Experimental Researches on the Two Pathways of Pollen Development in Oryza sativa L.

The present paper deals with the experimental researches on the gametophytic and sporophytic pathways of pollen development in Oryza sativa L. Subsp. Keng, Cultivar Jinghong No. 2. Three methods of culture were used: (1) The lemma, palea and pistil of excised spikelets were removed and the pedicel was inserted vertically into the medium with the intact stamens standing freely above the medium surface (vertical culture). (2) The spikelets were manipulated similarly but placed horizontally on the medium so that their anthers were directly contacted with the latter ('horizontal culture'). (3) The anthers were excised and inoculated separately (anther culture). In all cases the pollen stage at inoculation was in late uninucleate. N6 basic medium supplemented with or without MCPA (2 ppm) was used. After inoculation the samples were collected periodically for cytological observation. In all cases the pollen passed a short stage of gametophytic development, forming a vegetative and a generative cell, then various pathways commenced in different cultures. In vertical culture, most of the pollen went on .along. the gametophytic pathway up to normal 3-celled stage, but some showed anomalous divisions of vegetative or/and generative nuclei, indicating an initiation of sporophytic development. In horizontal culture, the sporophytic deve]opment went on further, producing some calluses, though the main pollen population remained as gametophyte. In anther culture, the gametophytic pathway to a mature 3-celled pollen was blocked, the unique pathway being sporophytic. In rice, the pollen developed along sporophytic path- way mainly via A route. These comparative investigations indicate that there are two chief factors concerning the switch of pollen development from one pathway to another: first, to be freed from the in vivo restrictions, which, as suggested by Sunderland and as sup- ported by the results of vertical culture in our experiments, is sufficient to trigger the first sporophytic division, and second, 'direct contact with the medium, which is necessary to support the successive growth of multicellular grains and calluses. As to the exogenous hormone, rather than functioning as an agent triggering sporophytic development, it plays an important role in increasing eventual induction frequency, growth rates and differentiating ability of calluses.     

Gene expression during the male gametophytic phase

Abstract

In recent years a number of experimental findings have indicated that in higher plants the gametophytic phase is able to express its own genetic information, a large part of which it shares with the sporophytic generation. Quantitative estimates of haploid and haplodiploid gene expression have been obtained by mRNA and isozyme analysis in several plant species: 60-70% of the genes are expressed in both pollen and plant, about 10% are pollen-specific, and 20% represent the sporophytic domain. Moreover, it has been demonstrated that stage-specific genes are expressed in the gametophytic generation: at least two sets of genes are activated during pollen development, others are expressed only in the postshedding period, during germination and tube growth. Studies have been made to ascertain the role played by gametophyte-expressed genes in pollen development; the in vivo and in vitro pollen tube growth rate has been revealed to be controlled by the gametophyte genome itself. Differential effects of specific chromosomal deficiencies on the development of maize pollen grains have indicated that components of normal microspore development are controlled by genes located in specific parts of the genome. For single gene analysis, gene transfer can be used; on the contrary, for traits with a multifactorial genetic control, direct proof of gene expression both in the gametophytic and the sporophytic generation can be obtained when selection is applied to the pollen population of a hybrid plant, and response to selection is observed in the resulting sporophytic progeny. Response to selection, applied at different stages of the gametophytic phase, has been described in the sporophytic progeny and this with regard to many adaptive traits; thus the phenomenon can have an important bearing on the genetic structure of natural populations and on higher plant evolution, it can also be used as a breeding tool to increase the efficiency of conventional selection methods.     

Pollen competitive ability in maize: within population variability and response to selection

Summary Male gametophytic selection can play a special role in the evolution of higher plant populations. The main assumption — gametophytic-sporophytic gene expression of a large portion of a plant's genes — has been proven by a number of studies. Population analyses have revealed a large amount of variability for male gametophytic fitness. However, the data available do not prove that at least a portion of this variability is due to postmeiotic gene expression. This paper reports the analysis of a synthetic population of maize based on a gametophytic selection experiment, carried out according to a recurrent scheme. After two cycles of selection, the response was evaluated for gametophytic and sporophytic traits. A parameter representing pollen viability and time to germination, although showing a large amount of genetic variability, was not affected by gametophytic selection, indicating that this variability is largely sporophytically controlled. Pollen tube growth rate was significantly affected by gametophytic selection: 21.6% of the genetical variability was released by selection. Correlated response for sporophytic traits was observed for mean kernel weight: 15.67% of the variability was released. The results are a direct demonstration that pollen competitive ability due to pollen tube growth rate and kernel development are controlled, to a considerable extent, by genes expressed in both tissues. They also indicate that gametophytic selection in higher plants can produce a higher evolution rate than sporophytic selection; it can thus serve to regulate the amount of genetic variability in the populations by removing a large amount of the genetic load produced by recombination.     

Variation in sporophytic and gametophytic vigor in wild and cultivated varieties of Cucurbita pepo and their F1 and F2 generations

 This study examines sporophytic and gametophytic vigor in wild and cultivated varieties of Cucurbita pepo L. and their hybrids in order to determine whether hybrid vigor extends to the microgametophyte generation. It also examines the variation in sporophytic and gametophytic vigor to discern the non-genetic influences of pollen provisioning by the sporophyte on pollen performance from the genetic influences of the microgametophyte’s own genotype on pollen performance. A cultivated and a wild C. pepo and their F1 and the F2 generations were grown under field conditions and flower and fruit production were monitored over one summer. We found that the four types of plants differed significantly in the number of male and female flowers and the number of fruits they produced. The F1 plants produced significantly more male flowers and marginally more female flowers and fruits than the parental lines. To estimate gametophytic vigor pollen was germinated in vitro and pollen tube length measured after 30 min. We found that pollen tubes from the F1 plants had significantly greater growth than tubes from the parental lines or the F2 generation, indicating that hybrid vigor extends to the microgametophytic generation. By partitioning the variance of pollen tube growth into ’within’ and ’among’ plant components of variation, we were able to show that the genotype of the microgametophyte influences pollen performance in vitro, but that expression of hybrid vigor in the microgametophyte is likely to be due to an environmental effect related to provisioning of the pollen grains during development. Received: 16 April 1998 / Revision accepted: 18 August 1998     

Isolation and Early in Vitro Development of Young Pollen Protoplasts in Nicotiana tabacum

For isolating young pollen protoplasts in Nicotiana tabacum. The authors had established two efficient enzymatic methods via anther preculture or pollen starvation pretreatment. Procedure of the first method included the following steps: 1. Cold pretreatment of flower buds with pollen at late unicellular to early bicellular stage; 2. Anther floating culture for pollen shedding into the culture medium followed by dehiscence of exine; 3. Enzymatic maceration of exine-dehisced pollen resulting in degradation of intine and release of pollen protoplasts in large quantity. Procedure of the second method involved the following steps: 1. Culture of pollen at middle bicellular in Kyo and Harada' B medium for starvation: 2. Enzymatic maceration of starvated pollen resulting in release of pollen protoplasts and subprotoplasts. Factors affecting the results of both methods as well as early in vitro developmental events of young pollen protoplasts were studied. The protoplasts could be induced either to trigger the first sporophytic division or to continue the gametophytic pathway leading germinatation of pollen tubes !ndicating their potentiality of inducing both sporophytic and gametophytic development of pathway. In rare instance a quite interesting phenomenon was observed that a pollen protoplast first divided into two daughter cells and one of which then germinated a pollen tube. It may insinuate that such pollen protoplasts initially induced a sporophytic pathway could reverse induce a gametophytic pathway.     

Effects of Alternaria alternata f.sp. lycopersici toxins on pollen

Summary Effects of the phytotoxic compounds (AAL-toxins) isolated from cell-free culture filtrates of Alternaria alternata f.sp. lycopersici on in vitro pollen development were studied. AAL-toxins inhibited both germination and tube growth of pollen from several Lycopersicon genotypes. Pollen from susceptible genotypes, however, was more sensitive for AAL-toxins than pollen from resistant plants, while pollen of species not belonging to the host range of the fungus was not significantly affected by the tested toxin concentrations. AAL-toxins elicit symptoms in detached leaf bioassays indistinguishable from those observed on leaves of fungal infected tomato plants, and toxins play a major role in the pathogenesis. Apparently, pathogenesis-related processes and mechanisms involved in disease resistance are expressed in both vegetative and generative tissues. This overlap in gene expression between the sporophytic and gametophytic level of a plant may be advantageously utilized in plant breeding programmes. Pollen may be used to distinguish susceptible and resistant plants and to select for resistances and tolerances against phytotoxins and other selective agents.     

Environmental control and evidence for predetermination of pollen embryogenesis inNicotiana tabacum pollen

Summary The effect of daylenght and temperature for the donor plants (Nicotiana tabacum var. Badischer Burley) on the formation of pollen competent for embryogenesis (P-pollen) by the three possible routes (during normal flower developmentin situ (pollen dimorphism), during cold-treatment of excised flower buds, in cultured anthers) was studied. In all three routes, P-pollen frequency (premitotic pollen, before 1. sporophytic division, PPF) was affected in essentially the same way. At 24 °C and long days, PPF was low and short days had only a slightly increasing effect. At 18 °C and long days, PPF was higher and short days further increased it. Correlated with PPF under the different growth regimes was the percentage of units with more than one vegetative-type nucleus (normal embryos + abortive embryos + multinucleate pollen) in 3 weeks old anther cultures. Under greenhouse conditions, PPF was generally higher than at 24° in growth rooms and showed a maximum in the winter months. Plant age did not affect PPF. These results give further evidence that pollen embryogenesis is predetermined before excision and culture of the pollen or anthers.     

Identification, subcellular localization, and developmental studies of oleosins in the anther of Brassica napus

mRNAs encoding putative oleosins have been detected in the tapetum of developing anthers in Brassica and Arabidopsis, but the authentic proteins have not been previously documented. Antibodies against a synthetic 15-residue polypeptide that represents a portion of the putative tapetum oleosins encoded by two cloned Brassica napus genes were raised. Using these antibodies for immunoblotting after SDS-PAGE of the sporophytic extracts of B. napus developing anthers, two oleosins of ～ 48 and 45 kDa were detected. These two oleosins were judged to be the putative oleosins encoded by cloned Brassica genes because of their identical N-terminal sequences. The two oleosins were present in the anthers only during the developmental stage when the tapetum cells were packed with organelles. A fraction of low-density organelles was isolated from the developing anthers by flotation centrifugation. The fraction contained plastoglobule-filled plastids and lipid-containing particles. The structures of these two isolated organelles were similar to those in situ in the tapetum cells. Of subcellular fractions of the anther homogenate, the two oleosins were present exclusively in the low-density organelle fraction. They were absent in the surface fractions of the developing microspores and the mature pollen, although fragmented oleosin molecules were earlier reported to be present on the pollen. By immunocytochemistry, immunogold particles were found largely on the periphery of the plastoglobuli inside the plastids in the tapetum cells. The antibodies also detected oleosins on the surface of storage oil bodies inside the maturing microspores. Apparently, the gametophytic microspore oil-body oleosins share common epitopes at the generally non-conserved C-terminal domain with the sporophytic tapetum oleosins.     

Effects of pollination intensity on the vigor of the sporophytic and gametophytic generation of Cucurbita texana

 We examined the effects of pollen competition (pollen load size) on sporophytic vigor and gametophytic performance in Cucurbita texana, a wild gourd, while controlling for alternative interpretations of the data. Under field conditions we compared the vigor of progeny produced from large and small pollen loads and examined the in vitro performance of the pollen produced by the progeny. We found that the progeny from large pollen loads germinated faster and had a greater reproductive output (male flowers and fruits) than progeny produced from small pollen loads. In addition, we found that the pollen produced on plants derived from large pollen loads grew faster in vitro than the pollen produced on plants derived from small pollen loads. These findings indicate that pollen competition affects the performance of the resulting sporophytic generation and the microgametophytes they produce. Received: 26 January 1997 / Revision accepted: 25 June 1997     

Nonradioactive in situ localization of poly(A)+ RNA during pollen development in anthers of tobacco (Nicotiana tabacum L.)

Summary A method is described for non-radioactive labeling of total mRNA [poly(A)+ RNA] in plastic-embedded plant tissue sections. Oligo-deoxythymidylic acid (oligo-dT) labeled with digoxigenin-conjugated dUTP was used for in situ hybridization to poly(A)+ RNA in sections of tobacco (Nicotiana tabacum) anthers. The digoxigenin was immuno-stained using antidigoxigenin IgG and gold-labeled protein-A, followed by silver enhancement of the gold label. Reproducibly similar positive staining patterns were obtained with digoxigenin-labeled oligo-dT and polyuridylic acid [poly(U)], but not with a similarly labeled sense probe, poly(A). In the developing anthers, from the onset of meiosis to the production of pollen grains, labeling patterns were compatible with a gradual depletion of nuclear and chromosome-associated sporophytic mRNA molecules during prophase of meiosis, followed by postmeiotic production of gametophytic mRNA in microspore nuclei and the vegetative nuclei of the pollen grains.Abbreviations BSA bovine serum albumin - DIG digoxigenin - IgG immunoglobulin-G - oligo-dT oligo-deoxythymidylic acid - PAS-ABB periodic acid Schiff-aniline blue black - PBS phosphate buffered saline - poly(A) polyadenylic acid - poly(U) polyuridylic acid - SSC standard saline citrate     

Embryogenesis induction, callogenesis, and plant regeneration by in vitro culture of tomato isolated microspores and whole anthers

In this work, some of the different in vitro developmental pathways into which tomato microspores or microsporocytes can be deviated experimentally were explored. The two principal ones are direct embryogenesis from isolated microspores and callus formation from meiocyte-containing anthers. By means of light and electron microscopy, the process of early embryogenesis from isolated microspores and the disruption of normal meiotic development and change of developmental fate towards callus proliferation, morphogenesis, and plant regeneration have been shown. From microspores isolated at the vacuolate stage, embryos can be directly induced, thus avoiding non-androgenic products. In contrast, several different morphogenic events can be triggered in cultures of microsporocyte-containing anthers under adequate conditions, including indirect embryogenesis, adventitious organogenesis, and plant regeneration. Both callus and regenerated plants may be haploid, diploid, and mostly mixoploid. The results demonstrate that both gametophytic and sporophytic calli occur in cultured tomato anthers, and point to an in vitro-induced disturbance of cytokinesis and subsequent fusion of daughter nuclei as a putative cause for mixoploidy and genome doubling during both tetrad compartmentalization and callus proliferation. The potential implications of the different alternative pathways are discussed in the context of their application to the production of doubled-haploid plants in tomato, which is still very poorly developed.     

Isolated pollen culture: plant reproductive development in a nutshell

Abstract

Isolated pollen can develop into two different directions when cultured in vitro. In a rich medium, microspores and young pollen grains develop into mature pollen that is fertile. When pollen is treated by a stress treatment such as a hunger treatment in a sucrose - and/or nitrogen - free medium, embryogenic pollen is formed that, after transfer to a rich medium, develops into embryos and haploid plants. This system of isolated pollen culture offers an opportunity to study two developmental processes, i.e. pollen development and embryogenesis, as well as a basic regulatory event, i.e. the transition from the gametophytic to the sporophytic phase in the alternation of generations in higher plants. In addition, both systems offer various application-oriented possibilities, such as production of doubled haploids, to overcome self-incompatibility, to rescue sterile pollen, pollen selection and pollen transformation. An understanding of the cell biological and molecular events during embryogenic induction may promote a wider application of doubled haploid breeding and the use of such plants for gene transfer.     

Heat-shock proteins during pollen development in maize

In contrast to sporophytic tissues, mature pollen of higher plants does not synthesize the typical set of heat-shock proteins (HSPs) in response to a marked temperature upshift. Immature grains, however, seem able to do so, at least partially. We investigated the characteristics of HSP synthesis throughout the male gametophytic phase in maize and compared gametophytic and sporophytic heat-shock responses. One-dimensional Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis technique (SDS-PAGE) of newly synthesized proteins revealed that immature pollen synthesizes HSPs, some of which are not induced in sporophytic tissues. The heat-shock response appeared to be related to microgametophytic developmental stages. The strongest response was found in uninucleate microspores: at this stage, in addition to the sporophytic 102, 84, 72, and 18 kD HSPs, three other polypeptides of 74, 56, and 46 kD were observed. In the binucleate and trinucleate stages, only a reduced synthesis of few HSPs could be induced, and differences between genotypes were observed. In germinating pollen, HSP synthesis was not induced under a voriety of heat-stress conditions; however, the consti-tutive synthesis of two polypeptides of the same molecular weight, 72 and 64 kD, as two HSPs was observed. The biological significance of these results is discussed.     

Characterization of genes expressed during the development ofBrassica napus pollen

Summary The study of the formation of pollen in plants has been the focus of extensive morphologic and cytologic observations. This complex developmental process requires the coordinated activity of both gametophytic and sporophytic tissues. The events that occur during microspore development represent a carefully orchestrated program of physiologic, biochemical, and genetic activities. Genes expressed specifically in pollen or in sporophytic tissues that support pollen development have only recently been identified and desribed. In the present paper we describe several genes expressed during pollen development in the important oil seed speciesBrassica napus (oil seed rape/canola). The characterization of three gene families expressed during microspore development is reviewed which provides a basis for comparison with other genes expressed during pollen maturation. The, potential value of these genes for the development of novel plant breeding strategies and hybrid seed production is discussed. Presented in the Session-In-Depth In vitro, Gametophyte Biology at the 1991 World Congress on Cell and Tissue Culture held in Anaheim, CA, June 16–20, 1991.     

Intra- and extracellular lipid composition and associated gene expression patterns during pollen development in Brassica napus

Pollen development in angiosperms is regulated by the interaction of products contributed by both the gametophytic (haploid) and sporophytic (diploid) genomes. In entomophilous species, lipids are major products of both sporophytic and gametophytic metabolism during pollen development. Mature pollen grains of Brassica napus are shown to contain three major acyl lipid pools as follows: (i) the extracellular tryphine mainly consisting of medium-chain neutral esters; (ii) the intracellular membranes, particularly endoplasmic reticulum, mainly containing phospholipids; and (iii) the intracellular storage lipids, which are mostly triacylglycerols. This paper reports on the kinetics of accumulation of these lipid classes during pollen maturation and the expression patterns of several lipid biosynthetic genes and their protein products that are differentially regulated in developing microspores/ pollen grains (gametophyte) and tapetal cells (sporophyte) of B. napus. Detailed analysis of three members of the stearoyl-ACP desaturase (sad) gene family by Northern blotting, in situ hybridization and RT-PCR showed that the same individual genes were expressed both in gametophytic and sporophytic tissues, although under different temporal regulation. In the tapetum, maximal expression of two marker genes for lipid biosynthesis (sad and ear) occurred at a bud length of 2–3 mm, and the corresponding gene products SAD and EAR were detected by Western blotting in 3–4 mm buds, coinciding with the maximal rates of tapetal lipid accumulation. These lipids are released following tapetal cell disintegration and are relocated to form the major structural component of the extracellular tryphine layer that coats the mature pollen grain. In contrast, in developing microspores/pollen grains, maximal expression of the lipid marker genes sad, ear, acp and cyb5 was at the 3–5 mm bud stages, with the SAD and EAR gene products detected in 4–7 mm buds. This pattern of expression coincided with accumulation of the intracellular storage and membrane lipid components of pollen. These results suggest that, although the same genes may be expressed in the sporophytic tapetal cells and in gametophytic tissues, they are regulated differentially leading to the production of the various contrasting lipidic structures that are assembled together to give rise to a viable, fertile pollen grain.     

In-situ seed production after pollination with in-vitro-matured,isolated pollen

Immature tobacco (Nicotiana tabacum L.) pollen has been isolated from anthers in three distinct stages of development, including the microspore stage. In in-vitro cultures, fully functional, mature pollen was obtained. In a germination medium, this pollen produced pollen tubes. After application to stigmas in situ, the in-vitro-matured pollen fertilized ovules, and seeds were produced. Genetic tests with seedlings obtained from pollinations with in-vitro-matured pollen from a transgenic plant revealed normal Mendelian segregation of two marker genes, the neomycin-phosphotransferase II gene and the nopaline-synthase gene. These results are of interest with respect to the control of self-incompatibility, cytoplasmic male sterility and pollen-allergen formation, and it offers an alternative route for gene transfer in those plants which cannot be regenerated in vitro.Abbreviations cms cytoplasmic male sterility; AMGLU, MS, M2S, MR26 - GK culture media, see Material and methods     

Microspore-derived embryos in Brassica: the significance of division symmetry in pollen mitosis I to embryogenic development

Summary An attempt has been made to manipulate the cytological processes regulating the switch from gametophytic to sporophytic development induced by culturing the microspores of higher plants. Previous studies have indicated that sporophytic development, which leads to the formation of haploid embryos, normally follows the symmetrical division of the microspore rather than the asymmetric mitosis characteristic of normal development. To determine whether symmetry of division is a key factor in the determination of subsequent development, cells were supplied with the antimicrotubule drug colchicine to disrupt elements of the microtubular cytoskeleton believed to be involved in nuclear positioning. The treatment resulted in a highly significant increase in the numbers of cells turning to sporophytic development; further, timed applications indicated that the cells were sensitive to the drug over a 12-h period immediately prior to pollen mitosis. The results suggest that alteration of division symmetry is sufficient to switch the developmental pathway from gametophytic to sporophytic. These findings are discussed in the perspective of current models proposed for the regulation of development in eukaryotic cells.     

Disposition of Pollen in situ and its Relevance to Anther/Pollen Culture

Disposition of pollen in immature anthers of Hordeum vulgareis illustrated by scanning and transmission electron microscopy.Freeze-fracture confirms that the pollen is confined to a uniseriatecolumn aligned against the tapetum. There is no free pollenin the lumen of the anther loculi. In contrast, in Nicotianatabacum and Paeonia lactlflora the pollen is disposed at randomand occupies the whole of each loculus. Freezing preserves thefluid content of the loculi, appearing in fracture profilesas an amorphous matrix in which the pollen is embedded. Thematrix, which generally obscures the tapetum, is present throughoutthe microspore phase but diminishes as the spores enlarge. Itis still present in N. tabacum and H. vulgare at the first pollendivision, fragmentary at this stage in P. lactiflora, but isno longer discernible in any of the species at the onset ofpollen-grain maturation. Pretreatment of excised Hordeum spikes at 4 ?C during the microsporephase, a prerequisite for anther/pollen culture, disrupts thenormal developmental sequence but does not alter the uniseriatedisposition. Before the spores start to divide, however, thetapetum degenerates and the fluid phase is dispersed. The observations are discussed in relation to isolated pollenculture, float culture of anthers and the switch in programmefrom gametophytic to sporophytic development. Key words: Pollen, Tapetum, Freeze-fracture