AGROBACTERIUM - MEDIATED TRANSFORMATION OF SECONDARY SOMATIC EMBRYOS FROM ROSA HYBRISA L. AND RECOVE

AGROBACTERIUM - MEDIATED TRANSFORMATION OF SECONDARY SOMATIC EMBRYOS FROM ROSA HYBRISA L. AND RECOVE

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70 AGROBACTERIUM - MEDIATED TRANSFORMATION OF SECONDARY SOMATIC EMBRYOS FROM ROSA HYBRIDA L. AND RECOVERY OF TRANSGENIC PLANTS A. Borissova, T. Hvarleva, I. Bedzhov, V. Kondakova, A. Atanassov, I. Atanassov AgroBioInstitute, Sofia, Bulgaria ABSTRACT A genetic transformation protocol, based on co-cultivation of Rosa hybrida L. secondary embryos with Agrobacterium was established. Transgenic phosphinothricin (ppt) - resist- ant plants were obtained through application of interrupted selection procedure. Follow- ing the Agrobacterium inoculation step, the somatic embryos were initially cultivated on ppt - free media containing cefatoxim for elimination of the bacteria and later transferred on media containing the selective agent. The ppt - resistant secondary somatic embryos were matured and regenerated on ppt - free media and the putative transgenic plants were subsequently selected on media contained ppt and chlorphenol red. The performed PCR - and PAT protein - assays demonstrated presence and expression of bar gene encoding the phosphinothricin acetyltransferase (PAT) in part of the selected plants. The possibilities for application of the established transformation protocol are discussed. Introduction Contemporary commercial requirements on roses are focused on a change of decorative qualities including color, size, shape, hue, higher yield, longer vase life, as well as biotic and abiotic stress resistance (1). Rose improvement through the classical genetic and breeding is limited because the com- plex structure of rose genome and difficul- ties for recovery of the desired phenotype, changed at crossing. Recently established technologies for genetic transformation of Rosa sp. offer an attractive alternative for introducing of genes of interest (2, 3). Both transformation approaches co-cultivation with Agrobacterium (4) and biolistic trans- formation (5) were successfully applied. In spite of the obtained results, the reported transformation protocols are genotype de- pendent and confer relatively low trans- formation efficiency. That’s why the de- velopment of new transformation proce- dures based on different genotype and re- generation system, as well as utilization of easy reproducible starting plant material will further enhance the application of gene transfer technology in rose improvement. In the previous studies on rose somatic em- bryogenesis at AgroBioInsitute, we deve- loped efficient system for reproducible secondary somatic embryogenesis and plant regeneration for two R. hybrida cvs. “Anny” and “Saltze Gold”. In this paper we report the development of efficient transformation protocol based on co-cultivation of secondary somatic em- bryos with Agrobacterium and recovery of the ppt -resistant transgenic plants through interrupted selection scheme. Materials and Methods
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This note was uploaded on 08/01/2009 for the course HORT hor-11-12 taught by Professor Park during the Spring '09 term at A.T. Still University.

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AGROBACTERIUM - MEDIATED TRANSFORMATION OF SECONDARY SOMATIC EMBRYOS FROM ROSA HYBRISA L. AND RECOVE

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