GENETIC ENGINEERING FOR RESISTANCE, QUALITY AND PLANT HABIT

We are now able to obtain transgenic shoots of chrysanthemum cv ‘1581’ within 6 weeks. The efficiency varies from 10 to 60 %, depending on unknown factors such as the construct used. This level of efficiency was reached step by step by continuously improving the media and the selection procedures. Other cultivars are transformed at lower rates and about 50 % of the cultivars tested cannot be transformed at all with the protocol used. Cultivar specificity remains an obstacle and alternative protocols, particularly those that do not rely on regeneration, are much needed.

Initially we failed to obtain a measurable level of expression of the crystal protein gene in chrysanthemum, even though control transformations in tobacco gave the resistance expected. The problem was solved by replacing the 35S promoter by a chrysanthemum specific promoter and the use of a fully synthetic CryIC gene for better transcription and translation by the plant. Designing constructs for optimal expression in the plant will be the challenge for the coming decade. The use of constitutive promoters will be restricted in favor of promoters that control time and site of expression. Genes will be redesigned for improved expression in the plant.

Genetic modification, as it is done up to now, with random insertion of the transgene, results in variation in expression and selection is needed afterwards to identify the desired genotype. Variation in expression of the transgene may be reduced and expression increased by flanking the transgene with Matrix Attached Regions. Still, variation remains and selection is required. Site directed insertion is the next step on the road to controlled expression.

The gene itself may be improved considerably over the wild type as work with the Crystal protein genes from Bacillus thuringiensis has shown. Initially, codons were changed for better expression in the plant, later, gene fragments were recombined for improved toxicity. Site directed mutagenesis, coupled with efficient selection procedures is the next step in protein breeding

A major obstacle in commercial application of the CryIC based resistance to Florida moth is the complicated patent situation in the Bt technology. It requires a major effort to identify the relevant patents and obtain licenses from the patent holders. Other genes of interest are, at this time patented in less detail, but they will ultimately all reach the level and complexity of the Bt gene.

Not only is molecular breeding coming of age, the classical aims of resistance to herbicides, insects, or viruses and in the case of flowers, color are now being replaced by other applications such as quality or more sophisticated control of expression. The latter is especially important in biotechnological approaches to modify plant form. Plant architecture is to a large extent controlled by the plant hormones. Changes in hormone dynamics usually have a multitude of effects and controlled expression of the iaa, ipt, etr

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