Clonal propagation of Cnidium officinale by shoot tip culture

Multiple shoots were induced from the apical domes of shoot tips of Cnidium officinale Makino (Apiaceae) by culturing them on Murashige and Skoog (MS) 1 static media solidified with 0.2% gelrite and supplemented with 6-benzylaminopurine (BAP) 10 (-6)M. An average of 5.3 shoots per segment were obtained within 6 weeks and this ability did not decline even after two years of subculture. Subsequent transfer of these regenerated shoots on MS media supplemented with alpha-naphthaleneacetic acid (NAA) 10 (-7)M and BAP 10 (-7)M resulted in root formation. Rooted plantlets were able to grow in soil after a short period of acclimatization. Cytological observation in root tip cells of cultivated, as well as in vitro propagated plantlets revealed that in both cases the cells had 2n = 22 chromosomes indicating the homogeneity of the clonally propagated plants.     

Rutin and Scopoletin Content and Micropropagation of Fabiana imbricata

An HPLC method for the simultaneous quantitative determination of rutin and scopoletin in the aerial parts of FABIANA IMBRICATA is presented. The results showed high variability in the scopoletin (240-2,400 mg%) and rutin (195-1,950 mg%) content in the populations surveyed. A micropropagation method for F. IMBRICATA plants was established by culturing shoot tips on Murashige-Skoog (MS) medium containing 1.0 mg/l benzylaminopurine (BAP), 0.01 mg/l naphthaleneacetic acid (NAA), and 0.1 mg/l gibberellic acid (GA3). Auxin addition to the rooting medium, especially 0.5 mg/l indolebutyric acid (IBA) or 0.5 mg/l IBA and 0.1 mg/l NAA, enhances root formation. The micropropagation method presented allowed the obtention of regenerated plantlets in six weeks from shoot tips.     

Micropropagation of Polygonum multiflorum THUNB and quantitative analysis of the anthraquinones emodin and physcion formed in in vitro propagated shoots and plants

An efficient and rapid protocol for in vitro induction and complete plant regeneration of Polygonum multiflorum THUNB has been developed. Nodal explants were grown in vitro on Murashige and Skoog's (MS) basal medium containing different concentrations of alpha-naphthaleneacetic acid (NAA) and benzyladenine (BA). The nodal explants (97%) produced multiple shoots (4.7 shoots per explant) on MS basal medium supplemented with 0.2 mg/l NAA and 2.0 mg/l BA after 6 weeks of culture. Eighty-eight percent to 100% of the shoots (1.0 cm in length) elongated (about 3.02-4.28 cm) and rooted on MS basal medium supplemented with NAA or indole-3-butyric acid (IBA). All the rooted shoots were transferred to pots containing autoclaved soil, vermiculite, and peat moss (1 : 1 : 1). The plantlets were successfully acclimatized under greenhouse conditions with high humidity before transferring to the field. The anthraquinone contents were determined using HPLC. Analysis revealed that the contents of the major medicinal compounds-emodin and physcion in the 6 weeks old in vitro grown shoots and three month old in vitro propagated plants grown in greenhouse were higher than those of the marketed crude drug (processed underground or stem parts of P. multiflorum).     

Helenalin Acetate in in vitro Propagated Plants of Arnica montana

Propagated "IN VITRO" shoots and plantlets of ARNICA MONTANA L. (Asteraceae) have been shown to produce sesquiterpene lactones, i.e. helenalin and 11,13-dihydrohelenalin esters. The compounds were detected in green organs only; roots of the plantlets contained no sesquiterpene lactones. The helenalin acetate content in leaves of the plantlets (0.073% dry wt) was 4-times higher than in proliferated shoots (0.016% dry wt). The best rate of shoot multiplication was achieved on MS medium, supplemented with NAA 0.5mg/l and Kn 2.5 mg/l (formation of 22 shoots within 8 weeks).     

Studies on the tissue culture of Stevia rebaudiana and its components; (II). Induction of shoot primordia

Shoot primordia, which were able to propagate vegetatively with a very high rate and to redifferentiate easily to new plants, were induced from shoot tips of STEVIA REBAUDIANA Bertoni on Gamborg B5 medium containing 6-benzylaminopurine (BAP) and alpha-naphthaleneacetic acid (NAA) under light. The propagation of the shoot primordia of STEVIA REBAUDIANA is rapid, and they are highly stable in chromosome number and karyotype. The shoot primordia can propagate at a high rate for a long time without differentiation. At any time, the shoot primordia readily developed into plantlets with shoots and roots within 2 or 3 weeks in static culture on B5 medium containing 0.02 mg/l BAP and 2% sucrose. The plantlets were transplanted to sterilized soil to grow to normal adult plants.     

Tropane alkaloid production and shoot regeneration in hairy and adventitious root cultures of Duboisia myoporoides-D. leichhardtii hybrid

Co-culture conditions for Duboisia myoporoides-D. leichhardtii hybrid hairy root induction were investigated using leaf explants and Agrobacterium rhizogenes ATCC 15834. The bacteria density and duration of co-culture greatly affected the induction rate; the highest rate of 50% was obtained when the leaf explants were co-cultured for 2 d with 10(6) bacteria. One hairy root clone that showed the fastest root growth was selected and used for comparison study with adventitious roots cultured with 0.5 mg/l indole-3-acetic acid (IAA). The hairy roots cultured in Murashige and Skoog (MS) liquid medium grew well and yielded much more tropane alkaloids (35 mg/l scopolamine and 17 mg/l hyoscyamine) than adventitious roots cultured in 0.5 mg/l IAA after 6 weeks of culture at 25 degrees C in the dark. The hairy and adventitious roots (2.5 cm) grown in liquid media were divided into 5 parts (each 0.5 cm) along the root axis. Distribution of scopolamine and IAA was then determined by enzyme-linked immunosorbent assay (ELISA). Inverse relationship between contents of scopolamine and IAA was observed in the hairy roots; increase of scopolamine and decrease of IAA were proportional to the distance from the root meristem. In contrast, the contents of scopolamine and IAA were relatively constant in the adventitious roots. In shoot regeneration experiments, the hairy and adventitious root segments (1 cm) were placed onto 1/2 MS solid medium containing various concentrations of IAA and BA cultured at 25 degrees C under 16 h light. In adventitious roots, the shoots regenerated on media containing 6-benzyladenine (BA) (0.5 to 5 mg/l), and 100% regeneration was observed in medium with 0.1 mg/l IAA and 2 mg/l BA. On the other hand, shoot regeneration was only observed in 33% of hairy roots cultured on medium containing 5 mg/l BA.     

Clonal Multiplication of Aconitum carmichaeli by Tip Tissue Culture and Alkaloid Contents of Clonally Propagated Plant

Shoot tips and axillary buds of ACONITUM CARMICHAELI Debx. cultured in Murashige and Skoog medium supplemented with 5 ppm BAP at 25 +/- 1 degrees C under continuous light for 6 weeks formed multiple shoots with an average of 7 shoots per culture. Multiple shoot forming ability does not decrease at least for 6 generations. Subsequent culture of these shoots on MS medium supplemented with 0.5 ppm IAA at 20 +/- 1 degrees C under continuous light for 6 weeks resulted in root formation. The plantlets were transplanted successfully in the soil. Alkaloid contents of clonally-propagated plants showed less variation than those of the normally grown plants.     

In vitro plant regeneration from leaf-derived callus of Cimicifuga racemosa

Cimicifuga racemosa (L.) Nutt., also known as Black Cohosh, is among the top 10 selling medicinal herbs in the United States. The rhizomes have been used to relieve menopausal discomfort. This plant is wild crafted and conservationists have expressed concerns with the sustainability of C. racemosa. Excised tissues from young leaves of C. racemosa were cultured on Murashige and Skoog's medium (MS) supplemented with various concentrations of NAA and TDZ for production of callus. The optimum callus growth and maintenance was in 1.0 microM NAA plus 0.5 microM TDZ. Two-month-old calli were sub-cultured on different concentrations of cytokinins (BA, kinetin, 2ip, TDZ) or in combination with GA(3) for shoot induction. The rate of shoot induction and proliferation was higher in MS media supplemented with 2.0 or 4.0 microM of TDZ. Concentrations of TDZ greater than 4.0 microM suppressed shoot growth. Adding 3.5 microM of GA(3) into media containing BA increased shoot growth. The presence of GA(3) with kinetin or TDZ did not affect shoot production. For rooting, shoots were transferred to MS medium with activated charcoal supplemented with various auxins (IAA, IBA and NAA), roots were noticed 20 days after transference. Activated charcoal was an essential component for vigorous rooting formation. Our results suggest that conservation of C. racemosa is possible through in vitro multiplication of leaf-derived callus.     

Micropropagation and in vitro flowering of Rauvolfia tetraphylla; a potent source of anti-hypertension drugs

A simple protocol for in vitro mass multiplication of Rauvolfia tetraphylla (Apocynaceae) has been developed. The endophytic microflora was controlled by adopting integrated measures. Multiple shoot development was achieved on MS + Kin (0.1-0.2 mg/l) + BAP (0.4-0.5 mg/l) media. Rooting from in vitro shoots occurred on NAA containing media. In vitro flowering was induced in shoot multiplication media.     

In vitro propagation of Stevia rebaudina plants using multiple shoot culture

A multiple shoot culture was induced from nodal segments on MS medium containing half concentration of macroelements, 1% sucrose, and supplemented with NAA (0.01 mg/l). A bioreactor with hormone-free MS medium (300 ml) was inoculated with 1.5 g of the multiple shoot culture and cultivated for a month. The cultivating process of the multiple shoot culture in the bioreactor and the transfer into ex vitro conditions took about 8-9 weeks and produced approx. 600 new seedlings, that could be transferred from greenhouse to field conditions.     

Cultures ofGinkgo biloba,effect of nutritional and hormonal factors on the growth of cultured cells derived fromGinkgo biloba

Calli and suspension cultures were obtained following inoculation of the explant from leaves ofGinkgo biloba L. on the supplemented MS basal medium. The obtained calli and suspension cultured cells were able to produce detectable amounts of ginkgolides which are known as natural specific PAF antagonists. The production of ginkgolides in the calli and suspension cultured cells were identified using GC/MS, GC and HPLC with authentic compounds. Since the production of ginkglides A and B in the calli and suspension cultured cells had been confirmed, effects of types and concentration of plant growth regulators, media and illumination on the induction and growth of the callus were studied. The concentrations of growth regulators for optimal callus induction were 1.0 to 2.0 mg/L for NAA and 0.1 mg/L for kinetin. The growth of the callus seemed to be more stimulated with the combination of NAA and kinetin than NAA and BA with illumination at all concentration ranges of 1.0 to 4.0 mg/L for NAA and 0.1 to 1.0 mg/L for kinetin or BA studied. Among 8 different media used, the induction rate of callus on Anderson, Eriksson, and Schenk and Hildebrant at 4 weeks after the innoculation was almost the same as that of MS. However, callus was rarely induced on Heller or White medium. Suspension cultures were easily initiated with 3 g of callus (fresh weight) derived from ginkgo leaves on supplemented MS medium. A typical growth curve of suspension cultured cells could be obtained by measuring the fresh weight of the suspension cultured cells at every 3 days. To improve the growth of suspension cultured cells of ginkgo, effects of concentrations of NAA, sucrose, phosphate ions and molar ratio of NH₄ ⁺ to NO₃ ^? ions in the culture medium were studied. The maximum growth of the cells was achieved when the culture medium contained 1.0 mg/L of NAA, 30 g/L sucrose, 1.75 mM phosphate ions and 1∶5 molar ratio of NH₄ ⁺ to NO₃ ^? ions.     

Transformation of ipecac (Cephaelis ipecacuanha) with Agrobacterium rhizogenes

Transformed root cultures of ipecac (Cephaelis ipecacuanha A. Richard), one of the recalcitrant woody plant species for Agrobacterium-mediated transformation, were established by co-culturing of in vitro petiole segments with Agrobacterium rhizogenes ATCC 15 834. Southern blot analysis of the established roots revealed that only the TL-DNA was integrated into the plant genome without incorporation of the TR-DNA. The transformed roots grew slowly on phytohormone-free solid medium and adventitious shoots were regenerated after over 6 months of culture on HF, half-strength Murashige and Skoog (1/2 MS) medium in the dark. The individually separated transformed shoots developed into plantlets on phytohormone-free solid medium at 25 degrees C under 16 h/day light, and the plants demonstrated wider leaves, shorter internodes and vigorous root growth compared to non-transformed plants. Effects of basal media and auxins on the growth and the ipecac alkaloid production of the transformed roots were investigated either under light or in the dark. The roots cultured in the dark grew well in Gamborg B5 (B5) liquid medium containing 0.5 mg/L IBA and yielded 112 mg/L of cephaeline and 14 mg/L emetine after 8 weeks of culture.     

Cryopreservation of Digitalis lanata Shoot Tips

A method for the preservation in liquid nitrogen of shoot tips (meristems) of D. LANATA is described. It includes the following steps: (a) hardening of shoots by cultivation at 4 degrees C for 8 weeks, (b) treatment of the explanted shoot tips with cryoprotectors, e.g., 2 mol DMSO l (-1) for 2 h, (c) either ultrarapid cooling (ca. 4000 K min (-1)) of the shoot tips by submerging in liquid nitrogen or slow cooling (ca. 0.5 K min (-1)) of the shoot tips to -40 degrees C using a suitable freezer, (d) storage of the shoot tips at -196 degrees C in liquid nitrogen, (e) ultrarapid rewarming of the ultrarapidly cooled shoot tips by placing them directly into nutrient medium or rapid rewarming of the ampoules containing the slowly cooled shoot tips with water at 40 degrees C, and (f) recultivation of the shoot tips at the surface of a solidified nutrient medium containing 2.5 micromol BA 1 (-1). About 70% of the shoot tips survived this procedure and about 30% of the shoot tips regenerated shoots.     

Auxins affected ginsenoside production and growth of hairy roots in Panax hybrid

Hairy roots of interspecific hybrid ginseng (Panax ginseng x P. quinquefolium), induced by Agrobacterium rhizogenes ATCC 15834, grew well in B5 liquid media supplemented with 2.5 microM auxins (3-indole butyric acid (IBA), 1-naphtaleneacetic acid (NAA) and 3-indoleacetic acid (IAA)). The hairy roots cultured in B5 liquid medium supplemented with 2.5 microM IBA showed best growth (6.39 g fresh weight per a flask, at week 8). The highest content of the total ginsenosides was 1.63% as dry weight at week 8 when cultured with 2.5 microM NAA. The different auxins affected the numbers and lateral branching roots. Especially, 2.5 microM IBA promoted the lateral root formation (43.7+/-4.0 roots, at week 8), and 2.5 microM NAA promoted the lateral root growth (45.3+/-5.6 mm, at week 8). The growth and ginsenosides production of 8-week old hairy roots cultured in B5 liquid media supplemented with IBA and NAA combinations were also investigated. Hairy roots produced higher amounts of ginsenosides in B5 liquid media supplemented with 0.5-1.0 microM IBA and NAA combinations than that cultured in B5 liquid media supplemented with only IBA and NAA. The highest yield of ginsenoside was obtained when cultured with 0.5 microM IBA and 1.0 microM IBA combination (6.38 mg per a flask, at week 8).     

Somatic Embryogenesis and Clonal Multiplication of Panax ginseng

Embryogenesis of PANAX GINSENG was induced from young flower buds via callus during a 3 months culture period. Matured embryos could be germinated on the 1/2 Murashige and Skoog's medium supplemented with GA (3) (1.4 microM)-BAP (2.2 microM) and 1.5% sucrose. In the medium containing 1.4 microM GA (3) and 11.1 microM BAP, a multiple shoot complex having 8 shoots per segment was formed from a single shoot set. On the other hand, the addition of 11.1 microM BAP and 1.4 microM GA (3) to the medium stimulated the flower bud formation. For root formation of shoots, the MS medium supplemented with 5.4 microM NAA was the most favourable. Subsequently the plantlet was transferred to vermiculite.     

Efficient protocol for rapid Aloe vera micropropagation

Context: Aloe vera Linn. (Liliaceae) is a medicinal plant and has a number of curative properties. Vegetative propagation has not enough potential for supplying market demand. However, via in vitro propagation makes possible the mass production of Aloe plants.

Objective: The current study was conducted to investigate growth regulators’ effects on proliferation of A. vera.

Materials and methods: In this study, for comparison of plant growth regulators’ effects on proliferation, the shoot tips and auxiliary buds of A. vera were cultured in the Murashige and Skoog (MS) medium. Rooted plantlets were transferred to garden soil, compost, and sand in the proportion of 1:1:1, respectively, after hardening.

Results: The maximum number of shoots was obtained on the medium supplemented with 1?mg/L IAA?+?4?mg/L BAP and 0.2?mg/L IAA?+?0.8?BAP mg/L. Rooting was also achieved in the same media composition proliferation of shoot. The acclimatized plants showed 100% of survival. The regenerated plants looked healthy, and they were morphologically similar to that of stock plants.

Conclusion: These results suggest that in vitro culture may be used as a technique for rapid propagation of A. vera.     

Micropropagation of a Thai medicinal plant for women’s health, <Emphasis Type="Italic">Curcuma comosa</Emphasis> Roxb., via shoot and microrhizome inductions

We studied the effects of explant types, plant growth regulators, and sucrose concentrations on shoot and microrhizome inductions of Curcuma comosa Roxb., an important Thai medicinal plant for women’s health. Explant types significantly affected shoot induction of the plant. The maximum shoot multiplication rate of 11.82 ± 1.03 shoots/responding explant was obtained when culturing terminal bud explants on semi-solid Murashige and Skoog (MS) medium supplemented with 18.16 μM thidiazuron for 8 weeks. Subsequently, they were transferred to a semi-solid MS medium without plant growth regulators for 4 weeks. The regenerated shoots produced roots spontaneously. Rooted plantlets were successfully transferred to the soil. Microrhizome induction was significantly influenced by sucrose concentrations, but not by 6-benzyladenine (BA). Liquid MS medium with a combination of 17.76 μM BA and 50 g L⁻¹ sucrose was optimal for microrhizome induction of C. comosa. After 12 weeks of culture, the microrhizome induction rate was 3.36 ± 0.44 microrhizomes/responding explant. Starch accumulation in microrhizomes increased with higher sugar concentration and with longer duration of culture. The microrhizomes were allowed to germinate under greenhouse conditions and further developed into normal plants. The protocols established will be used for the production of uniform plantlets suitable for field plantation for the herbal industry.     

Formation of Digitalis lanata Clone Lines by Shoot Tip Culture

Propagation of DIGITALIS LANATA by shoot tip culture made possible (a) the rapid multiplication of elite plants with the formation of plant clones and (b) the long-term cultivation of these plants which under normal growth conditions would die at the end of the second vegetation period. Optimum conditions were established for the regeneration of shoots from shoot tips, for daughter shoot formation and rooting as well as for the adaptation of the regenerated plants to the open ground. Gene banks of valuable clones were built by keeping shoots at 4 degrees C on media with high sucrose concentration (maximum period of storage 2 years) or by growing juvenile clone plants in the greenhouse at temperatures preventing the induction of flowering. The clone plants were in the juvenile state even if they were derived from flowering mother plants. They showed normal growth and development.     

Development of in vitro techniques for the important medicinal plant Veratrum californicum

VERATRUM CALIFORNICUM (Liliaceae) is an important monocotyledonous medicinal plant which is the only source of the anticancer compound cyclopamine. An IN VITRO culture system for somatic embryogenesis and green plant regeneration of VERATRUM CALIFORNICUM was developed. Embryogenic calli were induced from mature embryos on induction medium. Five basal media supplemented with different growth regulators were evaluated for embryogenic callus induction, modified MS medium with 4 mg/L picloram showing the best result for embryogenic callus production. Fine suspension cell lines were established by employing friable embryogenic calli as starting material and AA medium and L2 medium as culture media. The suspension cell lines cultured in AA medium with 4 mg/L NAA appeared to be fresh yellow and fast growing. The suspension cells were cryopreserved successfully and recovered at a high rate. Green plants were regenerated from embryogenic calli maintained on solid medium with 73 % regeneration ability (green plants/100 calli) in 27-month-old culture. The IN VITRO plantlets contained the steroid alkaloids cyclopamine and veratramine. This IN VITRO system will form the basis for metabolic engineering of VERATRUM cells in the context of biotechnological production of pharmaceutically important secondary metabolites. DMSO:dimethyl sulfoxide fw:fresh weight NAA:naphthaleneacetic acid 2,4-D:2,4-dichlorophenoxyacetic acid picloram:4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid dicamba:3,6-dichloro-2-methoxybenzoic acid.     

Clonal propagation of Isoplexis canariensis

Isoplexis is a plant genus closely related to Digitalis. Members of this genus contain cardenolides considered more "primitive" than those present in Digitalis. Isoplexis plants, tissue cultures, and isolated cardenolides may thus be used to elucidate the biosynthesis of cardenolides in the Scrophulariaceae. Therefore, a method was developed to cultivate and propagate Isoplexis canariensis (L.) Lindl. ex. G. Don in vitro. Seeds were germinated in liquid modified MS medium and shoot cultures were established and propagated in liquid modified MS medium containing 0.1 mg/l IAA and 1 mg/l BAP. Shoot cultures were also established from excised axillary buds and propagated on solid culture medium containing 0.1 mg/l IAA and 1 mg/l BAP. Shoots of either origin were rooted in medium containing 1 to 5 mg/l IAA and 0.5 to 4 mg/l IBA. Rooted plantlets were cultivated for 2 to 3 weeks in hormone-free modified MS medium and then transferred to the greenhouse, where they developed into healthy plants.