4 DISCUSSION
The anther development in wheat fallows a pattern similar to other members of Poaceae family, including Brachypodium distachyon (Sharma et al., 2015) and rice (Zhang et al., 2006). Resemblances also exist in Arabidopsis thaliana (Chang et al., 2011) but some distinct differences were recorded in wheat anther development. The critical step in the anther development is the balance of cell sizes (width, length and depth) and the cell counts of EPI, EN, SPL, ML, TA, PMC, MP and PG cells. Therefore, we investigated the effects of T. laevis and T. controversa on above cell sizes and number on comparison with control anthers.
4.1 The timeline of cell development of normal wheat anther
In this study, we firstly construct the timeline of wheat anther development using confocal microscopy. The AR cells appear from L2 layer of anthers < 150 µm and differentiate into EN, SPL and PMC cells of anthers. In this range, vascular tissues developed more clearly. The SPL cells differentiate into ML and TA almost in 400 µm long anthers. PMC cells differentiate into Tds almost in 700 µm anthers, where ML start to be wrinkled between TA and EN cells and disappear almost in 1100 µm anthers. Tds start to change into MP almost from 720-1000 µm anthers. Here ML is scarcely present, and TA is at its most swollen and largest size. However, near to 1200 µm, MP become vacuolate towards the TA walls. An exine layer start to develop on the MP and TA began to reduce in size (Browne et al., 2018). At 1300 µm, TA cells disappear, and microspore grow large and began to change in PG cells. At 2300 µm, mature PG formed and which are starch filled. EPI cells at stomium start to degrade, causing locules to open and PG start to be released (Figure 1 ). RAFTIN1 and TaMYB80 play a crucial role in TA development and TA program cell death (Gao et al., 2013; Wang et al., 2003; Murray et al., 2003; Millar et al., 2005). We approved the anther timeline by analyzing the expression of TA development and TA program cell death in different anther length. Results showed that the expression of RAFTAN1 was high in 200-900 µm anther compared to other sized anthers. While, TaMYB80 showed maximum expression in 1100-1300 µm anthers. The expression results suggested that TA start to develop from 400 µm anthers and almost disappear in 1300 µm anthers (Figure S1 ). Confocal results showed that PMC cells were surrounded by three somatic cell layers: namely EPI, EN and SPL from interior to outside (Figure 2B ). Except EPI, the three inner cells were differentiated after mitotic division in AR cells. Hence, earliest and most important, steps in anther development are the production of AR cells. The final structure results in the PMC enclosed by a serious of somatic cells, which are EN, SPL and EPI (Figure 2B ). Finally, after meiosis PMC cells differentiate into PG almost from 1300 to 2300 µm, which released from pollen sac by opening the stomium (Figure 2I ). However, according to Browne et al. (2018), AR cells generated primary parietal cells (PPC) and primary sporogenous cells (PSC) after mitosis. Then PPC divide into EN and ML and PSC divide into PMC cells, which produce Tds. In our results, AR cells directly differentiate into EN, SPL and PMC cells and then SPL cells divide into ML and TA and PMC cells divide into Tds that differentiate into MP and PG in short time. Moreover, the difference in wheat anther development was noted from model plant, Arabidopsis thaliana (Chang et al., 2011). However, according to Zhang and Wilson (2009) the EN, SPL and PMC cells differentiate from AR cells then it differentiate into other cells.