Regeneration, an all-inclusive term, embraces a wide variety of functions. In the context of animals regeneration refers to a tendency to replace the lost parts. However, unlike animal cells, plant cells even after undergoing differentiation retain the capacity to revert to meristematic state and are endowed with an inherent capacity to regenerate into entire plants. This capacity is described as totipotency.
Pteridophytes are known for their high regeneration potential. Practically every cell, of these plants, can be described as totipotent (Fig. 12.1). Regeneration of whole plants from isolated single cells of fern gametophytes was accomplished earlier than it was possible with flowering plants.
Pteridophytes also offer a possibility of studying regeneration from gametophytic as well as sporophytic generations. This chapter is concerned with a comparative account of regeneration of these two generations. Also in a dedifferentiated tissue (callus) one can notice the plasticity of regeneration potential and its control i.e., regeneration at will, either into a gametophyte or into a sporophyte.
Apogamy:
The development of a sporophyte from the gametophyte without fertilization is known as apogamy. The discovery of this phenomenon (apogamy) in ferns has been of considerable interest. It represents a deviation from the normal life cycle, and provides a favourable opportunity for investigations on the causal factors underlying alternation of generations.
In some ferns apogamous sporophyte is a rule, either because of the absence or non-functional nature of one or both sex organs – ‘obligate’ apogamy. Apogamous sporophytes can also be induced on gametophytes which have functional sex organs and form sporophytes by syngamy under normal conditions – ‘induced’ or ‘facultative’ apogamy. Induced apogamy is of greater interest because it does not appear to involve a genetic change and its occurrence, to some extent, at least, can be brought under experimental control.
Induction of Apogamy in Vivo:
A variety of factors favouring apogamy, in several species of ferns, have been reported by previous workers. Upright prothallial growth, i.e. growth away from the soil surface, was an important factor in the induction of apogamy in Doodia caudate.
It has been possible to successfully induce apogamy in several species and varieties of ferns by growing the prothalli exposed to direct illumination; resulting in general level of increase of photosynthesis and supplied with capillary water (through basal end which prevented opening of archegonia and consequent absence of fertilization).
Therefore, the ability of prothallial cells to become meristematic, prevention of fertilization, and exposure of prothalli to direct illumination were considered to be important factors in the induction of apogamy. In a race of Cystopteris fragilis apogamous sporophytes developed during summer, while normal embryos were formed during winter. High light intensity during summer was regarded as the causal factor for apogamy.
Induction of Apogamy in Vitro:
Apogamy can be readily induced on prothalli of Pteridium aquilinum, capable of forming sexual sporophytes, on medium enriched with glucose. There was no response from prothalli without glucose. At a concentration below 1.0 per cent there was only slight response, and striking increase occurred at 1.0 per cent glucose.
The number of apogamous sporophytes per culture steadily increased with an increase in glucose up to 2.5 per cent (Fig. 12.2). Further increase resulted in a decrease in the number of sporophytes per culture. Sucrose, maltose, and fructose could be substituted for glucose.
By the above method apogamy has been induced in a number of ferns. Sucrose (Fig. 12.2) is more effective than glucose. The osmotic effect of sucrose has no effect on the induction of apogamy.
High concentration of available sugar presumably modifies carbohydrate metabolism of the gametopyte whether this has anything to do with the increase in the level of respiratory substrate and, consequently, the available energy remains to be studied. Sucrose allows the normal, delicate, thalloid gametophytes to assume a thickened form and, ultimately, a complex and more massive sporophyte is produced.
Sucrose also promotes apogamy in obligate apogamous forms like Cyrotomium falcatum, and Cheilanthes tomentosa and C. farinose. A comparison made of gametophyte development between normal sexual species and obligate apogamous species of ferns, in particular Pellaea glabella, has revealed that gametophytes of apogamous forms grow faster than their corresponding sexual forms and produce apogamous sporophytes much earlier than sexual forms.
Apospory:
The origin of non-vascular gametophyte, vegetatively from vascular sporophyte, without the formation of spores is known as apospory. It was first described as a natural phenomenon in Athyrium filix-femina var. clarissima.
Since then it has been reported in a number of ferns and is reckoned to be one of the pathways of natural polyploidization in pteridophytes. An interesting instance of spontaneous occurrence of apospory is the production of gametophytic outgrowth from leaves in Phyllitis scolopendrium.
Apospory can be readily induced in homosporous ferns from root, stem and leaf segments but this phenomenon has not been reported in other pteridophytes; homosporous as well as heterosporous,. As for heterosporous pteridophytes it is opined that evolution of heterospory results in stabilization of sporophytic genome. There is simultaneous loss of aposporous development. In this text are considered the developmental aspects of apospory.
In root cells of Pteridium aquilinum on ‘starvation’ medium, which is conducive to apospory (Fig. 12.8), structural changes have been followed. These roots begin to differ from controls much before the appearance of gametophytic outgrowths. The root meristem appears abnormal and disorganized.
In sections, along with the normal viable cells are seen some cells undergoing degeneration. In degenerating cells vacuole losses definition and organelles begin to break up and protoplasmic connections also fade out. Of the viable cells, in region behind the meristem, some become intensely green, form protuberances, and divide to form filaments on which gametophytes differentiate.
These cells show more lamellae in their plastids than the normal root cells of this region. Such cells are dense; having about twice the number of mitochondria, more endoplasmic reticulum and more ribosomes. Cells showing these features are regarded as ‘transformed’.
The filamentous outgrowths developing from these cells at their earliest stage have vacuoles and numerous chloroplasts with lamellae extending across the profile and are also stacked in grana.
The origin of apospory in Pteridium, therefore, can be said to have followed steps:
(a) Disorganization of root meristem,
(b) Degeneration of cells,
(c) Transformation of remaining viable cells, and
(d) Differentiation of characteristic gametophytic cells from these cells.
Contrary to it in leaf segments of Pteridium aquilinum responsive to apospory there is neither any evidence of isolation of cells or death of cells. Instead, in responsive region of leaf is seen loss of vascular connection by an occlusion of tracheids, due to deposits of osmiophilic material. This results in interruption of vascular supply.
An increased frequency of aposporous response is possible in more hydrated medium. This is due to outflow of substances from leaf to the nutrient medium, and also explains the lack of response from older leaves having a relatively thick cuticle which inhibits this outflow.
An analysis of these two results points towards discrepancy which is apparent than real. In both the instances there is isolation of tissue, from the correlative influences, this is achieved in root cells by death of some of the cells.
In leaf cells it is evidenced by blockage of tracheids in leaf leading to its metabolic deprivation. Outflow of substances, in more hydrated medium, also contributes to metabolic deficiency. Hence, metabolic deprivation coupled with leaching of substances becomes additive and results in apospory.
In ferns the detachment of a sporophytic organ from the rest of the plant body causing ‘injury’ as well as ‘starvation’ of the intact organ are conducive to apospory. The reduced rate of metabolism seems to be the key factor in the initiation of aposporous gametophytes.
Low energy supply in the form of low concentration of sugar, or no sugar in the medium, results in apospory. Even in callus cultures only gametophytes differentiate on medium containing little or no sucrose.
Aposporous production of gametophytes by leaves seems to be a function of young leaves only (Fig 12.7D, E) Juvenile leaves up to the seventh order could regenerate and form aposporous gametophytes in young plants of Pteridium aquilinum. In Thelypteris paulistris the leaves up to the tenth order formed aposporous gametophytes.
In Adiantum pedatum juvenile leaves produced aposporus gametophytes while the adult leaves always regenerated sporophytes. Regeneration from mature leaves has been limited to horticultural and genetically abnormal varieties.
The regeneration from rhizome segments of Ampelopteris prolifera into gametophytes or sporophytes was conditioned by the length of segments, and energy level of medium. With 0.5 per cent or more sucrose, segments of larger size (1 cm or more) produced sporophytes. At a low level of sucrose, or on sucrose-free medium, only gametophytes developed. The smaller segments (3-4mm) regenerated only gametophytes even on medium enriched with two per cent sucrose.
From these results it can be concluded that following factors favour aposporous response, from sporophytic explants:
(a) Senescence of sporophytic tissue
(b) Isolation from the main body of plant
(c) Unfavourable nutritive condition
(d) Physiological isolation of cells within the explant
(e) Juvenile nature
(f) Size of explants.
Multiple Pathways of Cellular Differentiation:
A continuously growing tissue (callus) and control of its differentiation in Pteris cretica has been achieved. The callus formation, on excised young plants, was induced on mineral medium containing two per cent sucrose and 1.0 ppm 2, 4-D.
The tissue could be maintained as an undifferentiated mass on mineral medium containing either 2,4-D (0.1 ppm), or IAA (5.0 ppm), Differentiation occurred when the auxin concentration was decreased to 0.01 ppm, or it was omitted altogether.
With 0.1 per cent or higher concentrations of sucrose, glucose, or fructose sporophytes differentiated. Occasionally, gametophytes also differentiated. When the callus was grown on the sugar-free medium, instead of sporophytes gametophytes differentiated. Sugar, therefore, exerted the control over the type of differentiation. The auxin (2, 4-D) in the concentrations chosen did not alter the type of differentiation; gametophyte or sporophyte.
Similarly, on auxin-supplemented medium, the excised leaves of P. vittata formed callus. This differentiated into gametophytes on medium lacking both auxin and sucrose. Scaly hairs, a sporophytic feature, also developed. Sporophytes differentiated on medium lacking only auxin.