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Optimization of transformation in embryogenic cultures of oak (quercus robur L.) and sitka spruce (picea sitchensis Bong Carr; )

Dublin City University. School of Biotechnology
Publication Date
  • Biotechnology
  • Botany
  • Genetic Engineering (Botanical)
  • Cell Cultures
  • Antibiotic Resistant Transformed Cells
  • Chemistry


Methods were investigated for the transformation of embryo cultures of Oak and Sitka Spruce with Agrobacterium tumefaciens and for their subsequent maturation to embryo derived seedlings. This involved the optimization of a range of parameters which are known to have an effect on the production of transformed cells, the determination of selective conditions for the removal of Agrobacterium tumefaciens and for the selection of antibiotic resistant transformed cells, and investigations on the interactions of a wide range of parameters known to affect maturation of embryos to seedlings. The isolation of cultures derived from single cell and small cell aggregates (<14 cells) was also examined as a means to permit all cells to be accessible to Agrobacterium and reduce the proportion of chimeric cell colonies containing transformed and nontransformed cells. Embryogenic cell lines were inoculated with a supervirulent strain of Agrobacterium tumefaciens LBA4404 ::pB I121. The latter contained a gene for a visible selectable marker, (3-glucuronidase and a marker gene for antibiotic resistance, np t II gene. Transient gene expression was determined histochemically by recording the number of distinct areas of p-glucuronidase (GUS) activity. Experiments to optimize the transformation parameters (bacterial dilution, incubation time, co-cultivation time and acetosyringone concentration) were carried out. Maximum expression of the GUS gene • 9-3 was achieved with a bacterial suspension with an OD 6OO o f 0.8-1.1 (c.a. 10 cells cm ' ) diluted with an equal volume of MS or Sitka Spruce embryogenesis initiation medium for Oak and Sitka Spruce respectively, in oculation of cells with bacteria for 180 m in and 60 min for Oak and Sitka Spruce respectively with a 72 hco-cultivation period and exposure o f Agrobacterium and plant cells to 25 f iM acetosyringone. Cefotaxime is the most common ly used antibiotic for thee limination of residual Agrobacterium following transformation. The growth of Oak and Sitka Spruce embryogenic suspension cultures in liq u id medium containing the antibiotic cefotaxime was investigated. Agrobacterium was eliminated from cultures following co-cultivation with 500 mgdm '3 cefotaxime antibiotic. This concentration had no effe ct on the plant ce ll g rowth. Growth of Oak and Sitka Spruce embryogenic suspension cultures in liquid medium containing kanamycin and paromomycin antibiotic s was examined as a method of selecting cultures expressing the nptII••3 gene following incubation with the Agrobacterium strain. Paromomycin at 30 mgdm' and at 3 mgdm'3 was strongly inhibitory to the growth of embryogenic suspension cultures of Oak and Sitka Spruce respectively and these levels were used for the selection of transformed cells following infection with Agrobacterium. Regeneration o f Oak embryogenic callus with a root and shoot was achieved o n P24 maturation medium 1 in the light. This contained 1% Activated charcoal without plant growth regulators. Cultures were previously cultured on P24 medium plus 0.9 |xM BAP. Sitka Spruce embryonal suspensor masses were easily regenerated in to seedlings using culture of embryos on embryo germination medium and embryo development medium from previously published reports. Cultures derived from single cells from bo th Oak and Sitka Spruce embryogenic • * “3 suspension cultures were produced by exposing cultures to a pectinase (0.07gdm‘ ) digestion. Sitka Spruce single cells required a Percoll density separation (19%) step to separate the single cells derived from the embryogenic head from those derived from the suspensor cells. Oak single cell cultures could be more easily achieved by reducing the BAP concentration in MS medium by 100-fold to 0.01 mgdm 3. Single cells were regenerated back to micro-calliby increasing the BAP concentration to 1.0 mgdm'3. Oak single cells were transiently transformed in a 50% Agrobacterial dilution for 60 min with a 72 h co cultivtion period with both Agrobocterium and plant cells exposed to 25 | jM acetosyringone. Agrobacterium was eliminated with 500 mgdm'3 cefotaxime antibiotic. Cultures derived from single cells provide a source of cells for transformation which can be regenerated to micro-calli and subsequently to embryos without the problems associated with protoplast cultures.

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