**2. The mechanism of AIC appearance and its development**

#### **2.1 The process of AIC appearance**

Four apomictic bahia grass varieties and two obligate sexual varieties were chosen for this study. These materials were kindly provided from Osumi breeding branch, Kagoshima Prefecture Agricultural Experiment station, Japan. For the 2 obligate sexual materials, Nangoku and C 1, the former is a diploid (2n = 20) variety [31], and the latter is a diploid (2n = 20) trace. About four apomictic varieties, they are tetraploid [32].

One hundred to over 300 buds or flowers before and at anthesis were collected for each variety for embryo sac analysis. The buds and flowers were fixed in FPA50 (formalin propionic acid: 50% ethanol = 5: 5: 90) for 5–7 days at 4°C [20, 21, 33, 34]. Ovaries were picked out carefully from fixed buds or flowers under microscope by using needle and tweezers, placed in 70% ethanol followed dehydration series (70, 80, 90, 100% ethanol), and cleared in Herr's benzyl-benzoate-four-and-a-half fluid [35] for over 2 h at 0–4°C. The observations were conducted using DIC.

The frequencies of apospory and sexual were calculated at anthesis according to the schematic of **Figure 1** [36]. Here, apospory was classified into two types, *Panicum* type (PN) and *Paspalum* type (PS). PN type represents 4-nucleate embryo sac with one polar nucleus, one egg cell and two synergids [20, 37–39]. PS type represents 4-(or 5-) nucleate embryo sac with two polar nuclei, one egg cell and one (or two) synergid(s). Sexual means *Polygonum* type (S) 8-nucleate embryo sac with one egg cell, two polar nuclei, two synergids and three antipodals [40]. Therefore, the frequency of apospory in this study was estimated according to the total percentage of PN and PS types.

Until megasporogenesis there showed similar behaviors in both sexual and apospory varieties. After megasporogenesis, however, different events from sexual varieties were observed in apospory varieties. While the formed megaspore became almost unfunctional and degenerated with the membrane disappeared, AIC-derived from enlarged unreduced nucellar cells appeared from a different direction and usually entered the space around the degenerated or surviving megaspore (**Figure 2**(1), [36]). In **Figure 2**(2) and (3), there showed coexistence of AIC and degenerated or surviving megaspore.

#### **2.2 The process of AIC-derived aposporous embryo sac formation**

In general, the AIC forms the embryo sac through a special process. The AIC undergoes mitosis two times and forms 2-, 4-nucleate. And no antipodal was found (**Figure 2**(4)). The cell division only occurred in the half-space of the embryo sac in the micropylar end. It is different from the sexual one which usually occupied the whole space of the sac. And then, the 4 nucleates developed to complete their parts in order (**Figure 2**(5)). In common, a mature embryo sac formed with one egg cell, one synergid and two polar nuclei (**Figure 2**(6)). In some rare cases, *Panicum* type 4-nucleate embryo sac with one polar nucleus was observed. In most ovules multiple apomictic embryo sacs were observed from one to six (**Figure 2**(7)). The distinctive features of mature embryo sacs were observed. 1) There is one egg cell, the nucleolus, ca 8 μm in diameter, being visible and surrounded with bright starch grains. With which sometimes the nucleus and its membrane could be distinguished, and the

#### **Figure 1.**

*Schematic representation of embryo sac development during sexual gametogenesis and the apospory forms in*  P. maximum *and* P. notatum.

cytoplasm was denser than the other cells. 2) In most of the embryo sacs, there is one synergid cell with the nucleolus, ca 4 μm in diameter, usually observed. And the cytoplasm was very few and the cell was occupied with bigger vacuoles. Moreover, near the synergid filiform apparatus usually were observable. 3) Two polar nuclei were almost observed with the nucleolus, ca 12 μm in diameter, surrounded by a nuclear membrane, and the cytoplasm was few; 4) No antipodal. The appearance of filiform apparatus is also evidence of embryo sac maturity in both of sexual and apomictic plants. The nucleoli are stained deeply with the clearing fluid, so that we can easily

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

#### **Figure 2.**

*Aposporous embryo sac initial cell (AIC) appearance and AIC-derived embryo sac maturity in apomictic bahia grass (P. notatum). (1) AIC appearance (big arrow) and degenerating megaspore remained (small arrow). Big arrow indicates AIC derived from nucellar cells, with a pointed (sharp) cell wall inserting into the space where only one megaspore without nuclear membrane remaining. 2 and 3 are 2 focal planes of same specimen. (2) Megaspore degenerated (small arrow). (3) Functional AIC formation (big arrow) with a circular cell wall in chalazal end in one ovule. (4) Four-nucleate aposporous embryo sac formation after AIC underwent two times of division. The nucleates were gathered up from each other in the micropylar end. (5) Differentiation of four-nucleate nuclei, an egg cell firstly formed (e, one synergid cell (s) and two polar nuclei (p). (6) Aposporous embryo sac maturity with one egg cell (e, two nuclei (p) and one synergid cell (usually invisible) in micropylar end. (7) Polyembryonic ovule containing three aposporous embryo sacs marked with single, double and third arrows, respectively. e: egg cell; p: polar nucleus; s: synergid cell; mi: micropylar end. Bar = 30 μm in Fig. 2(1)–(3), Bar = 45 μm in Fig. 2(4) and (5), Bar = 25 μm in Fig. 2(6) and (7).*

distinguish the stage of embryo sac formation by counting the numbers of nucleoli. Apomictic embryo sac in the micropylar end, matured about one day before anthesis, but in sexual varieties embryo sac did at the day of anthesis.

In most ovules of apomictic plants, AICs appeared continuously as the ovary length increased. The numbers of AICs were increased between the formations of functional megaspore and mature 4- or 8-nucleates according to the ovary length. For example, the ovary length in "competitor " was ca 520 μm when the first AIC appeared, and ca 496 μm when the third one did. However, 5 AICs appeared between 520 μm to 624 μm (688 μm) in ovary length. So were the other varieties. These values of ovary length indicated that AICs in the same ovule did not seem to differentiate synchronously.

From the length of ovaries, the stages of the ovary containing degenerated embryo sac were from 4-nucleate to their embryo sac maturity in apomictic plants, and from 4- and 8-nucleate to embryo sac maturity in sexual plants, respectively. On the other hand, the stage of functional megaspore showed a range of ovary length so wide it became very close to the value of degenerated ovaries.
