The main path of morphogenesis processes in plant cells culture

Various forms of morphogenesis have been observed in cell suspension cultures; this morphogenesis is apparently dependent upon the development in the cultures of relative large aggregates of cells, often several hundred in number, associated in a symplast. The morphogenesis can take the form of root initiation, shoot bud initiation or the development of somatic embryos (often referred to as embryoids) (Konar, Thomas & Street, 1972). Usually only one form of morphogenesis is expressed; sometimes the situation is more complex but in these cases one form is usually dominant—carrot cell cultures can be manipulated to show predominantly root initiation or exclusively embryoid development (Kessel & Carr, 1972).

15.5.1—
Somatic Embryogenesis

Work on embryogenesis in carrot cultures illustrates clearly the problems in cell physiology raised by the morphogenetic potential of plant cell cultures. These studies date from the report in 1958 (Steward, Mapes & Mears, 1958; Steward, 1958) of embryo-like plantlets in liquid carrot cultures and their continuing development when the cultures were plated out on agar-solidified medium. Further work quickly established the presence in such cultures of structures strikingly similar to the globular, heart-shaped and torpedo-shaped stages of normal embryology from the zygote.

Halperin and coworkers (Halperin & Wetherell, 1964; Halperin, 1967; Halperin & Jensen, 1967) and Street and coworkers (Smith & Street, 1974; McWilliam, Smith & Street, 1974) have shown that the embryos in carrot cultures arise from single cells at the surface of the cellular aggregates and are released, at various stages of development, as free-floating structures which, if they have already reached the advanced globular stage, are capable of completing their development in isolation from the parent embryogenic clump. These cultures grow actively, the embryogenic clumps proliferating and fragmenting due to enlargement and separation of cells in their interior, and do not form embryos in a synthetic medium containing sucrose, inorganic salts, thiamine, meso -inositol, kinetin and auxin (2, 4-dichlorophenoxyacetic acid). To initiate embryogenesis they are transferred to a similar medium, lacking auxin, which contains nitrate and ammonia (or urea or glutamine) and is at pH 5.0–5.4. Despite earlier claims, coconut milk is neither essential for growth nor for embryogenesis. Evidence that immature embryos have exacting nutritional

requirements (isolated embryo culture—see Street, 1969) and the contrasted simplicity of the culture medium which supports prolific embryogenesis in carrot cultures suggests that the embryogenic cell aggregate fulfils a 'nurse' role to the embryogenic surface cells and that the embryos must remain attached to the aggregate to complete their early development to the point where they can survive and mature into plantlets when released.

This phenomenon of somatic embryogenesis raises a number of questions of cellular physiology. The term totipotency has been introduced to describe the embryogenic competence of the single cells from which the embryos arise. At present it is only possible to establish new plants, whether via embryogenesis of via shoot bud initiation, followed by adventitious root development, from the cell cultures of a limited (if now quite large) number of species or varieties within species. Cultures which show this morphogenetic potential support the view that the pathways of cytodifferentiation which result in living tissue cells do not involve any loss or permanent inactivation of the genome and that they are, under appropriate environmental stimuli, completely reversible. Whilst however some cell cultures remain recalcitrant, this cannot be established as a universal principle. It may be that in such cases the conditions of culture fail to provide or permit the synthesis of an essential morphogen. Halperin (1967) has, on the other hand, advanced the very interesting hypothesis that the achievement of totipotency occurs during the initiation of the carrot culture from the primary explant (storage root or seedling organ) and that embryogenesis is expressed by cell clumps derived from such 'induced' cells, the primary culture consisting of both these and 'non-induced' cells. Retention of high embryogenic capacity in the cultures will then depend upon culture conditions favouring the active proliferation of the induced cells. On this hypothesis, failure to obtain embryogenesis in culture would have as its primary cause inappropriate conditions of callus initiation; the conditions of initiation would have effectively activated cell division and growth in the explant cells but failed to achieve the necessary 'dedifferentiation' to obtain cells with the competence of the zygote (a concept already raised here in previously discussing cytodifferentiation in cell cultures).

In classical plant embryology it has been considered that the early segmentations of the zygote (at least up to the 16-celled proembryo) follow a precise and species specific sequence which has phylogenetic significance (Johansen, 1950). During these divisions the cells of the proembryo are considered to inherit different cytoplasmic potentialities from the different regions of the zygote and these differences are regarded as determining from the beginning the exact role they and their daughter cells will play in constructing the embryo and its parts. This concept is often referred to as the theory of precise mosaic organization. The early segmentations involved in somatic embryogenesis have now been followed in a limited number of species (Street, 1976). Figure 15.9 illustrates the sequence of early embryology in carrot cultures. These segmentation sequences involved in somatic embryogenesis show more uniformity one with another than is depicted in the published accounts of the zygote embryology

of the species concerned. In summary, early development of somatic embryos involves enlargement and regular segmentation in a subspherical cell mass by walls of minimum surface. This supports the concept of D'Arcy Thompson (1942), based upon his studies in animal embryology, that surface tensions are important in determining the early segmentations of embryology and that only at a later stage do localized growth centres emerge whose functioning gives rise to the divergences in the morphology and anatomy of embryos of different species. This regulative theory of organization regards the early segmentations as being controlled by physical factors and as not involving any 'determination' of the early formed cells. With this background, the greater diversity and specificity recorded for zygotic embryogenesis can be interpreted as resulting from the physical restrictions and polar chemical gradients imposed upon the embryo as it develops within the ovule; when these influences are removed, as in cell cultures, the embryology reverts to a more basic or 'primitive' type of segmentation. This interpretation is supported by observations on the segmentations observed in natural polyembryony and by recent reports that indeed much more variable patterns of segmentation occur in ovule embryology than has hitherto been recognized (Jensen, 1965; Brown & Morgensen, 1972).

15.5.2—
Polarity of Embryogenic Cells

The recognition that the embryos arising in cell culture have their origin in superficial cells of the cell aggregates raises the question of whether such cells have any unique cytological characters and whether their observation can yield information on the physical basis of cell polarity. In carrot cultures proliferating in the presence of 2, 4-D (Street & Withers, 1974) these cells are small, rich in cytoplasm and with a large diffusely-staining nucleus containing a prominent nucleolus. Small vacuoles are clustered round the nucleus and each cell contains several amyloplasts containing prominent starch grains (Fig. 15.10). Study of these cells in the electrorn microscope shows the presence of numerous round and oval mitochondria and Golgi bodies and the regular presence of small numbers of lipid bodies (spherosomes). As might be expected from their meristematic activity, these cells are frequently observed in mitosis (in contrast to the expanded interior cells of the aggregates) and show numerous wall microtubules and limited arrays of cytoplasmic and nuclear microfibrils. When the cultures are transferred to auxin-free medium there is a transient increase in proliferation prior to the initiation of embryogenesis and the superficial cells show a change in segmentation pattern leading to the origin of 4-celled groups (Fig. 15.10). The individual cells in these groups either initiate an embryo, or by their further division promote the growth of the aggregate or undergo expansion (and senescence?) and become involved either in the release of the developing embryo or the break-up of the proliferating embryogenic aggregate. Associated with this changed segmentation pattern the densely cytoplasmic superficial cells

show changes in fine structure. They have increased numbers of E.R. profiles (parallel arrays of rough E.R. profiles become particularly prominent) and of ribosomes. The Golgi bodies are more numerous and more compact. Additional large mitochondria (discs with swollen rims in outline) make their appearance and may come to occupy a considerable volume of the cytoplasm. The cells of the young proembryos show similar fine structure (Fig. 15.11). Although the first division of the embryogenic cells is by a wall parallel to the surface of the aggregate and at right angles to the longer axis of the cell (giving rise to an apical and a basal cell; the former being the first cell of the proembryo proper and the latter of the very variable suspensor), nevertheless fine structure studies do not reveal any prior asymmetry in the distribution of cytoplasm or cell organelles. Rather disappointingly, these studies have not revealed any unique features of the embryogenic cells or exposed any structural polarity. Perhaps this is not unexpected when we bear in mind the lack of any uniformity of fine structure in those angiosperm zygotes which have been studied (Jensen, 1965; Schulze & Jensen, 1969; van Went, 1970; Morgensen, 1972). Such studies only serve to emphasize that the special nature of embryogenic cells must now be approached at the level of molecular biology.

17)Suspension culture: obtaining and methods of cultivation of suspension culture. The adventage of suspension culture
A type of culture in which intact cells are maintained in suspension in the culture medium so that they are distributed evenly within it

There are two types of suspension cultures, i) Bat culture ii) Continuous Culture

A) Batch Culture:

a.Slowlyrotatingculture
b.Shakeculture
c.Spinningculture
d. Stirred culture

B) Continuous Culture:

a.Chemostats
b. Turbidostats.

A) Batch Culture:

These cultures are maintained continuously by propagating a small aliquot of inoculum in the moving liquid medium and transferring it to fresh medium (5 x dilution) at regular intervals. Generally cell suspensions are grown in flasks (100-250 ml) containing 25-75 ml of the culture medium. Batch suspension cultures are most commonly maintained in conical flasks incubated on orbital platform shakers at the speed of 80-120 rpm. The biomass growth in batch culture follows the fixed pattern. When the cell number in suspension cultures is plotted against the time of incubation, a growth curve is obtained depecting that initially the culture passes through a lag phase, followed by a brief exponential growth phase- the most fertile period for active cell division. The growth declines after three to four cell generations, signalling that the culture has entered the stationary phase.

For subculture, the flask containing suspension culture is allowed stand still for a few seconds to enable the large colonies to settle down. A pipette or syringe with orifice fine enough to hold aggregate of two to four cells or only single cells is used. The suspension is taken from the upper part of the culture and transferred to a fresh medium.

i) Slowly Rotating Cultures:

Single cells and cell aggregates are grown in a specially designed flask, the nipple flask. Each nipple flask possesses eight nipple-like projections. The capacity of each flask is 250 ml. Ten flasks are loaded in a circular manner on a large flat disc of a vertical shaker. When the flat disc rotates at the speed of 1-2 rp, the cell within each nipple of the flask are alternatively bathed in a culture medium and exposed to air.

ii) Shake Culture:

It is very simple and effective system of suspension culture. In this method, single cells and cell aggregates in fixed volume of liquid medium are placed in conical flask. Conical flasks are mounted with the help of clip on a horizontal large square plate of an orbital platform shaker. The square plate moves by a circular motion at 60-180 rpm.

iii) Spinning Culture:

Large volume of cell suspension may be cultured in 10L,bottles which are rotated in a culture spinner at 120 rpm at an angle of 45 0.

iv) Stirred Culture:

This system is also used for large scale batch culture. In this method, the large culture vessel is not rotated but the cell suspension inside the vessel is kept dispersed continuously by bubbling sterile air through culture medium. The use of an internal magnetic stirrer is the most convenient way to agitate the culture medium safely. Magnetic stirrer revolves at 200-600 rpm. The culture vessel is a 5-10 litres round bottom flask.

B) Continuous Culture System:

In this system, the old liquid medium is continuously replaced by the fresh liquid medium to stabilize the physiological stage of the growing cells. Normally, the liquid medium is not changed until the depletion of some nutrients in the medium and the cells are kept in the same medium for a certain period. As a result, the active growth phase of the cell declines the depletion of nutrient. The cells passing through out flowing medium are separated mechanically and reintroduced in the culture.

i) Chemostats:

In this system, cultures vessels are generally cylindrical or circular in shape and posses inlet and outlet pores for aeration and for introduction of and removal of cells and medium. The liquid medium containing the cell is stirred by a magnetic stirrer. The introduction of fresh sterile medium, which is pumped in at a constant rate into the vessel is balanced by the displacement of an equal volume of spent or old medium and cells.
Such a system can be maintained in a steady state so that new cells are produced by division at a rate which compensate the number lost in outflow of spent medium.

ii) Turbostats:

In this system, the input of medium is intermittent as it is mainly required to control the rise in turbidity due to cell growth. The turbidity of a suspension culture medium changes rapidly when cells increase in number due to their steady state growth. The changes in turbidity of the culture medium can be measured by the changes of optical density of the medium. In Turbostats an automatic monitoring unit is connected with the culture vessel and such unit adjusts the medium flow in such a way as to maintain the optical density or PH at chosen, present level.

19 The immobilized plant cells. The adventage of immobilized cells culture. Cell immobilization is a technique to fix plant cells in a suitable matrix. Cell immobilization is different from cell entrapment in that immobilized cells can be entrapped cells but also the cells are absorbed onto support materials. Plant cells grow slowly, they produce targetted compounds slowly, they are more easily disrupted by physical stress and their behaviour (growth and synthesis) is influenced by chemical signals by neigbouring. Then by immobilization, the plant cells are protected from liquid shear forces. Moreover, immobilization facilitates the importance of cellular cross talk, which can establish inter-cellular communication by the action of signalling molecules. This should enhance the biosynthetic of plant cells. Freely suspended plant cells mostly accumulate their secondary metabolistes in the stationary phase of their growth cycle, at the point of time their growth stop. Entrapment of plant cells is one the means to create non-growth condition under which the production of secondary metabolites may be improved. Advantages: retention of biomass enables its continuous reutilization as a production system. The high cell density allows a reduction in contact in packed bed catalyst leading to an increased volumetric productivity; separates cells from medium and the product is extra cellular, which will simplify downstream processing compared to extract from tissue; allows a continuous process, which increase volumetric productivity; compatible with non-growth associated product formation; reduces some problems associated with the cultivation of plant cells such as the formation of aggregates, and susceptibility to mechanical damage (shear stress) are problems which do not affect immobilized system compared to cell culture.