Journal of Experimental Botany, Vol. 51, No. 348, pp. 1277-1287,
July 2000
© 2000 Oxford University Press
Original papers |
Developmental changes and water status in tulip bulbs during storage: visualization by NMR imaging
1 Department of Biomolecular Sciences, Wageningen University, Wageningen NMR Centre, Wageningen, The Netherlands
2 Department of Ornamental Horticulture, ARO, The Volcani Centre, Bet Dagan, Israel
3 Department of Chemical Services, MR Centre, Weizmann Institute of Science, Rehovot, Israel
Received 10 November 1999; Accepted 14 March 2000
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Abstract |
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Magnetic Resonance Imaging (MRI) and light and scanning electron microscopy (SEM) were used to follow time-dependent morphological changes and changes in water status of tulip bulbs (Tulipa gesneriana L., cv. ‘Apeldoorn’) during bulb storage for 12 weeks at 20 °C (non-chilled) or 4 °C (chilled) and after planting. MR images reflecting the water content, the relaxation times T1 and T2 (or their reciprocal values, the relaxation rates R1 and R2), and the apparent self-diffusion coefficient of water molecules (ADC), were obtained for intact bulbs. After planting, scape elongation and flowering occurred only in chilled bulbs, while elongation in non-chilled bulbs was retarded. Microscopic observations showed different structural components and high heterogeneity of the bulb tissues. MRI revealed the elongation of the flower bud during storage, which was significantly faster in the chilled bulbs. In addition, MRI demonstrated a redistribution of water between different bulb organs, as well as significant differences in the pattern of this redistribution between the chilled and non-chilled bulbs. Generally, R2 relaxation rates became faster in all bulb organs during storage. At the same time, ADC values remained constant in the chilled bulbs, while exhibiting a significant increase in the non-chilled bulbs.
Key words: Tulip bulb, floral development, storage, temperature treatment, geophytes, MRI, SEM.
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Introduction |
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Evolution of geophytes in climatic areas with marked seasonal changes has led to their adaptation to the periods of high and low temperatures and/or drought. One of these adaptations is a state of rest (dormancy), in which bulbs do not exhibit any visible external growth (De Hertogh and Le Nard, 1993
). In many bulbs, the processes of organogenesis inside the bulb during the rest period, growth and flowering are temperature-dependent (Hartsema, 1961). In tulip, low temperatures simultaneously induce stem elongation of the current year and formation of the lateral buds for the next year (Le Nard and Cohat, 1968). Many morphological and biochemical changes are known as initial responses to cold induction in flower bulbs (Le Nard and De Hertogh, 1993). However, none of the known parameters correlates significantly with the completion of chilling and flowering ability (Banasik et al., 1980; Hobson and Davies, 1977; Lambrechts et al., 1994; Le Nard et al., 1988; Rebers et al., 1995; Walch and Van Hasselt, 1991).
In most geophytes, the initial response to cold treatment occurs within the storage organ. Therefore, research into these processes requires destructive measures, which prohibits the continuous developmental analysis of individual plants, and of post-planting response studies. This can be avoided by using Magnetic Resonance Imaging (MRI), which is a powerful, non-destructive tool for visualizing morphological structures in living organisms. MRI can also be used to measure characteristic magnetic resonance parameters, such as the relaxation times T1 (longitudinal or spin-lattice relaxation) and T2 (transverse, or spin-spin relaxation), and the apparent water self-diffusion coefficient, ADC, which provides information on water status at the cellular level, such as the permeability of the membranes between subcellular water compartments and the distribution of water over these compartments (Callaghan et al., 1994; Chudek and Hunter, 1997; Clark et al., 1997; Donker and Van As, 1999; Donker et al., 1997; MacFall and Van As, 1996; Ratcliffe, 1994; Ratkovic et al., 1982). The methodology for obtaining parameter-specific images and its application to different plant systems was presented in numerous previous reports (Callaghan et al., 1994; Donker and Van As, 1999; Edzes et al., 1998; Ishida et al., 1997; MacFall and Van As, 1996; Reinders et al., 1988). MRI has been used to study various phenomena in plant systems, such as water distribution in fruits, freezing damage in buds, the effect of cold treatment on alterations of water status in plant tissues, and water flow in plant root and vascular systems (Chudek and Hunter, 1997; Clark et al., 1997; MacFall and Van As, 1996; Ratcliffe, 1994). MRI was also applied to investigate the water status and the effect of cold storage in bulbs such as tulips (Iwaya-Inoue et al., 1996; Okubo et al., 1997) and Allium (Yamazaki et al., 1995; Zemah et al., 1999). These latter studies measured only a single NMR parameter, and (in the case of the tulip bulbs) only at a single time-point at the end of the storage process. In this study, a broad MRI survey of four parameters (R1 (1/T1), R2 (1/T2), proton density, ADC) was conducted on tulip bulbs using two different storage protocols, at five time-points during the 4 month storage, as well as after planting.
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Materials and methods |
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Plant material and temperature treatments
Bulbs of Tulipa gesneriana L., cv. Apeldoorn, 10/11 cm in circumference, were obtained from the Bulb Research Centre in Lisse, the Netherlands. One hundred mature bulbs were harvested in June 1998, and subsequently sorted, cleaned and dried prior to storage at 20 °C for 2 months. On 6 September, after the first MRI measurements, 50 bulbs were randomly picked and placed at 4 °C for 4 months (chilled bulbs). Fifty additional bulbs were kept at 20 °C (non-chilled bulbs). Sixteen intact bulbs per treatment were sampled, tagged and studied by MRI at Wageningen NMR Centre (Wageningen University, the Netherlands). MRI experiments were performed five times at 0, 2, 4, 8, and 12 weeks of storage and twice after planting (4 d and 10 d after planting).
After storage, 12 bulbs of each treatment were planted in 15 cm diameter pots (one bulb per pot). The pots were placed in the Phytotron in the Volcani Centre, Bet Dagan, Israel, 20/12 °C day/night, respectively, under a 10 h photoperiod. Plant growth analysis was performed from planting to the completion of flowering.
Bulb histology: microscopic observations
During storage, morphological and developmental analyses of bulbs were performed twice a month. For light microscopy, fresh material sections were stained with IKI (iodine in the KI solution) for starch and with Sudan red for lipids (Jensen, 1962). In addition, the material was fixed in FAA (5 : 5 : 90) a mixture of glacial acetic acid : formalin (40%) : ethanol (70%) and embedded in paraffin (Jensen, 1962). Paraffin sections, 12 µm thick, were stained for polysaccharides with Alcian Green Safranin (Joel, 1983).
Scanning electron microscopy (SEM) was performed by JEOL (Jsm-5410LV, Japan), at low vacuum.
MRI experiments
MRI experiments were conducted at field strength of 0.47 T, (20.35 MHz 1H frequency) on a system consisting of an electromagnet (Bruker, Karlsruhe, Germany) and a SMIS spectrometer (Surrey Medical Imaging Systems, Guilford, Surrey, UK). A custom-engineered coil/gradient set, containing a transmitter/receiver coil with a diameter of 50 mm and gradients in three directions with strengths up to 500 mT/m (Doty Scientific Inc., Colombia, South Carolina, USA) was used. The cylindrical probe bore had a diameter of 45 mm and was accessible from both ends. The tulip bulbs closely fitted the bore and were inserted into the magnet on top of a sample holder. The open structure of this magnet allowed the insertion and correct positioning of planted bulbs.
Images representing the proton density and the relaxation time T2 (or their reciprocal value, the relaxation rate, R2=1/T2)1 were obtained by use of a multi-echo imaging sequence described by Edzes et al. (Edzes et al., 1998). In this study 48 echoes were collected, with the first detected echo at 7.1 ms, and later inter-echo delays (IED) of 4.7 ms. Other acquisition parameters were: TR 1.0 or 1.5 s, FOV 45 mm, 128x128 matrix, slice thickness 3 mm, two averages.
The apparent diffusion coefficient (ADC) was obtained by using the pulsed field gradient (PFG) turbo-spin-echo sequence as described by Scheenen et al. (Scheenen et al., 1999): TR 1.5 s, first detected echo (and effective TE) at 29.3 ms, later inter-echo delays 4.6 ms, 
=4 ms, 
=20 ms, six diffusion gradient steps from 68 to 410 mT/m, bmax 3.61x109 s m-2, slice thickness 3 mm, FOV 45 mm, 128x128 matrix, four averages. In this sequence 16 phase-encoding steps were included in a single multi-echo scan, thereby reducing the acquisition time needed for a 128x128 matrix to only eight multi-echo scans. Image acquisition was repeated six times, with increasing diffusion weighting along the y-direction (parallel to the central axis of the bulb).
T1 relaxation (or their reciprocal value, the relaxation rate R1=1/T1) data were obtained using the turbo-spin-echo sequence with varying repetition times (TR 200, 350, 550, 900, 1500, 2500, and 5000 ms, first detected echo at 29.4 ms including a pulse train of nine 180° pulses with delays of 1.9 ms, and later inter-echo delays of 4.6 ms, slice thickness 3 mm, FOV 45 mm, 128x128 matrix, 8 averages). These data were only obtained at two time points, after 12 weeks of storage and after planting.
Three-dimensionally resolved images of the morphological structure of the planted bulbs were obtained using a turbo-spin-echo sequence with two phase-encoding gradients and the usual read gradient (FOV 60x60x45 mm3, TR 1 s, first detected echo at 6 ms, later inter-echo delays 5 ms, 128x128x128 matrix, four averages, total acquisition time about 1 h).
Data processing
The 48 echoes from the multi-echo experiments were Fourier transformed, and the absolute value images were fitted on a pixel by pixel basis to a mono-exponential decay function. This resulted in spin density images (from the calculated extrapolated intercept at TE=0), and images reflecting the R2 value at each pixel. R1 images and ADC images were generated by the appropriate fitting of the phase corrected real images from the corresponding experiments, assuming a single value of R1 or ADC for each pixel.
The parameters R1, R2, or ADC are ‘intrinsic’ parameters, meaning that their values should not depend, in principle, upon slight day-to-day (or in our case, week-to-week) fluctuations in the instruments technical performance. On the other hand, the calculated spin densities are arbitrary, computer-generated numbers, whose absolute values are meaningless. In order to enable a meaningful comparison of results between different bulbs and different examination dates, the reported spin density results were therefore normalized with respect to the highest signal intensity in the acquisition data of each individual bulb experiment (the centre of k-space). This value represents, to a good approximation, the total integrated water content of the bulb.
The parameter images were segmented by visual inspection into different regions of interest (ROI) encompassing the storage scales, the basal plate, and the flower bud. The mean parameter values in each ROI, as well as parameter histograms (number of pixels versus small range of parameter value) were calculated for each experiment. Average values of these mean values, and average histograms, were calculated for each treatment group. Student t-tests were used to test for significant changes in these averages over time, and between treatment groups.
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Results |
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Bulb morphology, development and histology
In August, before the storage period, bulbs were dissected and checked for the developmental stage. ‘Stage G’, indicating initiation of the gynoecium was found in all dissected bulbs. During subsequent bulb development, changes in size and shape of the storage scales, leaf and floral scape elongation, and the enlargement of the central daughter bulb were all detectable on MR images. MRI permits visualization of the basal plate, which consists of heterogeneous components and root primordia, the four storage scales, and the developing monocarpic shoot with several leaf primordia and the developing flower (Fig. 1
).
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Microscopic observations showed that the storage scales contained large cells about 125 µm in diameter (Fig. 2a
, d). In the central part of the scale, the starch granules of different sizes were heavily packed in the internal cell spaces (Fig. 2a, d, e). In the lateral, subepidermal layer of the scales, cells contained an amount of a gel-like substance, which was identified as polysaccharides by staining with Alcian Green Safranin (Fig. 2b, c). At the end of the storage period, in December, starch degradation was observed in the scales of both chilled and non-chilled bulbs (Fig. 2e).
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Root primordia were clearly visible in the basal plate in July-August, even before storage treatments (Fig. 3a
). Starch granules and oil drops were identified in the basal plate by staining with Sudan red (Fig. 3a). Different tissues were visible in the longitudinal section of the basal plate: large storage cells, small compressed cells and vascular bundles (Fig. 3b).
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During storage, the progressive development and elongation of the central flower bud was observed by bulb dissections and visible on MR images. MRI enabled a quantitative measurement of bud elongation. The average bud length was initially 8.2±0.3 mm (n=31) and then increased linearly during the first eight weeks of storage at 2.0±0.1 mm week-1 (n=15) for the chilled bulbs, but only 1.6±0.1 mm week-1 (n=15) for the non-chilled bulbs. After the first eight weeks, elongation became slower, and after 12 weeks, the bud length of the chilled bulbs was 26.1±0.5 mm (n=15) and that of the non-chilled bulbs 23.5±0.6 mm (n=16).
In December, the central bud contained leaf primordia, floral scape and the flower. Developing flower consisted of different tissue types, small compact cells (c. 10 µm) and large amount of intracellular air (Fig. 3c
).
MR images of a chilled and non-chilled bulb 10 d after planting are shown in Fig. 4. The bud in the chilled bulb appears more developed, with only the stem remaining within the sensitive region of the coil, while the developing flower (seen on the image of the non-chilled bulb) is already above the detectable region. Moreover, the scales in the chilled bulb seem to have lost a significant amount of water compared to the non-chilled specimen.
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Two weeks after planting, chilled bulbs sprouted and produced 15–18 cm long leaves. The floral scape was visible at the end of January, and reached 45 cm in length in February. Flowering commenced at the end of February and continued through March. By comparison, non-chilled bulbs sprouted at the end of February and produced very short (2–3 cm) leaves. The short floral scape initials reached only 2 cm in length and aborted.
MR parameter images
R1 maps of two representative bulbs at the end of storage for 12 weeks at 4 °C and 20 °C, respectively, are shown in Fig. 5. The most obvious difference between the two bulbs is in the lateral parts of the scales, which show much lower R1 values in the non-chilled, compared to the chilled bulb.
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The images in Fig. 6
display proton density and R2 maps for two representative bulbs at five time points during the storage period at 4 °C and 20 °C, respectively. The R2 images show a general trend towards faster relaxation in the scales and in the basal plate for both bulbs. At the same time, the proton density images show a conspicuous difference between the two storage protocols, which is again (like for the R1 maps above) apparent in the lateral regions of the scales which contain gel-like polysaccharides (Fig. 2b). In the non-chilled bulb, this region is richer in water compared to the central part of the scales, and this contrast does not change significantly throughout the storage period. On the other hand, for the chilled bulb, the contrast between the central part and the subepidermal layer, containing a gel-like substance reverses. The water content in the central part scales increases, while it decreases in the lateral parts of the scales.
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Figure 7
contains graphs of the proton densities in the basal plate and the scales, averaged over the entire populations of the chilled and non-chilled bulbs, respectively. In the basal plate there is a clear decrease in water content during storage time, with no significant difference between the two storage protocols. In the scales, the mean water content remains constant, and the values for the chilled and non-chilled populations are again similar. One should recall that the values representing the scales encompass the entire scales area, including the central and lateral regions. The redistribution of intensities apparent on the images in Fig. 6 is therefore not reflected in the graph on Fig. 7. According to this graph, however, there is no significant overall uptake or loss of water from the scales during storage, for both protocols.
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The corresponding mean values of R2, for scales and basal plate are shown in Fig. 8
. Overall, the relaxation becomes faster during the storage time, for both storage protocols. In the basal plate (but not in the scales), this increase in relaxation rate is significantly more pronounced in the chilled bulbs.
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The histograms in Fig. 9
show the R2 distribution in the scales, as well as the R2 and proton density distributions in the basal plate, averaged over the entire population of each storage protocol. The overall shift towards higher R2 and lower proton density values during storage is well seen on these histograms. In the basal plate, there is a significant difference between the two storage protocols for the R2 (but not for the proton density) distributions.
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The results for the ADC values are displayed in Figs 10
and 11. Figure 10 shows images for a representative bulb from each storage protocol, and Fig. 11, graphs of population averages in the different organs. There is a significant trend towards increased ADC values in the non-chilled bulbs, compared to the chilled bulbs where the ADC values remained constant during the storage period.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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Active developmental processes occurring within tulip bulbs during post-harvest storage include cell division and elongation, development of newly formed organs, and degradation of the storage materials (De Hertogh and Le Nard, 1993
). In the present study, microscopic and MRI observations showed similar processes of intra-bulb development in both chilled and non-chilled tulip bulbs. However, after planting, scape elongation and flowering occurred only in the chilled bulbs. Recently, various parameters have been investigated as markers for the fulfilment of the low temperature requirement in tulips: starch and amylase content (Gorin and Heidema, 1985; Lambrechts et al., 1994), protein and carbohydrate content and respiration (Higuchi and Sisa, 1967; Kanneworff and Van der Plas, 1994), membrane lipids (Walch and Van Hasselt, 1991), and endogenous hormones (Rebers et al., 1995; Rakhimbaev et al., 1978; Aung and De Hertogh, 1979). However, none of them can explain the influence of the cold period on further bulb development and flowering ability.
Under the experimental conditions and after data processing as used in the present study, the proton density predominantly represents water, and moreover so-called ‘free’ water, implying water molecules which are not incorporated into macromolecules (Donker and Van As, 1999; Donker et al., 1997). The importance of producing images of other parameters, such as R1, R2, or ADC, is 2-fold: first, the values of these parameters are intrinsic and not arbitrary, and should therefore allow comparison of results between different experimental sessions, and also between studies from different researchers on different instruments. Second, these parameters may be a sensitive probe of the biophysical water status, at the cellular level. For example, R2 was shown to be a sensitive marker of vacuole size in mushrooms (Donker et al., 1997). However, both these statements must be met with some caution and reservations. First, R1 (T1) values of biological tissue are strongly dependent on the magnetic field. R2 (T2) values derived from MRI are also affected by the magnetic field strength, as well as the details of the applied pulse sequences (Donker and Van As, 1999; Donker et al., 1996). Second, it must be kept in mind that in many cases (including the present study), the spatial resolution is such that individual image voxels, and certainly whole organs, contain a heterogeneous distribution of different cell types, cell sizes, and intercellular space. This leads to a broad distribution of parameter values, as apparent on the histograms (Fig. 9). In such cases, changes in the mean parameter values may not be directly interpretable in terms of the biophysical water status at the cellular level.
It is widely accepted (Chudek and Hunter, 1997; Clark et al., 1997; MacFall and Van As, 1996; Ratcliffe, 1994; Ratkovic et al., 1982) that an increase in free water content is accompanied by a decrease in the relaxation rates R1 and R2 (increase in the relaxation times T1 and T2). This relationship was confirmed in our present study. For example, in the thin subepidermal layers of the individual scales, one clearly observes (for the chilled bulbs) a decrease in proton density (decrease of water content) concomitant with an increase in R2 (Figs 6, 9). While R1 was not measured as function of storage time, it is evident that the R1 of these regions in the chilled bulbs (after 12 weeks of storage) is higher than in the non-chilled bulbs (Fig. 5), in accordance with the lower water content. In the same manner, the decrease in water content in the basal plate is concomitant with an increase in R2 (Figs 7, 9).
In several previous studies (Okubo et al., 1997; Faust et al., 1991) changes in local water content were interpreted as local transitions in water status from bound water to free water. The results of this study suggest that this may not necessarily be the correct or only possible interpretation. Rather, local free water concentrations may also change due to redistribution of free water between different organs or tissues in the plant, and this mechanism may, in some cases, dominate the values of the measured NMR parameters. In the bulbs studied in these experiments, the flower bud is growing, i.e. increasing in mass, and this mass is mostly water. Since there is no external supply of water, this water must come from other parts of the bulb. According to these results, at least some of the water accumulated in the growing bud originates from the basal plate where a significant loss of water is evident (Fig. 7). In the scales, the mean water content seems to remain constant for both treatment protocols (Fig. 7). However, this does not imply conclusively that there was no net loss of water from the scales as well. Since the total volume of the scales is much bigger than that of either the basal plate or the bud, the loss of a significant total amount of water may correspond to a very small (i.e. non-detectable) change in per-pixel proton density. The increase of R2 in the scales during storage can either suggest that the scales are also supplying water to the growing bud or that membrane permeability for water increases (Donker and Van As, 1999; Hills and Snaar, 1992). From the images taken after planting (Fig. 4), the loss of water from the scales is very obvious, particularly for the chilled bulb.
The gel-like substance in the subepidermal layer of the individual scales appears to contain non-structural polysaccharides (e.g. glucomannan), which probably affect cell-water relations (Meier and Reid, 1982) and the susceptibility of the bulb to dehydration (Matsuo and Mizuno, 1974). Low temperature storage has been reported to enhance degradation of glucomannans in Lillium longiflorum scales (Miller and Langhans, 1990). From the results of this work it appears that these regions serve as storage areas for water (and maybe other substances dissolved in this water), which is supplied to the inner regions of the scales during the storage period. It seems that this water redistribution is a key process in the healthy development of the flower buds, and that it is somehow inhibited when the storage temperature is too high.
The present results agree with those of previously published, comparable measurements. The T2 values in another geophyte, Allium aflatunense, were shown to decrease during storage in both the scales and basal plate, with a significantly stronger decrease in the basal plate of chilled bulbs (Zemah et al., 1999). In another study on tulip bulbs, Iwaya-Inoue et al. (Iwaya-Inoue et al., 1996) reported on the T1 values measured in chilled and non-chilled bulb at the end of the storage period. Their results indicate that the T1 in the scale epidermis of the non-chilled bulbs was much longer than for the chilled bulbs, which agrees with the findings of this study.
The results of the ADC experiments seem to diverge from the concomitant trend observed for the other parameters. As Figs 10 and 11 demonstrate, the ADC values and their spatial distribution remain constant in the chilled bulbs, while they significantly increase in the non-chilled population. The ‘redistribution’ between the inner and outer parts of the scales is not observed here. These results therefore suggest that, in this system, the ADC, under these measurement conditions, is not correlated with the free water content, and may be a true measure of water molecular mobility at the cellular level. It may be speculated that the ADC may be an indicator of water balance between different subcellular (vacuole and cytoplasm) compartments, including the effect of water membrane permeability. In chilled bulbs a healthy or normal balance may be maintained (no change in ADC), while the increasing ADC in the non-chilled bulbs may reflect a disruption in this balance.
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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It was demonstrated that NMR parameter imaging (proton density, R1, R2, ADC) is a sensitive and non-destructive indicator of internal bulb development during storage. Significant differences in the mean values of certain NMR parameters, and their spatial distribution, between chilled and non-chilled bulbs, were detected from 4 weeks of storage. Changes in R1 and R2 (but not ADC) were correlated with changes in the free water content. The most significant local changes in these parameters seemed to be associated with redistribution of water between different parts of the bulb. The growing flower bud is an obvious sink of water, which is supplied by the basal plate and the scales, in both chilled and non-chilled bulbs. During the storage process the chilled (but not the non-chilled) bulbs exhibited a transfer of water from the lateral (subepidermal) to the central part of the storage scales.
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Acknowledgments |
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This research was conducted at the large-scale NMR facility in Wageningen, supported by the EU TMR programme (contact ERBFMGECT950066). The authors would like to acknowledge the Wageningen NMR Centre (Wageningen University, The Netherlands) for the funds and assistance to make the MR experiments possible. Also we would like to thank Dr H Franssen of the Bulb Research Centre in Lisse, The Netherlands, for supplying the tulip bulbs for the project, and Professor A Fahn and Dr E Werker of the Department of Botany, the Hebrew University of Jerusalem, Israel, for their help in light microscopy.
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Notes |
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4 Present address to which correspondence should be addressed: Department of Ornamental Horticulture, ARO, The Volcani Centre, Bet Dagan, Israel. Fax: +972 3 966 0589. E-mail: rmgold{at}agri.huji.ac.il and vhrkamen{at}agri.gov.il
1 Fast relaxation corresponds to low values of T1 or T2 (the relaxation time) or high values of R1 or R2 (the relaxation rate). The use of either T or R values to describe relaxation is completely equivalent, and is a matter of semantics or convenience. While the use of the T value is more popular, the use of the R value is sometimes more appropriate, because when several mechanisms contribute to the relaxation, the R2 values from each mechanism can simply be added to obtain the overall relaxation rate. The same applies to spin lattice relaxation (T1 or R1).
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