Agrobacterium rhizogenes ATCC 15834 wild type strain was transformed with the binary vector pBI121 using the heat shock method. The transformed Agrobacterium was then tested for virulence through tobacco leaf explant transformation. Compared to the non-transformed Agrobacterium, the transformed Agrobacterium showed reduced virulence, producing significantly lower number of hairy roots in tobacco leaf explants. Although the transformed Agrobacterium showed reduced virulence, it was able to transfer the T-DNA of the binary vector into the plant genome, resulting in stable GUS expression in the generated hairy roots. This indicated that in addition to the transfer DNA (T-DNA) from its root inducing (Ri) plasmid, the transformed Agrobacterium is also capable of transferring the binary vector T-DNA and allowing the integration of a foreign gene. Results also showed that hairy root generation efficiency of the transformed Agrobacterium varied with the concentration of the selection agent (kanamycin). Hairy root generation efficiency (hairy roots·explant-1) progressively increased with decreasing concentrations of kanamycin; and the efficiency was highest in the absence of kanamycin. Generated hairy roots showed very strong to tiny GUS expression even those that grew under the highest concentration of the kanamycin (50 mg·L-1). This indicated that co-transformation and efficient transgene expression does not always occur.
Capsicum (pepper) species have high economic values as vegetable crops and medicinal plants. Most of the Capsicum is known to be recalcitrant to plant regeneration in vitro, and to genetic transformation with Agrobacterium tumefaciens. However, genetic improvement against pathogens requires discovering new pest resistance genes and revealing their functions and mechanism in vitro. The development of improved transformation methods serves this purpose, which needs a binary vector technology carrying the gene of interest to be transferred into the host plants. Agrobacterium rhizogenes mediated transformation serves as a useful alternative way for the Capsicum transformation. The A. rhizogenes transformation compared to the A. tumefaciens transformation has the advantage that the method needs no regeneration step in vitro.
Our goal is to obtain a highly efficient transformation system that can be used to study the functions of different genes in Capsicum annuum varieties. Our study’s further goal is to validate and describe the candidate gene (Me1) involved in resistance against root-knot nematode species.
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