Traditional gladiolus propagation methods are now supplemented with in vitro propagation to meet the demands of modern floriculture in terms of quick production of disease-free, quality planting material. Due to virus infections, vegetative propagation in gladiolus in the field is slow, and is a serious concern in the propagation of gladiolus. In vitro propagation provides an enormous increase in propagation rate and the ability to produce disease-free plant material. Numerous elements, including cultivars, explant type, size of explants, position of explants on medium, plant growth regulators and certain additives, incubation conditions, and sub-culturing time, all have a significant impact on in vitro clonal propagation of gladiolus plants as well as the development of in vitro cormel efficiency. There are certain obstacles and challenges that arise in the in vitro development of plants and the cormels of gladiolus. However, numerous studies and review reports on gladiolus for in vitro propagation have been reported, but very little is known about the factors influencing gladiolus’ in vitro effectiveness. In the present review, we focused on and analyzed research data accumulated over 50 years on diverse strategies for in vitro propagation such as direct, indirect organogenesis, and somatic embryogenesis, as well as various factors such as physical, nutritional, and hormonal influences on in vitro propagation, in vitro cormel formation efficiency, difficulties that arise, and new insights into in vitro development in gladiolus from the available literature worldwide. Future possibilities for further improvement in the in vitro propagation of ornamental gladiolus are also discussed. The current review provides insight into a comprehensive protocol for gladiolus in vitro propagation and emphasizes the importance of continuously advancing tissue culture techniques and factors influencing the in vitro efficiency towards improving in vitro plantlets and cormels in gladiolus (Gladiolus spp.).