Origin of Multicellular Pollen and Pollen Embryos in Cultured Anthers of Pepper (Capsicum Annuum)

Anthers of Capsicum annuum L. were cultured on Murashige and Skoog (MS) medium containing 0.1 mg l⁻¹ NAA and 0.1 mg l⁻¹ kinetin. Inoculated anthers were subjected to 31 °C and development of microspores in anthers of varying stages was observed cytologically using 4′-6-diamidino-2-phenylindol-2HCl (DAPI). Pepper was characterized by a strong asynchrony of pollen development within a single anther. Percentage of pollen at different stages changed with the culture period, and the proportion of dead pollen increased drastically from day 2 after culture. Microspores that were cultured at the late-uninucleate stage followed one of two developmental pathways. In the more common route, the first sporophytic division was asymmetric and produced what appeared to be a typical bicellular pollen. Embryogenic pollen was formed by repeated divisions of the vegetative nucleus. In the second pathway, which occurred in fewer microspores, the first division was symmetric and both nuclei divided repeatedly to form embryogenic pollen. In early-bicellular pollen, sporophytic pollen was produced through division of the vegetative nucleus. In mid-bicellular pollen, the generative nucleus may undergo division to produce two or more sperm-like nuclei. However, division of the generative nucleus alone to form the embryo was never observed. The anther stage optimal for embryo production contained a large proportion (>75%) of early-binucleate pollen. Associations were found among the percentage of early-binucleate pollen, the frequency of embryogenic multinucleate pollen, and the yield of pollen embryos.     

Embryology of <Emphasis Type="Italic">Cardiopteris</Emphasis> (Cardiopteridaceae,Aquifoliales), with emphasis on unusual ovule and seed development

Cardiopteris (Cardiopteridaceae), a twining herb of two or three species distributed from Southeast Asia to Northern Australia, requires an embryological study for better understanding of its reproductive features. The present study of C. quinqueloba showed that the ovule and seed development involves a number of unusual structures, most of which are unknown elsewhere in angiosperms. The ovule pendant from the apical placenta is straight (not orthotropous), ategmic, and tenuinucellate, developing a monosporic seven-celled/eight-nucleate female gametophyte with an egg apparatus on the funicular side. Fertilization occurs by a pollen tube entering from the funicular side, resulting in a zygote on the funicular side. The endosperm is formed by the cell on the funicular side in the two endosperm cell stage. While retaining a (pro)embryo/endosperm as it is, the raphe (differentiating late in pre-fertilization stages) elongates toward the antiraphal side during post-fertilization stages, resulting in an anatropous seed. The two-cell-layered nucellar epidermis (belatedly forming by periclinal divisions), along with the raphe, envelops the embryo/endosperm entirely as the seed coat. The possibility was discussed that the arrested integument development triggers a series of the subsequent unusual structures of ovule and seed development. The fertilization mode in Cardiopteris underpins the hypothesis that the Polygonum?type female gametophyte comprises two four-celled archegonia.     

A high throughput <Emphasis Type="Italic">Brassica napus</Emphasis> microspore culture system: influence of percoll gradient separation and bud selection on embryogenesis

Microspore culture for the purpose of developing doubled haploid plants is routine for numerous plant species; however, the embryo yield is still very low compared with the total available microspore population. The ability to select and isolate highly embryogenic microspores would be desirable for high embryo yield in microspore culture. To maximize the efficiency of canola microspore culture, a combination of bud size selection and microspore fractionation using a Percoll gradient was followed. This approach has consistently given high embryo yields and uniform embryo development. Microspores isolated from buds 1.5 to 4.4 mm in length of Brassica napus genotypes Topas 4079, DH12075, Westar and 0025 formed embryos at different frequencies. The most embryogenic bud size range varied with each cultivar: Topas 4079 3.5–3.9 mm, DH12075 2.0–2.4 mm, and Westar and 0025 2.5–2.9 mm. When the microspores from 2.0 to 2.4 mm buds of DH12075 were carefully layered on top of a discontinuous Percoll gradient of 10, 20 and 40%, and subsequently spun through the Percoll layers by centrifugation, bands were formed containing populations of microspores of uniform developmental stage. The middle layer of the gradient contained the late uninucleate and early binucleate microspores that were the most embryogenic. In addition, the relationship between the bud size, developmental stage of isolated microspores, Percoll gradient concentration and the embryogenic frequency of each cultivar were studied. Optimization of these factors is required for each genotype evaluated.     

极小种群野生植物崖柏的生殖物候、传粉及胚胎发育研究

以濒危植物崖柏（Thuja sutchuenensis Franch.）为对象,对其生殖物候、传粉机制进行观察,并采用石蜡切片法对其胚胎发育过程进行研究。结果显示：崖柏于8月分化出大、小孢子叶球,次年3月传粉,为花粉无气囊、具传粉滴、胚珠直立型传粉机制,球果于10月开裂;显微观察发现,传粉期花粉进入珠孔后,贮藏在珠心上方的贮粉室内,同时珠心组织中分化出孢原细胞,进入雌配子体发育阶段,5月中旬,花粉管开始萌发,6月初完成受精,进入胚胎发育阶段,10月初,胚胎发育成熟。研究表明崖柏从大、小孢子叶球形成至种子成熟的整个发育过程中均存在败育,而胚珠败育及雌配子体游离核时期至幼胚发育期间的败育是其生殖障碍的主要原因。本研究获得了崖柏生殖生物学的基础资料,为其人工繁育和制定保护策略提供了重要依据。     

Factors influencing induction and maintenance of <Emphasis Type="Italic">Vitis rotundifolia</Emphasis> Michx. embryogenic cultures

Unopened leaves, petioles and fully opened leaves from micropropagation cultures of five Vitis rotundifolia Michx. varieties were cultured on induction medium to study their embryogenic response. Among the various explants tested, the maximum number of varieties produced embryogenic cultures from unopened leaves followed by fully opened leaves and petioles. Based on morphological differences, two types of embryogenic cultures were identified. Friable cultures typically arose as proembryonic masses (PEM) on induction medium, whereas somatic embryo production without an intervening PEM stage was observed in compact cultures. Of the five varieties tested, the highest frequency of embryogenic response was observed from fully opened leaves of ‘Supreme’ and unopened leaves and petioles of ‘Delicious’. Attempts to initiate suspension cultures from varieties resulted in proliferation and maintenance of ‘Alachua’ and ‘Carlos’ cultures in liquid medium for 16 weeks. Embryogenic potential of varieties was studied on cultures growing on embryo development medium. The maximum number of cotyledonary stage somatic embryos from 0.2 g proembryonic masses were observed in ‘Carlos’ (379.3) followed by ‘Alachua’ (350.0) and ‘Delicious’ (305.0). Cotyledonary stage somatic embryos germinated when cultured on Murashige and Skoog medium containing 1 μM Benzyladenine (BA). Although high embryo germination rates (80–100%) were observed in the varieties tested, plant recovery from germinated somatic embryos ranged from 6–47%. Embryogenic cultures could be maintained on X6 medium and used in genetic engineering studies.     

Crucial stages of embryogenesis of <Emphasis Type="Italic">R. arvalis</Emphasis>: Part 1. Linear measurements of embryonic structures

Investigations of individual variability have allowed us to reveal the crucial (=nodal) stages in embryogenesis of the moor frog (Rana arvalis Nills.). These crucial stages are: the late gastrula stage (stages 18–20), the hatching stages (stages 32–33) and, apparently, early metamorphosis (stage 39). Moreover, we have found that each embryonic structure passes through its specific crucial stages. For example, stage 34 is crucial for the trait “tail width” but is internodal for all other embryonic traits. At this stage, larva passes from an attached to a free-swimming life style. We also found considerable differences between the different frog populations in the the level of developmental variability. These differences were associated with internodal developmental stages.     

Embryological peculiarities of interspecific hybridization in <Emphasis Type="Italic">Pinus sibirica</Emphasis>

Cytoembryological research of the ovules in experiments with interspecific hybridization of Pinus sibirica (pollination be the pollen of P. koraiensis, P. armandii, P. parviflora, P. strobus, P. hokkaidensis, P. wallichiana, P. monticola, and P. сembra) revealed that the development of megagametophytes occurred in them by the usual scenario and resulted in the formation of mature archegonia. Pollen successfully germinated on the nucellus of ovules. However, disturbances were observed in the process of male gametophyte development, and pollen tubes on the nucellus were not visible by the period of archegonia maturation. Fertilization was usually absent. The development of embryonic channel is determined by egg cell maturity. The only exception was the variant of the controlled pollination of Pinus sibirica × P. сembra, in which the embryo has been formed.     

An acidic esterase as a biochemical marker for somatic embryogenesis in orchardgrass (Dactylis glomerata L.) suspension cultures

 The isoenzyme pattern of esterases (EC 3.1.1.2) secreted into the medium of orchardgrass (Dactylis glomerata L.) embryogenic suspension cultures during defined stages of somatic embryogenesis was compared with that of non-embryogenic suspension cultures during unorganised cell proliferation. Isoelectric focusing revealed the presence of 7–14 predominantly acidic isoforms. Comparison with the corresponding cell-wall isoenzyme pattern showed minor, mainly quantitative differences. The pattern of intracellular soluble esterases did not change markedly during somatic embryo development. A unique esterase whose migration in two-dimensional gel electrophoresis corresponds to an apparent molecular mass of 36 kDa and pI=3.8 was detected only in embryogenic cultures at very early stages of development. Since this isoform appeared long before morphological changes had taken place, it could possibly be used as a biochemical marker for embryogenic potential in D. glomerata L. suspension cultures. Received: 6 June 2000 / Revision received: 17 July 2000 / Accepted: 17 July 2000     

Late steps of egg cell differentiation are accelerated by pollination in Zea mays L.

 Egg cells were analysed cytologically during the female receptivity period in maize (Zea mays L., line A 188). Three classes of egg cell were distinguished: type A – small, non-vacuolated cells with a central nucleus; type B – larger cells with small vacuoles surrounding the perinuclear cytoplasm located in the middle of the cell; type C – big cells with a large apical vacuole and the mid-basal perinuclear cytoplasm. The less-dense cytoplasm of the vacuolated egg cells usually contained numerous cup- or bell-shaped mitochondria. The three egg types appear to correspond to three late stages of egg cell differentiation. The frequencies of each of the three egg types were monitored in developing maize ears before and after pollination. In young ears, with the silks just extending out of the husks, small A-type cells were found in about 86% of ovules. Their frequency decreased to about 58% at the optimum silk length, remained unchanged in non-pollinated ears, and fell to 16% at the end of the female receptivity period. However, after pollination and before fertilisation the frequency of these cells decreased to about 33%, and the larger vacuolated egg cells (types B and C) prevailed. At various stages of the receptivity period, pollination accelerated changes in the egg population, increasing the number of ovules bearing larger, vacuolated egg cells. Experiments with silk removal demonstrated that putative pollination signals act immediately after pollen deposition and are not species-specific. Received: 5 February 1999 / Accepted: 28 August 1999     

Immature embryos culture in Italian red chicory

Chicory (Cichorium intybus L.) embryo maturation in vitro was achieved in B5 medium without growth regulators. Immature embryos were collected at different developmental stages 4–168 h after pollination. Only a few plantlets were obtained from ovules collected shortly after pollination since the embryos at these early stages developed abnormal leaves and roots which regenerated shoots ‘via’ organogenesis. Zygotic embryos collected 24–72 h after pollination needed one month of culture in vitro before acclimation. Embryos collected at the heart or early torpedo stage of development (three days after pollination) produced green plantlets after two weeks of in vitro culture and thus they could be transferred to the greenhouse for acclimation. Culture of zygotic embryos in vitro has shown to be a suitable technique to accelerate the breeding process in biennial cultivated Italian red chicory. In fact it is possible to obtain plantlets ready for performing the selection for the desired agronomic traits one month after pollination. Since flowering occurs after vernalization, the selection can be performed in the correct season before flowering. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fertilization and embryogeny in Agapanthus praecox ssp. orientalis (Leighton) Leighton

Fertilization and embryogeny in Agapanthus praecox ssp. orientalis are described for the first time, and embryogenic characters of Agapanthus are discussed. The main results are: (1) The pollen tube enters the embryo sac and discharges two sperm 44?C48?h after pollination. (2) The sperm fuse with the egg cell and polar nuclei, forming zygote and primary endosperm nucleus, approximately 50?h after pollination. The zygote then enters a short period of dormancy. (3) Seven days after pollination, the zygote starts division. The first division of the zygote is transversal. (4) The embryo undergoes globular stage, rod-shaped stage, and finally forms a monocotyledonous embryo. (5) The suspensor cells are ephemeral and degenerate at the globular embryo stage. (6) Endosperm cells contain massive starch grains as nutrition for embryo development. (7) Embryogeny conforms to the Onagrad type, and endosperm formation is of the nuclear type; the whole process of embryogeny and endosperm development needs approximately 60?days in A. praecox ssp. orientalis. (8) Dicotyledonous together with monocotyledonous forms of embryo morphogenesis in Agapanthus supports the concept of homology of monocots and dicot cotyledons.     

Low external pH replaces 2,4-D in maintaining and multiplying 2,4-D-initiated embryogenic cells of carrot

A mixed culture comprised of both embryonic globules and nonembryogenic callus. was derived from seedling hypocotyls of Daucus carota cv. Scarlet Nantes on 2,4-D-containing medium using well-established methods. Then the mixed cultures were transferred to, and serially subcultured on, a hormone-free medium near pH 4. The medium contained 1 m M NH+ as the sole nitrogen source. When cultured in this way, embryonic globules were able to multiply without development into later embryo stages Nonembryogenic callus did not survive. Continuous culture of embryonic globules on this low pH hormone-free medium yielded cultures consisting entirely of preglobular stage proembryos (PGSPs). PGSP cultures have been maintained as such with continuous multiplication for nearly 2 years without loss of embryogenic potential. These hormone-free-maintained PGSPs continue their development to later embryo stages when cultured on the same hormone-free medium buffered at pH 5.8. We show that hormone-free medium near pH 4 can replace 2.4-D in its ability to sustain multiplication of 2,4-D-initiated embryogenic cells of carrot at an acceptable growth rate without their development into later embryo stages. This procedure provides selective conditions that do not permit the growth of nonembryogenic cells while providing an adequate environment for embryogenic cell proliferation and should prove invaluable in studying habituation.     

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。     

Endosperm response to pollen irradiation in kiwifruit

 Cytological details of endosperm development after pollination with irradiated pollen were studied in Actinidia deliciosa (kiwifruit) cultivar Hayward. Pollinations were carried out involving five different sources of pollen (Matua, Tomuri, Burt, Berryman, and fruiting male) irradiated with gamma rays at doses of 700 and 900 Gy. Non-irradiated crosses were used as controls. Irradiated pollen induced development of approximately 25–30% of the ovules. Two types of ovules were observed: (1) with both embryo and endosperm and (2) with endosperm only. No mitotic abnormalities were found in control or irradiated endosperms. Mitotic divisions were regular and nuclei spherical and evenly spaced. However, the cells of irradiated endosperm usually contained low amounts of storage products. Ploidy level of the endosperms was evaluated by nuclear size (volume) with the use of image analyzis. Mean nuclear size in control and irradiated endosperms was 1598.3 and 750.9 μm³, respectively. It is concluded that endosperm produced after pollination with irradiated pollen is autonomous and represents the 2n level. Received: 14 October 1998 / Revision accepted: 10 March 1999     

On the Embryonic Development and Ovule Culture of Interspecific Hybrids Between Populus simonii Carr and P.pyramidalis Borkh

The embryonic development following P. simonii Cart. × P. pyramidalis Borkh. is described in the present paper. The majority of pollen grains of P. pyramidalis Borkh. may germinate on the stigma of P. simonii Cam and the pollen tubes grow normally through the style and enter the embryo sac from the micropyle. Fertilization occurs as usual 4–7 days after pollination. A lot of proembryos and heart-shaped embryos are abortive; however, the others may develop normally and grow into mature embryos. Some of the endosperms appear normal and others may degenerate at free nuclear stage or cease to develop further at cellular stage. The ovules containg immature hybrid embryos of 19 days, 22 days, 26 days and 29 days after pollination at various developmental stages (heart-shaped stage, torpedo-stage and cotyledonary elongation stage) are excised and inoculated on nutrient agar for culture. The results show that: ( 1 ) Mll0 medium ( 1/2 MS+IAA 0.01 mg/L+BA 0.1 mg/L+sucrose 2% ) is the best of all the media used; (2) immature hybrid embryos of various developmental stages contained in ovules cultured in vitro may grow into normal plantlets.     

Kinetics of double fertilization in Torenia fournieri based on direct observations of the naked embryo sac

Torenia fournieri Lind. has a naked embryo sac that protrudes from the micropyle. The precise time course of the entire process of double fertilization and the kinetics of fertilization events were determined in this species by the following methods: (i) without squashing, pollen tubes on the torn stylar canal were observed by fluorescence microscopy after staining with both 4′,6-diamidino-2-phenylindole (DAPI) and aniline blue; and (ii) large numbers of living embryo sacs were observed directly by differential interference microscopy before and after fertilization. The pollen began to germinate 5 min after pollination and extruded pollen tubes which elongated at a constant rate of 2.3 mm · h⁻¹. At 4.0 h after pollination, the mitotic index of the generative cell within the pollen tube reached 88% and the two sperm cells were formed. Pollen tubes began to arrive at ovules 8.9 h after pollination and directly entered one of two synergids in the naked embryo sac. The time required for transport of sperm cells in the degenerated synergid was estimated statistically to be 1.9 ± 1.8 min for transport of the first cell and 7.4 ± 1.6 min for the second. In the nucleus of the fertilized egg cell, the male nucleolus began to emerge 10 h after pollination and the female nucleolus often decreased in size. The two nucleoli fused together prior to elongation of the zygote, which began 28 h after pollination. In the central cell, the secondary nucleus migrated to a region adjacent to the egg apparatus after pollination but prior to the arrival of the pollen tube. The primary endosperm nucleus rapidly returned to the inner region after fertilization. Prior to embryogenesis, the first division of the primary endosperm began about 15 h after pollination, at a defined site, to form the chalazal haustorium. Received: 24 October 1996 / Accepted: 13 March 1997     

Embryo abortion and histological features in the interspecific cross between <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L. and <Emphasis Type="Italic">P. coccineus</Emphasis> L

In this paper, the causes of early embryo abortion in the reciprocal crosses between Phaseolus vulgaris L. (a cultivar) and Phaseolus coccineus L. (a wild form) were studied. Methacrylate resin sections, 3–5 μm thick, of 3 to 14 day-old seeds were used to examine the embryo developmental stages and the state of seed tissue. It was observed that, embryos aborted at different developmental stages (globular to early cotyledon) depending on the maternal parent. The use of P. coccineus cytoplasm resulted in a higher number of abortion than in reciprocal crosses. Many of them took place between 5 and 6 days after pollination (DAP). Histological analyses permitted to observe that the embryo development was slower in the cross between P. coccineus and P. vulgaris, compared to parental seeds. It would be related to a deficient endosperm development in reciprocal crosses and, in some extent, hypertrophy of the suspensor might be the main cause of early embryo abortion. Then, it would be practical to overcome this incompatibility by rescuing the embryo at the globular stage of development.     

Changes in protein pattern during different developmental stages of somatic embryos in chickpea

Mature embryonic axes were used for chickpea (Cicer arietinum L.) regeneration via somatic embryogenesis. Qualitative and quantitative estimation of protein profile during somatic embryogenesis by SDS-PAGE and densitometric analysis showed differential expression of various storage proteins at different stages of somatic embryo development, which was compared with the profile of developing seeds. Total protein content in somatic embryos of chickpea increased from globular stage [2.9 μg mg⁻¹(f.m.)] to cotyledonary stage [4.8 μg mg⁻¹(f.m.)] and then started decreasing during onset of maturation and germination [up to 1.5 μg mg⁻¹(f.m.)]. Differential expression of seed storage proteins, late embryogenesis abundant (LEA) proteins and proteins related with stress response were documented at different stages of somatic embryogenesis. Germinating somatic embryos showed degradation products of several seed storage proteins and the appearance of new polypeptides (76.8, 67.6, 49.9 and 34.2 kDa), which were absent during differentiation of somatic embryos. A low molecular mass (17.7 kDa) polypeptide was uniformly present during all stages of somatic embryogenesis and it may belong to a group of stress-related proteins. This study describes the expression of true seed storage proteins like legumin, vicilin, convicilin and their subunits at different stages of somatic embryogenesis, which may serve as excellent markers for embryogenic pathway of regeneration in chickpea.     

Flowering and pollination patterns of Magnolia denudata with emphasis on anatomical changes in ovule and seed development

Magnolia denudata is an excellent ornamental and ecologically important tree that exhibits low fecundity because of seed abortion. To better understand the cause of this low sexual reproduction, pistil receptivity, and pollen quality were investigated using culture medium and histochemical staining, with emphasis on pollen viability at different flowering stages. Cross-pollination, geitonogamy, and self-pollination were undertaken on flowers of sample trees. We found that flowers produce a large number of pollen grains available for effective pollination. Pollen viability varied at different stages, but was highest during stage 3, in which the stamens detach from the axis, the anthers dehisce, and the gynoecium stigma starts to fade. The duration of pistil receptivity was approximately 5 h. These results indicate that poor fertilisation may occur if the optimum period for pollination is missed or the stigma receives poor-quality pollen grains from stages other than stage 3. Although dichogamy avoids selfing, it has led to considerable geitonogamy. Our anatomical analysis of ovule and seed development further revealed that fertilisation occurred in samples with geitonogamy, but that the embryo degenerated in the torpedo stage, demonstrating that aborted seeds were produced by fertilisation, rather than by pseudogamy. Our results provide new insights into the mechanism of reduced seed set under natural conditions.