打碗花生殖细胞,精细胞及卵细胞中的细胞质类核

已有不少超微结构的资料阐明被子植物双亲和单亲母系质体遗传的细胞学基础。近年应用DAPI荧光染色的方法,可快速地从检测质体DNA存在的状况确定被子植物中具双亲遗传潜能的种。从质体的类核存在与否判断质体遗传方式为母系遗传或双亲遗传与已有的遗传分析结论基本一致,只有少数种类是矛盾的。DAPI荧光技术可以认为是研究细胞质遗传机理的一个重要手段。我们曾证明旋花科牵牛属植物生殖细胞、精细胞中存在细胞质类核,确定其具双亲或单亲父系质体遗传的潜能,并用RFLP技术进一步确定其为质体父系遗传型。本研究证明旋花科的打碗花属生殖细胞、精细胞和卵细胞中细胞质类核存在的状况与牵牛属的相似,提供了打碗花可能在质体遗传上与牵牛属 具相同的遗传方式的资料。     

Cytoplasmic Inheritance of Sweet Potato: with Respect to the Study of Plastids and Mitochondria and the Existence of Their DNA in Sperm Cells

The mature pollen of sweet potato ( Ipomoea batatas lam. ) was bicellular. After pollination generative cell divided into a pair of sperm cells before its germination. The pair of sperm cells remained in the hydrated pollen was similar in their shape and volume with enriched cytoplasmic plastids and mitochondria. The specific fluorescence of cytoplasm DNA indicated that the sperm cells and the generative cell contained numerous organelle nucleoids. The pair of sperm cells had no significant difference in their numbers of organelle nucleoids. Two kinds of organelle nucleoids existed in the pair of sperm cells. Tile ones as big and strong fluorescent dots appeared to be the plastid nucleoids and the others as tile small and weak fluorescent dots could be the mitochondrial nucleoid. Few of the angiosperms were of biparental or paternal plastid inheritance. The result of this study has provided the cytological evidence for another genus, Ipomoea, which is of biparental or paternal plastid inheritance besides Pharbitis and Calystegia in Convolvulaceae.     

Studies of Generative Cell Development in Pisum sativum--With Special Reference to Cytological Basis of Plastid Maternal Inheritance

It was proved by ultrastructural observations that few plastids and abundent mitochondria were ever present in the generative cell of Pisurn sativurn L. from its initiation to maturation. Fluorescence observations of DNA showed that many cytoplasmic DNA nucleoids were present in generative cell of mature pollen, but none in the early developing generative cell. During the germination of mature pollen in vitro, the cytoplasmic DNA nucleoids of the generative cell in the pollen tube degenerated gradually following the growth of the pollen tube and disappered completely 24 h after germination. The results provided a cytological basis for confirming the conclusion of plastid maternal inheritance in P. sativurn obtained from genetic study, and resolved the contradiction between results from cytological observation and genetic or RFLP analysis.     

豌豆生殖细胞的发育—着重论述质体母系遗传的细胞学基础

超微结构的研究证明,豌豆（Ｐｉｓｕｍ ｓａｔｉｖｕｍ Ｌ．）生殖细胞自形成直至成熟花粉时期,始终存在少量质体和较多的线粒体。ＤＮＡ 荧光的观察表明,在发育早期的生殖细胞中不含细胞质ＤＮＡ 类核,但在成熟花粉的生殖细胞中有许多的类核。在花粉离体萌发过程中,随着花粉管的生长,生殖细胞中的类核逐渐降解。在花粉培养２４ ｈ 后,生殖细胞的类核全部消失。研究结果确定了豌豆质体母系遗传的细胞学基础,支持遗传分析及ＲＦＬＰ研究的结论,阐明了过去在细胞学上认为是双亲遗传的判断不正确的原因     

Changes of Plastids Mitochondria and Their DNA in the Egg Cell Before and After Fertilization in Pharbitis

The ultrastructure and cytoplasmic DNA in the egg cell and zygote of Pharbitis purpurea, (L.) Voyght and P. limbata Lindl. which were studied with electron microscopy and DNA epifiuorescence microscopy. The egg cell before fertilization was highly vacuolated with only a few cytoplasmic plastids and mitochondria. Plastids were spherical and/or rod- shaped containing 1 ～ 2 large starch grains. Most of the mitochondria were cup and/or circular. The cytoplasm in the zygote was much more abundant than that in the egg cell. The number of plastids and their electronic density were greatly increased, in most of which containing osmiophilic bodies. The mitochondria were rich and spherical-shaped in the zygote. Two types of cytoplasmic DNA nucleoids were detected in the egg cell, the more abundant one being big and circle-shaped and the other dot-shaped. Only dot-shaped nucleoids were present in the zygote. The content of nucleoids in the zygote was much less than that in the egg cell. Authors propose that some cytoplasmic DNA may degenerate after fertilization. The ultrastructural characteristics of the egg cell and the reduction of cytoplasmic DNA in the zygote may related to the mechanisms of plastid unipaternal inheritance in Pharbitis.     

Study on Organelle DNA Within the Generative Cell and Sperm Cells in Pharbitis

The organelle DNA in generative cell and its behavior during spermatogenesis in Pharbitis limbata and P. purpurea were observed by epifluorescence microscopy stained with 4',-6-diamidino-2-phenylindole (DAPI). In these two species, the generative cell is long and thin in which a great amount of cytoplasmic DNA is present. Most pairs of sperm cells are isomorphic, in which one end is obtuse and the other is elongate, but in a few pairs dimorphi sperms are present. The nucleus is located at one end of the cell. A lot of cytoplasmic DNA are distributed randomly throughout the cytoplasm. The size of organelle nucleoids and their fluorescence intensity are different in a sperm cell. The features of generative cell and sperm cell, and behavior of cytoplasmic DNA are similar in P. limbata and P. purpurea. The obvious differences between them are that the size and fluorescence intensity of organelle nucleoids in P. purpurea are respectively smaller and weaker than in P. limbata. The results showed that morning glory has potential of biparental or paternal cytoplasmic in heritance. Isomorphism and dimorphism of sperms, and the relationship between the ratio of nucleus and cytoplasm in sperm cell and the plastid biparental inheritance are discussed.     

Cytoplasmic DNA apportionment and plastid differentiation during male gametophyte development in Pelargonium zonale

In the male gametophyte of Pelargonium zonale, generative and sperm cells contain cytoplasmic DNA in high density compared to vegetative cells. Cytoplasmic DNA was examined using the DNA fluorochrome DAPI (4'6-diamidino-2-phenylindole) and observed with epifluorescence and electron microscopy. The microspore cell contains a prominent central vacuole before mitosis; mitochondria and plastids are randomly distributed throughout the cytoplasm. Following the first pollen grain mitosis, neither the vegetative cell nor the early generative cell display a distributional difference in cytoplasmic DNA, nor is there in organelle content at this stage. During the maturation of the male gametophyte, however, a significant discrepancy in plastid abundance develops. Plastids in the generative cell return to proplastids and do not contain large starch grains, while those in the vegetative cell develop starch grains and differentiate into large amyloplasts. Plastid nucleoids in generative and sperm cells in a mature male gametophyte are easily discriminated after DAPI staining due to their compactness, while those in vegetative cells stained only weakly. The utility of the hydrophilic, non-autofluorescent resin Technovit 7100 in observing DAPI fluorescence is also demonstrated.     

玉竹雄配子的发育——着重阐明质体在生殖细胞和营养细胞中的分布和变化

玉竹(Polygonatum simizui Kitag)小孢子在分裂前,质体极性分布导致分裂后形成的生殖细胞不含质体,而营养细胞包含了小孢子中全部的质体。生殖细胞发育至成熟花粉时期,及在花粉管中分裂形成的两个精细胞中始终不含质体。虽然生殖细胞和精细胞中都存在线粒体,但细胞质中无DNA类核。玉竹雄性质体的遗传为单亲母本型。在雄配子体发育过程中,营养细胞中的质体发生明显的变化。在早期的营养细胞质中,造粉质体增殖和活跃地合成淀粉。后期,脂体增加而造粉质体消失。接近成熟时花粉富含油滴。对百合科的不同属植物质体被排除的机理及花粉中贮藏的淀粉与脂体的转变进行了讨论。     

Male gametophyte development inPlumbago zeylanica: cytoplasm localization and cell determination in the early generative cell

Summary The behavior of the generative cell during male gametophyte development inPlumbago zeylanica was examined by epifluorescence microscopy and electron microscopy with organelle nucleoid as a cytoplasm marker. When the thin sections stained with 4,6-diamidino-2-phenylindoIe (DAPI) were observed under an epifluorescence microscope, two types of fluorescence spots were detected in the cytoplasm of the pollen cells before the second mitosis. The spots emitting stronger fluorescence were confirmed as plastid nucleoids and those emitting dimmer fluorescence were mitochondrial nucleoids. Before the first mitosis, both plastid and mitochondrial nucleoids distributed randomly in the cytoplasm of the microspore. A small lenticular generative cell formed with attachment to the interior of the intine after the mitosis. Small vacuoles were found in the lenticular cell. In the cytoplasm of the lenticular cell, both plastid nucleoids and the small vacuoles were distributed randomly at the very beginning but began to migrate in opposite directions immediately. Plastid nucleoids aggregated to the side of the cell that faces the pollen center and the small vacuoles aggregated to the side of the cell that attaches to the inline. As the result, the lenticular generative cell appeared highly polarized in cytoplasm location soon after the first mitosis. In accordance with the definition of the cytoplasm polarization, the primary wall between the generative and the vegetative cells began to flex and the lenticular generative cell started to protrude towards the pollen center. When the generative cell peeled away from the inline, it was spherical in shape with the pole that aggregated plastids towards the vegetative nucleus. But the cell direction appeared to be transformed immediately. The pole that aggregated small vacuoles turned to the position towards the vegetative nucleus and the pole that aggregated plastid nucleoids turned to the position countering to the vegetative nucleus. A cellular protuberance formed at the edge of the pole that aggregated small vacuoles and elongated into a tapered end that got into contact with the vegetative nucleus. The polarization of the cytoplasm kept constant throughout the second mitosis. The small vacuoles that apportioned to the sperm cell which attached the vegetative nucleus (the leading sperm cell) disappeared during sperm cell maturation. Plastid nucleoids were apportioned to the other sperm cell (the trailing sperm cell) completely. Mitochondrial nucleoids became undetectable after the second mitosis.     

Cytological Basis of Plastid Inheritance in Phaseolus vulgaris-Investigations of Plastids,Mitochondria and Their DNA Nucleoids During Pollen Development

Electron microscopic and DNA fluorescence microscopic observations of the plastids, mitochondria and their DNA in the developing pollen of Phaseolus vulgaris L. have demonstrated that the male plastids were excluded during microspore mitosis. The formed generative cell was free of plastids because of regional localization of plastids in early developing microspore and the extremely unequal distribution during division. The fluorescence observations of DNA showed that cytoplasmic (plastid and mitochondria) nucleoids degenerated and disappeared during the development of microspore/pollen, and were never presented in the generative cell at different development stages. These results provided precise cytological evidence of maternal plastid inheritance in Phaseolus vulgaris, which was not in accord with the biparental plastid inheritance identified from early genetic analysis. Based on authors' previous observations in a variety of common bean that the organelle DNA of male gamete was completely degenerated, the early genetic finding of the biparental plastid inheritance was unlikely to be effected by genotypic difference. Thus those biparental plastid inheritance might be caused by occational male plastid transmission, and plastid uniparental maternal inheritance was the species character of Phaseolus vulgaris.     

Heritable paternal cytoplasmic organelles in alfalfa sperm cells: ultrastructural reconstruction and quantitative cytology.

Sperm cells within pollen grains and pollen tubes of alfalfa (Medicago sativa L.) were observed at the ultrastructural level, and their plastid DNA was detected by DAPI (4,6-diamidino-2-phenylindole) staining. One sperm pair within the pollen grain and three sperm pairs within pollen tubes were reconstructed in three-dimensions from serial ultrathin sections. The two sperm cells are linked by cytoplasmic bridges in both pollen grains and tubes, and the vegetative nucleus is closely associated with the sperm cells within the pollen tube. The number of plastids and plastid nucleoids (DNA aggregates) in the sperm cell pair, collectively, is not significantly different from that in the generative cell; however, over 60% of the sperm cell plastids contain no DNA detectable with DAPI. The mean number of mitochondria in sperm cells is reduced from that in the generative cell (from 54 to 17), which suggests that paternal mitochondrial inheritance probably does not occur in the genotype investigated. Sperm cells of a pair may vary in their shape within the pollen grain and tube, but the number of plastids and mitochondria is not significantly different between the sperm cells. Therefore, heterospermy is not a factor determining cytoplasmic inheritance patterns in this species.     

Monitoring by epifluorescence microscopy of organelle DNA fate during pollen development in five angiosperm species

The fates of mitochondrial and plastid nucleoids during pollen development in six angiosperm species (Antirrhinum majus, Glycine max, Medicago sativa, Nicotiana tabacum, Pisum sativum, and Trifolium pratense) were examined using epifluorescence microscopy after double staining with 4',6-diamidino-2- phenylindole (DAPI) to stain DNA and with a potentiometric dye (either DiOC7 or rhodamine 123) for visualization of metabolically active mitochondria. From the pollen mother cell stage to the microspore stage of pollen development, mitochondria and plastids both contained DNA detectable by DAPI staining. However, during the further maturation preceding anthesis, mitochondrial DNA became undetectable cytologically in either the generative or the vegetative cell of mature pollen; even in germinated pollen tubes containing hundreds of metabolically active mitochondria undergoing cytoplasmic streaming, vital staining with DAPI failed to reveal mitochondrial DNA. By the mature pollen stage, plastid DNA also became undetectable by DAPI staining in the vegetative cell. However, in the generative cell of mature pollen the timing of plastid DNA disappearance as detected by DAPI varied with the species. Plastid DNA remained detectable only in the generative cells of pollen grains from species known or suspected to have biparental transmission of plastids. The apparent absence of cytologically detectable organelle genomes in living pollen was further examined using molecular methods by hybridizing organelle DNA-specific probes to digests of total DNA from mature pollen and from other organs of A. majus and N. tabacum, both known to be maternal for organelle inheritance. Mitochondrial DNA was detected in pollen of both species; thus the cytological alteration of mitochondrial genomes during pollen development does not correspond with total mtDNA loss from the pollen. Plastid DNA was detectable with molecular probes in N. tabacum pollen but not in A. majus pollen. Since the organelle DNA detected by molecular methods in mature pollen may lie solely in the vegetative cell, further study of the basis of maternal inheritance of mitochondria and plastids will require molecular methods which distinguish vegetative cell from reproductive cell organelle genomes. The biological effect of the striking morphological alteration of organelle genomes during later stages of pollen development, which leaves them detectable by molecular methods but not by DAPI staining, is as yet unknown.     

Divergent potentials for cytoplasmic inheritance within the genus Syringa. A new trait associated with speciogenesis

Epifluorescence microscopic detection of organelle DNA in the mature generative cell is a rapid method for determining the potential for the mode of cytoplasmic inheritance. We used this method to examine 19 of the known 22 to 27 species in the genus Syringa. Organelle DNA was undetectable in seven species, all in the subgenus Syringa, but was detected in the 12 species examined of the subgenera Syringa and Ligustrina. Therefore, species within the genus Syringa display differences in the potential cytoplasmic inheritance. Closer examination revealed that the mature generative cells of the species in which organelle DNA was detected contained both mitochondria and plastids, but cells of the species lacking detectable organelle DNA contained only mitochondria, and the epifluorescent organelle DNA signals from the mature generative cells corresponded to plastid DNA. In addition, semiquantitative analysis was used to demonstrate that, during pollen development, the amount of mitochondrial DNA decreased greatly in the generative cells of the species examined, but the amount of plastid DNA increased remarkably in the species containing plastids in the generative cell. The results suggest that all Syringa species exhibit potential maternal mitochondrial inheritance, and a number of the species exhibit potential biparental plastid inheritance. The difference between the modes of potential plastid inheritance among the species suggests different phylogenies for the species; it also supports recent conclusions of molecular, systematic studies of the Syringa. In addition, the results provide new evidence for the mechanisms of maternal mitochondrial inheritance in angiosperms.     

Fluorescence microscopy of plastid nucleoids and a survey of nuclease C in higher plants with respect to mode of plastid inheritance

Summary Plastid nucleoids (pt nucleoids) were observed during pollen formation, or in generative cells of mature pollen grains using fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI). Nuclease C activity was surveyed using SDS-PAGE and agarose gel nuclease assay methods. InMirabilis jalapa, pt nucleoids were observed both in pollen mother cells and the monocellular pollen grains after meiosis, followed by the complete disappearance both in the generative and vegetative cells at the bicellular pollen grain stage. This observation is a direct evidence of maternal plastid inheritance. By contrast, in the generative cells of mature pollen grains fromRhododendron kaempferi, Zygocactus truncatus, Oenothera laciniata, andO. speciosa, pt nucleoids were clearly observed. Thus cytological evidence convinces the mode of biparental plastid inheritance. Nuclease C activity was clearly detected both in the stamen and pistil ofM. jalapa. InR. kaempferi low nuclease C activity was detected in both organs, but the activity in the stamen was much less than in the pistil. InZ. truncatus, O. laciniata, andO. speciosa, the activities were difficult to detect in both organs. These results suggest a significant role of nuclease C for the digestion of pt nucleoids in the generative cells.Abbreviations EGTA ethylene-glycol-bis-(2-aminoethyl ether)-N, N, N, N-tetraacetic acid - DAPI 4,6-diamidino-2-phenylindole - Nuclease C Ca²⁺ dependent nuclease - SDS-PAGE SDS-polyacrylamide gel electrophoresis - pt nucleoids plastid nucleoids     

Variation in generative cell plastid nucleoids and male fertility in Medicago sativa

Biparental inheritance of plastids has been documented in numerous angiosperm species. The adaptive significance of the mode of plastid inheritance (unior biparental) is poorly understood. In plants exhibiting paternal inheritance of plastids, DNA-containing plastids in the microgametophyte may affect survival or growth of the gametophyte or the embryo. In this study the number of plastids containing DNA (nucleoids) in generative cells and generative cell and pollen volumes were evaluated in a range of genotypes of Medicago sativa (alfalfa). M. sativa exhibits biparental inheritance of plastids with strong paternal bias. The M. sativa genotypes used were crossed as male parents to a common genotype and the relationships between the gametophytic traits measured and male reproductive success were assessed. Generative cell plastid number and pollen grain size exhibited opposing associations with male fertility. Path analysis showed that generative cell plastid number was negatively associated with male fertility. This study provides evidence that there may be a competitive advantage at fertilization afforded sperm that have minimized their organelle content. The apparent lack of strong selection for reduced plastid number in generative cells of M. sativa may be a reflection of the diminished importance of reproductive success due to its perenniality or its long use in cultivation.     

Disappearance of plastid and mitochondrial nucleoids during the formation of generative cells of higher plants revealed by fluorescence microscopy

Summary The fate of plastid and mitochondrial nucleoids (pt and mt nucleoids) ofTriticum aestivum was followed during the reproductive organ formation using fluorescence microscopy after staining with 4'6-diamidino-2-phenylindole (DAPI). This investigation showed a drastic morphological change of pt nucleoids during the differentiation of reproductive organs from the shoot apex. Dot-shaped pt nucleoids grew into ring-shaped ones, which divided into small pieces in the monocellular pollen grain, as observed in this plant's earlier stage of leaf development. During the development of mature pollen grain from monocellular pollen grain, pt and/or mt nucleoids disappeared through the division of the male generative cell ofT. aestivum. Cytologically, this observation is direct evidence of the maternal inheritance of higher plants. Thus far, cytological evidence of this phenomenon has been found mostly by morphological criteria using electron microscopy, which admits some ambiguity. In the plants exemplified byLilium longiflorum, pt and/or mt nucleoids disappeared after the first pollen grain mitosis, which precededT. aestivum. In the plants exemplified byTrifolium repens, pt and/or mt nucleoids existed in the generative cells of the mature pollen grain.The significance of these observations was discussed in relation to the interaction between nuclear and organelle genomes during plant development.Abbreviations DAPI 4'6 diamidino-2-phenylindole - Mt DNA Mitochondrial DNA - Mt nucleoid Mitochondrial nucleoid - Pt DNA Plastid DNA - Pt nucleoid Plastid nucleoid On leave from Department of Biology, Nagoya University, Furocho, Chikusaku, Nagoya 464, Japan.     

Potential cytoplasmic inheritance in Wisteria sinensis and Robinia pseudoacacia (Leguminosae)

We examined pollen cells of Wisteria sinensis and Robinia pseudoacacia (Leguminosae) to determine a possible mode for cytoplasmic inheritance in these species. Epifluorescence microscopy revealed distinct mature generative cells. Mature generative cells of W. sinensis were associated with large numbers of punctuated fluorescent signals corresponding to cytoplasmic DNA aggregates, but no fluorescent signals were observed in the generative cells of R. pseudoacacia. Closer examination showed that the punctate fluorescent signals corresponded to plastid but not mitochondrial DNA. These results suggest a strong potential for paternal transmission of the plastid genome in W. sinensis. Electron microscopy confirmed the presence of plastids in the generative cells of W. sinensis and the absence of plastids in R. pseudoacacia cells due to an unequal distribution of plastids during the first pollen mitosis. Mitochondria were present and intact in the mature generative cells of both species. The lack of fluoresced mitochondrial DNA suggests a very low level of mitochondrial DNA in the cells. Immunoelectron microscopy demonstrated that the labeling of mitochondrial DNA in these cells was reduced by nearly 90% during pollen development. Such a dramatic reduction suggests an active degradation of paternal mitochondrial DNA, which may contribute greatly to the maternal inheritance of mitochondria. In short, we found that W. sinensis exhibits a strong potential for paternal transmission of plastids and that both W. sinensis and R. pseudoacacia appear to share the same mechanism for maternal mitochondrial inheritance.     

Potential for biparental cytoplasmic inheritance in Jasminum officinale and Jasminum nudiflorum

 Mature Jasminum officinale and J. nudiflorum pollen grains were stained with 4′,6-diamidino-2-phenylindole (DAPI) and examined by epifluorescence microscopy. The pollen grains were found to be trinucleate, and the sperm cells in both species contained a large number of epifluorescent spots that corresponded to cytoplasmic DNA aggregates (nucleoids). The nucleoids of J. nudiflorum were observed to be dimorphic under the epifluorescence microscope, indicating that the sperm cells might contain both plastid and mitochondrial DNA. The nucleoids of J. officinale presented a similar appearance when stained with DAPI, but electron microscopic examination of the sperm cells revealed that they contained both plastids and mitochondria. When analyzed by DNA immunogold electron microscopy, gold particles were detected on both plastids and mitochondria. These findings demonstrated the preservation of plastid and mitochondrial DNA in mature sperm cells and thus the potential for biparental cytoplasmic inheritance in J. officinale and J. nudiflorum. Received: 8 August 1997 / Revision accepted: 25 February 1998     

Microgametophytic plastid nucleoid content and reproductive and life history traits of tribeTrifolieae (Fabaceae)

Microgametophytic plastid nucleoids were quantified for 18 species representing the four core genera of the tribeTrifolieae (Fabaceae),Medicago, Melilotus, Trigonella, andTrifolium. Generative cells of all taxa contained nucleoids, establishing that biparental plastid inheritance is common in theTrifolieae. Nucleoid number and volumes of pollen grains and generative cell nuclei differed among taxa. Nucleoid number was positively correlated with pollen grain and generative cell nuclear volumes, flower size and style length. These relationships disappeared after adjusting nucleoid number for pollen grain and generative cell nuclear volumes. Adjusted nucleoid numbers provided no evidence to support hypotheses that plastid content is associated with ploidy level, mating system, perenniality or size of the reproductive apparatus.     

Ultrastructures of the Egg Apparatus and the Central Cell of Calystegia hederacea (Convolvulaceae) Before and After Fertilizati on

Ultrastructures of the mature embryo sac of Calystegia hederacea Wall. and its changes after fertilization are described. The positional organization of the egg cell, the two synergids and the central cell, as well as their interrelationships were studied. Some regions of the cell boundaries between the egg cell and the central cell, as well as between the egg cell and the synergids were devoid of typical cell wall before fertilization, displayed a feature quite similar to the characteristic absence of the cell wall in the fertilization target zone occurred in most angiosperms. Besides the genera ultrastructural characteristics of the egg apparatus and the central cell, there were several unusual aspects in C. hederacea, such as the egg nucleus located above the large vacuole near the chalazal end of the cell, many polyribosomes in the cytoplasm of the egg cell and wall ingrowths on both sides of the hooks of the central cell. All these unusual characteristics seemed to be closely associated with the short duration of the fertilization and the absence of antipodal cells in the mature embryo sac. It is concluded that the female germ unit of C. hecleracea is considered to be a topographical and physiological unit to realize their functions for successful double fertilization.