In vitro propagation of Gentiana scabra Bunge – an important medicinal plant in the Chinese system of medicines

Background: Gentiana scabra Bunge commonly known as `Long dan cao\u27 in China has been used in traditional Chinese medicines for more than 2000 years. Dry roots and rhizome of the herb have been used for the treatment of inflammation, anorexia, indigestion and gastric infections. Iridoids and secoiridoids are the main bioactive compounds which attribute to the pharmacological properties of this plant. The species is difficult to mass propagate by seed due to the low percentage of germination and limited dormancy period. Wild populations in some locations are considered to be in the endangered category due to over exploitation. Results: In the present study, we report an efficient micropropagation system. Shoot apices of six weeks old in vitro grown G. scabra plants were used as explants for the in vitro propagation. Induction of multiple shoots (9.1/explant) was achieved on the culture of shoot apices on half strength Murashige and Skoog\u27s basal medium (MSBM) containing 2.0 mg/L 1 6-benzylaminopurine (BA), 3% sucrose and 0.9% Difco agar. In vitro shoots induced profuse rooting on half strength of MSBM supplemented with 0.1 mg/L 1 1-naphthaleneacetic acid (NAA), 3% sucrose and 0.3% gelrite. A two-stage ventilation closure procedure during the in vitro culture, and transparent sachet technique enhanced the survival rate of G. scabra plantlets to 96% in the greenhouse. Tissue culture plants flowered after 5 months of transfer to pots. Conclusions: A simple and an efficient in vitro propagation protocol of Gentiana scabra Bunge by optimizing the medium composition and ventilation closure treatments has been developed. The protocol can be very useful in germplasm conservation and commercial cultivation of G. scabra plants

Development of pollen mediated activation tagging system for Phalaenopsis and Doritaenopsis

Abstract In the present study, a novel plant transformation system for Doritaenopsis and Phalaenopsis has been developed. The pollen-mediated activation tagging system was established by artificial pollination. The pollens, co-cultured with Agrobacterium tumefaciens strain EHA105 harbouring an activation tagging vector (pTAG-8), were used for pollination. In order to optimize the transformation efficiency, several factors (concentration of A. tumefaciens, concentration of acetosyringone during co-cultivation and the duration of co-cultivation) known to influence Agrobacterium-mediated DNA transfer were examined. A concentration of 0.5-1 x 108 CFU/ml for A. tumefaciens, 0.1 mM acetosyringone, and 6 hrs of co-culture period were found to be the optimal condition for high transformation efficiency. Integration of T-DNA into the genome of putative transgenic plants was confirmed by PCR and DNA blot analyses. Single copy of the transgene was observed in all transgenic plants analyzed. Most of the transgenic plants had a morphologically normal phenotype and the overall capsule formation efficiency was similar to control plant. Our results showed a new approach of genetic transformation in orchids and this method can be employed for genetic improvement of the orchids

Wild bitter gourd improves metabolic syndrome: A preliminary dietary supplementation trial

<p>Abstract</p> <p>Background</p> <p>Bitter gourd (<it>Momordica charantia </it>L.) is a common tropical vegetable that has been used in traditional or folk medicine to treat diabetes. Wild bitter gourd (WBG) ameliorated metabolic syndrome (MetS) in animal models. We aimed to preliminarily evaluate the effect of WBG supplementation on MetS in Taiwanese adults.</p> <p>Methods</p> <p>A preliminary open-label uncontrolled supplementation trial was conducted in eligible fulfilled the diagnosis of MetS from May 2008 to April 2009. A total of 42 eligible (21 men and 21 women) with a mean age of 45.7 ± 11.4 years (23 to 63 years) were supplemented with 4.8 gram lyophilized WBG powder in capsules daily for three months and were checked for MetS at enrollment and follow-up monthly. After supplementation was ceased, the participants were continually checked for MetS monthly over an additional three-month period. MetS incidence rate were analyzed using repeated-measures generalized linear mixed models according to the intention-to-treat principle.</p> <p>Results</p> <p>After adjusting for sex and age, the MetS incidence rate (standard error, <it>p </it>value) decreased by 7.1% (3.7%, 0.920), 9.5% (4.3%, 0.451), 19.0% (5.7%, 0.021), 16.7% (5.4%, 0.047), 11.9% (4.7%, 0.229) and 11.9% (4.7%, 0.229) at visit 2, 3, 4, 5, 6, and 7 compared to that at baseline (visit 1), respectively. The decrease in incidence rate was highest at the end of the three-month supplementation period and it was significantly different from that at baseline (<it>p </it>= 0.021). The difference remained significant at end of the 4th month (one month after the cessation of supplementation) (<it>p </it>= 0.047) but the effect diminished at the 5th and 6th months after baseline. The waist circumference also significantly decreased after the supplementation (<it>p </it>< 0.05). The WBG supplementation was generally well-tolerated.</p> <p>Conclusion</p> <p>This is the first report to show that WBG improved MetS in human which provides a firm base for further randomized controlled trials to evaluate the efficacy of WBG supplementation.</p

Tissue Culture Technology of Medicinal Herbs and Its Application in Taiwan

藥用植物對人類健康極為重要，我國傳統醫學用藥以植物性藥物為主，歷經數千年而不衰，為我國醫藥文化之重要資源，值得加以發揚光大。從生藥研究開發新藥製品之市場，在公元2,000年預期將會有大幅度的成長。目前所有藥物中，自生藥取得成分者約佔25%，而其應用範圍仍不斷增加。然而許多藥用植物之繁殖，常面臨世代過長、果實結實率極低、種子發芽不良及因異花授粉產生後代嚴重變異等困難；而部分已瀕臨絕種之珍稀藥用植物更應立刻加以保存，組織培養技術提供了解決這些問題的機會。過去十年中農業試驗所在國科會補助下，進行本土藥用植物-台灣金線連及大陸珍貴藥材-當歸之組織培養技術研究，本文敘述這兩種藥用植物利用組織培養之大量繁殖技術。又懸浮細胞除可做為植物大量繁殖的材料外，亦可做為二次代謝物生產的工廠。由於植物懸浮細胞不具色素，而且生合成之二次代謝物純化過程極為簡單，如此不但大大降低了生產成本，而且提供週年生產的機會，因此利用植物懸浮細胞生產人類有用的重要醫療成分，前景亦大有可為。本文也報導農業試驗所在國科會支持下，進行紅豆杉、柴胡、竹葉柴胡、當歸、山藥、銀杏及白芷等藥用植物細胞懸浮培養之初步研究成果。 Medicinal herbs are the most important sources of medicines and many other pharmaceutical products, comprising about 25% of prescribed drugs. However, many of these plants when propagated by conventional methods take a long time for multiplication. They either have a low rate of fruit set, or have a poor seed germination and sometimes the clonal uniformity cannot be maintained through seed. Most of all, those endangered medicinal herb germplasms need to be conserved. The rapid multiplication of medicinal herbs by tissue culture techniques can help to solve these problems. Mass propagation of the medicinal herbs such as Anoectochilus formosanus (台灣金線連 ) and Angelica sinensis (當歸) has been the main research subjects of Taiwan Agricultural Research Institute during the last ten years. Current research in this area and their development, achievements during the process of mass propagation will also be discussed. Cell suspension systems could be used for the large-scale culture of plant cells from which secondary metabolites can be extracted. The principal advantage of this technology is that it may ultimately provide a continuous, reliable source of natural products year-round. In addition, compounds from tissue cultures may be more easily purified because of simple extraction procedure and the absence of significant amounts of pigments, thus possibly reducing production and processing costs. This paper described the methods that we have established for the production of useful compounds of the Angelica dahurica (白芷) , Dioscorea doryophora (山藥)Bupheurum falcatum (三島柴胡) Bupleurum marginatum (竹葉柴胡) and Angelica sinensis (當歸) by callus and cell suspension culture

Selection of cell lines with high DAPI stainability from rice (Oryza sativa L.) suspension culture for protoplast isolation and regeneration

Suspension culture plays an important role in high yield of protoplast isolation. From our routine maintenance of rice suspension lines, we found growth rates of most suspension lines were not quite stable even after subculture for 3-4 months. A DNA specific binding fluorochrome-DAPI(4',6-diamidino-2-phenylindole) was used to elucidate the nuclear status of cell lines during culture. Suspension lines derived from anther calli were used for this study. The results show that cell lines tend to have differential DAPI stainability during culture. The DAPI stainability is classified into high, medium and low groups based on percentage of suspension cells exhibiting nuclear fluorescence immediately after staining. Variations in regeneration ability and protoplast yield exist among three groups. High DAPI stainability cell lines tend to have higher regeneration ability and higher protoplast yields than the others. The average protoplast yields of high DAPI stainability lines are 2.5 x 10(7) protoplasts/g.fresh wt. cells and 7.8 x 10(6) protoplasts/g.fresh wt. cells for cultivars Hsin-Chu 56 (HC56) and Taipei 309 (TP309) respectively. A maximum yield up to 2-3 x 10(8) protoplasts/g.fresh wt. cells was also noted occassionally. Plating efficiency apparently increased when selection for high DAPI stainability and medium size of nucleus were applied. The non-stained cells were believed to be nonvital or aged cells in a contrast study on FDA test

Determination of the components in a Chinese prescription, Yu-Ping-Feng San, by RAPD analysis

In this study, the RAPD (random amplified polymorphic DNA) technique was employed for the first time to determine the components in a Chinese herbal prescription. Forty decamer oligonucleotide primers were screened in the RAPD analysis to identify three Chinese medicines, the dried root of Astragalus membranaceus (Fisch.) Bge., the dried root of Ledebouriello seseloides Wolff, and the dried rhizome of Atractylodes macrocephala Koidz, in a Chinese prescription. Only primer OPP-10 simultaneously generated three distinct markers were each specific to one component. The marker with 200 bp is specific to Astragalus membranaceus; the 440 bp marker is specific to Atractylodes macracephala; and the remaining marker with 500 bp was present in Ledebouriello seseloides. The presence of the three herbal medicines in the mixed sample, the Chinese prescription, was determined when the primer OPP-10 RAPD reaction was performed. The technique was proved to contribute to the identification of components in the Chinese medicinal preparations

Identification of Anoectochilus formosanus and Anoectochilus koshunensis Species with RAPD Markers

RAPD (random amplified polymorphic DNA) markers were developed to distinguish Anoectochilus formosanus from Anoectochilus koshunensis and their putative hybrids. Morphological differentiation of these two species beyond the flowering period is difficult. RAPD markers provide a rapid and easy tool for identification of the two Anoectochilus species. In the study, forty arbitrary decamer primers were screened, and nineteen species-specific RAPD markers generated from polymerase chain reactions (PCR) with eight random primers were obtained. Nine were specific to A. formosanus and ten to A. koshunensis. Two primers, OPC-08 and OPL-07, produced two markers, one specific to A. formosanus and the other specific to A. koshunensis, which simultaneously appeared in the hybrids pattern. The RAPD markers can be applied both to identification of A. formosanus and A. koshunensis species and to assessment of the extent fo hybridization in hybrids between them. This information facilitates the breeding program process