‘香榧’雌配子体发育和原胚形成的组织学观察
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摘要
【目的】明确‘香榧’授粉到越冬前雌配子体的发育、受精和原胚形成过程,丰富和完善榧属植物的生殖发育学。【方法】人工授粉后,定期连续采集雌球花,应用常规石蜡切片技术,获得‘香榧’雌球花的纵切片,在光学显微镜下观察。【结果】‘香榧’雌球花由4层鳞片包被,由外向内依次为假种皮、珠被和珠心。珠心中下部分化雌配子体。‘香榧’在授粉期尚处于大孢子母细胞时期;授粉后1周内,完成减数分裂形成功能大孢子;6月初到8月底完成雌配子体的发育,包括游离核时期、细胞化阶段和颈卵器时期;‘香榧’有2个颈卵器母细胞,每个颈卵器分裂形成4个颈细胞和1个卵核,腹沟核很快消失。9月中上旬精卵融合形成合子并发育形成原胚,排列成4层,并以原胚越冬。【结论】‘香榧’的雌配子体发育过程基本上遵循了红豆杉科植物的发育规律,只在原胚类型和颈卵器数目上与其他种类有一定差异。‘香榧’原胚为松型,2个颈卵器,而红豆杉科其他种原胚为标准型,绝大多数仅1个颈卵器。
【Objective】The purpose of this research was to determine anatomically the megagametophytedevelopment, fertilization process and proembryo forming in Torreya grandis‘Merrillii'during the peri-od 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 pro-embryo. 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 ac-quire 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.
【Objective】The purpose of this research was to determine anatomically the megagametophytedevelopment, fertilization process and proembryo forming in Torreya grandis‘Merrillii'during the peri-od 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 pro-embryo. 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 ac-quire 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.
引文
[1]康宁,汤仲勋.榧属分类学研究[J].植物研究,1995,15(3):349-362.KANG Ning,TANG Zhongxun.Studies on the taxonomy of the genus Torreya[J].Bulletin of Botanical Research,1995,15(3):349-362.
[2]黎章矩,戴文圣.中国香榧[M].北京:科学出版社,2007.LI Zhangju,DAI Wensheng.Zhongguoxiangfei[M].Beijing:Science Press,2007.
[3]WILLIAMS C G.Conifer reproductive biology[M].Springer Science&Business Media,2009.
[4]王莉,陆彦,金飚,林明明,陈鹏.银杏雌雄配子体发育及胚胎形成的研究进展[J].植物学报,2010,45(1):119-127.WANG Li,LU Yan,JIN Biao,LIN Mingming,CHEN Peng.Gametophyte development and embryogenesis in Ginkgo biloba:a current view[J].Chinese Bulletin of Botany,2010,45(1):119-127.
[5]COULTER J M,LAND W J G.Gametophytes and embryo of Torreya taxifolia[J].Botanical Gazette,1905,39(3):161-178.
[6]汤仲埙,陈祖铿,王伏雄.香榧有性生殖周期的研究[J].植物分类学报,1986,24(6):447-453.TANG Zhongxun,CHEN Zukeng,WANG Fuhsiung.Investigation on sexual reproductive cycle in Torreya grandis‘Merrillii’[J].Acta Phytotaxonomica Sinica,1986,24(6):447-453.
[7]YU X M,ZHAO G F.Female gametogeny,fertilization,embryogeny of Larix chinensis and it’s systematic significance[J].Acta Botanica Boreali-occidentalia Sinica,2004,4(6):1024-1034.
[8]王莉,王永平,汪琼,潘烨,金飚,徐小勇,陈鹏.银杏胚珠发育进程的解剖学研究[J].西北植物学报,2007,27(7):1349-1356.WANG Li,WANG Yongping,WANG Qiong,PAN Ye,JIN Biao,XU Xiaoyong,CHEN Peng.Anatomical study of development of ovule in Ginko biloba L.[J].Acta Botanica Boreali-Occidentalia Sinica,2007,27(7):1349-1356.
[9]陈祖铿,王伏雄.白皮松受精作用的研究[J].植物学报,1982,24(1):10-16.CHEN Zukeng,WANG Fuhsiung.Fertilization in Pinus bungeana[J].Acta Botanica Sinica,1982,24(1):10-16.
[10]SOGO A,TOBE H.Mode of pollen-tube growth in pistils of Myrica rubra(Myricaceae):a comparison with related families[J].Annuals of Botany,2006,97:71-77.
[11]SOGO A,NOGUCHI J,JAFFRE′T,TOBE H.Pollen-tube growth pattern and chalazogamy in Casuarina equisetifolia(Casuarinaceae)[J].Journal of Plant Research,2004,117:37-46.
[12]SOGO A,TOBE H.Intermittent pollen-tube growth in pistils of alders(Alnus)[J].Proceedings of the National Academy of Sciences of the USA,2005,102:8770-8775.
[13]郑万钧,傅立国.中国植物志(第七卷)[M].北京:科学出版社,1978.ZHENG Wanjun,FU Liguo.Flora of China(Ⅶ)[M].Beijing:Science Press,1978.
[14]胡志昂,王洪新,刘长江,潜忠兴.裸子植物的生化系统学(三)——从种子蛋白多肽和针叶过氧化物酶探讨红豆杉科的系统位置[J].植物分类学报,1986,24(4):260-263.HU Zhiang,WANG Hongxin,LIU Changjiang,QIAN Zhongxing.Biochemical systematics of gymnosperms(3)——on the systematic position of taxaceae from their seed protein peptides and needle peroxidases[J].Acta Phytotaxonomica Sinica,1986,24(4):260-263.
[15]陈祖铿,王伏雄.从胚胎发育看穗花杉属的系统位置[J].植物分类学报,1984,22(4):269-276.CHEN Zukeng,WANG Fuhsiung.On the systematic position of Amentotaxus from its embryological investigation[J].Acta Phytotaxonomica Sinica,1984,22(4):269-276.
[16]PENNELL R I,BELL P R.Megasporogenesis and the subsequent cell lineage within the ovule of Taxus baccata L.[J].Annals of Botany,1987,59:693-704.
[17]中国科学院植物研究所形态细胞研究室比较形态组.松树形态结构与发育[M].北京:科学出版社,1978:92-119.Institute of Botany of The Chinese Academy of Sciences.Morphological structure and development of Pinus[M].Beijing:Science Press,1978:92-119.
[18]陈祖铿,王伏雄.白豆杉的配子体发育和受精作用[J].植物学报,1979,21(1):19-29.CHEN Zukeng,WANG Fuhsiung.The gametophytes and fertilization in Pseudotaxus[J].Acta Botanica Sinica,1979,21(1):19-29.
[19]陈祖铿,王伏雄.穗花杉的胚珠结构与雌、雄配子体的发育[J].植物学报,1985,27(1):19-25.CHEN Zukeng,WANG Fuhsiung.The ovule structure and development of male and female gametophytes in Amentotaxus[J].Acta Botanica Sinica,1985,27(1):19-25.
[20]ANDERSON E D,OWENS J N.Megagametophyte development,fertilization and cytoplasmic inheritance in Taxus brevifolia[J].International Journal of Plant Science,1999,160(3):459-469.
[21]于永福,傅立国.杉科植物的系统发育分析[J].植物分类学报,1996,34(2):124-141.YU Yongfu,FU Liguo.Phylogenetic analysis of the family Taxodiaceae[J].Acta Phytotaxonornica Sinica,1996,34(2):124-141.
[22]王伏雄,陈祖铿.银杉的胚胎发育[J].植物学报,1974,16(1):64-69.WANG Fuhsiung,CHEN Zukeng.The embryogeny of Cathaya(Pinaceae)[J].Acta Botanica Sinica,1974,16(1):64-69.
[2]黎章矩,戴文圣.中国香榧[M].北京:科学出版社,2007.LI Zhangju,DAI Wensheng.Zhongguoxiangfei[M].Beijing:Science Press,2007.
[3]WILLIAMS C G.Conifer reproductive biology[M].Springer Science&Business Media,2009.
[4]王莉,陆彦,金飚,林明明,陈鹏.银杏雌雄配子体发育及胚胎形成的研究进展[J].植物学报,2010,45(1):119-127.WANG Li,LU Yan,JIN Biao,LIN Mingming,CHEN Peng.Gametophyte development and embryogenesis in Ginkgo biloba:a current view[J].Chinese Bulletin of Botany,2010,45(1):119-127.
[5]COULTER J M,LAND W J G.Gametophytes and embryo of Torreya taxifolia[J].Botanical Gazette,1905,39(3):161-178.
[6]汤仲埙,陈祖铿,王伏雄.香榧有性生殖周期的研究[J].植物分类学报,1986,24(6):447-453.TANG Zhongxun,CHEN Zukeng,WANG Fuhsiung.Investigation on sexual reproductive cycle in Torreya grandis‘Merrillii’[J].Acta Phytotaxonomica Sinica,1986,24(6):447-453.
[7]YU X M,ZHAO G F.Female gametogeny,fertilization,embryogeny of Larix chinensis and it’s systematic significance[J].Acta Botanica Boreali-occidentalia Sinica,2004,4(6):1024-1034.
[8]王莉,王永平,汪琼,潘烨,金飚,徐小勇,陈鹏.银杏胚珠发育进程的解剖学研究[J].西北植物学报,2007,27(7):1349-1356.WANG Li,WANG Yongping,WANG Qiong,PAN Ye,JIN Biao,XU Xiaoyong,CHEN Peng.Anatomical study of development of ovule in Ginko biloba L.[J].Acta Botanica Boreali-Occidentalia Sinica,2007,27(7):1349-1356.
[9]陈祖铿,王伏雄.白皮松受精作用的研究[J].植物学报,1982,24(1):10-16.CHEN Zukeng,WANG Fuhsiung.Fertilization in Pinus bungeana[J].Acta Botanica Sinica,1982,24(1):10-16.
[10]SOGO A,TOBE H.Mode of pollen-tube growth in pistils of Myrica rubra(Myricaceae):a comparison with related families[J].Annuals of Botany,2006,97:71-77.
[11]SOGO A,NOGUCHI J,JAFFRE′T,TOBE H.Pollen-tube growth pattern and chalazogamy in Casuarina equisetifolia(Casuarinaceae)[J].Journal of Plant Research,2004,117:37-46.
[12]SOGO A,TOBE H.Intermittent pollen-tube growth in pistils of alders(Alnus)[J].Proceedings of the National Academy of Sciences of the USA,2005,102:8770-8775.
[13]郑万钧,傅立国.中国植物志(第七卷)[M].北京:科学出版社,1978.ZHENG Wanjun,FU Liguo.Flora of China(Ⅶ)[M].Beijing:Science Press,1978.
[14]胡志昂,王洪新,刘长江,潜忠兴.裸子植物的生化系统学(三)——从种子蛋白多肽和针叶过氧化物酶探讨红豆杉科的系统位置[J].植物分类学报,1986,24(4):260-263.HU Zhiang,WANG Hongxin,LIU Changjiang,QIAN Zhongxing.Biochemical systematics of gymnosperms(3)——on the systematic position of taxaceae from their seed protein peptides and needle peroxidases[J].Acta Phytotaxonomica Sinica,1986,24(4):260-263.
[15]陈祖铿,王伏雄.从胚胎发育看穗花杉属的系统位置[J].植物分类学报,1984,22(4):269-276.CHEN Zukeng,WANG Fuhsiung.On the systematic position of Amentotaxus from its embryological investigation[J].Acta Phytotaxonomica Sinica,1984,22(4):269-276.
[16]PENNELL R I,BELL P R.Megasporogenesis and the subsequent cell lineage within the ovule of Taxus baccata L.[J].Annals of Botany,1987,59:693-704.
[17]中国科学院植物研究所形态细胞研究室比较形态组.松树形态结构与发育[M].北京:科学出版社,1978:92-119.Institute of Botany of The Chinese Academy of Sciences.Morphological structure and development of Pinus[M].Beijing:Science Press,1978:92-119.
[18]陈祖铿,王伏雄.白豆杉的配子体发育和受精作用[J].植物学报,1979,21(1):19-29.CHEN Zukeng,WANG Fuhsiung.The gametophytes and fertilization in Pseudotaxus[J].Acta Botanica Sinica,1979,21(1):19-29.
[19]陈祖铿,王伏雄.穗花杉的胚珠结构与雌、雄配子体的发育[J].植物学报,1985,27(1):19-25.CHEN Zukeng,WANG Fuhsiung.The ovule structure and development of male and female gametophytes in Amentotaxus[J].Acta Botanica Sinica,1985,27(1):19-25.
[20]ANDERSON E D,OWENS J N.Megagametophyte development,fertilization and cytoplasmic inheritance in Taxus brevifolia[J].International Journal of Plant Science,1999,160(3):459-469.
[21]于永福,傅立国.杉科植物的系统发育分析[J].植物分类学报,1996,34(2):124-141.YU Yongfu,FU Liguo.Phylogenetic analysis of the family Taxodiaceae[J].Acta Phytotaxonornica Sinica,1996,34(2):124-141.
[22]王伏雄,陈祖铿.银杉的胚胎发育[J].植物学报,1974,16(1):64-69.WANG Fuhsiung,CHEN Zukeng.The embryogeny of Cathaya(Pinaceae)[J].Acta Botanica Sinica,1974,16(1):64-69.