Microscopic techniques remain an integral tool which has allowed for the better understanding and manipulation of in vitro plant culture systems. The recent advancements will inevitably help to unlock the long-standing mysteries of fundamental biological mechanisms of plant cells. Beyond the classical applications in micropropagation aimed at the conservation of endangered and elite commercial genotypes, plant cell, tissue and organ cultures have become a platform for elucidating a myriad of fundamental physiological and developmental processes. In conjunction with microscopic techniques, in vitro culture technology has been at the centre of important breakthroughs in plant growth and development. Applications of microscopy and plant tissue culture have included elucidation of growth and development processes, detection of in vitro-induced physiological disorders as well as subcellular localization using fluorescent protein probes. Light and electron microscopy have been widely used in confirming the bipolarity of somatic embryos during somatic embryogenesis. The technique highlights basic anatomical, structural and histological evidence for in vitro-induced physiological disorders during plant growth and development. In this review, we discuss some significant biological insights in plant growth and development, breakthroughs and limitations of various microscopic applications and the exciting possibilities offered by emergent in vivo live imaging and fluorescent protein engineering technologies.