Preliminary studies on callus induction in the moss: Hyophila involuta

Department of Botany, University of Delhi, Delhi 110007, India

Preliminary Studies on Callus Induction in the Moss: Hyophila involuta KAVITA RAHBAR and R. N. CHOPRA With 4 figures Received February 1, 1980 . Accepted March 6, 1980

Summary Callus has been induced in Hyophila involuta grown on MURASHlGE and SKOOG'S medium. Ammonium nitrate, chela ted iron, vitamins together with sucrose are the constituents responsible for callus induction, and 1 per cent sucrose is optimum. Addition of 2,4-D (2,4dichlorophenoxyacetic acid) at 10-7 and 10-6 M to MS medium significantly prepones callus initiation and stimulates callus growth. Kinetin (6-furfurylaminopurine) at lower concentrations (10- 6 and 10-5 M) does not affect callus induction ;;,nd growth, but at 10-4 M it is inhibitory to callus induction. At the levels tried, 2,4-D does not overcome the inhibitory effect of 10-4 M kinetin. At 10-5 M kinetin in combination with 2,4-D (10- 6 M) is optimum for callus induction and growth. Key words: Moss, Hyophila involuta, sucrose, 2,4-D, kinetin, callus induction.

Introduction Studies on callus induction and its controlled differentiation have been helpful in understanding the mechanism underlying morphogenesis. Much work has been done in this respect on higher plants and pteridophytes, but there are only a few reports of induction of callus in bryophytes. Callus has been induced from the spores of three liverworts: Cephaloziella (MEYER, 1953), Fossombronia pusilla and Reboulia hemisphaerica (ALLSOPP, 1957) and of a moss, Funaria hygrometrica (WETTSTEIN, 1953). In Polytrichum commune and Atrichum undulatum callus has been induced from protonemata (WARD, 1960) on Knudson's basal medium supplemented with micro nutrients (NITSCH, 1951) and 0.25 per cent sucrose. Callus induction has also been possible from the thalli of some liverworts: Marchantia nepalensis (KAUL et al., 1962), Riccardia (ALLSOPP and ILLAHI, 1969), Asterella angusta (RASHID, 1971), Riccia crystallina (CHOPRA and SOOD, 1973), Athalamia pusilla (MEHRA and PENTAL, 1976) and Fossombronia himalayensis (MEHRA and PAHWA, 1976). Z. Pflanzenphysiol. Bd. 99. S. 199-205. 1980.

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LAL (1961) observed that mature gametophytes of Physcomitrium coorgense produced callus masses from the stem and venter wall of archegonia on medium containing Knop's mineral salts C/2 of the original concentration), White's minor salts (lec/l) and 2 per cent sucrose. When leaves and apical regions of aberrant plants of Polytrichum commune were cultured on medium containing casamino acids and nutrients, they exhibited the development of undifferentiated growth (WARD and FREDRICK, 1967). MENON (1974) observed callus formation from the gametophytes as well as from the secondary protonema in Physcomitrium pyriforme with 2 per cent sucrose. ONO (1973) reported callus formation from the gemmae of the female gametophytes of Marchantia polymorpha on a medium containing half strength Knop's mineral salt solution, IAA (4 ppm), GA3 (6 ppm) and 2 per cent sucrose. BAUER (1957,1961) observed callus formation from the sporogonial tips of Physcomitrium pyriforme. RASHID and CHOPRA (1969) reported callus formation in Funaria hygrometrica from the young, apogamously produced sporophytes on basal medium with increased carbohydrate levels. We have made an attempt to induce callus in Hyophila involuta and the preliminary observations are being recorded below. Material and Methods Hyophila involuta (HOOK.) JAEG. is a dioecious moss of the family Pottiaceae. It was collected from Dalhousie (N-W. Himalayas) during 1977, and aseptic cultures were raised from spores on basal medium comprising Knop's major and Nitsch's minor salt solutions, 10 ppm ferric citrate and 1 per cent sucrose (NBM). The medium employed for spore germination was solidified with 0.8 per cent agar. The culture tubes with the medium were autoclaved at 15 Ibs/square inch for 15 minutes. The cultures were maintained at 25 ± 2°C in 3,500-4,000 lux light obtained from a combination of incandescent bulbs and fluorescent tubes. Twenty-day-old proton em a growing on NBM was transferred to MURASHIGE and SKOOG'S medium (MS) containing mineral salts (i/5th of original concentration), chelated iron 15th of original concentration) and vitamins. Protonema was also transferred to NBM containing the following supplements of MS medium, alone and in various combinations: NH4 NO a (3'30 mg/I), chelated iron (i/5th of original concentration) and vitamins. Cultures were also raised on MS medium supplemented with various concentrations of 2,4-D (10-7 , 10-6 and 10-5 M), kinetin (10-6 , 10-5 and 10-4 M) and sucrose (0.5, 1, 2 and 4010).

e

Results

The protonema grew normally on NBM. On MS medium, good protonemal growth was followed by callusing. During the course of callus induction from protonema, the elongated chloronemal cells started dividing into smaller cells. These cells eventually became rounded and the chloronemal filaments gave a beaded appearance (Fig. 1). Some of the cells in this chain later got separated (Fig. 2). Further divisions in such units resulted in the formation of fragile callus masses, which comprised un-

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differentiated, parenchymatous cells (Fig. 3). After 9 weeks of growth, 80 per cent cultures revealed brownish, rounded, friable callus masses (Fig. 4). In order to find out which particular component of MS medium is effective in callus induction, cultures were raised on MS medium devoid of its various components (Tabe 1). From the data it is evident that complete MS medium is essential for callus induction in Hyophila involuta. In another experiment, various components of MS medium ware individually added to NBM, but no callusing took place .

•

3

•

4

Figs. 1-4: Development of cdlus from chloronemal filam ents in Hyophila involuta (HOOK.) on MS med ium. - Fig. 1: chloronemal filaments with rounded cells, 6-week-old culture. X 260. - Fig. 2: Isola ted cells from the same, 9-week-old. X 260. - Fig. 3: Individual cclls dividing to form a n unorganized cell-mass, 9-week-old. X 260. - Fig. 4: Callus in culture, 10-week-old. 2.3.

JAEG.

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Table 1: Effect of various components of MURASHIGE and SKOOG'S medium on callus induction in Hyophila involuta. Treatments

Growth Response

Cultures showing call us response

(010) Nitsch's Basal Medium (NBM) MURASHIGE and SKOOG'S Medium (MS) MS (Major + Minor salts only) MS (Major + Minor salts + vitamins only) MS (Major + Minor salts +chelated iron only) MS (Major + Minor salts + FeS04 only) NBM + 330 mg/l NH4 NO a NBM + (Vitamins + Chelated iron of MS) NBM + NH4 NO a + (Vitamins + chelated iron of MS medium)

Normal protonema Callusing Chlorosis of protonema Chlorosis of protonema Normal protonema Chlorosis of protonema Normal protonema Normal protonema Callusing

0 80 0

0 0 0 0 0 60

The chief difference between MS and NBM is in the nitrogen source. However, when ammonium nitrate (330 mg/l) of MS was added to NBM, no callusing was observed, but when ammonium nitrate was added together with chelated iron and vitamins of MS medium, 60 per cent cultures showed callusing after 9 weeks. All further experiments were therefore carried out on MS medium. Effect of sucrose, 2,4-D and kinetin: In order to study the effect of sucrose concentration on callus induction, cultures were supplemented with 0.5, 1, 2 and 4 per cent sucrose. At 1 per cent sucrose, callus induction was maximum in terms of percentage of cultures showing response, as well as the amount of callus induced. After 9 weeks, 80 per cent cultures revealed callus masses, whereas no callusing was observed in the control cultures (MS medium without sucrose). 2,4-D played an important role in callus induction. At lower levels of 2,4-D (10-1 and 10-6 M), 75 per cent cultures showed callus induction within 6 weeks of growth (Table 2). Kinetin at 10-6 and 10- 5 M did not have any appreciable effect on callus induction. At 10-4 M, callus induction was inhibited (Table 2). 2,4-D and kinetin interaction: Since 2,4-D and kinetin elicited different responses, their interaction was studied. Lower concentrations of kinetin, in combination with 2,4-D did not increase the percentage response of cultures (Table 2). Callus growth, however, increased significantly when 2,4-D (10- 7 and 10- 6 M) and kinetin (10- 6 and 10- 5 M) were combined. 2,4-D at the concentrations tried was not able to overcome the inhibitory effect of 10-4 M kinetin. In the interaction of 10-6 M 2,4-D with 10-5 M kinetin 83 per cent cultures showed maximum amount of callusing. Therefore, this combination is optimal for callus induction and growth in this moss. When callus was transferred to fresh medium of same composition, initial protonemal growth was followed by renewed callus formation after 2 weeks. Z. Pjlanzenphysiol. Bd. 99. S. 199-205. 1980.

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Table 2: Effect of 2,4-D, kinetin and their interaction on callus induction and growth in

Hyophila involuta on MS Medium. Treatments

Control (0 kinetin)

10-6 M kinetin

1O-5 M kinetin

10-4 M kinetin

Control (02,4-D)

12"')

25

25

0

(+)

(+)

(+)

10-7 M 2,4-D 10- 6 M 2,4-D 10-5

M 2,4-D

75

75

75

8

(++)

(+++)

(++++)

(+)

75

75

83

8

(++)

(+++)

(++++)

(+)

16

33

75

0

(+)

(++)

(++)

".) The figure denotes percentage of cultures showing response. + Indicates the relative amount of callus growth.

Discussion The present investigation has revealed that in Hyophila involuta callus could be induced on MS medium, or on NBM supplemented with ammonium nitrate (330 mg/ 1), chelated iron and vitamins of MS medium. No callus induction is observed on NBM supplemented with chelated iron and vitamins of MS medium in the absence of ammonium nitrate. Therefore, ammonium nitrate appears to play an important role in callus induction. MEDORA et al. (1979) have also reported that ammonium nitrogen is essential for papaya callus growth. However, in the present work, when chela ted iron and vitamins were deleted from the MS medium, no callus could be induced. It is clear from these observations that the combination of ammonium nitrate, chelated iron, vitamins and sucrose is important for callus induction. According to BUNNING (1952), polarity is necessary for differentiation. Suppression of polarity, either by the direct effect of various treatments on the protoplasm or by neutralization of internal gradients of nutrients, growth substances etc., prevents normal differentiation, but does not necessarily result in the cessation of cell division. In some hepatics and mosses enhancement in sugar concentration alone is sufficient to induce callus (ALLSOPP, 1957; ALLSOPP and ILLAHI, 1969; ILLAHI and ALLSOPP, 1970; CHOPRA and SOOD, 1973; WARD, 1960; LAL, 1961; MENON, 1974). RASHID (1971) reported that addition of mannitol results in the formation of stable callus tissue, indicating that callusing has been brought about by an increase in osmotic potential. He observed that cell elongation is a prerequisite for differentiation, but mannitol is known to prevent cell elongation and consequently inhibits differentiation (CLELAND, 1967). It is probable that sucrose might also be influencing callus induction by bringing about changes in osmotic potential, besides its metabolic effects.

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In the present system sucrose alone is ineffective in bringing about callus induction. It needs to be supplemented with ammonium nitrate, chelated iron and vitamins. In some higher plants callus formation requires the presence of fairly high concentration of hormones including auxins and cytokinins. OSWALD et al. (1977) reported callus induction in Glycine max and Trifolium repens with 50 : 1 auxin to cytokinin (w/w) ratio. The auxins added were 2,4-D and 2,4-5-T, and the cytokinin was kinetin. MALMBERG (1979) induced callusing in Pisum sativum on MS medium supplemented with NAA and benzyladenine. MEDORA et al. (1979) reported that in papaya callus, of the growth regulators tried, only 2,4-D promoted growth whereas benzylaminopurine and gibberellic acid were inhibitory. In Hyophila 2,4-D plays a significant role in callus induction. Lower concentrations (10-7 and 10- 6 M) of 2,4-D greatly enhance the percentage response of cultures. Kinetin is not effective at lower concentrations (10- 6 and 10- 5 M), and at 10-4 M it is inhibitory for callus induction. In this moss optimal response is had with a combination of 10-6 M 2,4-D and 10- 5 M kinetin. MENON and LAL (1974) reported that in Physcomitrium kinetin is ineffective in causing cell differentiation, and its role is to bring about cell multiplication. In Hyophila involuta also kinetin appears to enhance cell multiplication, and 2,4-D diverts the morphogenic path of protonemal growth towards undifferentiated cell masses. Acknowledgements

The authors are thankful to the University Grants Commission, New Delhi, for the financial assistance.

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ALLSOPP, A. and r. lLLAHI: Studies in the Metzgeriales (Hepaticae). I. The effect of certain sugars on the growth and morphology of some thalloid species. Phytomorphology 19, 242-253 (1969).

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ILLAHI, r. and A. ALLSOPP: Studies in the Metzgeriales (Hepaticae). VI. Regeneration and callus formation in some thalloid species. Phytomorphology 20, 126-136 (1970). KAUL, K. N., G. C. MITRA, and B. K. TRIPATHI: Responses of Marchantia in aseptic cultures to well known auxins and anti-auxins. Ann. Bot. 26, 447-466 (1962). Z. Pflanzenphysiol. Bd. 99. S. 199-205. 1980.

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LAL, M.: In vitro production of apogamous sporogonia in Physcomitrium coorgense BROTH. Phytomorphology 11,263-269 (1961). MALMBERG, R. L.: Regeneration of whole plants from callus cultures of diverse genetic lines of Pisum sativum L. Planta 146, 243-244 (1979). MEDORA, R. S., D. E. BILDERBACK, and G. P. MELL.: Effect of media on growth of papaya callus cultures. Z. Pflanzenphysiol. 91, 79-82 (1979). MEHRA, P. N. and M. S. PAHWA.: Phenotypic variations in Fossombronia himalayensis KASH. in vitro-Effect of sugars, auxins, growth substances and growth inhibitors. J. Hattori bot. Lab. No. 40, 371-395 (1976). MEHRA, P. N. and D. PENTAL: Induction of apospory, callus, and correlated morphogenetic studies in Athalamia pusilla KASH. J. Hattori bot. Lab. No. 40,151-183 (1976). MENON, M. K. c.: Morphogenetic studies on apogamy in the moss Physcomitrium. Ph. D. Thesis, Univ. Delhi. India, 1974. MENON, M. K. C. and M. LAL: Morphogenetic role of kinetin and abscisic acid in the moss Physcomitrium. Planta 115, 319-328 (1974). MEYER, D. E.: Tumorartige Zellwucherungen bei einem Lebermoos. Naturwissenschaften 40, 297-298 (1953). NITSCH, J. P.: Growth and development in vitro of excised ovaries. Am. J. Bot. 38, 566576 (1951). ONO, K.: Callus formation in liverwort, Marchantia polymorpha. Jap. Jour. Genet. 48, 69-70. (1973). OSWALD, T. H., A. E. SMITH, and D. Y. PHILLIPS: Callus and plantlet regeneration from cultures of Ladino clover and soyabean. PI. Physiol. 39, 129-134 (1977). RASHID, A.: Callusing and regeneration potential of cell-aggregates and free-cells of liverwort: Asterella angusta AUST. Experientia 27,1948-1949 (1971). RASHID, A. and R. N. CHOPRA: The apogamous sporophytes of Funaria hygrometrica and their cultural behaviour. Phytomorphology 19,170-178 (1969). WARD, M.: Callus tissues from the moss Polytrichum and Atrichum. Science 132, 14011402 (1960). WARD, M. and S. E. FREDRICK: Propagation of aberrant gametophytes from aggregates and from single cells of moss callus. Phytomorphology 17, 371-374 (1967). WETTSTEIN, D. VON.: Beeinflussung der Polaritiit und undifferenzierte Gewebebildung aus Moossporen. Z. Bot. 41, 199-226 (1953).

KAVITA RAHBAR and R. N. CHOPRA, Department of Botany, University of Delhi, Delhi 110007, India.

Z. Pf/anzenphysiol. Bd. 99. S. 199-205. 1980.