The life cycles of archegoniate plants in general, and of pteridophytes in particular, reveal that two cells -spore and fertilized egg- initiate two separate phases of growth by giving rise to two dissimilar and independent plants.
Each plant is characteristic of its generation. While gametophyte and sporophyte, are two dissimilar phases, as well as two separate plants in a pteridophyte, there is little direct evidence to account for the causal differences of these two generations.
Basically, it requires a comprehension as to how one and the same genome can produce plants of two diverse morphologies. It remains to be ascertained whether differential behaviour of the germ cells results in the alternation of generations.
As for history, a regular alternation of an asexual sporophytic generation with a sexual gametophytic generation was elucidated long ago. This alternation of generations is associated with a reduction in chromosome number from diploid (2n) to haploid (In) level. There is, therefore, an alternation of morphologies as well as levels of ploidy.
This phenomenon of alternation of generations, in its phylogenetic implications, has attracted considerable attention and remains a fascination for botanists. For its understanding, the efforts made also reflect the change of interests and developments in science and technology. An alternation between two generations has stimulated research in comparative morphology and prompted the interpretation of morphology in causal terms.
Modifications of Alternation of Generations:
The controversy concerning the interpretation of alternation of generations attracted even more attention when apogamy and apospory were described in ferns. Thus, while the alternation of levels of ploidy was not obligatory, there was only an alternation of morphologies.
Thus the alternation of generations exists with two modifications, apogamy and apospory. The apospory or production of gametophytes from the leaves of ferns is possible under starvation (low concentration of sucrose), and maltreatment (injury).
In obligate apogamous forms the sporophyte develops vegetatively from a gametophyte because the archegonia are either missing or non-functional. Apogamy can also be readily induced on fern gametophytes by supplying higher concentration of sucrose than needed for vegetative growth.
Prothalli of sexually reproducing Pteridium can be induced to form haploid apogamous sporophytes on sucrose-enriched medium. Aposporous gametophytes develop from the leaves of apogamous sporophytes on culture to mineral-agar-medium only. These aposporous gametophytes on transfer to sugar-enriched medium, form apogamous sporophytes. Thus apogamous-aposporous life cycle can be continued generation after generation.
It does not seem unreasonable that normal two-dimensional growth of gametophytes, is related to limited energy supply normally available to the gametophyte. It contrasts sharply with three-dimensional pattern of sporophyte related to high energy supply. These two phenomena are represented by a reversible reaction (Fig. 13.1).
It is also substantiated by experiments revealing multiple pathways of cellular expression, the genetic component imposing no restriction on differentiation. Leaf callus of Pteris cretica differentiated into sporophytes on medium containing high levels of sucrose, and gametophytes at low levels (Bristow, 1962).
However, difficult to explain are the results of the experiments in which diverse growth patterns, both gametophytic and sporophytic, differentiate at the same time under similar cultural conditions. The rhizome and petiole segments of fern Phlebodium aureum regenerated gametophytes as well as sporophytes.
Differential Behaviour of Germ Cells: Spore and Egg:
The life cycle of pteridophytic plants reveals that two cells, spore and fertilized egg, initiate two separate phases by giving rise to two dissimilar plants characteristic of each generation. While their products are two dissimilar phases, there is not much direct evidence to account for the differences between the gametophytic and sporophytic generations.
Considerations for the differential behaviour of the cells; spore and zygote, resulting in the alternation of generations have been engaging attention since the beginning of the 20th century. It was proposed that characteristic differences in a gametophyte and a sporophyte are due to extrachromosomal nuclear components of spore and zygote.
Further it was pointed out that one must look for answers in causal morphology, rather than heredity or chromosome number. In other words, we should consider the range of possibilities within which a plant can express itself, rather than insist upon a fixed routine of alternation by which it must express itself.
It was suggested that either the spore or zygote are inherently different structures which result into distinct developmental patterns. Or, the initial cells are similar but the environment (physical and nutritional) in which they develop determines their response.
Biophysical and Biochemical Environment of Germ-Cells:
There are significant differences in the initial environment of two germ-cells. The spore germinates as a free cell, whereas the fertilized egg develops within the confines of an archegonium. A germinating spore is subject to losses of metabolites by diffusion into the substrate whereas the zygote within the confines of gametophytic tissue has no corresponding drain on its resources.
Rather, lying in a gradient of growth regulating environment; it enjoys the nutritional advantages not available to a spore. It was reasoned quite long time ago that restraint or pressure exerted on the fertilized egg by the adjacent prothallial cells in some way influences the characteristic growth pattern of egg.
It is in contrast to freely germinating spore on a substrate external to the plant. It was envisaged that the initial cell is a neutral or uncommitted cell but its immediate environment controls its development to an appropriate path. The proponents also suggested inserting a spore in the archegonium, and placing the zygote under environmental conditions normal for spore gemination.
Cultural Behaviour of Isolated Zygote:
In experiments when four to five-day-old undivided zygote (35-55 microns) was isolated (Fig. 13.2A) and grown in liquid mediuim containing three per cent sucrose and sorbitol or inositol. During the first week in culture the zygote divided followed by division corresponding to the usual sequence of ‘contained embryo’.
However, during the second week the sequence of divisions was unlike that of the normal embryo. As the growth continued, the cultured embryo lost three-dimensional appearance and developed into an unorganized callus-like mass consisting of loosely-arranged parenchymatous cells. After one month there was no further growth.
When these cultures were transferred to liquid medium on a rotary shaker, free-cells and small cell-aggregate dispersed from the cell mass. However, on agar culture after three to four months the renewed activity was noticed and the embryos developed into asymmetrical, two-dimensional thalloid plants (Fig. 13.2B).
These structures not only resembled a gametophyte but also produced structures similar to antheridia (Fig. 13.2C) which, however, did not differentiate spermatogenous tissue. This experiment indicated that thalloid structure results from an excised zygote (on isolation from the confines of archegonium).
Continued experimentation revealed:
(a) Younger the excised embryo, greater is the degree of variation in its development. The older excised embryo shows much less deviation from the development of the ‘contained embryo’,
(b) Nutritional requirements of younger embryos are far more complex and exacting than those of older embryos, and
(c) Embryos entering the ‘differentiation-phase’, or already in advanced, stage of growth, are not influenced in their development by the restrictions of the surrounding prothallial tissue.
In a follow up of these experiments the prothalli bearing fertilized eggs on culture to medium supplemented with 1.0 per cent sucros and 10-6M NAA, remained indefinitely in the division phase of growth characterized by rapid, unorganized cell division and enlarged calyptra. Addition of NAA not only interferes with timing of the embryological events but also with morphological form.
Interference in RNA metabolism of developing embryos of P. aquilinun is reported to prevent embryogenesis, or retard it. Gametophytes fed with thiouracil, this analogue is known to disturb RNA metabolism, after fertilization produced deformed sporophytes which often showed a tendency to produce a gametophytic form.
A prolonged feeding with low concentration of thiouracil killed the embryos, but embryos allowed to develop normally for seven days before feeding showed only transient effects even at the highest concentration of the analogue.
All these experiments indicate that normal development of the embryo is subject to both hormonal and physical control.
The conclusions arrived at, lend support to hypothesis that different morphologies of gametophyte and sporophyte can be explained in terms of different environments in which two initial cells – spore and zygote- initiate their development. These experiments however, fail to explain whether or not egg and spore are two different specialized cells or unspecialized, neutral cells.
Differential Activation of Genes in Germ-Cells:
Contrary to the point of view presented above it was pointed out that a germ-cell is not a neutral structure, instead it is considered to inherit a specific programme to develop into a particular pathway. It was proposed that a life cycle be regarded as a system of cyclic correlation, in which the concluding stage of one phase influences the development of the next. In other words germs-cells are specialized cells.
Further, it was hypothesied that characteristic differences in gametophyte and sporophytes are due to extrachromosomal nuclear components of spore and zygote. In an extension of this point of view it has been pinpointed (that the subcellular differences in the spore and zygote other than ploidy, may control their development, and account for the alternation of generations.
It was found that deoxyribosenucleic acid (DNA) is detectable not only in the nucleus but also in the cytoplasm of the egg in Pteridium, and the egg cytoplasm contains large amounts of RNA and basic proteins in comparison with the somatic cell.
Subcellular studies of egg during oogenesis revealed that old organelles descended from spore degenerate, and new ones arise de novo with the active participation of the nucleus. Furthermore, an additional membrane which is acetolysis-resistant and surrounds the egg may act as a barrier, during early embryogenesis, to any information passing from the gametophyte to fertilized egg and disturbing its newly-acquired potential.
Before these changes are apparent, to begin with there is extensive vesiculation it is followed by attenuation of egg cytoplasm resulting in loss of ribosomes (as revealed by reduction in affinity for basic dyes). This stage is ascribed to result in loss of information molecules i.e., elimination of RNA, protein (Fig. 13.3) and other macromolecules, which are associated with preceding phase.
A change in the number and variety of these macromolecules in egg; substantial level of nucleic acid and basic proteins in its cytoplasm and zygote may well explain the capacity for new mode of development.
Also during sporogenesis of Pteridium there are striking parallels with oogenesis.
These are briefly summarised as:
(a) Spore-mother-cells are isolated structures, surrounded by thick callose wall,
(b) There is attenuation of cytoplasm arising out of cellular autophagy; resulting in decline in frequency of ribosomes (Fig. 13.4), plastids and mitochondria becoming indistinct,
(c) Nucleocytoplasmic interaction takes the form of tubular extension of nucleus which penetrates the cytoplasm.
On the basis of above information three inevitable conclusions are:
(a) Isolation of a cell is an essential requirement for its differentiation into a specialized cell, be it spore-mother-cell or egg.
(b) Prior to differentiation of cell there is elimination of information from earlier phase and
(c) During sporogenesis as well as oogenesis there is extensive nucleo-cytoplasmic interaction resulting in acquiring of new information.
Based on these findings, it is possible to interpret pteridophyte life cycle in terms of differential activation of genes (Fig. 13.5). During oogenesis activation of genes for sporophytic growth takes place and during sporogenesis activation of genes for gametophytic growth takes place. The scheme is applicable to both sexual and apogamous or aposporous cycles.
However, for apogamous cycle in Cheilanthes neither there is an evidence that cells destined to produce a sporophyte are isolated from the rest of the cells of gametophyte nor these cells are in any way different from rest of the cells of gametophyte. They are also identical with other cells of gametophyte in their cytology.
The difference if any is likely to be cytoplasmic. This is in fact the case. In apogamous ferns such as Dryopteris borreri the spores produced are about half the frequency but much bigger than those in corresponding sexual species.
The spore size in D. borreri is 70 micron as against 55 micron in D. filix-mas. This greater volume is occupied by cytoplasm. It is very likely that in apogamous forms, during sporogenesis the process of cellular autophagy fails to eliminate the entire information for sporophytic growth.
As for aposporous cycle two crucial factors, of production of gametophytes from sporophytes in an aposporous way, are:
(a) Widespread autolysis of cells, under starvation- condition favourable for apospory and
(b) Only a few of the cells grow out to form aposporous gametophytes, the remaining cells degenerate.
Autolysis is a situation similar to what occurs during sporogenesis in spore-mother-cells. An extensive degeneration of cell might be of significance in isolation of a few cells from their natural environment, to change them for a new cycle of growth.
On the basis of current knowledge, the concept of cyclical correlation has been redrawn (Fig. 13.6) in terms of cyclical alternation. It essentially conforms to the condition that concluding stage of one phase conditions the development of the next.
