**2.4 The general discussions concerning the appearance of AIC and AIC-derived embryo sac formation**

There are no differences observed between obligate sexual and apomictic plants until megasporogenesis in bahia grass. After megasporogenesis, however, different events are followed in embryo sac formation. Sexual ovules proceeded in a manner typical of the Gramineae family, i.e., functional megaspores divided and formed a mature 8- nucleate embryo sac as reported in *P. notatum* [6] and in *P. maximum* [20, 40]. In contrast, megaspore does not divide in apomictic ovules and becomes degenerated (**Figure 2**(2) and (3)). Consequently, AIC (2n) derived from nucellus tissue, different from megaspore (n) appeared, divided, and directly formed mature 4-nucleate embryo sac. Most of the embryo sacs contain one egg cell, one synergid cell and two polar nuclei. It is different from that reported in *P. maximum*, i.e., one egg cell, two synergids and one polar nucleus [20, 38]. Here, it was called as *Paspalum* type of 4-nucleate embryo sac. Koltunow [2] indicated that in *Hieracium* MMC (mother megaspore cell) and AIC appeared together. It is different from *Paspalum* reported here and *Panicum* [20]. That means apospory has different reproductive process in different species, genus, or families. In this study, however, when megaspore developed normally, AIC appearance was not observed. By the way, after AIC appeared megaspore coexisted with AIC for a movement, and finally, degenerated. In any case, once AIC appeared, megaspore did not develop. This also differs from *Panicum* type reported by Chen and Kozono [20]. AICs appearance stage is distinctly different from sexual ones, and AIC occurs only in apomictic varieties. So, the stage


**Table 1.** *Number of ovules with different types of embryo sacs at anthesis in apomictic varieties of* P. notatum*.*

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

could be considered as a stage related to apomixis gene expression. Here, we can set up a hypothesis that, AIC gene exited and usually waited for a chance to express, only when the megaspore gave out a signal not fulfilling its mission to form embryo sac. AIC appearance and the embryo sac formation also have an important evolution meaning to protect from any unforeseen happenings.

The earliest AIC that appeared in ovule always located in micropylar end, as the ovary grows, the later appeared are located along with the first AIC and being apart from it. To understand the mechanism of AIC appearance, we selected ovary length as an index and measured the ovaries when they were observed in different AIC appearance. From the range of ovary length, AICs do not appear together in same time. Instead, they seemed following a continuous course and appeared one by one during the period from megasporogenesis even to the first embryo sac maturity. According to the ovary length compared with the morphology of spikes, AICs appeared in the period of spike emerging to open at anthesis. With regard to the ovary length measured we could collect every stage of embryo sac to apply apomixis gene cloning. Sterile ovules with degenerated embryo sac appeared in both sexual and apomictic varieties based on the observation and quantitative analysis of ovary length. The ovary length of the ovary in which the first AIC appeared was longer than that of ovary the functional megaspore appeared in all varieties, indicating that the aposporous phenomenon of AIC appearance is initiated after megasporogenesis. Further, the ovary length of the ovary staged in functional megaspore was wide and close to the ovary lengths of the ovaries showing degeneration of megaspores in different developed stages. These results indicated indirectly that the development of sexual embryo sac derived from megaspore is often terminated accompanied by AIC appearance in many aposporous apomicts around the stage of megasporogenesis [41, 42]. Which one of megaspore or AIC firstly showed the signal to terminate or to appear will be interesting to further researches of apomixis. In the present study, 10 types of embryo sac formation were observed in *Paspalum* notatum (**Table 1**). Here we must issue that "S" types (sexual embryo sacs) observed were almost 4- or 5- nucleate embryo sacs. Except S type, the ovules with over 2S types contain only the same, 8-nucleate ones were not observed. For the case of ovules containing one, two or more 4-, 5- (8) nucleate embryo sacs in one ovule, two pathways could be considered as follows. 1) The sexual embryo sac formation results from the direct division of one, two or over two megaspore(s) though the AIC(s) appeared (or not) in the same ovules. 2) They are derived from AIC(s). In particular, as the ovules with two megaspores in chalazal end were not observed in this study while AIC(s) appeared in the micropylar end, the former pathway could be hardly considered as a putative one. So, the later pathway seems reasonable based on that AICs develop into not only 4-nucleate [39] but also, at a low frequency, 5- nucleate embryo sacs in *Panicum* [38], or 8-nucleate ones in *Hieracium* [42]. In *Panicum*, the 4- nucleate embryo sac formation with an egg cell, one synergid and two nuclei, was reported by Bashaw and Hanna [37]. And 5- nucleate one with an egg cell, two synergies and two nuclei reported by Nakajima and Mochizuki [38]. For the mechanism of 5-nucleate embryo sac formation in *Panicum*, Chen and Kozono [20] set up a hypothesis. That is, after megaspore or AIC, whether which is located in micropylar end or not, divided firstly into two nuclei, only the micropylar nucleus continued to divide twice secondly and to form 4-nuclei, and in final, 5 nuclei formed totally in an embryo sac. For the distribution of the 5 nuclei, the chalazal nucleus derived from the first division of megaspore or AIC, and one of four micropylar nuclei derived from the second division of megaspore or AIC, pair with each other to form two polar nuclei, and for the remaining three nuclei, one becomes

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

one egg cell and two being synergies. From the above reports, we could conclude that facultatively apomictic bahia grass prefers to produce *Paspalum* type 4-(or 5-) nucleate (one egg cell, one (or two) synergid(s), and two polar nuclei) rather than to produce *Panicum* type 4-(or 5-) nucleate (one egg cell, two synergids, and one (or two) polar nucleus). Especially, there may be no *Polygonum* type (8-nucleate) embryo sac in polyembryonic ovules. Why did the two different apospory reproductive processes in the same 4-nucleate types of *Paspalum* and *Panicum* occur? This question means what should be clarified in the next future experiments.
