**3.3 The mechanism of seed forming embryo development in polyembryonic embryo sacs**

At 6 DAA, the developed embryo and endosperm of the sac in micropylar end occupied the embryo sac it located in, and at the same time, the space of the whole ovules was almost occupied by the developed sac (**Figure 3a**). The embryo usually exists with near globular shape, and it is surrounded by well-developed endosperm. For the embryo sacs in the other ends, they usually were squeezed out to outside of the developed sac. However, those sacs showed continuous development. Some egg cells divided well, and formed embryos usually located in opposite side to micropylar end (**Figure 3a**), or the neighbor (**Figure 3b** and **c**). At 10 DAA, the formed 2 embryos showed the same morphology (**Figure 3c**), and it is difficult to distinguish their origins between them. From **Table 3** [36], we can find that about 4–17% of ovules observed contain two or more embryos in the same ovule.

In emasculated ovules observed at 4 DAA, no developed embryo sac distinguished in 60 ovules of 4 varieties. The parthenogenesis rate was 0% in all the 4 varieties. At 15 DAA, the inflorescences that were emasculated and then isolated from any pollen source failed to produce seed. So, pseudogamous is essential for seed set in *P. notatum*.

## **3.4 The general discussion concerning the dominant development of the sac in micropylar end and seed formation by parthenogenesis**

In facultative apomictic bahia grass, AICs appeared one by one, and then, they became multiple embryo sacs in same ovule, as the ovary length increased [36]. And as the first AIC usually appeared and located in the micropylar end, 92.5 to 100% of embryo sacs closest to micropylar end of ovule matured at anthesis observed in this study (**Table 2**). On the other hand, the embryo sacs located in other end showed 40.4 to 86.0% mature rates at anthesis. The AIC appearance age (order) maybe influence

#### *The Cytological Mechanism of Apospory in* Paspalum notatum *Analyzed by Differential… DOI: http://dx.doi.org/10.5772/intechopen.104575*

the mature of apomictic embryo sacs themselves. It could be considered that the first appeared AIC in micropylar end has the temporal dominant in formation and maturity of the embryo sac when compared with the other sacs. And for the fertilization chance, the sac has also the positional dominant, as it was closest to synergid cell through which pollen tube penetrates and finishes fertilization. Therefore, the sac derived from first AIC located in micropylar end has the advantage of fertilization. On the observations of ovules at 4 DAA, the rates of developed embryo sacs with embryo and endosperm were from 56 to 87% in the sacs of micropylar end (**Table 3**). On the other hand, the other sacs were 0% in 3 varieties, and one variety was 3%. Therefore, the sac in micropylar end has the advantage of seed set. This result also supported the hypothesis that the embryo of developed sac in micropylar end, in final, became a seed-forming embryo [20]. Using this method, we can estimate the degree of apomixis or sexual of any facultative apomictic materials used, based on the analysis of embryo sacs in micropylar end at anthesis.

Different events were observed on the seed set between guinea grass and bahia grass. In guinea grass, the other embryo sacs were crowed out to the chalazal end by the developed micropylar sac, and in final, they were completely degenerated after 10 DAA [21]. In contrast, the rates of embryos formed in the other embryo sacs were 4–17% in 4 accessions of bahia grass used in this study. These are higher than that (0%) in 5 accessions and that (2%) in one accession of guinea grass [21]. This evident was also observed from embryo sac analysis (**Figure 3**). As the sac derived from AIC contains 2n level reproductive cells, egg cell does not need fertilization. However, for the endosperm formation, fertilization between central cell and sperm cell is needed. And egg cell usually starts division followed the endosperm cell formation. The other sacs also follow the same manner. As the embryo sac developed advantageously in micropylar end, it could be considered that the egg cells in the other sacs were developed vigorously in different places of ovules. In that case, only embryo formed but no endosperm. In the polyembryonic ovules, the embryos located in the other sacs usually presented close to the well-developed endosperm of the micropylar sac. The 2 kinds of embryos in the same ovule seemed sharing the endosperm of the micropylar sac. Maybe that is why the embryos in different embryo sacs could coexist in the same ovule. At the germination experiment, twines, or multiple seedlings (>5%) were observed (data not shown). This means the different embryos have the same germination capacity. For the endosperm balance number, some reporters have discussed the requirement for balance between maternal and paternal contributions to the endosperm formation [44–48]. When they used different ploidies and sexual materials, endosperm balance in terms of maternal to paternal ratio, 2:1 was considered balanced and should produce normal endosperm. And they indicated that unreduced embryo sacs with one central-cell nucleus (4 factors), when fertilized by a sperm (2 factors), would result in the proper endosperm ratio of 2m:1p, and the closed percentages were obtained between embryo sacs with a single central-cell nucleus and ovaries with endosperm developing 6 and 8 d after pollination. The result of aposporous guinea grass reported by Chen and Kozono [21] also supported the above explanation. In grasses, the most common type of unreduced embryo sac is the 4-nucleates *Panicum* type (PN) developing into one egg, two synergids, and one polar nucleus or, more rarely, one egg, one synergid, and two polar nuclei [2, 42]. In *Paspalum notatum*, Chen et al. [36] reported that the percentages of unreduced embryo sacs with 2 central-cell nuclei (PS) were 60.7–90.0%, and that with one central cell nucleus (PN) were 5.0–30.3% in 4 varieties observed, respectively. However, the percentages of ovaries with endosperm developing 4 DAA

were 56–87% in the same 4 varieties in this study. If we follow the report of Morgan et al. [47], only 5.0–30.3% of developed endosperm should be obtained. Recently, Quarin [8] reported the related information about the endosperm balance number that, apomictic 4x *P. notatum* is a pseudogamous species with effective fertilization of the 2 unreduced (2n = 4x) polar nuclei by a reduced (n = 1x) sperm. In that case, endosperm development and seed set occurred independently in the species. In that case, endosperm development and seed set occurred independently of the species or the ploidy level of the pollen donor. In his explanation, as sexual *Paspalum* plants fit the endosperm balance number (EBN), the EBN insensitivity is observed in apomictic apomixis. The EBN insensitivity could have arisen as an imprinting consequence of a high maternal contribution.

Recently, the mechanisms of reproductive characterization [49], molecular and genetic regulation [50–52], and its utilization [53, 54] have been discovered consequently in *P. notatum*. Together with the clarification of the mechanisms of aposporous embryo sac initial cell appearance and the cell-derived aposporous embryo sac formation descripted, here, in *P. notatum* and previous and similar report in *P. maximum* [19], this study will provide the essential and important information for successful cloning of apospory genes in *P. notatum*. And therefore, the *P. notatum* as one of the main players will be chosen in apomixis research and give another extension for the breeding program and productive utilization in agriculture and forage grasses.
