Hatice Nurhan BÜYÜKKARTAL, Hatice ÇÖLGEÇEN, Ufuk KOCA ÇALIŞKAN

Ultrastructural observations in somatic embryogenesis ofnatural tetraploid Trifolium pratense L.

Previous reports of plant regeneration of natural tetraploid T. pratense L. Elçi could be realized only through the apical meristem calli. In order to proceed to the production stage, other regeneration methods need to be tried. Aseptic seedlings were used for the production of somatic embryos through various 2,4-D and kinetin trials. Nonuniform external callus cells with translucent cytoplasm were observed in various developmental stages of somatic embryos. Beneath these cells, there were uniformly aligned, dark-stained embryo cells with dense cytoplasm. Despite the similar developmental stages and cell characteristics of zygotic and somatic embryos, the walls of somatic embryo cells revealed a highly wavy pattern. The nucleus generally contained only one nucleolus, which was spherical, dark stained, and electron-dense. Electron-dense droplets were seen in vacuoles. The cytoplasm consisted of starch-containing amyloplasts, mitochondria, plastids, ribosomes, endoplasmic reticulum, dictyosomes, lipid, and protein bodies. In some of the somatic embryos at the globular and heart stages, vacuole or electron-translucent zones were observed in the nucleolus. Additionally, a few embryo degenerations were recorded during developmental stages of the zygotic embryo. For the first time, the somatic embryos of natural tetraploid T. pratense were produced from hypocotyl (85%), cotyledon (75%), and apical meristem (60%) explants in 0.3 mg/L 2,4-D and 2 mg/L kinetin-containing MS medium. Our study developed an effective and efficient in vitro production method for using natural tetraploid T. pratense in biotechnological studies.

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Algan G, Bakar HN (1996). Light and electron microscopic examination of the embryo and endosperm development in the natural tetraploid Trifolium pratense L. Isr J Plant Sci 44: 273-288.
Algan G, Bakar HN (1997). The ultrastructure of the mature embryo sac in the natural tetraploid of red clover (Trifolium pratense L.) that has a very low rate of seed formation. Acta Soc Bot Pol 66: 13-20.
Algan G, Bakar Büyükkartal HN (1999a). Ultrastructure of seed coat development in the natural tetraploid Trifolium pratense L. J Agron Crop Sci 184: 205-213.
Algan G, Bakar Büyükkartal HN (1999b). Tetraploid çayırüçgülü (Trifolium pratense L.) nde apomiktik gelişme. Turk J Agric For 23: 519-525 (article in Turkish with an abstract in English).
Babaoğlu M, Gürel E, Ozcan S (2001). Bitki Biyoteknolojisi I. Doku Kültürü ve Uygulamaları. Birinci Baskı, Konya, Türkiye: Selcuk Universitesi Basımevi (in Turkish).
Bakar Büyükkartal HN (2008). Causes of low seed set in the natural tetraploid Trifolium pratense L. (Fabaceae). Afr J Biotechnol 7: 1240-1249.
Bakar HN, Algan G (1998). Ultrastructure of the endosperm haustorium in Trifolium pratense L. Acta Biol Cracov Bot 40: 15-19. Brisibe EA, Nishioka D, Miyake H, Taniguchi T, Maeda E (1993). Developmental electron microscopy and histochemistry of somatic embryo differentiation in sugarcane. Plant Sci 89(1): 85-92.
Çölgeçen H, Büyükkartal HN, Toker MC (2008). In vitro germination and structure of hard seed testa of natural tetraploid Trifolium pratense L. Afr JBiotechnol 7: 1473-1478.
Çölgeçen H, Toker MC (2008). Plant regeneration of natural tetraploid Trifolium pratense L. Biol Res 41: 25-31.
Elçi Ş (1982). The utilization of genetic resource in fodder crop breeding. Eucarpio, Fodder Crop Section, 1316 September, Aberystwyth, UK, pp. 28.
Fernando JA, Vieria MLC, Geraldi IO, Gloria BA (2002). Anatomical study of somatic embryogenesis in Glycine max (L.) Merrill. Braz Arch Biol Techn 45(3): 277-286.
Jasik J, Salajova T, Salaj J (1995). Developmental anatomy and ultrastructure of early somatic embryos in European black pine (Pinus nigra Arn.). Protoplasma 185: 205-211.
Karlson SB, Basil IK (1986). Morphology and ultrastructure of embryogenic cell suspension cultures of Panicum maximum(guinea grass) and Pennisetum purpureum (napier grass). Am J Bot 73(6): 894-901.
Konieczny R, Pilarska M, Tuleja M, Salaj T, Ilnicki T (2010). Somatic embryogenesis and plant regeneration in zygotic embryos of Trifolium nigrescens (Viv.). Plant Cell Tiss Org 100: 123-130.
Konieczny R, Sliwinska E, Pilarska M, Tuleja M (2012). Morphohistological and flow cytometric analyses of somatic embryogenesis in Trifolium nigrescens Viv. Plant Cell Tiss Org 109: 131-141.
Luft JH (1961). Improvements in epoxy resin embedding methods. J Biophys Biochem Cy 9: 409.
Luo Y, Koop HU (1997). Somatic embryogenesis in cultured immature zygotic embryos and leaf protoplasts of Arabidopsis thaliana ecotypes. Planta 202: 387-396.
Maheswaran G, William EG (1984). Direct somatic embryoid formation on immature embryos of Trifolium repens, T. pratense and Medicago sativa and rapid clonal propagation of T. repens. Ann Bot-London 54: 201-211.
Maheswaran G, William EG (1985). Origin and development of somatic embryoids formed directly on immature embryos of Trifolium repensin vitro. Ann Bot-London 56: 619-630.
Maheswaran G, William EG (1986). Clonal propagation of T. pratense, T. resupinatum and T. subterraneum by direct somatic embryogenesis on cultured immature embryos. Plant Cell Rep 3: 165-168.
Moura EF, Ventrella MC, Motoike SY (2010). Anatomy, histochemistry and ultrastructure of seed and somatic embryo of Acrocomia aculeata (Arecaceae). Sci Agr (Piracicaba, Braz.) 67: 399-407.
Mclean NL, Nowak J (1998). Inheritance of somatic embryogenesis in red clover (Trifolium pratense L.). Theor Appl Genet 97: 557-562.
Monteiro M, Appezzato-da-Gloria B, Valarini MJ, Alberto de Oliviera C, Vieira MLC (2003). Plant regeneration from protoplast of alfalfa (Medicago sativa) via somatic embryogenesis. Sci Agr 60: 683-689.
Murashige T, Skoog FA (1962). Revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plantarum 15: 473-497.
Pederson GA (1986). In vitro culture and somatic embryogenesis of four Trifolium species. Plant Sci45: 101-104.
Phillips GC, Collins GB (1980). Somatic embryogenesis from cell suspension cultures of red clover. Crop Sci 20: 323-326.
Puigderrajols P, Mir G, Molinas M (2001). Ultrastructure of early secondary embryogenesis by multicellular and unicellular pathways in cork oak (Quercus suber L.). Ann Bot-London 87: 000-011.
Radionenko MA, Kuchuk NV, Khvedynich OA, Gleba YY (1994). Direct somatic embryogenesis and plant regeneration from protoplast of red clover (Trifolium pratense L.). Plant Sci 97: 75-81.
Ramakrishnan K, Granam R, Sivakumar P, Manickani A (2005). Developmental pattern formation of somatic embryos induced in cell suspension cultures of cowpea (Vigna unguiculata (L.) Walp). Plant Cell Rep 24: 501-506.
Shang H, Liu C, Zhang C, Li F, Hong W, Li FG (2009). Histological and ultrastructural observation reveals significant cellular differences between Agrobacterium transformed embryogenic and non-embryogenic calli of cotton.J Integr Plant Biol 7: 1-10.
Snedecor GW, Cochran WG (1967). Statistical Methods. 6th ed. Iowa, USA: The Iowa State University Press, pp. 327-329.Stempak JG, Ward RT (1964). An improved staining method for electron microscopy. J Cell Biol 22: 697.
Wang YF, Cui KR, Wang LH, Wang LS (1994). Ultrastructural studies of somatic embryogenesis in wheat. Acta Bot Sin 36: 418-422.