Agrobacterium-mediated gene transfer to chrysanthemum

M.F. van Wordragen

Research output: Thesisexternal PhD, WU


<p>Genetic manipulation of plants is a technique that enables us to add to the plant genome, in a precise and well controlled manner, one or a few new genes, coding for desirable traits. In contrast to this, the conventional method for the introduction of new properties in plants, by cross breeding, is a random process in which two complete genomes are mixed and the desired phenotype has to be regained by repeated back crossing with the cultivated parent line. Despite these differences, both procedures basically accomplish the same; the addition of new inheritable characteristics to the genome of a plant. If both are available, the choice between molecular or conventional breeding for the introduction of a trait is often determined by the unique advantages and disadvantages of the techniques. Though the development of protocols is very laborious, genetic manipulation is in principal faster than cross breeding, because of the reduced need for back crossings. On the other hand, cross breeding is still very successful in introducing traits, which can only be recognized by phenotypic expression. Genetic modification requires precise knowledge of the gene involved, and as this knowledge is still very limited, only a few genes are available. The most important advantage of genetic modification is the fact that it is not hampered by crossing barriers. Therefore, the technique opens the possibility to introduce genes even from outside the plant kingdom into crops. This offers new opportunities to develop crop genotypes, resistant to pests and diseases, that formerly could only be controlled by the (often excessive) use of chemical pesticides and insecticides. Therefore, much research effort has been put in the development of genetic modification protocols for a wide range of plants. Examples of genes that have been successfully applied in this respect are: viral coat protein genes, which confer resistance to various viral diseases, proteinase inhibitor genes and <em>Bacillus</em><em>thuringiensis</em> toxin genes, which both confer resistance to feeding by a wide variety of pest insects. Other options, which are less interesting from an environmental point of view, but important for growers, are e.g. the introduction of new flower colours and elevation of food value by directing the synthesis of nutritious proteins.<p>The aim of the research described in this thesis, was the development of a geneticmanipulation protocol for the ornamental crop chrysanthemum, employing the natural gene transfer capacity of the soil bacterium <em>Agrobacterium tumefaciens,</em> and the introduction of insect resistance genes derived from the insecticidal bacterium <em>Bacillus thuringiensis</em> (Bt). Though, until now, no Bt crystal proteins are known that are specifically toxic against the major pests in chrysanthemum culture (e.g. thrips, leaf miner and red spider mite), some minor pests, e.g. the Florida moth ( <em>Spodoptera</em><em>exigua)</em> are within reach. Moreover the development of genetic modification protocols for an ornamental crop in itself is important, in view of the large arrearage compared to applied biotechnology in vegetable crops (chapter 7).<p>All genetic manipulation protocols, including the <em>Agrobacterium</em> mediated transformation, must fulfill two conditions: it should be possible to stably introduce a new gene in a plant cell and to regenerate a complete plant from that single altered cell. The genotype 'Parliament' that we chose as starting material seemed to meet both prerequisites. Gene transfer by several <em>Agrobacterium</em> strains was demonstrated by tumour induction in vivo and in vitro (chapter 2) and several direct regeneration protocols starting from diverse types of tissue had already been developed.<p>A difficulty was the fact that induced tumourous outgrowths were sometimes not really tumours, or were the result of a gene transfer process, with a very low efficiency (chapter 3, chapter 4). It appeared that tumour-like tissue resulted even from a slight disturbance of the apparently very narrowly tuned hormonal status of 'Parliament'. Thus, very few gene transfer events, which needed not to be stable, were sufficient to induce cell proliferations. These findings were done by utilizing a newly developed reporter gene, the intron containing β-glucuronidase gene. This gene allowed the rapid analysis of transformation events, shortly after infection. Previously this type of analysis was done by counting the number of transformed shoots or calli that were formed, a time consuming process and moreover a process that is virtually useless when the transformation efficiency is very low, as was the case in transformation of 'Parliament'. The new reporter gene allowed the screening of a range of chrysanthemum genotypes and the investigation of the effects of changes in the protocol and of the use of different <em>Agrobacterium</em> strains (chapter 4, chapter 5).<p>This work resulted in the selection of a few readily transformable genotypes and the preferential use of the supervirulent <em>Agrobacterium</em> strain A281 or its disarmed derivative EHA101. This part of the research is still being continued at the Centre for Plant Breeding and Reproduction Research (CPRO).<p>A second problem we met was the fact that the efficient regeneration of adventitious shoots in 'Parliament' was severely or even completely inhibited by infection with <em>Agrobacterium. A</em> recent publication, by Ledger et al., in which a different chrysanthemum variety was transformed, also stressed the importance of highly efficent regeneration for succesful <em>Agrobactetium</em> -mediated gene transfer. Further investigations revealed that inhibition of regeneration due to infection was a general problem in chrysanthemum. However, the phenomenon turned out to be partly genotype dependent, which enabled us to select for less sensitive cultivars. Also, procedures were developed to diminish the repressive effect of infection on the regeneration (chapter 5). It was recognized that the detrimental effect on regeneration was caused by the superimposement of infection stress and wound stress. Therefore, adaptations of the procedure were aimed at the reduction of stress, either by omitting brushing of the leaf explants prior to cocultivation, or by separating explant excision and infection in time, by preculturing the explants for eight days before infection. the results of this research suggest that the inhibitory effect of <em>Agrobacterium</em> infection might be partly responsible for low transformation efficiencies obtained in recalcitrant crops. However, since the control for regeneration often consists of uninfected explants instead of explants infected with disarmed strains, this phenomenon might have escaped attention in many studies.<p>In the course of our studies a successful transformation protocol for chrysanthemum was reported by Dr. C. Lemieux, at DNAP, California. The protocol has been reported on congresses, but is not yet published in literature. Though detailed information is unfortunately not available, which hampers comparison of their procedure with ours, it did become clear that the explant source might be of crucial importance. In the procedure described in this thesis we used leaf explants, taken from in vitro grown plants. Regeneration of shoots is very efficient and shoots develop directly from the explants, without intermediate callus production. From the results of Lemieux, it was apparent that a callus phase before regeneration was essential. This is best achieved if explants from greenhouse grown plants are used. This option is now being explored at the CPRO.<p>Expression of transgenes in plants is influenced by many factors. This also holds for Bt toxin genes, for which recent analyses in several plant species have revealed poor expression of the protein. This might be caused by the presence of poly-adenylation signals and other plant regulatory sequences within the coding sequence, leading to mRNA instability and reduced translation efficiency. This latter phenomenon may be deteriorated by the bacterial codon use, which is different from the preferential codon use in plants. This information, necessitated the investigation of the level of expression and biological activity of Bt genes in chrysanthemum, even though a transformation/ regeneration protocol was not yet available. To explore the attainability of insect resistance in chrysanthemum by the introduction of Bt genes, we introduced the <u>cry</u> IA(b) gene in <em>Agrobacterium</em> induced tumours. In that way it was possible to analyse the expression and translation of the foreign gene in chrysanthemum cells, and moreover to assess the insect resistance of the transgenic tissue.<p>A bioassay was developed for larvae of the tobacco budworm, <em>Heliothis virescens,</em> with which the effect of feeding with tumourous chrysanthemum tissue on the growth and development of larvae could be measured accurately. In view of the expression problems pointed out above, the bioassays were remarkably successful. In some tumour lines a complete resistance to feeding by larvae of <em>Heliothis virescens</em> (tobacco budworm) was reached (chapter 6). Several other lines showed intermediate growth inhibition of the larvae and some lines were not resistant at all. These results indicate that it will eventually be possible to introduce insect resistance in chrysanthemum by utilizing the toxin genes from <em>Bacillus thuringiensis.</em>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • van Kammen, A., Promotor, External person
  • Dons, J.J.M., Promotor, External person
Award date19 Nov 1991
Place of PublicationS.l.
Publication statusPublished - 1991


  • ornamental plants
  • genetic engineering
  • recombinant dna

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