**1. Introduction**

Apomixis provides a method for cloning plants through seeds, so that it is of value for agriculture used to fix hybrid vigor and other hopeful breeding materials that are positioned in the middle breeding process [1, 2]. Apomixis is usually classified into three major mechanisms, apospory, diplospory and adventitious embryogeny [1]. Among the three mechanisms, apospory is considered as the most important one in agriculture because it does not undergo meiosis to propagate through the seed. Bahia grass (*Paspalum notatum* Flugge) is an apoamphimictic perennial that sets seed by apospory, a form of gametophytic apomixis [3–6]. Studies on chromosome, cytology, cross-compatibility, and colchicine treatment have been done in bahia grass [7–9]. However, studies in the field of molecular level have not been developed yet and still need more research.

Recently, some challenges using differential interference-contrast microscopy (DIC) technology have been conducted in all organs of plants. For example, in the meiotic chromosome [10], in petal development and ethylene biosynthesis [11], in nucleolus morphological changes [12], in pharmacology and cell biology [13], in xylem differentiation [14], in single microtubules [15]. In particular, near field DIC provides the ability to illuminate two neighboring points on the sample simultaneously, which shows that by modulating the two wavelengths employed in exciting such a probe, phase difference information can be retrieved through measuring the near field photoinduced force at the difference of the two modulation frequencies [16]. And more, two phases (cell structure and fluorescence) that appeared concurrently in the same sample and could be observed in *ASG-1* transgenic rice [17], and *Arabidopsis* (Chen et al. in contribution) while using the general DIC system. From the above description, it is understood that DIC shows a bright future for the clarification of not only the structures but also the mechanisms, and not only in plants but also in animals, as well as the microbes.

We choose bahia grass as a monocotyledons species as that would be particularly amenable to a molecular study of apomixis. It is shorter a plant and easily to cultivate among the important forage grasses. Recently, the somatic embryogenesis and plant regeneration system of bahia grass has been established in developing gene introduction techniques [18]. To clarify the molecular process controlling apomixis in *P. notatum*, it is important to determine the developmental timing and location of apomictic events in suitable laboratory strains that are currently available. It is a fact that the timing of apomictic gene expression, the key to cloning the apomixis genes has not been identified in bahia grass. Recently, it is reported in guinea grass (*Panicum maximum*) analyzed ultrastructurally and cytologically by DIC and Transmission electron microscopy (TEM) that, aposporous embryo sac initial cell (AIC) appearance is related with the increasing of ovary length [19–21]. And based on the ovary length as an index, AIC-specific clones have been obtained named as Apomixisspecific gene-1 (*ASG-1*) [22–24]. In the other species, molecular approaches to apomixis research have also been reported, i.e., *Citrus aurantium* [25], *Arabidopsis thaliana* [26], *Brachiaria brizantha* [27], *Tripsacum* [28], *Pennisetum* [29]. That bahia grass has or has not the same mechanisms should be understood for the molecular studies. In *P. notatum*, Quarin [8] reported the method to observe the effect of pollen source and pollen ploidy on endosperm formation and seed set in pseudogamous apomict. However, an efficient embryo sac analysis method cannot be found in bahia grass. Therefore, it is essential for the analysis of genetic and breeding in apomixis that the mechanism of embryo sac formation in apomict gets clear using an efficient analysis method [30].

In this study, the major objectives were, using the microscopy method of DIC I) to make it clear the cytological and quantitative observations of AIC appearance and its development in bahia grass, and to estimate the period of AIC appearance using ovary length as an index; and II) to clarify the process of polyembryonic seed set in facultatively apomictic ovules, and to provide information for estimation of the degree of apomixis or sexual of *P. notatum*. And the multiple embryo formation and the balance of maternal and parental to endosperm formation were also discussed.
