Multicellular Pollen Research Articles

First stages of microspore reprogramming to embryogenesis through anther culture in Prunus armeniaca L.

Prunus armeniaca L. is a worldwide known species, very important particularly in the Mediterranean basin. Microspore embryogenesis through in vitro anther culture is a widely used method to obtain haploid and doubled haploid (DHs) plants which are being routinely used in breeding programmes for new superior cultivar development in many crops. Haploid–diploidization through gametic embryogenesis allows single-step development of complete homozygous lines from heterozygous parents. In the case of fruit crops, with long reproductive cycle, a high degree of heterozygosity, large size, and, often, self-incompatibility, there is no way to obtain haploidization through conventional methods. Induction of microspore embryogenesis in vitro is switched by a stress treatment. In many species, heat or cold stress has been reported to trigger pollen embryogenesis, the response being genotype dependent. In the present work we analyzed whether microspore reprogramming could be induced in apricot cultivars by cold stress through anther culture. We report the development of an in vitro anther culture protocol in P. armeniaca L. and analyse the response of several cultivars to stress treatments and culture media for inducing pollen embryogenesis. Results showed the formation of multicellular pollen and proembryos. The effect of two culture media in the embryogenic response was also analyzed, being the responses genotype-dependent. Monitoring of the cellular changes on the microspores was performed by structural and confocal microscopy analyses. Results indicated that the reprogramming of the microspore and the first steps of the embryogenic pathway have been achieved in different varieties of P. armeniaca, which constitutes a crucial step in the design of protocols for the regeneration of microspore-derived embryos and DH plants, for future potential applications in breeding programmes of this economically important fruit tree.

INDUCTION OF POLLEN EMBRYOGENESIS IN SEVEN CULTIVARS OF PRUNUS ARMENIACA L.

Haploids and doubled haploids (DHs), very important for their potential use in breeding and genetic analysis, can be induced mainly by two strategies, i.e. by regeneration from the female gamete or from the male gamete. Microspore embryogenesis through in vitro anther culture is a widely used method to obtain haploidization. In this research, the first stages of the gametic embryogenesis through the anther culture of seven apricot genotypes have been described and characterized through microscopical analysis, carried out at different times of culture. The switch of the developmental microspore programme from the gamethophytic to the sporophytic pathway has been indicated by the presence of multicellular pollen, proembryos and globular embryos in the in vitro system.

Development of multicellular pollen of Eriobotrya japonica Lindl. through anther culture

Eriobotrya japonica Lindl. is a worldwide known tree important for its use as horticultural and ornamental plant, especially in sub-tropical and Mediterranean countries. Microspore embryogenesis through in vitro anther culture is a widely used method to generate genetic variability by obtaining gametic or somatic embryos with many applications for plant breeding. In this work, a protocol has been set up for anther culture in loquat which resulted in the formation of multicellular pollen as a first step to further attempt haploid–plant production via pollen-derived structures. The response of nine of the most widely grown loquat cultivars to anther culture has been evaluated, and four cultivars being selected due to their higher response. The occurrence of anther swelling and the development of calli were analyzed as typical morphological features and potential markers that accompany pollen induction and reprogramming in many systems. Microscopical analysis in responsive anthers and the comparison with the normal gametophytic pollen development was carried out to characterize the cellular changes promoted by the treatment in the anther. The presence of multicellular pollen in the in vitro system developed here indicated the switch of developmental programme which constitutes a crucial step in the design of protocols for the regeneration of microspore-derived embryos and plants. Monitoring structural analysis at different times of the culture revealed specific features of the early microspore embryogenic pathway as well as the cellular organization changes.

Influence of Fe concentration in the medium on multicellular pollen grains and haploid plants induced by mannitol pretreatment in barley (Hordeum vulgare L.)

This study aims to clarify the short- and long-term effects of the iron concentration in the medium on androgenesis induced in barley by isolated microspore culture. The ultrastructural features and pectin composition of the intine wall were studied in the initial stages of androgenesis. The evolution of electron-dense iron deposits on the intine was analysed in multicellular pollen grains obtained by isolated microspore culture performed for 3, 6, and 9 days using various concentrations of FeNa(2) EDTA. Finally, the number of embryo-like structures and green plants obtained by microspore culture using different Fe concentrations was evaluated in order to estimate the optimum concentration for isolated microspore culture.

Isolation and culture of soybean (Glycine max L. Merrill) microspores and pollen grains

In the last three decades, research on soybean microspore embryogenesis was restricted to anther culture, which presents limitations such as the small number of responsive microspores and the high embryogenic potential of sporophytic tissues. Therefore, a sequence of studies was performed to establish appropriate conditions for the isolation and culture of soybean microspores and pollen grains as an alternative to anther culture. First, a pollen and microspore isolation technique was developed using floral buds from four soybean cultivars (Bragg, IAS 5, MG/BR-46 Conquista and BRSMT Uirapuru). This technique allowed the establishment of cultures with satisfactory density and characteristics. Subsequently, different culture conditions were tested. Although B5 and MS media have been currently recommended for soybean anther culture, the best result was obtained in PTA-15 modified medium, with the formation of enlarged microspores and 0.4% of multicellular pollen grains in the cultivar BRSMT Uirapuru.

Effect of temperature shock on soybean microspore embryogenesis

In this study the effect of cold and heat shock on androgenetic induction in soybean anther culture was tested. Anthers of soybean were subjected to 4 and 33ºC, while the anthers used for control were maintained at 25ºC. Cytological analysis done during the 30 days of culture showed that frequencies of symmetrical binucleated and multicellular pollen grain did not differ among temperature treatments. Multicellular pollen grain could be formed by symmetrical division, as well as by assymetrical division. In relation to the embryo induction, the results of the treatments did not differ too. These results suggested that these treatments did not induce a sporophytic pathway in soybean.

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.

Ontogenetic events in androgenesis of Brazilian barley genotypes.

This paper describes a simple procedure for obtention of barley (Hordeum vulgare L.) doubled haploid plants from Brazilian hybrid genotypes. Anatomical and histological examinations showed the reversion of barley pollen to an sporophytic mode of development. A sequence of mitosis led to the formation of multicellular pollen grains. Regeneration of plants occurred either by direct embryogenesis or callus formation followed by differentiation through direct embryogenesis or organogenesis. Plants were formed in the same medium used for induction dispensing an additional regeneration step. This procedure makes doubled haploid production simpler and faster. Plantlets were transferred to another medium for rooting and after that planted in pots with vermiculite and nutrient solution.

In situ characterization of the late vacuolate microspore as a convenient stage to induce embryogenesis inCapsicum

Pollen embryogenesis has been induced inCapsicum annuum L., selecting the late vacuolate microspore as the suitable developmental stage for the induction. Various modern in situ methods including cytochemistry, immunocytochemistry, and in situ hybridization have been used to characterize the functional organization of the microspore nucleus at this specific stage of development in which the change to the sporophytic program can be induced. The results on the chromatin pattern, interchromatin structures and nucleolar subcompartments reveal a transcriptionally very active nucleus in the late vacuolate microspore and define characteristic cellular features of this specific period. The first stages of the microsporederived embryos have also been studied identifying the main ultrastructural features exhibited by the nuclei of two-cell and multicellular pollen grains. The approach used, providing information on the subcellular location and expression time of key molecules involved in gene expression, presents a high potential for studying the complex process of pollen embryogenesis induction.

Microspore development in the anther culture of wheat(Triticum aestivum L.).

A cultivar (Chris) of hard red spring wheat (Triticum aestivum L.) and a one-step haploid generation medium were used to study microspore development and callus formation by using an anther culture technique. Cytological observations on microspore development were made 7, 10, 13 and 16 days after the anther was placed on the medium. The first mitotic division of the uninucleate microspore formed two nuclei, which were similar or different in size. Further divisions of the vegetative nucleus produced multicellular pollen grains that continued to grow and then were released from the exine. This extensive division of the vegetative nucleus could be the source of embryoid or callus generation.

Ultrastructural studies on pollen embryogenesis in maize (Zea mays L.)

Maize anthers have been induced on modified N6 medium to produce embryoids. Different stages from the cultures were sampled and prepared for microscopical examination. The microspores at the onset of culture were in an early developmental stage, with the nucleus and numerous organelles centred in the middle, surrounded by many small vacuoles with a lipid content. The binuclear pollen grains contained small vesicles and much starch. The partially condensed vegetative nucleus indicated participation of the vegetative component in the formation of multicellular pollen grains (MPGs). Several MPGs have been observed which differed in morphology. We suggest, on the basis of these ultrastructural observations, that in maize mainly the vegetative cell contributes to the MPG which further develops directly into embryoids.

Effect of anther orientation on microspore-callus production in barley (Hordeum vulgare L.)

Barley anthers from cold pretreated spikes produced no or few calluses when plated with both loculi in contact with the medium (‘flat’). When anthers were plated with only one loculus in contact with the medium (‘up’), a high proportion of the anthers produced calluses. The top loculus of the ‘up’ anthers was most productive. ‘Flat’ anthers, when compared with ‘up’ anthers, were not only slower to produce multicellular pollen grains (MCPs) and microcalluses, but also produced fewer of them and ceased production earlier. The MCPs and microcalluses in ‘flat’ anthers grew more slowly and few developed beyond the 30 cell stage. These results establish the importance of anther orientation for barley anther culture.

Some factors affecting the in vitro propagation of gladiolus

Callus tissue cultures have been established from the excised segments of the inflorescence, flower stalks, denuded flower, bract, perianth and leaf segments of 2 cultivars of Gladiolus grandiflorus. Of all the explants and the media tested, the best callus was obtained from the segments of the flower stalks, cultured on a basal medium supplemented with naphthalene acetic acid and kinetin. The callus mostly underwent rhizogenesis, and occasionally differentiated shoots. Complete plants were regenerated from the in vitro cultured cormels, cormel tips and the axillary buds, and 6 plants were formed from the segments of 1 cormel, whereas in nature only 1 plant is obtained per cormel. Cultured young anthers callused and developed leaf-like petaloid structures, and occasionally showed multicellular pollen.

Pollen Dimorphism and the Frequency of Inductive Anthers in Anther Culture of Triticum monococcum

Summary The frequenry of in vitro cultured Triticum monococcum anthers showing multicellular pollen development was with the exception of one instance significantly lower than the estimated frequency of anthers exhibiting pollen dimorphism at anthesis. A compamble frequency would support the hypothesis that multicellular pollens develop from the smaller form of dimorphic pollen. There was circumsumtial evidenre however in that the highest and lowest proportion of the smaller pollen in sampled dimorphic populations corresponded more or less to those of multicellular pollens observed in inductive anthers. Interaction between culture medium and spike pretreatment precluded any generalization on their inductive efficacy. One line of T. monococcum, designated no. 34, was used throughout the study. Plants were vernalized as seedlings for 4 weeks and raised in the glasshouse through autumn and winter in 1980; anthesis occurred 7 months after sowing. The frequency of anthers showing pollen dimorphism at anthesis was estimated from a sample of 10 plants from which 2 spikes were randomJy sampled per plant and likewise 3 spikelets ( = 9 anthers) per spike. Pollen grains within individual anthers were removed on microscope slides and stained for at least 5 min with 1% acetocarmine. A minimum of 500 grains was scored at low magnification(s) for percent of small(er) non-staining pollen (see Tan and Halloran 1980) per anther sample. For anther culture, spikes at the uninucleate stage, which corresponded to the awns emerging 1-2 cm from the flag leaf, were harvested with about 10 cm of the stem intact. These were subdivided into four treatment groups: (1) no pretreatment (control); (2) chilling at 12 °C dark for 5 d; (3) centrifugation of the spike at ca. 1,000 g for 10 min prior to anther inoculation; and (4) preatment (2) followed by (3). The efficacy of three culture media were compared: (1) Nitsch and Nitsch's (1969) basal medium solidified with 0.9% agar and containing 6% sucrose, 0.75 mg/l each of NAA (naphthalene acetic acid) and 2,4-D (2,4-dichlorophenoxyacctic acid) plus 0.15 mg/l each of kinetin and zeatin, with the pH of the medium adjusted to 5.8 (medium designated NN); (2) NN medium as in (1) but incorporating in addition 30 ppm Ethrel (2-chlorethylphosphoric acid) (designated NN + E); and (3) the “New Potato Medium” (designated P2) as described by Chuang et al. (1978). Disposable 55 × 15 mm plastic petridishes were used as culture vessels. The media were sterilized by autoclaving at 1.05 kg/ cm2 for 15 min. Chilled spike-tillers in groups of fives were stood in 50-ml Ehrlmeyer flasks containing either liquid basal NN or NN + E medium. Each group of spikes was hooded with a plastic bag to minimize desiccation. Those stood on basal NN medium were assigned to both NN and P2 media, whilst those on NN + E medium only to same but containing the phytohormones. Before anthers were removed for inoculation, the awns were clipped and the whole spike was surface- sterilized with 70% ethanol. Cytological examination for m.c.p. development in cultured anthers was undertaken after 3-4 weeks' incubation at 25 °C/dark Anthers were removed and individually quashed in 1% acetocarmine.

Glutamine, inositol and conditioning factor in the production of barley pollen callus in vitro

A growth supplement consisting of L-glutamine (800 mg/l) and m-inositol (5000 mg/l), beneficial in the culture of isolated tobacco pollen, was tested in barley anther culture as a substitute for the anther-conditioning factor (CF.) Unlike the CF, the supplement had no influence on the formation of multicellular pollen grains (MPGs), but it partially replaced the CF, depending on the inoculation density of the anthers, in sustaining growth of MPGs into callus. Inositol proved to be the active component and was more effective at 1000 than at 5000 mg/l. Glutamine at 160 or 800 mg/l was either ineffective or inhibitory. The combined influence of inositol and CF exceeded the sum of their separate influences. The influence of the CF and hence the synergism with inositol was destroyed by heat. The role of compounds such as amino acids and inositol in the growth of morphogenic pollen is seen as subsidiary to that of the CF.

Multicellular Pollen Formation in Cultured Barley Anthers

Multicellular Pollen Formation in Cultured Barley Anthers

Multicellular Pollen Formation in Cultured Barley Anthers

Multicellular pollen units partitioned into embryo- and endosperm- or possibly suspensor-like components are formed in barley (Hordeum vulgare cv. Sabarlis) anthers cultured from spikes excised during the free spore phase of microsporogenesis. The embryo-like component may be derived from the generative cell, the vegetative cell, or from contributions of both cells, and appears to be usually, though not invariably, haploid. The endosperm- or suspensor-like component is derived from the vegetative cell and rapidly becomes non-haploid or mixoploid. The initial pattern of division often simulates that in the formation of 4-celled and 7-celled embryo sacs. The time of excision during the free-spore phase is critical. Partitioning occurs only with excision during the mid-unicellular stage (stage 2) when the nuclei are probably still in the pre-DNA replication phase, but excision at the early unicellular stage (stage 1) in ineffective and leads to rapid degeneration of the spores. With excision at the late-unicellular stage (stage 3), independent contribution by the generative cell is blocked and the pollen develops by the known A, B, or C pathways. Temperature stress given to the excised spikes before culture of the anthers enhances the frequency of partitioned units but is thought not to be causal. It is suggested that the multiplicity of developmental pathways may have some bearing on whether the plantlets ultimately produced are albino, variegated, or green.

Effects of Sucrose and Growth Substances Concentration of Medium on the Formation of Multicellular Pollen Grains in Anther Culture of Tea Plant

1. 葯培養によるチャの半数体を育成するための基礎的な知見を得るために,培地の糖濃度が葯内花粉粒に与える影響と培地の糖濃度ならびに植物生長調節物質が葯内花粉粒に与える影響について検討した。2. 培地の糖濃度が葯内花粉粒に与える影響について,異常分裂につながる3核(栄養核2+生殖核1)の花粉粒が認められたのは,糖濃度5%,10%と15%区であった。しかし糖濃度15%区は,Fig.2のように枯死率が高かったので,培地の糖濃度は5%と10%が適当であると考えられた。3. 培地の糖濃度ならびに2.4-Dが葯内花粉粒に一与える影響は,Fig.5に示すように,培地の糖濃度は5%よりも10%が有効であり,2.4-Dの有無については,培地に添加することが分裂花粉粒を含む葯の頻度を高くした。4. Kinetinが葯内花粉粒の分裂に与える影響については(Fig.6),培地にkinetinがなくとも培養7日目まで分裂花粉粒が認められたが,花粉粒の分裂にとって,培地のkinetin濃度は10pplnが適当であると考えられた。5. 糖濃度10%のkinetin 10ppmで,培養21日目に花粉粒は約30細胞まで分裂した(Plate 7)。6. 形成されたカルスの染色体数を調査した結果,2n=30であった。葯の切片の調査より,カルスは葯の結合組織から形成され(Plate 2),花粉粒起原でなかった。