Miscanthus ×giganteus Greef et Deu. (Poaceae), a hybrid of Miscanthus sinensis and M. sacchariflorus native to Japan, is an ornamental and a highly lignocellulosic bioenergy crop, cultivated in the European Union as an alternative source of energy. This grass reproduces exclusively vegetatively, by rhizomes or via expensive in vitro micropropagation. The present study was aimed at finding the barriers that prevent sexual seed production, based on detailed embryological analyses of the whole generative cycle, including microsporogenesis, pollen viability, megasporogenesis, female gametophyte development, and embryo and endosperm formation. Sterility of M. ×giganteus results from abnormal development of both male and female gametophytes. Disturbed microsporogenesis (laggard chromosomes, univalents, micronuclei) was further highlighted by low pollen staining. The frequency of stainable pollen ranged from 13.9% to 55.3% depending on the pollen staining test, and no pollen germination was observed either in vitro or in planta. The wide range of pollen sizes (25.5-47.6 μm) clearly indicated unbalanced pollen grain cytology, which evidently affected pollen germination. Only 9.7% of the ovules developed normally. No zygotes nor embryos were found in any analyzed ovules. Sexual reproduction of M. ×giganteus is severely hampered by its allotriploid (2n=3x=57) nature. Hybrid sterility, a strong postzygotic barrier, prevents sexual reproduction and, therefore, seed formation in this taxon.
ADATI S. 1958. Studies on the genus Miscanthus with special reference to the Japanese species suitable for breeding purposes as fodder crops. Bulletin of the Faculty of Agriculture, Mie University 17: 1-96.
ADATI S, and MITSUISHI S. 1955. Wild growing plants of the Far East, especially Japan, suitable for breeding purposes. Part I. Karyological study in Miscanthus (1). Bulletin of the Faculty of Agriculture, Mie University 12: 1-10.
BHANWRA RK. 1988. Embryology in relation to systematics of Gramineae. Annals of Botany 62: 215-233.
CARLOS A, MARTINEZ EJ, and QUARIN CL. 2004. Apospory followed by sterility in hypotriploid hybrid (2× X 4×) of Paspalum. Caryologia 57: 373-378.
CHOU CH, CHIANG YC, and CHIANG TZ. 2000. Genetic variability and phytogeography of Miscanthus sinensis var. condensatus, an apomictic grass, based on RAPD fingerprints. Canadian Journal of Botany 78: 1262-1268.
CHOU CH. 2009. Miscanthus plants used as an alternative biofuel material: the basic studies on ecology and molecular evolution. Renewable Energy 34: 1908-1912.
DAFNI A. 1992. Pollination Ecology: A Practical Approach (Practical Approach Series). Oxford University Press. U.S.A.
DE CESARE M, HODKINSON TR, and BARTH S. 2010. Chloroplast DNA markers (cpSSRs, SNPs) for Miscanthus, Saccharum and related grasses (Panicoideae, Poaceae). Molecular Breeding 26: 539-544.
GŁOWACKA K, JEŻOWSKI S, and KACZMAREK Z. 2009. Polyploidization of Miscanthus sinensis and Miscanthus ×giganteus by plant colchicine treatment. Industrial Crops and Products 30: 444-446.
GŁOWACKA K, JEŻOWSKI S, and KACZMAREK Z. 2010. The effects of genotype, inflorescence developmental stage and induction medium on callus induction and plant regeneration in two Miscanthus species. Plant Cell, Tissue and Organ Culture 102: 79-86.
GUOHUA M, XUELIN H, QIUSHENG X, and BUNN E. 2009. Multiporate pollen and apomixis in Panicoideae. Pakistan Journal of Botany 41: 2073-2082.
HER JM JR. 1971. A new clearing-squash technique for the study of ovule development in angiosperms. American Journal of Botany 58(8): 785-790.
HODKINSON TR, CHASE MW, TAKAHASHI CH, LEITCH I, BENETT MD, and RENVOIZE SA. 2002. The use of DNA sequencing (ITS and TRNL-F), AFLP, and fluorescent in situ hybridization to study allopolyploid Miscanthus (Poaceae). American Journal of Botany 89: 279-286.
KHAN N, ZHOU S, RAMANNA MS, ARENS P, HERRERA J, VISSER RGF, and VAN TUYL JM. 2009. Potential for analytic breeding in allopolyploids: an illustration from Longiflorum × Asiatic hybrid lilies (Lilium). Euphytica 166: 399-409.
LAFFERTY J, and LELLEY T. 1994. Cytogenetic studies of different Miscanthus species with potential for agricultural use. Plant Breeding 113: 246-249.
LEWANDOWSKI I. 1999. Propagation method as an important factor in the growth and development of Miscanthus ×giganteus. Industrial Crops and Products 8: 229-245.
LEWANDOWSKI I, SCURLOCK JMO, LINDVALL E, and CHRISTOU M. 2003. The development and current status of perennial rhizomatous grasses as energy crops in the US and Europe. Biomass and Bioenergy 25: 335-361.
LINDE-LAURSEN I. 1993. Cytogenetic analysis of Miscanthus 'Giganteus', an interspecific hybrid. Hereditas 119: 297-300.
MA GH, and HUANG XL. 2007. Cytological and embryological studies on apospory in Bothriochloa ischaemum L. Acta Biologica Hungarica 58(4): 421-429.
MOYLE LC, and NAKAZATO T. 2008. Comparative genetics of hybrid incompatibility: sterility in two Solanum species crosses. Genetics 179: 1437-1453.
NISHIWAKI A, MIZUGUTI A, KUWABARA S, TOMA Y, ISHIGAKI G, MIYASHITA T, YAMADA T, MATUURA H, YAMAGUCHI S, RAYBURN AL, AKASHI R, and STEWART JR. 2011. Discovery of natural Miscanthus (Poaceae) triploid plants in sympatric populations of Miscanthus sacchariflorus and Miscanthus sinensis in southern Japan. American Journal of Botany 98: 154-159.
PŁAŻEK A, and DUBERT F. 2010. Improvement of medium for Miscanthus ×giganteus callus induction and plant regeneration. Acta Biologica Cracoviensia Series Botanica 52(1): 105-110.
PROSCEVIÈIUS J, RANÈELIENË V, and DAMBRAUSKAITË D. 2007. Cytogenetic analysis of progeny derived from allotriploid inter-specific hybrids of Lilium. Biologija 53: 8-12.
RAYBURN AL, CRAWFORD J, RAYBURN CHM, and JUVIK JA. 2009. Genome size of three Miscanthus species. Plant Molecular Biology Reports 27: 184-188.
ROUNSAVILLE TJ, TOUCHELL DH, and RANNEY TG. 2011. Fertility and reproductive pathways in diploid and triploid Miscanthus sinensis. HortScience 46: 1353-1357.
SEOK KIM H, ZHANG G, JUVIK JA, and WIDHOLM JM. 2010. Miscanthus ×giganteus plant regeneration: effect on callus types, ages and culture methods on regeneration competence. Global Change Biology: Bioenergy 2: 192-200.
SŁOMKA A, KAWALEC P, KELLNER K, JĘDRZEJCZYK-KORYCIŃSKA M, ROSTAŃSKI A, and KUTA E. 2010. Was reduced pollen viability in Viola tricolor L. the result of heavy metal pollution or rather the tests applied? Acta Biologica Cracoviensia Series Botanica 52(1): 123-127.
TONDINI F, TAVOLETTI S, MARIANI A and VERONESI F. 1993. A statistical approach to estimate the frequency of n, 2n and 4n pollen grains in diploid alfalfa. Euphytica 69: 109-114.
WANG J, KANG X, and ZHU Q. 2010. Variation in pollen formation and its cytological mechanism in an allotriploid white poplar. Tree Genetics & Genomes 6: 281-290.
WĘDZONY M. 1996. Mikroskopia Fluorescencyjna dla Botaników. Polska Akademia Nauk, Zakład Fizjologii Roślin im. Franciszka Górskiego, Kraków.
YEON YU CH, SEOK KIM H, RAYBURN AL, WIDHOLM JM, and JUVIK JA. 2009. Chromosome doubling of the bioenergy crop, Miscanthus ×giganteus. Global Change Biology: Bioenergy 1: 404-412.
YUANJIE Z, JIANXIONG J, MINGXI L, XIN A, and ZILI Y. 2009. Comparison of pollen viability determining methods for Miscanthus sinensis Anderss. Chinese Agricultural Science Bulletin 25: 147-150. (In Chinese).