CYTOTAXONOMY, ULTRASTRUCTURE, and SYSTEMATIC RELATIONSHIPS in BRYOPHYTES (Organizers: B

Journ. Hattori Bot. Lab. No . 64 : 169-175 (June 1988)

SYMPOSIUM 5-16: CYTOTAXONOMY, ULTRASTRUCTURE, AND SYSTEMATIC RELATIONSHIPS IN BRYOPHYTES (Organizers: B. CRANDALL-STOTLER, W. FREY, B. E. LEMMON and A. J. E. SMITH)

IN VITRO PRODUCTION OF APOGAMY AND APOSPORY IN BRYOPHYTES AND THEIR SIGNIFICANCE

R. N. CHOPRA1

ABSTRACT : It is possible to induce apogamous sporophytes and aposporous gametophytes in some bryophytes under cultural conditions. Among the factors which favour apogamy are reduced hydration, low light level, high sugar concentration, chloral hydrate, low concentration of hormones, coconut milk, 'sporogon factor', high chromosome number and heritable variations. Apospory is favoured by suitable temperature and light, wounding, removal of apical dominance, sufficient humidi· ty and lack of sugar in the medium. Being rare in nature, these phenomena do not have much signifi­ cance in the life cycle of bryophytes. However, these have resulted in increased ploidy level in some mosses. These phenomena support the 'homologous' theory of the origin of alternation of generations, and have also helped in the understanding of some aspects of differentiation.

In addition to the regular, well defined heteromorphic alternation of generations in bryophytes the sporophytic generation can arise from the vegetative cells of a gameto­ phyte, and the gametophyte may develop from the vegetative cells of a sporophyte. These phenomena are called apogamy and apospory, respectively. In these pathways then, the gametes and spores are dispensed away with. When one generation arises directly from the other the genome remains unaltered. This results in the production of haploid sporophytes and diploid gametophytes. How­ ever, when callus formation precedes differentiation of sporophytes the ploidy level is likely to be enhanced, and it should be determined by cytological studies.

ApOGAMY Apogamous sporophytes in mosses were first observed by Springer (1935) on the leaf tips of a naturally occurring diploid Phascum cuspidatum. Since then several in­ stances of apogamy in vitro, both in the diplophase and haplophase, have been re­ ported (see Chopra & Kumra 1988). The main exogenous and endogenous factors which influence production of apogamous sporophytes are being considered below:

1 Department of Botany, University of Delhi, DeJhi-llOOO7, India. 170 Journ. Hattori Bot. Lab. No. 64 198 8

Hydration Springer (I935) observed that the swellings on the leaf tips of the diploid Phascum cuspidatum produced protonema in the presence of abundant moisture. Under drier conditions these swellings developed into apogamous sporophytes. Addition of 3 % agar to the medium also increased the number of apogamous sporophytes per unit area of the protonemal patch in this moss (Wettstein 1942). Subsequent investigations revealed that relative dryness of the nutrient medium also favours differentiation of apogamous sporophytes in Tetraphis pellucida (Bauer 1956), Physcomitrium pyriforme (Bauer 1957, Menon 1974), Desmatodon ucrainicus (Lazarenko 1960), Splachnum ovatum (Lazarenko 1961), and Funaria hygrometrica (Chopra & Rashid 1967). Light In Physcomitrium coorgense the gametophytic callus produces sporophytes and gametophytes in diffuse light, whereas in dark only apogamous sporophytes differ­ entiate. Lal (1963) suggested that light plays a formative role in determining the be­ haviour of apical cell. The frequency of apogamy on aposporous protonema of Phascum cuspidatum is low in day light, whereas in yellow filtered fluorescent light it is greatly increased (Hughes 1969). In Physcomitrium pyriforme apogamy is suppressed in high light level (5,000 to 6,000 lux), whereas in low light (50 to 60 lux) apogamous sporophytes as well as gameto­ phytes differentiate from protonemal filaments (Menon 1974). Sugars Physcomitrium coorgense and P. pyriforme produce apogamous sporophytes only on sucrose-supplemented medium (Lal 1961a, Menon 1974). In Funaria hygrometrica increase in sucrose concentration in the medium from I to 4 % promotes induction of apogamous sporogonia from the stem axes (Rashid & Chopra 1969). Apogamous sporophytes also differentiate from callus obtained from the protonema of this moss on media containing higher concentrations of sucrose (Kumra & Chopra 1980, Kumra 1981). The sporophytic generation seems to require more sugar because of its higher respiratory rate and greater osmotic concentration of its cells. The increased energy level shifts the balance of differentiation to the sporophytic development. On the basis of their work on Physcomitrium pyri/orme Menon and Lal (1972, 1977) postulated that sucrose may be exercising some sort of 'hormone-like' control over the production of a factor for apogamy, possibly by interacting with the endogenous growth substances. Chloral Hydrate The protonema of Splachnum luteum produces a low percentage of apogamous sporophytes spontaneously in addition to normal buds. With the addition of chloral hydrate to the medium, capability for apogamy is increased to 80 % (Bauer 1959a). The isolated aposporous protonema from the hybrid P. pyriforme X F. hygrome­ triea produces apogamous sporophytes only when cultured on a medium containing R. N. CHOPRA: In vitro production ofapogamy and apospory in bryophytes 171 chloral hydrate (Bauer 1966). Growth Hormones The number of sporogones per unit area of protonema in Tetraphis pellucida increases with the addition of low concentration of IAA (Bauer 1956). In Splachnum application of bryokinin (2iP) enhances apogamy (Bauer 1961). At low levels GAa, IAA, kinetin and a combination of kinetin and IAA also appreciably increase the num­ ber of apogamous sporophytes in Funaria hygrometrica (Rashid & Chopra 1969). In Physcomitrium pyriforme IAA and NAA act somewhat synergistically with sucrose in inducing apogamy on protonema (Menon 1974). Interaction of hormones and sucrose is also known during bud formation and regeneration in bryophytes (see Chopra & Kumra 1988). Growth Adjuvants Coconut milk stimulates differentiation of apogamous sporophytes from the callus of Physcomitrium coorgense (Lal 1961 b). In P. pyriforme as well, coconut mil k favours apogamy (Men on & Lal 1977). In Funaria hygrometrica casein hydrolysate enhances apogamy at lower concentra­ tions, but at higher levels sporophyte production is differentially inhibited (Rash id & Chopra 1969). Sporogon Factor Apart from the age of the tissue (Bauer 1963), some other endogenous factors af­ fect apogamy. Bauer (1959 b, c) observed that diploid protonema from the hybrid sporophyte P. pyriforme x F. hygrometrica produces apogamous sporophytes only when it is in organic contact with the parent sporophyte. If the connection is removed only gametophytes are produced. This led Bauer to postulate that a factor elaborated by the parent sporophyte is translocated to the aposporous protonemal filaments, and it induces differentiation of fresh a pogamous sporophytes. The concept of sporogon factor was subsequently supported by Lazarenko (1960), Lal (1963), Rashid and Chopra (1969) and Menon and Lal (1977, 1981). High concentration of sugar in the medium and dry conditions favour the pro­ duction of this factor, whereas high light level and absence of sucrose in the medium prove inhibitory (Menon & Lal 1981). It has been suggested by Lal (1963) that in Physcomitrium coorgense differentiation of gametophytes and apogamous sporophytes is controlled by two distinct substances. The one responsible for gametophytic induction is formed only in light, whereas the 'sporogon factor' is produced in light as well as dark. The sporogon factor seems to be of hormonal nature. The application of bryokinin (known to occur endogenously) enhances apogamy in Splachnum (Bauer 1961). Hart­ mann (1970) observed that red light increases synthesis of bryokinin in the callus derived from the hybrid sporophyte (P. pyriforme x F. hygrometrica), and it also pro­ motes apogamy. 172 Journ. Hattori Bot. Lab. No. 64 1 988

Genetic Constitution Chromosome number seems to play an important role in apogamy. In cultures apogamy is induced with comparative ease in diploid systems or in mosses with high chromosome numbers. Whereas, there are no reports of apogamy in species in which chromosome number is monoploid (true haploid). In the members of Pottiaceae, which readily show apogamy, the gametophytic number (N) reaches up to 50 or 52. In Physcomitrium pyrijorme (Funariaceae) as many as 72 chromsomes have been observed in some cells of callus derived from protonema (Menon 1974). Bauer (1959c) proposed that apogamy is preceded by diploidization of chromo­ somes, since in his work on Funaria hygrometrica only spontaneous diploids formed apogamous sporophytes on regeneration. Lazarenko et al. (1961) obtained apogamous sporophytes in the haplophase as well as diplophase in an allopolyploid species of Desmatodon randii. The view that polyploidization is an important factor in apogamy is further supported by the fact that in liverworts, which have lower chromosome num­ bers, there are no reports of apogamy. On the other hand, this phenomenon is more common in ferns which in general have higher chromosome numbers. On the basis of his investigations on Pottia intermedia Ripetsky (1979a, b, 1980, 1983) holds a different view. He considers that apogamy is a consequence of changes in gene activity (heritable variations), and is not a result of simple chromosome duplica­ tion. The aposporic gametophytes of this moss obtained from a young spore sac wall lacked the capacity for apogamy, whereas those from other parts of the sporogonium did give rise to apogamous sporophytes. Ripetsky (1985) further adds that even though the capacity to produce apogamous sporophytes is maintained at the cellular level, the actual formation of apogamic structures takes place only in suitable environmental conditions. Thus, the three factors important for apogamy are: chromosome number, heritable variations, and the environment.

ApOSPORY Pringsheim (1876) discovered apospory in Amblystegium serpens, and Hypnum cupressiforme. In the same year Stahl observed this phenomenon in Ceratodon purpureus. Marchal and Marchal (1907, 1909) raised polyploid races in some mosses by this method. Since then diploid gametophytes have been obtained from the capsule wall and seta cuttings in several mosses, and in some liverworts with varying degree of suc­ cess (see Mehra & Pental 1976, Chopra & Kumra 1988). In general, the factors which influence apospory are the same which favour re­ generation i.e. suitable temperature and light, wounding and removal of apical dom­ inance. Sufficient humidity and lack of sugar in the medium are two other factors which promote apospory, and these are reverse of conditions which favour apogamy. In some liverworts degeneration of tissues is a prerequisite for apospory. Matzke and Raudzens (1968) suggest that when sufficient number of cells die in the seta of Blasia pusil/a the integration within the organ fails. The surviving cells get isolated and behave as in- R. N. CHOPRA: In vitro production of apogamy and apospory in bryophytes 173 dividuals and not as a part of the whole.

SIGNIFICANCE Apogamy is extremely rare in vivo. Apogamous sporophytes produced in vitro are mostly atypical (being outside the confines of calyptra), and are also usually sterile. The aposporously formed gametophytes are often slow to grow and may not bear gametangia (see Kumra & Chopra 1980, Lal 1984). These phenomena, therefore, have very little importance as alternative pathways in the life cycle of bryophytes. However, some other aspects of apogamy and apospory are significant. It has been suggested that increase in ploidy could have come about, at least oc­ casionally, in mosses as a result of apospory (Matzke & Raudzens 1969). Since the formation of diploid gametophytes is rare in hepatics, it explains the almost com­ plete absence of polyploidy in this group (Berrie 1960). Since effective control of differentiation is possible in vitro, some aspects of mor­ phogenesis have been better understood through a study of these phenomena in cultures. The gametophytic and sporophytic generations have some basic similarities, like the ability to photosynthesize and to regenerate, as also the presence of apical domin­ ance and polarity. The direct origin of one generation from the other further sup­ ports the 'homologous' rather than the 'antithetic' concept of the origin of alternation of generations. Even though work on apogamy and apospory had its beginning more than a century ago, very few taxa have so far been investigated, and our understanding of these phenomena is far from complete. A study of the physiological, biochemical and ultrastructural changes accompanying apogamy and apospory would also be very rewarding.

ACKNOWLEDGEMENTS: I am thankful to the University of Delhi, the Ministry of Educa­ tion and Culture, New Delhi, and the Indian National Science Academy for very kindly providing financial assistance to me for attending the XIV International Botanical Congress.

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