In Vitro Cell.Dev.Biol.—Plant (2016) 52:45–55 DOI 10.1007/s11627-016-9746-9
MICROPROPAGATION
Induced polyploidization increases 20-hydroxyecdysone content, in vitro photoautotrophic growth, and ex vitro biomass accumulation in Pfaffia glomerata (Spreng.) Pedersen João Paulo Oliveira Corrêa 1 & Camilo Elber Vital 2 & Marcos Vinícius Marques Pinheiro 1 & Diego Silva Batista 1 & Cleber Witt Saldanha 3 & Ana Cláudia Ferreira da Cruz 1 & Marcela Morato Notini 1 & Débora Márcia Silva Freitas 1 & Fábio Murilo DaMatta 4 & Wagner Campos Otoni 1
Received: 2 August 2015 / Accepted: 14 January 2016 / Published online: 29 January 2016 / Editor: David Duncan # The Society for In Vitro Biology 2016
Abstract The present study aimed to verify the effects of induced polyploidization on Pfaffia glomerata regarding its 20-hydroxyecdysone (20E) production both in vitro and under greenhouse conditions, its in vitro photoautotrophic potential, and its ex vitro biomass accumulation and photosynthetic performance. Synthetic polyploidization efficiently produced individuals with increased in vitro photoautotrophic potential and ex vitro biomass accumulation, although photosynthetic rates per leaf area did not vary between diploids and tetraploids. Among the five tetraploids tested (P28, P60, P68, P74, and P75), P28 showed significantly increased biomass both in vitro and ex vitro when compared with diploid plants, whereas the other tetraploids did not differ significantly from the diploids in terms of biomass accumulation. Although photosynthetic
rates per unit leaf area remained constant among all the plants tested, P28 showed a significantly greater total leaf area, which may have resulted in an increase in net photosynthesis on a whole-plant basis. Under greenhouse conditions, the 20E content in the tetraploid P28 was 31% higher than that in diploid plants, and the final 20E mass per plant produced by P28 ex vitro was approximately twice that produced by diploid plants. Accumulation of 20E in vitro did not follow the same pattern observed among the plants ex vitro; instead, greater accumulation was observed in diploid plants. The induction of polyploidy in P. glomerata appears to be a promising strategy for producing plants with higher biomass accumulation and 20E production ex vitro, in addition to its higher in vitro photoautotrophic potential.
* Wagner Campos Otoni
[email protected]
Keywords Autopolyploidy . Brazilian ginseng . Leaf gas exchange . Photoautotrophic . Micropropagation . Synthetic polyploidy
1
Laboratório de Cultura de Tecidos (LCTII)/BIOAGRO, Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Campus Universitário, Avenida Peter Henry Rolfs s/n, 36570-900 Viçosa, MG, Brazil
2
Laboratório de Fisiologia Molecular de Plantas, Departamento de Biologia Vegetal, Universidade Federal de Viçosa, 36570-900 Viçosa, MG, Brazil
3
Centro de Pesquisas em Florestas, Fundação Estadual de Pesquisa Agropecuária, BR 287, Acesso VCR 830, km 4,5, Boca do Monte, CP 346, 97001-970 Santa Maria, RS, Brazil
4
Laboratório de Nutrição e Metabolismo de Plantas, Departamento de Biologia Vegetal, Universidade Federal de Viçosa, 36570-900 Viçosa, MG, Brazil
Introduction Induced polyploidy in medicinal plants is generally a successful strategy for obtaining highly productive plants (Dhawan and Lavania 1996; Caruso et al. 2013). Although the impacts of polyploidy on biomass accumulation and secondary metabolite production are variable, ploidy level manipulation in many species has been suggested as an interesting strategy for increasing plant biomass accumulation and the production of high-value secondary metabolites (Lavania 2005; Caruso et al. 2013; Xu et al. 2014). Increases in ploidy level often lead
46
CORRÊA ET AL.
to structural changes in traits such as leaf size, stomatal density, cell size, and number of chloroplasts per cell (Dhawan and Lavania 1996), and these changes may affect physiological and biochemical activities such as photosynthesis (Warner and Edwards 1989). Pfaffia glomerata (Amaranthaceae), also known as Brazilian ginseng, is a medicinal species native to South America that produces a wide variety of triterpenic saponins, including the phytoecdysteroid 20-hydroxyecdysone (20E; also known as β-ecdysone), considered to be its main active compound (Vigo et al. 2004). This compound is used in many commercial anabolic preparations for athletes (Lafont and Dinan 2003). In addition, various properties attributed to the Korean ginseng (Panax ginseng) are also attributed to the Brazilian ginseng, including adaptogenic and aphrodisiac effects as well as contributions to physical and mental stress relief (Lorenzi and Matos 2002; Vigo et al. 2003; Carulo 2012). The species is largely traded in Brazil and Japan (Nascimento et al. 2007). Typically, the roots are sold in powder form by intermediaries or producers to wholesalers or exporters. Each harvest yields a maximum of 5–7 tons for a field of 1 ha, and the average field size is 24 ha (Carulo 2012). P. glomerata tetraploids were previously obtained from nodal bud thin-layer sections treated with colchicine. Five tetraploids were regenerated from independent polyploidization events, and the characterization of one of these polyploids (P68) demonstrated that 90-d-old plants, although having reduced root and stem biomass in comparison with diploids, showed a 50% increase in root 20E content (Gomes et al. 2014). The other four P. glomerata tetraploids obtained were not characterized in that study. Previous findings showed that photoautotrophic micropropagation is a very promising system for both in vitro production of secondary metabolites from P. glomerata and mass propagation of the species: the plants displayed higher photosynthetic rates and produced more 20E in vitro when grown under photoautotrophic conditions (Iarema et al. 2012; Saldanha et al. 2012, 2013, 2014). In addition, Corrêa et al. (2015) previously surveyed other accessions from an in vitro germplasm bank and demonstrated that they had different in vitro photoautotrophic potentials and biomass accumulation. Because polyploidization may increase photosynthetic performance, it may also have positive effects on the photoautotrophic potential of the species. Given the facts discussed above, the goals of the present study were to examine a diploid accession (accession 22) and five autotetraploids independently generated from this accession to study the effects of induced polyploidization in P. glomerata on the following characteristics: (1) in vitro photoautotrophic potential, (2) biomass accumulation and photosynthetic rates ex vitro, and (3) 20E production by the species under both greenhouse and in vitro photoautotrophic conditions.
Materials and Methods Plant material and treatments The P. glomerata plants used in the experiments were taken from the Plant Tissue Culture Laboratory (LCT-BIOAGRO-UFV) germplasm bank, where 71 diploid accessions and five synthetic polyploids derived independently from accession 22 (Gomes et al. 2014) were maintained in vitro by means of monthly subcultures. Cultures from the germplasm bank were kept in a growth room at 25 ± 2°C air temperature and 60 μmol m−2 s−1 photon irradiance provided by two tubular cool-white fluorescent lamps (Daylight, F96T 12/D/HO, 110 W, OSRAM, Mississauga, Canada) with a 16-h photoperiod. Subculture was performed using either apical or single-node dissection in MS semi-solid medium plus vitamins (Murashige and Skoog 1962), supplemented with 3% (w/v) sucrose and 100 mg L−1 myo-inositol (Sigma-Aldrich® Co, St. Louis, MO) and solidified with 7 g L−1 of granulated agar (Merck®, Darmstadt, Germany). All culture media were adjusted to pH 5.7 and sterilized by autoclaving it at 121°C and 152 kPa for 15 min. All experimental plants were subcultured for 30 d under the same conditions described for the germplasm bank plants. Two experiments were conducted, with the ploidy level of each event being the only known source of variation between them. The plants used were diploid plants from P. glomerata accession 22 and five synthetic polyploids (tetraploids): events P28, P60, P68, P74, and P75. Experiment I aimed to evaluate the influence of ploidy level on in vitro photoautotrophic growth and 20E production in vitro, whereas experiment II aimed to assess the effects of ploidy level on ex vitro growth, leaf gas exchange, chlorophyll a fluorescence, and 20E production. Nodal segments without leaves excised from subcultured plants were used as explants in experiment I. Four explants were placed in each 750-mL vessels containing 100 mL of liquid basal MS medium with myo-inositol (100 mg L−1) and without sucrose (experimental design is specified further). Instead of a gelling agent, a 2:1 (w/w) mixture of vermiculite and ground cellulose pulp was used as supporting material (Corrêa et al. 2015). The 750-mL vessels were constructed by using couplers (Sigma-Aldrich®) to join two Magenta® vessels (Fig. 1). Two 10-mm-diameter holes were made in the top of each 750-mL vessel and covered with a Fluoropore hydrophobic membrane (PFTE; MilliSeal® Air Vent, Tokyo, Japan) to provide gas exchange between the vessel and the outside environment. The cultures were incubated for 45 d under the conditions described above. For experiment II, subcultured plants were acclimatized using hydroponics with macronutrient solution (101.10 mg L −1 KNO 3 , 27.11 mg L −1 MgSO 4 ·7H 2 O, 188.93 mg L −1 Ca(NO 3 ) 2 ·4H 2 O, and 42.56 mg L −1 NH4H2PO4) for 15 d. Plants were then transferred to 5-L pots containing substrate (Tropstrato Florestal Vida Verde®, Mogi-
POLYPLOIDIZATION OF PFAFFIA GLOMERATA
47
Fig. 1 Pfaffia glomerata diploid and tetraploid plantlets propagated in vitro at 45 d of culture. From left to right: diploid accession 22 (A), P28 (B), P60 (C), P68 (D), P74 (E), and P75 (F). Bar = 6 cm
Mirim, SP, Brazil) made of Pinus bark, vermiculite, charcoal, calcium nitrate, and coconut fiber, and they were maintained under greenhouse conditions for 160 d. Growth parameters In experiment 1, plant height (cm), number of nodal segments, and dry weight (g per experimental unit) were assessed. Experiment 2 included these same measurements except that dry weight was measured separately for root, stem, and leaves. Also in experiment 2, the leaf area (cm2) was assessed using an Area Meter (model Li-Cor® 3100, Li-Cor® Biosciences, Lincoln, NE), and stem diameter (mm) was assessed with a tape measure. Total chlorophyll content Total chlorophyll was estimated in both experiments using a SPAD chlorophyll meter (SPAD502, Minolta, Osaka, Japan). Measurements were taken from the second and third fully expanded leaves from the shoot tip. High-performance liquid chromatography determination of 20E content Methanolic extracts were prepared in 15-mL conical polypropylene tubes with 100 mg of powdered dried plant material in 10 mL of methanol, and they were incubated at 27°C under agitation for 7 d. The extracts were then centrifuged at 2795×g for 20 min in 10-mL conical polypropylene tubes, after which the supernatants were transferred to 1.5-mL microcentrifuge tubes and centrifuged again under the same conditions. After the second centrifugation, the supernatant was transferred to 1.5-mL vials and the 20E content in the methanolic extract was quantified using high-performance liquid chromatography (HPLC) in a Shimadzu LC-10Ai (Shimadzu Co., Tokyo, Japan) instrument, coupled to a SPD-10AI detector and a Bondesil C18
(5.0 μm × 4.6 mm × 250 mm) column. The HPLC detection was performed at 245 nm, and the mobile phase used was a water/methanol 1:1 (v/v) mixture, with a flow rate of 0.7 mL min−1. The linear equation for the calibration curve was obtained by adding the 20E standard (Sigma-Aldrich®) to methanol at 10, 20, 40, 60, and 80 mg L−1 and performing a linear regression. Data for 20E are expressed both in concentration (percentage by mass) and in total mass per plant/organ. Stomatal density calculation Whole leaves (second and third fully expanded leaves from the shoot tip) of ex vitro plants were processed by diaphanization with 10% (w/v) sodium hydroxide, bleached with 10% (v/v) sodium hypochlorite, stained with 0.001% (w/v) basic alcoholic fuchsin (SigmaAldrich®), and mounted in glycerinated gelatin, as described by Dalvi et al. (2013). Slides were sealed with clear nail polish. Images of both abaxial and adaxial epidermis of each leaf were captured with an Olympus AX70TRF microscope (Olympus Optical, Tokyo, Japan) with a U-Photo Camera System (Spot Insight Color 3.2.0, Diagnostic Instruments Inc., Sterling Heights, MI). Stomatal density was calculated using the software ANATI QUANTI (Aguiar et al. 2007). Leaf gas exchange and chlorophyll a fluorescence of ex vitro plants The net CO2 assimilation rate (A), stomatal conductance to water vapor (gs), internal-to-ambient CO2 concentration ratio (Ci/Ca), and transpiration rate (E) were determined simultaneously with measurements of chlorophyll a fluorescence using the open gas exchange system Li6400XT (Li-Cor®) equipped with an integrated fluorescence chamber head (Li-6400-40, Li-Cor®). Measurements were
48
CORRÊA ET AL.
taken between 08:00 and 13:00 at 25°C ambient temperature, 55–65% humidity, and approximately 39 Pa CO2 partial pressure under artificial photosynthetically active radiation of 1, 000 μmol photons m−2 s−1. Previously dark-adapted (8 h) leaf tissues were illuminated with weak modulated measuring beams (0.03 μmol m−2 s−1) to obtain the initial fluorescence (F0). Saturating pulses of white actinic light (Li-Cor®) at 8000 μmol photons m−2 s−1 were applied for 0.8 s to ensure maximum fluorescence emissions (Fm), from which the variable-to-maximum chlorophyll fluorescence ratio, Fv/Fm = ((Fm − F0)/Fm)), was calculated. This ratio expresses the maximum photosystem II (PSII) photochemical efficiency. In light-adapted leaves, the steady-state fluorescence yield (Fs) was measured following by a saturating pulse of white light (8000 μmol m−2 s−1; 0.8 s) that was applied to achieve the light-adapted maximum fluorescence (Fm′). The actinic light was then turned off, and far-red illumination was applied (2 μmol m−2 s−1) to measure the lightadapted initial fluorescence (F0′). Using these parameters, the coefficient for photochemical quenching (qP) was calculated as qP = (Fm′ − Fs)/(Fm′ − F0′), and that for non-photochemical quenching (NPQ) was calculated as NPQ = (Fm/Fm′) − 1. The actual quantum yield of PSII electron transport (ΦPSII) was computed as ΦPSII = (Fm′ − Fs)/Fm′, from which the apparent ele ctr on tr an spo rt ra t e (ETR) w as calc ula ted as ETR = ΦPSII*PPFD*f*α, where f is a factor that accounts for the partitioning of energy between PSII and photosystem I (PSI) and is assumed to be 0.5 (indicating that the exCitation energy is distributed equally between the two photosystems),
Fig. 2 Growth parameters of Pfaffia glomerata of different ploidy levels propagated in vitro under photoautotrophic conditions, after 45 d of culture. (A) Plant height (cm). (B) Number of internodal segments per plant. (C) Dry weight (g) per replicate. (D) Total chlorophyll content (SPAD readings). Means marked with the same letter do not differ significantly from each other (P > 0.05, Tukey’s test). Error bars represent standard deviation
and α is the leaf absorptance of the photosynthetic tissues and is assumed to be 0.84. Experimental design and statistical analysis The experiments were arranged in a completely randomized design. The ploidy level was the only source of variation in each experiment and was analyzed as a single factor. The diploid accession 22 and the four polyploid events each represented a treatment. Experiment I was assembled with eight replicates per treatment, each represented by one culture vessel containing four plants, whereas each of the six replicates in experiment II consisted of a single plant per pot. All variables were examined by ANOVA, and means were compared by Tukey’s test at 5% probability using the software SISVAR (Ferreira 2003). Data on 20E content were analyzed using square root transformation.
Results Synthetic polyploidization increased in vitro photoautotrophic potential In vitro photoautotrophic growth varied significantly both among polyploid plants and between diploids and polyploids (Figs. 1 and 2). The highest in vitro dry weight was observed in the polyploid P28 (mean of 472 mg/replicate; Figs. 1B and 2C) and was 32% higher than that of diploid accession 22 (Figs. 1A and 2C); in contrast, P75 (Fig. 1F) displayed the lowest dry weight value (255 mg). The other polyploids (P60, P68, and
POLYPLOIDIZATION OF PFAFFIA GLOMERATA
P74; Fig. 1C–E, respectively), like diploid accession 22, showed intermediate biomass accumulation (Fig. 2C). The number of internodes per plant did not vary significantly across different ploidy levels (Fig. 2B). Total chlorophyll also varied significantly among the polyploid plants in vitro, with SPAD readings ranging from 22.5 to 29 (in P75 and P60, respectively), and accession 22 showed an intermediate content (26.3) (Fig. 2D). Fig. 3 Growth parameters of 150-d-old Pfaffia glomerata plants of different ploidy levels grown under greenhouse conditions. (A) Plant height (cm). (B) Number of internodal segments per plant. (C) Stem diameter (mm). (D) Leaf area (cm2). (E) Dry weight per plant (g). (F) Dry weight by plant organ (g). (G) Stomatal density (stomata mm−2). Means marked with the same letter do not differ significantly from each other (P > 0.05, Tukey’s test). For (F), uppercase letters are used to denote differences among organs within the same accession, and lowercase letters are used to denote differences between treatments. Error bars represent standard deviation
49
Synthetic polyploids displayed higher biomass accumulation ex vitro but showed unchanged photosynthetic performance The lowest absolute biomass accumulation was found in the diploid plants, although the only polyploid that differed significantly from accession 22 was P28 (Fig. 3E), as previously observed in vitro. P28 displayed a total dry weight of 44.1 g, a value 72% higher than that of its diploid counterpart. Polyploids P60, P68, P74, and P75 showed intermediate
50
CORRÊA ET AL.
dry weight values that did not differ significantly from either diploid accession 22 or P28 (Fig. 3E). The same pattern as that found for biomass was observed for leaf area (Fig. 3D). In addition, leaf shape and size also varied, as visually noted through the higher width/length ratio of leaves in polyploids compared with the diploid (Figs. 4 and 5). Additionally, abnormal leaf development was observed in P75, in which curly leaves were noted both in vitro (Fig. 6A) and ex vitro (Figs. 4E and 6B). Diploid plants allocated similar amounts of biomass to roots, stems, and leaves, whereas polyploids tended to allocate more biomass to stems (Fig. 3F). Root biomass did not vary significantly among different ploidy levels, although stem and leaf biomass in P28 were significantly higher than in accession 22. Although the average internode number and plant height varied among genotypes, no significant difference was observed for any of these parameters (Fig. 3A, B) due to the high variance observed within treatments. In general, polyploids showed larger stem diameters, particularly P28 and P68 (8.2 mm for each; Fig. 3C), whereas the average stem diameter of diploid plants was 5.6 mm. The stomatal density decreased due to the polyploidization process, with the highest value observed in diploid plants (Fig. 3G). Differences in biomass accumulation were unrelated to varying photosynthetic performance, as shown by the fact that both the chlorophyll a fluorescence parameters (Fv/Fm, ΦPSII, NPQ, and ETR; Fig. 7A–D) and the gas exchange parameters (A, gs, Ci/Ca, and E) (Fig. 8A–D) did not differ significantly among plants of different ploidy levels. Nonetheless, chlorophyll contents ex vitro were highest in P28, intermediate in the other polyploids, and lowest in diploid plants (Fig. 7E). Fig. 4 Pfaffia glomerata leaves from the fourth lowest node segment (counting from the bottom) from plants of different ploidy levels grown in a greenhouse for 150 d after acclimatization from in vitro conditions. From left to right: P28 (A), P60 (B), P68 (C), P74 (D), P75 (E), and diploid accession 22 (F). Bar = 5 cm
Fig. 5 Pfaffia glomerata plants of different ploidy levels grown in a greenhouse for 150 d after acclimatization from in vitro conditions. From left to right: P28 (A), P60 (B), P68 (C), P74 (D), P75 (E), and diploid accession 22 (F). Bar = 25 cm
POLYPLOIDIZATION OF PFAFFIA GLOMERATA
51
Fig. 6 Views of curly leaves of tetraploid P75 plants grown in vitro for 45 d (A) and grown in greenhouse for 150 d after acclimatization from in vitro conditions (B). Bars = 1.5 cm (A); 5 cm (B)
Synthetic polyploidization increased 20E production ex vitro Under greenhouse conditions, the highest 20E content per plant was found in polyploid P28 (0.8%), while diploid plants contained 0.61% 20E (Fig. 9A), which represents an increase of 31% in 20E production on a whole-plant basis. Fig. 7 Chlorophyll fluorescence parameters and chlorophyll levels in 150-d-old Pfaffia glomerata plants of different ploidy levels grown under greenhouse conditions. (A) PSII maximum quantum yield (Fv/Fm). (B) Actual quantum yield of PSII photochemistry (ΦPSII). (C) Nonphotochemical quenching (NPQ). (D) Electron transport rate (ETR). (E) Total chlorophyll content (SPAD readings). Means marked with the same letter do not differ significantly from each other (P > 0.05, Tukey’s test). Error bars represent standard deviation. The y-axis on (A) starts on 0.8 rather than 0 due to the fact that Fv/Fm is a parameter that normally shows low variation rates, normally ranging from 0.79 to 0.85 in plants not exposed to stresses
The lowest 20E content was found in P68 (0.56%) (Fig. 9A). Regardless of treatment, the 20E accumulated primarily in leaves; the highest leaf 20E content was found in P28 (1.5%), and the lowest concentration was found in P68 and diploid accession 22 (approximately 1.1%) (Fig. 9B). Root
52
CORRÊA ET AL.
Fig. 8 Gas exchange parameters from 150-d-old P. glomerata plants of different ploidy levels grown under greenhouse conditions. (A) Net CO2 assimilation rate (A). (B) Stomatal conductance to water vapor (gs). (C) Internal-to-ambient CO2 concentration ratio (Ci/Ca). (D) Transpiration rate (E). Means marked with the same letter do not differ significantly from each other (P > 0.05, Tukey’s test)
and stem 20E concentrations did not differ significantly within polyploid plants, but diploids showed higher levels in roots than in stems (Fig. 9B). Polyploid P28 also showed the highest total mass of 20E per plant (293 mg), approximately twice that produced by diploid plants (146 mg) (Fig. 9C). The mass of 20E did not differ among organs in any plant except for P68, which displayed a lower 20E mass in the roots than in the other organs. Regardless, the highest levels of 20E in roots, stems, and leaves were noted in P28 (Fig. 9D). The 20E production in vitro (Fig. 9E, F) did not follow the same pattern observed ex vitro. The highest content in vitro was observed in diploid plants (1.24%), in contrast to the ex vitro observations (compare Fig. 9E with 9A) although the differences between accession 22 and most of the polyploids were not significant in the in vitro test P75 showed the lowest values for concentration and total mass of 20E in vitro. The highest masses per plant of 20E were found in P74, diploid accession 22, and P28 (1.45, 1.37, and 1.17 mg, respectively; Fig. 9F).
Discussion The synthetic polyploidization of P. glomerata was effective in producing individuals with increased biomass accumulation and secondary metabolite production, which is a highly desirable feature in the commercial production of the species. Although similar results were previously reported for other medicinal species (Gao et al. 1996; Kim et al. 2004; Dehghan et al. 2012), this is the first report on the
improvement of in vitro photoautotrophic growth through induced polyploidization. Corrêa et al. (2015) demonstrated that different P. glomerata accessions have different in vitro photoautotrophic potentials, and the accessions that accumulated more biomass in vitro also displayed higher biomass accumulation and higher photosynthetic capacities per unit leaf area ex vitro. In the present study, variation in in vitro photoautotrophic potential between diploid and polyploid plants was demonstrated by the significant increase in both in vitro biomass accumulation and dry weight ex vitro of the tetraploid P28 relative to the diploid accession 22. Nevertheless, in sharp contrast with previous results (Corrêa et al. 2015), this increased biomass accumulation was clearly unrelated to differences in photosynthetic performance per unit leaf area, given that net CO2 assimilation rate (A) and photochemical parameters did not differ significantly among the plant materials that were assessed (Figs. 7 and 8). It is possible that factors other than photosynthetic capacity (e.g., growth and maintenance respiration) contributed more to the differences in biomass accumulation between the diploid accession 22 and the tetraploid P28. Polyploidy affects three interacting factors that determine the photosynthetic rate per unit leaf area: DNA content per cell, cell size, and number of cells per unit leaf area (Warner and Edwards 1989, 1993). Photosynthetic rate per cell is positively correlated with DNA content per cell, but when the number of chromosomes doubles, cell size also increases, thus reducing the number of cells per unit leaf area (Warner and Edwards 1993). Therefore, the polyploidy-driven changes in the photosynthetic rate per unit leaf area depend on the cell packing in the leaves and the ratio of DNA content to cell
POLYPLOIDIZATION OF PFAFFIA GLOMERATA
53
Fig. 9 A–D Levels of 20E per plant (A) and per plant organ (B) of P. glomerata grown under greenhouse conditions (ex vitro) for 150 d, as well as total 20E mass per plant (C) and per plant organ (D). E–F 20E content per plantlet (E) and total 20E mass per plantlet (F) grown in vitro for 45 d. Means marked with the same letter do not differ significantly from each other (P > 0.05, Tukey’s test). For (B) and (D), uppercase letters are used to denote differences among organs within the same accessions and lowercase letters are used to denote differences between treatments. Error bars represent standard deviation
volume. If cell volume also doubles with a doubling in DNA amount, the number of cells per leaf area decreases by half, and although the photosynthetic rate per cell doubles, carbon assimilation per leaf area will remain the same. This occurs with induced polyploids of Medicago sativa (Molin et al. 1982) and Pennisetum americanum (Warner and Edwards 1988), and the similar photosynthetic rates per leaf area observed in diploids and polyploids in the present study suggests that this is also the case in P. glomerata. The higher biomass produced by some P. glomerata polyploids appears to have resulted simply from the increased organ size frequently caused by chromosome duplication (Osborn et al. 2003), as depicted in Figs. 4 and 5. Consequently, the invariant A per unit leaf area coupled with increased total leaf area in some polyploids would lead to increased A on a whole-plant basis to fuel the higher biomass accumulation. The polyploids P28, P60, and P74 showed the highest levels of 20E ex vitro, in contrast to in vitro observations showing that diploid plants tended to have higher 20E contents. This is most likely related to the fact that secondary
metabolic pathways are induced routes that are generally triggered by environmental stresses such as intense light, microorganisms, herbivores, and heat stress (Bennett and Wallsgrove 1994). None of these environment-related factors are normally present in in vitro cultivation systems, and one of the most successful strategies used to increase in vitro secondary metabolite production is elicitation, which consists of applying stresses (e.g., autoclaved mycelium of pathogenic fungi, protein extracts, temperature, UV light, heavy metal salts, altered pH) to the in vitro cultures, triggering the production of secondary metabolites that are normally not produced (Borgaud et al. 2001). Given that P. glomerata displayed better growth, higher photosynthetic rates, and higher 20E yield in vitro when grown in photoautotrophic systems than when grown under in vitro heterotrophic conditions (Iarema et al. 2012; Saldanha et al. 2013, 2014), the optimization of photoautotrophic culture conditions concomitantly with studies of elicitation may be a promising step toward achieving viable in vitro production of 20E from this species. Although P28 plants did not
54
CORRÊA ET AL.
produce more 20E than the diploids in vitro, they showed the highest dry weight in vitro and the highest 20E content ex vitro, and they remain a promising polyploid for further studies of in vitro 20E production with elicitation. As reported elsewhere for other species, the induced polyploidization of P. glomerata could successfully generate more productive material in terms of secondary metabolites and biomass. However, not all of the polyploids produced independently from accession 22 were as productive as P28, and some of them did not differ significantly from accession 22. The differences observed among the polyploids, including the curly leaves that resulted from abnormal tissue growth in P75, may be a result of epigenetic changes during genome duplication. Other studies have provided evidence for frequent epigenetic changes in new polyploids, including DNA methylation, histone modification, and chromatin packaging (Osborn et al. 2003). Polyploids of Eragrostis curvula obtained with colchicine presented different methylation patterns, which possibly led to dramatic changes in gene expression (Martelotto et al. 2007; Mecchia et al. 2007). Polyploids of this species also showed polymorphisms in 28% of the detected loci compared with the diploid plants, but tetraploids obtained from parallel polyploidization events did not show any genetic polymorphisms relative to one another. Furthermore, spontaneously generated wheat synthetic allopolyploids showed the same pattern of sequence modifications as the polyploids generated by tissue culture or colchicine treatment (Ozkan et al. 2001).
Conclusions Herein, it is demonstrated that, when compared with diploid parental plants, induced polyploidization of P. glomerata can result in plants with higher in vitro photoautotrophic potential, in addition to displaying higher biomass accumulation and 20E production ex vitro, as was the case for P28. These characteristics may improve P. glomerata production from propagation to ex vitro cultivation, thus ultimately producing genotypes with a highly increased total 20E mass produced per plant, and of greater interest from an industrial perspective. To further explore the potential of the generated polyploid accessions, a field trial is currently running to evaluate the performance of the diploid accession as compared with the synthetic polyploid material derived from it, since active constituents are very much dependent on the external environment where the plants are actually growing. Acknowledgments The authors thank the National Council for Scientific and Technological Development (CNPq) [MCT 480675/ 2009-0; PDJ 500874/2012-3; PQ 303201/2010-10, and MCTI 459529/ 2014-5 to WCO], the Minas Gerais State Research Foundation (FAPEMIG) [CAG-APQ-01036-09; CRA-APQ-01651-13; CRA-BPD00046-14], and CAPES (PNPD) for financial support. We also thank
Dr. Roberto Vieira and Dr. Rosa das Neves Alves (National Center for Genetic Resources and Biotechnology—Embrapa/Cenargen, Brasília, DF, Brazil) for providing Pfaffia glomerata accessions.
Conflicts of Interest The authors declare that there are no conflicts of interest. Authors’ Contributions JPOC, CEV, DMSF, MMN, MVMP, DSB, CWS, and ACFC performed the experiments. JPOC and ACFC raised the in vitro plants for the experiments. JPOC, FMD, and WCO contributed to the design and interpretation of the research and to the writing of the paper.
References Aguiar TV, Sant’anna-Santos BF, Azevedo AA, Santos RF (2007) Anati Quanti: Software de análises quantitativas para estudos em Anatomia Vegetal. Planta Daninha 25:649–659 Bennett RN, Wallsgrove RM (1994) Secondary metabolites in plant defense mechanisms. New Phytol 127:617–633 Borgaud F, Gravot A, Milesi S, Gontier E (2001) Production of plant secondary metabolites: a historical perspective. Plant Sci 161:839– 851 Carulo MF (2012) Use of SFC in extraction of adaptogens from Brazilian plants. Am J Analyt Chem 3:977–982 Caruso I, Dal Piaz F, Malafronte N, De Tommasi N, Aversano R, Zottele CW, Scarano MT, Carputo D (2013) Impact of ploidy change on secondary metabolites and photochemical efficiency in Solanum bulbocastanum. Nat Prod Commun 8:1387–1392 Corrêa JPO, Vital CE, Pinheiro MVM, Batista DS, Azevedo JFL, Saldanha CW, Cruz ACF, DaMatta FM, Otoni WC (2015) In vitro photoautotrophic potential and ex vitro photosynthetic competence of Pfaffia glomerata (Spreng.) Pedersen accessions. Plant Cell Tiss Organ Cult 121:289–300 Dalvi VC, Meira RMSA, Azevedo AA (2013) Extrafloral nectaries in neotropical Gentianaceae: occurrence, distribution patterns, and anatomical characterization. Am J Bot 100:1779–1789 Dehghan E, Häkkinen ST, Oksman-Caldentey KM, Shahriari Ahmadi F (2012) Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.). Plant Cell Tiss Organ Cult 110:35–44 Dhawan OP, Lavania UC (1996) Enhancing the productivity of secondary metabolites via induced polyploidy: a review. Euphytica 87:81– 89 Ferreira DF (2003) Sisvar 5.0. Available online: http://www.dex.ufla.br/ ~danielff/programas/sisvar.html Gao SL, Zhu DN, Cai ZH, Xu DR (1996) Autotetraploid plants from colchicine treated bud culture of Salvia miltiorrhiza Bge. Plant Cell Tiss Organ Cult 47:73–77 Gomes SSL, Saldanha CW, Neves CS, Trevizani M, Raposo NRB, Notini MM, Santos MO, Campos JMS, Otoni WC, Viccini LF (2014) Karyotype, genome size, and in vitro chromosome doubling of Pfaffia glomerata (Spreng.) Pedersen. Plant Cell Tiss Organ Cult 118:45–56 Iarema L, Cruz ACF, Saldanha CW, Dias LLC, Vieira RF, Oliveira EJ, Otoni WC (2012) Photoautotrophic propagation of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]. Plant Cell Tiss Organ Cult 110:227–238
POLYPLOIDIZATION OF PFAFFIA GLOMERATA Kim YS, Hahn EJ, Murthy HN, Paek KY (2004) Effect of polyploidy induction on biomass and ginsenoside accumulations in adventitious roots of ginseng. J Plant Biol 4:356–360 Lafont R, Dinan L (2003) Practical uses for ecdysteroids in mammals including humans: an update. J Insect Sci 3:1–30 Lavania UC (2005) Genomic and ploidy manipulation for enhanced production of phyto-pharmaceuticals. Plant Genet Res 3:170–177 Lorenzi H, Matos FJA (2002) Plantas Medicinais do Brasil: Nativas e exóticas cultivadas. Instituto Plantarum, Nova Odessa, SP, Brazil Martelotto LG, Ortiz JPA, Stein J, Espinoza F, Quarin CL, Pessino SC (2007) Genome rearrangements derived from autopolyploidization in Paspalum sp. Plant Sci 172:970–977 Mecchia MA, Ochogavia A, Selva JP, Laspina N, Felitti S, Martelotto LG, Spangenberg G, Echenique V, Pessino SC (2007) Genome polymorphisms and gene differential expression in a ‘back-andforth’ ploidy altered series of weeping lovegrass (Eragrostis curvula). J Plant Physiol 164:1051–1061 Molin WT, Meyers SP, Baer GR, Schrader LE (1982) Ploidy effects of isogenic populations of alfalfa: II. Photosynthesis, chloroplast number, ribulose-1,5-bisphosphate carboxylase, chlorophyll, and DNA in protoplasts. Plant Physiol 70:1710–1714 Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15:473–497 Nascimento EX, Mota JH, Vieira MC, Zárate NAH (2007) Produção de biomassa de Pfaffia glomerata (Spreng.) Pedersen e Plantago major L. em cultivo solteiro e consorciado. Ciência Agrotecnol 31:724– 730 Osborn TC, Chris-Pires J, Birchler JA, Auger DL, Chen ZF, Lee H, Comai L, Madlung A, Doerge RW, Colot V, Martienssen RA (2003) Understanding mechanisms of novel gene expression in polyploids. Trends Genet 19:141–147 Ozkan H, Levy AA, Feldman M (2001) Allopolyploidy-induced rapid genome evolution in the wheat (Aegilops–Triticum) group. Plant Cell 13:1735–1747
55
Saldanha CW, Otoni CG, Azevedo JLF, Dias LLC, Rêgo MM, Otoni WC (2012) A low-cost alternative membrane system that promotes growth in nodal cultures of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]. Plant Cell Tiss Organ Cult 110:413–422 Saldanha CW, Otoni CG, Notini MM, Kuki KN, Cruz ACF, Neto AR, Dias LLC, Otoni WC (2013) A CO2-enriched atmosphere improves in vitro growth of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]. In Vitro Cell Dev Biol Plant 49:433–444 Saldanha CW, Otoni CG, Rocha DI, Cavatte PC, Detmann KSC, Tanaka FAO, Dias LLC, DaMatta FM, Otoni WC (2014) CO2-enriched atmosphere and supporting material impact the growth, morphophysiology and ultrastructure of in vitro Brazilian-ginseng [Pfaffia glomerata (Spreng.) Pedersen] plantlets. Plant Cell Tiss Organ Cult 118:87–99 Vigo CLS, Narita E, Marques LC (2003) Validação da metodologia de quantificação espectrofotométrica das saponinas de Pfaffia glomerata (Spreng.) Pedersen—Amaranthaceae. Rev Bras Farmacogn 13:46–49 Vigo CLS, Narita E, Milaneze-Gutierre MA, Marques LC (2004) Caracterização farmacognóstica comparativa de Pfaffia glomerata (Spreng.) Pedersen Hebanthe paniculata Martius—Amaranthaceae. Rev Bras Plant Med 6:7–19 Warner DA, Edwards GE (1988) C4 photosynthesis and leaf anatomy in diploid and autotetraploid Pennisetum americanum (pearl millet). Plant Sci 56:85–92 Warner DA, Edwards GE (1989) Effects of polyploidy on photosynthetic rates, photosynthetic enzymes, contents of DNA, chlorophyll, and sizes and numbers of photosynthetic cells in the C4 dicot Atriplex confertifolia. Plant Physiol 91:1143–1151 Warner DA, Edwards GE (1993) Effects of polyploidy on photosynthesis. Photosynth Res 35:135–147 Xu CG, Tang TX, Chen R, Liang CH, Liu XY, Wu CL, Yang YS, Yang DP, Wu H (2014) A comparative study of bioactive secondary metabolite production in diploid and tetraploid Echinacea purpurea (L.) Moench. Plant Cell Tiss Organ Cult 116:323–332
