In Vitro Developmental Pathways

Explants Sterile pieces of a whole plant from which cultures are generally initiated Types of explant: Generally all plant cells can be used as an explant, however young and rapidly growing tissue (or tissue at an early stage of development) are preferred.

Root tip: Root cultures can be established from explants of the root tip of either primary or lateral roots. Shoot tip: The shoot apical meristem from either axillary or adventitious buds can be cultured in vitro. Embryo: Both immature and mature embryos can be used as explants to generate callus cultures or somatic embryos. Immature, embryo-derived callus is the most popular method of monocot plant regeneration. Haploid tissue Male gametophyte (Pollen in anthers) or female gametophyte (the ovule) can be used as an explant. Haploid tissue cultures can produce haploid or di-haploid plants due to doubling of chromosomes during the culture periods.

Cell suspension culture Culture Types Callus Cell suspension culture Protoplast Hairy root

Callus Definition: It is an unspecialized and unorganized, growing and dividing mass of cells, produced when explants are cultured on the appropriate solid medium, with both an auxin and a cytokinin and correct conditions. During callus formation there is some degree of dedifferentiation both in morphology and metabolism, resulting in the lose the ability to photosynthesis.

Callus Callus cultures may be compact or friable. Compact callus shows densely aggregated cells Friable callus shows loosely associated cells and the callus becomes soft and breaks apart easily. Habituation: it is the lose of the requirement for auxin and/or cytokinin by the culture during long-term culture.

Cell-suspension cultures When friable callus is placed into the appropriate liquid medium and agitated, single cells and/or small clumps of cells are released into the medium and continue to grow and divide, producing a cell-suspension culture. The inoculum used to initiate cell suspension culture should neither be too small to affect cells numbers nor too large too allow the build up of toxic products or stressed cells to lethal levels. Cell suspension culture techniques are very important for plant biotransformation and plant genetic engineering.

Three stages of callus culture Induction: Cells in explant dedifferentiate and begin to divide Proliferative Stage: Rapid cell division Morphogenesis stage: Differentiation and formation of organized structures; specifically processes that lead to plant regeneration from somatic cells

Induction

Division

Plant morphogenesis Organogenesis The formation of organs (such as leaves, shoots, roots) on a plant organ, usually of a different kind. 1. Enhancement of axillary bud proliferation/ development 2. Adventitious shoot formation 3. Adventitious root formation Somatic embryogenesis Embryo initiation and development from somatic cells

Organogenesis Shoot initiation and development with subsequent formation of adventitious roots Enhancement of axillary bud proliferation and development - stimulation of the shoot apical meristem in vitro that includes proliferation of lateral buds Adventitious shoot formation - dedifferentiation and/or differentiation and development of shoots from non-meristematic cells (one or more than one) either directly or indirectly Adventitious root formation - roots are initiated adventitiously at the base of the shoot apex and a vascular continuum is established to complete plant regeneration.

Somatic Embryogenesis Dedifferentiation is typically minimal but a meristemoid-like tissue can be formed in the latter case Histogenesis of somatic embryogenesis is characterized by the formation of a bipolar structure, in contrast to adventitious organogenesis Single cell origin of somatic embryos makes chimerism infrequent; adventitious shoots can arise from more than one cell

Somatic Embryos Bipolar Not connected to explant or callus cells by vascular tissue In most woody plants, tissue must be juvenile or reproductive

Cell Developmental Phases Leading to Morphogenesis