Breeding & Engineering Shatter Resistance

There are a number of characteristics during oilseed development that have the potential to improve both yield and/or production efficiency. The creation of sufficient variation of a characteristic in model crop plants (ideotype) has been shown to enhance the value of a wide range of crops. Some of the features suggested for potential ideotype breeding programmes are as follows (Mendham et al, 1991; Thurling, 1991):

Rapid, early growth, even at low temperatures (ie. winter).
Early flowering.
Short, strong stem (eg. reduce lodging).
Apetalous flowers to improve photosynthetic light interception.
Resilient to desiccation or nutrient depletion (ie. reduce pod/seed abortion).
Upright pod orientation (ie. improve radiation distribution & reduce knocking of pods).
Reduced lower branching & enhanced pod production on mainstem & upper branches.
Increased shatter resistance.

Breeding Shatter Resistance

Breeding programmes for B. napus show little variation for shatter resistance, although resistant lines have been isolated from its diploid parents (B. oleracea & B. rapa) and other members of Brassicae (eg. B. juncea, B. carinata & B. nigra). So far the breeding programmes have been unable to introduce this characteristic from other species, but as plant biotechnology develops, such techniques as embryo rescue and marker assisted breeding may hold the key to new variation.

Oilseed rape breeding requires new genetic variability which can be obtained by interspecific hybridisations from wide crosses. There have been many such examples, with one of the initial attempts conducted by Roy (1983) who showed partial transfer of shatter resistance from B. juncea to B. napus. Prakash & Chopra (1988) also used these species, although they carried out a number of backcrosses to maintain the high quality B. napus characteristics in conjunction with increased shatter resistance. Unfortunately seed fertility was far too low for commercialisation.

Strategies also include anther and microspore culture which enable the rapid production of highly variable populations for use in conventional and genetically engineered breeding programmes. A further aspect is by irradiation to induce mutant strains which may show improved shatter resistance, such as the gamma irradiation experiment carried out by Luczkiewicz (1987), resulting in highly fertile pollen grains and improved shatter resistance of the mutant siliqua.

Generally breeding programmes are assessed on the overall level of shatter resistance achieved, whereas targeting specific areas for improvement may hasten the process. Many examples exist, including the increase in size or number of vascular strands within the dehiscence zone (DZ), increasing the area of the DZ or by modification of pod wall thickness (ie. reduce desiccation induced stresses).

Thurling (1991) believed that the production of shatter resistant lines of B. napus, coupled with maintained agronomic qualities depends on a crucial number of architectural characters. This was devised based on many canopy factors, such as 'pod knocking' that enhances pre-harvest shatter which may be influenced by pod angles, length and width.

Engineering Shatter Resistance

Much of the molecular control regarding dehiscence is yet to be determined, so that manipulation at such a fine level is currently inconceivable, but approaches based for example on offsetting the initiation of pod shatter relative to seed maturation are being developed. Therefore all the maturation and senescence events can occur normally, but the co-ordination between seed dehydration and pod opening can be altered to prevent or delay pod shatter.

At present there are two main areas that are being investigated for delaying pod opening relative to seed maturation:

Altering the enzymic action on the middle lamella within the DZ as to reduce/prevent the associated loss of cohesion, followed by cell separation.
Altering the hormonal regulation (eg. phytochromes such as auxin and ethylene) of dehiscence.

These opportunities for manipulation can be augmented to produce transgenic crop plants via transformation of oilseed rape. Currently investigations are underway utilising an efficient transformation procedure, coupled with spectomycin resistance as a selectable marker:

Polysaccharide Hydrolase Antisense Investigation:
Jenkins et al (1996) identified an up-regulated endo-polygalacturonase (endo-PG, pectinase) gene localised to the cells of DZ and is responsible for the loss of pectic material (pectin depolymerisation) from their cell walls prior to dehiscence (Josefsson, 1968). This approach is aimed to prevent the increase in PG activity by antisense mRNA down-regulation. This involves the production of transgenic oilseed plants (via transformation) that contain a copy of a gene encoding the complementary sequence (ie. antisense) of the PG gene, thus their respective mRNA's are also complementary and so interact within the cell to effectively remove translatable PG mRNA from circulation. The antisense gene is placed under the control of a DZ-specific promoter to ensure selective suppression so that only the dehiscence related cells are affected.

As well as PG, cellulase (Beta-1,4-glucanase) is also known to increase in activity and is also associated with DZ cell wall degradation, so it too is an ideal enzyme for the study of its antisense down-regulation with respect to dehiscence.

Hormone Regulation:
Plant hormones, such as the phytochromes, auxin and ethylene are associated with the DZ. Auxin is known to play an important role in the timing of dehiscence (Chauvaux et al, 1997). The seeds produce auxin, the high levels of which correlate with the maintenance of the DZ, but during desiccation the auxin transport mechanism ceases, thereby removing auxin support which results in the DZ enzyme activation to cause middle lamella dissolution. Further experiments indicated that sustained auxin exposure (ie. sustained high levels in the pod) causes a delay in DZ cell separation, thus increasing their resistance to shatter. It has been proposed that the fall in auxin may act to enhance ethylene sensitivity, as ethylene can act to accelerate both maturation and senescence. Therefore one approach based on these findings is to sustain the level of auxin within the DZ by producing transgenic plants (via transformation) with prokaryotic genes integrated that code for auxin biosynthesis. Once again the expression of these genes will be regulated by a DZ-specific promoter (eg. PG promoter).

DZ Hydrolase Interference:
The increased enzymic activity associated with DZ degradation is another mechanism set for manipulation because inhibiting the cell activity would prevent the activation of such enzymes, consequently reducing middle lamella dissolution and improving dehiscence resistance. Investigations into the controlled expression of an RNAase (eg. barnase) following the differentiation of pod tissues are underway, as it is hoped it will prevent the cell wall breakdown, although the effects of total cell RNA removal could still be fatal!