Even if widely adopted throughout the world, conventional photomixotrophic micropropagation systems are inefficient owing to high rates of mortality upon transfer of plantlets from in vitro to ex vitro conditions. Exogenous medium sugar is suggested to be the major cause of this problem. Classical biochemical and enzymological studies did not provide a satisfactory explanation to this phenomenon. To investigate the central role of sucrose supply on the regulation of plantlets physiology, we measured growth parameters and used a comprehensive nonbiased approach providing a thorough image of the metabolic profile of in vitro potato plantlets subjected to different tissue culture conditions consisting of Murashige and Skoog (MS) medium and without sucrose (photoautotrophic condition) or with 3% sucrose (photomixotrophic condition). Using GC-MS, a set of 51 differing metabolites were identified in leaf tissues during the rooting phase. Most growth parameters, such as shoot length, leaf fresh weight, leaf number, and leaf area/plant were significantly lower under photomixo- than under photoautotrophic conditions, and photosynthesis was inhibited due to partial stomatal closure under photomixotrophic conditions. Metabolomics methods and bioinformatics tools, such as Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA), revealed distinct metabolic signatures for the two treatments. Photoautotrophic leaves were characterised by the accumulation of urea and erythritol. Photomixotrophic leaves were characterized by the accumulation of metabolites belonging to the primary metabolism and catecholamines as well as compounds related to abiotic stress conditions such as proline, hydroxyproline, asparagine, GABA, soluble sugars and myo-inositol. Metabolomics provide comprehensive information on plant physiological status, demonstrating the usefulness of this technique as a diagnostic tool for the tissue culture conundrum.
Keywords Metabolomics. Gas chromatography-mass spectrometry. Principal Component Analysis. Hierarchical Cluster Analysis. Tissue culture.
Abbreviations
MS Murashige and Skoog (1962) medium
PAT Photoautotrophic
PMT Photomixotrophic
GC-MS Gas chromatography-mass spectrometry
HCA Hierarchical Cluster Analysis
PCA Principal Component Analysis
Introduction
Micropropagation is used extensively to rapidly propagate many plant species and special cultivars or accessions. Despite its potential, this technique has not lived to expectations and is still plagued with many problems. One of its principal limitations is the poor survival of plantlets once transferred from in vitro conditions to the natural uncontrolled environment (Pospisilova et al. 1999). These problems have restricted the application of this technology to the mass production of high-priced plants like fruit trees and some flower crops.
Plantlets growing in vitro are continuously exposed to atypical culture conditions, characterized by high relative humidity, poor ventilation, low level of light, low CO2 concentration during the photoperiod and presence of high concentrations of sugar, nitrogen and phytohormones in the culture medium. These nutritional and environmental conditions are responsible for the abnormal morphology, anatomy and physiology of in vitro grown plantlets (Desjardins 1995). In many cases, these aberrations hamper the acclimation to ex vitro conditions.
A major cause for the poor acclimatization success of in vitro plantlets is their low photosynthetic capacity, most probably provoked by the presence of sucrose in the culture medium (Hdider and Desjardins 1994). For instance, the activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) was reduced by adding sugar in the medium (Grout 1988). Furthermore, Premakumar et al. (2001) found that micropropagated oak and strawberry plantlets had a reduced quantity of both large and small Rubisco subunits. In avocado, Rubisco content was noticeably diminished in leaves of plantlets cultured with high sucrose supply (87.6 mM) and maximum photosynthetic rate was significantly decreased when plants grew in the presence of both high sucrose concentration and elevated CO2 (de la Viña et al. 1999). At the beginning of acclimatization stage, mixotrophic Rauvolfia tetraphylla plantlets were not photosynthetically active, but the photosynthetic capacity increased in newly formed leaves over time during ex vitro period (Faisal and Anis 2009). In addition, Xiao et al. (2010) showed that growth of tissue cultured plantlets can be improved by using the sugar-free media, and by elevating photosynthetic photon flux (PPF) and the CO2 concentration in the vessel in a culture system designated as photoautotrophic (PAT). PAT plants are defined as those that use CO2 as their only carbon source for growth and development (sugar-free medium) whereas photomixotrophic (PMT) plants use sucrose, but may satisfy some of their carbon requirements by photosynthetic fixation of CO2. For example, Kubota et al. (2001) reported that the dry weight of photoautotrophically cultured tomato plantlets (under high light, high CO2 concentration and higher number of air exchanges) was more than twice as large as that of plantlets cultured photomixotrophically (under low light intensity, low CO2 concentration, and low number of air exchanges). Additionally, Nguyen et al. (1999) found that, the fresh weight, shoot length, root length, leaf area and photosynthesis ability of coffee plantlets when cultured on Florialite with sugar-free medium and with high ventilation rate were greater than those cultured in MS agar medium with 20 g l−1 sucrose.
The presence of exogenous sugar in the culture medium may also cause osmotic stress to the plantlets. Javed and Ikram (2008) found that raising sucrose concentrations above a certain level caused osmotic stress in the tissue culture medium of two wheat genotypes (S-24 and MH-97). Plantlets accumulated osmotic compatible solutes such as proline and total soluble carbohydrates in greater amounts and grew much less. Finally, Desjardins et al. (2007) suggested that in vitro plantlets are continually intoxicated by high concentration of sucrose and nitrogen from the medium, and they have to adjust their metabolism in order to survive under these artificial stressful conditions.
Although the presence of sugar in the culture medium reduced photosynthesis and disturbed in vitro plantlets hardening (Deng and Donnelly 1993), the opposite effect has also been observed (Tichá et al. 1998). For instance, sugar appears to be an essential component of the medium for many species. In some cases, independent growth could not be achieved on a medium devoid of sugar. Wainwright and Scrace (1989) found that higher shoot length, fresh and dry weights were obtained in vivo when Ficus lyrata and Potentilla fruticosa plantlets were cultured in a liquid medium with 2 or 4% sucrose. Plantlets establishment success declined when sucrose was not used. In apple shoots without sucrose did not survive their transfer to greenhouse (Zimmerman 1983). Sucrose linearly increases the level of reducing sugars, starch and total chlorophyll in citrus plantlets (Hazarika et al. 2000). Moreover, Tichá et al. (1998) reported that plant growth, dry matter accumulation and total leaf area were higher under photomixotrophic than photoautotrophic conditions. Not only biomass accumulation was higher, but photosynthesis capacity was also positively affected by exogenous sucrose.
Until recently, the understanding of the causes of acclimatization problem was curbed by the lack of suitable analytical methods. Yet, to meaningfully study the global changes in metabolism, a comprehensive, high-throughput and unbiased analysis is required. Metabolite profiling using gas chromatography-mass spectrometry (GC-MS) was first successfully used in plant biology by Roessner et al. (2000). This approach is powerful and allowed to measure broad-scale metabolites changes between two culture conditions, in vitro and soil-grown potato tubers (Roessner et al. 2000), to characterize the response to different concentrations of nitrate in the culture solution (Okazaki et al. 2008) and to investigate developmental aspects of sink-to-source transition of quaking aspen leaf (Jeong et al. 2004). The technique has a very high sensitivity for detecting both genetic and environmental effects on biological systems (Fiehn 2002).
In this study, we used a GC-MS-based metabolite profiling approach to distinguish dynamic metabolic responses exhibited by fully expanded potato leaves developed under PAT or PMT conditions. This study of the leaf metabolome provided a global image of the physiological and biochemical status of the plantlets and documented compositional differences during this key stage of the tissue culture process. The aim of this work was thus to define a metabolic phenotype of two contrasting artificial growth conditions (PAT and PMT), an information that could then provide plant tissue-culturists with physiological biomarkers or signatures of a well adapted plantlets, leading to a high survival rate to the acclimatization conditions.
