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.

Agrobacterium mediated transformation of chickpea (Cicer arietinum L.) embryo axes.

Embryo axes of four accessions of chickpea (Cicer arietinum L.) were treated with Agrobacterium tumefaciens strains C58C1/GV2260 carrying the plasmid p35SGUSINT and EHA101 harbouring the plasmid pIBGUS. In both vectors the GUS gene is interrupted by an intron. After inoculation shoot formation was promoted on MS medium containing 0.5 mg/l BAP under a selection pressure of 100 mg/l kanamycin or 10 mg/l phosphinothricin, depending on the construct used for transformation. Expression of the chimeric GUS gene was confirmed by histochemical localization of GUS activity in regenerated shoots. Resistant shoots were grafted onto 5-day-old dark-grown seedlings, and mature plants could be recovered. T-DNA integration was confirmed by Southern analysis by random selection of putative transformants. The analysis of 4 plantlets of the T1 progeny revealed that none of them was GUS-positive, whereas the presence of the nptII gene could be detected by polymerase chain reaction.

Plant regeneration via somatic embryogenesis in chick pea (Cicer arietinum L.).

Five genotypes of chickpea (Cicer arietinum L.) PG1, PG5, PG12, N59 and C235 were evaluated for induction of somatic embryogenesis. Somatic embryogenesis was induced from immature cotyledons of genotypes PG12 and C235 and immature embryo axes of genotypes PG5, PG12 and C235. Genotypes N59 and PG1 showed no response. The maximum frequency of globular embryo formation occurred in cotyledonary segments on MS medium with 3.0 mg/l 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Further embryo development was achieved only in somatic embryos derived from cotyledonary segments of genotype PG12. Globular-stage embryos derived from immature embryo axes of PG5, C235, PG12, and cotyledonary segments of C235 dedifferentiated and formed callus. The cotyledonary stage embryos of genotype PG12 germinated on half-strength MS medium supplemented with 1 mg/l zeatin. The regenerated plants were transferred to soil and grown to maturity.