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Anatomical variation in the dehiscence zone of oilseed rape pods and its relevance to pod shatter

Robin Child and Alison Huttly

IACR – Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol, BS41 9AF UK. email: robin.child@bbsrc.ac.uk ; alison.huttly@bbsrc.ac.uk

Abstract

Changes in the structure of the DZ, which are associate with susceptibility to shatter, have been investigated in an irradiation – induced mutant and a population of its parent cultivar, Jet Neuf. Much lignification of groups of cells was found throughout the DZ of the most resistant Jet Neuf plants and in the mutant. In addition, vascular traces are situated close to the inner edge of the DZ in the mutant and help to secure the valves, making it more difficult to open the pods. The changes in pod structure in the mutant appear to be associated with differences in flower structure and raceme architecture. These are likely to be pleiotropic effects caused during initial irradiation treatment.

Introduction

Fully mature pods of current commercial cultivars are extremely sensitive to opening. Sutures situated on both sides of the bi-valved pod contain dehiscence zones (DZ’s) which are composed of a layer, 3-4 cells wide, of simple, parenchymatous cells. The DZ is situated between the pod valve edge and a central replum that contains the main vascular bundle to the stalk or pedicel. Dissociation of the cells in the dehiscence zone (DZ) takes place during pod senescence and is complete by the time that the pods reach full maturity (Meakin and Roberts, 1990). Valve separation can then take place. It is assumed that DZ cell separation takes place uniformly throughout the DZ and there are no reports of failure to separate, which might be the basis of selection for breeding for increased resistance. Current work which aims to engineer resistance to cell separation by inhibiting the breakdown of the middle lamella through inhibition of enzyme and hormone activity is described by Child et al., (1999).

However, the DZ also contains vascular traces, which pass from the pod wall to the pedicel (stalk) and the replum. The process of pod shatter takes place only after external force fractures the delicate vascular threads, allowing the valves to separate and the seeds to fall to the ground. This occurs during disturbance of the canopy and contact with the combine during harvesting. The vascular tissue contains thickened, lignified cells which form the collenchymatous groups of cells found adjacent to the conductive cells (Meakin and Roberts, (1990). This provides rigidity to the tissue and presumably, some resistance to fracturing. Lignification elsewhere in the pod may also be relevant to the shatter susceptibility of the pod. Josefsson (1968) believed that shatter resistance might be associated with increased lignification at the junction of the valves with the replum.

Thick - walled pods of irradiation - induced mutants produced at the University of Poznan, Poland, are known to be more resistant to opening (Luczkiewicz, 1984). The anatomical characters of one of these mutants have been compared with the parent cultivar (Jet Neuf) from which it was derived, and are outlined in this paper. The objective of this work was to identify those anatomical characters of the pod and the DZ which were associated with increased tendency to shatter and to estimate the possibility of introgressing the characters into breeding lines of oilseeed rape.

Materials and methods

Plants used in analyses: Irradiation-induced mutants of the cultivar Jet Neuf, (produced by Dr T. Luczkiewicz at the Agricultural University of Poznan, Poland) and the recent commercial cultivars, Tapidor and Apex were grown in pots under cold glass at IACR – Long Ashton

Assessment of pod shatter: Comparisons of susceptibility to pod shatter between cultivars have mainly relied upon visual observations of the crop in the field or upon hand tests of pods. We devised a random impact test (RIT) procedure that consisted of agitating fully mature pods with steel balls in a cylinder 20cm diameter, on a reciprocal shaker for a standard time and amplitude. Two replicates of 20 intact mature pods were tested for each line or cultivar. The numbers of opened pods were counted at intervals. This test aimed to subject pods to conditions that were similar to those experienced in the canopy where, during harvesting, pods impact with other pods, racemes and harvesting machinery randomly at all points on their surface. We also used a cantilever-bending test described by Kadkol (1984) to measure the force required to initiate and propagate a crack along the dehiscence zone of the pod.

Examination of DZ in separated valves: Fully mature pods were opened by hand and one-cm portions of the two valves from the pedicel end of each pod were mounted on aluminium specimen stubs using plastic/carbon cement. Preparations were coated with gold under reduced pressure for eight minutes using a Poleron series II Sputter coater and the structure of the valve edges (which included the DZ) examined in a scanning electron microscope (SEM) using an electron beam at 5kV. Images were viewed on a television monitor and photographed for later evaluation.

Cell separation in the DZ. Slices of pod tissue of the cultivar Fido containing the DZ were fixed in a mixture of 4% formaldehyde and 5% gluteraldehyde and post-fixed in 1% osmium tetroxide before being embedded in epoxy resin. The process of cell separation in the DZ was studied using ultrathin sections, which were examined using a transmission electron microscope (TEM). The primary cell wall and middle lamella remained intact whilst the pod was still photosynthesising. After pod senescence had begun, dissolution of the cell wall was visible and by the time senescence was complete, total separation had taken place.

Results

Shatter susceptibility in some commercial cultivars.

Individual plants in cultivars that have been developed for specific characters such as disease resistance or glucosinolate level may vary genetically for other characters such as pod structure. Although in current breeding lines, there does not appear to be useful variation that might be the basis of increased resistance to pod shatter, these estimates are generally based on values obtained for the crop in the field. Values for individual plants have not been reported.

Random impact tests: Fully mature pods from each of 100 plants of the cultivar Jet Neuf were subjected to the standard RIT procedure. Continuous variation between values of 20%- 80% shatter was recorded. Similar variation appeared to be present in the modern cultivars Tapidor and Apex although only 10 plants of each were examined. Although the range of variation in these commercial cultivars appeared to be quite wide it was not known whether this included useful resistance of practical significant which would result in increased seed recovery during combining.

Pod anatomy: The DZ of pods from the most shatter susceptible plants (greater than 60%) consisted of parenchymatous cells covering approximately 80 % of the valve surface. The remaining area consisted of a large vascular trace at the pedicel end and several small, discrete, traces widely spaced throughout the rest of the DZ surface. The DZ of pods from the more shatter-resistant plants (less than 25% opened) appeared to contain increased thickening in cells adjacent to the vascular traces. It was not known whether the small amount of structural variation within cultivars is entirely genetic in origin or whether it can be environmentally influenced.

Shatter resistance in the irradiation-induced mutant of Jet Neuf

Cantilever bending tests: Collaborative work carried out with David Bruce at Silsoe Research Institute confirmed the increased resistance to opening in the irradiation mutant when compared with its parent cultivar, Jet Neuf. The results (Table 1) show that the mutant requires more than four times the energy necessary to open pods of the parent cultivar.

Table 1. Cantilever bending tests with pods (8% moisture) of an irradiation mutant and the parent oilseed rape cv Jet Neuf.

Kadkol et al., (1984) found that values of 0.05 - 0.21 mJ were necessary to open the pods of a range of Brassica napus cultivars. These very small energy values represented the range of susceptibility, as judged by visual scoring. All would appear to be below the threshold required for significant resistance in the crop because little variation in susceptibility to shedding is found amongst present day cultivars (Thompson and Hughes, 1986). The values obtained for Jet Neuf appear to be within the range found by Kadkol et al., (1984). However the value for the mutant is approximately four times that obtained for the parent cultivar and indicates significant increase in shatter resistance.

Anatomical analysis of the mutant showed that there was much thickening of cells in the DZ with the formation of ‘bridges’ of lignified cells which connected the valves to the central replum.

In addition, prominent vascular traces, which connect the pod wall with the vascular bundles of the pedicel, are situated close to the inner edge of the DZ. It was clear that these structural modifications were present in all plants and that they were associated with the significantly greater values for shatter resistance compared with the parent line. However, although this ‘pure line’ material provided a unique source of shatter resistance, fertility was low. This was apparently associated with deformed styles. We were unable to separate this character from shatter resistance in crosses of the mutant with other cultivars. This less desirable architectural character appeared to preclude the use of this material in breeding programmes. These apparently pleiotropic characters will be present as a result of damage to genes during the radiation treatment of the parent plant.

Acknowledgements

We acknowledge with thanks, the technical assistance given by Karen John and Vicky Child in the preparation and examination of material with the TEM and the SEM. Our work is supported by the Biotechnology and Biological Sciences Council.

References

Child, R.D., Ulvskov, P., Van Onckelen, H.A. and Botterman, J. (1999). Strategies for engineering shatter resistance into oilseed rape pods. In: Proceedings of 10th International Rapeseed Congress, Cambridge.

Josefsson, E., (1986). Investigations into shattering resistance of cruciferous crops. Zeitschrift fur Pflanzenzuchtung, 59, 384 – 396.

Kadkol, G.P. , MacMillan, R.H. , Burrow, R. P. and Halloran, G. M. (1984). Evaluation of Brassica genotypes for resistance to shatter. 1. Development of a laboratory test. Euphytica 33: 63-73.

Meakin, P. J. and Roberts, J. A. (1990a). Dehiscence of fruit in oilseed rape (Brassica napus.). I. Anatomy of pod dehiscence. Journal of Experimental Botany 41, 995 – 1002.

Luczkiewicz, T. (1987). Winter rapeseed mutant with decreased tendency to shattering. In: Proceedings of the 7th International Rapeseed Congress, 2, 463 – 467, GCIRC, Poznan.

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