CORE Metadata, citation and similar papers at core.ac.uk Provided by Springer - Publisher Connector González-Gutiérrez et al. SpringerPlus 2014, 3:575 http://www.springerplus.com/content/3/1/575 a SpringerOpen Journal RESEARCH Open Access Embryo sac formation and early embryo development in Agave tequilana (Asparagaceae) Alejandra G González-Gutiérrez, Antonia Gutiérrez-Mora and Benjamín Rodríguez-Garay* Abstract Agave tequilana is an angiosperm species that belongs to the family Asparagaceae (formerly Agavaceae). Even though there is information regarding to some aspects related to the megagametogenesis of A. tequilana, this is the first report describing the complete process of megasporogenesis, megagametogenesis, the early embryo and endosperm development process in detail. The objective of this work was to study and characterize all the above processes and the distinctive morphological changes of the micropylar and chalazal extremes after fertilization in this species. The agave plant material for the present study was collected from commercial plantations in the state of Jalisco, Mexico. Ovules and immature seeds, previously fixed in FAA and kept in ethanol 70%, were stained based on a tissue clarification technique by using a Mayer’s-Hematoxylin solution. The tissue clarification technique was successfully used for the characterization of the megasporogenesis, megagametogenesis, mature embryo sac formation, the early embryo and endosperm development processes by studying intact cells. The embryo sac of A. tequilana was confirmed to be of the monosporic Polygonum-type and an helobial endosperm formation. Also, the time-lapse of the developmental processes studied was recorded. Keywords: Agavaceae; Chalazal haustorium; Helobial endosperm; Hypostase; Megagametogenesis; Megasporogenesis; Polygonum-type Background et al. 2012). In each ovule meiosis of the megaspore One of the main characteristics of Angiosperms is that mother cell produces four haploid cells called mega- they possess seeds enclosed inside a fruit derived from the spores. In the monosporic pattern, three of these mega- ovary of flowers (Li and Ma 2002). Another outstanding spores degenerate while the closest cell to the chalazal characteristic of angiosperms is that they present alterna- region remains viable and gives rise to a single func- tion of generations in their life cycle (as in many other tional megaspore. plants), that is divided in two phases: one dominant dip- During the megagametogenesis process, the functional loid phase, which is called sporophytic, and one haploid megaspore passes through one or more mitotic divisions phase known as gametophytic (Haig 1990; Rodríguez- without cytokinesis forming a multinucleate coenocyte. Garay et al. 2000; Fan et al. 2008; Ma and Sundaresan Latter, cell walls are formed around the nuclei resulting 2010). The main function of the gametophyte phase is the in a mature embryo sac (Rabiger and Drews 2013). The production of haploid gametes whether they are male or embryo sacs may present a diversity of developmental female (Reiser and Fischer 1993; Yadegari and Drews pathways, however, the most common is the monosporic 2004; Fan et al. 2008). Polygonum-type, in which the functional megaspore The female gametophyte, also named megagametophyte passes through three mitotic divisions producing a seven or embryo sac, is developed within the carpel, which con- celled embryo sac (Chasan and Walbot 1993; Li and Ma sists of three elements: the stigma, the style and the ovary, 2002; Maheshwari 1937) consisting of three antipodal which can contain one or several ovules (Gutiérrez-Mora cells, one central cell formed by two polar nuclei, two synergid cells, and the egg cell (Dresselhaus 2006; Kägi and Groß-Hardt 2007; Yang et al. 2010). * Correspondence: [email protected] Unidad de Biotecnología Vegetal, Centro de Investigación y Asistencia en Some studies have characterized the female gameto- Tecnología y Diseño del Estado de Jalisco, A.C. (CIATEJ), Av. Normalistas No. phyte of different species belonging to the Asparagaceae 800, Col. Colinas de la Normal, Guadalajara, Jalisco 44270, México © 2014 González-Gutiérrez et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. González-Gutiérrez et al. SpringerPlus 2014, 3:575 Page 2 of 11 http://www.springerplus.com/content/3/1/575 family formerly Agavaceae (APG III 2009), where the cells with a chalaza-micropylar orientation (Figure 2b, majority of them have been described as Monosporic c), the average size of the tetrad is about 58 μm long and Polygonum-type. Among these species, Yucca rupicola 21 μm wide (for the rest of measurements mentioned on (Watkins 1937); Y. aloifolia (Wolf 1940); Y. filamentosa this paper see Additional file 1: Table S1). (Reed 1903); Agave lechuguilla (Grove 1941), A. virgi- Once the meiotic tetrad is formed, the three mega- nica (Regen 1941), Hesperocallis undulata, Leucocrinum spores closest to the micropylar pole degenerate (Fig- montanum (Cave 1948) and Comospermum yedoense ure 2d) and their remains could be observed as highly (Rudall 1999) are found. However, Piven et al. (2001) re- stained spots which were frequently observed in the ported the embryo sac development of Agave fourcroydes two-nucleated embryo sacs. Only the megaspore cell and A. angustifolia as bisporic Allium-type. closest to the chalazal pole remains viable, becoming the Only one study could be found that was centered on functional megaspore (FM), and its size is bigger than the Agave tequilana female gametophyte development the rest of the cells in the tetrad with dimensions of where Escobar-Guzmán et al. (2008) reported that the about 33 μm long and 23 μm wide. megagametophyte is originated from the megaspore that is located closest to the chalazal region, forming an em- Megagametogenesis bryo sac of seven cells (Polygonum-type). However, these The process of megagametogenesis starts with the increase authors did not report or describe the whole mitotic div- in size of the FM, which is about 43 μmlongand25μm ision process that gives rise to this sac with seven cells, wide.Atthisstage,thehypostasestartstobevisiblejust nor the early embryogenesis. Even though there is infor- above the FM in the nucellar tissue; this structure was de- mation regarding to some aspects related to the megaga- tected as a well defined and intensely stained group of cells, metogenesis of A. tequilana, there is no published which have a poor cytoplasmic content and thickened cell information describing the embryo and endosperm de- walls (Figure 3a) (also see Additional file 2: Figure S1). velopment in this species and the distinctive morpho- The first mitotic division of the functional megaspore logical changes of the embryo sac after fertilization. produces two nuclei, one located at the chalazal pole and The objective of this work was to study and characterize the other one at the micropylar pole, both separated by a the process of megasporogenesis, megagametogenesis, the large vacuole in the center of the embryo sac without mature embryo sac formation and the early embryo deve- cytokinesis. At this stage, the mean size of the embryo sac lopment in Agave tequilana Weber which is the raw ma- is about 50 μmlongand35μm wide (Figure 3b). Later, terial for the production of Tequila in Mexico, in order to these two nuclei divided again forming four nuclei, two lo- get basic knowledge useful for plant systematics and evo- cated at the chalazal end and the other two at the micro- lution studies and plant breeding programs, which may in- pylar end of the embryo sac (Figure 3c). At the same time, clude in vitro fertilization and the production of haploid the embryo sac continues expanding its size to about plants among others. 60 μmlongand42μm wide, and in both stages of embryo sac development its morphology is ovoid. A third and last Results mitotic division gives place to an eight-nucleated embryo A total of 5,000 ovules were taken from floral buds at di- sac (Figure 3d). These three mitotic divisions occur in a verse stages, receptive flowers and immature fruits were synchronized manner at both extremes of the embryo sac. analyzed. Different developmental stages of the collected ovules were studied from megasporogenesis to the first The mature embryo sac division of the embryo. The plant material used in this The study of mature embryo sacs was carried out in study consisted of inflorescences collected from May to ovules from flowers with mature and receptive stigma. June in the years 2010–2013 (Figure 1). It was difficult At the mature stage, the size of the embryo sacs is about to know the specific timeframe of each developmental 247 μm long and 106 μm wide and they are wider at the stage of the embryo sac, however, it could be observed chalazal pole than at the micropylar end, having a bulb- that it takes about 15 days for the floral buds since their ous form with a small and narrow haustorial tube at the appearance to reach the maturity of the embryo sac. chalazal end which connects to the hypostase (Figure 4a). At this stage, the embryo sac is already cellularized and Megasporogenesis consists of seven cells: three antipodal cells situated at The process of megasporogenesis was observed using the chalazal haustorium, which degenerate rapidly (fre- ovules in different developmental stages. The first ob- quently only their residues in the form of three highly served stages showed a megaspore mother cell (MMC) stained spots could be observed, however, also non- differentiation from the nucellar tissue (Figure 2a). This degenerated antipodal cells could be observed); the MMC goes through a meiotic cell division that first re- central cell formed by two polar nuclei was observed just sults in a dyad and finally in a linear tetrad of haploid below the antipodals and they were beside each other, González-Gutiérrez et al.