In Vitro Cell.Dev.Biol.—Plant (2011) 47:710–718 DOI 10.1007/s11627-011-9379-y
MICROPROPAGATION
In vitro propagation of North American ginseng (Panax quinquefolius L.) Esther E. Uchendu & Gopinadhan Paliyath & Dan C. W. Brown & Praveen K. Saxena
Received: 6 April 2011 / Accepted: 30 May 2011 / Published online: 21 June 2011 / Editor: John Finer # The Society for In Vitro Biology 2011
Abstract 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 E. E. Uchendu (*) : G. Paliyath : P. K. Saxena Department of Plant Agriculture, University of Guelph, Guelph, ON, Canada N1G 2WI e-mail:
[email protected] D. C. W. Brown Agriculture and Agri-Food Canada, London, ON, Canada N5V 4T3
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. Keywords Antioxidant . Bioreactor . Liquid medium . Micropropagation . Node . Phenolic
Introduction Panax quinquefolius L. is commonly referred to as North American ginseng (NAG) (Proctor and Bailey 1987). NAG is a perennial herb of the family Araliaceae (Harlan and de Wet 1971) grown for its highly valued bioactive chemicals, including ginsenosides that are useful for the treatment of several medical ailments (Yuan et al. 1999; Wang et al. 2008). Ginsenosides have been detected in various parts of the ginseng plant with higher levels found in roots (Shi et al. 2007; Christensen 2008). NAG seeds are heterogeneous, and propagation via seed is slow because of a growth cycle of up to 7 yr from seed to harvest under commercial production conditions (Choi et al. 1994; Fournier et al. 2008). In vitro techniques can significantly accelerate plant growth stages, eliminating the restrictions imposed by a lengthy field production cycle, as well as producing genetically uniform propagules. Although tissue culture of the genus Panax began in the 1960s (Luo et al. 1964), more recent micropropagation protocols are available for cell (Wang 1990; Mathur et al. 1994; Zhong et al. 1996; Feeney and Punja 2003), tissue, and organ cultures (Jhang et al. 1974; Proctor et al. 1996; Kevers et al. 1999; Hovius et al. 2007). Most of these
IN VITRO PROPAGATION OF NORTH AMERICAN GINSENG
reports focus on somatic embryogenesis as a basic method for the micropropagation of Panax species, and although substantial progress has been made, limitations still exist in the efficiency of somatic embryo (SE) maturation, plant production, and rooting. The process of plant production through somatic embryogenesis often involves frequent reinitiation of embryogenic cultures or repetitive secondary and tertiary embryogenesis, resulting in a gradual reduction in regeneration capacity and propagation efficiency (Shoyama et al. 1995). Direct organogenesis via adventitious bud and shoot formation was reported for Panax ginseng (Choi et al. 1998) with the best bud formation occurring on cotyledons treated with a combination of 0.05 mg/l indole-3-butyric acid (IBA) and 5 mg/l 6benzylaminopurine (BAP). Micropropagation using nodes could potentially speed plant production with the additional benefit of maintaining genetic stability of elite germplasm. However, an efficient procedure for the micropropagation of Panax species via nodal segments is currently unavailable. Shoot proliferation on a solid medium is a common practice in micropropagation. However, liquid-based systems are often more productive and result in moderate to high multiplication rates compared to solid- or semi-solid-based systems, possibly because of better nutrient uptake and aeration (Garcia et al. 1997; Kevers et al. 2000; Monteiro et al. 2002; Murch et al. 2004). Wang et al. (2010) found that improved cell growth and metabolite production correlated with nutrient uptake for cell suspension cultures of NAG in a liquid bioreactor system. A number of factors may affect proliferation of NAG nodes in liquid culture medium, including plant growth regulators and exudates produced by growing tissues. Ginseng cell and root cultures are known to produce and accumulate bioactive compounds such as saponins, polysaccharides, and polyphenolics (Wu and Zhong 1999; Hu et al. 2001; Hahn et al. 2003). These secondary metabolites have many beneficial effects; however, some are harmful to plant survival and growth. For example, Kevers et al. (1999) cultured hairy roots of P. ginseng and P. quinquefolius in a liquid medium in a bioreactor system and observed decreasing multiplication as root exudates increased. They attributed this reduction to the presence of a growthinhibiting compound released into the medium by the roots. In a similar study, Trautmann and Visser (1991) observed a significant reduction in cell number and percent viability of guayule (Parthenium argentatum Gray) suspension cultures that correlated with the accumulation of exudates in the culture medium. Analysis of exudates from the fibrous roots of a 4-yr-old field-grown P. quinquefolius plant identified several phenolic autotoxins, which might be responsible for reduced growth (He et al. 2009). The relationship between ginseng exudates and in vitro plant growth has not been documented.
711
Chemical additives such as activated charcoal (AC) are commonly applied to prevent or reduce the deleterious effects of chemical inhibitors and to improve the growth of in vitro cultured plants (Pan and van Staden 1998; Thomas 2008). For example, 1% AC was added in NAG tissue culture medium to improve SE maturation (Zhou and Brown 2006), and 0.5–1% AC was added for plant development (Tirajoh et al. 1998; Feeney and Punja 2003; Zhou and Brown 2006). Ascorbic acid (AA) is an antioxidant which has been reported to improve growth or stimulate embryogenesis in plants. Habibi et al. (2009) reported an improved SE production in fodder grass (Themeda quadrivalvis) using 0.15 mM AA. Melatonin (MEL) is an antioxidant present in many plant species (Dubbels et al. 1995; Chen et al. 2003; Murch and Saxena 2006), and affects the regulation of growth, flowering, photoperiodicity, and control of circadian rhythms in plants (Kolar et al. 1997; Kolar et al. 2003; Murch et al. 2009). Application of MEL increased the rate of seed germination (Posmyk et al. 2009) and protected plants against toxic pollutants (Tan et al. 2007). Polyvinylpolypyrrolidone (PVPP) is well-known for its effectiveness in reducing phenolics in culture (Castillo-Sanchez et al. 2008). The goal of the current study was to improve the efficiency of NAG micropropagation by developing a method to multiply single-node segments in a liquid culture medium. Specifically, we aimed to optimize the plant growth regulator (PGR) concentrations in liquid-based NAG culture medium; test the efficacy of this medium with a bioreactor system (temporary immersion) to determine the role of phenolic production on growth and development of in vitro cultures; and evaluate the effects of added AC, AA, MEL, and PVPP on phenolic production and growth of NAG.
Materials and Methods Plant propagation. In vitro shoot cultures of NAG originally derived from seeds were obtained from the Southern Crop Protection and Food Research Center (SCPFRC), Agriculture and Agri-Food Canada, London, ON, Canada, and multiplied on a modified culture medium (Zhou and Brown 2006, 2007). The shoots were multiplied in liquid medium containing half-strength Schenk and Hildebrandt (SH) (1972) basal medium, with 10 mg/l GA3 (Sigma-Aldrich Co., St Louis, MO), 5 mg/l BAP (Sigma-Aldrich Co.), 0.5 mg/l IBA (Sigma-Aldrich Co.), 30 g/l sucrose, and 100 mg/l AC (Life Technologies, Inc., Grand Island, NY) adjusted to pH 5.5. The medium was autoclaved at 121°C and 118 kPa for 20 min. The cultures were subcultured at 3-wk interval in 2.5×15-cm test tubes sealed with MicroporeTM tape (Fisher Scientific Inc., Ottawa, ON, Canada) and placed
712
UCHENDU ET AL.
on a gyratory shaker under standard growth room conditions at 22°C with a 16-h photoperiod (30 μmol m−2 s−1). Optimizations of plant growth regulators for shoot and root formation. Single-node segments (∼1.5–2 cm) from 3-wkold shoot cultures were transferred to liquid medium containing half-strength SH basal medium, 30 g/l sucrose, 10 mg/l GA3, 100 mg/l AC, 0.5 mg/l IBA with varying levels of each of the following five cytokinins: BAP, 6-γ,γdimethylallylaminopurine (2ip) (Sigma-Aldrich Co.), kinetin (KN) (PhytoTechnology, Shawnee Mission, KS), zeatin (Sigma-Aldrich Co.), and thidiazuron (TDZ) (Riedel-de Haen, Seelze, Germany). The cytokinin concentrations tested were; 0, 0.5, 1, 2.5, 5, 7.5, and 10 mg/l. The best concentration of each cytokinin was also evaluated with each of three auxins: IBA, α-naphthalene acetic acid (NAA), and indole-3-acetic acid (IAA) at 0, 0.5, and 1 mg/l. The medium was autoclaved after adjusting the pH to 5.5. Each experiment consisted of ten test tubes per treatment and one nodal segment (for cytokinin treatment) or shoot (for auxin treatment) per test tube. Experiments were repeated three times (n=30). Data for shoot production were recorded after 2 wk and after one additional week for root development. Data include the number of shoots produced per nodal segment and the number of explants that produced shoots or roots following the treatments. Evaluation of phenolic production and plant performance in a bioreactor system. Thirty fresh nodal segments (∼1.5– 2 cm), 3 wk from the date of last subculture, were transferred into the optimized liquid culture medium (determined from previous experiments), with and without 100 mg/l AC. These cultures were incubated in a Liquid LabTM Rocker temporary immersion bioreactor system (Southern Sun BioSystems, Inc. Hodges, SC). The rate of rocking motion (side-to-side rotation) was 30 s/cycle with a 1.2-min interval between cycles. Cultures were grown at 22°C with a 16-h photoperiod (25–40 μmol m−2 s−1). The exudates from these cultures were assayed for total phenolics at 0, 1, 2, and 4 wk. This experiment was conducted three times. Changes in the culture medium were evaluated weekly for 4 wk. The physical status of each explant was examined at 2 and 4 wk for changes in color and vigor. Data on plant growth were recorded including the number of shoots produced per single node and the number of nodes that produced shoots and roots. Determination of total phenolics. Total phenolics were measured according to the Folin–Ciocalteu method (Singleton et al. 1999). Briefly, to a 75 μl of sample (culture medium) was added 925 μl of 50% ethanol, 5 ml double distilled water, and 500 μl Folin-Ciocalteu reagent. The mixture was
centrifuged and incubated for 5 min at 25°C before 1 ml of 5% (w/v) sodium carbonate (Na2CO3) solution was added to each sample, followed by centrifugation and incubation in the dark for 1-h with intermittent agitation. After 60 min, the absorbance was measured at 725 nm using a spectrophotometer (Model DU-8000, Beckman Coulter, Lawrence, KS). A similar procedure was repeated for all standard gallic acid solutions. A curve was generated using gallic acid (0–1,000 μg/ml), and total phenolic contents of the samples were expressed as gallic acid equivalents. Effects of chemical additives. Effects of each chemical (AC, AA, MEL, and PVPP) (Sigma-Aldrich Co.) at five concentrations, ranging from 0 to 100 mg/l, added to the PGRoptimized liquid-culture medium, were evaluated for their effects on phenolic production, antioxidant capacity, and plant growth. MEL was filter sterilized using membrane filters (0.45 μm, 150-ml analytical filter unit, Corning Life Sciences Inc., Pittsburgh, PA) and then added to cooled culture medium after autoclaving. AC, PVPP and AA were added to the culture medium before adjusting the pH and autoclaving. All chemicals were tested separately. Cultures were maintained on a Liquid LabTM Rocker temporary immersion bioreactor system at 22°C with a 16-h photoperiod (10– 15 μmol m−2 s−1). Each experiment consisted of 20 treatments with 30 single-node segments each. Plant growth was evaluated at 4 wk by counting the number of nodes that formed shoots and roots. Fresh weight (FW) and dry weight (DW) of roots per treatment were also determined. The exudates from these cultures were assayed for total phenolic, and root samples were assayed for total antioxidant capacity at 4 wk. Each experiment was repeated three times (n=90). Antioxidant assay. Total antioxidant capacity was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent (Molyneux 2004). Roots (∼50 mg FW) of NAG were rinsed and homogenized in 5 ml methanol. The homogenate was tested for the ability to scavenge DPPH. DPPH has a stable free radical with a characteristic purple color in methanol, which is scavenged in the presence of the antioxidant components of the extract resulting in a loss of the purple color. The antioxidant capacity of the extract is evaluated as the inhibition percentage (the percentage reduction of the DPPH). Aliquots (5 and 10 μl/ml) of root homogenate were added to 800 μl of 0.1 mM DPPH. The mixture was incubated in the dark for 30 min at 25°C and then centrifuged at 15,000×g for 40 s. The absorbance of the extract was read at 517 nm. The inhibition (percentage) of DPPH was calculated as follows: (Abs [control]−Abs [sample]×100/Abs [control]). The Abs [control] reaction includes all reagents except the tested compound [root extract], and the Abs [sample] is the absorbance of the tested compound.
IN VITRO PROPAGATION OF NORTH AMERICAN GINSENG
Determination of FW and DW. Root FW were measured and 50 mg of fresh root per treatment were oven dried (102°C for 6 h). The percent moisture was calculated as [FW−DW]/FW×100. Post flask management. The in vitro-grown plants were rinsed after 2 wk of culture, separated, and potted in plastic trays (24×60 cm, with individual 4×4-cm cells) containing moistened potting mix (Sunshine mix no. 4 aggregate, Big Top Grow PRO, Thornton, CO) and covered with transparent plastic. A total of 315 plants were transplanted into six trays. The trays were transferred to a growth chamber with 75% relative humidity, 22°C/16-h light (40 μmol m−2 s−1) and 20°C/8-h darkness. Two weeks following transplanting, the plants were uncovered and transferred to larger pots (8.5×8.5 cm) in the greenhouse. Data on survival were taken after 2 wk of uncovering the plants and following transplanting. Statistical analysis. Data were analyzed using SAS version 9.2 for Windows (SAS Institute Inc., Cary, NC). ANOVA was done, and means were grouped according to the Duncan’s multiple range test. Data were considered significant if the value of probability of a type III error was ≤0.05.
Results and Discussion
14
A
100
B
a
12 10
b
b
8
c
c
6
c
c
c
d 4
c
d
2 0
half-strength SH medium with 2.5 mg/l kinetin (Fig. 1A); 81% of the cultured nodes produced shoots. The kinetin/ NAA pairing was the most effective PGR combination for shoot and root production from nodal segments of NAG cultured in liquid medium (Fig. 1B). Root formation was significantly better (P
