Morphology of Pollen Grains Obtained from Dried Specimens of Spiny and Spineless Sago Palms Grown at Distant Sites in Indonesia

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1 Jpn. J. Trop. Agr. 50 (3): , 2006 Morphology Pollen Grains Obtained from Dried Specimens Spiny and Spineless Sago Palms Grown at Distant Sites in Indonesia Hiroshi EHARA1*, Madeline M. HARLEY2, William J. BAKER2, John DRANSFIELD2, Hitoshi NAITO3 and Chitoshi MIZOTA4 1 Graduate School Bioresources, Mie University 2 Herbarium, Royal Botanic Gardens, Kew 3 College Life Science, Kurashiki University Science and The Arts 4 Faculty Agriculture, Iwate University Abstract The morphology grains obtained from dried specimens one spiny and one spineless varieties grown at distant sites, West Papua (Irian Jaya) and West Sumatra, in Indonesia was examined to investigate intra-specific differences in sago palm (Metroxylon sagu Rottb.). The two varieties displayed both perfect (hermaphrodite) and male (staminate) flowers on a single palm. The two flower types did not differ in size in eir varieties and gynoecium was reduced to a pistillode in male flower. Pollen grains were produced from hermaphrodite and staminate flowers in both varieties, and were examined by SEM. The grains were equatorially disulcate, i.e. y exhibited two germination apertures on short equatorial axis grains. In vitro, general shape grains was ellipsoid. There were variations in grain shape from hermaphrodite and staminate flowers in both varieties, presumably due to level contraction or expansion grain wall (exine). However, re were no distinct differences in shape between grains produced from hermaphrodite or staminate flowers in eir varieties. In hermaphrodite and staminate flowers both varieties, tectum grains was psilate (smooth), and ten sparsely perforated. We did not record any significant differences in exine characteristics or, in grain shape, in two varieties examined. Key Words: Folk variety, Hermaphrodite, Metroxylon sagu, SEM observation, Staminate flower Introduction Sago palm (Metroxylon sagu Rottb.) that is distributed in Souast Asia and in areas inhabited by Melanesian people grows in swampy, alluvial and peaty soils where almost no or crops can grow without drainage or soil improvement (Sato et al., 1979; Flach, 1977; Jong, 1995). This palm species stores a large amount starch in trunk, approximately 300kg (dry wt.) per palm (Ehara, 2005) and has long been cultivated like banana and taro (Barrau, 1959; Takamura, 1990). Sago palm as a staple food continues to be important in some areas Souast Asia and in areas inhabited by Melanesian people (Ehara et al., 2000). The carbohydrate (starch) can be furr processed into various basic raw materials for food, animal feed and for industrial uses. Sago palm is one most important crops for sustainable agriculture and for rural development in swampy areas tropics. Received Feb. 2, 2006 Accepted Jun. 3, 2006 * Corresponding author Kurimamachiya-cho, Tsu , Japan ehara@bio.mie-u.ac.jp Morphological data are generally useful to identify plant species and varieties. Sago palm had previously been classified by spine character, i.e. absence or presence spines and ir length and density (Rauwerdink, 1986). However, spines occasionally emerge in seedlings produced from seeds spineless sago palm (Ehara et al., 1998), and Jong (1995) reported opposite case, namely that not only spiny seedlings but also spineless seedlings grew from seeds spiny sago palm. Since identification sago palm varieties using only presence or absence spines and ir characteristics may not be reliable, attention should be paid to or characteristics. and grain morphological Floral studies are important for plant taxonomy, systematics, evolution and phylogeny. There are several studies on floral morphology in Metroxylon section Coelococcus: Metroxylon warburgii Heim. (Uhl and Dransfield, 1987), M. vitiense Benth. et Hook. (Tomlinson, 1990), and M paulcoxii McClay (M. upoluense Becc.) (McClay, 1998). Rauwerdink (1986) had examined grain morphology four species in section Coelococcus (M amicarum Becc., M. vitiense, M salomonense Becc. and M warburgii) and one species in section Metroxylon

2 122 Jpn. J. Trop. Agr. 50 (3) 2006 (Eumetroxylon) (M. sagu: section Metroxylon consists only M sagu). However, re are few studies on floral and grain morphology in section Met roxylon and information about grain morphology sago palm (M sagu) varieties is very limited. Therefore, in present study we examined morphology grains obtained from dried specimens one spiny and one spineless sago palms grown at distant sites, West Papua (Irian Jaya) and West Sumatra, in Indonesia to investigate intra-specific differences in Metroxylon sagu. Besides, morphological characters flowers were also observed. Material and Methods Flowers at pre-ansis stage were taken from herbarium specimens a spiny variety (20 flowers) grown in Fakfak, West Papua (Irian Jaya), (Baker 881 (K)) and a spineless variety `Rumbio' (30 flowers) grown in Padang, West Sumatra (Edison, Naito and Mizota s.n.). The term ` variety' was used instead variety in present report, based on conclusion drawn at Eighth International Sago Symposium (EISS 2005 held in Jayapura, Indonesia on 4-6, August 2005), according to which different types sago palm that show variable specific characters and are locally recognized as varieties or accessions, should be designated as varieties until significant evidence specification is gared. In case spiny variety, flowers were collected from mid- 2nd branch (80cm length) attached to 1st branch (1.5m length) in inflorescence. Flower samples were collected from mid-2nd branch attached to mid-1st branch that was cut from middle position inflorescence in spineless variety. The calyx, two petals and three stamens were removed under a dissecting microscope, to observe presence or absence a gynoecium, n flowers were identified as eir hermaphrodite staminate. Pollen grains were obtained from hermaphrodite and staminate flowers both varieties, and were examined by scanning electron microscopy (SEM). Samples were prepared according to acetolysis protocols for palm used in Palynology Unit Herbarium at Kew Gardens (Harley, 1990). Results and Discussion A flower spiny variety at ansis is shown in Fig. 1. Sago palm flowers both spiny and spineless varieties have three sepals and three or petals, as in M, warburgii (Uhl and Dransfield, 1987) and M vitiense (Tomlinson, 1990). Metroxylon bears both perfect (hermaphrodite) and male (staminate) flowers on a single palm (Tomlinson, 1990). Since it was difficult to identify hermaphrodite or staminate flowers by external appearance, calyx, two petals and three six stamens were removed from flowers, preserved at pre-ansis stage, for observation. Of twenty flowers examined for spiny variety, ten were hermaphrodite were staminate. flowers were hermaphrodite Fig. 2 shows hermaphrodite and ten In spineless variety, eighteen calyx, two petals and three stamens. and twelve were staminate. flowers with removed Staminate flowers with removed calyx, two petals and three stamens are shown in Fig. 3. According to Tomlinson (1990), two flower types do not differ in size and gynoecium is reduced to a pistillode in male flower in Metroxylon section Coelococcus. These characteristics were also observed in each varieties used in current study (cf. Figs. 2 and 3). The component parts each flower in both varieties were closely similar to those species in section Coelococcus (Uhl and Dransfield, 1987; Tomlinson, 1990). Pollen grain morphology Pollen grains were produced from both hermaphrodite and staminate flowers in both samples studied varieties M sagu. The grains were equatorially disulcate, i.e. y exhibited two germination apertures on short equatorial axis grain (Fig. 4). In vitro general shape grains was ellipsoid. Our results were in agreement with findings Rauwerdink (1986). However, according to Schuiling et al. (1993), Tuwan (1991) observed varietal differences in grain shape between three spiny varieties grown in Ambon, Maluku, Indonesia (differences between spiny varieties in strict sense according to symposium conclusion from EISS 2005). Of three varieties (three varieties), Tuwan (1991) indicated that grain Tuni was elliptic, while that Makanaru was round and that Ihur triangular. Jong (1995) examined grain morphology sago palms grown in Sarawak, Malaysia and reported that grains were mostly oval and, occasionally, round in spineless type, and consisted a mixture oval and triangular forms in spiny type. According to him, it remained to be determined wher triangular grains were defective or deformed by

3 Ehara et al.: Morphology dehydration. Our own data demonstrated 123 Pollen Grains Sago Palm that variable grain shape from hermaphrodite and staminate flowers both varieties was due to level contraction or expansion grain wall (exine). Fig. 5 shows, schematically, appearance a fully expanded grain in different planes, (5A, B, Ci, Cii); triangular appearance grain in alternative equatorial planes should also be noted (5B, Civ). Fig. 4 (A-D) and Fig. 6 (A-B) show grains spiny and spineless varieties in a slightly oblique distal polar plane (cf. Fig. 5 Ciii). Fig. 2 There was no distinct difference in shape between Fig. 1 Photomicrograph variety a flower spiny Fig. 3 Photomicrograph a staminate flower spiny (left) and spineless (right) varieties. Pe, petal; A, anr; Pi, pistillode. at ansis. A, anr; Pe, petal; C, calyx. Fig. 4 Electronmicrographs staminate flowers spineless spiny variety erumbio f grains Photomicrograph a hermaphrodite flower spiny (left) and spineless (right) varieties. Pe, petal; A, anr; G, gynoecium (i.e. pistil). variety; ( produced C) from hermaphrodite grains in slightly A) hermaphrodite and oblique D) and staminate distal polar flowers plane). B)

4 124 Jpn. J. Trop. Agr. 50 (3) 2006 Fig. 5 Schematic representation appearance a fully expanded equatorial disulcate grain in different planes. A: fully expanded disulcate grain, equatorial plane, long axis, B: fully expanded disulcate grain, equatorial plane, short axis, Ci: fully expanded disulcate grain, polar plane, distal face, Cii: fully expanded disulcate grain, polar plane, proximal face, Ciii: equatorial disulcate grain, aperture margins infolded, oblique polar plane, distal face, Civ: equatorial disulcate grain, aperture margins infolded, slightly oblique equatorial plane, long axis, Ei: position polar (P) and equatorial (E axes, equatorial disulcate grain, equatorial plane, long axis, Eii: position polar (P and equatorial (E) axes, equatorial disulcate grain, equatorial plane, short axis, Eiii: position equatorial (E) axis, equatorial grain, polar plane, distal face. Fig. 6 Electronmicrographs spineless polar grains produced flowers grain grains variety erumbio f; with aperture A) margins produced expanded from grain a with ( hermaphrodite infolded grain flower aperture in slightly margins, oblique B) distal plane). from hermaphrodite or staminate during dehydration, or due to acetolysis (this study) grains tended to collapse. in eir varieties. The wall (exine) palm grains is As a result, in disulcate grains, such as those in Metroxylon generally more resilient than that in most or monocotyledonous plants, hence its high fossilization spp., and most rattan palms, aperture potential acetolysed (Harley and Morley, 1995). Neverless, tended to fold inwards (or fossilized) margins (Fig. 4C-D, Fig. 6A). specimens, In delicate

5 Ehara et al.: Morphology Pollen Grains Sago Palm 125 membrane covering aperture (Fig. 4B) was usually destroyed (Fig. 6B). In an extensive survey palm based on light microscopy, Sowunmi (1972) recorded measurements exine thickness for M amicarum, M salomonense and M warburgii. She commented that exine was notably thicker than that in or palm taxa she examined. Ferguson and Harley (1993) who described grains M amicarum, M sagu, M salomonense and M upoluense Becc., stated that exine was very thick. Harley (1996) described and discussed incidence very thick exines palm grains. The exines M. sagu and M. amicarum were included in her exine ultrastructure Group 1A: palm species with outstandingly thick exines. In same study, exine M vitiense was included in exine ultrastructure Group 1C, a similarly proportioned but thinner exine. Interestingly, in palms, very thick Group la type exine is unusual widespread. but systematically Apart from subfamily Calamoideae, as in Metroxylon, this type was observed in a few species Daemonorps, a rattan palm, as well as in subfamilies Coryhoideae (Pritchardia) and Arecoideae: Arecaceae, subtribe Iguanuriae (Harley, 1996). In hermaphrodite and in staminate flowers, both varieties, tectum grains was psilate (smooth), and ten sparsely perforated (Fig. 4, Fig. 6), which is in agreement with findings Rauwerdink (1986) for M. amicarum, M sagu and M warburgii. Harley (1996) illustrated completely psilate grain M. sagu. Rauwerdink (1986) reported a reticulate exine for equatorial faces M salomonense and M vitiense. Ferguson and Harley (1993) observed and illustrated an extremely coarse reticulate exine for M salomonense. Sago palm formae had previously been classified by spine character (Rauwerdink, 1986), while spines occasionally emerged in seedlings produced from seeds spineless sago palm (Ehara et al., 1998). In contrast, spineless seedlings were occasionally produced also from seeds spiny sago palm (long, 1995). Since identification sago palm varieties using only presence or absence spines as a character is not reliable. Or morphological characters should be used to identify a variety formae in sago palm. However, as described above, we did not record any significant differences in exine characteristics varieties examined. or in grain shape, in two Variations in grain shape were probably due to previous misinterpretation. Needless to say, from data based on such a small sample, we could not draw final and meaningful conclusions as to wher morphological variations floral parts might become a valuable character set for distinguishing genetic groups sago palm. Neverless, results obtained so far suggest that re was no distinct difference in grain morphology between spiny and spineless varieties grown at isolated sites separated by a distance more than 3500km in a straight line. Acknowledgments This article is contribution No. 34 from Laboratory Crop Production and Ecology, Mie University. We are indebted to Dr. Edison Munaf, Andalas University for his co-operation in collection specimens. This study was supported by Anglo-Japanese Scientific Exchange Programme between Royal Society and Japan Society for Promotion Science, to which we express gratitude. References our Barrau, J The sago palm and or food plants Marsh Dwellers in South Pacific Islands. Econ. Bot. 13: Ehara, H Geographical distribution and specification Metroxylon palms. Jpn. J. Trop. Agr. 49(Ext.2): ** Ehara, H., C. Komada and O. Morita Germination characteristics sago palm seeds and spine emergence in seedlings produced from spineless palm seeds. Principes 42: Ehara, H., S. Susanto, C. Mizota, S. Hirose and T. Matsuno Sago palm (Metroxylon sagu, Arecaceae) production in eastern archipelago Indonesia: Variation in morphological characteristics and pith-dry matter yield. Econ. Bot. 54: Ferguson, I. K. and M. M. Harley The significance new and recent work on morphology Palmae. Kew Bull. 48: Flach, M The sago palm. FAO Plant Production and Protection Paper 47 AGPC/MIS/80, FAO (Rome) p.85. Flach, M Yield potential sago palm and its realization. In: Sago-'76: The Equatorial Swamp as a Natural Resource (Tan, K. ed.) The 1st International Sago Symposium (Kuala Lumpur) Harley, M. M Palm and fossil record. Ph.D. Thesis, University East London in collaboration with RBG, Kew. Harley, M. M Occurrence simple, tectate, monosulcate or trichotomosulcate grains within Palmae. Rev. Palaebot. & Palynol. 64: Harley, M. M. and R. J. Morley Ultrastructural studies some fossil and extant palm, with notes on geological history subtribes Iguanurinae and Calaminae. Rev. Palaebot. & Palynol. 85: Jong, E. S Research for development sago palm (Metroxylon sagu Rottb.) cultivation in Sarawak, Malaysia. Ph.D. Dissertation Agricultural University (Wageningen) p.139.

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