Effect of some Physical Factors on Growth and Fertility in the Male Clones of the Moss Microdus brasiliensis (Dub.) Ther.

Short Communication

Effect of some Physical Factors on Growth and Fertility in the Male Clones of the Moss Microdus brasiliensis (Dub.) Ther. R. N. CHOPRA and POONAM MEHTA Depanmcnt of Botany, University of Delhi, Delhi 110007, India Received January 12, 1987 . Accepted March 16, 1987

Summary Vegetative growth and antheridial formation in the male clones of Microdus brasiliensis are affected by light level (intensity), photoperiod, temperature and hydration of the medium. Increase in light level up to 4,500 lux increases the total number of gametophores as well as the number of fenilc gametophores per culture. Gametangia! induction and vegetative growth increase with increasing photoperiod, responding optimally with continuous illumination. A tempera(Ure of 18°C and a medium with 0.8% agar is optimal for gametangial induction and vegetative growth. With 2.4 % and 3.2 % agar, vegetative growth is reduced and no antheridia are induced.

Key words: Fertility, growth, male clones, moss, physical factors.

Introduction Bryophytes are easily maintained in culture in the vegetative phase, but induction of the reproductive phase is not always possible because the factors responsible for this change are not yet clearly understood. Therefore, we report in this paper effects of different exogenous factors [light level (intensity), light duration, temperature and agar concentration] on growth of and gametangial induction in the male clones of a dioecious moss of the family Dicranaceae, Microdus brasiliensis (Dub.) Ther. Materials and Methods The moss was collected from Pachruarhi (Madhya Pradesh) during October 1982. Aseptic cultures were raised from spores on one-half strength Knop's macro nutrients basal medium, with Nitsch's trace element solution (Nitsch and Nitsch, 1956). 10 rug/I ferric citrate as the iron source, 1 % sucrose and 0.8 % agar. Before autoclaving, the pH of the medium was adjusted to 5.8. Cultures were raised in rimless «Borosil .. glass tubes (15x2.5cm), each containing 20ml medium. To establish male clones of Microdus for the present experiments, leafy branches (gametophores) bearing antheridia were picked from stock cultures and planted individually on fresh medium. These gametophores regenerated secondary protonemata which developed numerous buds; of the resulting gametophores 18 % bore antheridia after 48 days.

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Cultures were usually maintained in a room at 25t2 °C in 3,500 lux. of continuous light from a combination of white light fluorescent tubes and incandescent bulbs of 40 W each (fluorescent tubes: incandescent bulbs: 3: 2). A 25-day-old, bud-free protonema was used as inoculum and these cultures were subjected CO different light levels (150-4,500 lux). light durations (8-24h photoperiod.) and temperatures (18-30°C) in incubacoI'S. Agar was added at concentrations of 0.4,0.8, 1.6,2.4, and 3.2%, w/v. Fertile gametophores were easily identified with a dissecting microscope. Experiments, each repeated once, lasted 60 days. Each treatment was replicated twelve times.

Results

Light level Cultures were subjected ta six levels af light: darkness {cantrall, and 150 ta 4,500 lux at 25 ±2 dc. In darkness the inoculum failed to regenerate. Under 150 and 1,100 lux antheridial heads were naticed after 50 days. Under higher light levels they were induced two days earlier. The percentage of fenile gametophores and total number of gametophores per culture (index of vegetative grawth) increased with increasing light level, with a maximum at 4,500 lux {Fig. 1). The number of antheridia per fenile gao metophore was 8-10 at 2,200 to 4,500 lux, whereas at 1,100 lux and belaw their number was reduced to 6 - 8.

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Fig. 1: Effect of light intensity on vegetative growth and gametangial induction in the male clones of Microdus brasiLiensis. In darkness inoculum failed to regenerate. % response indicates the percent of fertile gametophores. Each datum indicates the mean and standard error from 12 replicates. Data from 6O-day-old cultures.

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Vol. 130. pp. 477-482 {1987}

Growth and fertility in Microdus

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Fig. 2: Effect of light duration on vegetative growth and gametangial induction in the male clones of Microdus brasiliensis. Each datum indicates the mean and standard error from 12 replicates. Data from 6O-day-old cultures,

Light duration Cultures were subjected to three photoperiods: 8, 16, and 24-h with a light inten~

sity of 3,500 lux at 25±2 0c. Antheridia were induced after 55, 52, and 48 days in 8, 16, and 24-h photoperiod, respectively. The percentage of fertile gametophores and total number of gametophores per culture increased with increasing photoperiod, and maximum response was observed in continuous illumination {Fig.2}. The num~

ber of antheridia per fertile gametophore remained 8 -10 in all of the photoperiods. Temperature

In 3,500 lux of continuous light antheridia were induced after 46 days at 18°Cj at 25 and 30°C they appeared after 48 and 50 days, respectively. The highest number of antheridial heads was observed at 18°C (Fig. 3). The total number of gametophores per culture also decreased with increase in temperature (Fig. 3). The number of anthe-

ridia per fertile gametophore always remained 8 -10.

Hydration Cultures of Microdus were grown on media containing different concentrations of

agar. Those in liquid medium served as contro1. The percentage of fertile gametophores and total number of gametophores were maximum at 0.8 % agar. With 2.4 % and 3.2 % agar, no antheridia were induced (Fig. 4). The number of antheridia

J. Plant Plrysiol. Vol. 130. pp.

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R. N.

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Fig. 3: Effect of temperature on vegetative growth and gametangial induction in the male clones of Microdus brasiliensis. Each datum indicates the mean and standard error from 12 replicates. Data from 6O-day-old cultures.

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Fig. 4: Effect of agar concentration on vegetative growth and gametangia! induction in the male clones of Microdus brasiliensis. Each datum indicates the mean and standard error from 12 replicates. Data from 6O-day-old cultures.

! Plant PbysioL VoL 130. pp. 477-482 (1987)

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per fertile gametophore was reduced to six or seven in liquid medium as compared to 8 -10 on media containing 0.8 % or 1.6 % agar. Medium with 0.8 % agar was optimal for vegetative growth as well as for antheridial production, but the effect was more pronounced on the latter, Relative enhancement of antheridial production and vegetative growth at the optimal concentration of agar was 245 % and 155 % of the controls, respectively.

Discussion In Microdus brasiliensis increasing light intensity raises the number of fertile gametophores per culture, as also reported in four other moss species (see Kumra and Chopra, 1983). In the previously investigated liverworts as well increased light intensity favours production of gametangia (see Chopra and Bhatia, 1983), whereas in Bryum argenteum maximum response occurs at 1,800-2,000 lux (Chopra and Bhatia, 1981). Most of the investigated mosses are day-neutral (Chopra and Rahbar, 1982; Kumra and Chopra, 1983). Microdus brasiliensis also behaves as a day-neutral plant. Even if Microdus brasiliensis does not have a critical temperature requirement for gametangial induction, the lowest temperature induced maximum antheridia. The behaviour of Microdus is similar to that of Rieeia crystallina (Chopra and Sood, 1973 b) and Leptobryum pyriforme (Chopra and Rawat, 1977) in which the response per culture is inversely related to a rise in temperature, In contrast to the above taxa, some mosses require a specific temperature for gametangial induction: 7 °C for Funaria hygrometrica and Physcomitrium pyrifonne, 17°C Physeomityella patens (Nakosteen and Hughes, 1978) and 18 °C for Philonotis tumey.,na (Kumra and Chopra, 1983). In some species the temperature requirement for the onset of the reproductive phase in cultures corresponds to the temperature range in their natural environment. Philonotis occurs at high altitudes and requires a lower inductive temperature in culture (18 °C). Furthermore, Barbula gregaria and Bryum corona tum, which occur at low as well as high altitudes, develop gametangia over a wide range of temperatures in culture (Kumra and Chopra, 1983). Similar response is also observed in the two species of Riccia in Delhi: Riccia crystallina growing during winter; andR, gangetica growing in summer. In culture, gametangial induction is optimal at 8-15 °C for R. crystallina (Chopra and Sood, 1973 a) but it isso at 25 ±2 °C for R. gangelica (Dua et aI., 1982). The behaviour of Microduscan also be correlated with its natural environment. Acknowledgements We thank Prof. S. S. Kumar, Panjab University, Chandigarh, for the identification of the moss. Financial assistance from the Council of Scientific and Industrial Research, New Delhi, to one of us (P.M.) is gratefully acknowledged.

References CHOPRA, R. N. and S. C. BHhTI.A' New Phytol. 89, 439-447 (1981). - - Bot. Rev. 49, 29-63 (1983). CHOPRA, R. N. and K. RAHBA", New Phytol. 92, 251-258 (1982).

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CHOPRA, R. N. and M. S. RAwAT: Beitr. BioI. Pflanzen 53, 353-357 (1977). CHOPRA, R. N. and 5.5000: Bryologist 76, 278-285 (1973 a). - - Phytomorphology 23, 230-244 (1973 b). DUA, 5., N. SINGAL, and R. N. CHOPRA: Cryptogam. Bryol. Lichenol. 3, 189-199 (1982) KUMRA, P. K. and R. N. CHOPRA: Bot. Gaz. 144,533-539 (1983). NAXOSTEEN, P. C. and K. W. HUGHES: Bryologist 81, 307-314 (1978). NIT5CH, J. P. and C. NrrsCH: Am. J. Bot. 43, 839-851 (1956).

J. Plant Physiol. Vol. 130. pp. 477-482 (1987)