J. AMER.SOC.HORT.SCI. 140(1):12–18. 2015. Pollen Tube Growth and Double Fertilization in Camellia oleifera Chao Gao, Deyi Yuan1, Ya Yang, Bifang Wang, Dongming Liu, and Feng Zou Key Laboratory of Cultivation and Protection for Non-Wood Forest Trees of Ministry of Education and the Key Laboratory of Non-Wood Forest Products of Forestry Ministry, Central South University of Forestry and Technology, Changsha 410004, China ADDITIONAL INDEX WORDS. crossbreeding, fluorescence microscopy, hybridization, reproduction, scanning microscopy, angiosperms, plant ABSTRACT. Camellia oleifera is an important plant species that produces edible oils. Understanding the double fertilization of this plant is critical for studies concerning crossbreeding, self-incompatibility, and the biological mechanisms underlying hybridization. We aimed to characterize pollen tube growth and double fertilization in C. oleifera. The female and male parent cultivars (Huashuo and Xianglin XLC15, respectively) were used for artificial pollination. Growth of the pollen tube in the style, ovary, and ovule from pollination to fertilization and the cytological characteristics of female and male gamete fusion during double fertilization were observed using fluorescence and scanning electron microscopy (SEM). Numerous pollen grains germinated 2 to 4 hours after pollination. The pollen tubes entered the interspaces between the papillar cells, grew along the stylar canal, and aggregated at the one-third site of the style. They grew in the gradually narrowing stylar canal, entering the locule. The tubes turned 908 and entered the embryo sac through the micropyle; subsequently, they entered a degenerated synergid, where the spermatids were released. One sperm nucleus fused with the polar nucleus, forming the primary endosperm nucleus, whereas the other sperm fused with the egg, forming the zygote. The polar nucleus was fertilized earlier than the egg. Double fertilization of C. oleifera is characterized as pre-mitotic gametogony. The current results lay a theoretical foundation for studies concerning the crossbreeding and embryology of C. oleifera and provide fundamental data concerning the reproductive biology of the genus Camellia. The compatible fertilization of angiosperms is initiated being pollinated for 32 to 34 h. After being pollinated for 22 h, through the hydration of the pollen at the stigma and the primary endosperm nuclei were formed. After being pollinated germination of the pollen tube. After entering the interpapillar for 24 h, mitosis was observed for the first time (Peng and Shen, cell space, the tube grows in the stylar canal or transmitting 2005). Fertilization is an important biological event in plant tissue until it enters the ovary. Subsequently, the tube penetrates sexual reproduction and has become an important topic in the embryo sac through the micropyle and enters degenerated studies on plant embryology. Since the first report describing synergids, where it releases two spermatids: one sperm fuses double fertilization, great progress has been made in many with an egg to form a fertilized egg and the other sperm fuses studies. For the study of male and female germ units, the model with the central cell to form the endosperm, thereby fulfilling plant Arabidopsis thaliana has been selected as the main the process of double fertilization. Biological fertilization experimental model. Studies on the segregation and function events from pollen germination to gamete fusion are complex of male and female germ units have enriched theories of and delicate with strict timing and spatiality; the underlying angiosperm reproductive biology (Ge et al., 2011; Huang and laws and characteristics of these processes vary depending on Russell, 1992). To elucidate the interactions between pollen the plant (Ge et al., 2007; Weterings and Russell, 2004). For and stigma, Ma et al. (2013) comprehensively analyzed studies example, based on studies of double fertilization in Oryza examining the characteristics of Corylus plants after self- sativa (Ding et al., 2009), the egg and the sperm fuse to form crossing or outcrossing pollination, focusing on the affinity a zygote at 0.5 to 2.5 h after pollination. At 10.0 h, the zygote is between pollen and stigma. Their research has laid a theoretical split into the two-celled proembryo for the first time. Zygo- foundation for improving fruit quality and understanding the phase is referred to as a period of pollination that lasts from 2.5 evolution of Corylus plants. Higashiyama et al. (2000) exam- to 10.0 h. Because they are pollinated for 1.0 to 3.0 h, the sperm ined the events occurring after pollen tubes entered the embryo, nucleus and the two polar nuclei fuse. After being pollinated for describing the detailed process of spermatid release inside 5.0 h, the primary endosperm nuclei began to split. However, in degenerated synergids after the pollen tubes of Torenia four- Brassica campestris ssp. pekinensis, zygotes were formed after nieri entered the embryo. Berger et al. (2008) performed studies being pollinated for 24 h, and they split for the first time after on spermatids and ova, comprehensively exploring their inter- recognition and fusion with each other. These studies provided Received for publication 6 June 2014. Accepted for publication 7 Oct. 2014. vital information to allow control of plant fertilization to This work was supported by the National Natural Science Foundation of increase agricultural output and reproduction. China (31170639), the Graduate Scientific Research Foundation of Central Camellia oleifera is an evergreen shrub or small tree South University of Forestry and Technology (CX2013A01), and the Hunan belonging to the genus Camellia, a flowering plant in the Provincial Innovation Foundation of Postgraduates (CX2013A014). family Theaceae (Fig. 1A and B), and it is an important woody We thank Prof. Huiqiao Tian for performing the reproductive biology analyses and Dr. Ting Liao for assistance with the field work. tree plant producing edible oil in southern China. Camellia has 1Corresponding author. E-mail: [email protected]. a wide range of uses with a long cultivation history. C. oleifera 12 J. AMER.SOC.HORT.SCI. 140(1):12–18. 2015. Fig. 1. The tree body, floral organs, and pistil of Camellia oleifera:(A) the adult tree body, (B)thefloralorgan,(C) the pistil. (D) The papillar cells were densely distributed on the stigma (·100), and the surface that could not accept pollen comprised the epidermis (Ep). (E) Papillar cells were observed on the surface of the stigma. (F) The transection of the style shows three vascular bundles (·100). (G) A partially enlarged view of the area within the red circle in F, the stylar canal, showing transmitting cells with regular morphology and a large nucleus (·100). (H) The styles began to attach from the two-thirds site downward (·100). (I) The stylar canals converged to form an interconnected structure in the ovary, and the transmitting tissue cells in the innermost layer can be observed (·100). (J) Polar view (top) of the ovary: five locules are divided with straight lines (·25). (K) Equatorial view of the ovary: the ellipse shows one locule with two columns of ovules (·25). (L) One column of ovules inside a seed bud (·25); bars: E =20mm; D, F, H, I =40mm; G = 50 mm; J, K, L = 400 mm. CC = canal cell; Co = cortex; En = endodermis; Ep = epidermis; Ov = ovule; Pa = papilla; PT = pollen tube; TT = transmitting tissue; VB = vascular bundle. J. AMER.SOC.HORT.SCI. 140(1):12–18. 2015. 13 is mainly cultivated for its seed, and the manufacture of edible FLUORESCENCE MICROSCOPY AND PARAFFIN SECTIONING. The oils from Camellia seed is the primary cultivation objective; the pistils were collected before pollination and at 1, 2, 4, 12, 36, edible oil obtained from C. oleifera is referred to as ‘‘eastern 48, 54, 60, 66, 72, 78, 84, 90, 96, 108, 120, 144, and 168 h after olive oil’’ as a result of its high quality (Lee and Yen, 2006). pollination. The pistil samples were fixed in Carnoy’s solution Although researchers have conducted numerous studies on the (at an acetic acid:ethanol ratio of 1:3) for 5 h and subsequently double fertilization of model plants and crops (Ding et al., 2009; maintained in 70% alcohol (Ding et al., 2009). The pistils were Faure et al., 2002; Li et al., 2009; Mol et al., 1994), studies on softened for 5 h in 8 molÁL–1 NaOH before observation. Then, the sexual reproduction of woody plants are rare, particularly the pistils were washed with distilled water until no NaOH those of the genus Camellia. Furthermore, even studies on the remained. The softened style was torn open along the stigma sexual fertilization of the genus Camellia have primarily and stained for 5 h with 0.5% water-soluble aniline blue focused on the development of the anther, ovules, megaspores, (prepared in 0.15 N dipotassium hydrogen phosphate buffer). microspores, female gametocytes, and male gametocytes Routine pallet pressing was performed. The growth of the (Kapil and Sethi, 1963; Tsou, 1997; Zou et al., 2013a, 2013b); pollen tube in the style was observed (Kho and Baer, 1968) self-incompatibility (Chen et al., 2012; Wachira and Kamunya, under a fluorescence microscope (BX-51; Olympus, Tokyo, 2005); and seed growth and development (Mondal, 2011; Sethi, Japan), and images were captured. 1965). Currently, there are no reports concerning fertilization, The double fertilization process was observed using a con- which is essential for the sexual reproduction of C. oleifera and ventional paraffin sectioning method, which is a modified is closely associated with the breeding of this plant. version of the method described by Kapil and Sethi (1963). Therefore, in this study, we explored the processes of pollen Hematoxylin staining and iron alum–hematoxylin section tube growth and fertilization to more clearly understand sexual staining were performed; the section thickness was 8 mm. reproduction of C. oleifera after pollination. Fluorescence The sections were observed under a fluorescence microscope microscopy and SEM were used to observe the development (BX-51).