Embryogenic cell suspension (ECS) cultures consist of cells and cell aggregates ​that have the potential to form somatic embryos and regenerate into whole plants. ​As an expert in plant tissue culture and transformation, I have been working with ​ECS cultures for more than 12 years. ECS is an exceptional target tissue for ​Agrobacterium and microprojectile-mediated transformation, plant regeneration via ​somatic embryogenesis, protoplasts, and gene editing. Sufficient, regenerable, and ​competent cells can be bulked up in a very short time, enabling a myriad of ​scientific investigations.

Benefits for Genetic Transformation

Ample Target Tissue: ECS can be easily proliferated, providing ample target tissue.

Maximal Exposure: The small cell clumps allow maximal exposure to the transforming ​agent, facilitating the identification of independent transformation events within the ​dispersed cell clusters under selection.

Non-chimeric Plants: ECS allows the recovery of non-chimeric gene-edited and ​transgenic plants due to the unicellular origin of embryos.

Key Characteristics and Advantages of ECS

Heterogeneity: Cell suspension cultures are heterogeneous, containing both ​embryogenic and non-embryogenic cells. The non-embryogenic cells act as nurse ​or companion cells and can be removed to enrich the culture with embryogenic ​cells, which are competent for genetic transformation and have high regeneration ​potential.

Embryogenic Cells: These cells are small, cytoplasmically dense, and isodiametric ​in shape, often present in small multicellular clumps. Such cultures appear ​cream/yellow. When cultured on the appropriate medium, they form numerous ​somatic embryos.

Micropropagation and Gene Editing: Highly regenerable embryogenic cell cultures ​can produce thousands of somatic embryos per mL of cells, making it an attractive ​and efficient micropropagation system. Their unicellular origin makes them an ​ideal target tissue for gene editing and transformation.

Applications of ECS

Somatic Embryogenesis: ECS cultures are widely used for somatic embryogenesis, a ​process where somatic cells develop into plants without fertilization, providing a rapid ​means of clonal propagation (Fig. 1).

Protoplasts: ECS is an ideal source for protoplast isolation. Protoplasts are cells ​without cell walls, which can be used for fusion experiments to create hybrids and for ​direct DNA uptake in gene editing.

Gene Editing and Transformation: ECS is a preferred tissue for genetic transformation ​and CRISPR/Cas9 gene editing due to its high regenerability.

Secondary Metabolite Production: ECS can be optimized for the production of ​valuable secondary metabolites, making it useful for pharmaceutical and industrial ​applications.

Best Practices for Maintaining ECS

Regular Subculturing: To maintain high embryogenic potential and avoid ​somaclonal variation, ECS cultures should be subcultured regularly.

Optimal Growth Conditions: Maintaining optimal temperature, light, and medium ​composition is crucial for the success of ECS cultures. Typically, a temperature ​range of 22-25°C and a 16-hour photoperiod are ideal.

Monitoring and Selection: Regular monitoring of cultures for contamination and ​selection of highly embryogenic cell clusters ensures the longevity and stability of ​ECS lines.

Cryopreservation: For long-term preservation, ECS cultures can be cryopreserved. ​This technique involves freezing cells at ultra-low temperatures to maintain their ​viability over extended periods.

Limitations

Longevity and Stability: Over time, the proportion of cells that enter or complete ​embryogenesis decreases, reducing regeneration frequency and potentially making ​it impossible.

Somaclonal Variations: Prolonged time in culture can lead to somaclonal ​variations. Consequently, new cultures must be constantly initiated to maintain ​genetic integrity and regenerability. However, somaclonal variation could ​potentially produce plants with valuable traits, circumventing the need for ​intentional genetic transformation and lengthy, costly deregulation procedures.

Figure 1: Plant regeneration via somatic embryogenesis from embryogenic cell suspension in Taro (Colocasia ​esculenta var. esculenta)

Embryogenic cell clumps (a), immature somatic embryos formation (b), somatic embryo maturation (c), somatic ​embryo germination (d, e), plant development (f). (​Deo, 2008)