TRANSLEG-EU concerted action on grain legume transformation to develop commercial applications

Introduction
The concerted action, TRANSLEG was proposed to coordinate research efforts on the development of efficient methodologies for genetic engineering of grain legumes in Europe in order to provide suitable applications to meet the breeder s objectives. This included:

• the determination of the current status of genetic engineering in grain legumes
• identification of the priorities in grain legume improvement
• evaluation of the availability and suitability of agronomically important genes.

Third meeting - December 1995
E. Teoule (INRA) reported that the robust system for somatic embryogenesis available at their lab for regenerating pea plants apparently is not suitable for pea transformation. Although a similar protocol was applied for transformation of pea which successfully works in Arabidopsis thaliana, it was found that transformation influences somatic embryogenesis in a negative way. The problems, however, were found to be associated with the procedure used to remove Agrobacteria after co-cultivation than in the infiltration method.

T. Pickardt from Berlin reported a comparison of three different Agrobacterium-based methods for pea transformation, namely the methods reported by Schroder et al.(CSIRO, Australia), Jan Grant (New Zealand) and the one described at the Copenhagen-meeting by Samantha Bean (JIC). At this time, when the experiment had not been completed, no transgenics had been obtained applying the methods of Schroder et al. and that of Grant et al.. Still some candidate cultures with putatively transformed plantlets obtained following the procedure from Bean were under investigation. It became that researchers differed in their ways of handling the material. Even minor differences like the thickness of the explants were found to influence the outcome of the experiment. This supported the need for joint work in the lab.

Pickardt also reported on ongoing research on faba-beans in his lab and demonstrated, that the mannose-system works in his hands for the selection of faba-bean protoplasts. This led to further discussion of the need for the organisation of a ring-test was again discussed. In an initial phase, the University of Naples agreed to provide and demonstrate their know-how on tisuue-electroporation and the University of Hannover would do the same for their protoplast-based system.

G. Ramsey (SCRI, UK) reported on their attempts to develop an in vivo transformation system for Vicia faba. Previous work on factors which influence the transient expression of GUS has shown, that the apparent enhancement of the plant-Agrobacterium interaction is repeatable. However, the difficulties in maintaining Vicia faba tissues in vitro for protracted periods led to the consideration of in vivo methods for transformation. Increasing efficiencies of Arabidopsis in vivo transformation protocols and a published report of a similar successful report in the legume Arachis hypogaea encourage such an approach. Large-scale experiments have been carried out but persisting Agrobacteria were found to cause detrimental effects (swelling and disruption of meristem growth). A number of control methods were tried and copper-oxychloride (an agent used to control the disease caused by wild-type Agrobacterium tumefaciens, crown gall) was found to be effective and to allow relatively normal growth of meristems in seedlings following wounding and inoculation. Screening this material for transgene expression was in progress.

AP Goldsbrough reported on recent results on the production of transgenic pea plants. In average a transformation efficiency of 1% was calculated. However, under the selection procedure described, chimeric plants were also observed. The experiments were restricted to the genotype Puget and will be adapted for other varieties.

Clara Conicella (CNR, Portici), reported on the difficulties and achievements on wide crosses in pea. Crosses between P. fulvum and P. sativum were analysed cytogenetically. Following several back-crosses, lines were identified with agronomically interesting traits. The group now intended to use in-situ hybridisation techniques for further characterisation of hybrids.

Sergio Lucretti from Rome discussed the use of flow-sorting and high-resolution flow-karyotyping of faba-bean and pea chromosomes. He reported on his recent research, potential difficulties and recent achievements and explained the potential of this technique in setting up chromosomal gene-libraries and the mapping and isolation of agronomically important genes.

Alessandro Pellegrineschi introduced IITA's research on wide crosses and genetic engineering in cowpea. He pointed out, that this legume is a protein source for about 400 million people and that there is a high demand for introducing insect resistance. He reported on his attempts in developing a transformation system and presented promising results on both, protoplast as well as Agrobacferium-mediated transformation.

Edgardo Filippone presented recent results of his group on tissue electroporation in cowpea. The results presented also support the necessity of a ring-test, since results differed between the lab in Napoli and the cooperating partner in the USA.

Hans Weber from Gatersleben present data from his research group aimed to answer the question how carbohydrate and protein deposition is linked together and how embryo development is regulated by these processes. In this context he also presented the strategy and objectives of the EC-funded UNCLE-project, involving Gatersleben, John Innes Institute and INRA.

M. Pineda, reported on the isolation of genes encoding asparaginase and uricase, responsible for asparagine catabolism and allantoin synthesis, respectively. He explained, that the group is interested in the identification of molecular factors controlling the shift between ureide and amide-synthesis in legumes. Although, the advantage of these shifts is not apparently clear, one may speculate that this can affect biological nitrogen fixation.

The Coordinator, H-J Jacobsen reported on the characterisation of a novel cell wall protein, designated p40. This protein is located in the epidermal cell layer and is abundantly expressed in elongating tissue and repressed in non-elongating or elongated tissues. A recombinant antibody was produced, able to significantly inhibit elongation. The gene coding for this protein is suggested to be a good candidate to develop transgene approaches to control plant elongation.

During the general discussion it was concluded that, in addition to resistance to fungi, a better understanding of standing ability was of great interest to breeders. Increased stem stiffness by influencing lignin-biosynthesis or the mapping, isolation and introduction of dwarfing genes was suggested. In this respect, the potential of the p40 protein was also discussed. The general discussion closed with the decision to hold two ring sessions. In the first experiment, Prof. Filippone will host scientists from other participating labs in Portici to share his lab's experience with the in-vivo electroporation method, the second experiment will be carried out in Hannover to make the scientists from the TRANSLEG consortium familiar with the protoplast-based direct transformation system developed for pea in Hannover. As a mode to make the experiments feasible, it was proposed that each lab uses its own material and constructs, if possible. Following the experiments, each participant will carry his/her material back to the respective home lab for further evaluation. A first joint evaluation is scheduled for the next workshop meeting.