A Modified Carrot (Daucus carota L.) Somatic Embryogenesis Protocol to Elicit the Different Stages of Early Plant Development

Carrot callus was successfully induced on solid Murashige and Skoog (MS) medium supplemented with 0.1 mg/L 2,4-D and 3 mg/L BAP or 1 mg/L Kinetin. Calli from the BAP supplemented medium were friable whereas those from the Kinetin medium were compact. Explants exposed to light were green-pigmented while those in the dark were achromatic. To attain fine cell suspensions, calli were broken into smaller pieces prior to placement in the liquid media with the phytohormone supplements. The exponential growth phase was determined from the cell suspensions where the Kinetin supplemented medium in the dark was used. The observed data on higher cell density and propensity for cellular division was the basis for its use in the subsequent SE growth determination. Compared to the 90% purity from literature, the modified approach by subculturing with intermittent starvation cycles, using a low phytohormone concentration, and using the 700 mu M diameter blue tip was found to be efficient and cost-effective, resulting in 77.4% purity in terms of a particular cell developmental stage. Morphological stages following sequential transition through the earliest stages of SE were observed: single cells, dividing cells, cell clusters, globular stage, triangular stage, heart stage and torpedo stage. The transitory stages, triangular, and heart stages were documented.