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It is the purpose of this project to develop the technology required to engineering shatter resistance into oilseed rape using the tools of molecular biology. Transgenic oilseed rape with a reduced tendency to shatter may be prepared for exam-ple by offsetting the initiation of pod opening relative to the maturation of the seeds. In other words, all maturation and senescence processes may be allowed to proceed as in normal plants, only the timely coordination between seed dehydration and pod open-ing is severed and pod opening is delayed relative to seed maturation.

Two physiologically distinct approaches towards obtaining a selective delay of pod opening relative to seed maturation are being investigated:

- interference with the enzymes that bring about the physical separation of the carpels
- interference with the hormonal regulation of the pod opening process in broader terms.

These strategies are linked in theory by the fact that the enzymes studied in the first strategy are subject to the over-all regulation of the process as studied in strategy two. In practice, when it comes to engineering the traits, they become much more interdependent.
In a move to ensure exploitation of the results Plant Genetic Systems was made a partner of the project (originally there were no industrial partners in this project). Plant Genetic Systems stepped into the current AlR-project for a number of reasons, the most pertinent being a pressing need to submit a patent-application in order to ascertain the economical viability of the applied aspects of the project. This has now happened with a patent that amply reflects the broadening of the project as it developed during the first two years.


So far a solid foundation for two main approached aimed at engineering shatter resistance has been established. A rape transformation procedure is in place, and the first generation of are being developed.
Pod shatter denotes the opening of oilseed rape silques and dispersal of the seeds as they mature. Flowering and seed shatter is not synchronous in rape; hence there is no such thing as the "correct harvest time". Immature seeds contain more moisture and more chlorophyll both of which lead to reduced oil quality. Late harvest dates leading to less immature seeds is often accompanied by loss of yield and problems with fighting run-away rape with chemical herbicides in the following year. Swathing reduces the risk that wind bring about excessive shattering of seeds from the most mature pods while allowing the younger ones to catch up to some extent. Timing of swathing is very critical and it is not always that yields are greater than the potential yield from a crop harvested by direct combining.


These include an examination of the roles of endo-polygalacturonase in dissolution of load bearing structures holding the carpels together and a detailed examination of the roles of auxins and ethylene and its precursor on the initia-tion or stimulation of pod opening. Such studies have enabled the possibility of breeding greater shatter resistance in transgenic plants, based on key elements of the pod opening process, based on interference with pod opening.


Both main strategies, indicated above, aim to interfere with developmental events in the so-called dehiscence zone, a few layers of thin walled cells along the edges of the two valves of the pod. The cells in this zone eventually separate and allow the pod to open. It is possible to interfere in either late, or early events. Interference may be directly with a processes directly, or be in regulation. In either case the objective is to override develop-mental events in the dehiscence zone. Rape transformation is essential for all strategies. Hence, a rational and efficient transformation procedure, using spectomycin resistance as the selectable marker, has been developed.

Polysaccharide hydrolase-antisense approach
Anatomical studies have shown that the middle lamella between the dehiscence zone cells eventually dissolves while the primary cell walls of the same tissue softens and looses wall material. Biochemical studies have demonstrated the presence or accumu-lation late in development of a range of enzymes that may play a role in the weakening of the tissue so that the pods eventually open. Among these , most research efforts have been applied to endo-polygalacturonase (endo-PG) which is believed to take part in depolymerization of the middle lamella. One endo-PG, among the several that occur in rape has been cloned. It has been found that this isoform is expressed in the dehiscence zone immediately preceding and during pod opening. An antisense strategy, that entails production of transgenic rape plants in which the expression this enzyme is suppressed selectively, has been adopted so that only pod opening and not other processes that depend on related genes are disturbed. The gene in hand will be used to make the first generation of transgenic plants and thus establish the feasibility of the polysaccharide-antisense approach.

Hormonal regulation approach
Investigations of endogenous hormones in various pod tissues in combination with applications of synthetic growth regulators have produced a very interesting picture. Evidence have been produced that the dehiscence zone remains intact as long as the auxin concentration in the dehiscence zone cells is at a sustained high level. The use of par-thenocarpic pods (pods that have been induced to develop without seeds inside), has shown that the seeds is indeed the main source of the auxin influencing the dehiscence zone cells. Dur-ing dehydration auxin transport ceases from the seeds and the dehiscence zone cells respond accordingly. One of the responses may well be that the tissue becomes receptive to ethylene, an accelerator of maturation arid senescence processes. The strategy depends on equipping the dehiscence zone cells with their own auxin bio-synthetic machinery, so that they become independent of seeds as source of auxin, and hence will not acquire sensitivity to ethylene. Removal of the ability to respond to ethylene is expected to delay pod opening.

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