Light Microscope Study of Pollen Tube Growth, Fertilization and Early Embryo and Endosperm Development in the Avocado Varieties Fuerte and Hass

Pollen tube growth, fertilization and early embryo and endospermdevelopment were studied using light microscopy in the avocadovarieties Fuerte and Hass. The ovule was penetrated by a pollen tube by 24 h after pollination.On reaching the ovary, the pollen tube grew along the surfaceof the inner ovary wall. It then grew around the funicle, throughthe micropyle in the inner integument and between the papillatecells at the apex of the nucellus. It entered the embryo sacvia a synergid. Sperm nuclei were present in the embryo sacat 48 h after pollination and fusion of the polar and spermnuclei took place before fusion of the egg and sperm. The endospermnucleus was the first to divide and cell wall formation occurredfollowing division. The first division of the zygote occurredat 5 or 6 days after pollination. In the variety Fuerte less than 20 per cent of the 1- and 2-day-oldembryo sacs had been penetrated by a pollen tube although tubeswere often observed in the integument or nucellus. In the varietyHass over 60 per cent of the embryo sacs were penetrated. Inwas concluded that low yields of the variety Fuerte may be partlyattributable to the failure of the pollen tube to penetratethe embryo sac. Persea americana Mill, avocado, pollen tube, fertilization, embryo, endosperm     

Embryological study on gynogenesis in onion (Allium cepa L.)

Haploid induction in onion can, to date, be induced only via gynogenesis by culturing unfertilized flowers, ovaries or ovules. The process of haploid embryo induction has been macroscopically well studied, but only limited data exist from microscopic examination of ovule development status at the inoculation stage and of the origin of gynogenic embryos. Microscopic studies were carried out using individual donor plants with relatively high embryo induction frequencies (45.9 embryos formed per 100 flowers, on average, for 2 years). Ovaries from flower bud culture were fixed at 1 week intervals up to the 7th week of culture. These were compared with pollinated ovaries at 1 or 2 weeks after pollination. In total, 1428 unfertilized embryo sacs were examined. The results indicate that, at the time of inoculation, ovules within ovaries 2.0–3.0 mm in diameter contained two- or four-nucleate embryo sacs in the smallest ovaries to mature embryo sacs in the largest ovaries. It seems likely that the embryos are actually induced from ovaries cultured at the immature stage. After 1 or 2 weeks in culture, the egg apparatus primarily consisted of distinctly enlarged synergids and the egg cell, which was often detached from the micropylar pole. But free nuclear endosperm was also formed. From the 2nd to 7th week in culture, formation of haploid embryos (from globular to the almost mature cylindrical stage) was detected in 5.7% of the ovules. Their origin, for several reasons, was most likely the egg cell. In addition, ovules containing endosperm only (3.6%) and ovules containing the egg apparatus (0.5%) or both endosperm and embryo (0.4%) were detected. This observation is probably unique and has not yet been reported in other species studied. Received: February 2001 / Revision accepted: 20 April 2001     

In vitro Culture of Abscissed Immature Avocado Embryos

The efficiency of an avocado hybridization programme, usingsmall potted glasshouse plants, was reduced by a high rate ofabscission of immature fruitlets bearing embryos too young forconventional germination. This was overcome in part by culturein vitro of the shed embryos on a liquid medium supplementedwith 0.5 mg l^–1 benzyladenine. Most embryos younger than6 weeks did not survive in culture, but older embryos slowlyproduced multiple shoots, with axillary shoot growth being furtherstimulated by removal of both cotyledons. Embryo response wasnot related to cultivar. Shoots removed from culture could begrafted to seedling rootstocks. Grafting was considered morereliable than dependence on growth of the main root or adventitiousroots in vitro to produce established plants. Persea americana Miller, avocado, abscissed fruitlets, in vitro embryo culture     

Ovule and Embryo Ontogenesis in Bombacopsis glabra (Pasq.) A. Robyns

Ontogeny of the ovule and development of the embryo in Bombacopsisglabra (Pasq.) A. Robyns were examined. The ovule is bitegmic,crassinucellate, and anatropous. The exostome is eccentric relativeto the endostome; stomata occur on the outer integument. Thesingle archesporial cell functions directly as the megasporemother cell. The embryo-sac is bisporic. The organization ofthe nuclei in the mature embryo-sac is normal. The antipodalcells disintegrate soon after formation. Double fertilization takes place; the zygote undergoes a longperiod of dormancy, but the primary endosperm nucleus dividesimmediately to produce first a nuclear-type, later a cellular-typeendosperm. The zygote is of the caryophyllad type. Adventive embryos arise from single cells of the nucellus inthe vicinity of the micropyle, and develop faster than the sexuallyproduced embryo; this leads to anomictic renroduction.     

Development of fertilized ovules and their role in the growth of the pea pod

The histological development of fertilized ovules during fruit-set and development in pea ( Pisum sativum L. cv. Alaska) has been investigated. Killing the ovules on day 0 (anthesis) or day 1 prevented fruit-set and resulted in ovary degeneration. When the ovules were destroyed at later stages the ovaries developed, though the rate of growth of the pod was reduced significantly. Pollination in pea occurs normally the day before anthesis, and fertilization of the egg cell 32 to 48 h later. The first divisions of the zygote and endosperm nuclei started simultaneously (ca 48 h after pollination) but the endosperm developed more rapidly than the embryo; the embryo sac cavity was lined with free endosperm nuclei at the time of beginning suspensor elongation. Extracts of endosperm and ovule coats from ovules at day 7 after anthesis showed fruit-set activity in pea, the latter material having about 3 times more activity than the former per ovule basis. These results indicate that fertilization of the ovule is necessary for fruit-set in pea, and that compounds which induce fruit-set are probably synthesized in the ovules following fertilization.     

Carbohydrate Availability in Relation to Fruitlet Abscission in Citrus

Abscission of flowers and fruitlets in the Washington navelorange (Citrus sinensis [L.] Osbeck) has been characterizedin relation to carbohydrate availability. A main wave of flowerabscission occurs shortly after anthesis while the carbohydratereserves in the tree are high. Fruitlet abscission starts approx.30 d after the commencement of flowering, while carbohydrates(mainly starch) are being accumulated in the leaves. Flowerand early fruitlet abscission are not caused by carbohydrateshortage. During late fruitlet abscission sucrose concentrationin the leaves falls to a low value demonstrating a limitationin supply and competition among the developing fruitlets forcarbohydrates. Concentrations of sucrose and reducing sugarsin the peel of the fruitlets also fall to low values, and arelationship could be demonstrated between these free sugarlevels and abscission. Ringing increases carbohydrate supplyto fruit and reduces late fruitlet abscission, but only hasa marginal effect on the growth of the fruitlets, which seemsless sensitive than abscission to carbohydrate shortage. Thelimitation of carbohydrate supply to the fruitlets occurs whilestarch levels in the leaves remain high. Slow mobilization ofstarch reserves may be one factor limiting set in Citrus. Copyright2001 Annals of Botany Company Carbohydrate supply, citrus, fruit growth and abscission, ringing, navel orange, starch, sugar metabolism     

An ultrastructural study of early endosperm development and synergid changes in unfertilized cotton ovules

Excised, unfertilized cotton (Gossypium hirsutum L.) ovules were cultured for 1–5 days postanthesis and embryo-sac development was studied with the electron microscope. In some ovules the two polar nuclei fuse and the diploid endosperm nucleus goes through a limited number of free nuclear divisions after 2–3 days in culture. Each nucleus has two nucleoli, in contrast to nuclei of fertilized triploid endosperm which have three nucleoli. Precocious cell walls form between the endosperm nuclei on the 3rd day in culture. The morphology of the plastids, mitochondria, rough endoplasmic reticulum (RER), dictyosomes and microbodies, and the amount of starch and lipid in the diploid cellular endosperm are similar to those of the central cell. A few large helical polysomes appear close to plastids and mitochondria. After 2 days in culture, one of the two synergids in the unfertilized cultured ovules shows degenerative changes which in fertilized ovules are associated with the presence of the pollen tube, i.e., increase in electron density, collapse of vacuoles, irregular darkening and thickening of mitochondrial and plastid membranes, disappearance of the plasmalemma and the membranes of the plasmalemma and the membranes of the RER. The second synergid remains unchanged in appearance. The egg cell does not shrink or divide or show structural changes characteristic of the cotton zygote. Embryo-sac development is arrested on the 4th and 5th days in culture. The nucellus continues growth and at 14 days crushes the degenerate embryo sac.     

An embryological study ofPlatanthera bifolia (Orchidaceae)

Flowers ofPlatanthera bifolia were hand-pollinated and fixed in FPA₅₀ after 2, 5, 7, 14, and 21 days. Ovules, made transparent in Herr's clearing fluid, were investigated using confocal scanning laser microscopy. Pollination initiates the megasporogenesis. Two days after pollination dyads are frequent. Three days later most embryo sacs contain two nuclei. Seven days after pollination the embryo sacs are 4–8-nucleate and some are organized, and a week later all embryo sacs are organized and fertilization takes place. The embryo sac development follows thePolygonum type. Twenty-one days after pollination the egg nuclei have been fertilized and the embryo sacs contain 2- to many-celled embryos. A suspensor is formed during early stages of embryo development but degenerates later. Fertilization of the central nucleus does not lead to endosperm development.     

焦核与大核龙眼雌配子体发育、授粉受精及早期胚胎发育的比较研究

利用人工授粉,采用压片法对大核龙眼‘九月乌’和焦核龙眼‘闽焦64-1’、‘闽焦64-2’、‘白核’等的自交与杂交后花粉管的生长特性进行研究,同时应用常规石蜡切片技术对大核与焦核龙眼的雌配子体以及合子胚早期发育进行观察。结果表明,龙眼胚珠在单核胚囊形成前就开始败育,且焦核品种(系)的败育率显著高于大核品种。不同亲本组合的授粉率存在差异,所有授粉组合在授粉36～48 h后均能观察到1个花粉管生长并进入胚囊受精。焦核品种(系)的胚胎在谢花后10 d开始败育,且败育率明显高于大核品种。受精是龙眼子房发育的首要条件,胚珠败育的雌蕊在谢花后10 d不膨大,不能发育形成焦核果实。谢花后10～30 d的早期胚胎败育是形成焦核龙眼的主要原因。焦核品种‘白核’胚乳具有成胚能力。约有24%的‘闽焦64-1’胚珠在胚胎发育过程中,其助细胞、合点端细胞及胚乳发生异常,这可能与早期胚胎败育有关。     

Cytological study of pollen tube growth and early seed development inPetunia inflata

Pollen tube growth from the stigma into the ovule, and the early fruit and seed development following fertilization were examined using fluorescence microscopy, scanning electron microscopy and light microscopy inPetunia inflata. After growing intercellularly in the transmitting tract for 24–36 hr, the pollen tubes emerged into the top part of the ovary cavity and grew along the surface of the septum to reach the ovule. It grew around the furnicle and penetrated the micropyle to enter the embryo sac for fertilization. After fertilization, the endosperm nucleus divided first before the embryo, and the cell wall formation occurred following the division, exhibiting the pattern of cellular type of endosperm development. The first division of the zygote did not occur until 3 days after pollination. At 6 days after pollination, the seeds grew considerably and the endosperm has gone through multiple rounds of cell division. High starch formation in the integument, especially around the embryo sac, was also observed.     

Functional Anatomy of the Ovule in Broad Bean (Vicia faba L.): Ultrastructural Seed Development and Nutrient Pathways

Ovules of broad bean (Vicia faba L.) were studied to discloseultrastructural features, which can facilitate nutrient transportto the embryo sac from 10 d after pollination (DAP) to the matureseed. Fertilization occurs during the first 24 h after pollination.The endosperm is a coenocyte, which is eventually consumed bythe embryo. By 10 DAP the inner integument is degraded and theouter integument adjoins the embryo sac boundary. The heart-shapedembryo approaches the embryo sac boundary at two sites, whichhere are named contact zones. Small integument cells in theneighbourhood of the first formed contact zones become separatedby prominent intercellular spaces. A heterogenous scatteringmaterial, probably representing secretion products accumulatesin these spaces. By 14-16 DAP the integument exudate disappears,and the suspensor degenerates. As the contact zones increasein size, wall ingrowths form a bridging network in the narrowspace between the embryo sac boundary and the extra-embryonicpart of the endosperm wall. The epidermal cells of the embryoseparate adjacent to these zones, and develop conspicuous wallingrowths. At 20 DAP vacuoles showing various stages in formationof protein bodies appear in the cells of the embryo.Copyright1994, 1999 Academic Press Vicia faba, broad beans, ovule, seed, nutrient transport     

Disruption of endosperm development: an inbreeding effect in almond (Prunus dulcis)

A homozygous self-compatible almond, originated from self-fertilization of a self-compatible genotype and producing a reasonable yield following open pollination, exhibited a very high fruit drop rate when self-pollinated. To investigate whether fruit dropping in this individual is related to an abnormal development of the embryo sac following self-fertilization, histological sections of ovaries from self and cross-pollinated flowers were observed by light microscopy. Additionally, the presence of pollen tubes in the ovary and fruit set were determined for both types of pollination. Despite pollen tubes reached the ovary after both pollinations, differences in embryo sac and endosperm development after fertilization were found. Thus, while for cross-fertilized ovules a pro-embryo and an endosperm with abundant nuclei were generally observed, most self-fertilized ovules remained in a previous developmental stage in which the embryo sac was not elongated and endosperm nuclei were absent. Although 30 days after pollination fruit set was similar for both pollination types, at 60 days it was significantly reduced for self-pollination. These results provide evidence that the high fruit drop in this genotype is the consequence of a disrupted development of the endosperm, what could be an expression of its high level of inbreeding.     

Anatomy of Watermelon Embryo Sacs Following Pollination, Non-pollination or Parthenocarpic Induction of Fruit Development

There is little information on the fate of embryo sacs in plantovules if pollination is prevented. In this study embryo sacsfrom watermelon were observed over a 13 day period followingflowering with (a) normal pollination, (b) non-pollination and(c) induction of parthenocarpic fruit development with naphthaleneacetic acid. Following pollination, and prior to fertilizationapproximately 2 days later, the embryo sacs completed developmentand consisted of two synergids with prominent filiform apparatus,an egg cell, a central cell with two polar nuclei and threeantipodal cells. Sperm nuclei were observed within the embryosac at 2 days and by 4 days the endosperm was proliferating.In the non-pollination treatment the embryo sac was still intactafter 4 days although the antipodal nuclei were becoming hardto distinguish. By 7 days only the two synergids and the eggcell were still well defined, the polar nuclei appeared in somepreparations to be fused, and the antipodals had degenerated.By 10 days the embryo sac was a structure-less watery mass.In parthenocarpic fruit the fate of the embryo sac was similarto that in non-pollinated fruit except that final breakdownwas delayed past 10 days. Maturity of the majority of embryo sacs in an ovary appearedto be contemporaneous with penetration of the pollen tube, andon the basis of the anatomical results it seems possible thatembryo sacs could be fertilized up to 2 days beyond the normaltime. Citrullus lanatus, watermelon, embryo sac, anatomy, pollination, parthenocarpy     

Unfertilized ovules of Epilobium obcordatum (Onagraceae) continue to grow in developing fruits

To determine whether unfertilized ovules continue to grow when in an ovary containing fertilized ovules, we measured ovule lengths in developing fruits of Epilobium obcordatum that were harvested 4, 5, 8, and 10 d post pollination. We found that unfertilized ovules that were in the presence of fertilized ovules continued to grow and that there was a broad range of overlap in their sizes at all sampling times. This effect was found for two types of unfertilized ovules that occur throughout the length of the ovary: normal, unfertilized ovules, apparently bypassed by pollen tubes; and sterile ovules lacking an embryo sac. In addition, there is a position effect within developing fruits. Both fertilized and unfertilized ovules are larger at the stylar end. In six samples resulting from pollination with a single pollen tetrad, a total of 18 embryos were found, and the effect on unfertilized ovules, greatest at the stylar end, diminished with distance from the ovules with embryos. Our results are consistent with the interpretation that diffusible hormones produced by developing seeds cause nearby unfertilized ovules to grow. We conclude that caution is necessary when attempting to infer ovule fertilization histories from the appearances of ovules in developing and mature fruits. What are often inferred to be aborted seeds, in many cases, may not be seeds at all. They may be enlarged, unfertilized ovules.     

Observation of Pollen Tube Behaviour and Early Embryogenesis Following Interspecies Pollination Between Actinidia deliciosa and A. arguta

The pollen tube behaviour in the style and early embryogenesis following interspecies pollination between Actinidia deliciosa No. 26 and A. arguta were observed by means of fluorescence and light microscopy. Pollen grains germinated on the papillate stigma and pollen tubes grew along the V-shaped open-type style. Pollen tubes showed slower growth and reached the ovules 50--60 hours later than those of the control. Several abnormalities of pollen tubes have been observed at the base of the style, including wave-like pollen tubes, pollen tubes with swollen or pointed tips, with variable diameters, and a few with irregular growth. Random deposition of callose along pollen tube wall and even the whole wall was observed. About 26.74 % of the ovules were successfully fertilized and developed into seeds, among them 68.50% of the seeds were normal and 31.50% were abortive. About 11.41% were empty seeds without embryo and endosperm. Unfertilized small ovule was 61.45 %. Normal seed and its embryo were smaller than those of the control. The development of embryo was of the Soland type. The endosperm was cellular. The zygote remained quiescent for about 12-15 days before it started to divide, eventually forming a cotyledonary embryo 50 days after pollination.     

Seedlessness and Parthenocarpy inPistacia veraL. (Anacardiaceae): Temporal Changes in Patterns of Vascular Transport to Ovules

Pistacia vera‘Kerman’ (pistachio nut) typicallyproduces high numbers of seedless or blank fruits. Patternsof vascular transport into fruits and ovules were studied over3 years by following the movement of disodium fluorescein solutionfrom cut branches into developing fruitlets. Early in the season,vascular conductivity is intact through to the chalazal endof the ovule. Soon afterwards, the percentage of ovules withvascular conductivity through to the chalaza declines, and ina variable fraction of fruits, movement of the fluorochromesolution becomes blocked either at the placenta or in the funiculus.Six to 9 weeks after anthesis there is blockage in 90 (1 year)to 100% (2 years) of fruits. Subsequently, vascular conductivityresumes in 83.3% (3 year mean) of ovules, a percentage thatcorrelates well with the mean percentage of seeded nuts at harvest(77.5%). Ovules from fruits with dysfunctional vascular conductionearly in the season are smaller than those with fully functionalvascular tissue. At the time conductivity declines, a high percentageof those ovules with blocked vascular movement lack endospermand appear to be unfertilized; none of the ovules that retainfull vascular flow lack endosperm. Pollination using gamma-irradiatedpollen (⁶⁰Co, 1.0 kGy) led to a nearly three-fold increase inthe production of blank nuts. The results indicate that fluoresceintransport may be a valuable tool to predict the fate of ovules,and are consistent with the hypothesis that parthenocarpic fruitset may be an important factor in blank nut production in pistachio.Copyright1999 Annals of Botany Company Pistachio,Pistacia veraL., fluorescein, seed set, seedlessness, parthenocarpy, blanking, ovule, funiculus, chalaza, embryo.     

OVULE AND EMBRYO DEVELOPMENT IN DORITIS PULCHERRIMA (ORCHIDACEAE)

The placental ridge began to proliferate 10 days after pollination. Megaspore mother cell underwent meiosis to form two dyads at first division. At 50 days two megaspores and generating dyad were formed by second division. The functional megaspore divided successively three times to form an eight-nucleate embryo sac at 60 days. Double fertilization occurred forming the zygote and endosperm initial cell. However, the endosperm initial cell degenerate soon thereafter. The zygote divided to form a terminal cell, the middle cell and suspensor initial cell at 70 days. The terminal and middle cells successively divided to form a multi-celled embryo up to 120 days after pollination. Histochemical study showed that the stainability of DNA, RNA and total proteins were almost constant during ovule and embryo development. Stainability of total carbohydrates decreased.     

Role of Ethylene in Avocado Fruit Development and Ripening: I. FRUIT DROP

A survey of avocado (Persea americana Mill.) fruit drop andethylene production was carried out during fruit development.Natural drop of avocado fruitlets started soon after set (May)and continued at a gradually decreasing rate until September,except for a temporarily increasing rate in late July. Fruitletsweighing up to 0?2 g dropped at a rate of over 30 percent perweek. With larger fruits, the rate was under 1 percent per week.Fruit drop ceased after September, when fruit growth declinedand the seed coat began to shrivel. A positive correlation was found between the rate of fruitletand fruit drop and ethylene production. Fruitlets with defectiveseeds produced ethylene at a very high rate of 7–10 timesmore than apparently normal fruits. The high incidence of defectiveseeds might be the cause of the very high levels of ethyleneproduction by young avocado fruitlets. The seed was found to be the main site of ethylene productionin fruitlets. Abscisic acid (ABA) levels in young abscissing fruits were 7times as high as those in non-abscissing fruits.     

A Flower and Pod Staging System for Soybean

Flower and pod abscission limit soybean yield. A system forquantifying flower and pod development based on the morphologicalappearance of the flower prior to and following anthesis hasbeen developed to aid in studies of pod abscission. Changesin the appearance of the corolla, primarily the banner petal,are used to distinguish the different stages of the system.External pistil dimensions have been correlated with internalfeatures for each stage of development. From anthesis to podset, pistil length and weight increase almost two- and fivefold,respectively, and ovule development progresses from unfertilizedegg cells to embryos surrounded by cellular endosperm. Pod determinedare correlated with ovule length and width and embryo cell number.Flower and pod stages can be determined in situ, thus permittingnon-destructive observation and experimental manipulation offlowers or pods without necessarily impeding their development.Stages have been identified that indicate precisely when podset occurs and when young pods cease growing and ultimatelyabscise. This system of flower and pod staging is useful instudies designed to assess effects of abiotic or biotic stressand genetic factors on pod set and abortion. Abscission, anthesis, Glycine max (L.) Merr, embryo development, pod set