| Sexual reproduction is as important as a link between past and future in the life cycle of plant. There are a number of areas in the research of plant generation, including pollination、fecundation and embryonic development. Tarocco Xinxi (C. sinensis Osbeck) originated from a superior clone selected from of fsprings of Tarocco nucellar, which was introduced from Italy, by the Citrus Research Institute (CRI),the fruits have some advantages as seedless、big、late-maturing、better quality and cold resistance and so on. However, in the present paper we study a little cultivation technique about Tarocco Xinxi, and there is few study about sexual reproduction; and we also discovered that more seeds could produced by artificial selfing which is different from natural selfing. Therefore, studies on sexual reproduction and understands the seedless mechanism is great significance in the academic both Tarocco Xinxi’s study and all of citrus’s reproductive biology, and also can guide production practice on Tarocco Xinxi. This investigation made Tarocco Xinxi to be the object, revealed sexual reproduction and seedless mechanism by studied male and female gametogenesis、process of pollination and fertilization、identification and genetic analysis from progenies and so on. Main results are as follows.Observe the development of Tarocco Xinxi anther with paraffin slice technique. It showed that there are 4 pollen sacs in the anther, and looks like a butterfly. anther chamber is make up of 4 layers tissues,and from the inside to the outside is the tapetum(1-2 layers cells)、the endothecium (monolayer cells) and the middle layer(2-3 layers cells), the outside sheet is the monolayer epidermis of the anther. Deferred de-generation or without completely of tapetum cells、the development is too early of microspores、some mononuclear microspores couldn’t develop had been found in pollen development may will Influence of microspores maturation.Observe the development meiosis process of pollen mother cells(PMC) of Tarocco Xinxi with the method of flame-drying protocol after enzyme digestion of cell wall was used to prepare meiosis chromosome.It showed that there’re plenty of abnormal condition in PMC, high frequency of univalent、trivalent and multivalent at diplotene/diakinesis; the laggard chromosomes at MetaphaseⅠ; the laggard chromosomes、bridge of chromosome and the unequal separating of chromosomes at anaphaseⅠ/Ⅱor telophaseⅠ/Ⅲ; dyad and triad at the tetrad. Some polyploidy phenomenon also has been found at PMC. However, the most obviously abnormal phenomenon is high frequercy of univalent、trivalent and multivalent at diplotene diakinesis, and even could not find a normal 9 pair of bivalents at metaphase I. Then, we could conjecture the synapsis abnormal is the main cause of low fertility of Tarocco Xinxi.Male gamete of Tarocco Xinxi can develop into matured pollen grain, and split into two sperm cell finally. That is male gamete of Tarocco Xinxi could complete the whole development process.Scanning the mature pollen with the Scanning Microscope(SEM), it showed that the normal pollen grains are long spherical(27μm×19μm) or subcircular(25μm×23μm). There have 4 germinal apertures, at polar view could see 4 germinal apertures and 4 flaps fissure circular, at equatorial plane could see long spherical or subcircular,1-2 germinal aperture, apertures are long-narrow, hole diaphragm are obvious; the aperture length is 18μm. Ornamentation on the surface is thinnet, and mesh are round, more shallow and dimegaly; murus is more flat and wide. It is N4P4C5 according to NPC classification system. At the same time, we also found a number of malformation pollen(48.3%), and classfied these malformation pollen as’the germinal furrow distribution abnormal type’、’the surface unequality collapse type’、’the dished type’、’the carambola type’. We conjecture these malformation pollen are result of abnormal of development early.Chromosome number of Tarocco Xinxi was checked with the method of flame-drying protocol after enzyme digestion of cell wall was used to prepare meiosis chromosome. The result shows that Tarocco Xinxi is diploid(2n=2x=18),and make sure seedless reason isn’t resulted by abnormal chromosome number. Chromosome number of pollen also was checked by flow cytometry,and found the content of DNA were same between Tarocco Xinxi and control group, and they are both showed normal distribution, show that seedless reason isn’t resulted by abnormal male gamete, and pollen’s almost are n gamete.The amount of pollen grains are 1.24 X 104 in single anther, a flower has 22～24 anthers, then the total amount of pollen grains are 2.7 X 105～3.0 X 105 per flower, is more less than varieties that has more seeds. There are 2 kinds of determin methods be used to test the pollen vigor, such as I2-IK and Exvivo-germination. The pollen vigor of the Tarocco are showed as 36.8% and 0.61%. We can think that the pollen vigor is 1664.08～1815.36, is lower than other has more seeds variteties. It’s belongs to the lower fertility pollen. We think it is a reason of seedless.Observe the development of Tarocco Xinxi embryo sac with paraffin slice technique. It showed that there are 10～11 ventriculars in the ovary, each ventricular have 4-6 ovules in two rows parallel to the longitudinal arrangement of inverted born in the ventricular. Mature ovule was Polygonum type, Constituted by the funicle, nucellus, embryo sac, and two layers of beads. In addition, there has embryo self development in the mature ovary. One of the reasons of Tarocco Xinxi seedless is that part of the female gamete sterility.The observation of anther dehiscence、stigma and pollen germination on the stigma of Tarocco Xinxi showed that only part of the anthers can release of pollen the day after flowering; higher stigma receptivity in 2 day before flowering to same day of flowering; the stigma mucus to be air-dried in 1 day after flowering, at the same time the receptivity of stigma become lower. In addition, the pollen germination test was observed, and showed that the amount of pollen germination on artificial pollination of the stigma is much higher than the natural pollination; and pollen tubes would reach the ovary after artificial selfing and hybridization. Generally speaking, although pollen fertility was low, it still could complete the process of pollination and fertilization. In addition, the result of natural pollination or artificial pollination on Tarocco Xinxi showed that the fruit of natural pollination was seedless (mean 0.33 seeds per fruit), but the fruit of artificial pollination had more 4 seeds. Therefore we can infer, under natural conditions anthers release of pollen is too late, and the stigma mucus to be air-dried is too fast can reduce the stigma receptivity. It’s the reason of the result that different methods of pollination, and also is the seedless reason of Tarocco Xinxi.In a word, the abortion of female and male gametophyte could affect the process of sexual reproduction of Tarocco Xinxi in some extent, however, the man reason of seedless is that anthers release of pollen is too late, and the stigma mucus to be air-dried is too fast.We got 3 triploid plants by crossing of Tarocco Xinxi×Morocco blood, the reason needs further study. |
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