Somatic Embryogenesis

Abstract

Embryogenesis is a specialized mode of development by which a fertilized egg through a series of predetermined pattern of cell divisions and differentiation forms an embryo and the precursor of the next generation. In nature, embryogenesis is restricted to ovule and involves the fusion of male (sperm) and female (egg) gametes. Occasionally, as in some varieties of mango and citrus, the sporophytic cells of the nucellus form asexual or adventive embryos independent of fertilization. However, these embryos also mature only inside the embryo sac (female gametophyte). Fertilization-independent embryogenesis is also exhibited by some elements of the embryo sac (egg or synergid) in apomictic plants. Thus, in nature, sexual and asexual embryo formation is restricted to ovular cells and full development of the embryo occurs only inside the embryo sac. This led some scientists to suggest that the embryo formation requires a special environment available only inside the embryo sac. However, tissue culture studies during the past five decades have clearly demolished this myth and demonstrated that most plant cells, irrespective of their specialization and ploidy level, are capable of forming typical embryos that can germinate. The embryos formed by somatic cells are called somatic embryos, and the process by which a somatic cell differentiates into embryo is termed somatic embryogenesis. Since the first report of somatic embryogenesis in carrot in 1958, this phenomenon has been observed in over 500 plant species. The most potent explant to initiate embryogenic cultures is immature zygotic embryo. However, in plants such as alfalfa, buttercup and carrot almost all vegetative tissues have yielded embryogenic cultures. In vitro somatic embryogenesis has found wide applications in basic and applied areas of plant sciences. It is being used extensively to understand physiological, biochemical and molecular events underlying plant embryo development which is difficult to investigate using zygotic embryogenesis. Analysis of proteomes and transcriptomes has lead to the identification and characterization of genes involved in somatic embryogenesis. Scaling up the production of somatic embryogenesis in bioreactors has opened up the possibility of using it as an efficient system for rapid clonal propagation of plants.

Appendix

• 1. Protocol for inducing somatic embryogenesis in Daucus carota (after Smith and Street 1974).

  • (i) Surface sterilize seeds in 10 % calcium hypochlorite for 15 min and, after washing three times in sterile distilled water, germinate them on sterilized moistened filter paper in a Petri dish, in dark at 25 °C.

  • (ii) Cut 1 cm long segments of roots from 7-day-old seedlings and culture them individually on a semi-solid medium containing the inorganic salts of Murashige and Skoog’s medium, organic constituents of White’s medium 100 mg L⁻¹ myo-inositol, 0.2 mg L⁻¹ Kinetin, 0.1 mg L⁻¹ 2,4-D, 2 % sucrose, and 1 % agar (Difco bacto agar or of any other brand). Incubate the cultures in dark.

  • (iii) After 6–8 weeks, transfer pieces of root calli (0.2 g fresh weight) to fresh medium of the original composition and maintain the cultures in light at 25 °C. The tissue may be multiplied by subculturing every 4 weeks in a similar manner.

  • (iv) After the first passage initiate suspension cultures by transferring ca. 0.2 g of callus tissue to a 200 ml Erlenmeyer flask containing 20–25 ml of liquid medium of the same composition as used for callus growth (without agar). Incubate the flasks on a horizontal rotary shaker at 100 rpm in light at 25 °C.

  • (v) Subculture the suspension every 4 weeks by transferring 5 ml of it to 65 ml of fresh medium (1:13).

  • (vi) To induce embryo development, transfer callus pieces or portions of suspension to 2,4-D-free medium of otherwise the same composition as used before.

  • (vii) After 3–4 weeks the cultures contain numerous embryos at different stages of development.

• 2. Protocol for inducing somatic embryogenesis in Citrus sp. (after Tisserat and Murashige, 1977).

  • (i) Take a 6–8-weeks-old fruitlet of a local cultivar and surface sterilize it with 1 % sodium hypochlorite for 15–20 min. Follow the subsequent steps under aseptic conditions.

  • (ii) Bisect the fruit and transfer the ovules to a sterile Petri dish.

  • (iii) Excise nucellus tissue from the ovules using a dissecting microscope. Hold the chalazal region of the ovules with pointed forceps. Give a shallow incision longitudinally through the integuments. Cut the ovule transversely into two halves. From the micropylar half, remove the integuments, endosperm, and any embryo (especially zygotic), if present, transfer the nucellar section to the culture vessel in a manner that its cut end is in contact with the medium.

  • (iv) Use semi-solid medium containing inorganic salts of Murashige and Skoog’s medium, 100 mg L⁻¹ myo-inositol, 0.2 mg L⁻¹ thiamine-HCl, 1 mg L⁻¹ pyridoxine–HCl, 1 mg L⁻¹ nicotinic acid, 4 mg L⁻¹ glycine, 500 mg L⁻¹ malt extract, 5 % sucrose, and 1 % Difco bacto agar.

  • (v) Incubate the cultures in light (1000 lux) at 27 ± 1 °C.

  • (vi) Within 4–6 weeks multiple embryos should develop from the callused nucellar tissue. To stimulate full plant development, transfer the embryos to another medium which differs from the previous medium in having 1 mg L⁻¹ GA₃ in place of malt extract.