Transgenic carnations obtained byAgrobacterium tumefciens-mediated transformation of leaf explants

Abstract

For the development of an Agrobacterium-mediated transformation procedure of carnation (Dianthus caryophyllus L.), an intron-containing ~-glucuronidase (gus) gene was used to monitor the frequency of transformation events soon after infection of leaf explants. The efficiency of gene transfer was dependent on the carnation genotype, explant age and cocultivation time. Leaf explants from the youngest leaves showed the highest number of GUS-positive spots. After selection on a kanamycin-containing medium, transgenie shoots were generated among a relatively high number of untransformed shoots. The selection procedure was modified in such a way that the contact between explant and medium was more intense. This improved the selection and decreased the number of escapes. Kanamycin-resistant and GUS-positive plants were obtained from five cultivars after infection of leaf explants with the supervirulent Agrobacterium strain AGLO. A higher transformation frequency was observed with the binary vector pCGN7001 than with the p35SGUSint vector. Integration of the genes into the carnation genome was demonstrated by Southern blot hybridization. The number of incorporated T-DNA insertions varied between independent transformants from one to eight. Transformants were morphologically identical to untransformed plants. Segregation of the genes occurred in a Mendelian way.

Direct and reverse pollen-mediated gene flow between GM rice and red rice weed

“…(1987) but using a modified extraction buffer described by Van Altvorst et al . (1995) and reducing the ferrocyanide solution 10-fold.…”

Somatic Embryogenesis and Genetic Transformation of Carnation (Dianthus caryophyllus L.)

“…Genetic engineering is a valuable tool, currently used in classical breeding methods for the production of novel carnation varieties, and these technologies have been exploited to introduce new traits into commercial cultivars. In the last 20 years, Agrobacterium tumefaciens -mediated transformation systems have been developed using different plant explants like leaves, petals, and stems Van Altvorst et al 1995, 1996Yantcheva et al 1997b ;Nontaswatsri et al 2004 ). Direct gene transfer by using microprojectile bombardment or with Agrobacterium was also successfully conducted (Zuker et al 1995(Zuker et al , 1999 in creating transgenic carnations with novel traits.…”

Transgenic carnation plants obtained by Agrobacterium tumefaciens-mediated transformation of petal explants

“…The quality of regenerants was less compared to noninfected petal regenerants, which might be due to infection. Twelve weeks after infection, the remaining 70 plants were assayed for/3-glucuronidase activity (as described by Van Altvorst et al, 1995) of which two were positive. One GUS-positive plant, PT-1, resulted from infection with AGLO(p35SGUSint) and the other, PT-2, from infection with AGLO(pCGN7001).…”

“…To analyze the integration and copy number of the genes, a Southern blot analysis was performed described by Van Altvorst et al (1995). Genomic DNA was isolated and digested with EcoR1 or HindlII.…”

“…In spite of the potential of petal explants to obtain transgenic plants after A.tumefaciens infection, they are not suitable for transformation due to the hyperhydricity and premature flowering of the regenerants. Leaf explants should be used instead of petal explants forAgrobacterium transformation as the former are much more efficient and produce healthy plants that can be easily transferred to the greenhouse(Van Altvorst et al, 1995).Internal fragment Number of fragments…”