10.2.2 Types of differentiation

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Differentiation implies development of organised structures, usually from undifferentiated tissue but also from previously specialised cells that would not normally give rise to organised multicellular growth (e.g. epidermal cells, pollen grains). In plant tissue culture, undifferentiated tissue is referred to as callus (Figure 10.20a) although a callus can contain meristematic nodules that may not be obvious to the naked eye but which never develop further unless suitable conditions are supplied. Development of organised structures can follow one of three pathways:

1. shoot regeneration, based on a unipolar structure with a shoot apical meristem (Figure 10.20b)

2. root regeneration, essentially a unipolar structure with a root apical meristem (Figure 10.20c)

3. somatic embryogenesis in which there is a bipolar structure (Figure 10.20d).

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Figure 10.20 Growth in plant tissue cultures can take many forms ranging from (a) disorganised callus, in this case initiated from carrot hypocotyl on a nutrient medium supplimented with 0.45 μM 2,4-D. (b) Shoot regeneration from a callus derived from a single protoplast of Hyoscyamus muticus. (c) Root regeneration from a callus derived from a root explant of Arabidopsis thaliana. (d) A single somatic embryo removed from a callus derived from a carrot cotyledon explant.

(Photographs courtesy J. Gorst (a,c,d) and W. Wernicke (b))

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Figure 10.21 Develoment of haploid plants from immature pollen grains of an anther of Nicotinia tabacam. The normal developmental program leading to mature pollen grains has been diverted post-meiosis into an embryogenesis program. The dark-brown-coloured structure is the remains of the anther which has burst open.

(Photograph courtesy W. Wernicke)

Repeated division of immature post-meiotic pollen grains (microspores) leads to production of haploid plants, and is another type of embryogenesis (Figure 10.21). The pheno-m-enon was first discovered in 1966 by Guha and Maheshwari when studying meiosis in vitro in Datura innoxia anthers. Sometimes differentiation takes place in the absence of cell division: in tissue culture systems, such as Zinnia elegans, we commonly find xylem elements appearing among otherwise undifferentiated cells (Fukuda and Komamine 1980a, b). This form of xylogenesis represents the acquisition of a specific metabolic competence that is quite different from that of the parental cell. Clearly, differentiation in vitro can take several forms.

(a) Indirect organogenesis: plant growth regulators and differentiation

An understanding of the mechanisms underlying regeneration of whole plants, or parts of plants, from cells has come some way since the classic observations of Skoog and Miller that the direction of differentiation could be influenced by the ratio of the exogenously supplied growth regulators auxin and cytokinin. They observed in tobacco stem pith cultures that a high ratio of auxin to cytokinin led to initiation of roots whereas a low ratio led to development of shoots (Figure 10.22). Although there are many species for which this simple manipulation will not work, in general auxins (e.g. IAA (indoleacetic acid), NAA (a-naphthaleneacetic acid) and IBA indolebutyric acid)) will stimulate regeneration of roots, and cytokinins (e.g. BAP (6-benzylaminopurine) and kinetin) will promote regeneration of shoots or embryos.

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Figure 10.22 Tobacco leaf explants cultured on media with varying concentrations of an auxin (α-naphthaleneacetic acid; NAA) and a cytokinin (6-benzylaminopurine; BAP). Concentrations of NAA are from left to right, 0, 0.01 μM, 0.1 μM, 1.0 μM; concentrations of BAP are from top to bottom 0, 0.01 μM, 0.1 μM, 1.0 μM. At low auxin to cytokinin ratios shoot development predominates, whereas at high ratios profuse root initiation occurs. At intermediate ratios, callus is often the result. Not that type of response also depends on the explant source: tobacco stem pith under the same culture conditions usually shows no development whatsoever unless some auxin is supplied.

(Photograph courtesy W. Wernicke)

It is now obvious that the two groups of growth regulators play an important role in unlocking totipotent expression. Dedifferentiation and callus formation occur naturally in response to wounding. Indeed, wound responses involve auxin and cytokinins and seem to be the biological trigger for plant regeneration from somatic cells (Potrykus 1989). However, sustained callus growth in vitro requires addition of one or more growth regulators. Prior to the chemical characterisation of IAA in 1934, attempts to obtain long-term callus cultures failed. With very few exceptions, auxin is essential for dedifferentiation and commonly 2,4-dichlorophenoxyacetic acid (2,4-D) is used to promote callus; cytokinin often enhances this process. In tissues with a high endogenous level of auxin, culture of explants on a medium containing cytokinin as the only growth regulator may lead to development of shoots with very little callus. Not all living cells respond to auxin and this is particularly true of mature cells of grasses. Without dedifferentiation, it is not possible to move to the next stage of totipotent expression — plant regeneration. Until the late 1980s, grasses, especially the economically important cereals, were regarded as recalcitrant in vitro. Since that time, researchers have developed methods of regeneration for most of these species. This includes work on sugar cane, sorghum, wheat and barley in several Australian laboratories.

The first example of a single isolated cell dividing directly to produce an embryo was recorded in 1970 in a suspension culture of Daucus carota (Backs Hüsemann and Reinert 1970). However, even in this carrot system, the phenomenon is rare and normally somatic embryogenesis is indirect via an initial callus phase. Protoplasts (Figure 10.23a) capable of undergoing cell division seldom give rise directly to organised structures but first synthesise a new cell wall, then produce a callus (Figure 10.23b) from which shoots or embryos can later regenerate.

Although auxin stimulates initial cell division in quiescent cells, continued presence of auxin can inhibit organised out-growth. This is a typical example of the sequential functions of a single hormone through a developmental progression. In practical terms, cultures are usually transferred onto low or zero auxin media to permit or speed up shoot organogenesis. Sometimes ‘removal’ of auxin occurs when auxin in the medium is degraded either by the tissue itself or via chemical reactions such as photo-oxidation. Cytokinins promote out-growth of shoots but are normally kept at very low concentration when root regeneration is wanted.

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Figure 10.23 (a) Protoplasts of Triticum aestivum just after isolation from leaf mesophyll cell. (b) Cell division in two protoplasts of Hyoscyamus muticus has led to formation of two cell clusters or micro-calluses.

(Photograph W. Wernicke)

(b) Direct organogenesis: the role of growth regulators

Direct organogenesis bypasses the need for a callus phase. A good example is the formation of somatic embryos. Most evidence suggests that direct embryogenesis proceeds from cells which were already embryogenically competent while they were part of the original, differentiated tissue. These pre-embryogenic cells appear only to require favourable conditions (such as wounding or application of exogenous growth regulators) to allow release into cell division and expression of embryogenesis. Such cells tend to be much more responsive than those involved in indirect organogenesis and do not seem to require the same auxin ‘push’ to initiate division; indeed, the cells may never have left the cell cycle and growth regulator application has some more subtle role. In Trifolium repens hypocotyl epidermis, we see that BAP (a cytokinin) promotes reorientation of the plane of cell division, leading to initiation of a promeristemoid. An analogous response occurs in cotyledon explants of Abies amabilis where subepidermal cells develop into shoots. In haploid embryos developed from Brassica napus anther cultures, cytokinin actually suppresses secondary embryoid formation and instead promotes normal leafy shoots (Loh et al. 1983). This suggests a role for cytokinin in switching between shoot development and embryogenesis. Similarly, Tran Thanh Van et al. (1974), working with thin-layer explants only three to six epidermal cells deep from floral branches of Nicotiana tabacum, revealed an absolute effect of growth regulators on the direction of differentiation. Although sucrose concentration and light modify the response, structures produced depend mainly on the auxin to cytokinin ratio. At 0.1:1, vegetative shoot buds form and at 100:1 roots are generated, but a 1:1 ratio promotes floral bud initiation. Incidentally, this remains a classic example of formation of new floral meristems in vitro.

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