Highly efficient plant regeneration and <Emphasis Type="Italic">in vitro</Emphasis> polyploid induction using hypocotyl explants from diploid mulberry (<Emphasis Type="Italic">Morus multicaulis</Emphasis> Poir.)

Efficient plant regeneration is essential for successful transformation and in vitro polyploidy induction in mulberry. A high frequency (80%) of plant regeneration from hypocotyls occurred under in vitro conditions in mulberry (Morus multicaulis Poir.). We identified three key factors for enhancing successful regeneration based on earlier work: (1) hypocotyl position, (2) the combination and concentration of growth regulators, and (3) the addition of AgNO₃. The highest frequency of shoot regeneration was achieved using hypocotyl segments, which are proximal to apical meristems, and the optimal culture conditions were Murashige and Skoog’s (MS) (Murashige and Skoog, 1962) basal medium supplemented with 3.0 mg l⁻¹ 6-benzylamino purine, 0.3 mg l⁻¹ indole-3-acetic acid, 0.1% polyvinypyrrolidone, and 1.0 mg/l silver nitrate (AgNO₃) under subdued light at 25 ± 2°C. Treating the shoots with 0.2% colchicine (dipping for 72 h) resulted in a 14% tetraploid frequency, whereas a 20% tetraploid frequency resulted from using a 0.25% colchicine (dripping for 5 d) treatment, as determined by chromosome number counts. The diploid plant chromosome number was 28 (2n = 2x = 28) and that of tetraploid plants was 56 (2n = 4x = 56). Regenerated shoots rooted easily in 8–10 d using half-strength basal MS medium with 0.5 mg l⁻¹ indole-3-butyric acid and were successfully established in the soil.     

Development of a plant regeneration system based on friable embryogenic callus in the ornamental Alstroemeria

 Stem segments of seedlings from two Alstroemeria breeding lines, cultured on media supplemented with 4 mg/l 2,4-dichlorophenoxyacetic acid and 0.5–1.0 mg/l 6-benzylaminopurine (BA), initiated soft callus, which became compact after subculture on a medium with only 0.5 mg/l BA. Friable embryogenic calli were initiated from compact callus on a medium supplemented with 10 mg/l picloram. Proembryos developed from friable embryogenic calli via embryos into plants after subculture on medium supplemented with 0.1 mg/l BA. The proembryos formed friable embryogenic calli again after culture on medium supplemented with 10 mg/l picloram. The total time needed to regenerate a complete plantlet from friable callus was approximately 6 months. This system for the production of embryogenic material is considered to have valuable applications for genetic transformation in Alstroemeria. Received: 22 April 1999 / Revision received: 16 July 1999 · Accepted: 20 July 1999     

Transgenic plants of coffee Coffea canephora from embryogenic callus via Agrobacterium tumefaciens-mediated transformation

 Embryogenic calli were induced from leaf explants of coffee (Coffea canephora) on McCown's woody plant medium (WPM) supplemented with 5 μM N⁶–(2-isopentenyl)-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pIG121-Hm, containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransferase II genes. Selection of putative transgenic callus was performed by gradual increase in hygromycin concentration (5, 50, 100 mg/l). The embryogenic calli surviving on medium containing 100 mg/l hygromycin showed a strong GUS-positive reaction with X-Gluc solution. Somatic embryos were formed from these putative transgenic calli and germinated on WPM medium with 5 μM 2-iP. Regenerated small plantlets with shoots and roots were transferred to medium containing both 100 mg/l hygromycin and 100 mg/l kanamycin for final selection of transgenic plants. The selected plantlets exhibited strong GUS activity in leaves and roots as indicated by a deep blue color. GUS and HPT genes were confirmed to be stably integrated into the genome of the coffee plants by the polymerase chain reaction. Received: 14 December 1998 / Revision received: 12 March 1999 / Accepted: 24 March 1999     

Agrobacterium-mediated genetic transformation of a phalaenopsis orchid

 Genetically transformed plants of a phalaenopsis orchid [Doritaenopsis Coral Fantasy×Phalaenopsis (Baby Hat×Ann Jessica)] were regenerated after cocultivation of cell clumps with Agrobacterium tumefaciens strains LBA4404 (pTOK233) and EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (GUS) and hygromycin resistance. The efficiency of transformation was markedly increased by 10 h cocultivation of cell clumps with A. tumefaciens that had been induced with 200 μm acetosyringone, and by inclusion of 500 μm acetosyringone in the cocultivation medium. Hygromycin-resistant cell clusters (0.5–3 mm in diameter) were selected from the infected cell clumps after 4–6 weeks of culture on agar (8 g/l)-solidified new Dogashima medium (NDM) containing 20 g/l sucrose, 0.1 mg/l naphthaleneacetic acid, 1.0 mg/l benzyladenine (BA), 50 mg/l hygromycin and 300 mg/l cefotaxime. The cell clusters proliferated 4 weeks after transfer onto the same medium. To induce callus greening, the carbon source was changed from sucrose to maltose. The green calli obtained produced protocorm-like bodies (PLBs) after 4 weeks of culture on phytohormone-free NDM medium. Regeneration of transgenic plantlets was enhanced by incubating PLBs on NDM medium supplemented with 0.1 mg/l abscisic acid, followed by partial desiccation for 10–30 min. Successful transformation was confirmed by histochemical GUS assay, PCR analysis and Southern hybridization of transformants. With this transformation system, more than 100 hygromycin-resistant phalaenopsis plantlets were produced about 7 months following infection of the cell aggregates. Received: 10 November 1998 / Revision received: 4 June 1999 / Accepted: 22 June 1999     

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     

Production of transgenic gentian plants by particle bombardment of suspension-culture cells

 Cell suspension cultures were established from leaf explants of gentian (Gentiana triflora×G. scabra) for the generation of transgenic plants by particle bombardment. The parameters for the bombardment of suspension culture cells with a particle gun were examined by monitoring the transient expression of a gene for β-glucuronidase driven by the cauliflower mosaic virus (CaMV) 35S promoter. We found that prior culture of suspension culture cells for 5 days on solid medium was optimum for successful particle bombardment. Putative transformed calli were obtained from bombarded cells after a two-step selection procedure. Cells were cultured first with 30 mg l^–1 hygromycin in liquid MS medium that contained 10 mg l^–1 N-phenyl-N′-1,2,3-thiadiazol-5-yl urea, 1 mg/l 1-naphthaleneacetic acid and 30 g l^–1 sucrose and then on solid medium prepared from the same liquid medium plus 2 g l^–1 gellan gum. After 12 weeks of selection on solid medium that contained 30 mg l^–1 hygromycin, two transgenic gentian plants were regenerated from each selected callus. Analysis by the polymerase chain reaction and Southern blotting revealed the stable integration of transferred DNA. Received: 3 June 1999 / Revision received: 21 September 1999 / Accepted: 20 September 1999     

Micropropagation and induction of autotetraploid plants of <Emphasis Type="Italic">Chrysanthemum cinerariifolium</Emphasis> (Trev.) Vis

Rapid propagation technology was established and optimized in vitro for Chrysanthemum cinerariifolium (Trev.) Vis., an important botanical insecticide plant with a huge international market. A large number of buds could be induced directly from epicotyl and hypocotyl explants on Murashige T; Skoog F. J. Plant. Physiol. 15: 473–479; (1962) medium [Murashige and Skoog (MS) medium] supplemented with 0.3 mg l⁻¹ benzyladenine (BA) and 0.3 mg l⁻¹ α-naphthaleneacetic acid (NAA). Root induction and development could be observed within 15 d after inoculation on 1/2 MS medium supplemented with 0.2 mg l⁻¹ indole-3-acetic acid (IAA) and 0.1 mg l⁻¹ rooting powder (ABT). Furthermore, a polyploid breeding study in vitro was reported to obtain superior breeding lines with high yield and good quality. Autotetraploid lines of C. cinerariifolium were obtained by colchicine treatments and identified by root-tip chromosome determination and stoma observation. The chromosome number of the autotetraploid plantlet was 2N = 4x = 36. Obtained autotetraploid lines will be of important genetic and breeding value and be used for further selection and plant breeding.     

Plant regeneration in Arachis pintoi (Leguminosae) through leaf culture

Plant regeneration in Arachis pintoi was obtained via two developmental pathways: organogenesis and somatic embryogenesis. Organogenic callus cultures were initiated from pieces of leaf on MS medium supplemented with NAA or 2,4-D in combination with BA, KIN or 2iP. The most suitable combination for plant regeneration through organogenesis was an initial medium composed of 10 mg/l NAA+1 mg/l BA followed by transfer of the callus to a shoot induction medium (MS+1 mg/l BA). Rooting of regenerated shoots was readily achieved by culture on MS+0.01 mg/l NAA. Embryogenic callus cultures were initiated from pieces of leaf on MS medium supplemented with PICL in combination with KIN, ZEA, BA or 2iP, and the most suitable combinations were 20 mg/l PICL+1 mg/l BA or 2iP. When pieces of embryogenic callus were subcultured on MS+1 mg/l BA, somatic embryos were differentiated and developed further into well-developed plants in MS+1 g/l AC followed by MS medium devoid of plant growth regulators. Received: 29 April 1999 / Revision received: 24 November 1999 / Accepted: 18 December 1999     

Agrobacterium-mediated transformation of Brassica campestris ssp. Parachinensis with synthetic Bacillus thuringiensis cry1Ab and cry1Ac genes

 An effective plant regeneration procedure and a gene transfer system via Agrobacterium tumefaciens were developed in Brassica campestris ssp. parachinensis. Hypocotyls from 5-day-old seedlings with 2 days pre-culture were infected with Agrobacterium strain MOG301 harboring a binary vector containing a synthetic Bacillus thuringiensis (B.t.) cry1Ab or cry1Ac gene with full codon-modification. After culture and selection on MS medium supplemented with 4.0 mg/l BAP, 2.0 mg/l NAA, 70 μM AgNO₃ and 50 mg/l kanamycin, a number of kanamycin-resistant plantlets were regenerated. PCR and Southern blotting analysis were used to identify and characterize the transgenic plants with the integrated cry1Ab or cry1Ac gene. Western blotting analysis of the transgenic plants confirmed the expression of insecticidal proteins encoded by cry1Ab or cry1Ac. Subsequent bioassay with larvae of the Diamondback moth, Plutella xylostella, demonstrated that the transgenic plants were resistant to feeding damage. Received: 22 February 1999 / Revision received: 14 April 1999 / Accepted: 26 April 1999     

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. Received: 30 May 1997 / Revision received: 18 September 1997 / Accepted: 22 March 1999     

Agrobacterium-mediated transformation of cotyledonary explants of Chinese cabbage (Brassica campestris L. ssp. pekinensis)

 A procedure for producing transgenic Chinese cabbage plants by inoculating cotyledonary explants with Agrobacterium tumefaciens strain EHA101 carrying a binary vector pIG121Hm, which contains kanamycin-resistance and hygromycin-resistance genes and the GUS reporter gene, is described. Infection was most effective (highest infection frequency) when explants were infected with Agrobacterium for 15 min and co-cultivated for 3 days in co-cultivation medium at pH 5.2 supplemented with 10 mg/l acetosyringone. Transgenic plants of all three cultivars used were obtained with frequencies of 1.6–2.7% when the explants were regenerated in shoot regeneration medium solidified with 1.6% agar. A histochemical GUS assay and PCR and Southern blot analyses confirmed that transformation had occurred. Genetic analysis of T1 progeny showed that the transgenes were inherited in a Mendelian fashion. Received: 15 December 1998 / Revision received: 2 July 1999 · Accepted: 8 July 1999     

In vitro induction of autotetraploids from diploid yellow passion fruit mediated by colchicine and oryzalin

In vitro chromosome doubling from hypocotyl segments of yellow passion fruit (Passiflora edulis Sims.) was carried out in the presence of either colchicine (0, 25, 250 and 1,250 μM) or oryzalin (0, 5, 15 and 30 μM). Murashige and Skoog (in Physiol Plant 15:473–497, 1962)(MS)-based regeneration medium containing 250 or 1,250 μM colchicine markedly affected explant development leading to browning and death of the hypocotyl segments. Oryzalin has similar effect to colchicine in inducing polyploidy. In vitro regenerated autotetraploid plants induced by 25 μM colchicine or 15 μM oryzalin were further acclimatized and cultivated in hydroponics system in greenhouse. Autotetraploids plants were more vigorous than the control diploids. The chromosome number of diploid plants was 2n = 2x = 18, whereas that found on autotetraploid plants were 2n = 4x = 36. The stomata sizes of the autotetraploids were significantly larger than those on the diploid counterparts, while the frequency of stomata was significantly reduced. Similarly, the chloroplast number of guard cells of autotetraploid plants increased significantly. Two albino plants (4%) were generated in medium with 25 μM colchicine, indicating phytotoxic effects. These plants are being grown to full maturity in order to test their potential to use in a breeding program.     

Rosmarinic acid production by Lavandula vera MM cell-suspension culture

The time courses of growth and rosmarinic acid production by Lavandula vera MM cell suspension were investigated. The uptake of the main nutrients (sucrose, nitrogen, phosphorus, K, Ca, Mg) was followed during cultivation and the data on the physiology of the L. vera MM cell culture are presented. It was established that the cell culture synthesizes rosmarinic acid during the linear phase of growth for a relatively short period (between the 4th and 8th days of cultivation). The influence of sucrose concentration in the nutrient medium on cell growth and accumulation of rosmarinic acid by L. vera MM cell culture was investigated. The results showed that 7% sucrose in the nutrient medium ensured a steady growth of the cell suspension and increased the yield of rosmarinic acid (29.2 g/l dry biomass and 507.5 mg/l rosmarinic acid compared to 13.0 g/l dry biomass and 68.6 mg/l rosmarinic acid for the control cultivation with 3% sucrose). Received: 17 September 1996 / Received revision: 31 January 1997 / Accepted: 1 February 1997     

An evaluation of antibiotics for the elimination of Agrobacterium tumefaciens from walnut somatic embryos and for the effects on the proliferation of somatic embryos and regeneration of transgenic plants

Four antibiotics were evaluated for their effects on eliminating the hypervirulent Agrobacterium tumefaciens strain C58C1 ATHV Rif^R (pEHA101)/p35-gus-intron from walnut somatic embryos and on the production of secondary somatic embryos and the transformed somatic embryos. Exposure to 100–1000 mg l⁻¹ of ampicillin, carbenicillin or cefotaxime respectively for up to 60 days did not eliminate the A. tumefaciens while timentin at 500–1000 mg l⁻¹ eradicated it from somatic embryos. One-hour acidified medium treatments and the addition of 100 mg l⁻¹ kanamycin to 500 mg l⁻¹ ampicillin, carbenicillin, cefotaxime or timentin were of little help in eliminating the Agrobacterium. All four antibiotics reduced somatic embryo production, carbenicillin minimally and cefotaxime maximally, especially at higher concentrations, in comparison with antibiotic-free medium. Putative transformed embryos were selected for continued proliferation on a 100 mg l⁻¹ kanamycin-containing medium. Histochemical assessments indicated that more gus-positive somatic embryos, particularly fully gus-positive embryos, regenerated from timentin-containing medium than from other antibiotic-containing media under equivalent conditions. Transformed embryos have been grown and converted into plants and gus activity was observed in whole plants. Received: 13 July 1999 / Revision received: 2 December 1999 / Accepted: 6 December 1999     

Micropropagation of <Emphasis Type="Italic">Vitex agnus-castus</Emphasis>, (Verbenaceae)—a valuable medicinal plant

This study describes in vitro shoot induction and plant regeneration from a mature apical meristem and nodal explants of the endangered medicinal shrub Vitex agnus-castus. Multiple shoots were induced directly from the axis of nodal and apical meristem explants on Murashige and Skoog (MS) medium containing 3% sucrose and different concentrations (1.0, 1.5, 2.0, and 2.5 mg/l) of 6-benzyl aminopurine (BAP) in combination with Kinetin (Kin) and α-naphthalene acetic acid (NAA), both at 0.1 mg/l. BAP and Kinetin were used as supplements to MS basal medium, either individually or in combination with auxins. The optimal concentration of BAP for inducing bud break was found to be 2.0 mg/l when Kinetin was at 0.1 mg/l. Regeneration frequency was highest for both apical meristem and nodal explants (94.5% and 90.3%, respectively) when explants were cultured on MS medium supplemented with BAP (2.0 mg/l) and Kin (0.1 mg/l). A maximum of 7.7 ± 0.4 and 6.7 ± 0.2 shoots were obtained per explant for apical meristem and nodal explants, respectively. Regenerated shoots, transferred to MS medium supplemented with either 1.0 or 1.5 mg/l BAP combined with 0.1 mg/l GA₃, showed maximum elongation of 6.7 ± 0.4 and 6.0 ± 1.3 cm in apical meristem and nodal explants, respectively. In vitro regenerated shoots transferred to half-strength MS medium supplemented with 0.1 mg/l IBA induced 90.4% of the shoots to form roots after 30–35 d of culture. Up to 80% of the regenerated shoots were successfully established in soil in the greenhouse.     

Phenylacetic acid improves bud elongation and in vitro plant regeneration efficiency in Capsicum annuum L.

 A highly efficient three-stage protocol for the regeneration of chilli pepper (Capsicum annuum L.) from cotyledon explants was developed. This protocol used PAA in both the shoot-bud induction medium and the medium for elongation of the shoot buds. A superior medium for the induction of buds from the cotyledons was MS medium supplemented with BA (5 or 7 mg/l) + PAA (2 mg/l). Buds were elongated during the second stage on medium containing BA (2 or 5 mg/l) + PAA (2 mg/l). On this medium most of the buds elongated, and their number also increased due to the formation of new buds; bud elongation was achieved in 100% of the cultures provided the buds were induced in the primary stage on a medium supplemented with BA+PAA. The shoots that elongated in the second-stage rooted at 100% frequency on a medium supplemented with NAA (1 mg/l). The complete plantlets with well-developed root and shoot systems were transferred to field conditions where they grew to maturity, flowered and fruited normally. While shoot-bud induction from the cultured cotyledons was also observed on media supplemented with BA (5 or 7 mg/l) alone or in combination with IAA (0.2–2 mg/l), buds induced on these media were often distorted, with most not developing into normal shoots in the second-stage subculturing; a rosette of buds was seen in the second stage subculturing. On the other hand, PAA in combination with BA in the primary induction medium and second-stage medium promoted normal development and the elongation of shoot buds. Received: 28 July 1998 / Revision received: 22 December 1998 / Accepted: 19 February 1999     

Pilot-scale production of butanol by Clostridium beijerinckii BA101 using a low-cost fermentation medium based on corn steep water

To improve the economic competitiveness of the acetone/butanol/ethanol fermentation process, glucose/corn steep water (CSW) medium was used on a pilot scale for the production of solvents. The production of butanol by the Clostridium beijerinckii NCIMB 8052 parent strain and the solvent-hyperproducing BA101 mutant was compared. In a 20-l fermentation using 5% glucose/CSW medium,  C. beijerinckii 8052 produced 8.5 g butanol/l and 5 g acetone/l, while  C. beijerinckii BA101 produced 16 g butanol/l and 7.5 g acetone/l. Further studies were carried out on a larger scale using an optimized 6% glucose/CSW medium. In a 200-l pilot-scale fermentor,  C. beijerinckii 8052 produced 12.7 g butanol/l and 6 g acetone/l following 96 h of fermentation.  C. beijerinckii BA101 produced 17.8 g/l and 5.5 g/l butanol and acetone respectively, following 130 h of fermentation. These results represent a 40% increase in final butanol concentration by the C. beijerinckii BA101 mutant strain when compared to the 8052 parent strain. The total solvents (acetone, butanol, and ethanol) produced by C. beijerinckii NCIMB 8052 and BA101 in a 200-l fermentation were 19.2 g/l and 23.6 g/l respectively. This is the first report of pilot-scale butanol production by the solvent-hyperproducing C. beijerinckii BA101 mutant employing an inexpensive glucose/CSW medium. Received: 26 May 1998 / Received revision: 21 September 1998 / Accepted: 11 October 1998     

High-frequency plant regeneration via somatic embryogenesis and organogenesis and in vitro flowering of regenerated plantlets in Panax ginseng

 The morphogenesis ability of light yellowish globular callus derived from cotyledons of mature zygotic embryos of Panax ginseng was investigated. The optimal media for somatic embryogenesis and shoot organogenesis were MS medium containing 0.5 mg l^–1 2,4-dichlorophenoxyacetic acid, 0.1 mg l^–1 6-benzyladenine (BA), and 500 mg l^–1 lactoalbumin hydrolysate, and SH medium supplemented with 0.5 mg l^–1 α-naphthaleneacetic acid, 0.1 mg l^–1 BA, and 500 mg l^–1casein hydrolysate. The influences of glucose, mannose, fructose, and sorbose in the media on somatic embryogenesis and shoot organogenesis were revealed as differences in the numbers of somatic embryos and adventitious shoots per gram of morphogenic callus. The best regeneration of somatic embryos was obtained on medium containing glucose, with a mean of 8.7 somatic embryos per gram of callus. The best regeneration of shoots was observed on medium containing fructose, with an average of 12.2 adventitious shoots per gram of callus. Of the somatic embryos 95% were converted into regenerated plantlets, and 100% of adventitious shoots rooted to form regenerated plantlets. Regenerated plants were successfully established in soil. Flowering was observed in 5.7% of the regenerated plants derived from shoot organogenesis and in 1.4% of the regenerated plants derived from somatic embryogenesis. Received: 1 December 1998 / Revision received: 13 September 1999 / Accepted: 20 September 1999     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of North American ginseng (<Emphasis Type="Italic">Panax quinquefolius</Emphasis> L.)

North American ginseng (NAG) (Panax quinquefolius L.) is a medicinally important plant with multiple uses in the natural health product industry. As seed propagation is time-consuming because of the slow growth cycle of the plant, in vitro propagation using a bioreactor system was evaluated as an effective approach to accelerate plant production. An efficient method was developed to multiply nodal explants of NAG using liquid-culture medium and a simple temporary immersion culture vessel. The effects of plant growth regulators, phenolics, and chemical additives (activated charcoal, melatonin, polyvinylpolypyrrolidone, and ascorbic acid) were evaluated on in vitro-grown NAG plants. The highest number (12) of shoots per single node was induced in half-strength Schenk and Hildebrandt basal medium containing 2.5 mg/l kinetin, in which 81% of the cultured nodes responded. In a culture medium with 0.5 mg/l α-naphthalene acetic acid (NAA), roots were induced in 78% of the explants compared to 50% with a medium containing indole-3-acetic acid. All of the resulting plants appeared phenotypically normal, and 93% of the rooted plants were established in the greenhouse. Phenolic production increased significantly (P < 0.05) over a 4-wk culture period with a negative impact on growth and proliferation. Activated charcoal (AC; 50 mg/l) significantly reduced total phenolic content and was the most effective treatment for increasing shoot proliferation. Shoot production increased as the phenolic content of the cultures decreased. The most effective treatment for NAG development from cultured nodal explants in the bioreactor was 2.5 mg/l kinetin, 0.5 mg/l NAA, and 50 mg/l AC in liquid culture medium. This protocol may be useful in providing NAG tissues or plants for a range of ginseng-based natural health products.