Pollen tube - pistil interaction and fertilization in Lilium longiflorum

In this thesis the interaction between pollen tube growth and the pistil with the subsequent fertilization was studied both in intact flowers and after different flower manipulations and in vitro pollination. light and electron microscopy and electrophoresis were used.To achieve interspecific crosses in lily the cut-style pollination, in which the style is cut off just above the ovary and pollen grains are applied at the cut surface, is used. Just a few ovules are penetrated by a pollen tube. In an intraspecific compatible combination the penetration percentage of the ovules after cut-style pollination is however low as well. Complications occur in the interaction between the pollen tube and the ovule. It might be that the ovules lack an enhancement which takes place during pollination or germination at the stigma or during pollen tube growth in the style, which is absent after cut-style pollination.In chapter 1 a review is given of compatible, incompatible and interspecific pollen tube growth in Lilium, the guidance of the pollen tube and other aspects of the interactions between the pollen tubes and the pistil. Following this chapter interactions are first studied in an intact system of Lilium longiflorum .In chapter 2 the exudate production in the pistil, embryo sac development and pollen tube growth in L. longiflorum is studied and related to flower bud length and flowering stage. The exudate production on the stigma and in the style starts before the bud opens, as determined by cryo scanning electron microscopy. Just underneath the stigma the exudate first accumulates at the top of each secretory cell, followed by a merging of those accumulations as exudate production proceeds.After germination the pollen tubes grow across the stigma and enter the style in between the three stigma lobes. This growth over the stigma seems at least at first not directed. In the style the pollen tubes grow straight downward at a constant speed and are covered by exudate. As the pollen tube bundle reaches the ovary, the secretory pathway and thus also the pollen tube bundle is divided into three ovarian cavities. Hereby they spread out, but their growth is restricted to the area with secretory cells. The secretory cells covering the placenta are similar to those present in the stylar canal, although their surface shape is more spherical rather than elongated as in the style. The transfer wall of the placenta] cell is originating from fusing Golgi vesicles but the endoplasmic reticulum (ER) seems to have an important role as well.In between the two rows of ovules in one ovarian cavity a pollen tube bundle is formed in the exudate produced by the placental cells. After neglecting the first few ovules the pollen tubes bend from this bundle in between the ovules and grow towards the micropylar side. There they bend again to stay close to the secretory cells. At anthesis a part of the embryo sacs are in their seven-nucleate and six-cellate (the cell walls in the embryo sac were hard to detect after clearing) stage, i.e. mature. Penetration of the pollen tubes into the micropyle has only been observed in these ovules. About 8 days before anthesis exudate is observed in a flower bud. Pollen tube growth in the style is possible from seven days before anthesis. The pollen tube growth is then however strongly retarded compared with the pollen tube growth in a flower at anthesis. It seems that some pollen tubes are not covered by an exudate layer. Newly appearing pollen tube tips have a tendency to grow close to the secretory cells, resulting in a growth between these cells and preceding pollen tubes. If there is still little exudate produced it results in a lifting up of the pollen tubes, out of the exudate.The development of the embryo sacs in sector two (sector one is at the top and four is at the basis of the ovary) is ahead of the embryo sacs in the other sectors of the ovary. Pollination or pollen tube growth did not influence the development of the embryo sacs. When the pollen tubes formed after pollination of a flower bud finally reach the ovary, a part of the ovules have matured. From four days before till seven days after anthesis, pollination results in penetration of the ovules. The protein pattern of the ovules observed after electrophoresis did not show a consequent change in the period from anthesis till 9 days after anthesis or 7 days after pollination.In chapter 3 the ultrastructure of the embryo sac, the nucellus and parts of the micropyle of L.longiflorum is studied both before and after pollination. Before pollen tube penetration the three cells of the egg apparatus cannot be distinguished, neither in structure nor in their position. No filiform apparatus was detected, no degeneration of a synergid occurs without pollen tube penetration. The polar nuclei in the central cell do not fuse until fertilization. The metabolic activity of the cells of the egg apparatus and the central cell seems low. The nucleus of the most chalazal of the two antipodals has an irregular shape and in some embryo sacs a third antipodal cell, small in size and without a nucleus, is present. Pollen tube growth does not induce changes in the embryo sac.When the pollen tube arrives at the nucellus the cuticle surrounding this nucellus is lifted up. Enzymatic digestion of the cell wall of the at this place one cell layer thick nucellus has to take place to create a pathway for the pollen tube to enter the embryo sac. After entering the embryo sac, the pollen tube grows along the inside of the nucellus and finally penetrates one of the three cells of the egg apparatus, now distinguished as the degenerated synergid. Shortly after fertilization two enucleate cytoplasmic bodies of a different ribosome density were observed in the degenerated plasma of the synergid and the pollen tube. These structures border both the central cell, the egg cell and each other and are most likely the two empty sperm cells. The sperm nucleus in the central cell is probably transported by ER and first makes contact with the haploid polar nucleus which is, as the triploid polar nucleus, connected with ER as well. In the egg cell another process is more likely, because here strands of ER were not observed. Here the nuclei line up before fusion. The cells of the embryo sac become more metabolic active after pollen tube penetration. In this chapter an attempt is made to relate ultrastructure to function and processes.In chapter 4 the pollen tube growth in the ovary after cut-style pollination was observed with scanning electron microscopy. Different flower manipulations were carried out in an attempt to elucidate the interaction between the pollen tube growth and the pistil. Until the arrival of the pollen tube at the inner integument, the pollen tube growth did not show any difference between cut-style and stigmatic pollination, as studied in chapter 2. Using cut-style pollination the pollen tubes either grew past the inner integument and ignored it, or grew along but not into the micropyle or penetrated the micropyle.Grafting a stigma just above the ovary did not influence the penetration percentage, nor did a possible activation of the ovary induced by pollination or pollen tube growth in the style or even in the ovary itself, preceding or during cut-style, interstylar or placental pollination in a pistil. The percentage of penetration after cut-style pollination increased however when the stylar part present at the ovary was left longer.The presence of the ovary did not influence the pollen tube growth in the style as determined after isolation of styles from the ovary and comparing the pollen tube length.When pollen grains and stigmatic exudate were applied through a slit half-way down the style of an intact pistil the pollen tube growth was not influenced by a simultaneous pollination at the stigma.In chapter 5 placental pollination was carried out predominantly with L.longiflorum to study the interaction between the pollen tube and the placenta with ovules. The percentage of penetrated ovules is low when compared with compatible pollination at the stigma. After placental pollination the pollen tube growth between the ovules seems directed and the pollen tubes do find the inner integument. A reaction to the inner integument or the micropyle is observed, but hardly results in ovule penetration. Embryos were found, but did not develop vigorously. The similarities and differences with cut-style pollination, in which the percentage of ovule penetration is also low, are discussed. Grafting a style with pollen tubes to the placenta increased the penetration percentage obtained after placental pollination five times.In chapter 6 the data from chapter 2 and 3 are combined in a reproductive calendar.In chapter 7 the reproduction is considered as a regulated interaction process. Previous experimental results are considered as aspects of the system and are discussed in this context