Somatic Embryogenesis (SE)

Somatic Embryogenesis (SE) Defined

• SE involves somatic embryos developing from somatic cells
• This happens regardless of ploidy levels or specializations
• SE has significant applications in both basic and applied plant biotechnology

History

• Haberlandt proposed in the early 1900s that a single vegetative cell could become a complete embryo
• Two scientists, Steward (USA) and Reinert (Germany), successfully developed carrot plants through somatic embryos in 1958
• Since this discovery, over 500 monocot and dicot species have been explored
• Molecular cell biology and genetics made a significant contribution by deducing the complete genome sequence through understanding SE in Arabidopsis sp
• Experiments using different media have been conducted to produce efficient varieties
• Callus formation was seen by culturing root segments of Daucus carota in 2,4-D medium
• Somatic embryo formation was promoted by medium enriched with coconut milk and IAA from the callus of Ranunculus sceleratus
• Mesophyll tissues of Macleaya cordata were observed to produce somatic embryos from single cells

Modes of Somatic Embryogenesis

• Direct SE
• Indirect SE

Direct SE

• Plantlets produced are expected to mirror the mother plant (except for chimeras)
• The cell determines which pure/solid lines will be produced

Indirect SE

• Explants produce undifferentiated cells (callus)
• Callus then differentiates into somatic embryos
• The callus cells show variability, which will be reflected in the plantlets

Plant Regeneration Stages

• There are five major steps for plant regeneration by SE
• Initiation
• Proliferation
• Prematuration
• Maturation
• Plant Development

Factors Influencing SE

• Before somatic embryos develop, plant cells undergo biological re-differentiation
• Many factors can affect SE and its development
• A thorough selection of explants and nutrient media is needed (MS, B5, N6, and White media)
• Plant growth regulators and the physical environment during incubation (including light and temperature) are important
• The process involves stimulation, development of embryonic cells and their transformation through germination
• Explants commonly include shoot tips, immature embryos, and young floral parts
• Immature zygotic embryos are the most common explant in monocots and many dicots
• Proto dermic cells of cotyledons forms SE in mature embryos

Growth Regulators

• Growth regulators stimulate embryogenesis
• Auxins are most important for tissue regeneration; 2,4-D is used for callus induction in different concentrations (0.5-1.0 mg/L)
• Callus that proliferates on auxin media must be transferred to cytokinin-rich media for embryogenesis
• Zeatin (0.1µM) promotes regeneration in carrot
• BAP with IAA helps develop globular embryos in Podophyllum sp
• Combinations of growth regulators can effectively promote SE
• ABA, gibberellic acid, and zeatin, as well as 2iP and GA3, are required for grape vine growth
• pH modification and high sucrose can promote SE without plant growth regulators

Nitrogen

• Nitrogen is essential for inducing embryo formation, initiation, and maturation
• Ammonia (reduced nitrogen) triggers rapid embryogenesis
• Adding reduced nitrogen (casein hydrolysate/ NH4Cl/ NH4NO3/ amino acid etc.) helps induce embryogenesis once callus is shifted from auxin media to cytokinin
• Sucrose is observed as the most frequent carbon source

Other Factors

• Cell density, ethylene concentration, and light affect SE
• Initial cell density is critical for differentiating somatic embryos
• High-density carrot cultures on auxin-free medium inhibit somatic embryo formation
• Cells cultured at high density release high amounts of phenolic compounds (pHBA, benzoic acid, 4-hydroxy benzoic acid) that inhibit embryo formation
• Ethylene biosynthesis can increase SE in Daucus carota and Hevea brasiliensis
• Appropriate oxygen concentration is significant for developing somatic embryos; a six-fold improvement was observed in wheat cultures by reducing dissolved oxygen
• Co-culturing soyabean with Pseudomonas maltophilia enhances somatic embryo development
• Blue green algae Anabena extracts promote embryogenesis in Daucus carota cultures
• Different plant species respond differently to light intensity
• Somatic embryos grew well under light in Solanum melongena
• SE was promoted in complete darkness in Populus
• The quality of light also plays a role
• High-intensity white and blue light has inhibitory effects in Daucus carota

Induction and Cellular Polarity

• Dedifferentiation of cells is the primary requirement for embryonic development
• PGRs, pH, and heavy metals are known to promote cell dedifferentiation and embryonic responses
• Cellular polarity is linked to SE induction and altered by PGRs and other treatments to promote asymmetric division
• In a Daucus carota cell suspension, the first division is asymmetric

Development, PEMs, and Auxin

• Cell divisions recommence and proliferate, embryogenic cells are released, and two types of cells are present in suspended culture
• Proembryogenic masses (PEMs)
• Cytoplasmically rich cells
• PEMs comprise embryogenic cells of 400 – 800 µm
• Angular
• Connected via plasmodesmatas
• Small vacuoles
• Large starch grains (5-25%)
• High-density ribosomes
• ER
• Non-embryogenic cells holding the embryogenic cells are 1000 – 3000 µm3
• Rounded
• Large vacuoles (80% volume)
• Few starch grains (1-2%)
• Low ribosome population
• Adding auxin post-induction elongates the cells
• Enlarged cells permanently lose ability to form somatic embryos

Induction of Somatic Embryogenesis in Daucus Carota

• Leaf petiole or cambium explants are sterilized, placed on semi-solid Murashige and Skoog’s medium, and allowed to produce callus tissue
• Callus transfers to Erlenmeyer flask, then placed horizontally on a gyratory shaker to initiate a cell suspension culture
• Sub-culturing takes place every 4 weeks by transferring the cell suspension to liquid
• Cell suspensions are passed through stainless steel mesh sieves for embryo formation uniformity
• Numerous embryos develop within four weeks
• Somatic embryos are placed on agar medium to further develop
• Small plantlets are transferred to Jiffy pots to enhance growth and development

Physiology and Biochemistry of SE

• Cells gain embryogenic competence through physiological and biochemical changes before morphological differentiation of the somatic embryo
• Changes observed are in levels of phytohormones and amino-acid metabolism

Synchronization

• While embryos are single-cell derived, cells in liquid or semi-solid media show development at different stages
• Synchronizing embryo development helps optimize the number of embryos and can be achieved by:
• Physically separating embryos at different developmental stages
• Using growth regulators for physiological control

Molecular Markers

• Transcriptomics are carried out when somatic embryos develop
• LEC, WUS, FUS, auxins, and transcriptional factors induce SE and transfer the somatic cell to an embryogenic cell
• Epigenetics controls gene expression
• Auxins modify DNA methylation patterns of embryogenic cells
• Post-translation modifications, protein turnover, and protein-protein interactions regulate proteins

Molecular Markers and Calcium

• As somatic cells progress to embryogenic development, gene expression changes
• Molecular markers indicate this transition, and genes have been cloned to enhance response
• Callus and calcium in vacuoles are first signs of recognition
• Cell wall proteins participate in the embryogenic process
• Somatic embryogenesis can be induced in fresh cultures by adding cell-free conditioned medium via extra-cellular proteins (ECPs).

Maturation

• Zygotic embryos undergo maturation for desiccation tolerance, food storage, and ABA synthesis to induce dormancy
• Somatic embryos lack an embryo maturation stage, resulting in seedlings
• Mature embryos require an extra stage similar to zygotic embryo formation
• Desiccation increases via sucrose and ABA during maturation increase plantlet survival upon transplantation
• Reducing humidity mimics the embryo sac environment and enhances the survival of embryos in vivo

Applications: Micropropagation

• Somatic embryos form in large numbers
• If an efficient cloning method is developed, large numbers can develop at low cost
• SE can occur in bioreactors, reducing manpower/space

Applications: Synthetic Seeds

• Artificial or synthetic seeds are encapsulated somatic embryos
• Plants with sterile seeds produce synthetic seeds and are used for germplasm conservation.

Applications: Embryo Cloning

• Large numbers of somatic embryos are produced due to recurrent embryogenesis
• These embryos produce metabolites (proteins and α-linolenic acid) which are efficient for industrial usage

Applications: Genetic Engineering

• Using somatic embryos is a good method for gene transfer
• This method is better than others because transformation and its genetic stability are easier
• The process involves incubating somatic embryos on an Agrobacterium solution or subjecting cells to particle bombardment

Conclusion

• SE has been studied in many species, with zygotic embryos as the most potent explants
• SE is a cost-effective method for clonal propagation
• Bioreactors enhance SE for micropropagation
• SE has increased knowledge of the physiological, biochemical, and molecular events in the development from somatic to embryogenic cells