T-DNA Transfer and GUS Expression in Agrobacterium-mediated Transformation of C. annuum under a Range of in vitro Culture Conditions

Several factors that affect the survival rate and the effectiveness of Agrobacterium-mediated transformation which resulted in the expression of gus gene in transgenic C. annuum were investigated. The initial investigation on seed treatment has shown that intact seeds exhibited the highest rate of generation with 96.33%. Analysis of preculture treatment revealed that two days incubation on wet filter paper resulted in highest number of explants with GUS expression. No significant differences on percentage of GUS expression were observed on preculture with shaking at 120 rpm and exposure at 4°C which showed the highest of only 17% of explants with GUS activity recorded. Analysis of Agrobacterium and mature seeds interaction involved three Agrobacterium concentration for the inoculation phase, three co-culture and two incubation regimes. No differences in explant survival and GUS activity were observed between the two incubation time tested. The OD reading of 0.55 was shown to be the optimum inoculation condition for both explants survival and GUS activity. Analysis of co-culture regimes indicated that the shorter co-culture times resulted in higher explant survival and higher GUS activity, whereas the co-culture time of five days severely reduced the survival of explants and lowered the GUS potential. Selection of regenerated transgenic plants was performed on hygromycin-containing medium. The presence of the T-DNA was confirmed by Polymerase Chain Reaction (PCR) amplification of the internal fragment of gus and hpt genes. Enzymatic GUS assay confirmed the expression of the GUS-intron gene in the primary transformants as well as the progeny. The Chi-square analysis for segregation of Hyg-B resistance on at least 10 T1 progenies revealed that the transgene was inherited as a single or two mendelian locus.