GYNOECIAL DEVELOPMENT,POLLINATION, AND THE PATH OF POLLEN TUBE GROWTH IN THE TEPARY BEAN,PHASEOLUS ACUTIFOLIUS

The gynoecium of Phaseolus acutifolius var. latifolius, a self-compatible legume, is characterized by a wet non-papillate stigma, an intermeditae hollow/solid style type, and secretory cells on the ventral surface of the ovary which direct pollen tube growth. The stigma is initially receptive 5–6 days prior to anthesis. Production of stigmatic secretions, composed primarily of carbohydrates and lipids, fragment the cuticle covering epidermal cells of the stigma early in ontogeny; the lipidic aspect of the copious secretions apparently serves to inhibit desiccation after the cuticle is ruptured. Stylar canal development occurs as a combination of elongation of a basal canal present early in development, and dissolution of part of a solid transmitting tract tissue just below the stigma. Anthers dehisce and the tricolporate pollen is released onto the receptive stigma one day before anthesis. Following initial growth in intercellular spaces in the transmitting tract of the stigma, pollen tubes adhere to epidermal secretory cells along the ventral side of the stylar canal and upper ovary; here the transmitting tract is apparently limited in the number of tubes it can accommodate, providing a possible site of selection of male gametes.     

Divergent pollination systems in the cleistogamous species,Collomia grandiflora (Polemoniaceae)

Summary A structural study of pollination in the dimorphic flowers ofCollomia grandiflora, a cleistogamous species, reveals significant differences in stigma behavior during pollination, stylar structure, the timing of generative cell division, and pollen tube growth rate patterns. The cleistogamous flower shows a loss of protandry and the stigma is receptive only after reflexing and closing of its lobes. In contrast, the chasmogamous stigma is receptive when reflexed and closes when pollen has been deposited on the lobes. Pollen tube penetration of the dry stigma papillae and entry into the style is similar in the two morphs. The chasmogamous style is solid and the cleistogamous style partly hollow. The matrix of secretion produced by the transmitting tract cells is mainly carbohydrate with a trace of lipids. It is fibrillar in nature and appears to be partly comprised of wall material from the transmitting tract cells. In the chasmogamous pollen, the generative cell enters the tube before division, which occurs between 30 and 60 min after pollination. This division correlates with an increased growth rate for the pollen tube. In the cleistogamous pollen, contact with the stigma triggers generative cell division inside the hydrated pollen grain before germination. The two resulting sperm cells exit the grain 15–30 min after pollination when the pollen tube is in the stigma lobes. The cleistogamous pollen tube shows only one phase of growth which occurs at a rate similar to that of the slow, first phase of the chasmogamous pollen.Abbreviations CH chasmogamous - CL cleistogamous - DAPI 4, 6-diamidino-2-phenylindole     

INFLUENCE OF THE PISTIL ON POLLEN TUBE KINETICS IN PEACH (PRUNUS PERSICA)

Pollen tube growth has been studied in peach and has been related to changes in the pistil structures which the pollen tube has to traverse in its way from the stigma down to the ovule. Growth of the pollen tubes along the pistil is not continuous. While pollen tubes reach the base of the style 7 days after pollination, fertilization does not take place until 12 days later. Pollen tubes stop for 5 days at the top of the obturator and they further stop for 3 days before entering the ovule. The pollen tube growth is heterotrophic; starch, present all along the pistilar tract at anthesis, vanishes as the pollen tubes pass by. Discontinuous pollen tube growth appears to be controlled by the pistil. At anthesis the pistil is not fully matured. Maturation of the pistil implies a number of secretory processes that occur in a basipetal way starting from the stigma down to the style and ending in the ovule. Some of these secretions at the stigma and the style are triggered by pollination; others appear to be a maturative stage of the pistil and are produced in a discrete way. The fact that the pollen tube depends on these secretions together with the fact that these secretions are not continuously produced confer upon the pistil a role of controlling pollen tube kinetics and point out that, for a successful fertilization, male gametophyte development and pistil maturation need to by synchronized.     

COORDINATED TIMETABLES FOR MEGAGAMETOPHYTE DEVELOPMENT AND POLLEN TUBE GROWTH IN RHODODENDRON NUTTALLII FROM ANTHESIS TO EARLY POSTFERTILIZATION

Rhododendron nuttallii T. W. Booth (Ericaceae) was used to derive concurrent timetables for megagametophyte, pollen tube and early postfertilization development from anthesis through 3 wk after pollination, based on timed collections of self-pollinated pistils. Stages of development were determined for over 33,500 cleared ovules, including, for selected collection dates, stages on different portions of the placenta. Pollen tube information was obtained by fluorescence microscopy of pistil squashes stained with aniline blue. Because of the very large number of ovules observed, it was possible to recognize a much more closely graded series of stages in megagametophyte development than is usually the case. While a range of stages occurred on all days, development progressed steadily from a majority of functional spores and 2-nucleate gametophytes on the day of anthesis to mostly a late zygote-primary endosperm stage at 18 days, and some 2-celled endosperm stages at 21 days, after pollination. At all times the most advanced stages, including first pollen tube entries, occurred on the outer surface of the lower half of the placenta, and the youngest on the inner surface of the uppermost portion. Fertilizable ovules were not found in any frequency until 8 days after pollination (then in only about 34% of the ovules); a few fertilized ones were seen after 10 days but constituted less than 5% until 12 days after pollination, thereafter increasing to about 60%. Fertilization occurred in any one of three morphologically recognizable stages distinguished by position and state of fusion of polar nuclei. Pollen germinated on the stigma 1–2 hr after pollination, and pollen tubes grew at a rate of about 1–1.25 cm/day, reaching the top of the ovary in 8–9 days with the first ovule entries seen after 10 days. There was a close correlation between megagametophyte development and pollen tube growth, with large numbers of functionally mature ovules not being found until pollen tubes had reached the ovary. While nuclei within ovules could not be distinguished in the squashes, three gametophyte stages that could be recognized—unelongated, elongated either without or with a pollen tube—were tallied for almost 29,000 ovules. The progression in these general stages corresponded well with that documented in more detail from cleared ovules. Unpollinated pistils showed a similar progression of gametophyte stages until the time fertilization would start to occur, after which there was continued accumulation of functionally mature ovules. A variety of abnormally developed and/or collapsing(ed) ovules or gametophytes were seen; collectively, they averaged over 8.6% of all ovules.     

Asynchronous development of stigmatic receptivity in the pear (Pyrus communis; Rosaceae) flower

While stigma anatomy is well documented for a good number of species, little information is available on the acquisition and cessation of stigmatic receptivity. The aim of this work is to characterize the development of stigma receptivity, from anthesis to stigma degeneration, in the pentacarpellar pear (Pyrus communis) flower. Stigma development and stigmatic receptivity were monitored over two consecutive years, as the capacity of the stigmas to offer support for pollen germination and pollen tube growth. In an experiment where hand pollinations were delayed for specified times after anthesis, three different stigmatic developmental stages could be observed: (1) immature stigmas, which allow pollen adhesion but not hydration; (2) receptive stigmas, which allow proper pollen hydration and germination; and (3) degenerated stigmas, in which pollen hydrates and germinates properly, but pollen tube growth is impaired soon after germination. This developmental characterization showed that stigmas in different developmental stages coexist within a flower and that the acquisition and cessation of stigmatic receptivity by each carpel occur in a sequential manner. In this way, while the duration of stigmatic receptivity for each carpel is rather short, the flower has an expanded receptive period. This asynchronous period of receptivity for the different stigmas of a single flower is discussed as a strategy that could serve to maximize pollination resources under unreliable pollination conditions.     

Studies of pollen maturation in cotton: the storage reserve accumulation phase

Summary This study follows the maturation of the pollen grain of cotton (Gossypium hirsutum L.), particularly the development of the vegetative cytoplasm and the various storage products formed. CTEM, HVEM, stereoscopy, and cyto-histochemistry were used to examine the events occurring during the 9 days before anthesis. Starch began to accumulate in plastids at anthesis minus 9 days and reached a peak concentration shortly before anthesis; lipid deposition followed a similar pattern, but started at 6 days before anthesis. Lipid bodies were always seen closely oppressed to the endoplasmic reticulum (ER). Dictyosomes appear active during the entire 9 days; first producing vesicles involved in the formation of the intine and, later, producing vesicles stored in the pollen grain. The dictyosome vesicles appear to contain polysaccharides and concentrate in layers around the lipid bodies. Ribosomes increase in number from 6 days before anthesis and are particularly numerous in the mature pollen. From anthesis minus 6 days until anthesis, the ER cisternae become increasingly inflated and, in the hours immediately before pollen release, form pockets filled with lipid bodies and dictysosome vesicles. The mature pollen has a core region filled with ER pockets and a peripheral cytoplasm in which such pockets are generally lacking.This research was supported in part by NSF Grant BMS575-22-23 and Grant N.RR-00592 from the Division of Research Resources, National Institutes of Health     

The Development of Gynoecium After Anthesis and Fertilization in Eleutherococcus brachypus (Araliaceae)

Eleutherococcus brachypus Harms. is a protandrous plant because the female gametophyte delays its maturation until the fifth day after anthesis and pollen shelling. On the fifth day after anthesis, about 57.69% of the embryo sacs matured and the rest degenerated or failed to develop. Fertilization began in the embryo sac on the fifth day. On the tenth day fertilization took place in 53.37 % of the total of embryo sacs. The stigma became receptible after 3 to 4 days of anthesis. It took 2 to 3 days from the germination of pollen grains on stigma to the fusion of male and female nuclei. The process of fertilization in E. brachypus is not different from most other angiosperms. It belonged to the type of premitotic syngamy. The observations and statistical analysis were made on the number feature of male and female nucleoli in the zygote. The result indicated that it took three days or so from the appearance of male nucleolus in the zygote to its fusion with the female nucleotus. Refering to the number of free nuclei of the endosperm, the fusion of male and female nucleoli in most of the zygotes occurred in the stage of 32 to 128 nuclei of the endosperm. Most zygotes con-tained a big nucleolus resulting from the fusion of male and female nucleolus and proceeding to mitosis. A few without fusion could also proceed to the mitotic stage. Features of multiple sperms entering the embryo sac or entering the egg cell and the degeneration of mature embryo sacs were observed as well. The sign of the termination of fertilization in angiosperms was discussed.     

Breakdown of self-incompatibility during pistil development in Lycopersicon peruvianum by modified bud pollination

Summary Stylar self-incompatibility barriers in L. peruvianum can be avoided if pollen germination and growth through immature pistils is promoted under specific environmental conditions approximately 2–3 days before the initiation of anthesis. Since immature stigmas lack sufficient exudate for pollen germination, the sandwiching of a thin layer of pollen germination medium between the stigma and a mineral oil layer containing pollen allows precocious pollen germination and some compatible pollen tube growth through the style. This procedure is rapid, inexpensive, applicable in the field, and makes efficient use of pollen. Consistent though low seed yields have been obtained. A high proportion of aborted seed, seedling lethals, and aberrant seedling phenotypes in selfed progeny indicate the presence of strong post-zygotic barriers to such selfing. No evidence for a reduction in the strength of the SI response with increasing pistil age was observed.     

短柄五加开花后雌蕊的发育状态与受精作用的研究

短柄五加（ＥｌｅｕｔｈｅｒｏｃｏｃｕｓｂｒａｃｈｙｐｕｓＨａｒｍｓ．）开花当天,花药散粉,而雌配子体需经４～５ｄ才发育成熟。证实短柄五加为雄蕊先熟植物。开花第５天,成熟胚囊的比率为５７６９％,其余为退化和不育胚囊。开花第６天,胚囊开始受精。开花第１０天,受精胚囊占胚囊总数的５３５７％。柱头的可授期自开花后第４～５天开始,自花粉萌发至雌雄性核融合大约有２～３ｄ的间隔期。短柄五加受精过程与一般被子植物相同,其受精作用属于有丝分裂前配子融合类型。观察并统计了合子中雌性核仁的数目、存在状态,指出短柄五加合子中从雄性核仁出现到与雌雄性核仁融合为一个大核仁需经历３ｄ左右;如果以胚乳游离核数目为对照,大部分合子中雌雄性核仁的融合发生在３２～１２８个胚乳游离核时期。大多数合子是以雌雄性核仁融合为一个大核仁后进入合子分裂期;少数合子的雌雄性核仁不经融合也进入合子分裂期。观察到多精入胚囊、多精入卵以及成熟胚囊退化的现象。讨论了被子植物受精过程中有关受精终结的标志等问题。     

Sexual reproduction in the cork oak (Quercus suber L.). I. The progamic phase

 Cork oak (Quercus suber L.) is a monoecious wind-pollinated species with a protandrous system to ensure cross-pollination. To the best of our knowledge, this report provides the first insight into the sexual reproduction cycle in this species. The cork oak flowering season extends from April until the end of May. Our results show that, at anthesis, the pistillate flower is not completely formed and ovules are just starting to develop. Pollen reaching the dry stigmatic surface adheres to the receptive cells, germinates and penetrates the epidermis in aproximately 24 h, and grows through the intercellular spaces of a solid transmitting tissue. In cross-pollination, a sequential arrest of pollen tubes was observed along the style, providing preliminary evidence for a pollen tube competition mechanism. As a consequence, few pollen tubes reach the basal portion of the style. Furthermore, pollen tube growth is a discontinuous process since tubes are arrested in the basal portion of the style about 10–12 days after pollination. While tubes are latent, the ovarian loculus starts to develop from an emerging mass of sporogeneous cells which later will differentiate into the placenta and ovules. One and a half months after pollination ovules complete their differentiation, tubes resume growth and fertilisation occurs. Ovular abortion is frequent at this stage, and only one ovule will successfully mature during autumn into a monospermic seed. Received: 23 July 1998 / Revision accepted: 2 November 1998     

Studies on flower longevity in Digitalis

Flower lifespan was terminated by corolla abscission 5–6 days after stigma opening in the unpollinated flower. Increased pollen loads produced increased seed set and reduced flower longevity progressively to a minimum of one day after pollination with pure pollen. Weakening of the abscission zone was detectable 8 h after pollination, whilst the pollen tubes were still within the stigmatic zone, suggesting that a stimulus, moving at 4 mm h^–1 minimum, was transmitted through the style and ovary. Soon after pollination removal of the stigma prevented the pollination-induced corolla abscission. Later it was necessary to remove the stigma and upper style, and later still the whole style to delay abscission. The progressive induction of the stigma and style took place at a rate of 1.5 mm h^–1, in advance of the pollen tubes which grew at 0.75 mm h^–1. It was not possible to reproduce the pollination effects by application of indoleacetic acid (IAA) to the stigma or the style.Abbreviation IAA Indoleacetic acid     

Floral Viability and Pollen Tube Growth in Vicia faba L.

Durations of stigmatic receptivity, pollen viability and pollen tube growth were investigated in the largely entomophilous faba bean. Stigmas were examined for deposited and germinated pollen, and growth of pollen tubes was investigated using aniline blue-induced fluorescence. Entry of a pollen tube into a micropyle was found to be a reliable indicator of fertilization. After anthesis, stigmas remained receptive to pollination for six days, and pollen viable for five. Pollen tubes took up to three days to reach the ovules furthest from the stigma. Inspection of ovular development within pods showed that the incidence of fertilization had been accurately determined in the flowers.     

Stage-related expression of mRNAs during pollen development in lily and tobacco

Homogeneous populations of developing microspores and pollen from anthers of lily (Lilium longiflorum Thumb.) and tobacco (Nicotiana tabacum L.) show a continuous production of biomass, reaching a maximum in young pollen. The rate of RNA synthesis was 460 fg · h^–1 in young binucleate cells, 138 fg · h^–1 in late binucleate cells and 56 fg · h^–1 in microspores. The mRNA population in developing pollen can be separated into three groups. In the first group, certain types of mRNAs are present at a constant level during all stages of development. A second group is characteristic of young pollen and increases quantitatively until anthesis. A third group is seen transiently; to this belong mRNAs present only before mitosis or at a distinct cell stage after mitosis. Some of the translation products of this latter group of mRNAs showed similarities between lily and tobacco on two-dimensional gels in respect of molecular weight and isolectric point, indicating that those mRNAs and proteins play a role in the regulation of pollen development.Abbreviations cDNA copy DNA - pI isolectric point To whom correspondence should be addressed.     

Maturation of stamens and ovaries on cultured ear inflorescences of maize (Zea mays L.)

Observations were made on the maturation of stamens and ovaries from cultured maize (Zea mays L.) ear inflorescences. Immature ears (5.1–10.0 mm long) of maize were cultured in kinetin medium to study microsporogenesis and pollen maturation in developing stamens. Male spikelets developed on ears cultured in kinetin medium. Meiosis-I began by 7 days of culture in the developing anthers and the mature tri-nucleate pollen grains were developed by 20 days of culture. Further, kinetin was required in the culture medium for at least initial 5 days to obtain the microspores in differentiated stamens.To observe the embryosac formation in developed ovaries, ears were cultured in control, kinetin (10.1–15.0 mm long ears) medium, and kinetin + gibberellic acid (5.1–10.0 mm long ears) medium. Formation of embryosacs was noticed in the developed ovaries which were sampled after 20 days of culture. This differential flower development using two growth regulators provides an opportunity to uncover the biochemistry and physiology of micro- and mega-gametophyte development in maize.     

DEVELOPMENTAL MORPHOLOGY OF COTTON FLOWERS AND SEED AS SEEN WITH THE SCANNING ELECTRON MICROSCOPE

This report assembles and pictorially presents observations on the timing of relatively uniform and well-defined developmental events in the cotton flower and its component parts. The first floral bud occurs on the 7–9th node approximately 35–40 days postemergence; 20–25 additional days elapse until anthesis. Floral parts are morphologically well defined by two weeks preanthesis. In about 85 % of the flowers the basal, abaxial surface of two of the three bracts contains an outer involucral nectary; occasionally, none, one, or three nectaries are found. The maximum rate of increase in floral bud length occurs during the 24 hrs preceding anthesis. Flower opening occurs at about daylight, although light is not required. Multipored pollen grains germinate in about ½ hr after deposition on the stigmatic hairs. Fertilization is accomplished, for most ovules, by the end of the first day postanthesis. Stomata are abundant, particularly at the chalazal ends of ovules. Fiber initials (epidermal cells of the ovule) begin their elongation phase on the morning of anthesis and are bounded by a thin primary wall. Areas of contrast (spots) observed through the scanning electron microscope are speculated to be organelles “seen through” the relatively amorphous fiber wall, which lacks extensive fibrillar orientation of cellulose. Fiber elongation ceases by about 24–28 days postanthesis, and by 50–70 days postanthesis fibers are mature and exhibit a thickened secondary wall and spiral twisting. Concomitant with the time of fiber maturity, the ovary wall splits and opens along locular suture lines.     

CHEMOTROPIC ACTIVITY AND THE PATHWAY OF THE POLLEN TUBE IN LILY,

A study of the pollen tube pathway in Lilium leucanthum var. centifolium and in L. regale reveals that the entire pathway from stigma to ovule is lined with cytologically unique stigmatoid cells. Assays for chemotropic activity of tissues and exudates along the pathway of pollinated or unpollinated pistils showed that onset of chemotropic activity progressed basipetally (and, when pollinated, in advance of the pollen tubes), commencing at the stigma 3-5 days before anthesis and appearing in the ovules 1-2 days after anthesis. Activity persists about 10 days in ovules of pollinated pistils and for 14-16 days in ovules of non-pollinated pistils. Attempts to localize the source of the chemotropic factor showed that gynoecial tissues bearing stigmatoid cells are chemo-tropically active while slices of style or ovary wall lacking stigmatoid cells are inactive. When ovules were sliced transversely and the micropylar and chalazal halves assayed, only the micropylar half showed activity. We suggest that the ovules and the stigmatoid tissue along the pollen tube pathway are the sources of the chemotropic factor responsible for the directional growth of the pollen tube.     

Pollen Cryopreservation and Pollen Protoplast Isolation in Brassica carnpestris var.purpurea

The authors have investigated the factors affecting pollen cryopreservation in Brassica campestris var. purpurea, such as pollen development stages cryoprotectant and the process of freezing. A suitable procedure was established as follows :Pollen grains suspended in B5 medium containing 10X DMSO and 1SM sucrose were frozen by a three- –1℃/min – 1 ℃/min step method(0℃———→–10 ℃ ,standing for 15 min———→–40 ℃ , standing for 1 hr→liquid nitrogen)and later thawed in 40℃ water bath. During a period of 60, 90 days′preservation, the relative survival percentage of mature (at the day of anthesis)and nearly mature(2 days before anthesis, trinucleate stage)pollens maintained at ca. 91% that of young pollens(7-8 days before anthesis, late uninucleate stage to early binucleate stage)slightly declined from the original 91.6% to 84. 3%. Culture. experiment showed that the cryopreserved young pollen could be induced to cell division just as well as the fresh pollen. The method of isolating protoplasts from fresh mature pollen developed previously was improved and simplified. As a result, protoplasts were isolated more conveniently from mature pollen and young pollen for the first time. The protoplasts from cryopreserved mature and young pollen could be obtained as well with an isolation rate of 77.4% and 35.9% respectively. However, for isolation of protoplasts from preserved young pollen, an incubation in NLN medium at 35℃ after thawing was necessary.     

Reproductive biology of Nymphaea capensis Thunb. var. zanxibariensis (Casp.) Verdc. (Nymphaeaceae)

Anthesis in Nymphaea capensis var. zanzibariensis is diurnal with flowers opening and closing for three consecutive days. On the first day of anthesis, the stigmatic papillae secrete fluid and the outermost anthers are dehiscent. On the second day of anthesis the stamens form a cone above the dry stigmatic cup. The middle stamens open and turn outward. On the third day of flowering, all the stamens open and the dry stigmatic cup is exposed. The flowers are homogamous and not protogynous as the other Nymphaea. The gynoecium of the self-compatible N. capensis var. zanzibariensis , is characterized by a wet papillate stigma, a short hollow style, and secretory cells on the ventral surface of the ovary. The pollen is released on the receptive stigma. Following initial growth in intercellular spaces in the transmitting tract of the stigma, pollen tubes travel through the stylar canal and into the ovary.     

Wall-softening enzymes in the gynoecium and pollen of Hemerocallis fulva

Summary Variations in extractable cellulase and pectinase were followed during development of Hemerocallis (day lily) flowers. A peak in cellulase activity occurs in the pistil just prior to anthesis, followed by a 62% diminution in the enzyme activity at the time of anthesis. Cellulase activity, per mg protein, is about twice as high in the upper (stigma) portion as in the middle and lower one-third of the pistil tissues. No pectinase activity was detected in the pistil at all stages of development. Extractable pectinase is present at a maximum level in the very young ovary; it decreases rapidly as the ovary develops. Cellulase remains at a moderate level of activity throughout the development of the ovary, except for an increase of about 50% at pollination. Soluble cellulase and pectinase are found in mature pollen. The changes in the cell-wall hydrolytic enzymes in the pistil were pollen-tube growth. It may also promote changes in the cell walls of the pistil cells, although metabolism of the middle lamella during pollen germination is primarily controlled by pollen pectinases.A contribution of the Florida Agricultural Experiment Station, Journal Series No. 3070.