Agricultural Development and Environmental Research: American and Czechoslovak Perspectives: Proceedings of a Bilateral Workshop (1987)

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Gene Engineering and Transformation of the Plant Genome by Agrobacterium Vectors MILO§ ONDftEJ Institute of Experimental Botany (CSAV) Newly developed methods of gene engineering permit the analy- sis of the plant genome structure to the level of primary sequences of nucleotides in DNA. Such an analysis leads to the understanding of gene structure and the regulation of individual genes and gene bat- teries. An important part of such a complex study is the introduction of foreign genes into the plant genome. Among the several methods of introducing foreign genes, the most developed is the use of vectors derived from the Ti plasmid of Agrobacterium tumefaciens. This naturally occurring soil bacterium causes tumor growth in dicotyledenous plant tissues due to the integration of a particular segment of the Ti plasmid of A. tumefaciens (transferred DNA or T-DNA) into the plant genome (Chilton et al. 1977). The expression of T-DNA genes—in particular those coding for new pathways of auxin synthesis and cytokinin synthesis—causes dedifferentiation of plant tissues and their unlimited growth (Willmitzer et al. 1982). The dedifferentiating genes or oncogenes can be removed from T- DNA and replaced by other genes. These are then transferred to the plant genome, and the transformed tissues are capable of normal, undisturbed differentiation and morphogenesis. Several bacterial, viral, animal, and plant genes in intact or modified chimeric form have already been introduced into and expressed in the plant genome. In the study of the effect of T-DNA genes, coding for new path- ways of biosynthesis of phy tohormones provides a unique opportunity to study the effects of an endogenous increase of phytohormones on 165

166 several sites of plant hormone activity. Of interest are the tran- scription activity of the plant genome and the regulation of specific enzymes and many types of morphogenetic activities including initi- ation of flowering or differentiation of tubers. The study of the expression of introduced foreign genes other than T-DNA provides insights as to the process of regulation of individual genes. Of great importance are the practical outputs of this research, and particularly the possibility of enrichment of the plant genome by genes from unrelated organisms which can provide important properties to the cultivated plants. It is possible to construct new genomes of cultivated plants which are resistant to bacterial and fungal diseases and which provide higher yield quality, especially with regard to proteins. There are already encouraging results of the first field experiments with transformed tobacco plants, expressing the gene for delta-endotoxin from Bacillus thuringiensis. These plants show resistance to the moth Manduca sexto.. The number of cloned genes useful for improving the plant genome sharply increases, perhaps doubles each year. There is no doubt that this technology will change the concept of dicotyledonous cultivated plants for future plant breeding. The Department of Plant Breeding Theory of the CSAV Institute of Experimental Botany is studying the effects of integration of T- DNA into the genome of different dicotyledonous plant species from three points of view: • Introduction of the whole, unmodified T-DNA of different Ti plasmids of A. tumefaciens and A. rubi strains and integration of Ri plasmids of A. rhizogenes; • Introduction of single T-DNA genes; • Introduction of other genes. In most plant species, the introduction of T-DNA of A. tume- faciens to the plant genome induces undifferentiated tumors or teratomas—clumps of modified leaves and short, thick shoots—which never form roots but often form undifferentiated tumors. We have shown that the model cruciferous plant Arabidopsis thaliana is excep- tional from this point of view (Ondfej et al. 1984, Pavingerova et al. 1983, Pavingerova et al. 1984). Tumors derived by all Agrobacterium strains possess considerable ability to differentiate transformed plants and teratomas. They show the presence of T-DNA in their genomes by Southern blotting, and they possess opine synthesizing activities.

167 Opines are low molecular weight compounds produced by en- zymes which are coded by T-DNA. Plants differentiated from Ara- bidopsis thaliana with crown gall tumors never form roots but, due to their small size, they can be grown on the agar medium to the flower and seed stage. In the next generation, plants form roots, and they do not show phenotype deviations from controls. The opine synthesis markers (octopine and agropine) segregate in most of the clones in a 3:1 ratio which demonstrates that T-DNA is present in the Arabidopsis genome at a single site. In the next generation of opine positive plants, however, the proportion of opine synthesizing plants was always lower than theoretically expected at a 5:1 ratio. In still the next generation, opine synthesis was shown only by a small proportion of plants, but most opine negative plants showed the presence of T-DNA by the Southern blotting test. DNA methylation was therefore suspected as the probable cause of the disappearance of opine synthesizing activity. To test this hypothesis, a seed sample was sown on the agar medium containing different concentrations of the DNA demethylat- ing agent 5-azacytidine. This agent caused a sharp increase of the proportion of opine synthesizing individuals. It demonstrated that the methylation of cytosine in T-DNA is the cause of deactivation of opine synthesizing genes. The T-DNA of A. rhizogenes strains induces a proliferation of transformed roots (Chilton et al. 1982). These roots, like crown galls, are capable of unlimited growth on agar media without the addition of growth regulators, external auxines, or cytokinines. Roots are capable of regeneration of plants (Ondfej and Biskova 1986). We have used A. rhizogenes strains, adapted as binary vectors (Ondfej et al. 1986). In addition to their Ri plasmid, they also contain smaller vector plasmids, which possess T-DNA border regions necessary for the integration into the plant genome and chimeric in plant cells expressing kanamycin resistance gene (An et al. 1985). This gene was transmitted and integrated into the plant genome together with T-DNA of the plasmid Ri. Root cultures were derived in our laboratory from several plant species: tobacco, petunia, potato, pea, and Atropa belladonna. All roots contained high quantities of the opines agropine and mannopine. If the small plasmid of the binary vector pGA472 was also involved, most of the root cultures were able to grow on high concentrations of kanamycine. Atropa belladonna and tobacco roots spontaneously

168 formed plants. Atropa belladonna root cultures, which produced al- kaloids, were capable of plant regeneration even after two years of in vitro culture (Ondfej, Protiva 1987). In petunia roots, we have shown the karyotype stability of meristems (Ondfej, Biskova 1986). Tobacco plants regenerated from roots did not show any morpholog- ical deviations. Opine-synthesizing abilities were found in both anther calli and seedlings. Regenerated plants were studied from the point of view of the growth and photosynthetic activity. Growth was slower than in untransformed controls. Photosynthetic activity was not decreased, but the respiration rate was increased, which explains the observed growth retardation. The third species studied—Agrobacterium rubi, defined on the basis of numerical taxonomy—has Ti plasmid, which is comparable in size with those of A. tumefaciens (about 200 kb). The behavior of so-called "cane gall tumors" induced by A. rubi has never been studied before. This species was believed to be specific for the genus Rubus; however, we have induced tumors with differentiated plants on tobacco, petunia, and potato. We have also studied the properties of the opine synthesizing enzyme lysopine dehydrogenase. This work, with the use of full-length T-DNA, is nearly finished. At present, we are interested in vectors which do not integrate onco- gens into the plant genome, but only selectable genes for kanamycin resistance together with other cloned genes. We are interested in integrating virus cDNA sequences into the plant genome, which can introduce virus disease resistance to the genomes of cultivated plants by several mechanisms: • By transcription of anti-RNA, which is capable of forming double strands with mRNA tn vivo. These double strands cannot be translated, and virus reproduction is thus blocked. • By integration and expression of the coat protein gene. The coat protein has already been demonstrated to induce resistance by a mechanism related to cross-protection. • By integration of the cDNA sequence, which codes for satellite RNA of the virus. There is particular interest in potato viruses, viroid cDNA se- quences, and in cauliflower mosaic virus. Cooperation in this field with scientists from the United States would be welcome.

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