituent of many preparations in TCM formulations, but because there are insufficient studies on its clinical properties, its efficacy and safety cannot be established from a scientific point of view. We hope that this review will provide reference for further research and development of this unique plant.

Chemical constituents of Bletilla striata.

A new triphenanthrene compound named 2,2',2'',7,7',7''-hexahydroxy-4,4',4''-trimethoxy-[9,9',9'',10,10',10'']-hexahydro-1,8,1',6''-triphenanthrene (1), together with eleven known compounds (2－12), were isolated from tubers of Bletilla striata. Their structures were determined by analysis of spectroscopic data.

De Novo assembly, characterization and development of EST-SSRs from Bletilla striata transcriptomes profiled throughout the whole growing period.

Bletilla striata is an endangered orchid that has been used for millennia as a medicinal herb, in cosmetics and as a horticultural plant. To construct the first nucleotide database for this species and to develop abundant EST-SSR markers for facilitating further studies, various tissues and organs of plants in the main developmental stages were harvested for mRNA isolation and subsequent RNA sequencing. A total of 106,054,784 clean reads were generated by using Illumina paired-end sequencing technology. The reads were assembled into 127,261 unigenes by the Trinity package; the unigenes had an average length of 612 bp and an N50 of 957 bp. Of these unigenes, 67,494 (51.86%) were annotated in a series of databases. Of these annotated unigenes, 41,818 and 24,615 were assigned to gene ontology categories and clusters of orthologous groups, respectively. Additionally, 20,764 (15.96%) unigenes were mapped onto 275 pathways using the KEGG database. In addition, 25,935 high-quality EST-SSR primer pairs were developed from the 15,433 unigenes by MISA mining. To validate the accuracy of the newly designed markers, 87 of 100 randomly selected primers were effectively amplified; 63 of those yielded PCR products of the expected size, and 25 yielded products with significant amounts of polymorphism among the 4 landraces. Furthermore, the transferability test of the 25 polymorphic markers was performed in 6 individuals of two closely related genus Phalaenopsis and dendrobium. Which results showed a total of 5 markers can successfully amplified among these populations. This research provides a comprehensive nucleotide database and lays a solid foundation for functional gene mining and genomic research in B. striata. The developed EST-SSR primers could facilitate phylogenetic studies and breeding.