A CYTOLOGICAL ANALYSIS OF MICROSPORES OF TRITICUM AESTIVUM (POACEAE) DURING NORMAL ONTOGENY AND INDUCED EMBRYOGENIC DEVELOPMENT

Uninucleate microspores of Triticum aestivum cv. Pavon can be induced in vitro to alter their development to produce embryoids rather than pollen. Microspores expressed their embryogenic capacity through one of two division pathways. In the more common route, the first sporophytic division was asymmetric and produced what appeared to be a typical bicellular pollen grain. Here the generative cell detached from the intine, migrated to a central position in the pollen grain, and underwent a second haploid mitosis as the vegetative cell divided to give rise to the embryoid. In the second pathway, the first division was symmetric and both nuclei divided repeatedly to form the embryoid. This comparative analysis of normal pollen ontogeny and induced embryogenesis provided no evidence for the existence of predetermined embryogenic microspores in vitro or in vivo. Instead, microspores are induced at the time of culture, and embryogenesis involves continued metabolic activity associated with the gradual cessation of the gametophytic pathway and a redifferentiation into the sporophytic pathway. In conjunction with a previous study, it appears that embryogenic induction of wheat microspores involves switching off gametophytic genes and derepressing sporophytic genes.     

Identification of Embryoid-Abundant Genes That Are Temporally Expressed during Pollen Embryogenesis in Wheat Anther Cultures

Uninucleate microspores in anther cultures of bread wheat (Triticum aestivum cv Pavon) are capable of producing haploid pollen embryoids and plants. To gain an understanding of this alternate pathway of pollen development, we constructed a cDNA library to young pollen embryoids, isolated embryoid-specific genes, and analyzed their expression patterns during morphogenesis. Two embryoid-abundant clones, pEMB4 and 94, were expressed very early during culture, suggesting that these genes are associated with development and are not simply expressed as a consequence of differentiation. The accumulation patterns of five cloned mRNAs may indicate the activation of specific genes associated with the major morphological and physiological activities connected with the differentiation of embryoids in vitro. These results suggest that embryoid-abundant gene expression is causally related to this pathway because gene expression is spatially and temporally specific and is not observed when microspores are cultured under noninductive conditions.     

Microspore culture in wheat (Triticum aestivum) – doubled haploid production via induced embryogenesis

The inherent potential to produce plants from microspores or immature pollen exists naturally in many plant species. Some genotypes in hexaploid wheat (Triticum aestivum L.) also exhibit the trait for androgenesis. Under most circumstances, however, an artificial manipulation, in the form of physical, physiological and/or chemical treatment, need to be employed to switch microspores from gametophytic development to a sporophytic pathway. Induced embryogenic microspores, characterized by unique morphological features, undergo organized cell divisions and differentiation that lead to a direct formation of embryoids. Embryoids `germinate' to give rise to haploid or doubled haploid plants. The switch from terminal differentiation of pollen grain formation to sporophytic development of embryoid production involves a treatment that halts gametogenesis and initiates sporogenesis showing predictable cellular and molecular events. In principle, the inductive treatments may act to release microspores from cell cycle control that ensures mature pollen formation hence overcome a developmental block to embryogenesis. Isolated microspore culture, genetic analyses, and studies of cellular and molecular mechanisms related to microspore embryogenesis have yielded useful information for both understanding androgenesis and improving the efficiency of doubled haploid production. The precise mechanisms for microspore embryogenesis, however, must await more research.     

Ultrastructure of embryogenic pollen ofNicotiana tabacum var. Badischer burley

Summary Pollen grains capable of embryogenesis were selectively isolated from (a) near-mature buds from plants induced to flower in short days and low temperature (8 hours light and 18 °C) and (b) young buds from these plants with an additional low temperature treatment (10 °C for 10 days) and fixed for electron microscopy. The pollen from the former formed embryos at a very low frequency in culture, and at the subcellular level showed different degrees of regression of cytoplasm and mitochondria. On the contrary, cold-treated pollen were characterized by a high frequency of embryogenesis, up to 25% of the cultured pollen. They did not show regression of cytoplasm or organelles but had an attenuated cytoplasm which was not rich in ribosomes. Another noteworthy feature of embryogenic grains was the condensed nature of mitochondria. These characteristics of embryogenic grains indicate that they are repressed for gametophytic differentiation. The embryogenic pollen did not differentiate from gametophytic pollen which were very distinctive, having a thick exine, and dense cytoplasm rich in ribosomes. The close similarity of embryogenic grains with young microspores in terms of thin exine and sparse cytoplasm is suggestive of an indeterminate state and that determination into gametophytic or sporophytic (embryogenic) type is probably the function of differential gene activity. Of interest, in this context, is the condensation of mitochondria in embryogenic grains. The relationship, if any, between mitochondrial condensation and embryogenesis remains to be resolved.     

Scanning electron microscopy of microspore embryogenesis inBrassica spp.

Scanning electron microscopy was employed to study and compare microspore embryogenesis in vitro with pollen development in planta inBrassica napus andB. oleracea. An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. Upon in vitro culture, all microspores, i.e., embryogenic and nonembryogenic, initially showed the same morphological features. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture (type I), the other showed cell cluster volume expansion over the entire microspore surface (type II). Two-thirds of embryogenic microspores in bothB. napus andB. oleracea demonstrated type I development. When followed by fluorescence microscopy, in vitro culture of microspores revealed cultures with a high embryo frequency were those with a high frequency of symmetrical division.Abbreviations SEM Scanning electron microscopy - TEM Transmission electron microscopy     

Messenger-RNA and protein changes associated with induction ofBrassica microspore embryogenesis

Brassica napus L. microspores at the late uninucleate to early binucleate stage of development can be induced in vitro to alter their development from pollen to embryo formation. High temperatures or other stress treatments are required to initiate this redirection process. The critical period for induction of microspore embryogenesis is within the first 8 h of temperature-stress imposition. During this period, which precedes the first embryogenic nuclear division, the process regulating the induction and sustainment of microspore embryogenesis is activated. A number of mRNAs and proteins, some of them possibly heat-shock proteins, appear in microspores during the commitment phase of the induction process.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis     

Embryogenic Suspension Culture and Plant Regeneration from Suspension-Derived Proto-plasts of Cucumber (Cucumis sativus L.)

Fast growing embryogenic cell suspension culture was established when embryogenic callus derived from cotyledon protoplasts of cucumber was transferred into a liquid culture. So far the cell line has been subcultured for two years and retained the ability of embryogenesis and plant regeneration. Experimental data showed that the concentration of ABA or sucrose had a dramatic effect on embryogenesis and synchronization of embryoid development. Low level of sucrose concentration (1%) facilitated the precocious germination of the embryoids while 1 mg/l of ABA or 7–9% of sucrose was found to be effective for reducing callusing of the cultures and synchronisticly controlling the embryoids at globular or late globular stage. Embryogenic cells taken from 3–5 days after subculture were enzymatically digested. A large amount of viable protoplasts was isolated. Protoplasts were cultured in a DPDK1 medium either by means of drop or thin layer liquid culture or by means of sodium alginate encapsulation culture. Actively dividing cells formed cell colonies and globular embryoids which were transferred onto a solidified agar medium or directly into a liquid medium to form a shaken culture. The embryoids would proliferated continuously. Embryoids eventually developed into plantlets when they were transferred onto a 1/2 MSO medium devoid of phytohormones.     

A co-culture system leads to the formation of microcalli derived from microspore protoplasts ofBrassica napus L. cv. Topas

Summary This paper describes a procedure in which protoplasts are obtained from microspores and pollen of rapeseed to induce callus formation aided by a feeder cell system with embryogenic microspores. Microspores at late unicellular stage and pollen at early bicellular stage were isolated and precultured for 24 h at 32 °C before enzymatic treatment. Eleven enzymes were tested in various combinations and concentrations. The optimal enzyme combination was 1.0% cellulase, 0.8% pectinase, 0.3% macerozyme, and 0.02% pectolyase, in which 26.3% of the microspores released protoplasts. A successful co-culture system was set up by employing embryogenic microspores as feeder cells. To this end, microspores were cultured in a medium with high osmotic pressure at 32 °C. Up to 37% of the microspores exhibited cell division and embryos developed to the heart-shape stage without changing medium. Microspore protoplasts were cultured in Millicells surrounded by the embryogenic microspores as feeder. In growth-regulator-free medium 14.5% of the protoplasts divided but only formed budding-like multicellular structures. Only after pretreatment with 4 mg of 2,4-dichlorophenoxyacetic acid and 1 mg of naphthaleneacetic acid per liter protoplasts divided and formed microcalli. Pollen tubes or tubelike structures were not observed. The experiments reveal that selection of the specific developmental stage of microspores, which is a prerequisite for microspore embryogenesis, is also important in microspore protoplast culture. Compared to other methods used before, microculture fed with embryogenic microspores has obvious superiority.Abbreviations CPW basic protoplast washing medium according to Power and Chapman - CPW972 CPW basic medium supplemented with 9% mannitol and 7.2% sorbitol - DAPI 4,6-diamidino-2-phenylindole - NLN nutrient medium according to Lichter modified by Pechan and Keller - NLN13 NLN medium supplemented with 13% sucrose - NLNP NLN13 supplemented with 7.2% sorbitol     

Stress as the major signal controlling the developmental fate of tobacco microspores: towards a unified model of induction of microspore/pollen embryogenesis

Specific stress treatments (sucrose starvation, alone or combined with a heat shock) applied to isolated tobacco (Nicotiana tabacum L.) microspores irreversibly blocked normal gametophytic development and induced the formation of embryogenic cells, which developed subsequently into pollen-derived embryos by culture at 25°C in a sugar-containing medium. A cold shock at 4°C did not inhibit microspore maturation in vitro and did not induce cell division activity, even when combined with a starvation treatment. In the absence of sucrose, microspores isolated in the G1 phase of the cell cycle replicated their DNA and accumulated in G2. Late microspores underwent miotosis during the first day of culture which resulted in a mixed population of bicellular pollen grains and uninucleate microspores, both embryogenic. After the inductive stress treatments the origin of the first multicellular structures, formed in the sugar-containing medium, could be traced to divisions of the microspore cell or divisions of the vegetative cell of bicellular pollen, indicating that the symmetry of microspore mitosis in vitro is not important for embryogenic induction. These results represent a step forward towards a unified model of induction of embryogenesis from microspores/pollen which, within a relatively wide developmental window, are competent to deviate from normal gametophytic development and initiate the alternative sporophytic programme, in response to specific stress signals.Abbreviation DAPI 4,6-diamidino-2-phenylindole We acknowledge the help of Monica Boscaiu and Zarko Hrzenjak with the artwork, and Michaela Braun-Mayer for growing the tobacco plants. This project was financed by the Austrian Fonds zur Forderung der wissenschaftlichen Forschung, grant S6003-BIO.     

Stress-induced microspore embryogenesis in tobacco: an optimized system for molecular studies

Summary Specific stress treatments applied to isolated tobacco (Nicotiana tabacum L.) microspores efficiently induced haploid embryo formation in vitro. A heat shock at 33 or 37°C in the presence of sugar, as well as sucrose-starvation at 25°C, resulted in the formation of embryogenic microspores. A combination of both treatments had an additive effect. Under optimal induction conditions all viable microspores in the culture were embryogenic and developed subsequently into pollen embryos by culture at 25°C in a sugar-containing medium, with induction frequencies of more than 70% with respect to the initial microspore population. A high fraction of the early pollen embryos continued their development in vitro, giving rise to haploid plants. In contrast to other available systems for microspore/pollen embryogenesis, the new protocol allows the production of homogeneous populations of embryogenic microspores and early globular embryos in large-scale cultures, without any purification step, and is therefore well suited for biochemical and molecular work.Abbreviations EDTA ethylenediaminetetraacetate - DAPI 4,6-diamidino-2-phenylindole     

Influence of 2,4-d and lactose on pollen embryogenesis in anther culture of potato

Anthers of diploid genotypes of Solanum tuberosum capable of androgenesis were cultured on different media to examine the effect on induction of pollen embryogenesis of 2,4-d and lactose. Anthers cultured in callogenic medium with 2,4-d and sucrose produced pollen derived embryoids only exceptionally. When sucrose was replaced by lactose the frequency of embryogenesis was as high or higher than in embryogenic auxin-free medium. Substitution of lactose for sucrose in the embryogenic medium had no effect. Supplementing the embryogenic medium with 2,4-d strongly reduced the frequency of pollen embryoids in the presence of sucrose but not with lactose.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid     

The 35S-CaMV promoter is silent during early embryogenesis but activated during nonembryogenic sporophytic development in microspore culture

Summary The cauliflower mosaic virus 35S (35S-CaMV) promoter, which is generally used as a constitutive promoter in plants, is known to be silent during microspore and pollen development. Here we analyzed whether the 35S-CaMV promoter fused to thegus (-glucuronidase) gene can be used as a marker for early sporophytic development in embryogenic microspore cultures of tobacco andBrassica napus. In microspore culture ofB. napus, the 35S-CaMV promoter remained off from the start of embryogenic culture up to the mid-cotyledonary embryo stage. 35S-CaMV promoter activity was only present in those microspores that initiated sporophytic development, but failed to enter embryogenic development. Similar results were also obtained with shed-microspore cultures of tobacco, in which rapid, direct embryogenesis takes place. In isolated-microspore cultures, in which embryogenesis is delayed, an intermitting period of sporophytic development was observed, characterized by extensive 35S-CaMV promoter activity. Therefore, the 35S-CaMV promoter discriminates between two classes of sporophytic development: it is activated in microspores which change fate from gametophytic into (temporarily) nonembryogenic sporophytic development, whereas the promoter is silent in sporophytic microspores that enter embryogenic development directly. This mirrors our observation that the 35S-CaMV promoter is also silent in young zygotic embryos.     

Nuclear DNA synthesis during the induction of embryogenesis in cultured microspores and pollen of Brassica napus L.

The dynamics of nuclear DNA synthesis were analysed in isolated microspores and pollen of Brassica napus that were induced to form embryos. DNA synthesis was visualized by the immunocytochemical labelling of incorporated Bromodeoxyuridine (BrdU), applied continuously or as a pulse during the first 24 h of culture under embryogenic (32 °C) and non-embryogenic (18 °C) conditions. Total DNA content of the nuclei was determined by microspectrophotometry. At the moment of isolation, microspore nuclei and nuclei of generative cells were at the G1, S or G2 phase. Vegetative nuclei of pollen were always in G1 at the onset of culture. When microspores were cultured at 18 °C, they followed the normal gametophytic development; when cultured at 32 °C, they divided symmetrically and became embryogenic or continued gametophytic development. Because the two nuclei of the symmetrically divided microspores were either both labelled with BrdU or not labelled at all, we concluded that microspores are inducible to form embryos from the G1 until the G2 phase. When bicellular pollen were cultured at 18 °C, they exhibited labelling exclusively in generative nuclei. This is comparable to the gametophytic development that occurs in vivo. Early bicellular pollen cultured at 32 °C, however, also exhibited replication in vegetative nuclei. The majority of vegetative nuclei re-entered the cell cycle after 12 h of culture. Replication in the vegetative cells preceded division of the vegetative cell, a prerequisite for pollen-derived embryogenesis.     

Observation of Androgenesis in Cultured Wheat Anthers at Meiosis,Tetrad, Early Uninucleate and Trinucleate Stage

The obtaining of calluses and plantlets from cultured wheat anthersat the stages from pollen mother cell to trinucleate microspore has been reported previously. Haploids as well as diploids existed among the regenerated plantlets derivedfrom anthers at these stages. Present paper reports the study on androgenesis patter-ns of cultured anthers at meiosis, tetrad, early mid- and late uninucleate and trinucleate stage. Cytological evidence of pollen-origin of calluses produced by anthers atthese stages was given. Observation showed that meiosis of wheat anthers was able tocomplete under culture conditions, resulting in releasing microspores, from which multinucleate and multicellular pollen grains formed. In meiosis anthers, abnormal cells,including syncytium and two kinds of binueleate calls were sometimes observed. Theymight be products of abnormal meiosis and abnormal development of tapetum cells. Itwas noted that failure and/or uncomplction of forming callus wall and/or pollen wallin in vitro anthers at meiosis, tetrad and early uninucleate stage occured often. Itmight lead to the low frequency of callus induction. Mature wheat anthers (trinucleate stage) contained both normal and abnormal pollen grains (pollen dimorphism); onlythe abnormal pollen grains developed into embryoids while all the normai trinucleatepollen grains degenerated rapidly. However, the date of the frequency of equal divisionof microspores suggested that abnormal pollen (N pollen, small pollen) could not be theonly source of androgenic pollens in cultured anthers at late uninucleate and other earlier stages.     

The development of haploid embryoids from anther cultures of Atropa belladonna L.

Summary Development of haploid embryoids from the microspores of Atropa belladonna occurs with relatively high frequency when anthers are excised from buds in which the petals are shorter than the sepals (at this stage microspores are predominantly uninucleate) and cultured on a medium containing iron as the ferric salt of ethylenediamine-di-O-hydroxyphenylacetic acid (FeEDDHA). Additions of combinations of kinetin, auxin and casamino-acids to the culture medium induce callusing in both haploid and diploid tissues, lead to the origin of embryoids from somatic tissues of the anther and should be avoided. Simple techniques for the maintenance of haploid clones are described.Stages in early embryogenesis in the pollen grains have been observed and these indicate that embryogenesis is most frequently initiated by an equal division in the uninucleate spore. The frequency of grains showing embryoid formation is very low and it is estimated that plantlets are formed from up to 50% of these grains.     

Changes in synthesis and localization of members of the 70-kDa class of heat-shock proteins accompany the induction of embryogenesis inBrassica napus L. microspores

Elevation of the culture temperature to 32°C for approximately 8 h can irreversibly change the developmental fate of isolatedBrassica napus microspores from pollen development to embryogenesis. This stress treatment was accompanied by de-novo synthesis of a number of heat-shock proteins (HSPs) of the 70-kDa class: HSP68 and HSP70. A detailed biochemical and cytological analysis was performed of the HSP68 and HSP70 isoforms. Eight HSP68 isoforms, one of which was induced three fold by the stress treatment, were detected on two-dimensional immunoblots. Immunocytochemistry revealed a co-distribution of HSP68 with DNA-containing organelles, presumably mitochondria. Six HSP70 isoforms were detected, one of which was induced six fold under embryogenic culture conditions. During normal pollen development, HSP70 was localized in the nucleoplasm during the S phase of the cell cycle, and predominantly in the cytoplasm during the remainder. Induction of embryogenic development in late unicellular microspores was accompanied by an intense anti-HSP70 labeling of the nucleoplasm during an elongated S phase. In early bicellular pollen the nucleus of the vegetative cell, which normally does not divide and never expresses HSP70, showed intense labeling of the nucleoplasm with anti-HSP70 after 8 h of culture under embryogenic conditions. These results demonstrate a strong correlation between the phase of the cell cycle, the nuclear localization of HSP70 and the induction of embryogenesis. As temperature stress alone is responsible for the induction of embryogenic development, and causes an altered pattern of cell division, there might be a direct involvement of HSP70 in this process.Abbreviations HSP heat-shock protein - 2-D two-dimensional - DAPI 4,6-diamidino-2-phenylindole. 1-D = one-dimensional - pI isoelectric point     

In vitro differentiation of embryogenic pollen,control by cold treatment and embryo formation inab initio pollen cultures ofNicotiana tabacum var. badischer burley

Summary InNicotiana cold treatment causes differentiation of embryogenic pollen. This differentiation initiates on the plant and is completed in culture. Differentiation on the plant results in pollen dimorphism and differentiation in culture leads to pollen embryogenesis. An increased number of pollen capable of embryogenesis is possible on plants induced to flower in short days and low temperature (8 hours light, 18 °C). These embryogenic pollen on selective isolation, from buds at a petal length of 3.4±0.1 cm, fail to form embryos but do so in the cultures which receive cold treatment at 10 °C for 10 days. To some extent the differentiation of embryogenic pollen can be completed on plants induced to flower at 15 °C and embryogenic pollen from such plants form embryos at a low frequency which can be substantially increased on giving the cultures a cold treatment. The frequency of embryogenesis is higher in cultures of 15 °C plants than those of 18 °C plants. Low temperature requirements at two stages—to the plant and to the culture—are essential and complimentary for embryogenesis inab initio pollen cultures.Cold treatment causes repression of gametophytic differentiation and this results in the differentiation of embryogenic potential. The embryogenic pollen, unlike gametophytic pollen, are not fully differentiated structures. They are unable to divide and form embryos in presence of metabolic inhibitors such as actinomycin-D and cycloheximide.