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.     

鹤顶兰花粉管在子房中的生长途径

运用扫描电镜对鹤顶兰(Phaiustankervilliae(Aiton)Bl.)花粉管在子房内的生长途径进行了观察。结果表明:花粉管在子房中的生长途径可以分为3个阶段:(1)沿子房壁轴向生长阶段,从授粉开始至大孢子母细胞四分体时期,花粉管经过合蕊柱到达子房,经由胎座基部沿子房壁轴向生长;(2)沿子房径向生长阶段,二核胚囊之后,花粉管在胚珠之间穿梭,以径向生长为主;(3)朝珠孔定向生长阶段,胚囊成熟时,花粉管朝珠孔定向生长进入胚囊。实验结果说明花粉管的定向生长受胚珠的分子信号调控。     

中棉×戴维逊氏棉胚胎发育的研究

以棉花栽培种中棉作母本,野生种戴维逊氏棉作父本进行杂交试验,并用中棉自交作对照,比较研究了杂交情况下花粉粒的萌发、花粉管的生长、受精作用及胚和胚乳的发育过程,得到以下结果:(1)中棉×戴维逊氏棉花粉粒的萌发及花粉管在异己花柱中的生长基本正常,有花粉管胚珠的频率约20％,为中棉自交的1/4左右;(2)在杂交情况下,有花粉管进入的胚珠基本上能实现受精;(3)杂种胚乳在授粉后7天发育异常,11天开始解体,16天才有部分胚珠的胚乳开始形成细胞壁;(4)杂种胚不分化或畸形分化,在授粉后11—22天坏死。     

Investigation of Sexual Process in Interspecific Crosses Between Gossipium hirsutum and G. arboreum

The present investigation is the first of a series of cytological researches on inter-specific hybridization of cottons. The present report deals with the cytological behavior of sex and related cells revealed in crossing Gossipium hirsutum (female parent) with G. arboreum. Self-pollenated G. hirsutum served as control. The germination of pollen grains on the stigma and the growth of pollen tubes in the style and the entry of pollen tubes into the micropyles in crosses are in a fairly normal' manner when compared with controls. The percentages of pollen tubes entering ovules of the crossed and controls have no significant difference, being 48.2% in crosses and 54.7% in controls. Fertilization proceeds also normal in cross pollinated ovules. About 92.7% of pollinated ovules are found to have completed the fusion of sexual elements. It is thus: shown that the growth of foreign pollen tubes and fusion of female elements with foreign sperms are not the factors which cause failure in hybridization. The development of endosperm in crosses is abnormal. The number of free nuclei of hybrid endosperm is much smaller than that of controls; the formation of hybrid cellular endosperm is 6 or 7 days earlier than that of controls. The hybrid endosperm cells start to abort soon after their formation. About 14 days after pollination they become completely disintegrated. The hybrid embryo which thus far developed normally becomes arrested in its development and aborts also after that time. It may be concluded that the sterility of crosses between G. hirsutum and G. arboreum is primarily attributed to the abortion of hybrid endosperm. The sound development of the hybrids should be sought in the improvement nutrition of the hybrid embryo from resource other than their own endosperm.     

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.     

On the Fertilization of Oryza sativa L. × Sorghum vulgare Pets.

1.The pollen germination of Sorghum vulgate appeared normal on the stigma of the Oryza sativa, but the pollen tubes grew slowly in the style. Some of the pollen tubes may become enlarged in their tips or sometimes bursting, while others have continued to grow and entered the embryo sacs. 2. The growth rate of the pollen tubes varied widely. A few pollen tubes were observed in the embryo sacs of the materials 2 hours after pollination, but most of them entered the embryo sacs much later. 3. The zygote associated with a paucity of endosperm nuclei was observed in the materials 1 day after pollination. The double fertilization and 8–12-celled proembryo associated with a number of the free nuclei of the endosperm appeared with a rather high frequency (10.3%) in the materials 3 days after pollination. Some of them are normal in appearance and others may show more or less abnormalities. 4. No division figure was found except in one single case in which mitoses have occurred in both the proembryo and the endosperm. It is most likely that in such case the proembryo and the endosperm if left intact might develop further. 5. A 80-celled embryo was the biggest one which appeared in the materials 5 days after pollination. In general, no cells were ever formed in the endosperm, except in one instance among the 7 days materials the endosperm became cellular in micropylar end. In all other cases the endosperm either ceased to develop early or disorganized. The disorganized endosperm materials are considered to be utilized by the embryo. 6. In certain instances the free nuclei of the endosperm were not distributed at random. They were not equal in size and might fuse into giant nuclelei. 7. The most striking feature is that in the embryo sacs, in which double fertilization or proembryo and endosperm have occurred, a dark stained pollen tube was commonly present. This fact leads us to the conviction that in general only if a healthy pollen tube entered the embryo sac, double fertilization can take place and further development can proceed. 8. In certain cases the protoplasm of the embryo cells appeared scanty. It is apparently that the normal metabolism of the embryo was disturbed owing to the lack of nutrient, and the death of the embryo ensued. 9. No differentiated embryo was observed and no mature seeds were produced. The materials fixed 12 days after pollination showed a variety of abnormalities and collapses. The authors believe that the failure of seed production of rice X kaoliang was primarily due to the fact that the pollen tubes in the style grew too slowly to reach the embryo sacs in time. The consequence is that the double fertilization took place only in a late stage while the male and female gametes may have already become unhealthy. In addition, in this late stage the stored starch in the maternal tissues having gradually disappeared, the nutrient supply to the embryo sac was therefore limited and the young embryo and endosperm were finally in starvation.     

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     

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     

异叶苦竹花粉管生长及双受精过程

以异叶苦竹为材料,采用扫描电镜、荧光显微镜技术及传统的石蜡制片技术,解剖观察其花粉管生长途径及双受精过程。结果表明:(1)授粉后,花粉在柱头上吸水膨胀,约30 min即可萌发。(2)授粉1～2 h后花粉管可达到花粉长度的5～10倍,花粉管在柱头分支中进一步伸长,并开始伸入花柱中生长。(3)授粉后5 h,大量花粉管沿引导组织进入花柱基部与子房顶部之间的子房壁,有少量花粉管在子房壁与外珠被之间的缝隙中生长。(4)授粉后8 h,少量花粉管到达珠孔端。(5)授粉后15～18 h,精核与极核融合,形成初生胚乳核;精、卵核融合,形成合子。(6)授粉后20～30 h,仍可在花柱中见到大量呈束状的花粉管。(7)授粉后48 h,子房内的大部分花粉管出现解体,大多数花粉死亡。研究认为,精细胞到达胚珠的时间为8 h。     

油橄榄授粉受精和胚胎发育过程观察

该研究以油橄榄“鄂植-8”为材料,应用压片荧光观察花粉在柱头和花柱中的萌发及生长情况,采用石蜡制片法观察油橄榄的胚珠结构特点和授粉受精过程。结果表明：油橄榄为1子房2心室4胚珠,珠心较发达,由多层细胞构成,属于厚珠心椭圆型胚珠,胚珠直生;花开时花粉粒落到柱头上立刻萌发,随后花粉管在花粉通道中生长,再后进入子房经子房内表面,大部分花粉管不能到达胚珠,仅少数沿胎座生长经珠柄进入珠孔,释放2个精子,2个精核分别进入卵细胞和极核,并与卵核及极核相互融合;观察到合子中雌、雄性核仁融合的过程。授粉受精各阶段经历的时间为花粉落到柱头上立刻萌发;授粉后6d左右,花粉管长入胚珠的珠孔,随后释放精子;10 d左右完成授粉受精,30 d左右形成心型胚,40 d左右胚发育几乎成熟。在进行子房石蜡切片中,共观察到612个完整子房切片,发育完整的子房为97.53％,完成授粉受精的胚珠为15.52％。油橄榄在自然授粉下,约85％的胚珠授粉受精过程不能完成,在一定程度上影响其坐果。该研究结果为油橄榄授粉受精、高效生产等提供了理论依据。     

腊梅的受精作用及胚胎发生

腊梅 (Chimonanthuspraecox)花两性 ,离心皮雌蕊着生在杯状花托上 ,柱头线形 ,干性。花粉经昆虫传播 ,落在柱头上 1d后萌发 ,第 8d从珠孔进入 ,第 1 4d左右完成双受精 ,为珠孔受精。胚乳为核型胚乳 ;初生胚乳核经短暂休眠进行核分裂 ,位于合点端的游离核首先形成细胞 ,并从合点向珠孔端细胞化 ,第 37d胚乳充满整个囊腔。合子经过近 2周的休眠后开始分裂 ,随着胚的发育 ,大部分胚乳降解 ,为胚的发育提供营养。合点端的胚乳细胞则侵入合点珠心组织 ,为胚进一步发育提供营养。其胚胎发生为柳叶菜型。     

腊梅的受精作用及胚胎发生

腊梅(Chimonanthus praecox)花两性,离心皮雌蕊着生在杯状花托上,柱头线形,干性。花粉经昆虫传播,落在柱头上1 d后萌发,第8d从珠孔进入,第14d左右完成双受精,为珠孔受精。胚乳为核型胚乳;初生胚乳核经短暂休眠进行核分裂,位于合点端的游离核首先形成细胞,并从合点向珠孔端细胞化,第37d胚乳充满整个囊腔。合子经过近2周的休眠后开始分裂,随着胚的发育,大部分胚乳降解,为胚的发育提供营养。合点端的胚乳细胞则侵入合点珠心组织,为胚进一步发育提供营养。其胚胎发生为柳叶菜型。     

扇脉杓兰花粉超微结构及花粉管生长的观察

为明确扇脉杓兰花粉形态结构及雄性生殖特性,利用扫描电镜、透射电镜和荧光显微镜对花粉形态和超微结构及花粉管生长过程进行观察。结果表明:(1)扇脉杓兰单粒花粉长球形,表面光滑无特征纹饰,有少量胶黏物质,一些表面有2个或以上的深凹陷,凹陷内有球形突起的内容物。(2)花粉壁分为由棒状的基柱小单元组成的外壁和纤维素果胶组成的内壁,有覆盖层;生殖细胞近圆形,细胞核大而致密;营养细胞多弧形,核质分散;花粉粒细胞质含有大量的线粒体、质体和小泡等细胞器,淀粉、蛋白质和多糖含量丰富。(3)花粉管萌发后沿子房壁方向伸长,授粉20d花粉管伸长生长并不明显,授粉30d伸长的花粉管中出现大量胼胝质塞,并且伸长方向转为胚珠中间,花粉管逐渐接近胚珠,在授粉后50d基本完成受精作用。研究认为,扇脉杓兰花粉发育正常,不阻碍有性生殖过程。     

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.     

Fruit characteristics, seed production and pollen tube growth in the wild chilli pepper Capsicum flexuosum

Fruits formed after different pollination regimes (flowers hand pollinated, unpollinated, and open pollinated) and the seeds obtained were characterized in the wild chilli pepper Capsicum flexuosum Sendtn. Pollen tube development in vivo and ovary growth were also analyzed. Seedless fruits and empty seeds were abundant among the fruits from hand pollinated and open pollinated flowers, while no more than one seed with embryo was found in a low percentage of fruits from such pollination treatments. Parthenocarpic fruits were formed from unpollinated flowers. Pollen tube growth was arrested in the upper third of the style for almost all pollen tubes except for a single one that may continue elongating occasionally. The ovary size increased continuously after pollination, even without fertilization. The sum of the evidence registered may help to explain the low number of seeds with embryo harvested, the abundance of seedless fruits formed from pollinated flowers (possibly parthenocarpic), and the high rate of parthenocarpic fruits formed from unpollinated flowers.     

Gene transfer between canola (Brassica napus L. and B. campestris L.) and related weed species

Brassica species are particularly receptive to gene transformation techniques. There now exists canola genotypes with transgenic herbicide resistance for glyphosate, imidazolinone, sulfonylurea and glufosinate herbicides. The main concern of introducing such herbicide resistance into commercial agriculture is the introgression of the engineered gene to related weed species. The potential of gene transfer between canola (Brassica napus and B. campestris) and related weed species was determined by hand pollination under controlled greenhouse conditions. Canola was used as both male and female parent in crosses to the related weed species collected in the Inland Northwest region of the United States. Weed species used included: field mustard (B. rapa), wild mustard (S. arvensis) and black mustard (B. nigra). Biological and cytological aspects necessary for successful hybrid seed production were investigated including: pollen germination on the stigma; pollen tube growth down the style; attraction of pollen tubes to the ovule; ovule fertilisation; embryo and endosperm developmental stages. Pollen germination was observed in all 25 hybrid combinations. Pollen tubes were found in the ovary of over 80% of combinations. About 30% of the hybrid combinations developed to the heart stage of embryo development or further. In an additional study involving transgenic glufosinate herbicide resistant B. napus and field mustard it was found that hybrids occurred with relatively high frequency, hybrids exhibited glufosinate herbicide resistance and a small proportion of hybrids produced self fertile seeds. These fertile plants were found to backcross to either canola or weed parent.     

Evaluation of pollen tube growth ability in Petunia species having different style lengths

Pollen tube growth is essential for the fertilization process in angiosperms. When pollen grains arrive on the stigma, they germinate, and the pollen tubes elongate through the styles of the pistils to deliver sperm cells into the ovules to produce the seeds. The relationship between the growth rate and style length remains unclear. In previous studies, we developed a liquid pollen germination medium for observing pollen tube growth. In this study, using this medium, we examined the pollen tube growth ability in Petunia axillaris subsp. axillaris, P. axillaris subsp. parodii, P. integrifolia, and P. occidentalis, which have different style lengths. Petunia occidentalis had the longest pollen tubes after 6 h of culture but had a relatively shorter style. Conversely, the pollination experiments revealed that P. axillaris subsp. parodii, which had the longest style, produced the longest pollen tubes in vivo. The results revealed no clear relationship between the style lengths and the growth rate of pollen tubes in vitro. Interspecific pollinations indicated that the styles affected pollen tube growth. We concluded that, in vitro, the pollen tubes grow without being affected by the styles, whereas, in vivo, the styles significantly affected pollen tube growth. Furthermore, interspecific pollination experiments implied that the pollen tube growth tended to be suppressed in the styles of self-incompatibility species. Finally, we discussed the pollen tube growth ability in relation to style lengths.     

The progamic phase, embryo and endosperm development in an intraspecific Tulipa gesneriana L. cross and in the incongruent interspecific cross T. gesneriana × T. agenensis DC.

 The development of the embryo and endosperm has been investigated in an intraspecific Tulipa gesneriana cross and in the incongruent cross T. gesneriana ×T. agenensis at intervals of 10 days, from 12 to 82 days after pollination (DAP). In both tulip crosses, the zygote gives rise to an apparently undifferentiated cell mass, the proembryonal cell mass, on which a suspensor then develops. Subsequently, a globular embryo is formed on top of the suspensor. This embryo finally elongates, giving rise to a spindle-shaped embryo. The cellular endosperm fills the whole embryo sac in mature seeds, except for a region immediately around the embryo. In both crosses, aberrant developments were found. In the intraspecific T. gesneriana cross, the pollen tubes did not open in a number of ovules. In other ovules, the pollen tubes seemed to have opened, but an embryo or endosperm was not found or only endosperm was observed. In the cross T. gesneriana ×T. agenensis, fewer pollen tubes entered the ovules than in the intraspecific T. gesneriana cross. The ovules with embryo and endosperm formation of the incongruent interspecific cross showed, in general, retarded development in comparison with the intraspecific T. gesneriana cross. The first globular embryos and spindle-shaped embryos were found at the later fixation dates and the relatively lower number of spindle-shaped embryos at 82 DAP had a shorter average length. The number of ovules with deformations in embryo and/or endosperm development was also higher in the cross T. gesneriana × T. agenensis in comparison with the intraspecific T. gesneriana cross. Between 87% and 100% of the ovules with embryo and endosperm development showed normal development in the intraspecific T. gesneriana cross, while in the incongruent interspecific cross, from 22 DAP, between 17% and 56% of the ovules showed normal development. Of those ovules with aberrations in embryo and/or endosperm formation, about 80% had a deformed endosperm, of which more than 50% also contained a deformed embryo. Embryos of the incongruent cross might be saved by the application of embryo rescue techniques. Received: 10 December 1996 / Revision accepted: 23 April 1997     

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.     

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.