Callus capable of producing somatic embryos (embryogenic callus) is most reliably obtained from an explant during the initial phase of culture, and is frequently produced in conjunction with non-morphogenic tissue. Embryogenic callus can usually be distinguished by its nodular appearance, and is frequently produced preferentially from one part of an explant (e.g. the scutellum of a monocotyledon embryo), probably because only the cells of that part of the explant were embryogenically pre-determined. These may be the same tissues, which in another cultural environment are capable of producing embryos directly (Sharp et al., 1980). According to this hypothesis, although competent and non-competent cells may produce callus, only that which grows from competent cells will give rise to somatic embryos.
The expression of competence depends on the use of a suitable medium for the culture, containing requisite growth regulators at the correct concentration. The formation of somatic embryos in Lolium multiflorum, for example, was medium dependent (Dale et al., 1981). On the most suitable medium, immature inflorescence explants produced three types of callus, only one of which spontaneously formed embryo-like structures. Unless such different callus types are separated, cells of different regenerative capabilities may become mixed. Morphogenically competent cells could then be lost by competition in the combined callus tissue that results.
Stage I. Selection of an appropriate explant is most important. Embryogenic callus has been commonly gained from seed embryos, nucelli or other highly meristematic tissues such as parts of seedlings, the youngest parts of newly initiated leaves and inflorescence primordia. Within an inflorescence, staminoids [Theobroma cacao (Li et al., 1998)] and filaments [Aesculus hippocastanum (Radojevic, 1995)] have been reported to be adequate sources of explants. The initiation of embryogenic callus from root tissue is rare but has been reported in some monocotyledons e.g. rice (Inoue and Maeda, 1982; Toshinari and Futsuhara, 1985); oil palm (Paranjothy and Rohani, 1982), Italian ryegrass (Jackson and Dale, 1988) and Allium carinatum (Havel and Novak, 1988). Callus is usually commenced on a semi-solid medium incorporating a relatively high level of an auxin; compounds commonly used for this purpose are described in Chapters 11 and 12. Only a few tissues with a high natural embryogenic capacity do not require the addition of endogenous auxin for the development of embryogenic callus. Occasionally, primary callus arising from an explant may show no morphogenic capacity, but can be induced to give rise to new embryogenic tissue during later (secondary) subcultures by transfer to an inductive medium. Ahee et al. (1981) have used this method to propagate oil palms. On the medium used, calluses arising on the veins of young leaf fragments had no morphogenic capability. However, when primary calluses were subcultured onto appropriate media (unspecified), some of them gave rise to tissue that was different in structure and form, and grew at a much faster rate. These 'fast-growing calluses' could be induced to produce structures resembling embryoids, and afterwards plantlets, upon further subculture to other media.
One highly embryogenic tissue that has been extensively studied is that of the nucellus of the polyembryonic 'Shamouti' orange (Spiegel-Roy and Kochba, 1980). Here it seems that cells at just one end of the embryo sac (the micropylar end) are embryogenically predetermined and retain this capacity in subsequent cell generations. On subculture, proliferation of the nucellus cells proceeds without the addition of growth regulators to the medium, and results in the formation of an habituated callus. A tissue is said to have become habituated when it will grow without a growth regulator, or some other organic substance which is normally necessary, being added to the medium (see Chapter 7). Addition of auxins to the growth medium is inhibitory to the growth of auxin-habituated 'Shamouti' orange tissue, which has been thought to be composed (at least initially) of numerous proembryos and not of undifferentiated cells (Button et al., 1974). Embryogenic callus has also been obtained from the nucellus tissue of other plants, mainly tropical fruit species (Litz and Jaiswal, 1991).
Stage II. As a general rule, somatic embryos formed on a medium containing a relatively high concentration of an auxin, will only develop further if the callus culture is transferred to a second medium from which auxin has been omitted, another 'less active'' auxin has been substituted, or the level of the original auxin much reduced. This treatment is occasionally ineffective (Handley and Sink, 1985) and sometimes adding a cytokinin helps to ensure embryo growth. A further essential requirement is the need for a supply of reduced nitrogen in the form of an ammonium salt or amino acid. No change of nitrogen source is required if MS medium was used for Stage I, but if, for example, White's medium were used for Stage I, it would need to be supplemented with reduced nitrogen, or the culture transferred to MS.
Callus subculture. Once obtained, embryogenic callus can continue to give rise to somatic embryos during many subcultures over long periods. The continued production of somatic embryos in these circumstances depends either on the continued proliferation of pro-embryogenic nodules, and/or the de novo formation of embryogenic tissue from young somatic embryos during each subculture. Inocula for subcultures must be carefully selected. In wheat, callus with continued embryogenesis was only reinitiated from inocula taken close to somatic embryos; tissue from the same culture which did not contain embryos was not embryogenic in the next passage (Chu et al., 1987).
Sometimes the number of embryos produced per unit weight of callus rises during a few passages and then slowly falls, the capacity to form somatic embryos eventually being irrevocably lost. Callus derived from the nucellus of 'Shamouti' oranges increased in its capacity to form somatic embryos when subcultured at 10-15 week intervals, while transfer at 4-5 week intervals, reduced embryogenesis (Kochba and Button, 1974).
Somatic embryos can be formed relatively freely in callus tissue, but where they are to be used for large scale propagation, their numbers can often be increased more rapidly and conveniently by initiating an embryogenic suspension culture from the primary callus (see below).
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