Plant Biosystems, Vol. 141, No. 1, March 2007, pp. 75 – 81
Growth, yield and reproduction of dwarf tomato grown under simulated microgravity conditions
G. COLLA1, Y. ROUPHAEL1, M. CARDARELLI1, A. MAZZUCATO2 & I. OLIMPIERI2 1
Dipartimento di Geologia e Ingegneria Meccanica, Naturalistica e Idraulica per il Territorio (GEMINI), and 2Dipartimento di Agrobiologia ed Agrochimica (DABAC), Universita` della Tuscia, 01100 Viterbo, Italy
Abstract A closed hydroponic system combined with a horizontal uniaxial clinostat has been used to grow tomato plants (Solanum lycopersicum L.) under simulated microgravity conditions. The study was carried out to evaluate the quanti-qualitative traits (growth, yield and quality) of the dwarf tomato variety ‘Micro-Tom’ grown under simulated microgravity conditions and to determine if tomato plants would complete their life cycle (‘seed-to-seed’). Morphological and growth characteristics of ‘Micro-Tom’ were modified during clinorotation treatment. The ‘Micro-Tom’ plants grown under simulated microgravity exhibited a spreading growth and an increasing of the internode length. Total fruit yield, small fruit yield, leaf area, leaf dry weight, fruit dry weight, total dry weight and shoot – root ratio were lower in the clinorotated tomato plants than those grown in the control treatment. Foliar amount of carotenoids, and chlorophyll a and b were also substantially reduced under simulated microgravity conditions. Quality parameters (total soluble solids and fruit dry matter) of tomato plants were also negatively affected by clinorotation. The number of flowers per plant was increased by 32% in clinorotated plants versus controls. Fruit setting was reduced by 46% under clinorotation, while no significant difference was recorded for the pollen fertility and the seed number in small and large fruits. Clinorotation-exposed and control seeds were used in a germination trial in order to evaluate whether the seeds so formed were viable and if subsequent generations might be obtained in microgravity. Seeds formed under simulated microgravity proved to be biologically and functionally complete (germination ¼ 78.6%) showing that ‘Micro-Tom’ plants could realize complete ontogenesis, from seed to seed in microgravity.
Key words: Clinorotation, hydroponics, reproduction, seed, Solanum lycopersicum L.
Introduction Plants have been proposed as the basis for a Biological Life Support System that could be used alone or in concert with physical/chemical life support systems to provide food, atmospheric purification and water regeneration for crew members on long-duration space-flight missions. Therefore, it is of great interest to study the influence of microgravity on plant growth, development, and reproduction. Biological experiments under real microgravity conditions can be obtained only in sounding rockets or aboard the space fight and platforms, but the high costs of the missions and the few chances available tend to cause delays in the achievement of the results. On earth, the most usual technique employed to simulate microgravity in
plant experiments is clinorotation which consists of rotating plants at low angular speeds, aiming to disorient them similarly to what occurs under real microgravity (Kraft et al., 2000). Clinorotation has been widely used for in vitro and seedling studies (Paolicchi et al., 2002; Aronne et al., 2003). However, information is still lacking on growing plants under clinorotation due to the difficulties of supplying adequate nutrients and water to the root apparatus up to advanced phenological stages. Hydroponic systems that release the nutrient solution through microporous materials (Wright et al., 1988; Koontz et al., 1990; Steinberg et al., 2000) could be the most suitable systems for growing plants under simulated microgravity conditions. Many plant growth processes such as germination and seedling growth have been reported to proceed
Correspondence: G. Colla, Dipartimento di Geologia e Ingegneria Meccanica, Naturalistica e Idraulica per il Territorio (GEMINI), Universita` della Tuscia, Via S. De Lellis snc 01100 Viterbo (Italy). Tel.: þ39 (0)761 357536. Fax: þ39 (0)761 357531. E-mail:
[email protected] ISSN 1126-3504 print/ISSN 1724-5575 online ª 2007 Societa` Botanica Italiana DOI: 10.1080/11263500601153735
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normally for a short period under microgravity, but following this, plants exhibit a slowing or cessation of growth and frequently die (Halstead & Dutcher, 1987). Furthermore, studies have reported the failure of plants – Arabidopsis (Musgrave et al., 1997) and wheat (Strickland et al., 1997) – to complete their life cycle (‘seed-to-seed’) during long-term exposure to space flight. However, it was not clear whether the failure in seed production was due to microgravity effects or to inadequate control of other environmental conditions (light, humidity, CO2, ethylene) during the space-flight experiments. These facts suggest that detailed studies on the effects of microgravity on the complete life cycle of plants in controlled ground-based experiments are required. Starting from the above considerations, the aims of the current study were (i) to evaluate the quantiqualitative traits (growth, yield and quality) of a dwarf tomato variety (‘Micro-Tom’) grown under simulated microgravity conditions and (ii) to determine if tomato plants would complete their life cycle (‘seed-to-seed’) in microgravity.
Materials and methods Clinorotation system and growth conditions A uniaxial clinostat was used to simulate microgravity conditions. The rotatory movement was generated by an asynchronous single-phase motor plus planetary reduction unit with electronic velocity
regulator (0.4 – 7.0 rpm) (Mini Motor, Italy). Rotation rate was 1.5 rotations/min. The movement was transmitted through a roller chain to six 50-cm long growth modules supported by an aluminium structure and placed within a growth chamber. The growth chamber was programmed to maintain a 14:10 h light-dark cycle with corresponding 26/168C temperatures and 65% relative humidity. Average photosynthetic photon flux at canopy level was of 400 mmol/m2 s. A hydroponic porous approach, initially proposed by Dreschel & Sager (1989) and known as Porous Tube Plant Nutrient Delivery System, has been adopted in the current experiment due to the tubular shape and its ability to maintain the water content in the substrate, which is necessary for the application of clinorotatory movement. The growth module (GM) consists in a hydrophilic microporous polyethylene tube (Porex Technologies, Germany) with 8 – 20 mm of pore size, 38.1 mm external diameter and 6.1 mm of wall thickness inside a 6-cm internal diameter solid polyvinyl chloride (PVC) pipe. The external PVC tube of each growing unit had a row of nine holes (Ø 1.2 cm) 5 cm apart, in which were placed small rockwool capsules where the seeds were sown (Figure 1). The design created an almost totally enclosed root zone between the outer PVC shell and the inner microporous tube. The root zone was completely filled with perlite (Ø 1 – 2 mm) having the following physical and chemical properties: bulk density 105 kg/m3, pH 6.5, electrical
Figure 1. Cross section of the growth-module unit.
Clinorotation effect on dwarf-tomato production conductivity (EC) 0.03 dS/m, cation exchange capacity (CEC) 0.87 meq/100 g, and easily available water 13% v/v. The nutrient solution flowed through the microporous tubes at 180 cm3/min under slight negative pressure (70.59 kPa), which was maintained by use of a siphon. The siphon adopted was previously described by Tibbitts et al. (1995). Negative pressure inside the microporous tube was in the optimal range (70.5 to 71.5 kPa) as reported by previous studies (Dreschel & Sager, 1989; Steinberg & Henninger, 1997). Through capillary action the nutrient solution fills the entire tube porosity wetting the substrate where the plants developed their root apparatus. Because the system was operated in a siphon mode, it was extremely sensitive to air bubbles breaking the suction. We connected the porous tubes in series so that any air bubbles entering the system were pushed through the tubes and were unlikely to collect in the corners of a manifold. In addition, a positive pressure inside the system was generated daily to eliminate any air bubbles. During the experiment, the nutrient solution pressure within the tubes was daily monitored and adjusted using piezometers located at the beginning and the end of the porous tubes. Plant material, data collection and analysis ‘Micro-Tom’ tomato (Solanum lycopersicum L.) was selected as the experimental crop, because of its small size (miniature-dwarf-determinate cultivar), and short life cycle that make it ideal for groundbased and space experiments. Two treatments (presence and absence of clinorotation) were compared in a randomized complete block design with three replicates. There were six GMs (two clinorotation treatments 6 three replicates) each of them representing an experimental unit containing five plants. Seeds were sown directly on the rockwool capsules of the GMs on 15 April 2004. Three seeds per holes were sown and at cotyledonal stage seedlings were thinned out leaving one plant per hole. Plants were fertilized with the following complete nutrient solution (mg/l): N-NO3 (196), N-NH4 (14), S-SO4 (30), P-H2PO4 (31), K (234), Ca (160), and Mg (48), Fe (0.6), Mn (0.5), B (0.5), Cu (0.02), Zn (0.05), and Mo (0.01). The EC of the solution was 1.8 dS/m and the pH was 6.0. To prevent large fluctuations in EC, pH and ionic concentrations, a relatively high volume of nutrient solution per plant (3 l) was recirculated in all treatments. At flowering, stems were vibrated by a mechanical vibrator in order to facilitate self-pollination. Vibrations were applied daily, between 9:00 and 10:00 AM, with the same intensity for 10 s until all the flowers were open.
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During the growing cycle, the number of flowers on each plant was counted. Flowers spanning the stages from meiosis to open mature flower were collected from one plant for each plot (which was not taken into consideration for further measurements and analysis, i.e. fruit yield and grade, dry weight biomass and partitioning), fixed in FAA (10% commercial formaldehyde, 50% ethanol, 5% acetic acid) for 12 h and then stored in 70% ethanol at 48C until used. To observe the occurrence of male and female meiosis, flower buds measuring respectively 3 and 4 – 5 mm in length were used. The generative tissue was dissected from anthers and ovaries under a stereomicroscope and stained for callose with a drop of 0.005% (w/v) aniline blue solution in 0.15 M sodium phosphate buffer, pH 9.5. Specimens were observed under a fluorescence microscope as previously described (Mazzucato et al., 1998). To determine the percentage of stainable pollen, one flower from each of the clinorotated and control plants were used. One anther per flower was dissected, squashed on a microscope slide and the released pollen grains stained in a drop of 1% acetic orcein and 50% glacial acetic acid solution. Stainable pollen was calculated as the percentage of plump, purple pollen grains appearing in a sample of at least 200 grains. At maturity, red fruits were harvested and directly graded by calipers into two categories: small (Ø 5 15 mm) and large (Ø 4 15 mm) and the fresh weight and number of each category were recorded. The number of seeds in small and large fruits was also counted. Fruit setting was calculated as the percentage of total fruit number relative to the number of flowers. Fruits were dried in a forced-air oven at 808C for 72 h and weighed to determine dry matter (DM). Fifteen representative fruits of each plot (GM) were analysed for fruit quality parameters. From the liquid extract obtained from liquefying and filtering the fruit, total soluble solids (TSS) contents in juice was determined by an Atago N1 refractometer (Atago Co. Ltd., Japan) and expressed as 8Brix at 208C. Chlorophyll content of the leaves was determined spectrophotometrically after extraction of the fresh plant material with dimethylsulfoxide, as described by Blanke (1992). The absorption of the extracts was measured at 663 and 645 nm for chlorophyll a and b, respectively. Total leaf carotenoid content was measured through a spectrophotometer using the procedure described by Lichtenthaler & Wellburn (1983). At the end of the growing cycle (28 July 2004), the leaf area of each plant was measured using an electronic area meter (Delta-T Devices Ltd, Cambridge, UK). Each plant was separated into roots, leaves, and stems. Roots were carefully rinsed under water to remove as much substrate as possible. All plant organs were then
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dried at 808C for 72 h in a forced-air oven and then weighed. A germination trial was performed using the seeds collected from clinorotated and control plants in order to evaluate whether the seeds were viable and if a subsequent generation may be obtained in microgravity. Seeds collected from clinorotated plants were germinated under simulated microgravity conditions, while seeds obtained by control plants were germinated in the absence of clinorotation. Seeds were surface sterilized with a solution containing 80% of ethanol. After sterilization (10 min), the seeds were washed twice with sterile distilled water. Immediately after sterilization, seeds were placed in Petri dishes (Ø 10 cm) on filter paper wetted with distilled water. Each Petri dish contained 50 seeds. Petri dishes were then closed with Parafilm to maintain adequate environmental conditions for seed germination (moisture, sterility). Three Petri dishes were mounted on each GM of the clinostat previously described. There were three GM rotating with a total of nine Petri dishes containing seeds coming from the clinorotated plants grown in the previous cycle and three GM in stationary conditions with a total of nine Petri dishes containing seeds coming from the control plants grown in the previous cycle. Percentage germination was calculated based on the total number of germinated seeds per treatment after 14 days of incubation. The mean germination time (MGT) was calculated according to the following formula: MGT ¼
X
ni di =n
where, n is the total number of germinated seeds during the germination test, ni is the number of germinated seeds on day di and i is the number of days during the germination period. Statistical analysis of data was based on analysis of variance and data presented in percentages were subjected to arcsine transformation before analysis, and then converted back to percentage for presentation.
Results and discussion The total and small fruit fresh yield of tomato plants was significantly affected by clinorotation, while no significant difference was recorded on the large fruit yield (Table I). The lowest total yield observed under simulated microgravity was thus related to a reduction in the yield of small fruits, and not to that of large fruits. Total yield, small fruit yield, total and small fruit number were significantly reduced by 20, 52, 29 and 54%, respectively in the clinorotated plants in comparison with control treatments (Table I). Morphological and growth characteristics of ‘Micro-Tom’ were modified during clinorotation treatment. The ‘Micro-Tom’ plants grown under simulated microgravity exhibited a spreading growth and an increasing of internode length (data not shown). Leaf, fruit and total dry weight of tomato plants grown under simulated microgravity were significantly (p 0.01) reduced by 29, 28 and 10%, respectively than those grown in the absence of clinorotation (Figure 2). An opposite trend was observed for stems and roots dry weight (p 0.01). Shoot – root ratio was significantly (p 0.01) lower in clinorotated plants in comparison to that observed in plants of control treatment (7.05 and 9.91, respectively). Moreover, the effects of clinorotation on leaf area followed a similar trend to leaf dry weight, with significantly (p 0.01) lower values (170 cm2/plant) recorded on plants grown in the presence of clinorotation in comparison to those grown in the absence of clinorotation (219 cm2/ plant). Finally the fruit quality parameter: TSS content and fruit DM were significantly (p 0.05) lower in clinorotated plants (4.9 8Brix and 6.0%, respectively) than those grown in the absence of clinorotation (5.6 8Brix and 6.7%, respectively). The lower rates of growth and yield were probably caused by an impaired distribution of photosynthetic pigments in the ‘Micro-Tom’ leaves, with lower values recorded under simulated microgravity (Table II). It was already found that the spaceflight exposures reduced the carotenoid content and the levels of chlorophyll a and b without affecting their
Table I. Effect of clinorotation on fruit yield and yield components of ‘Micro-Tom’ tomato plants. Yield Small fruit (Ø 5 15 mm) Clinorotation Absence Presence Significance
Large fruit (Ø 4 15 mm)
Total
Fresh wt (g/plant)
Number (no/plant)
Fresh wt (g/plant)
Number (no/plant)
Fresh wt (g/plant)
Number (no/plant)
15.9 7.6 **
13.9 6.2 **
37.1 34.9 NS
8.6 9.6 NS
53.1 42.5 **
22.5 15.9 **
NS: non significant; **significant at p 0.01.
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Clinorotation effect on dwarf-tomato production ratio (Mashinsky et al., 1991, 1994). This evidence suggests that the photosynthetic inhibition is caused by destruction of the photosynthetic system. The results recorded in our experiment are in agreement with those reported by Miyamoto et al. (1995, 2001) who observed that simulated microgravity conditions enhanced the loss of chlorophyll in oat leaf segments due to alterations of the physiological process of leaf senescence (e.g. protease activity) and the dynamics of endogenous plant hormone levels (e.g. ABA). Overall the data indicated that the clinorotated plants have photosynthetized less than the control ones as observed by Tripathy et al. (1996) under real microgravity conditions. Nevertheless, the reduction of assimilation is clearly noticed from the lower
Figure 2. Effects of clinorotation on biomass production and partitioning of ‘Micro-Tom’ tomato plants. Vertical bars indicate + SE of means.
Table II. Effect of clinorotation on foliar amount of chlorophyll a, b (mg/g f.w.), and carotenoids (mg/g f.w.) of ‘Micro-Tom’ tomato plants.
Clinorotation Absence Presence Significance
Chlorophyll Chlorophyll Chlorophyll a b aþb Carotenoids 1.05 0.75 **
0.31 0.22 **
1.35 0.97 **
0.51 0.35 **
**Significant at p 0.01.
content of fruit TSS, a parameter directly connected to the photosynthetic activity. Under simulated microgravity conditions, ‘MicroTom’ plants developed normal flowers. However, the number of flowers per plant was significantly increased by 32% in clinorotated plants in comparison to those grown in absence of clinorotation (Table III). Such a phenotype has often been associated with altered auxin dynamics (Al-Hammadi et al., 2003; Mezzetti et al., 2004), which are a well-known effect of microgravity on plant growth (Miyamoto et al., 1999). Microscopic observations of clinorotated flowers at different stages did not reveal significant anomalies in the progression of male and female meiosis and on the formation of gametophytes. Fruit setting was significantly reduced (by 46%) under simulated microgravity, while no significant difference was recorded on the pollen fertility and the seed number in small and large fruits (Table III). It has been reported that plant growth under microgravity is poor and plants frequently die in the transition from the vegetative to the reproductive stage (Halstead & Dutcher, 1987). Our observations indicate that normal development of generative organs can occur under microgravity conditions. Because no difficulties were observed on seed formation under simulated microgravity (Table III), the lowest fruit setting in clinorotation treatment may be due to a deficiency in reserve substances, suggesting a lack of energy supply during the reproductive stage, especially in the last part of the growing cycle when source strength decreased (assimilate supply) and sink strength increased (assimilate demand) (Marcelis & Heuvelink, 1999). Clinorotation-exposed and control seeds collected from tomato plants during the previous trial were used in a germination trial in order to evaluate whether the seeds so formed were viable and if subsequent generations could be obtained in microgravity. No significant difference between treatments was observed for the MGT (avg. 10.5 d), while the percentage of germination was significantly (p 0.05) affected by clinorotation with a higher value in the
Table III. Effect of clinorotation on the total number of flowers, pollen fertility, fruit set and fruit seed number of ‘Micro-Tom’ tomato plants. Seed number (no/fruit) Clinorotation Absence Presence Significance
Flower (no/plant)
Pollen fertility (%)
Fruit set (%)
Small fruits
Large fruits
33.9 44.7 *
95.1 91.6 NS
66.4 35.9 **
15.8 13.7 NS
34.6 32.7 NS
NS: non significant; *significant at p 0.05; **significant at p 0.01.
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control treatment (87.3%) in comparison to that recorded under simulated microgravity (78.6%). No morphological alterations to the epygeal part of ‘Micro-Tom’ seedlings were observed in simulated microgravity conditions as reported for maize seedlings grown in space (Ueda et al., 1999) and for bean grown under simulated microgravity (Aronne et al., 2003). However, seedlings obtained by the germination under simulated microgravity of seeds collected in clinorotated plants exhibited a higher percentage of tricots in comparison to those grown in absence of clinorotation (10.9 and 4.4%, respectively). The higher occurrence of tricots may be the consequence of an alteration of hormone levels in clinorotated seedlings coming from seed produced under simulated microgravity because this phenotype has been associated with altered dynamics of the polar auxin transport (Benjamins et al., 2001; Al-Hammadi et al., 2003). Roots of tomato seedlings were also observed in aerial space of the Petri dishes. Thus, the seeds formed under simulated microgravity proved to be biologically and functionally complete, showing that ‘Micro-Tom’ plants could realize complete ontogenesis, from seed to seed in microgravity. This result is in agreement with the recent conclusion that none of the processes involved in plant sexual reproduction is dependent on gravity per se for its completion, but instead that the achievement of a ‘seed to seed’ life cycle in spaceflight depends on the proper management of the environmental constraints imposed by microgravity (Musgrave & Kuang, 2003). In addition, because previous studies of reproduction in microgravity were mainly based on species with dry fruits (Arabidopsis, Brassica, Triticum), the present report indicates that the set, development and ripening of a fleshy fruit like tomato is also possible, thus indicating new perspectives for the dietary and psychological needs of crew members in orbital flights or platforms. Acknowledgements This work was partially financed by the Italian Space Agency (ASI) within the projects ‘‘SpaceGreenHouse’’. We would like to thank Prof. Francesco Saccardo for his constructive comments to this paper. References Al-Hammadi ASA, Sreelakshmi Y, Negi S, Siddiqi I, Sharma R. 2003. The polycotyledon mutant of tomato shows enhanced polar auxin transport. Plant Physiol 133:113 – 125. Aronne G, De Micco V, De Pascale S, Ariaudo P. 2003. The effect of uni-axial clinostat rotation on germination and root anatomy of Phaseolus vulgaris L. Plant Biosys 137:155 – 162.
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