Effect of Agrobacterium co-cultivation time on recovery of transgenic Petunia hybrida plants

Carlson, Ceirra
Agrobacterium-mediated transformation genetically alters plant cells by transferring a DNA fragment (T-DNA) from the bacterial cell to the host species. This method is useful to insert traits of interest in plants for research and commercial purposes. Agrobacterium regrowth and subsequent culture contamination is a common problem leading to culture necrosis and poor recovery of transgenic plants. The goal of this research was to study the effect of Agrobacterium co-cultivation time on the recovery of transgenic cultures and plants in Petunia hybrida cultivar Mitcham. Petunia seeds were surface-sterilized in 50% bleach solution for 10 min followed by 3 washes in sterile distilled water and transferred to MS medium. After eight weeks of growth, leaf discs were obtained in vitro grown seedlings and used for co-cultivation. Agrobacterium strain 'EHA 105' harboring the green fluorescent protein (gfp) gene and MYBA1 gene coding for anthocyanin pigmentation, was grown overnight on a rotary shaker. Leaf discs were immersed in bacterial solution for 10 minutes and then transferred to Petri dishes containing MS medium. Leaf discs were co-cultivated for 24, 48 or 72 h and then transferred to MSP medium (MS medium containing 8.8 µM BA and 0.05 µM NAA) containing carbenicillin, cefotaxime and kanamycin antibiotics for inhibition of bacterial growth and selection of transgenic cells. Transient and stable gene expression frequency was recorded in various co-cultivation period treatments. A significant difference in transient gene expression frequency was observed between leaf discs co-cultivated for different periods of time. While no transient gene expression was observed for the 24 h treatment, significantly higher levels of transient gene expression frequency (60% and higher) were observed in leaf discs co-cultivated at 48 and 72 h. We are currently recording the number of transgenic shoots produced from each explant along with bacterial regrowth and subsequent contamination occurring in cultures. Transgenic plants obtained after rooting of shoots will be hardened and transferred to a greenhouse. Scanning electron microscopy will be used to study changes in plant morphology and anatomy between transgenic and non-transformed plants.
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