JXB Advance Access originally published online on July 1, 2003
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Journal of Experimental Botany, Vol. 54, No. 389, pp. 1919-1930,
August 1, 2003
© 2003 Oxford University Press
Increased resistance to pod shatter is associated with changes in the vascular structure in pods of a resynthesized Brassica napus line
Received 10 December 2002; Accepted 7 May 2003
1 Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton,Bristol BS41 9AF, UK
2 Silsoe Research Institute, Wrest Park, Silsoe, Bedford MK45 4HS, UK
* To whom correspondence should be addressed. E-mail: robin{at}child3209.freeserve.co.uk
Abbreviations: DZ, dehiscence zone; MFT, microfracture test; MVBV, main vascular bundle of the valve; MVBR, main vascular bundle of the replum; SEM, scanning electron microscopy.
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Abstract |
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The architecture of the pod wall and dehiscence zone (DZ) was studied in populations of a resynthesized, shatter-resistant, oilseed rape line, DK142, and the commercial cultivar Apex. The dimensions of the pod wall and its component tissues were significantly larger in DK142. However, the variation in the pod architecture of Apex, DK142 and F2 populations derived from crosses of DK142 and Apex was found to have little or no role in pod shatter. By contrast, variation in the dimensions of the DZ characters correlated strongly and positively with shatter resistance. The size of the main vascular bundle (MVBV) of DK142 as it exited the valve and joined the vascular tissue of the replum was, on average, 60% larger than in Apex, the DZ was 40% wider and there was a high preponderance of vascular tissue other than the MVBV. The variation in the size of the MVBV accounted for much of the variation in shatter resistance of all populations, including shatter-susceptible Apex. The DZ width was also found to be important in explaining the limited range of shatter values in Apex, but in populations of DK142 and F2, where the amount of vascular intrusion into the DZ was much greater, the variation in DZ width was not important. The importance of the vascular tissue to shatter resistance was further highlighted by a novel microfracture test (MFT). By contrast, no significant difference between DK142 and Apex in the ease of separation of the thin-walled DZ cells was detected using the MFT.
Key words: Brassica napus, cell separation, dehiscence zone, pod shatter, vascular tissue.
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Introduction |
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Fully mature pods of oilseed rape (Brassica napus L.) are extremely sensitive to opening, resulting in seed loss (pod shatter). This can take place prior to harvest due to disturbance of the canopy by wind or during harvesting as the combine harvester machinery moves through the crop. Typical losses vary between 8% and 12% of the potential yield (Kadkol et al., 1984), but reductions of up to 50% were estimated by MacLeod (1981) in seasons when weather conditions were poor prior to and during harvest. A reduction in the sensitivity of pods to opening would increase the proportion of the yield recovered by the combine harvester and thereby improve production efficiency.
Little variation in shatter susceptibility was thought to be present in current commercial breeding lines (Bowman, 1984), but assessments by breeders of susceptibility to pod shatter between lines had to rely mainly upon visual observations of the crop in the field or upon hand tests of pods. However, a test procedure has been devised that exposes pods to random impacts in a similar manner to those that occur in the crop canopy during harvest (Bruce et al., 2002). This random impact test (RIT) enables the rapid comparison of susceptibility to shatter in samples of fully mature pods from individual plants. For example, in a population of 100 plants of the cultivar Jet Neuf subjected to the RIT, individual values ranged from 20% to 80% of the pods per plant opened after testing (Child and Huttly, 1999).
Sources of resistance to pod shatter have been sought amongst related species. More resistance is apparently present amongst some lines of B. juncea, B. carinata and B. nigra as well as amongst the putative parents of B. napus (B. oleracea and B. rapa) (Kirk and Hurlstone, 1983). Attempts to introgress shatter resistance traits from related species into B. napus can be complicated by linkage with unwanted characters. However, synthesis of new B. napus lines derived from interspecific crosses between B. oleracea alboglabra and B. rapa chinensis has provided a range of useful variation amongst which increased shatter resistance has been identified (Morgan et al., 1998). By contrast with the current situation in commercial cultivars, several individuals were found to have high shatter-resistance values when measured using the RIT procedure. One of these high-shatter-resistant lines, DK142, showed similar ranges of resistance whether grown under glass or in the field, at sites in the UK and Belgium and the range was at least 3-fold greater than the values found in the commercial cultivar Apex (Summers et al., 2003).
Fully mature pods of B. napus consist of two valves which are separated by sutures that extend on each side and along the whole length of the pod. Summers et al. (2003) found that, individually, in Apex and DK142 populations, increased valve length correlated significantly with increased resistance. However, the shatter-susceptibile Apex pods were longer than the shatter-resistant DK142 pods indicating that length of the valve walls could not account for the differences in shattering between Apex and DK142. This suggested that increasing shatter resistance was more likely to be associated with differences in the pod wall and/or the dehiscence zone (DZ) structure rather than the overall pod size. The structure and development of the DZ in shatter-susceptible cultivars has been described by Picart and Morgan (1984) and Meakin and Roberts (1990a).
In fully developed pods, the DZ consists of a layer of 2–3 thin-walled cells which separate the heavily lignified cells of the pericarp edge from the replum which consists of thickened schlerenchyma and is heavily vascularized. Degradation of the thin-walled cells is followed by their separation at about 7–8 weeks after anthesis (WAA). The process coincides with pod wall senescence and significant changes in enzyme and hormone activity (Meakin and Roberts, 1990b; Chauvaux et al., 1997; Child et al., 1998). Dissociation of thin-walled DZ cells initiates the process of valve separation, but alone does not cause the detachment that results in seed loss. Separation of the valve from the replum takes place only after impact with other pods which severs the vascular connections between the valves and the replum.
Child and Huttly (1999) examined the surface of detached valves of the commercial B. napus cultivars Jet Neuf and Fido with a scanning electron microscope. They estimated that the large vascular traces at the pedicel end and the small, discrete traces that were widely spaced throughout the rest of the DZ surface accounted for a significant proportion of the total separation surface in some plants. It was not known whether these structural variations were genetic in origin or whether they were environmentally influenced. In this paper it is shown how the structure and dimensions of the DZ differ in DK142 from those in Apex and how these differences contribute quantitatively towards susceptibility to valve separation.
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Plant material
Plants of Apex and DK142 were grown under glasshouse conditions at Long Ashton Research Station (LARS), Bristol, UK in 2000. F2 plants were grown in the field at the John Innes Centre (JIC), Norwich, UK, during 2000. Full details of the growing conditions and the methods of collection of fully mature pods for shatter assessment by RIT have been described elsewhere (Summers et al., 2003).
Light microscopy
Flowers were labelled at anthesis and, 5 weeks later, pods that were fully developed but still green were collected for microscopy. Fresh material was prepared for determination of pod wall dimensions as follows. Transverse sections approximately 10 µm in thickness were cut by hand using a single-sided razor blade, from the middle of a fresh pod from each of 100 plants per parent line and from each of 107 F2 plants. Sections were cut through the middle of a seed so that the tissue slice included the thinnest part of the wall adjacent to a seed and the opposite, thicker wall on the non-seeded side. Each section was immersed for 1 min in an aqueous 0.01% solution of crystal violet that stained the thickened cells in the endocarp and the vascular tissue violet. All other parts of the wall remained unstained. The sections were surface-lit and measurements of the pod wall were made using a low power dissection microscope. The minimum width of the pod was measured in the line of the septum between the base of each suture. The maximum pod width and the width of the thickened and unthickened portions of the wall of each loculus were measured at 90° to the septum. The number of rows of cells on both sides of the pod were also recorded in the thickened portion.
The structure of the DZ and the arrangement of the vascular tissue in the replum were determined in embedded material. Transverse slices of pods approximately 1 mm thick were fixed in a mixture of 4% paraformaldehyde and 5% gluteraldehyde for 1 h under reduced vacuum. The material was washed in water and dehydrated in a series of solutions of ethanol of increasing strength over a period of 2 d, and then infiltrated and embedded in LR white acrylic resin (TAAB laboratories, Berkshire, UK) over a period of 3 d. Transverse sections 2 µm in thickness were cut on an Ultracut microtome (Reichert-Jung, Vienna, Austria), mounted on slides and stained with an aqueous mixture of 0.1% methylene blue and 0.1% Azure A. Unthickened cell walls stained dark blue. Parallel studies with phloroglucinol identified lignified cells which stained turquoise when treated with the methylene blue/Azure A mixture. The sections were examined using a Leica DMRB microscope.
Scanning electron microscopy
Mature pods from each of 100 plants of DK142 and Apex and from 38 F2 plants were collected after the entire plant had senesced and dried out. The F2 plants were selected from 107 plants to represent the full range of shatter values based on RIT results. Gentle pressure was applied by hand to up to eight pods from each plant to separate the valves along the suture line. The basal end of one of the valves was cut from each pod, approximately 7–8 mm in length, mounted on aluminium stubs and coated under vacuum with a film of fine-grain gold using a Polaron E5000 sputter coater. The separated surface of the pericarp edge was examined under a Phillips 505 scanning electron microscope (SEM). Images of standard fields of view for each valve were recorded at a magnification of x300 after calibration with an SIRA SEM scanner (Agar Scientific, Stansted, UK) and dimensions of the vascular tissue and the DZ were measured. The width of the DZ was measured at its widest point at the pedicel end, which included the fractured surface of the main vascular bundle of the valve (MVBV) and along the edge of the valve where the loculus was at its full width. The width and breadth of the MVBV was also measured and the records used to calculate its approximate area. The amount of vascular tissue, excluding the MVBV was estimated as a percentage of the DZ total area. For F2 material, only the area of small, vascular bundles adjacent to the MVBV was estimated as a percentage. When present, the length of longitudinal bundles lying along the edge of the DZ in the same field of view was also measured.
Fracture analysis
An analysis to establish the contribution to resistance by the vascular tissue of the DZ was carried out using pods from selected plants. A micro-fracture test (MFT) was developed at the Silsoe Research Institute to establish the contribution of the MVBV to the amount of energy needed to separate the valve from the replum. Pods from six plants of DK142 and six of Apex were selected to represent the full range of shatter resistance for each population. The range of RIT values for 50% of pods to open for Apex was <10–32 s and for DK142 was <10–99 s. Thus plants with similar resistance values were included from both lines as well as DK142 plants with RIT values which were greater than the maximum value of 32 s for Apex.
Pod wall tissue was excised with a small, circular saw (Minicraft, Spennymooor, Co. Durham, UK) to isolate areas for testing that were approximately 1 mm in length. These isolated areas consisted of the septum and valve, between which the DZ remained intact, and were prepared either from the pedicel end of the valve including the MVBV or from the middle of the pod about half-way between the pedicel and the beak and did not contain any vascular tissue.
With sections at the pedicel end of the valve, cuts were made at approximately 45° to the longitudinal axis of the pod to isolate the tip from the remainder of the valve (Fig. 1A). The pedicel was held vertically in a small chuck and the pod beyond the test zone was cut away using the saw. An L-shaped, steel device in which the horizontal projection was 0.5 mm, was mounted on a force transducer of range 2 N, and positioned so that it contacted just the valve tip (Fig. 1B). The device was raised by a Universal Test Machine (Model DN-10, Davenport-Nene, Wellingborough, Northants, UK) until fracture of the specimen occurred. After fracturing, the broken section of valve remained in place because a ‘peg’ of vascular tissue projecting from the valve part of the test piece remained located in the corresponding hole in the replum. This attachment allowed energy stored elastically to be recovered.
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For sections from the middle of the pod, the MFT required the firm bonding of pods to the base of the test machine via micro translation stages (Fig. 1C). The L-shaped steel device was manoeuvred under the isolated section and then raised at a rate of 1 mm min–1, while force and vertical movement were recorded (Fig. 1D). When a break was detected by a sudden drop in force, the vertical movement was reversed to lower the section of pod wall until the force reached zero. This action allowed the energy stored by elastic deflection of the test section to be recovered and quantified. The peak force needed to break the sample and the net energy expended were then calculated.
After completion of the MFT, the fracture surface of the piece of valve removed during testing was coated with gold and the total area of the DZ and that of the MVBV was determined during examination under the SEM.
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Morphology of pods in DK142 and Apex
Although the number of ovules was similar in DK142 and Apex at anthesis, the number that developed into mature seeds in DK142 was approximately 50% lower than in Apex. Anthers did not dehisce until 2 d after anthesis when they opened only at the distal end. Pollen production was much less than in Apex although cross-pollinations by hand with DK142 pollen resulted in Apex pods with full complements of seeds whilst selfing of DK142 or cross-pollination with Apex resulted in approximately half the number of ovules developing to seeds. It was concluded that although DK142 pollen was fertile, successful pollination may have been impeded either by reduced ovule fertility or by changes in receptacle structure which obstructed pollen tube growth.
Individual pods were fully mature in Apex between 9 and 10 WAA, but DK142 pods took up to 4 weeks longer. The time taken for individual plants of DK142 to senesce fully did not correlate with shatter resistance. The length of fully developed DK142 pods varied from 35–65 mm between plants and on average were 15% shorter than those of Apex, in which pod lengths were more consistent. Pod shape was generally similar in both lines (Fig. 2) although in all DK142 plants, the valves were on average 29% wider when measured between the sutures along the septum and 18% wider at 90° to the septum (Table 1). The surface of the pod was smoother, but less regular in width, in DK142 because of the widely spaced seeds, which were approximately 50% fewer.
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Pedicels were consistently thicker in DK142 than in Apex. In the pods of some DK142 plants, the gynophore, situated between the valve and the floral organ abscission zone located at the distal end of the pedicel, was up to 10 mm in length (Fig. 2). The base of each valve was rounded in both lines, but was blunt and obtuse in Apex and more tapered in DK142. The replum was also thick in DK142, causing each valve base to arch away from the replum forming a ‘lip’ at an angle of approximately 30°. In Apex, the valve base did not protrude and lay flat against the replum. Valve edge definition was clearer and the suture wider in DK142 than in Apex. The beaks of DK142 were similar in length to those of Apex, but were wider at the intersection with the valves.
In Apex, the outline of the main vascular bundle (MVBV) was clearly visible in the middle of each valve and, along its length, smaller vascular traces joined it at irregular intervals. This is shown diagrammatically in Fig. 2D. One or two longitudinal vascular bundles connected the small traces along the length of the valve and appeared to terminate near the junction of the valve with the pedicel close to the MVBV. Other vascular traces passed into the replum near the valve edge at regular intervals. All the vascular bundles were raised on the surface of the pods forming clear ‘ribs’ in Apex, but in DK142 an apparently similar arrangement of vascular tissues was much more difficult to identify externally because of the thicker pod wall. When the valves were detached in fully mature pods the main vascular bundles of each replum (MVBR) to which seeds were attached by their funicle could be seen at the base of each suture. A papery false septum joined the two MVBRs and contained no vascular tissue.
Pericarp wall structure in fully developed pods
Tissue architecture and the extent of thickening in the pod wall were determined in fresh, hand-cut sections taken from the middle of the pod (Fig. 3). Three WAA, when pods had extended to their final length, thickening was present in the endocarp of DK142, but not in Apex. By five WAA, thickening was complete in both lines and significant differences in internal architecture were established. The total width of the pericarp and the width of the band of cells with thickened walls varied in relation to position in the pod. In both lines, cell wall thickening was greatest in the non-seeded areas and least on the seeded side of the pod (Fig. 3A, B). In Apex, only a single row of endocarp cells with thickened walls was often present, although the average was 1.7 rows on the seeded side and 2.2 on the unseeded side of the pod. The thickened zone was, on average, 0.17 mm wide and was surrounded by the non-thickened zone 0.24 mm wide. In DK142, the thickened zone included mesocarp cells adjacent to the endocarp and was 165% wider than Apex, not only because it contained, on average, an extra row of cells, but because the radial length of each cell was 67% greater. Up to 35 vascular traces were present in the mesocarp of both lines adjacent to the unthickened exocarp (Fig. 3). At maturity, after drying down and the shrinkage of the unthickened exocarp these traces were prominent on the surface of the pod in Apex, giving it the ‘ribbed’ appearance described above. In DK142, the thickened zones on both sides of the pod subsumed the vascular traces and, after drying down, the surface of these pods was smooth and not ribbed.
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Correlations between the dimensions of the pericarp wall and its components in green pods were subsequently determined with shatter-resistance values in an RIT of subsets of fully mature pods (Table 1). Overall, correlations with RIT values were low, but were statistically significant for the pod diameter measured at 90° to the septum, for pod wall thickness and for radial cell length. The correlation of pod wall thickness with resistance was mediated in Apex through the variation in the width of the thickened zone, whereas in DK142 it was associated with the width of the non-thickened zone. There was no significant correlation with RIT for the number of rows of cells in the thickened zone in DK142 or Apex.
Vascular architecture in fully developed pods
The tissue embedded at 5 WAA was used to determine the architecture of the vascular system and, in particular, its spatial distribution at the valve edges. Just before the junction with the two valves the pedicel was swollen by scar tissue of the abscinded anthers, petals and shrivelled remains of the sepals. Just after this position the replum tissue consisted of an outer cortex composed of thickened collenchyma cells immediately below the epidermis and an inner layer of loose parenchyma cells. Groups of fibrous cells were associated with a ring of collateral vascular bundles (Fig. 4A, F). The fibrovascular tissue of the stele was arranged in two large groups of bundles that were separated from two opposite, single bundles by parenchymatous medullary rays that connected the cortex to the central medulla. The two separate, single bundles diverged from the stele into the cortex at the intersection with the base of the valve (Fig. 4B, G). These passed through the thin-walled cells of the DZ into the valves forming connecting ‘bridges’ of vascular tissue (Fig. 4C, D, H) forming the main vascular bundle of each valve (MVBV). Later examination of mature valves under the SEM, confirmed that in Apex this vascular ‘bridge’ was formed at an angle of divergence 70–90° to the vascular tissue of the stele. In DK142, the angle of divergence was much less and when the distance between the scar tissue and the junction of the valve with the septum was greatest the angle was less than 20°.
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After the divergence of the single bundles to the two MVBVs, the two remaining groups of bundles passed into opposite sides of the edge of the septum to form the main vascular bundles of the replum (MVBR). Vascular bundles could be seen passing between the replum and the valve for a distance of 5 mm (Fig. 4E). The number and angle of these bundles differed between Apex and DK142. This was shown later and with greater clarity in the SEM examination (see below). In Apex, the valves widened rapidly at approximately 5–7 mm from their base, after which the loculi were wide enough to contain seed. However, in DK142, the valves remained narrow in some plants for up to 1 cm. From this point onwards sections showed that the vascular architecture in the DZ, replum and MVBR was similar in both lines. The two replums were joined by the thin, papery septum composed of unthickened, loosely associated cells. No vascular tissue was present in the septum, which forms a partition in the pod cavity between the two loculi.
In transverse sections of embedded tissue taken from the middle of the pod 5 WAA (Fig. 3C, D), it was clear that there were no differences between DK142 and Apex in the structure of the DZ at the junction with the suture and around the replum. The number of thin-walled cells that were later to separate was the same in both lines. Vascular traces were occasionally found adjacent to the pericarp edge.
Fracture surfaces of detached pods
Examination of the fracture surfaces of detached valves under the SEM showed that the DZ was wider at the junction with the pedicel than along each of the valve edges bordering the loculi. In DK142 (Fig. 5A), the pedicel end of the DZ was 91% wider than in Apex (Fig. 5B) and the range of values in individual plants was 365% greater (Table 2). In the DZ bordering the loculi, the average width was greater by 40% and the range greater by 45%. In both lines, there was a high level of correlation between DZ width at the pedicel end of the valve with the variation in shatter resistance measured by RIT, but the correlation where the loculus was at its full width was only significant for Apex.
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In both DK142 and Apex, the dissociated DZ cells had usually separated along the line of their middle lamellas and had dispersed, leaving an imprint of their walls on the pericarp edge that they had lined. At the pedicel end, the imprint suggested that the cleanly separated and now dispersed cells were arranged indeterminately, forming an irregular surface (Fig. 5C, E, F). However, in DK142 at the junction with the pedicel, the valve arched away from the rest of the pod at an angle of about 30° and the thin-walled cells were orientated at a more oblique angle to the surface of the DZ than in Apex. Along the edge of the loculus, the remaining imprints of both lines were flat in appearance and were orientated along the surface of the DZ, for example, DK142 in Fig. 5D. Remnants of torn walls could clearly be seen at the junction of the inner edge of the DZ with the lining of the pericarp wall. In Apex there were one or two rows, but in DK142 four or five rows of torn cells could be seen.
Cracked surfaces of vascular bundles were clearly visible and sometimes associated with tearing of adjacent thin-walled cells. The MVBV was the largest of the vascular bundles, situated towards the inner edge of the DZ at the pedicel end in both lines. In Apex (Fig. 5B, F) it fractured cleanly, exposing a single, discrete, transverse section. In DK142, the MVBV was usually much wider, lay at a shallower angle and was apparently composed of several vascular tissue groups each of which fractured separately, often at different levels, resulting in a multi-faceted stump (Fig. 5A, B, C). All MVBV size parameters were significantly larger in DK142 than in Apex (Table 2). However, the size of the MVBV correlated positively with resistance and with a similarly high degree of statistical significance in both DK142 and Apex, despite the lower between-plant variation in populations of Apex.
In DK142, longitudinal bundles arose from the MVBV and ran parallel with the fractured surface of the DZ (Fig. 5A). In the DZ bordering the loculus in mid-pod sections, longitudinal vascular bundles, smaller than those near the MVBV, were found at irregular intervals (see also Fig. 3C). These were also seen in Apex, but vascular bundles in longitudinal orientation at the fracture surface adjacent to the MVBV were never seen. By contrast, the longitudinal bundles of the Apex valve arose from the main vascular tissue of the replum and crossed the DZ surface close to the MVBV at an angle of approximately 90° (Fig. 5B). Thus, the longitudinal bundles of Apex valves have minimal contact with the DZ and do not contribute to the size of the MVBV, but in DK142 these bundles have maximal DZ intrusion whilst also contributing to the size of the MVBV.
Fracture analysis
The peak force and energy required in the MFT to separate the isolated piece of pod wall from the replum at the pedicel end varied considerably within and between lines of Apex (Table 3). On average, line DK142 required 118% more force and 161% more energy than Apex. For Apex, the variation in the peak force and total fracture energy required to separate the small pieces of pod did not correlate significantly with the shatter-resistance values measured on whole pods, but for DK142 the correlations were highly significant.
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After carrying out the MFT, the detached valve pieces were examined under the SEM (Fig. 5E, F), and the area of the fractured surface of the MVBV and the non-vascular DZ cells calculated. The total DZ area in DK142 was larger than in Apex because the DZ was wider and the MVBV of both lines comprised a similarly small percentage of the total test area (approximately 9.7% for Apex and 11.3% for DK142). However, as found with previous SEM analyses of the DZ (Table 2), the average MVBV was larger than in Apex.
For Apex, the variation in the area of the MVBV and, particularly, the much larger area of the non-vascular DZ tissue, correlated significantly with the peak force and total fracture energy required to break open these small sections, but neither area related significantly with shatter resistance of entire pods. For DK142, despite the fact that the non-vascular tissue was larger than the MVBV, only the variation in the area of MVBV correlated significantly with peak force and total fracture energy and this area also correlated significantly with shatter resistance of whole pods.
Isolated sections taken from the middle of the pod were also microfracture tested, examined under the SEM (Fig. 5D) and the few containing vascular tissue discounted in the analyses. The DZ in Apex was 0.23 mm wide, but in DK142 was 48% wider (Table 4). A peak force of 0.25 N and fracture energy of 18 µJ was required to break open Apex sections, whereas corresponding values in DK142 were 132% and 72% greater. However, the energy per unit area was similar for both lines (P=0.67), but peak force per unit area was 50% greater for DK142 compared with Apex (P <0.05).
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Transmissibility of DZ characters and resistance into F2 populations
The range of values for pod wall dimensions in the F2 population derived from crossing DK142 with Apex, was closest to that seen in the resistant parent (DK142), but the mean for most parameters was intermediate between the two parent lines (cf. Tables 1 and 5). The range in shatter resistance measured by RIT was similar to DK142 and values ranged from <10 to 324 s. However, when the variation in the range of the pod parameters was correlated with shatter resistance, the only relationship that was significant was with the width of the non-thickened zone and this was at a low level of significance.
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Sections of the valves from a subsample of the F2 plants, whose shatter resistance spanned the full range, were examined under the SEM. The mean and range of values found for the length, width and area of the MVBV were more similar to those found in DK142 than Apex (cf. Tables 2 and 6). Correlations of the variation in these parameters with RIT values confirmed the importance of the size and, in particular, the width of the MVBV to shatter resistance. Longitudinal vascular bundles situated near the MVBV and parallel to the DZ were present in many of the valves as in DK142. The length of these bundles in the F2 valves correlated with a high degree of significance with shatter resistance, but by contrast, there was no significant correlation with the area occupied by bundles that had fractured in transverse section.
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Discussion |
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The range of shatter values recorded in populations of DK142 is wide and continuous indicating polygenic variation. This contrasts with Apex which shows a much narrower range of shatter susceptibility (Summers et al., 2003). Although some correlations of this variation in shatter susceptibility with that measured in several pod wall characters were significant (Table 1), the level of significance was low. Moreover, in the F2s only the width of the non-thickened zone 5 WAA showed any significant correlation with shatter values recorded in this population. This correlation was also at a low level of significance, which is not surprising, because during senescence the cells within this zone collapse forming only a thin layer around the thickened zone, contributing little to the overall pod dimension. Thus, it was concluded that pod wall dimensions play only a small part, if any, in shatter resistance. A similar conclusion was found in a comparable study with sesame seed capsules. As with oilseed rape, sesame seed loss before harvest is a major economic problem, but none of the anatomical features of the capsule correlated significantly with seed retention (Day, 2000).
Josefsson (1968) believed that lignification in the pod at the junction of the valves with the replum in B. napus was relevant to shatter susceptibility. It provided a firm edge to the valve against which the thin-walled cells formed the line of weakness. Genes expressed in the valve margins have been identified in Arabidopsis, for example, SHATTERPROOF1 and SHATTERPROOF2 appear to regulate lignification of the valve margins (Liljegren et al., 2000). Spence et al. (1996) have suggested that tension can develop in the pericarp wall of B. napus and B. juncea and in Arabidopsis during desiccation as a result of differential thickening and that this contributes towards the separation of the thin-walled cells in the DZ. However, the significant increase in the amount of thickening in the endocarp of DK142, compared with Apex, did not correlate with RIT and it seems unlikely that changes in the pod wall architecture play an important role in determining shatter susceptibility.
By contrast, overall pod dimensions of mature oilseed rape pods, particularly the length and weight of the septum and valves, have been shown to correlate with a high degree of significance with shatter resistance (Summers et al., 2003). Thus, this study concentrated on the role of the DZ, the tissue that interfaces between these organs and which is ultimately responsible for determining how readily the valves separate from the septum. For all DZ characters, the range of values found in Apex was narrower and the mean values lower than the wider ranges and higher means found in populations of DK142. This distribution of variation was similar to that found in the RIT scores (Summers et al., 2003) and the variation of many of the DZ features correlated significantly with the variation in shatter scores (Table 2). In particular, the highly significant correlations of MVBV size with RIT values in both parent lines and, subsequently, in the F2 populations indicated and then established that the size of this tissue plays a key role in shatter resistance. The new microfracture technique applied to sections in which the MVBV with some surrounding undifferentiated cells was isolated from the majority of the DZ, confirmed this relationship in the resistant parent. In both lines, the MVBV comprised approximately 10–11% of the DZ area of these sections and each section was no more than 2% of the entire pod. Despite this, the force and energy required to break open small sections of DK142 correlated with high significance with shatter resistance of whole pods. Moreover, peak force and energy required in these sections correlated significantly with the size of the MVBV and not the area of non-vascular tissue. For Apex, the correlation of shatter resistance with MVBV size was not significant and the peak force and energy required in these sections correlated with greater significance with the area of non-vascular tissue. Tests further down the pod confirmed the relationships that had previously been seen in the SEM examination (Table 3) between DZ width and shatter resistance. For the susceptible parent (Apex) with its small MVBV and narrow range in shatter susceptibility the relationship was highly significant, but in contrast, the variation in DZ width in populations of DK142 was not important to its wide variation in shatter susceptibility. Thus for resistant pods, the size of the large MVBV determines the upper limit to pod strength and influences from the variation in DZ width are less important. Weak pods, not only of Apex but including weak pods of DK142 and F2, always had a small MVBV and because of this, the width of the DZ was able to contribute proportionately more to pod strength.
Resistant DK142 and F2 pods are stronger than susceptible pods because of the larger MVBV, but it is possible that other structural changes contribute to the overall increase in shatter resistance. These arise from (a) the angle at which the MVBV and adjacent vascular tissue pass through the DZ into the replum and (b) the longitudinal bundles between the undifferentiated cells and the replum which present additional disruption to the separation of the valve. Apex had very little additional vascular tissue surrounding the MVBV and that which was present was transversely orientated and the amount did not correlate significantly with shatter resistance (Table 2). By contrast, longitudinally orientated bundles as well as transverse bundles commonly surrounded the MVBV of DK142. For this line, the amount of vascular tissue surrounding the MVBV correlated with shatter resistance with a high degree of significance. Moreover, in F2 populations, where a more detailed analysis of the surrounding vascular tissue was performed, correlations indicated that only the longitudinal bundles were important in determining resistance. If longitudinal bundles are stronger because of their direction, it is likely that the more longitudinally orientated MVBV of DK142 is likely to be stronger per unit area than that of Apex.
Views of the separated valve surface under the SEM suggested that tearing in more rows of cells might have taken place in DK142 than in Apex and the microfracture test on side sections confirmed that sections of DK142 required 50% more force per unit area to separate them. This was because the DZ of DK142 was 40–50% wider. However, a similar amount of energy per unit area was required to separate the undifferentiated, simple cells in both lines as the extra number of torn rows of cells in DK142 was in proportion to its wider DZ compared with Apex. Thus, although the wider DZ of DK142 gives additional resistance, between-line differences in cell separations are not important. Furthermore, no reduction in cellulase activity has been found in the DZ of DK142 (DJ Osborne, private communication). Thus, in the absence of a significant difference in hydrolase activity, it is not surprising that differences in the pattern of cell separation were not large in the fully mature, separated valves of DK142 and Apex when examined under the SEM. This contrasts with loss of shedding competence in a non-abscinding mutant of Lupinus angustifolius cv. Danja associated with loss of cellulase function (Henderson et al., 2001). It seems clear, therefore, that the increase in shatter resistance in DK142 is due to the size of its DZ and DZ components, particularly the size of its vascular tissue.
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This work was sponsored by DEFRA and HGCA through the Sustainable Arable LINK Programme and by the Biotechnology and Biological Sciences Research Council. We would like to acknowledge the contributions of the Glasshouse and Controlled Environment Section at Long Ashton and the Horticultural Services at the John Innes Centre.
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