Differentiation implies development of organised structures, usually from undifferentiated tissue but also from previously specialized cells that would not normally give rise to organised multi-cellular growth (e.g. epidermal cells, pollen grains). In plant tissue culture, undifferentiated tissue is referred to as callus  although a callus can contain meristematic nodules that may not be obvious to the naked eye but which never develop further unless suitable conditions are supplied. Development of organised structures can follow one of three pathways:

  1. shoot regeneration, based on a uni-polar structure with a shoot apical meristem
  2. root regeneration, essentially a uni-polar structure with a root apical meristem
  3. somatic embryo-genesis in which there is a bipolar structure

Dedifferentiation and callus formation occur naturally in response to wounding. Indeed, wound responses involve auxin and cytokinins and seem to be the biological trigger for plant regeneration from somatic cells (Potrykus 1989). However, sustained callus growth in vitro requires addition of one or more growth regulators. Prior to the chemical characterisation of IAA in 1934, attempts to obtain long-term callus cultures failed. With very few exceptions, auxin is essential for dedifferentiation and commonly 2,4-dichlorophenoxyacetic acid (2,4-D) is used to promote callus, cytokinin often enhances this process. In tissues with a high endogenous level of auxin, culture of explants on a medium containing cytokinin as the only growth regulator may lead to development of shoots with very little callus.

Direct organogenesis bypasses the need for a callus phase. A good example is the formation of somatic embryos. Most evidence suggests that direct embryogenesis proceeds from cells which were already embryogenically competent while they were part of the original, differentiated tissue. These pre-embryogenic cells appear only to require favourable conditions (such as wounding or application of exogenous growth regulators) to allow release into cell division and expression of embryogenesis. Such cells tend to be much more responsive than those involved in indirect organogenesis and do not seem to require the same auxin ‘push’ to initiate division; indeed, the cells may never have left the cell cycle and growth regulator application has some more subtle role.

The process of formation of an embryo is called embryogenesis. Embryogenesis starts from a single embryogenic cell, that can be a zygote (the product of the fusion of an egg and a sperm during fertilization), or an undifferentiated callus cell. Embryos developing from zygotes are called zygotic embryos, while those derived from somatic cells are called somatic embryos. During the embryonic development, the polar axis of the plant is established, domains that set up the organization of the plant body are defined, and the primary tissue and organ systems are delineated. Somatic embryogenesis is another important way to regenerate new plants in plant tissue culture.

Embryo development occurs through an exceptionally organized sequence of cell division, enlargement and differentiation. Zygotic and somatic embryos share the same gross pattern of development. Both types of embryos develop as passing through typical developmental stages . Embryo development is bipolar, having a shoot pole and a radicular pole at opposite ends. Embryos are not organs because they are structurally independent from their parent body (i.e. they do not have a vascular system connecting them with their parent plant body).