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Morphogenic Investigations on Parasitic Angiosperms
Flora, Abt. A, Bd. 160, S. 448-456 (1969) From the Department of Botany, University of Delhi, India
Morphogenic Investigations on Parasitic Angiosperms II. Striga angustifolia (DON) Saldhana (Scrophulariaceae)l) By N. S. RANGASWAMY and T. S. RANGAN With 2 figures (Received April 21, 1969)
I. Introduction The parasitic habit of Striga angustifolia (DON) Saldhana [= S. euphrasioides (VAHL) BENTH.] is debated. In his flora DUTHIE (1911) listed this species as a parasite but SRINIVASAN (1946) failed to discover any haustorial connection between it and any autotrophic plant growing in its vicinity and, therefore, declared the species an autotroph. According to WILLIAMS (1958) S. angustifolia is one of the "unlikely exceptions to the group of obligate parasitic species". This paper describes our studies on seed germination and embryo morphogenesis of Striga ang~tstifolia and compares the data with those on species of Stl'1:ga that art' unequivocally recognized as root parasites. II. Material and Methods Striga angustifolia is a common weed in sugarcane and sorghum fields. It produces oblong eapsl1.les which contain tiny seeds. The seed shows scanty endosperm and a dicotyledonous embryo. Capsules were collected from crop fields in and around Bangalore (Sol1.th India), surfacesterilized in chlorine water and placed on dry heat-sterilized filter paper in a petridish. In 4-6 days the capsules shed the seeds. To lessen the chances of microbial infection the seeds were surface-sterilized following RANGASWAMY'S (1963) technique. The sterilized seeds were sown on nutrient agar media, either untreated, or after soaking in aqueous solutions of gibberellic acid (5 and 10 p.p.m.) or kinetin (5 and 10 p.p.m.) for 24 or 48 hr, or after subjecting to dripping tap water for 48 hr. Each culture received 30-40 seeds. A modified White's medium (RmGASWAMY 1961), WB for short, served as the basal medium. In various experiments WB was supplemented with gibberellic acid, kinetin, 2,4-dichlorophenoxyacetic acid (2,4-D), adenine sulfate, casein hydrolysate, yeast extract, coconut milk and juice of watermelon (01:trullus vulgaris SeRRAD.), either individually or in different combinations. In one experiment Tepfer's HI medium rl'EPFER et al., 1963) was also used. All supplement were added while preparing the final medium which was invariably gelled with 0.8 % Difco Bactoagar, adjusted to pH 5.8, and autoclaved at ca. 1.06 kg/sq. cm for 15 min. 1) Part of a thesis by TSR entitled "Morphogenetic Investigations on the Parasitic Angiosperms - Cassytha, Oistanche and Striga".
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Each experiment was done 3 times, and each time 24 cultures were raised. All cultures were 2 DC and 50 -60 % relative humidity. grown in diffuse day light (400 -600 Luc) at 25 C Samples of the cultured material were periodically fixed in formalin-acetic-alcohol for preparing microtome sections. Following the customary methods of paraffin embedding, sections were cut 10-15 microns thick, stained with safranin and fast green, and mounted in Canada balsam. In addition squash preparations of growing tissues, and whole mounts of germinated seeds, s.tained with 1 0 0 acetocarmine, dehydrated through glacial acetic acid - n-butyl alkohol series and mounted in Canada balsam were studied.
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III. Results Untreated seeds sown on WB did not germinate although the cultures were maintained for 8 weeks. Likewiese, seeds treated with gibberellic acid or kinetin, and then sown on WB also failed to germinate. Neither did the addition of these substances individually to WB induce germination of untreated seeds. Because moisture treatment of seed induced seed germination of the scrophulariaceous root parasites Striga lutea (BROWN and EDWARDS 1946) and Alectra vogelii (BOTRA 1950 a, b), in our studies also we subjected the seeds to dripping water for 48 hr and then sowed them on nutrient media. On WB, like untreated seeds, the water-dripped seeds also did not germinate. Addition of gibberellic acid (5 p.p.m.) or kinetin (5 p.p.m.) to WB did not induce seed germination, whereas gibberellic acid (10 p.p.m.) or casein hydrolysate (400 p.p.m.) + coconut milk (15% v/v) induced only 6% germination in 3 weeks. At 10 p.p.m., kinetin had a dramatic effect; 10 days after culture als high as 69 % seeds germinated by releasing the embryos. The released embryos enlarged considerably (Fig. 1 A, B). In 18-20 days after culture the cotyledons and the hypocotyl both produces a callus; eventually the embryo lost its identity and became a mass of unorganized tissue. When the cultures were continually maintained on WB + kinetin (10 p.p.m.), the callused embryo did not shwo capacity for continuous growth, and turned brown 20-30 days after culture. To establish continuous tissue cultures and to induce morphogenesis in them the callused embryos were transferred to WB supplemented individually or in combinations, with 2,4-D yeast extract, cassein hydrolysate, and coconut milk. Upon transfer to WB + 2,4-D (2 p.p.m.) or WB + yeast extract (500 p.p.m.) the callus failed to renew growth. A concert of 3 substances (2,4-D 2 p.p.m. + yeast extract 0.25% + kinetin 5 p.p.m.) also did not promote the growth of embryo callus. If the transfers were made to WB + casein hydrolysate (400 p. p.m.) + coconut milk (15 %vjv) the growth of embryo callus was stimulated, and the whole culture presented a group of several callus masses of varied sizes derived from individual embryos which were no longer identifiable (Fig. 1 C). The newly formed tissue was friable; acetocarmine squash preparations showed both free cells and cell aggregates (Fig. 1 D). The tissue could be readily grown into 4-6 subcultures of nearly 400 mg each. About
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Fig. 1 A-D. Morphogenesis in seed cultures of Striga angustifolia - A. Embryo collected from 3-week-old seed culture on WB + kinetin (10 p.p.m.) showing overall enlargement. 90 x. B. Wholemount of embryo as in A. Observe further enlargement of radicle, and cotyledons, and formation of root hairs. Provascular strands are distinct in the hypocotyl. 47 x. C. Four-weekold transfer of embryo calluses on WB + casein hydrolysata (400 p.p.m.) + coconut milk (15 % vjv). 1.5 x. D. Acetocarmine squash preparation of embryo callus grown on WB + cacein hydrolysate (400 p.p.m.) + coconut milk (15 % vjv) showing a few free cells and cell aggregates. 108x.
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Fig.2A-E. Morphogenesis in seed cultures of Striga angustifolia - A. Histology of 4-week-old embryo callus grown on WB + casein hydrolysate (400 p.p.m.) + coconut milk (Hi % vjv) showing several "endogenous nests". 76 x. B. Transection througk an "endogenous nest" 118 X. C. Ten-week-old embryo callus showing differentiation of vessel members. 173 x. D.Eight-weekold embryo callus grown on WB + casein hydrolysate (400 p.p.m.) + coconut milk (15 % vjv) showing root formation. 3 x. E. L.s. 10-week-old callus showing origin of root. 7 x.
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2 weeks after subculture fresh mounds of tissue appeared and the subcultures showed ability for continued growth. As the subculture continued to grow, 2 regions were distinguishable in the mound: a core of compact cells and a peripheral region of loosely arranged vacuolated cells (Fig. 2A, B). Thus, the callus showd "endogenous nests" of cells rich in cytoplasm (Fig. 2 B). As growth progressed, cell layers contiguous between the mounds necrosed leading to separation of mounds from one another, and only vessel members but no organs differentiated in the callus (Fig. 2 C). Further investigations were, therefore, directed to a morphogenic study of the embryo callus. Seeds were as usual germinated on WB + kinetin (10 p.p.m.), and from the germinated seeds embryo callus was raised on WB + casein hydrolysate (400 p.p.m.) + coconut milk (15% v/v). The callus was grown into subcultures on WB + casein hydrolysate + coconut milk as well as on WB + adenine sulphate (40 p.p.m.), WB + watermelon juice (20% v/v), and Tepfer's HI media whIch were reported to induce shoot bud formation in embryo callus of certain other parasitic angiosperms (see RANGASWAMY 1963, RANGAN and RANGASWAMY 1968). Let alone inducing shoot bud formation, WB fortified with watermelon juice, and Tepfer's HI medium, both inhibited the growth of the subcultured embryo callus. On WB + casein hydrolysate + coconut milk, 40 % of the isolates showed root formation in 5 weeks after subculture (Figs. 2D, E). On WB + adenine sulphate the growth of the callus was slight, but 55 % of the isolates showed rooting. Usually the roots differentiated on the callus surface away from the culture medium. Instances of root development on tissue surface in contact with the culture medium were not wanting, but rare. Histologic preparations showed that the roots originated from "endogent)Us nests". The root comprised an epidermis. a cortex of 6-8 layers of parenchymatous cells, and a core of 3 or a few more xylem elements; endodermis and pericycle were not discernible. Those roots which differentiated on the surface of the embryo callus in contact with the culture medium continued to grow 1-2 cm long into the medium. The roots that originated on the free surface of the embryo callus proliferated as a callus in turn and lost their identity. Anatomical preparations showed that the callus originated from the cortical cells of the parent root. Eventually, however, this root callus also produced new roots which grew into the agar medium. IV. Discussion It is generally believed that for successful seed germination obligate root para-
sites, such as Cistanche and Striga, require a stimulus from the root of host plants. However, recent experiments have demonstrated that the host stimulus can be effectively replaced by kinetin (WORSHAM et al. 1959, WILLIAMS 1961), gibberellic acid (WILLIAMS 1961), and some coumarin derivatives (WORSHAM et al. 1962) for
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Striga asiatica; by casein hydrolysate or coconut milk or by both for Orobanche aegyptiaca (RANGASWAMY 1963, USHA 1968), Cistanche tubulosa (RANGAN 1965, RANGAN and RANGASWAMY 1968), Santalum album RANGASWAMY and RAO 1963), and Exocarpus cupressiformis (JOHRI and BHOJWANI 1965). In Btriga angustifolia kinetin (10 p.p.m.) induced seed germination, whereas gibberelic acid (5 p.p.m.) did not. Our observations on kinetin-induction of seed germination confirms those of WORSHAM et al. (1959) and of WILLIAMS (1961) on
Striga asiatica. That a variety of substances (cytokinins, gibberellic acid, coumarin, casein hydrolysate, and coconut milk) can induce seed germination in the root parasites, Cistanche, Orobanche, Btriga, Santalum and Exocarpus, suggestes a diverse chemical nature of the host stimulant. Although highly purified host-root extracts have been analyzed seed germination-stimulants have not been isolated and identified. Chemical analyses of the root secretion of Linum usitatissimum (host for Orobanche) indicated the presence of a lactone fraction in it (BROWN et al. 1951 a, b), and of the germination stimulant formed in the roots of Zea mays seedlings indicated it to be a coumarin (WORSHAM et al. 1964). Of the physical factors known to affect seed germination, BROWN and EDWARDS (1946) found that for Striga lutea a moisture treatment of seeds (by incubating the seeds in dark on moist filter paper) for 3 weeks considerably increased the germination percentage (82 % as against 30 %in control). VALLALCE (1950) also demonstrated a similar response for S. hermonthica seeds, and observed that seeds stored for 6 months required a longer period (15 days) of moisture treatment than seeds stored for 16 months which required only 4-day-treatment. Working on another scrophulariaceous parasite, Alectra vogelii, BOTHA (1950a, b) showed that by subjecting the seeds to dripping water their germination sensitivity to host stimulant was enhanced. In our present investigation on Striga angustifolia we found that seeds not dripped in water lay ungerminated, whereas the seeds washed in trickling water for 48 hr promptly germinated. According to KUMAR (1940) Striga angustifolia showed a high percentage of seed germination in light. OKONKWO'S (1964) work on S. senegalensis (= S. hermonthica) implies that seed germination occurred irrespective of light or dark treatments; this is true for S. angustifolia as well (present work). In root parasites, like seed germination, seedling morphogenesis also seems to be governed by factor resident in the host plant. For example, in Striga lutea upon germination the radicle grown 2-4 mm long, but the plumule does not emerge until a contact is made with a host plant (KUMAR et al. 1942, UTTAMAN 1950). A similar phenomenon was observed also in S. densiflora and S. euphrasioides (KUMAR 1940). OKONKWO implied that in S. senegalensis also the shoot originates only after the host-parasite connection is established through the primary haustorium. In Oro-
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banche hederae also a contact with the root of its host Hedera helix was indispensable for the initiation of shoots in culture (PRIVAT 1960). S. angustifolia (present work) does not seem to be an exception either; although roots were formed in vitro, shoot buds did not differentiate. In our present experiments on Striga angustifolia germination occurred only of the moistened, kinetin-treated seeds, and seedlings failed to develop. Thus a considerable morphogenic similarity exists between S. angustifolia and the 2 parasitic species S. asiatica (WORSHAM et al. 1959, 1962; WILLIAMS 1961) and S. senegalensl:s OKONKWO 1966). Such a morphogenic resemblance of S. angustifolia to 2 parasitic species of the same genus provides evidence to WILLIAMS' (1958) belief that S. angustifolia is one of the "unlikely exceptions to the group of obligate parasitic species". Like S. senegalensis (OKONKWO 1966), S. angustifolia also is probably a facultative parasite, and it may establish haustorial connections only during the subterranean (total parasitic) growth of its seedling. The lack of haustorial connections between S. angustifolia and any autotroph (SRINIVASAN 1946) is therefore not surprising, because only the flowering soecimens of S. angu,stifolia (i. e. plants that were almost in the final phase of their life cycle) were examined. Thus, parasitism in S. angustifolia is confined to early life of its seedling. Summary 1. Striga angustifolia (DoN) Saldhana is a herbaceous weed in sugarcane and sorghum fields. Some taxanomists consider this species a true parasite, others a pseudo-non-parasite, and still others an autotroph. 2. Morphogenesis of its seed germination was studies to seek a clue about the habit of this species. 3. Untreated seeds, water-dripped seeds, and seeds soaked in solutions of gibberellic acid and kinetin were cultured. 4. Untreated seeds as well as seeds soaked in solutions of gibberellic acid or kinetin (5 p.p.m.) failed to germinate. 5. Only the seeds subjected to dripping water for 48 hr followed by kinetin (10 p.p.m.) treatment germinated. Upon germination the embryos formed a callus tissue. 6. Subcultures of this embryo callus were raised to study its morphogenesis. In subcultures on White's medium + casein hydrolysate (400 p.p.m.) +coconut milk (15 o~ vjv) the callus differentiated roots. Shoot buds did not develop in any of the treatments. 7. Similarities in seed germination and in embryo morphogenesis between S. angusiifolia and some well-proven parasitic species of Striga are discussed. 8. From morphogenic viewpoint it is concluded that S. angustitolia may be a facultative root parasite and its parasitism is confined to the early subterranean life of its seedling.
Acknowledgements Our sincere thanks are to Professor B. M. JOHRI for his interest in this work. We are obliged to the authorities of the United States Department of Agriculture for granting one of us (TSR)
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a research fellowship in the scheme entitled "Physiology of Reproductive Organs of Seed Plants" under the PL 480 program, during- which tenure this research was conducted.
Literature BOTHI, P. J., 1950a. The germination of the seeds of angiospennous root parasites. 1. The natire of the changes occurring during pre-exposuxe of the seed of Aleclra vogelii BENTH. J. S. Afr. Bot. 16, 23-28. 1950b. The germination of the seeds of angiospennous root parasites. 11. The effect of time of pre-exposure, temperature of pre-exposure and concentration of host factor on the germination of the seed of Alectra vogelii BENTH. J. S. Afr. Bot. 16, 29-38. BROWN, R., and EDWARDS, M., 1946. The germination of the seeds of Siriga lutea. II. The effect of time of treatment and of concentration of the host stimulat. Ann. Bot. 10, 133-142. GREENWOOD, A. D., JOHNSON, A. W., and LONG, A. G., 1951a. The stimulant involved in the germination of Orobnnche minor SM. 1. Assay technique and bulk preparation of the stimulant. Biochem. J. 48, 559-564. - - - and TYLER, O. J., 1951 b. The stimulant involved in the germination of Orobnnche minor SM. 2. Chromatographic purification of crude concentrates. Biochem. J 48, 564-568. DrTITIE, J. F., 1911. Flora of the Upper Gangetic Plain, and of the Adjacent Siwalik and SubHimalayan Tracts. Calcutta. Vol. II, page 157. JOHRI, B. M., and BHOJWANI, S. S., 1965. Growth responses of nature endosperm in cultures. ;Xuture (London) 208, 1345-1347. KU~IAR, L. S. S., 1940. Flowering plants which attack economic crops. 1. SIr·iga. Indian Fmg. 1, 593-595. - ABR,IHAM, A., and SOLmIAN, L., 1942. A technique for the anatomical study of root parasitism. Ann. Bot. 6, 177 -182. OKONKWO, S. N. C., 1964. In vitro culture of seedlings of Striga senegnlensis BENTH. Nature (London) 204, 1108-1109. - 1966. Studies on Strign senegnlensis BENTH. I. Mode of host-parasite union and haustorial structure. Phytomorphology 16, 453-463. PRIVAT, G., 1960. Recherches sur les phanetogame parasites (Etude d'Orobal/che hederar DFBY). AnnIs. Sci. Nat. Bot. Soc. Ser. 12, 721-821. RANGAN, T. S., 1966. Morphogenesis of the embryo of Cistnnche tubulosa W1GRT in vitro. Phytomorphology 10,180-182. - and RANGASWA~IY, N. S., 19G8. Morphogenic investigations on parasitic angiosperms. 1. Oistnnche tubulosn (Orobanchaceae). Canad. J. Bot. 46, 263-26G. R.~NGASWAMY, N. S., 1961. Experimental studies on female reproductive structures of Oitrus microcnrpn BUNGE. Phytomorphology 11,109-127. 1963. Stdies on culturing seeds of Orobnnche negyptincn PERS. In Plant Tissue and Organ Culture - A Symposium, Eds P. Maheshwari and N. N. Rangaswamy, Int.. Soc. PI. Morphologists, Delhi. and RAO, P. S., 1963. Experimental studies on Snntnlum nlbum L. - Establishment of tissue culture of endosperm. Phytomorphology 13, 450-454. SRINIVASAN, A. R., 1946. Morphological and cytological st.udies in Serophulariaceae V. Striga euphmsioides BENTH. Proc. Indian Acad. Sci., Sect. B 24, 21-33. TEPFER, S. S., GREYSON, R. I., CRAIG, W. R., and HINDMAN, J. L., 1963. In vitro culture of floral buds of AquUegin. Amer. J. Bot. 00, 1035 -1045.
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USHA, S. V., 1968. Morphogenesis in aseptic seed cultures of the holoparasite - Orobanche aegyptiaca PERS. Ph. D. thesis, Univ. Delhi. UTTAMAN, P., 1950. A study on the germination of Striga seed and on the mechanism and nature of parasitism of Striga lutea (LOUR.) on rice. Proc. Indian Acad. Sci, Sect. B 32, 133-142. VALLANCE, K. B., 1950. Studies on the germination of the seeds of Striga hermonthica I. The influence of moisture-treatment, stimulant dilution and after ripening on germination. Ann. Bot. 14, 347-363. WILLIAMS, C. N., 1958. The parasitism of witchweed - A review. W. Afr. J. BioI. Chern. 2, 57 -73. - 1961. Growth and morphogenesis of Striga seedlings. Natu.re (London) 189,378-381. WORSHAM, A. D., KLINGMAN, G. C., and MORELAND, D. E., 1962. Promotion of germination of Striga asiatica seed by coumarin derivaties and effects on seedling development. Nature (London) 190, 199-201. MORELAND, D. E., and KLINGMAN, G. C., 1959. Stimulation of Striga asiatica (witchweed) seed germination by 6-substituted pu.rines. Science 130, 1654-1656. - 1964. Characterization of the Striga asiatica (witchweed) germination stimulant from Zea mays L. J. expo Bot. 10, 556-567. Authors' addresses: Dr. N. S. RANGASWAMY, Department of Botany, University of Delhi, Delhi 7, India, and Dr. T. S. RANGAN, Department of Horticultural Science, University of California, Riverside, California 92502, U.S.A.
Morphogenic Investigations on Parasitic Angiosperms II. Striga angustifolia (DON) Saldhana (Scrophulariaceae)l) By N. S. RANGASWAMY and T. S. RANGAN With 2 figures (Received April 21, 1969)
I. Introduction The parasitic habit of Striga angustifolia (DON) Saldhana [= S. euphrasioides (VAHL) BENTH.] is debated. In his flora DUTHIE (1911) listed this species as a parasite but SRINIVASAN (1946) failed to discover any haustorial connection between it and any autotrophic plant growing in its vicinity and, therefore, declared the species an autotroph. According to WILLIAMS (1958) S. angustifolia is one of the "unlikely exceptions to the group of obligate parasitic species". This paper describes our studies on seed germination and embryo morphogenesis of Striga ang~tstifolia and compares the data with those on species of Stl'1:ga that art' unequivocally recognized as root parasites. II. Material and Methods Striga angustifolia is a common weed in sugarcane and sorghum fields. It produces oblong eapsl1.les which contain tiny seeds. The seed shows scanty endosperm and a dicotyledonous embryo. Capsules were collected from crop fields in and around Bangalore (Sol1.th India), surfacesterilized in chlorine water and placed on dry heat-sterilized filter paper in a petridish. In 4-6 days the capsules shed the seeds. To lessen the chances of microbial infection the seeds were surface-sterilized following RANGASWAMY'S (1963) technique. The sterilized seeds were sown on nutrient agar media, either untreated, or after soaking in aqueous solutions of gibberellic acid (5 and 10 p.p.m.) or kinetin (5 and 10 p.p.m.) for 24 or 48 hr, or after subjecting to dripping tap water for 48 hr. Each culture received 30-40 seeds. A modified White's medium (RmGASWAMY 1961), WB for short, served as the basal medium. In various experiments WB was supplemented with gibberellic acid, kinetin, 2,4-dichlorophenoxyacetic acid (2,4-D), adenine sulfate, casein hydrolysate, yeast extract, coconut milk and juice of watermelon (01:trullus vulgaris SeRRAD.), either individually or in different combinations. In one experiment Tepfer's HI medium rl'EPFER et al., 1963) was also used. All supplement were added while preparing the final medium which was invariably gelled with 0.8 % Difco Bactoagar, adjusted to pH 5.8, and autoclaved at ca. 1.06 kg/sq. cm for 15 min. 1) Part of a thesis by TSR entitled "Morphogenetic Investigations on the Parasitic Angiosperms - Cassytha, Oistanche and Striga".
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Each experiment was done 3 times, and each time 24 cultures were raised. All cultures were 2 DC and 50 -60 % relative humidity. grown in diffuse day light (400 -600 Luc) at 25 C Samples of the cultured material were periodically fixed in formalin-acetic-alcohol for preparing microtome sections. Following the customary methods of paraffin embedding, sections were cut 10-15 microns thick, stained with safranin and fast green, and mounted in Canada balsam. In addition squash preparations of growing tissues, and whole mounts of germinated seeds, s.tained with 1 0 0 acetocarmine, dehydrated through glacial acetic acid - n-butyl alkohol series and mounted in Canada balsam were studied.
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III. Results Untreated seeds sown on WB did not germinate although the cultures were maintained for 8 weeks. Likewiese, seeds treated with gibberellic acid or kinetin, and then sown on WB also failed to germinate. Neither did the addition of these substances individually to WB induce germination of untreated seeds. Because moisture treatment of seed induced seed germination of the scrophulariaceous root parasites Striga lutea (BROWN and EDWARDS 1946) and Alectra vogelii (BOTRA 1950 a, b), in our studies also we subjected the seeds to dripping water for 48 hr and then sowed them on nutrient media. On WB, like untreated seeds, the water-dripped seeds also did not germinate. Addition of gibberellic acid (5 p.p.m.) or kinetin (5 p.p.m.) to WB did not induce seed germination, whereas gibberellic acid (10 p.p.m.) or casein hydrolysate (400 p.p.m.) + coconut milk (15% v/v) induced only 6% germination in 3 weeks. At 10 p.p.m., kinetin had a dramatic effect; 10 days after culture als high as 69 % seeds germinated by releasing the embryos. The released embryos enlarged considerably (Fig. 1 A, B). In 18-20 days after culture the cotyledons and the hypocotyl both produces a callus; eventually the embryo lost its identity and became a mass of unorganized tissue. When the cultures were continually maintained on WB + kinetin (10 p.p.m.), the callused embryo did not shwo capacity for continuous growth, and turned brown 20-30 days after culture. To establish continuous tissue cultures and to induce morphogenesis in them the callused embryos were transferred to WB supplemented individually or in combinations, with 2,4-D yeast extract, cassein hydrolysate, and coconut milk. Upon transfer to WB + 2,4-D (2 p.p.m.) or WB + yeast extract (500 p.p.m.) the callus failed to renew growth. A concert of 3 substances (2,4-D 2 p.p.m. + yeast extract 0.25% + kinetin 5 p.p.m.) also did not promote the growth of embryo callus. If the transfers were made to WB + casein hydrolysate (400 p. p.m.) + coconut milk (15 %vjv) the growth of embryo callus was stimulated, and the whole culture presented a group of several callus masses of varied sizes derived from individual embryos which were no longer identifiable (Fig. 1 C). The newly formed tissue was friable; acetocarmine squash preparations showed both free cells and cell aggregates (Fig. 1 D). The tissue could be readily grown into 4-6 subcultures of nearly 400 mg each. About
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Fig. 1 A-D. Morphogenesis in seed cultures of Striga angustifolia - A. Embryo collected from 3-week-old seed culture on WB + kinetin (10 p.p.m.) showing overall enlargement. 90 x. B. Wholemount of embryo as in A. Observe further enlargement of radicle, and cotyledons, and formation of root hairs. Provascular strands are distinct in the hypocotyl. 47 x. C. Four-weekold transfer of embryo calluses on WB + casein hydrolysata (400 p.p.m.) + coconut milk (15 % vjv). 1.5 x. D. Acetocarmine squash preparation of embryo callus grown on WB + cacein hydrolysate (400 p.p.m.) + coconut milk (15 % vjv) showing a few free cells and cell aggregates. 108x.
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Fig.2A-E. Morphogenesis in seed cultures of Striga angustifolia - A. Histology of 4-week-old embryo callus grown on WB + casein hydrolysate (400 p.p.m.) + coconut milk (Hi % vjv) showing several "endogenous nests". 76 x. B. Transection througk an "endogenous nest" 118 X. C. Ten-week-old embryo callus showing differentiation of vessel members. 173 x. D.Eight-weekold embryo callus grown on WB + casein hydrolysate (400 p.p.m.) + coconut milk (15 % vjv) showing root formation. 3 x. E. L.s. 10-week-old callus showing origin of root. 7 x.
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2 weeks after subculture fresh mounds of tissue appeared and the subcultures showed ability for continued growth. As the subculture continued to grow, 2 regions were distinguishable in the mound: a core of compact cells and a peripheral region of loosely arranged vacuolated cells (Fig. 2A, B). Thus, the callus showd "endogenous nests" of cells rich in cytoplasm (Fig. 2 B). As growth progressed, cell layers contiguous between the mounds necrosed leading to separation of mounds from one another, and only vessel members but no organs differentiated in the callus (Fig. 2 C). Further investigations were, therefore, directed to a morphogenic study of the embryo callus. Seeds were as usual germinated on WB + kinetin (10 p.p.m.), and from the germinated seeds embryo callus was raised on WB + casein hydrolysate (400 p.p.m.) + coconut milk (15% v/v). The callus was grown into subcultures on WB + casein hydrolysate + coconut milk as well as on WB + adenine sulphate (40 p.p.m.), WB + watermelon juice (20% v/v), and Tepfer's HI media whIch were reported to induce shoot bud formation in embryo callus of certain other parasitic angiosperms (see RANGASWAMY 1963, RANGAN and RANGASWAMY 1968). Let alone inducing shoot bud formation, WB fortified with watermelon juice, and Tepfer's HI medium, both inhibited the growth of the subcultured embryo callus. On WB + casein hydrolysate + coconut milk, 40 % of the isolates showed root formation in 5 weeks after subculture (Figs. 2D, E). On WB + adenine sulphate the growth of the callus was slight, but 55 % of the isolates showed rooting. Usually the roots differentiated on the callus surface away from the culture medium. Instances of root development on tissue surface in contact with the culture medium were not wanting, but rare. Histologic preparations showed that the roots originated from "endogent)Us nests". The root comprised an epidermis. a cortex of 6-8 layers of parenchymatous cells, and a core of 3 or a few more xylem elements; endodermis and pericycle were not discernible. Those roots which differentiated on the surface of the embryo callus in contact with the culture medium continued to grow 1-2 cm long into the medium. The roots that originated on the free surface of the embryo callus proliferated as a callus in turn and lost their identity. Anatomical preparations showed that the callus originated from the cortical cells of the parent root. Eventually, however, this root callus also produced new roots which grew into the agar medium. IV. Discussion It is generally believed that for successful seed germination obligate root para-
sites, such as Cistanche and Striga, require a stimulus from the root of host plants. However, recent experiments have demonstrated that the host stimulus can be effectively replaced by kinetin (WORSHAM et al. 1959, WILLIAMS 1961), gibberellic acid (WILLIAMS 1961), and some coumarin derivatives (WORSHAM et al. 1962) for
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Striga asiatica; by casein hydrolysate or coconut milk or by both for Orobanche aegyptiaca (RANGASWAMY 1963, USHA 1968), Cistanche tubulosa (RANGAN 1965, RANGAN and RANGASWAMY 1968), Santalum album RANGASWAMY and RAO 1963), and Exocarpus cupressiformis (JOHRI and BHOJWANI 1965). In Btriga angustifolia kinetin (10 p.p.m.) induced seed germination, whereas gibberelic acid (5 p.p.m.) did not. Our observations on kinetin-induction of seed germination confirms those of WORSHAM et al. (1959) and of WILLIAMS (1961) on
Striga asiatica. That a variety of substances (cytokinins, gibberellic acid, coumarin, casein hydrolysate, and coconut milk) can induce seed germination in the root parasites, Cistanche, Orobanche, Btriga, Santalum and Exocarpus, suggestes a diverse chemical nature of the host stimulant. Although highly purified host-root extracts have been analyzed seed germination-stimulants have not been isolated and identified. Chemical analyses of the root secretion of Linum usitatissimum (host for Orobanche) indicated the presence of a lactone fraction in it (BROWN et al. 1951 a, b), and of the germination stimulant formed in the roots of Zea mays seedlings indicated it to be a coumarin (WORSHAM et al. 1964). Of the physical factors known to affect seed germination, BROWN and EDWARDS (1946) found that for Striga lutea a moisture treatment of seeds (by incubating the seeds in dark on moist filter paper) for 3 weeks considerably increased the germination percentage (82 % as against 30 %in control). VALLALCE (1950) also demonstrated a similar response for S. hermonthica seeds, and observed that seeds stored for 6 months required a longer period (15 days) of moisture treatment than seeds stored for 16 months which required only 4-day-treatment. Working on another scrophulariaceous parasite, Alectra vogelii, BOTHA (1950a, b) showed that by subjecting the seeds to dripping water their germination sensitivity to host stimulant was enhanced. In our present investigation on Striga angustifolia we found that seeds not dripped in water lay ungerminated, whereas the seeds washed in trickling water for 48 hr promptly germinated. According to KUMAR (1940) Striga angustifolia showed a high percentage of seed germination in light. OKONKWO'S (1964) work on S. senegalensis (= S. hermonthica) implies that seed germination occurred irrespective of light or dark treatments; this is true for S. angustifolia as well (present work). In root parasites, like seed germination, seedling morphogenesis also seems to be governed by factor resident in the host plant. For example, in Striga lutea upon germination the radicle grown 2-4 mm long, but the plumule does not emerge until a contact is made with a host plant (KUMAR et al. 1942, UTTAMAN 1950). A similar phenomenon was observed also in S. densiflora and S. euphrasioides (KUMAR 1940). OKONKWO implied that in S. senegalensis also the shoot originates only after the host-parasite connection is established through the primary haustorium. In Oro-
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banche hederae also a contact with the root of its host Hedera helix was indispensable for the initiation of shoots in culture (PRIVAT 1960). S. angustifolia (present work) does not seem to be an exception either; although roots were formed in vitro, shoot buds did not differentiate. In our present experiments on Striga angustifolia germination occurred only of the moistened, kinetin-treated seeds, and seedlings failed to develop. Thus a considerable morphogenic similarity exists between S. angustifolia and the 2 parasitic species S. asiatica (WORSHAM et al. 1959, 1962; WILLIAMS 1961) and S. senegalensl:s OKONKWO 1966). Such a morphogenic resemblance of S. angustifolia to 2 parasitic species of the same genus provides evidence to WILLIAMS' (1958) belief that S. angustifolia is one of the "unlikely exceptions to the group of obligate parasitic species". Like S. senegalensis (OKONKWO 1966), S. angustifolia also is probably a facultative parasite, and it may establish haustorial connections only during the subterranean (total parasitic) growth of its seedling. The lack of haustorial connections between S. angustifolia and any autotroph (SRINIVASAN 1946) is therefore not surprising, because only the flowering soecimens of S. angu,stifolia (i. e. plants that were almost in the final phase of their life cycle) were examined. Thus, parasitism in S. angustifolia is confined to early life of its seedling. Summary 1. Striga angustifolia (DoN) Saldhana is a herbaceous weed in sugarcane and sorghum fields. Some taxanomists consider this species a true parasite, others a pseudo-non-parasite, and still others an autotroph. 2. Morphogenesis of its seed germination was studies to seek a clue about the habit of this species. 3. Untreated seeds, water-dripped seeds, and seeds soaked in solutions of gibberellic acid and kinetin were cultured. 4. Untreated seeds as well as seeds soaked in solutions of gibberellic acid or kinetin (5 p.p.m.) failed to germinate. 5. Only the seeds subjected to dripping water for 48 hr followed by kinetin (10 p.p.m.) treatment germinated. Upon germination the embryos formed a callus tissue. 6. Subcultures of this embryo callus were raised to study its morphogenesis. In subcultures on White's medium + casein hydrolysate (400 p.p.m.) +coconut milk (15 o~ vjv) the callus differentiated roots. Shoot buds did not develop in any of the treatments. 7. Similarities in seed germination and in embryo morphogenesis between S. angusiifolia and some well-proven parasitic species of Striga are discussed. 8. From morphogenic viewpoint it is concluded that S. angustitolia may be a facultative root parasite and its parasitism is confined to the early subterranean life of its seedling.
Acknowledgements Our sincere thanks are to Professor B. M. JOHRI for his interest in this work. We are obliged to the authorities of the United States Department of Agriculture for granting one of us (TSR)
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a research fellowship in the scheme entitled "Physiology of Reproductive Organs of Seed Plants" under the PL 480 program, during- which tenure this research was conducted.
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USHA, S. V., 1968. Morphogenesis in aseptic seed cultures of the holoparasite - Orobanche aegyptiaca PERS. Ph. D. thesis, Univ. Delhi. UTTAMAN, P., 1950. A study on the germination of Striga seed and on the mechanism and nature of parasitism of Striga lutea (LOUR.) on rice. Proc. Indian Acad. Sci, Sect. B 32, 133-142. VALLANCE, K. B., 1950. Studies on the germination of the seeds of Striga hermonthica I. The influence of moisture-treatment, stimulant dilution and after ripening on germination. Ann. Bot. 14, 347-363. WILLIAMS, C. N., 1958. The parasitism of witchweed - A review. W. Afr. J. BioI. Chern. 2, 57 -73. - 1961. Growth and morphogenesis of Striga seedlings. Natu.re (London) 189,378-381. WORSHAM, A. D., KLINGMAN, G. C., and MORELAND, D. E., 1962. Promotion of germination of Striga asiatica seed by coumarin derivaties and effects on seedling development. Nature (London) 190, 199-201. MORELAND, D. E., and KLINGMAN, G. C., 1959. Stimulation of Striga asiatica (witchweed) seed germination by 6-substituted pu.rines. Science 130, 1654-1656. - 1964. Characterization of the Striga asiatica (witchweed) germination stimulant from Zea mays L. J. expo Bot. 10, 556-567. Authors' addresses: Dr. N. S. RANGASWAMY, Department of Botany, University of Delhi, Delhi 7, India, and Dr. T. S. RANGAN, Department of Horticultural Science, University of California, Riverside, California 92502, U.S.A.