TY - JOUR AU - Kollöffel, C. AB - Abstract After storage and subsequent planting of flower bulbs, the flower bud frequently appears to be aborted. This physiological aberration is probably caused by a change in the water status of the bulb and may be initiated during storage. The development of bud abortion in tulip bulbs was studied during long‐term dry storage of the bulbs at 5 °C. The anatomy of individual tulip bulbs was followed non‐invasively with T2‐weighted NMR imaging, which allowed the monitoring of the growth of the shoot and daughter bulbs. Quantitative maps of T1 and T2 relaxation times of individual bulbs were used to assess regional changes in the water status of different tissues. Parallel to the NMR measurements, bulbs were planted to assess the ultimate flower quality. Moreover, water content, osmolality of tissue sap and ion leakage of excised shoot and scale tissues were determined to obtain information about the water status and viability of the bulbs. Significant decreases during long‐term storage were found in T1 and T2 relaxation times in the shoot and particularly in the stamens. An increase in the osmolality of tissue sap and the decrease in relaxation times in the shoot below a certain threshold value attained after 24 weeks of storage, could be indicative for the emergence of bud abortion in tulips. Key words: Bud abortion, MRI, storage, T1, T2, tulip bulb, water status. Received 19 November 2001; Accepted 11 March 2002 Introduction In horticultural practice, bud abortion (blasting), i.e. ceasing of the development of the shoot in flower bulbs, is a substantial problem in several crops, including hippeastrum, nerine and tulip. The cause and course of this physiological disorder are unknown. In addition, its presence can only be visibly detected shortly before, or after planting. To assess the development of bud abortion in bulbs, the tulip bulb is an attractive model system. The physiology of tulip bulbs has been studied widely in the past and bud abortion can be induced in tulip bulbs during storage. Moreover, failures in tulip forcing are often due to abortion of the flower‐bud, which may occur during various phases of development (De Munk and Hoogeterp, 1975). Bud abortion can be induced by long, cold dry storage (Le Nard and De Hertogh, 1993), storage at high temperatures (De Munk and Hoogeterp, 1975; Rees, 1973) or by exposure to ethylene (De Munk, 1973; De Munk and Hoogeterp, 1975). The physiological disorder is a result of the cessation of the development of the flower bud and may manifest itself in tulips with a short upper internode of the stem, also termed ‘neck’, with an approximately 1 cm long bud with papery white tepals (De Munk, 1973; De Munk and Hoogeterp, 1975; De Munk and Gijzenberg, 1977) and yellow‐brownish stamens. Flower‐bud abortion has been attributed to a lack of substrate supply to the bud (De Munk and Gijzenberg, 1977; Moe, 1979) or an impaired hormonal activity (De Munk and Gijzenberg, 1977). Nevertheless, desiccation of the tepals and stamens and a deficient stem elongation are expected to be the final result of a change in the water status. NMR‐imaging (MRI) enables non‐invasive, longitudinal assessment of the local water status of plant tissues (Donker et al., 1997; Kuchenbrod et al., 1995; Millard et al., 1995). It has been used to study several physiological disorders in fruits and vegetables (Chudek and Hunter, 1997; Clark et al., 1997; Faust et al., 1997). Therefore, MRI might be useful to study the water status of flower bulbs, which could give insight into the development of the bulbs and related disorders like bud abortion. The magnetic properties of water, which can be measured by MRI, include the longitudinal and transverse relaxation times (T1 and T2, respectively). In plant studies T1 and T2 are often used as markers for the water status as they are primarily correlated with the water content and the mobility of water (Ratcliffe, 1994; Ruan and Chen, 1998). Tulip bulbs (cv. Apeldoorn) require 12 weeks of storage at 5 °C to obtain proper growth and development of the shoot (Boonekamp et al., 1990; Moe and Wickstrøm, 1973). This cold requirement for tulip bulbs has been studied frequently (Kanneworff and Van der Plas, 1994; Lambrechts et al., 1994; Rietveld et al., 2000) also with the use of MRI (Bendel et al., 2001; Iwaya‐Inoue et al., 1996; Okubo et al., 1997; Van der Toorn et al., 2000). However, the process of bud abortion has not been studied by means of NMR techniques. When dry‐stored at 5 °C for a period longer than approximately 20 weeks the flower within the tulip bulb starts to abort (Le Nard and De Hertogh, 1993). In the present study long‐term storage was used to induce bud abortion. T2‐weighted images were obtained at several time points during the storage period to monitor changes in bulb morphology and anatomy. Furthermore, T1 and T2 relaxation time maps were calculated to monitor longitudinal changes in the water status during the same period. The relaxation times of the scale and the whole shoot and of specific areas within the shoot were analysed in order to evaluate developmental processes at tissue level. To obtain information about the emergence of flower bud abortion, bulbs were planted parallel to the NMR measurements. Water content and the osmolality of tissue sap of bulbs were determined during the storage period to obtain information on the physiological processes influencing the water status. Ion leakage experiments were carried out to test for viability of the tissue (Bonnier et al., 1992, 1994). The present study aimed to investigate whether MRI, in combination with other (classic) assays of tissue water status, can give an insight into the process of flower abortion in tulip bulbs. The ultimate goal of these experiments is to identify a parameter, which is indicative of the initiation of bud abortion at an early stage of its development. Materials and methods Plant material Tulip bulbs (Tulipagesneriana L., cv. Apeldoorn), 12–13 cm in circumference, were harvested in July 1998, and stored at 20 °C until 2 weeks after flower differentiation stage G (Rees, 1973). Subsequently, the bulbs were dry‐stored at 5 °C and 70–90% relative humidity, for 34 weeks. NMR imaging NMR images were obtained on a 200 MHz NMR instrument (Varian, Palo Alto, CA) interfaced to a 4.7 T, 40 cm horizontal bore magnet. An 85 mm Helmholtz volume coil was used. Images were measured with a multislice spin echo sequence, using a Field of View (FOV) of 6×6 cm2; 256×256 matrix (0.2×0.2 mm2 in plane resolution); 2.5 mm slice thickness; five slices were chosen such that the middle slice intersected the bud; two transients were averaged. T2‐weighted images were measured with the following parameter set: echo times (TE) of 5, 7, 10, 15, and 20 ms; repetition time (TR) of 5 s, to calculate T2 maps. T1‐weighted images were obtained with the inversion recovery technique using inversion times (TI) of 0.001, 0.2, 0.5, 1, and 5 s; TE of 5 ms; TR of 5–10 s. T1‐ and T2‐weighted images of five bulbs were collected over a period of 34 weeks. After 12 weeks of storage three of these bulbs were planted to determine the quality of the flowers. Three other bulbs were measured after 13 weeks of storage and followed throughout the rest of the protocol. To minimize discontinuities in the storage conditions, NMR measurements were done at 5 °C by blowing cold air via a heat exchanger into the cylinder in which the bulbs were fixed. Data processing T1 and T2 maps were obtained by mono‐exponential fitting of the T1‐ and T2‐weighted images on a pixel‐by‐pixel basis using a program written in Interactive Data Language (IDL). Regions‐of‐Interest (ROIs) as chosen in the five slices are depicted for the central slice in Fig. 1 and include an area in the second outermost scale, the whole shoot, an area in the basal plate, two regions in the stem (first and upper internode), and a region including the stamens and pistil. ROIs were placed in the T2‐images with the shortest TE, which have the highest signal intensity. T1 as well as T2 values of the calculated maps were averaged for each ROI and averaged over the relevant slices through the bulb. Assessment of flower quality Bulbs were planted after 12–28 weeks of dry storage at 5 °C. At each time point 20 bulbs were potted and grown in a ventilated room at 17 °C, 75% RH and with a 16 h light period (200 µmol m–2 s–1). Tulips with a bud of approximately 1 cm length, with papery white tepals, and a short upper internode of the stem were designated as aborted. Flowers, which did not have completely red coloured tepals and did not open completely, were designated aberrant. Water content The shoot and three punches of tissue (7 mm diameter) taken from the second outermost scale were weighed and dried for at least 4 weeks at 70 °C. The second outermost scale was used in this investigation because the first scale was often (mechanically) damaged. The water content of scale and bud tissue was calculated from the difference between fresh and dry weight and expressed on a fresh weight basis. The water content of normal and aborted flowers after planting (at anthesis) was also determined. Ion leakage A disc (13 mm diameter) from the second outermost scale was punched out, weighed and incubated in 50 ml distilled water g–1 tissue. After 24 h of incubation at 20 °C the conductivity of the incubation media was determined with a Radiometer Copenhagen conductivity meter. To determine the total amount of ions present, the tissue samples were boiled for 1 h and the conductivity of the medium was measured again at 20 °C. The same procedure was followed for the whole shoot (until 12 weeks of storage) and for the shoot, divided into two parts, i.e. the basal plate plus first internode of the stem and the bud (from 12–34 weeks of storage). At every time point during storage 10 bulbs were dissected and measured. A reference curve was obtained with KCl solutions. Osmolality of tissue sap Pieces (of approximately 1 g) of the second outermost scale and shoot of 10 bulbs were stored at –20 °C for 2 h. After 14 weeks of storage the shoot was divided into three parts, i.e. the basal plate plus first internode of the stem, the second until fifth internode and the floral tissue (bud). After thawing, the tissue was placed in a tube with a pin on top to squash the tissue during 10 min of centrifugation at 1000 g. Subsequently, the supernatant was centrifuged for 5 min at 15 800 g. The osmolality (in mmol kg–1) of 10 µl of the supernatant was determined with a thermocouple psychrometer (Wescor Vapor Pressure Osmometer), a technique based on a dew point measurement. Results Flower development Flower development was normal after 12 weeks of dry storage at 5 °C and subsequent planting. None of the flowers appeared to be aborted (Fig. 2). Flower abortion was apparent to a small extent after 18–24 weeks of storage increasing steadily beyond this to 35% after 26 weeks and to 95% after 28 weeks of storage. Storage longer than 28 weeks resulted in abortion of the shoot directly after planting. In these bulbs the shoot failed to emerge after planting. NMR measurements Anatomy: NMR‐images of one and the same bulb monitored during 34 weeks of dry storage at 5 °C are shown in Fig. 3. The shrivelling and separation of the scales in time is very clear as well as the presence of bruises in the scale tissue. Furthermore, shoot development in time is evident and seems normal. The stamens, pistil, upper internode of the stem, and vascular tissue, especially in the basal plate, can be distinguished. In Fig. 3 a daughter bulb is visible after 26 weeks of storage, but in most bulbs the appearance and growth of daughter bulbs were observed after approximately 14 weeks. T1relaxation times: Quantitative T1 and T2 maps of the five bulbs were calculated from series of T1‐ and T2‐weighted images. Typical examples are shown in Fig. 4. Within the T1 maps different areas within the scales can be distinguished: the central, (sub)epidermal and in between the ‘lateral’ area. The central part of the scale showed a lower T1 value compared to the lateral tissue (0.45 s versus 0.52 s at the beginning of storage) until approximately 12 weeks of storage (quantitative data not shown). The (sub)epidermal layer appeared to have a very long T1. To follow the development of the individual bulbs, average T1 and T2 values of the ROIs of the five measured slices were calculated for each bulb as a function of storage time (Fig. 5). The ROI in the second scale used for Fig. 5 included the central as well as lateral tissue but not the (sub)epidermal tissue. A decrease in T1 values of the ROI of the second scale was observed in all bulbs during storage. The decrease was mainly found in the lateral tissue of the scale. After approximately 12 weeks of storage the T1 relaxation time of the lateral tissue decreased to similar values as for the central part making distinction of both tissues impossible. The average T1 value of the whole shoot also decreased throughout the storage period. However, when focusing on different areas within the shoot, various trends could be distinguished. The T1 of the basal plate tissue displayed a minimum at 20 weeks of storage and slightly increased thereafter. The T1 values in the first internode were more variable, but the changes in the values through storage time were similar. The T1 values started to decrease after 12–18 weeks of storage. The T1 values of the upper internode as well as the stamens region showed a pronounced decrease throughout the storage period, similar to those in the entire shoot. T2relaxation times: In the second outermost scale, the mean T2 value started to decrease after 9 weeks of storage (Fig. 5). Within the scale a distinction between central and lateral tissue was not possible on the basis of T2 relaxation values. The average T2 value in the whole shoot decreased considerably until 18 weeks whereupon the decrease diminished. The average values of T2 relaxation times in the basal plate decreased during development. In the ROI in the first internode, every bulb showed a different value and pattern in time. A distinct trend in T2 relaxation times throughout development was not observed in the region of the first internode. In the region of the upper internode a major decrease occurred during the first 7 weeks of storage. However, after 3 weeks only two shoots were sufficiently large to distinguish the upper internode. From 12 weeks until the end of storage the T2 relaxation times within the ROI of the upper internode were essentially constant except for one bulb that exhibited a further decrease in T2. The mean T2 values in the stamens and pistil area decreased strongly (34%) during storage. Water content and growth of the shoot During the first 18 weeks of storage the water content of the second scale (Fig. 6) hardly changed because the dry matter and amount of water of the punches decreased to the same extent (data not shown). The decrease in water content in the scale after that period was the result of a further decrease in the amount of water while the dry matter mass remained the same. The shoot increased in mass and length throughout the storage period. Until approximately 14 weeks the water content of the shoot decreased as a result of the larger increase in dry matter compared to the amount of water per shoot. After anthesis the water content of normal flowers was 88.6±1.1% (n=34), while aborted flowers only contained 48.0±5.4% (n=17) water. Ion leakage The ion leakage from the scale, measured as the conductivity of the medium after 24 h of incubation, increased during the first 12 weeks of storage and then reached a plateau at approximately 130 µS cm–1 (Fig. 7). By contrast, ion leakage from shoot tissue decreased until 12 weeks of storage to a value of approximately 16 µS cm–1 and then remained constant. The total amount of ions in both tissues was constant during storage, whereas the conductivities of the solutions after boiling remained approximately 220 µS cm–1 for scale tissue and 260 µS cm–1 for shoot tissue. This corresponds to 85 µmol ‘KCl‐equivalents’ g–1 tissue and 101 µmol ‘KCl‐equivalents’ g–1 tissue, respectively. Osmolality of tissue sap The osmolality of tissue sap, which is a measure of the concentration of total solutes, increased considerably during the first 12 weeks of storage in scale as well as in shoot tissue (Fig. 8). Between 12 and 26 weeks of storage the osmolality values remained the same, whereupon they started to increase again both in shoot and in scale tissue. Discussion As was expected for cv. Apeldoorn, growth and shoot development after planting were normal when the bulbs were dry‐stored for about 12 weeks at 5 °C (Boonekamp et al., 1990; Moe and Wickstrøm, 1973). However, the percentage of bulbs showing bud abortion after planting increased suddenly after 26 weeks of storage, implying that there is a critical storage period regarding the flower potential of the bulb. This is in agreement with the results of Le Nard and De Hertogh (1993), who found bud abortion emerging after 6 months of cold storage for cv. Paul Richter. Although the shoots were not able to develop into normal flowers after planting, their growth within the bulb continued at about the same rate over the whole storage period of 34 weeks as appears from the increase in fresh weight and size (Fig. 6). The T2‐weighted images (Fig. 3) showed that the morphology/anatomy of the shoots also changed gradually over the whole storage period. A sudden morphological change around 26 weeks of storage, which might have been related to the emergence of bud abortion, was not observed. Daughter bulbs were visible in T2‐weighted images of bulbs stored for about 14 weeks. They clearly increased in size over the subsequent 20 weeks of storage. Le Nard and De Hertogh (1993) also found that after very long cold storage and subsequent planting the only developmental process taking place was the growth of the daughter bulbs. This occurred after 28 weeks of cold storage in this study. During long cold storage the daughter bulbs obviously grew at the expense of the shoot. The amount of water in the second outermost scale declined over a period of 34 weeks (data not shown). Since the bulbs were dry‐stored, water will have been distributed to the growing shoot and daughter bulbs, both acting as competing sinks for water and metabolites (De Munk and Gijzenberg, 1977; Le Nard and De Hertogh, 1993). Despite of the availability of sufficient external water to sustain growth after planting, aborted flowers had a lower water content (48%) than normal flowers (89%). The water status of the scale and shoot changed profoundly during storage, as evident from the T1 and T2 relaxation times (Fig. 5). T2 values in the whole shoot decreased and a minimal level was reached at 22 weeks of storage, which may indicate that the normal development was disturbed from 22 weeks onwards. Changes in the water status of the stem were also expected because stem elongation, especially in the upper internode, decreased considerably as a result of bud abortion. However, T1 and T2 relaxation times in the first internode of the stem were erratic and apparently did not change as a result of the induction of bud abortion. T2 values in the upper internode did not change during storage and thus do not seem to be indicative of bud abortion either. The stamens are known to be most sensitive to improper preparation and the first tissue in the bulb to show symptoms of blasting (De Munk, 1973). The T1 and T2 values in the stamens decreased continuously (by 38% and 34%, respectively) during the storage period. All bulbs stored for 28 weeks or longer showed bud abortion after planting. This suggests that relaxation times in the stamens below a certain threshold value of T1 and T2, which is attained around 26 weeks of storage, could be related to the emergence of bud abortion. When drawing conclusions from the presented relaxation data, one should keep in mind that the ROIs used for determining the average relaxation value in the different slices bring a risk of partial volume effects due to the slice thickness used and tissue heterogeneities, especially in the basal plate and the stamens. Nevertheless, the variation between the five bulbs in T1 and T2 values in the different ROIs is relatively small. Furthermore, it can be concluded that the information provided by these two parameters is different. The T1 and T2 parameters of water in biological tissues depend among others on the water content, interactions with surrounding molecules, diffusion processes and flow. Apart from tissue‐intrinsic relaxation processes, the applied temperature, magnetic field strength, and pulse sequence variables also affect the measured T1 and T2. For example, Van der Toorn et al. (2000) found after 12 weeks of storage of tulip bulbs a T2 value in the scales of 83 ms measured with a multi‐echo sequence at 0.47 T and 20 °C. In the present study an average T2 value of 15 ms was found in the second outermost scale using a spin echo sequence at 4.7 T and 5 °C. Although, a direct comparison of absolute relaxation times should only be done within a similar experimental set‐up, trends in relaxation times could be compared. However, most MRI studies on flower bulbs mainly focused on scale tissue of bulbs stored for only a short period of time (Bendel et al., 2001; Iwaya‐Inoue et al., 1996; Okubo et al., 1997; Van der Toorn et al., 2000; Yamazaki et al., 1995; Zemah et al., 1999). The increase of the osmolality in the scale and shoot tissue during storage was probably the result of degradation of starch into sugars and the transport of the sugars from the scales to the shoot (Haaland and Wickstrøm, 1975). Furthermore, the decrease in water content of the shoot and scales will have contributed to an increase in the osmolality. Although Heidema et al. (1985) rejected osmolality as a parameter to indicate the fulfilment of the cold requirement, a distinct increase in the osmolality was found during the first 12 weeks of storage (Fig. 8). In addition, the substantial increase in osmolality in scale and shoot tissue after 26 weeks of storage may reflect aberrations, which appeared in the bulb and will have resulted in bud abortion after long‐term storage. The osmolality showed an overall increase in scale as well as in shoot tissue. Concurrently, T1 and T2 relaxation times decreased. This coherence might partially be explained in terms of the motional properties of the tissue water. An increase in the osmolality, for instance as a result of an increased sugar content may have decreased the translational and rotational mobility of the water and thus caused a decrease in T1 and T2 (Clark et al., 1997; MacFall and Johnson, 1994; Robinson et al., 2000). Changes in the membrane permeability, assessed via ion leakage measurements might be indicative for the viability of cells (Bonnier et al., 1992). Ion leakage of excised shoot tissue was expected to increase as a result of a decreasing viability accompanying bud abortion. However, the ion leakage changed during the first 12 weeks of storage, while in the subsequent weeks no changes in ion leakage were found in scale or shoot tissue. This suggests that bud abortion is not associated with changes in tissue viability. In summary, the development of tulip bulbs during long‐term cold storage is accompanied by changes in the water status, indicated by changes in osmolality of tissue sap and T1 and T2 relaxation times of the shoot and especially of the stamens. The passing of a threshold value of these parameters might be used as an indication of an increased risk of bud abortion. Acknowledgements This research was supported by the Technology Foundation STW, Applied Science Division of NWO and the technology programme of the Ministry of Economic Affairs. The Research Unit Flower Bulbs and R van Sluis, G van Vliet, WHC Huibers, and J Koerselman‐Kooij are acknowledged for their technical support and AC Borstlap for help with writing the manuscript. View largeDownload slide Fig. 1. Regions‐of‐Interest in the slices (shown for the central slice), which were chosen to evaluate the NMR relaxation parameters during storage in five bulbs. View largeDownload slide Fig. 1. Regions‐of‐Interest in the slices (shown for the central slice), which were chosen to evaluate the NMR relaxation parameters during storage in five bulbs. View largeDownload slide Fig. 2. Bud abortion after storage and subsequent planting. View largeDownload slide Fig. 2. Bud abortion after storage and subsequent planting. View largeDownload slide Fig. 3. Development of a tulip bulb during 32 weeks of dry storage at 5 °C, monitored by T2‐weighted imaging (TE=0.005 s, TR=5 s). A single slice through the centre of the shoot of the same bulb is shown. View largeDownload slide Fig. 3. Development of a tulip bulb during 32 weeks of dry storage at 5 °C, monitored by T2‐weighted imaging (TE=0.005 s, TR=5 s). A single slice through the centre of the shoot of the same bulb is shown. View largeDownload slide Fig. 4. T1 (upper row) and T2 maps (lower row) of the bulb shown in Fig. 3, as collected during storage. Bright areas represent long relaxation times, dark areas short times. View largeDownload slide Fig. 4. T1 (upper row) and T2 maps (lower row) of the bulb shown in Fig. 3, as collected during storage. Bright areas represent long relaxation times, dark areas short times. View largeDownload slide Fig. 5. Mean T1 (left column) and T2 values (right column) of ROIs within the second outermost scale, whole shoot, basal plate, first internode, upper internode and stamens plus pistil area of five bulbs determined as a function of storage time. Error bars represent standard deviations. For further details see Materials and methods. View largeDownload slide Fig. 5. Mean T1 (left column) and T2 values (right column) of ROIs within the second outermost scale, whole shoot, basal plate, first internode, upper internode and stamens plus pistil area of five bulbs determined as a function of storage time. Error bars represent standard deviations. For further details see Materials and methods. View largeDownload slide Fig. 6. Water content (wc) of the second outermost scale and the shoot, and the fresh weight of the shoot (fw), indicating the growth of the shoot as a function of storage time. Error bars represent standard deviations. View largeDownload slide Fig. 6. Water content (wc) of the second outermost scale and the shoot, and the fresh weight of the shoot (fw), indicating the growth of the shoot as a function of storage time. Error bars represent standard deviations. View largeDownload slide Fig. 7. Ion leakage from excised tissue of the second outermost scale and the shoot measured as the conductivity of the incubation medium as a function of storage time. The bud contained the floral tissue above the first internode of the stem. Error bars represent standard deviations. View largeDownload slide Fig. 7. Ion leakage from excised tissue of the second outermost scale and the shoot measured as the conductivity of the incubation medium as a function of storage time. The bud contained the floral tissue above the first internode of the stem. Error bars represent standard deviations. View largeDownload slide Fig. 8. Osmolality of the press sap from the second outermost scale and the shoot as a function of storage time. The bud contained the floral tissue above the upper internode of the stem. Error bars represent standard deviations. 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