Histological studies on megagametophyte and embryogeny development in Torreya grandis‘Merrillii’

Abstract: 【Objective】The purpose of this research was to determine anatomically the megagametophytedevelopment, fertilization process and proembryo forming in Torreya grandis‘Merrillii’during the period from pollination to overwintering. It is noteworthy that gymnosperm differs from angiosperm is the inter⁃val from pollination to fertilization is longer than 3 months. Other characteristics are archegonium and proembryo. This is a basic research aim to observe these characteristics and find its special rules to differ fromother gymnosperms. It is also a research for more reproductive biology research, such as cytoplasm inheri⁃tance, meiosis relevant research.【Methods】Artificial pollination was done on April 24 of 2014 when pol⁃lination drop appears. Ovules were collected every 3 to 4 days from the middle of April to end Septemberin 2014. Length and diameter were measured and scales were removed. They were fixed in FAA (Vformalin∶Vglacial acetic acid∶V50% ethanol=5∶5∶90) at least 24 h, then hydrated in grated ethanol series, displaced by xyleneand then paraffin. Paraffin filled ovules were sectioned in 8 μm thick and placed in 37 °C about 12 h.Sections were dyed by safranine and fast green and observed with SEM.【Results】Structure of the ovuleincludes aril, integument, nucellus and megagametophyte. The aril develops its length in May mainly tosurround the nucellus for protection and formed micropyle for pollen entering into the ovule. About15 to 20 cells filled with tannins located on the inner side of the integument. Sporogenous tissue locatesbelow the middle of the nucellus. One of these cells differentiates the mega gametophyte and other cellsnourish it. The megagametophyte of T. grandis‘Merrillii’is still at megasporocyte stage during the polli⁃nation. One week after pollination, the megasporocyte undergoes meiosis and gives rise to functional mega⁃spore. Other three megaspores are small in volume and degenerated soon. As the first cell of megagameto⁃phyte, functional megaspore’forming means the beginning the megagametophyte development. The func⁃tional megaspore needs about one month to develop itself in the volume, nuclei size and cytoplasm abun⁃dance. From June to August, megagametophyte is fully developed experiencing free nuclear stage, cellular⁃ization stage and archegonium stage. The functional megaspore divides about eight times continually. Thedivision needs nutrition from spongy tissue directly. The spongy tissue is consumed as its volume is on thedecrease. Cellularization stage means free nuclei turned into cells with cell wall. The outer free nuclei acquire cell wall first, while the inner free nuclei last. Meanwhile, the megagametophyte forms its thick cellwall. The spongy tissue almost disappears. Moreover, T. grandis‘Merrillii’possess two archegonial ini⁃tials, each of which can divide into four neck cells and one egg cell while ventral canal cell disappearesquickly. Archegonium includes four neck cells and one egg cell. Neck cell can guide the pollen tube’s di⁃rection into the egg cell. Unfortunately, only two neck cells can be seen in the longitudinal cutting direc⁃tion. The pollen’s stored in a tapered place, which located in the end of the integument and the top of thenucellus. More than one pollen germinates and their trace can be seen clearly, as they move fast and ruinnucellus cells. Tip of the pollen tube stays at the top of the megagametophyte waiting for mature egg cell.It inflates much that formed a big cavity. Spermatophyte divides into two sperms and a sterile nucleus, atube nucleus. Around early to mid-September, a sperm fuses with the egg cell to form a zygote, which de⁃velops into proembryo in the form of four layers. When one egg cell combines with one sperm of two, theother one stopped. So we get only a zygote, the other egg cell stopped to develop and degenerated. In thisprocess, protein vacuole has a change in its volume, amount and location. Protein vacuole gives nutritionfor fertilization. We also find the change of the cytoplasm. After fertilization, cytoplasm around the zygoteturned dense. New cytoplasm means male cytoplasm may participate in the fertilization and cytoplasm in⁃heritance is worthy. Before next spring comes, T. grandis‘Merrillii’live through the winter in the condi⁃tion of proembryo. The proembryo includes 16 cells arranged in four layers, called standard proembryo.The zygote nucleus divides three times into eight nuclei. Eight nuclei acquired their cell walls and all di⁃vide one time. Diameter can measure stage of megagametophyte to some extend while length cannot clear⁃ly distinguish every stage. Diameter and length increase obviously in the stage of free nuclei and cellular⁃ization. And diameter has an obvious increase in the stage of megaspore forming, archegonial initials’divi⁃sion and fertilization, while length increase little compared the previous stage.【Conclusion】The develop⁃ment of megagametophyte in T. grandis‘Merrillii’mostly follows the development pattern of Taxaceae,only with certain differences in the proembryo type and the number of the archegonium. Unique character⁃istics of T.grandis‘Merrillii’include interval of four months from pollination to fertilization, two archego⁃nia and standard proembryo.