Genetic transformation of Chrysanthemum using wild type Agrobacterium strains; strain and cultivar s

Genetic transformation of Chrysanthemum using wild type Agrobacterium strains; strain and cultivar s

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Plant Cell Reports (1991) 9:505-508 Plant Cell Reports Springer-Verlag 1991 Genetic transformation of Chrysanthemum using wild type Agrobacterium strains; strain and cultivar specificity Monique E van Wordragen 1, Jan de Jong 1, Hans B.M. Huitema 1,2, and Hans J.M. Dons 1 z Centre for Plant Breeding Research (CPO), P.O.box 16, 6700 AA Wageningen, The Netherlands z Present address: University of Agriculture, Department of Plant Physiology, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands Received November 5, 1990 - Communicated by H. L6rz Summary. To develop an Agrobacteriurn mediated transformation protocol for chrysanthemum we studied the transformation efficiency of commonly used A.tumefaciens strains on 14 genotypes by com- paring tumour size and frequency. One genotype was analyzed in detail using 14 strains of both A.tume- faciens and A.rhizogenes. Only a few genotype/strain combinations resulted in significant tumour for- mation. Especially 0-type strains were highly efficient. An 0-type strain was used to transfer genes for neo- mycine phosphotransferase (NPT II) and 13-glucu- ronidase (GUS) to a susceptible cultivar. Transfer of the GUS gene was confirmed by using Polymerase Chain Reaction (PCR). Key words: Agrobacterium Tumour induction - Dendranthema grandiflora - Polymerase chain reaction -13-Glucuronidase - Neomycine phosphotransferase Introduction The aim of our research is the genetic engineering of the cutflower chrysanthemum, which is globally one of the most important ornamental crops. Genetic transformation of dicotyledonous plants is still most efficiently achieved by using the natural gene transfer system of Agrobacterium. The susceptibility chrysanthemum to Agrobacterium has already been demonstrated (Miller 1975; DeCleene DeLey 1976), but there is no information on genetic variation in susceptibility among chrysanthemum cul- tivars and within a cultivar for various Agrobacterium strains. This is of interest because both the Agrobac- terium strain and the cultivar used might influence efficiency in recalcitrant species (Puonti-Kaerlas et al. 1989). Recently, the super~ virulent strain A28l was found to be very useful for transforming recalcitrant crops (Fillipone and Lurquin Offprint requests to." M. E van Wordragen 1989; Raineri et al. 1990). Strain A281 has a very broad host range and induces tumours that appear faster and are larger than tumours induced by other strains (Hood et al. 1986). The efficiency of poplar was enhanced 14 times with strains harbouring the essential vir genes of A281 (Pythoud et al. 1987). Because of these supervirulent characteristics we have included A281 in our experi- ments. The aim of the investigation presented here was: to study genetic variation in tumour formation, to analyse the susceptibility of a sensitive cultivar for 14 Agrobacteriurn strains and to investigate whether tumours are stably transformed. For this latter purpose the transfer and expression of the NPT II and GUS genes was studied. Material and Methods Plant material. For greenhouse experiments 6 week old cuttings of (Dendranthema grandiflora, TzveL) grown under long day conditions were used. For leaf explant transformations
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Genetic transformation of Chrysanthemum using wild type Agrobacterium strains; strain and cultivar s

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