Micromorphological analysis of Aranda Broga Blue orchid PLBs for Agrobacterium-mediated transformation system

Abstract

Aims: The aim of this work was to analyse the morphological aspects of ABB’s PLBs and interaction between ABB’s PLBs and Agrobacterium. Methodology and results: Aranda Broga Blue’s (ABB) protocorm-like bodies (PLB) proliferated on Vacin and Went medium by incubating at 25 ± 2°C under 16 h semi-photoperiod was used to study the micromorphology of PLB, bacterial attachment and chemotaxis studies. The anatomy and micromorphology study indicates that ABB’s PLBs provide the platform for attachment of Agrobacterium tumefaciens strain LBA 4404. Successful propagation of ABB’s PLBs was achieved on modified Vacin and Went solid media supplemented with organic homogenates. Histological analysis of the development of ABB’s PLBs shows it is a meristematic tissue and contains actively dividing cells. Transmission electron microscope (TEM) studies signify the presence of specialized organelles to indicate PLB is similar to a mature plant. Scanning electron microscope (SEM) observation on ABB’s PLBs and bacterial attachment studies proves that trichomes and stomata on the surface facilitate the interaction with A. tumefaciens. Quantification of the attachment of Agrobacterium to ABB’s PLBs using spectrophotometric system displayed higher affinity compared to the attachment mutant Escherichia coli DH5α. The interaction of Agrobacterium to ABB’s PLBs was also evaluated based on chemotaxis studies. Highest motility respond of Agrobacterium was recorded (1.6 unit) towards severely wounded PLBs indicating wounding is necessary for the interaction between bacterium and PLB. Conclusion, significance and impact study: Micromorphology of protocorm-like bodies (PLBs) enhances the physical interaction with bacteria proved that PLB is a suitable explant for the Agrobacterium-mediated genetic transformation.