De novo regeneration of Scrophularia yoshimurae Yamazaki (Scrophulariaceae) and quantitative analysis of harpagoside, an iridoid glucoside, formed in aerial and underground parts of in vitro propagated and wild plants by HPLC.

A protocol for de novo regeneration and rapid micropropagation of Scrophularia yoshimurae (Scrophulariaceae) has been developed. Multiple shoot development was achieved by culturing the shoot-tip, leaf-base, stem-node and stem-internode explants on Murashige and Skoog (MS) medium supplemented with 4.44 microM N6-benzyladenine (BA) and 1.07 microM alpha-naphthaleneacetic acid (NAA). Stem-node and shoot-tip explants showed the highest response (100%) followed by stem-internode (74.4%) and leaf-base (7.7%) explants. The shoots were multiplied by subculturing on the same medium used for shoot induction. Shoots were rooted on growth regulator-free MS basal medium and the plantlets were transplanted to soil and acclimatized in the growth chamber. The content of harpagoside, a quantitatively predominant iridoid glycoside, in different plant material was determined by high performance liquid chromatography (HPLC). The analysis revealed that the content of harpagoside in the aerial and underground parts of S. yoshimurae was significantly higher than the marketed crude drug (underground parts of Scrophularia ningpoensis).

Formation of protoberberine-type alkaloids by the tubers of somatic embryo-derived plants of Corydalis yanhusuo.

The effects of 0.5 - 5 mg/l abscisic acid [ABA], 0.5 - 10 mg/l (2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol [paclobutrazol] and 0.5 - 2 mg/l alpha-cyclopropyl-alpha-(4-methoxyphenyl)-5-pyrimidinemethanol [ancymidol], 0.5 - 5 mg/l gibberellic acid [GA(3)] and 15 - 100 mg/l polyethylene glycol [PEG] 4000 supplemented in half-strength Murashige and Skoog's (MS) medium on the production of the two major protoberberine-type alkaloids (D,L-tetrahydropalmatine and D-corydaline) by the tubers of somatic embryo-derived plants of Corydalis yanhusuo were examined. Somatic embryo derived plants were also maintained for 6 months on half-strength MS medium containing 0.1 mg/l GA(3) or 0.5 mg/l paclobutrazol. The alkaloid contents were determined by high performance liquid chromatography (HPLC). The analysis revealed that the contents of D,L-tetrahydropalmatine and D-corydaline in the tubers of somatic embryo-derived plants were higher than the marketed crude drug and varied with growth regulator/PEG-4000 treatment and the age of the plant.

Quantitative determination of secoiridoid glucosides in in vitro propagated plants of Gentiana davidii var. formosana by high performance liquid chromatography.

A simple protocol for in vitro mass propagation of Gentiana davidii var. formosana (Gentianaceae) has been developed. Multiple shoot development was achieved by culturing the stem node explants on Murashige and Skoog (MS) medium supplemented with 4.44 microM N6-benzyladenine (BA). The shoots were multiplied by subculturing on MS medium supplemented with 1.07-10.74 microM alpha-naphthaleneacetic acid (NAA) and 8.88 microM BA. Shoots were rooted on MS basal medium supplemented with various auxins. Shoots rooted on growth regulator-free medium were transferred to peat moss:vermiculite mixture and acclimatized in the growth chamber. The contents of gentiopicroside and swertiamarin, the two important secoiridoid glucosides, in different plant material were determined by high performance liquid chromatography (HPLC). The analysis revealed that the content of gentiopicroside and swertiamarin in the aerial and underground parts of G. davidii var. formosana was higher than the marketed crude drug (underground parts of G. scabra) and varied with the age of the plant.

Cytokinin-induced somatic embryogenesis and plant regeneration in Corydalis yanhusuo (Fumariaceae) - a medicinal plant.

An efficient method has been developed for regeneration of complete plants via somatic embryogenesis in Corydalis yanhusuo (Fumariaceae), an important medicinal plant, using tuber-derived callus. Primary callus was induced by culturing mature tuber pieces on Murashige and Skoog's (MS) medium supplemented with 2.0 mg l(-1) N(6)-benzyladenine (BA) and 0.5 mg l(-1) alpha-naphthaleneacetic acid (NAA) in darkness. Somatic embryos were induced by subculturing the primary callus on MS medium supplemented with 0.5-4.0 mg l(-1) BA, kinetin, or zeatin, within 2 weeks of culture in light. Embryos with well-developed cotyledonary leaves were transferred in half-strength liquid MS medium supplemented with 1.0 mg l(-1) zeatin riboside for the development of roots. Converted somatic embryos were cultured on half-strength MS medium supplemented with 6% sucrose, and with 0.5-10.0 mg l(-1) abscisic acid (ABA), paclobutrazol, or ancymidol, 0.5-5.0 mg l(-1) GA(3) and 15-100 mg l(-1) polyethylene glycol (PEG) 4000 for further development of plantlets and in vitro tuber formation. The development of somatic embryos over the surface of tuber and/or cotyledonary leaf base region of the converted primary somatic embryo was observed. Before ex vitro establishment of somatic embryo-derived plants, plants with well-developed tubers were cultured on half-strength MS medium with 2% sucrose and 0.1 mg l(-1) GA(3) for 3 weeks.

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 Ledebouriella 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 macrocephala; and the remaining marker with 500 bp was present in Ledebouriella 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.

Somatic embryo formation and germination from immature embryo-derived suspension-cultured cells of Angelica sinensis (Oliv.) Diels.

Embryogenic callus was induced from immature embryos of Angelica sinensis cultured on Murashige and Skoog (MS) basal medium. Embryogenic callus growth was more rapid on MS basal medium than on B5 or White medium. Embryogenic callus was used to establish a suspension culture and somatic embryos and germinating embryos developed during the culture. A shaking speed of 80 rpm was found to be optimal for establishing suspension cultures, while 100 rpm produced more somatic embryos and germinating embryos with an initiation cell density of 0.2 ml packed cell volume/25 ml medium. Adding 0.3% agar to the liquid medium also stimulated the formation of somatic and germinating embryos. While no plant growth regulators were needed for culture initiation and plant regeneration, the addition of 0.5-1 mg/l 2,4-dichlorophenoxyacetic acid was needed to maintain the embryogenic suspension culture by preventing embryo germination. Forty percent of the germinating embryos survived after culturing on filter paper moistened with liquid half-strength MS medium containing 3% sucrose. The plants were successfully transferred into soil.

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.

Embryogenesis and plant regeneration from anther culture of bamboo (Sinocalamus latiflora (Munro) McClure).

Embryogenic callus was initiated from bamboo (Sinocalumus satiflora (Munro) McClure) anthers cultured on N6 medium supplemented with 1 mg/l 2,4-D, 1 mg/l BA, 2 g/l charcoal, 0.8% agar (Sigma) and 9% sucrose. Anthers with microspores at miduninucleate to early-binucleate stages showed better rate of response for callus induction. Prolonged culture of these embryogenic calli on the original medium or subculture to an auxin-free medium resulted in embryoid formation and their subsequent germination to form rooted plantlets. Chromosome counts from root-tip cells of anther-derived plant indicated that they were haploid (N=36).

Rapid clonal propagation of Pinellia ternata by tissue culture.

Adventitious buds or protocorm-like bodies were regenerated directly from excised explants without intervening callus. Differences in the ability of regeneration were observed among different plant organs with bulbils showing the highest regenerative ability followed by leaf blade and petiole. Ability of vegetative propagation of bulbil could be maintained by alternate solid-and liquid-medium culture. Theoretically, 1.7×10(27) plantlets could be produced from a single bulbil by this technique within one year based on the production and rapid growth of protocorm-like bodies and adventitious buds. Concentration of MS salts, NAA and sucrose influenced not only root formation from the differentiated adventitious buds, but also root number and length. For root formation, the best combination was one-half strength MS salts with 3-5% sucrose and 1 mg/l NAA. The high survival rate of 96% was recorded when plantlets were transplanted into a mixture of vermiculite:loam soil:peat moss (1:2:1). Plants from in vitro culture were morphological similar to field-grown plants. The acute toxicity of crude extracts from protocorm-like bodies was about one-fourth that of extracts from tubers of field-grown plants when tested with white mice. Tissue culture has potential for clonal propagation of Pinellia ternata plants for commercial use.

Cytochemical studies of callus development from microspore in cultured anther of rice.

Cytochemical studies of androgenic anthers of Oryza sativa picked from the culture at 2 day intervals from 0 to 40 days have been carried out. Glutaradehyde-OsO4-fixed and plastic-embedded sections were stained with TBO, SBB and PAS for acidic polymers, lipids and polysaccharides respectively. Among the population only 4% of microspores, which accumulate abundant amorphous lipid in the first few days of culture, are androgenic. Less than 30%, which have many lipid granules and some amorphous lipid, become nutritive microspores. Starch grains also accumulate in these nutritive microspores which degenerate at the stage when the androgenic multicellular microspores are in rapid development. The remaining microspores, which have no or little lipid, degenerate early. At about the 100-cell stage, each multicellular unit consists of two cell types, large and small. The large cells contain abundant amorphous lipid and starch grains which the small ones stain intensely with TBO.Our results indicate that the epidermis and endothecium of the cultured anthers are not quiescent. They can accumulate and transport lipid and polysaccharides at certain stages during the cultural period. Globular embryoid appearing structures and leaf-like protrusions can be observed at the surface of the callus in about 40-day old culture, indicating that both embryogenesis and organogenesis may take place in rice callus.

Anther culture of papaya (Carica papaya L.).

Papaya (Carica papaya L.) anther containing microspores in tetrad to early-binucleate stages were successfully cultured on 1/2 strength MS salts and vitamins with full strength Na-Fe-EDTA supplemented with 2 mg/l NAA, 1 mg/l BA and 6% sucrose for callus initiation and formation. Highest frequencies of callus induction were obtained when anthers at the uninucleate stage were cultured in the dark. Haploid plantlets and pollen-derived embryoids were obtained from anthers cultured at the uninucleate stage on solidified MS medium containing 3% sucrose without any growth regulators under a low light intensity (1,500 lux). Large quantities of embryoids were obtained when the original embryoids were transferred to MS medium with 3% sucrose and no growth regulators. Cytology of root tips of embryoid-derived plants confirmed the haploid chromosome number of 9 indicating that the embryoids originated from pollen.