SOMATIC EMBRYOGENESIS

HISTORY The capacity of flowering plants to produce embryos is not restricted to the development of the fertilized egg; embryos (embryoids) can be induced to form in cultured plant tissues. This was first observed in suspension cultures of carrot (Daucus carota) by Steward, Mapes and Mears(1958) and in carrot callus grown on an agar medium by Reinert (1959). It has been reported in more than 30 plant families.

SOURCES Somatic embryoids may arise in vitro from three sources of cultured diploid cells. Vegetative cells of mature plants Reproductive tissues other than the zygote, And hypocotyl and cotyledons of embryos and young plantlets without any intervening callus development

PLANT REPRODUCTION

Figure 1. Callus induction, embryogenic callus initiation, and plantlet regeneration in Moso bamboo and levels used to produce these images of each compound.

Somatic embryogenesis may be initiated in two different ways. In some cultures embryogenesis occurs directly in the absence of any callus production from “pre embryonic determined cells” that are programmed for embryonic differentiation. The second type of development requires some prior callus proliferation, and embryos originate from “induced embryogenic cells” within the callus.

The embryoid-forming cells are characterized by Embryoids are initiated in callus from superficial clumps of cells associated with highly vacuolated cells that do not take part in embryogenesis. The embryoid-forming cells are characterized by Dense cytoplasmic contents Large starch grains Large nucleus with a darkly stained nucleolus Staining reagents indicated that these embryogenic cells have high concentrations of protein and RNA. These cells also exhibited high dehydrogenase activity with tetrazolium staining.

Stages of embryo formation Globular Heart stage Torpedo stage

ABNORMALITIES Embryonal budding Embryogenic clump formation Due to high level of auxin present in medium Auxin and reduced nitrogen are required Organic forms of nitrogen are much more effective than inorganic forms

CYTOKININS The inhibitory effect of exogenous cytokinins may result from the increase in endogenous cytokinins in the developing embryoids.

CHARCOAL The induction of embryogenesis was successful in Daucus carota cultures containing charcoal when auxin depletion failed to produce the desired results. Charcoal was a requirement for embryogenesis in English ivy (Hedera helix) cultures. Evidence indicates that charcoal may absorb a wide variety of inhibitory substances as well growth promotors.

DURATION OR TIME PERIOD Embryogenesis occurs most readily in short-term cultures, and this ability decreases with increasing duration of culture.

GLASS BEADS A technique has been developed for the physical separation of the globular, heart and torpedo stages of embryogenesis by using glass beads to screen the cultures. This procedure should prove useful for further biochemical studies of the developmental process.

SYNCHRONIZATION A high degree of synchronization of embryogenesis was achieved in a carrot suspension culture by; Sieving the initial cell populations, Employing density grdaient centrifugation in Ficoll solutions, and Using repeated low-speed centrifugation for 5-sec The resulting cell clusters, cultured in an auxin-free medium containing zeatin, gave a greater than 90% frequency of embryoid formation.

COMMERCIAL MICROPROPAGATION In recent years the multiplication of somatic embryos in bioreactors has begun for commercial micropropagation. The U.S. biotechnology company DNA Plant Technology (DNAP) has applied bioreactor micropropagation via embryogenesis to coffee, banana, and pineapple.

Somatic embryos Primary Callus Shoot production Embryo germination