CHAPTER 5
PHOTOMIXOTROPHIC GROWTH AND
ROSMARINIC ACID PRODUCTION IN BASIL
(OCIMUM BASILICUM L.)
MICROPROPAGATED PLANTLETS.
5.1 INTRODUCTION
Cell and tissue culture constitute an alternative to the conventional agriculture for the production of high-value plant metabolites, because it may provide an useful tool to regulate their biosynthetic pathways in a controlled environment (Matkowski, 2008, Karuppusamy, 2009). The application of tissue culture for all-year round production of high-standard plant material with predictable RA content has been demonstrated in different species, for instance Lavandula vera, Coleus blumei, Salvia officinalis and other plants in the Lamiaceae and Boraginaceae (Matkowski 2008; Park et al., 2008). In the Lamiaceae family, sweet basil (Ocimum basilicum L.) is an important source of RA, as it is able to produce and accumulate large quantity of this bioactive compound (Jayasinghe et al., 2003; Juliani et al., 2008).
Different basil species were found to accumulate larger quantities of RA in cell, callus, hairy roots and micropropagated shoots than in vivo plants (Kintzios et al., 2003, 2004, Rady and Nazif., 2005; Hakkim et al., 2007; Kiferle et al., 2011). Identifying and understanding the effects of in vitro environment on biomass growth and secondary metabolism is essential for scaling-up the production of bioactive compounds by plant cell and tissue culture (Georgiev et al., 2009).
In vitro plants generally grow under heterotrophic conditions and the absence of photosynthesis is due to low irradiance and CO2 concentrations within the culture
vessel and the high sugar concentration in the growing medium (Xiao and Kozai, 2011). When explants are cultured under conditions promoting photosynthetic
competence (photoautotrophic or photomixotrophic conditions), they may grow and develop better compared to heterotrophic explants (Lucchesini et al. 2001; Afreen et al. 2002; Xiao and Kozai, 2011). For instance, the occurrence of some physiological disorders, such as hyperhydricity or the callus formation at the base of explants, was found to decrease (Xiao and Kozai, 2011).
In vitro environment and in particular gaseous composition of the headspace in the culture vessel play a major role in the acquisition of photosynthetic competence in micro-propagated plants (Kozai 1991, Xiao and Kozai, 2011). Photosynthesis can be stimulated when the plantlets are cultured in vessels with high ventilation rate, as they facilitate CO2 supply from the external atmosphere and reduced the
accumulation of ethylene synthesized by the plant tissue and/or released by abiotic sources as agar or plastic materials (Panizza et al, 1993; Xiao and Kozai, 2011). High ethylene concentration can influence morphogenesis and also induce chlorophyll breakdown in the explants, thus affecting negatively their photosynthetic activity (Hazarika, 2006).
Many studies have been conducted to investigate the influence of ethylene and CO2
concentration in the vessel atmosphere on growth and development of in vitro plantlets (Hazarika, 2006; Xiao and Kozai, 2011). Notwithstanding, much less attention has been paid on the effect of the gaseous composition of the headspace in the culture vessel on the production of secondary metabolites (e.g. Ikemeyer et al., 1989; Mosaleeyanon et al 2005; Zobayed and Saxena, 2004), and, to our knowledge, no paper focused on the accumulation of RA in vitro plantlets. Therefore, a study was conducted to investigate the effect of different vessel types on growth and RA accumulation of in vitro nodal explants of sweet basil genotypes (green-leaved Genovese and purple-leaved Dark Opal).
Five different vessels with different ventilation rates were compared in order to stimulate explants photosynthesis and the diffusive escape of ethylene from the culture vessel, which may affect explants growth and secondary metabolism (Cho et al, 1988; Songstad et al., 1989; Kim et al., 1991; Jeong et al., 2006). Along with
three vessels (Magenta™, Microbox Eco2™, PCCV25™) largely used for plant micro-propagation and filled with agarized medium, we tested two bioreactors with liquid medium for temporary (Rita™; Pavlov and Bley, 2006) or stationary (Growtek™; Bhanja et al., 2007) immersion. Growtek™ was modified in order to achieve a ventilation rate similar to that of RITA™. Bioreactors allow to scale-up and control efficiently the production of secondary metabolites in plant cell and tissue culture (Paek et al, 2005; Lucchesini and Mensuali-Sodi, 2010; Weathers et al., 2010). The headspace gaseous composition was analyzed during all the cultivation period and CO2 concentration was used to calculate the net photosynthetic
rate of the explants.
5.2 MATERIALS AND METHODS
CULTURE VESSELS
Five types of vessels with different shape (Fig. 5.1) and properties (Tab. 5.1) were compared for the experimental procedures.
Magenta™, Microbox Eco2™, PCCV25™ vessels were filled with agarized medium and equipped with a filter on their lips in order to ensure aseptic venting. RITA™ and Growtek™ bioreactors were employed for temporary and stationary immersion culture respectively. Microfiltered air pressure was applied to RITA™ systems to push the liquid medium up and immerse temporarily the explants; 1-min immersion was applied every 12 h. Growtek™ was modified by the application of a round filter (diameter 19 mm) on the side tube to permit sterile air exchanges.
All culture vessels were equipped with a gas-sampling device to allow the analysis of headspace air, as described by Lucchesini et al. (2001). The hourly number of gas exchanges (E) in each type of vessel was determined using ethylene as tracer gas according to Lucchesini et al. (2001).
Figure 5.1. Plantlets of basil (Ocimum basilicum L., cv. Genovese and cv. Dark Opal) in the different cultivation vessels during the multiplication phase. A: Temporary Immersion System (RITA™), B: cylindrical bioreactor (Growtek™), C: GA-7 box (Magenta™), D: round box (PPCV25™), E: rectangular box: (MicroboxEco2™).
PLANT MATERIAL AND GROWING CONDITIONS
The plantlets were originated from seeds purchased from SAIS (Cesena, Italy) and grown in hydroponics (floating system). Nodal segments were used as explants source and in vitro cultured as previously described (Kiferle et al. 2011). Initial basil explants for this experiment, were obtained from shoot stock cultures grown in PCCV25™ boxes (six explants per box) filled with 50 ml of the agarized MS medium (Murashige and Skoog, 1962) with the following modifications: 300 mg L-1
of reduced gluthatione (GSH), 500 mg L-1 of 2-(N-morpholino) ethanesulfonic acid (MES), 30 g L-1 of sucrose and 0.25 mg L-1 6-benzylaminopurine (BA). From basil
stock cultures, the explants were excised and sub-cultured in different types of vessel using agarized (containing 7 g L-1 of Difco Bacto agar) or liquid medium with the same nutrient composition. Fresh and dry weights were recorded at the beginning of the transplant (T-0), at the second, third and fourth week of culture while the total height, leaf number, pigments (chlorophylls and anthocyanins) and RA content of both cultivars were determined in plant tissue at the end of the culture period (4 weeks).
DETERMINATIONS
For estimating CO2 and ethylene concentration in the head-space of the culture
vessels gas sampling were analyzed on the first day of sub-culture and 14, 21 and 28 days later. Three 2-ml air samples were consecutively withdrawn with hypodermic syringe from three replicates, each consisting of an individual vessel. Samples were collected just before the beginning of the light period and 1, 3, 6 and 8 h later. Ethylene concentration was determined only in the air samples collected at 8 h.
Ethylene and CO2 concentrations were measured using an HP 5890 gas
chromatograph (Hewlett Packard, Milano, Italy) equipped with a stainless steel column (1.5 m length and 0.04 m i.d.) packed with HaySep® T (Agilent Technologies, Milan, Italy), a flame ionization detector (ethylene determination) and a thermal conductivity detector (CO2 determination). Column and detector
temperatures were 70 and 350 °C (ethylene analysis) or 70 and 200 °C (CO2
analysis). Nitrogen was used as a carrier gas at a flow rate of 30 cm3 min-1 and as reference gas at 15 cm3 min-1 for TCD detector.
Net photosynthetic rate (PN) (μmol g-1 s-1 DW) was calculated as reported by
Fujiwara et al. (1987) and expressed on the basis of total shoot dry biomass (DW) in the vessel:
PN = (CO2in - CO2out)t × E ×V / DW
where (CO2in - CO2out)t is the difference between CO2 concentration inside and
number of gas exchanges of the vessel, respectively (Table 5.1).
Total content of chlorophylls and anthocyanins were determined spectrophotometrically in shoot samples that were extracted overnight at 4°C in the dark using, respectively, ethanol 95% (v/v) and methanol 80% (v/v) containing HCl 1.2 M (Kiferle et al., 2011). Pigments concentration was calcultated on a FW basis; anthocyanins were expressed as cyanidin-3-glucoside equivalents.
For RA analysis, shoot samples were rapidly washed in tap water, rinsed in deionised water and gently dried with a towel; each sample consisted of one or two shoots removed from the same vessel in order to reach approximately 0.5 g FW. Samples were frozen in liquid nitrogen and stored at -80 °C before laboratory analysis, which were performed within a few weeks after sampling. The content of RA in MeOH:H2O:HCl (70:29:1 v/v) extracts was determined by means HPLC and
expressed per gram dry weight (DW) on the basis of the dry matter content determined in an aliquot of each sample after desiccation in a ventilated oven at 85°C. In addition to RA, the content of others caffeic acid derivatives (caffeic acid, caftaric acid, chlorogenic acid, cicoric acid, cynarin, ferulic acid, t-cinnamic acid, p-coumaric acid) was determined in the plantlets tissues. Peak identification was accomplished by LC-MS and LC-MS-MS, as previously reported (Kiferle et al., 2011). The detection limit of the analytical method was 0.05 mg g-1 DW.
STATISTICAL ANALYSIS
A completely randomized design was adopted. Data were subjected to two-way analysis of variance (ANOVA) using the Statgraphics Centurion XV.II (Manugistic Co., Rockville, Maryland, U.S.A.) software. The experiment was repeated twice with similar findings; the paper reports the results from a representative run.
Table 5.1. Main characteristics of the culture vessels and growing media used for in vitro culture of nodal explants of sweet basil
(Ocimum basilicum L., cv. Genovese and cv. Dark Opal). The number of explants per vessel and the air exchange rate (E, as determined using ethylene as tracer gas) are also shown. The headspace volume was calculated as the remaining vessel volume excluding the culture medium.
Vessel type Company Material Size (mm)
Vessel volume (ml) Medium volume (ml) Headspace volume (ml) Medium state Number of explants per vessel E (h-1)
PPCV25™ TQPL Co., New Milton, UK PP Ø:90 H:70 141.7 50.0 91.7 agar 6 4.2 SE
Magenta™ Sigma-Aldrich, IT PC L 77 W 77 H 97 575.1 50.0 525.1 agar 6 0.7 SE
MicroboxEco2™ Ducefha, Micropoli, IT PP L125 W:65 H:80 650.0 100.0 450.0 agar 12 7.1 SE
RITA™ Vitropic France PSU Ø180 H:150 980.0 150.0 830.0 liquid 12 2.0 SE
Growtek™ Scienceware NJ, USA PC+PP Ø113 H:160 1178.0 150.0 1028.0 liquid 12 1.0 SE
PSU: polysulfonate; PP: polypropylene; PC: polycarbonate; Ø: diameter; L: length; W: width; H: height; E: hourly number of gas exchanges of the vessels.
5.3. RESULTS
PHOTOSYNTHETIC ACTIVITY
The explants of both cultivars showed different trends in photosynthetic activity (PN)
in relation to the cultivation vessel used for their micropropagation (Fig. 5.2, A-B). For the sake of clarity, the results for the agarized media (Fig. 5.2, A) are presented separately from those found in the bioreactors (Fig. 5.2, B).
When grown in the liquid medium, the explants of both cultivars did not shown any appreciable photosynthesis whereas the plantlets cultured in agarized medium were able to assimilate CO2 (Fig5 5.2, A). In both cultivars, the CO2 production was
generally higher in RITATM than in GrowtechTM (Fig. 5.2, B).
When cultured in agarized medium, Genovese plantlets acquired photosynthetic competence earlier than Dark Opal, as a positive PN was observed in the former
cultivar already at day 14 when PN was negative in the latter (Fig. 5.2, A); in both
genotypes, the highest PN was recorded at day 21.
At least six hours of lighting were required to trigger the CO2 assimilation in Dark
Opal (Fig. 5.2, A), while positive PN was observed just at the first hour in the light
period in. Genovese (Fig. 5.2, A). The plantlets grown in the most ventilated vessel Microbox Eco2™) showed the highest PN in both cultivars (Fig. 5.2, A), whereas the
lowest PN was generally found in Magenta™, which was characterized by low E
(Fig. 5.2, A; Table 5.1).
ETHYLENE ACCUMULATION
The level of ethylene in the headspace of the culture vessels remained relatively constant during the experiment in both cultivars and regardless of the vessel type, apart from a large increase observed in RITATM (Fig. 5.3). In this vessel, with respect to the values detected at the beginning of the culture, ethylene concentration increased significantly already after 14 d in Genovese, while in the other cultivar a significant increase was found only at the end of the experiment (Fig. 5.3).
Figure 5.2. Net photosynthetic rate (PN) of the shoots of two cultivars of sweet basil
(Ocimum basilicum L., cv. Genovese and cv. Dark Opal) cultured in vitro using different types of vessels, which were filled either with agarized medium (A) or liquid medium (B). The values were determined 14, 21 and 28 days after the beginning of the sub-culture. Air samples were collected from individual vessel for CO2 analysis in the dark just before the beginning of the light period (T-0) and after
Figure 5.3. Time-course of the ethylene concentration in the headspace volume of different cultivation vessels used for in vitro culture of two cultivars of sweet basil (Ocimum basilicum L., cv. Genovese and cv. Dark Opal). Magenta™, Microbox Eco2™, PCCV25™ vessels were filled with agarized medium whereas RITA™ and Growtek™ bioreactors were employed for temporary and stationary immersion culture respectively. Means ± SE (n = 3).
GROWTH AND MORPHOLOGY
The type of vessel showed evident effects on growth parameters as measured at the end of culture (Table 5.2). The highest shoot length was observed in Magenta™ and RITA™ for Genovese, and in PPCV™ and Growtek™ for Dark Opal (Table 5.2).
Table 5.2. Growth parameters and pigments concentration in the shoots of two cultivars of sweet basil (Ocimum basilicum L., cv. Genovese and cv.
Dark Opal) cultured in vitro for 28 days using different types of vessel. Two-way ANOVA was performed (** P ≤ 0.01; *** P ≤ 0.001; n.s. = not significant) and mean values were separated using LSD test: values (n = 12) followed by different letters differ significantly (P ≤ 0.05).
Vessel type Medium
state
Shoot number
Shoot length
(cm) Leaf number Shoot FW (g)
Shoot DW (g) Chorophyll (mg g-1 FW) Anthocyanins (mg g-1 FW) cv. Genovese PPCV25™ agar 2.25 a 1.37 bd 12.3 ab 0.522 b 0.035 bc 1.33 a 0.04 c Magenta™ agar 2.33 a 2.00 ab 9.5 de 0.826 b 0.079 a 0.81 b 0.03 c M.boxEco2™ agar 1.92 c 0.87 d 9.5 de 0.442 b 0.035 bc 0.87 b 0.04 c RITA™ liquid 2.25 a 2.00 a 7.0 f 0.507 b 0.029 c 0.39 cd 0.02 c Growtek™ liquid 2.00 bc 1.28 cd 7.8 ef 1.212 a 0.079 a 0.25 cd 0.04 c cv. Dark Opal PPCV25™ agar 2.00 bc 2.04 a 14.3 a 0.697 b 0.053 b 1.60 a 0.95 a Magenta™ agar 2.00 ac 1.25 cd 11.3 bd 0.543 b 0.043 bc 0.57 bc 0.71 ab M.boxEco2™ agar 2.00 bc 1.37 bd 11.6 bc 0.673 b 0.050 bc 1.31 a 0.50 b RITA™ liquid 2.00 bc 1.39 bd 9.3 de 0.508 b 0.034 bc 0.36 cd 0.53 b
Growtek™ liquid 2.18 ab 1.56 ac 9.3de 1.458 a 0.083 a 0.10 d 0.07 c
Significance:
Cultivar: (A) n.s. n.s. ** n.s. n.s. n.s. ***
Vessel type: (B) n.s. *** *** *** *** *** **
In both cultivars the number of leaves tended to be smaller in the vessel with liquid medium (Table 5.2). Shoot number per plantlet was low in all growing conditions (it averaged 2.09) and was not affected by genotype and vessel type (Table 5.2).
Both genotypes showed a continued growth regardless of vessel type, as the FW and DW of single plantlet tended to increase till the end of cultivation with the exception of the DW of Dark Opal cultured in RITA (Fig. 5.4): in this vessel, DW did not differ significantly when the plantlets were sampled at day 21 and 28 (Fig. 5.4). In both genotypes the highest DW was observed in Growtek™ although in Genovese no significant differences were found between this vessel type and Magenta™ (Fig. 5.4; Tab. 5.2).
In general, at the end of the growing period the chlorophyll contents was significantly higher in the vessels with agarized medium (expecially in PPCV™ and Microbox Eco2™) than in those with liquid medium (Table 5.2). Dark Opal and Genovese contained similar amounts of chorophylls (Table 5.2). As expected, the content of anthocyanins in Dark Opal explants was much higher than the levels detected in Genovese irrespective of the vessel type (Table 5.2). The vessel type did not influence the content of these pigments in Genovese shoots. In contrast, in Dark Opal the use of bioreactors reduced considerably the content of anthocyanins as compared to the other types of vessel (Table 5.2), although the values determined in RITATM, MicroboxEco2™ and Magenta™ did not differ significantly. In the GrowtekTM system, anthocyanins appeared to accumulate in the callus that developed at the base of the explants (Fig. 5.5, F) which showed a purple color; no callus was observed in other vessels.
Generally, leaf morphology was not affected by vessel type in Genovese; the leaves maintained their oval shape and dark green color (Fig. 5.5, B-C). In contrast, the leaves of Dark Opal (Fig. 5.5, E-F) showed a tendency to greening in comparison to the plants grown in vivo (Fig. 5.5, D). Furthermore, some morphological alterations were evident when the plantlets were grown in GrowtechTM, as they lost the typical typical ruffled edge and showed hyperhydricity symptoms (Fig. 5.5, F).
Figure 5.4. Time-course of shoot dry weight (DW) and fresh (FW) of two cultivars of sweet basil (Ocimum basilicum L., cv. Genovese and cv. Dark Opal) cultured in vitro using different types of vessels. Magenta™, Microbox Eco2™, PCCV25™ vessels were filled with agarized medium whereas RITA™ and Growtek™ bioreactors were employed for temporary and stationary immersion culture respectively. Means ± SE (n = 12).
Figure 5.5. The aspect of two cultivar (Genovese, A; Dark Opal, D) of sweet basil (Ocimum basilicum L.) cultivated in vivo (hydroponic system). Nodal explants of the same genotypes at the end of the multiplication phase in different in vitro culture vessels: Genovese shoots grown in PPCV™ vessel (B) or RITA™ bioreactor (C); Dark Opal shoots grown in PPCV™ vessel (E) or Growtek™ bioreactor (F).
ROSMARINIC ACID CONTENT
At the end of the cultivation period, the level of RA was significantly higher in the shoots growing in RITA™ compared with other vessel types (Fig. 5.6): it was 154.8 and 100.2 mg g-1 DW in Genovese and Dark Opal, respectively, against average values of 43.4 and 44.4 mg g-1 DW in the other culture vessels (Fig. 5.6). The content of RA was not affected by the genotype.
In all the analyzed samples, among the CADs of interest RA was the only compound found in plant tissues at concentrations higher than the detection limit, as found previously (Kiferle et al., 2011).
Figure 5.6. Rosmarinic acid content (RA) in the shoots of two cultivars of sweet basil (Ocimum basilicum L., cv. Genovese and cv. Dark Opal) cultured in vitro using different types of vessels. The values were determined 28 days after the beginning of the sub-culture. Magenta™, Microbox Eco2™, PCCV25™ vessels were filled with agarized medium whereas RITA™ and Growtek™ bioreactors were employed for temporary and stationary immersion culture respectively. Two-way ANOVA was performed (*** P ≤ 0.001; n.s. = not significant) and mean values were separated using LSD test: values (n = 4) followed by different letters differ significantly (P ≤ 0.05).
5.4. DISCUSSION
The type of culture vessel could affect some growth parameters, such as shoot proliferation, elongation, fresh weight as well as the occurrence of hyperhydricity in micropropagated plants (Kavanagh et al., 1991; Hazarika, 2006; Kaçar et al., 2010; Xiao and Kozai, 2011). The effect of the vessel type was evident for all the morphologic features, except the shoot number. As reported in a previous work (Kiferle et al., 2011), the proliferation aptitude of the explants in terms of number and the length of new-formed shoots was lower in comparison with previous findings in sweet basil (e.g. Sahoo et al., 1997; Begum et al. 2002). Nevertheless, the adopted micropropagation protocol allowed to obtain large biomass production, which was up to 80 mg DW per plantlet at the end of the multiplication phase in Magenta™ and Growtek™. This value exceeded by far those reported by Kintzios et al. (2004), which produced about 0.13 mg DW/plantlet in bioreactor nodal cultivation of this species.
Although the use of Growtek™ stimulated growth of both cultivars, it caused some morphological alterations in Dark Opal, such as the loss of pigments, the modification of leaf shape and the occurrence of hyperhydricity. In vitro plants grown in a very artificial environment and frequently show morphological differences with respect to in vivo plants (Majada et al., 2000).
Plantlets may achieve autotrophic growth under favorable in vitro conditions (Kozai et al. 2000; Xiao and Kozai, 2011) and many papers report that higher growth values were obtained when vessel E was increased in order to stimulate photoautotrophic or mixotrophic growth (e.g. Lucchesini et al. 2001; Afreen et al. 2002). In this work, the highest chlorophyll content and photosynthetic rate were associated with the highest E of the vessels with agarized medium (Microbox Eco2™ and PPCV™), which were specifically designed to improve aeration. A positive correlation between the degree of aeration in the culture vessels and the photosynthetic pigment concentration was hypothesized by Pospíšilová et al. (2000).
and their photosynthetic competence and/or the ventilation rate of the culture vessel, on other side. In fact, the largest dry biomass accumulation was observed in Growetek™ (fully heterotrohic growth; E = 1.0 h-1) and in Magenta™ (low photosynthesis; E = 0.7 h-1).
In many cases, heterotrophic and mixotrophic conditions could yield better results than photoautotrophic conditions (Hazarika, 2006; Lucchesini et al. 2006). We may speculate that in Magenta™ vessel, which had a low E, the reduction of explants photosynthetic activity, as compared with other agarized systems, allowed a better utilization of exogenous carbohydrates, resulting in larger biomass accumulation. This result are in accord with those reported by Chanemougasoundharam et al. (2004) who cultured potato plantlets in tubes equipped with hermetic or non-hermetic closure types; he found that the systems less permeable to gas exchanges stimulated shoot growth.
Liquid culture systems are generally characterized by a heterotrophic behavior (Jackson, 2003). These systems facilitate the nutrient uptake by the plantlets in comparison with agarized systems (Dey, 2005; Afreen et al, 2002) and it is known that higher carbohydrates availability can decrease the photosynthetic ability of micro-propagated plantlets (Kozai, 1991). Moreover, the temporary or constant submersion of the plantlets with the culture medium in RITA™ and Growtek™ could have altered stomata functionality, thus affecting negatively their photosynthetic capacity, as reported by Afreen et al. (2002).
Both FW and DW of the explants grown in the Growtek™ system were higher in comparison with other vessels, probably because in this bioreactor the plantlets were partially submerged by the medium, thus allowing a constant feeding supply.
Dey (2005) reported that Growtek™ bioreactor facilitates nutrient uptake by the explants due to the presence of a floating explant holder. Moreover, the high availability of nutrients can explain the reduction of the anthocyanins content (greening) of the explants cultured in Growtek™ system, as the red pigments synthesis is inversely correlated to the sugar and mineral availability (Steyn et al.
2002). Moreover, the explants developed in Growtek™ showed hyperhydricity symptoms, in particular in Dark Opal shoots, as confirmed by higher FW/DW ratio compared to other vessels (data not shown). Hyperhydricity occurs more frequently in liquid than agarized media (Hazarika, 2006; Baggio Savio et al., 2012) and may inhibit photosynthesis (Chakrabarty et al., 2005).
The level of ethylene concentration in the vessel headspace did not change substantially during the in vitro culture in all vessels, apart from the RITA™ system. In this kind of vessel, ethylene concentration increased with time in both genotypes. As the production of this hormone from abiotic sources (e.g. gelling agent, plastic material etc.) was excluded in a preliminary experiment, ethylene build-up in the RITATM system was ascribed to genuine release from plant tissues. Different reasons can explain greater ethylene synthesis in RITATM-cultured sweet basil shoots.
Firstly, the higher CO2 concentrations in the RITATM system with respect to other
containers (data not shown) might have promoted the synthesis of ethylene. Although CO2 is an antagonist of ethylene action and retards ethylene-mediated
responses, it was found to increase ethylene production in many plant systems by affecting the step of ACC conversion to ethylene (Philosoph-Hadas et al., 1986). Secondly, ethylene can be synthesized in response to stress conditions (Morgan and Drew, 1997). The brief temporary immersion adopted in our study for the RITATM system may have induced a nutrient starvation and elicited a stress response.
Ethylene accumulation in the RITATM system vessel was associated to lower accumulation of dry biomass, especially in Genovese cultivar, and to increased RA content in shoot tissues at the end of culture. Ethylene may either stimulate or inhibit growth (Biddington, 1992) and secondary metabolism in various plant tissue and cell cultures: for instance, ethylene was found to promote the production of alkaloids in cell suspension cultures of Coffea arabica, Thalictrum rugosum (Cho et al., 1988) and Thalictrum minus L. (Kobayashi et al., 1991 a, b). In contrast, ethylene decreased the production of L-DOPA (L-3,4-dihydroxyphenylalanine), in Stizolobium hassjoo hairy roots (Sung and Huang, 2000) and the accumulation of
paclitaxel in cell cultures of Taxus cuspidate (Linden et al., 2001).
It is known that the nutritional mode (photoautotrophy, photomixotrophy or heterotrophy) of the explants could influence growth, morphology and secondary metabolism of in vitro cultured plants (Ikemeyer and Barz, 1989; Lucchesini and Mensuali-Sodi, 2010). The results obtained with Magenta™, Microbox Eco2™, PCCV25™ and Growtek™ system suggests that photomixotrophic and heterotrophic conditions influenced explants growth, without any significant effect on RA production however. Therefore, we hypothesized that lower growth and higher RA content in the explants cultured in the RITA™ system were due to the nutrient starvation and the large accumulation of the ethylene released from plant tissues and entrapped in the vessel, which was scarcely ventilated (E = 2.0 h-1).
In agreement with previous work (Kiferle et al., 2011), the levels of RA detected in sweet basil shoots were relatively high (31.8 to 154.8 mg g-1 DW) regardless of vessel type and cultivar, as they exceeded by far the concentrations reported in the literature for in vitro-grown sweet basil. For instance, the RA content of sweet basil nodal explants grown in bioreactor was approximately 0.18 mg g−1 dry DW (Kintzios et al., 2004). Much higher RA content was found in suspension cultures (10 mg g−1 dry DW; Kintzios et al., 2003) or immobilized cells (20 mg g−1 dry DW; Moschopolou and Kintzios, 2011).
In conclusion, both growth and RA production of nodal explants of sweet basil culture in vitro were affected by the type of vessel, which influenced the gaseous composition of internal atmosphere and the degree of photosynthetic capacity developed by the explants during the culture. The largest amount of dry biomass was produced under the heterotrophic conditions provided by GrowtekTM, although some morphological alterations occurred in the explants cultured in this system. The highest RA content in shoot tissues was recorded in the RITATM system and this results was associated to higher ethylene accumulation in the vessel headspace as compared to other culture systems. Regardless of vessel type, the in vitro culture of nodal explants developed in this work proved to be a more suitable system for RA
production from sweet basil than those reported in the literature (Kintzios et al., 2004; Kintzios et al., 2003; Moschopolou and Kintzios, 2011).
5.5 LITERATURE
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