<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of Persian poppy (<Emphasis Type="Italic">Papaver bracteatum</Emphasis>)

Persian poppy (Papaver bracteatum Lindl.) is an important commercial source of medicinal opiates and related compounds. In this research, calli were induced from seeds, roots, cotyledons and hypocotyls of P. bracteatum at a high efficiency. The optimized callus induction media consisted of the Murashige and Skoog (MS) basic media supplemented with 1.0 mg/L 2, 4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg/L kinetin and 15 mg/L ascorbic acid. The concentrations of 2,4-D and ascorbic acid were found critical to callus induction and proliferation. Subsequent subcultures resulted in excellent callus proliferation. Ascorbic acid at concentration 15 mg/L increased the callus proliferation significantly. Maximum callus growth was achieved when the explants were incubated at 25°C. MS salts at full strength were found inhibitory for callus induction, while ľ MS salts were found to favor callus induction. Shoot regeneration of calli in vitro was achieved on ľ MS medium containing 0.5 mg/L benzylamine purine and 1.0 mg/L naphthalene acetic acid. Analysis of alkaloid extracts from Persian poppy tissues by high-performance liquid chromatography showed that thebaine accumulated in the tissues of plants. The thebaine alkaloid profile of the Persian poppy is a well-defined model to evaluate the potential for metabolic engineering of thebaine production in P. bracteatum.     

Effects of genotype, explant source, and plant growth regulators on indirect shoot organogenesis in Dieffenbachia cultivars

The capacity for indirect shoot organogenesis of leaf and root explants of four Dieffenbachia cultivars were examined on a modified Murashige and Skoog (MS; Physiol Plant 15:473–495, 1962) medium supplemented with different plant growth regulators in 112 combinations. Callus formation was only observed from leaf explants on MS supplemented with 1–10 μM thidiazuron (TDZ) and 0.5–1.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D) regardless of cultivars. The combination of 5 μM TDZ and 1 μM 2,4-D resulted in the greatest callus formation frequency among the four cultivars tested. Significant differences in callus and shoot formation from leaf explants were also observed among cultivars. Cultivars Camouflage, Camille, Octopus, and Star Bright produced green nodular, brown nodular, yellow friable, and green compact calli with corresponding maximum callus formation frequencies of 96%, 62%, 54%, and 52%, respectively. A maximum of 6.7 shoots/callus was observed in cv. Camouflage, followed by cvs. Camille and Star Bright at 3.7 and 3.5, respectively. Calli of cv. Octopus displayed no capacity for shoot organogenesis. Regardless of cultivar, callus formation was not observed on root explants. Regenerated shoots were successfully acclimatized in a shaded greenhouse condition with 100% survival.     

Tropane Alkaloids from Callus Cultures, Differentiated and Shoots of Hyoscyamus muticus L.

Alkaloid production has been observed in cotyledonary leaf derived callus tissues, and also in in vitro differentiated shoots, and roots of Hyoscyamus muticus. The callus tissue was developed form cotyledonary leaf explants on Murashige and Skoog medium enriched with 2 mg 1^-1 2, 4-D and 0.5 mg 1^-1 BAP. Cotyledonary leaf derived callus was proliferated in the same medium for 2 passages (1 passage 28-30 days). Green and compact callus was used for alkaloid extraction. Shoots and roots formed on MS medium containing 0.05 mg 1^-1 NAA and 0.5 mg 1^-1 BAP, and also compact, nodular and embryogenic calli from which these shoots and roots differentiated, were used for alkaloid extraction. Chromatographic studies performed with TLC showed the presence of hyoscyamine as the major alkaloid present in the callus tissues, differentiated shoots and roots. However, alkaloid content varied in different tissues. Differentiated roots were found to contain maximum amount of hyoscyamine.     

ABA enhances plant regeneration of somatic embryos derived from cell suspension cultures of plantain cv. Spambia (<Emphasis Type="Italic">Musa</Emphasis> sp.)

Creamy friable calli were induced from meristems (scalps) of proliferating shoots of plantain (Musa sp.) cv. Spambia (genome AAB) incubated on a semi-solid modified Murashige and Skoog (MS) medium supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 μM zeatin. About 25% of shoot-tip explants formed scalps, and about 98% of scalps developed embryogenic calli. Small dense aggregates of cells, were obtained when these calli were transferred to liquid MS medium supplemented with 4.5 μM 2,4-D and 1.0 μM zeatin. Upon transfer to semi-solid MS medium of the same composition as described above, aggregates of cells formed somatic embryos. In the presence of 2.5 μM abscisic acid (ABA), maturation of somatic embryos was 2.6-fold higher than that of control (lacking ABA), and regardless of the type of cytokinin used in the medium. Upon transfer to MS medium supplemented with 1.25 μM 6-benzyladenine (BA), 80% of germinated embryos developed into plantlets.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration through somatic embryogenesis and direct shoot organogenesis in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.

An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.     

Overcoming phenolic accumulation during callus induction and in vitro organogenesis in water chestnut (Trapa japonica Flerov)

Summary Procedures for callus induction and subsequent organogenesis in the aquatic plant, water chestnut (Trapa japonica Flerov), were established. Phenolics exuded from explants at the callus-induction stage adversely affect callus growth. For cotyledonary node-derived callus cultured in Murashige and Skoog (MS) medium (full, half or quarter strength) containing 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with benzyladenine (BA), the accumulation of phenolics was reduced and callus induction increased by the addition of 10.8 μM phloroglucinol (PG) to the medium. Ascorbic acid was also effective in reducing phenolic accumulation, but less effective for callus induction than PG. Half-strength MS medium supplemented with 2.7 μM 2,4-D, 108.0 μM casein hydrolyzate, and 10.8 μM PG supported maximum callus induction. Plant organogenesis was increased by addition of vitamins (0.27 μM biotin and 2.7 μM folic acid) to half-strength MS medium supplemented with 0.27 μM BA. Many shoots developed from the regenerated nodal shoot explants in liquid half-strength MS salts medium supplemented with 1.08 μM BA and 0.27 μM naphthaleneacetic acid. Individual shoots were excised and cultured in liquid half-strength MS medium supplemented with 5.4 μM IBA and rooted plantlets (108) were transferred and acclimatized in plastic pots. After 3 wk, the plantlets were transplanted in a water chestnut field and the survival rate was 100%.     

High frequency plant regeneration and accumulation of tropane alkaloids in regenerated plants of Scopolia parviflora

An efficient in vitro plant regeneration system was established from callus culture of Scopolia parviflora. Callus was induced from adventitious roots on B5 medium with 0.45–9.04 μM 2,4-dichlorophenoxyacetic acid (2,4-D). In vitro plantlet regeneration was achieved on B5 medium supplemented with 44.38 μM benzyladenine (BA), 3% sucrose, and 0.38% gelrite. Plantlets were transplanted to artificial soil and grown to maturity successfully in a greenhouse. The tropane alkaloid contents in regenerated plants were analyzed using high-performance liquid chromatography (HPLC), and were found to be higher than those of adventitious roots, native growing plants, and acclimated plants. Regenerated plants from organogenic callus cultures produced a greater amount of tropane alkaloids.     

In vitro propagation of Cassia angustifolia through leaflet and cotyledon derived calli

High efficiency shoot regeneration was achieved through leaflet and cotyledon derived calli in Cassia angustifolia - an important medicinal plant. Dark brown compact callus was induced at the cut ends of the explants on Murashige and Skoog's (MS) medium augmented with 1 μM N⁶-benzyladenine (BA) + 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Such callus pieces on transfer to cytokinins (BA or kinetin) supplemented medium differentiated shoots within 10 – 15 d. Of the two cytokinins, 5 μM BA was optimum for eliciting morphogenic response in 83.33 and 70.83 % cultures with an average of 4.16 ± 0.47 and 3.70 ± 0.56 shoots in cotyledon and leaflet derived calli, respectively. The addition of 0.5 μM α-naphthaleneacetic acid (NAA) to MS + 5 μM BA further elevated the maximum average number of shoots to 12.08 ± 1.04 and 5.37 ± 0.52 for cotyledon and leaflet calli, respectively. The excised shoots were transferred to a rooting medium containing either IAA (indole-3-acetic acid), IBA (indole-3-butyric acid) or NAA. Nearly 95 % shoots developed an average of 5.4 ± 0.41 roots on half strength MS medium supplemented with 10 μM IBA.     

In vitro organogenesis and plant regeneration from unpollinated ovary cultures of Azadirachta indica

A novel method of organogenesis in neem (Azadirachta indica A. Juss.) from unfertilized ovaries is described. The Murashige and Skoog’s (MS) medium with 9 % sucrose, 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 μM 6-benzylaminopurine (BAP) was the best for callus induction from unfertilized ovaries. However, further proliferation of callus occurred better on MS medium supplemented with 0.5 μM 2,4-D either alone or in combination with 4.5 μM kinetin. Maximum shoot regeneration (78 %) was observed when calli, induced from ovaries of 4 mm size flower buds and proliferating on MS + 0.5 μM 2,4-D, were subcultured to MS medium containing 5 μM BAP. Histological analysis revealed that 4 mm sized flower bud corresponds to a 2-nucleate stage of embryo sac. The shoots were then multiplied by forced axillary branching on MS medium supplemented with 1.0 μM BAP and 250 mg dm⁻³ casein hydrolysate. The shoots could be rooted on 1/4 strength MS medium supplemented with 0.5 μM indole-3-butyric acid (IBA) at a frequency of 79 %. Cytological analysis by root tip squash preparations revealed that all the plantlets were diploids. These plants were subsequently hardened and established in soil with transplantation rate of 81.8 %.     

Production of chlorogenic acid and isoorientin hypoglycemic compounds in Cecropia obtusifolia calli and in cell suspension cultures with nitrate deficiency

The antidiabetic properties of Cecropia obtusifolia are attributed to chlorogenic acid (CGA) and isoorientin (ISO) phenolic compounds; both compounds possess hypoglycemic, hypolipidemic, and antioxidant properties. As a potential strategy for an adequate supply of authentic plant raw material, the aim of this study was to establish in vitro conditions for the development of cell suspension cultures that produce these bioactive compounds. Callus cultures of leaf explants from acclimatized tree and in vitro plantlets were set up using different auxin levels; treatments with 2,4-dichlorophenoxyacetic acid (2,4-D) and α-naphthalene acetic acid (NAA) to 8.92 μM with 6-benzylaminopurine (BAP) at 2.22 μM stimulate highest callus production. Seedling cotyledon, hypocotyl, leaf, and stem explants developed calli bearing roots with 2,4-D. With NAA, hypocotyl, cotyledon, and leaf explants developed morphogenic calli; 75% of stem explants formed calli, and the remaining calli developed shoots. Determined CGA concentrations in calli were similar to those detected in the leaves of wild trees, and ISO was not produced. Cell suspension cultures were established from leaf explants friable calli with 8.92 μM 2,4-D in combination with 2.22 μM BAP, employing 4 and 5% inocula in fresh weight; CGA levels were maintained and ISO was produced only at the end of logarithmic growth. On diminishing nitrate content in Murashige and Skoog (MS) medium to 8.0 mM, maximum cell biomasses diminished, CGA production is increased and twice with 16.0 and, instead of CGA production is tripled and quadrupled with 16.0 and 8.0 mM nitrates, respectively, and ISO synthesis was induced earlier and for a longer time period, increasing its levels at the end of culture. Two compounds with ultraviolet spectra similar to those of caffeic and ferulic acids were formed. Our results offer a protocol of cell suspension cultures for C. obtusifolia bioactive production and hypoglycemic property conservation.     

Unfertilized ovary: a novel explant for coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.) somatic embryogenesis

Unfertilized ovaries isolated from immature female flowers of coconut (Cocos nucifera L.) were tested as a source of explants for callogenesis and somatic embryogenesis. The correct developmental stage of ovary explants and suitable in vitro culture conditions for consistent callus production were identified. The concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) and activated charcoal was found to be critical for callogenesis. When cultured in a medium containing 100 μM 2,4-D and 0.1% activated charcoal, ovary explants gave rise to 41% callusing. Embryogenic calli were sub-cultured into somatic embryogenesis induction medium containing 5 μM abscisic acid, followed by plant regeneration medium (with 5 μM 6-benzylaminopurine). Many of the somatic embryos formed were complete with shoot and root poles and upon germination they gave rise to normal shoots. However, some abnormal developments were also observed. Flow cytometric analysis revealed that all the calli tested were diploid. Through histological studies, it was possible to study the sequence of the events that take place during somatic embryogenesis including orientation, polarization and elongation of the embryos.     

Somatic embryogenesis from mature zygotic embryos of commercial passionfruit (<Emphasis Type="Italic">Passiflora edulis</Emphasis> Sims) genotypes

Mature zygotic embryos of three genotypes of Passiflora edulis Sims, including ‘FB-100’, ‘FB-200’, and ‘FB-300’ were incubated on a Murashige and Skoog (MS) (1962) medium supplemented with different concentrations (18.1–114.8 μM) of 2,4-diclorophenoxyacetic acid (2,4-D) and 4.4 μM of 6-benzyladenine (BA). MS basal medium and MS with BA induced germination of P. edulis embryos. The highest frequencies of embryogenic calli were observed when explants were incubated on MS medium supplemented with 72.4 μM 2,4-D and 4.4 μM BA for ‘FB-200’, which showed the highest potential for embryogenic callus formation. Cytological and histological analyses of pro-embryogenic callus revealed two distinct cell types: thin-walled, small, isodiametric cells with large nuclei and dense cytoplasm, typical of intense metabolic activity; and elongated and vacuolated cells, with small nuclei and less dense cytoplasm. Differentiation of somatic embryos was promoted on MS medium supplemented with activated charcoal and indole-3-acetyl-l-aspartic acid (IAA-Asp) either with or without 2,4-D. However, no conversion of somatic embryos into plantlets was observed.     

Histological observations of morphogenesis in petiole derived callus of Amorphophallus rivieri Durieu in vitro

The initiation and development of somatic embryos and organogenic shoots and corm-like structures (CLSs) from petiole-derived calli of Amorphophallus rivieri Durieu were observed histologically. The petioles were cultured on Murashige and Skoog (MS) medium supplemented with 5.37 μM α-naphthaleneacetic acid (NAA) and 4.44 μM N⁶-benzylaminopurine (6-BA) for callus induction. The shoot and corm organogenesis occurred from the compact calli when they were transferred to a medium containing 0.54 μM NAA and 4.44 μM 6-BA. A combination of 13.57 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 8.88 μM 6-BA or 24.18 μM NAA and 6.66 μM 6-BA was optimum for induction of somatic embryos, which failed to produce plantlets because of their structural abnormalities. Shoot regeneration predominantly happened through organogenesis although somatic embryogenesis infrequently occurred. The subepidermal cells of the compact callus converted to competent cells and started divisions, which resulted in formation of the meristemoids. The meristemoid cells continued division to develop into bud primordia. Subepidermal cells could also form the globular structures. Subsequently, these globoids developed into CLSs from which plantlets regenerated during subculture. Meanwhile, the CLSs were capable to form cormels, which could be a promising way for the propagation of A. rivieri.     

Production of transgenic plants via Agrobacterium rhizogenes-mediated transformation of Panax ginseng

 Hairy roots of Panax ginseng were obtained after root disks were infected with wild-type strain Agrobacterium rhizogenes 15834. Three lines of hairy roots with different pigmentation were selected. Embryogenic callus was induced on Murashige and Skoog medium containing 1.0 mg/l 2,4-D. The frequency of embryogenic callus formation was 37.4% in a line with red pigmentation. Somatic embryo development from embryogenic callus was efficiently achieved by lowering the concentration of 2,4-D (0.5 mg/l). After the germination of somatic embryos on medium with 10 mg/l GA₃, the embryos were transferred to 1/2-MS medium without GA₃. The transformed ginseng plantlets had an actively growing root system with abundant lateral roots. The phenotypical alteration of transformed ginseng plants might be valuable character for increasing root yield. Received: 27 March 1999 / Revision received: 18 May 1999 / Accepted 8 July 1999     

Effects of cytokinin on adventitious root formation in callus cultures ofVigna unguiculata (L.) walp

Summary Yellowish compact callus, induced from cowpea hypocotyls on Murashige and Skoog(MS) medium (1962) containing 0.2 mg/l(0.93 μM) kinetin and 0.4 mg/l (1.81 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), was subcultured on MS medium containing cytokinin alone, auxin alone, or auxins plus cytokinins in order to determine the effect of cytokinins on root organogenesis in callus cultures. The callus actively proliferated on the same medium but did not show any organogenic activity macroscopically as well as microscopically. On medium with N⁶-benzyladenine (BA) and 1-naphthaleneacetic acid (NAA), the yellowish compact callus first changed to pale green compact callus and then many green spots appeared on its surface under light culture. But the yellowsih compact callus remained yellowish and white spots appeared on its surface in dark culture. These spots gradually became white nodular structures. Adventitious root formation from the nodular structures occurred not only on the same medium, but also on medium with either auxin or cytokinin but not both. Yellowish compact callus on medium with auxin alone was transformed to yellowish friable callus, which did not develop adventitious roots. The yellowish friable callus could gain rhizogenic activity only after morphological modification to pale green compact callus on medium with auxin plus cytokinin. The modified callus did not form adventitious roots on medium with auxins but only with cytokinins. Therefore, it is suggested that cytokinins have stimulating effects on root formation from callus that previously did not show rhizogenic activity on medium with auxins alone. In addition, the rhizogenic potential of cowpea callus was discriminated from that of leaf explants, which formed adventitious roots directly on medium with auxin alone.     

The initiation of callus culture ofMichelia champaca for essential oil production

Summary TheMichelia champaca callus was induced from rachises of theM. champaca flowers on 1/2 MS medium containing 3% sugar and 0.9% agar. The medium was also supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and 6-benzylaminopurine (BAP) as the growth regulators. It was observed that the calli ofM. champaca could be induced on media containing 0–50 M 2,4-D and 0.1–1 M BAP. The calli were grown well on media on media containing 0.1–1.0 M BAP and 2,4-D up to 50 M. As 1 M BAP was used, a lower concentration of 2,4-D was associated with a fast initiation of calli, but the culture of these calli turned brown quickly. To the contrary, a higher concentration of 2,4-D led to a slower rate of callus formation and the culture hardly turned brown. The optimum pH for the cell culture was about 5.6 as 1 M of 2,4-D and BAP were present in the medium.     

Development of an embryogenic callus induction method for centipede grass (<Emphasis Type="Italic">Eremochloa ophiuroides</Emphasis> Munro) and subsequent plant regeneration

The present study demonstrates the establishment of embryogenic tissue from seeds and (seedling-derived hypocotyls) shoot base explants derived from seedlings of Eremochloa ophiuroides. The highest percentage of callus induction obtained from seed and young shoot base explants was 52.0% and 66.6% on Murashige and Skoog (MS) basal media supplemented with 9.0 μM and 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. The type of callus obtained from both types of explants was off-white to yellow in color and non-friable and shiny in texture. Excised callus from the explants was subcultured onto fresh media of the same recipe for further proliferation. After 10–12 d of subculture, a yellow, globular, friable embryogenic callus was obtained from the initial callus. The highest percentage of embryogenic calli obtained at 40.0% was observed on media containing 2.2 μM 2,4-D. The highest regeneration rate of 46.6% was observed on MS media supplemented with 0.4 μM 2,4-D and 2.2 μM benzylaminopurine (BA). Regenerated shoots were rooted in MS basal medium. Plants with well-developed roots were transferred to pots containing a soil mix and acclimatized in greenhouse conditions. Four weeks post-transfer, acclimatized plants showed 100% survival and remained healthy and green. This is the first report of a successful method for induction of somatic embryogenesis with subsequent plant regeneration in centipede grass and demonstrates the establishment of embryogenic callus and efficient plant regeneration with potential application in the development of genetic transformation systems for centipede grass.     

In vitro plant regeneration from organogenic callus of <Emphasis Type="Italic">Curcuma kwangsiensis</Emphasis> Lindl. (Zingiberaceae)

A novel protocol for callus-mediated shoot regeneration was established for an important medicinal and ornamental plant native to South China, Curcuma kwangsiensis, using shoot base sections excised from seedlings in vitro as explant sources. The frequency of callus formation reached 91% for explants cultured on MS medium containing 1.4 μM TDZ, 4.4 μM BA and 2.3 μM 2,4-D. 8.2 shoots per callus was achieved on MS medium supplemented with 1.4 μM TDZ, 17.8 μM BA and 2.7 μM NAA. Single shoots transferred into MS medium free of plant growth regulator rooted well. Regenerated plants acclimatized ex vitro at 100%, and grew vigorously under shaded greenhouse conditions.     

In vitro culture of Phyllanthus caroliniensis (Euphorbiaceae)

An efficient micropropagation protocol was developed for the medicinal plant Phyllanthus caroliniensis (Euphorbiaceae) using nodal segments for axillary shoot proliferation. Maximum multiplication (21–23 shoots per explant) was achieved on MS or AR media supplemented with either 5.0 μM BA, 1.25–5.0 μM kinetin or 2.5–5.0 μM 2iP. Rooting was achieved with 80–100% of the microshoots on MS medium without growth regulators, although 1.25 μM NAA and 1.25–5.0 μM IAA promoted significant increases in the number of roots per explant. Regenerated plants were successfully acclimatized and about 88% of plantlets survived under ex vitro conditions. Flowering was observed on in vitro grown plantlets and after 3–4 weeks of acclimatization. High frequency callus initiation and growth was achieved when nodal segment explants were inoculated in the vertical position on MS medium supplemented with 5.0 μM 2,4-D. Root cultures were successfully established on MS medium containing 1.1 μM NAA. The optimized micropropagation, callus and root culture protocols offer the possibility to use cell/root culture techniques for vegetative propagation and secondary metabolism studies. This revised version was published online in June 2006 with corrections to the Cover Date.