Abstract: During pollen development each product of meiosis undergoes a stereotypical pattern of cell divisions to give rise to a three-celled gametophyte, the pollen grain. First an asymmetric mitosis generates a larger vegetative cell and a smaller generative cell, then the generative cell undergoes a second mitosis to give rise to two sperm cells. It is unknown how this pattern of cell divisions is controlled. We have identified an Arabidopsis gene, SIDECAR POLLEN, which is required for the normal cell division pattern during pollen development. In the genetic background of the NoO ecotype, sidecar pollen heterozygotes have about 45% wild-type pollen, 48% aborted pollen and 7% pollen with an extra cell. Homozygous sidecar pollen plants have about 20% wild-type pollen, 53% aborted pollen and 27% extra-celled pollen. Similar ratios of sidecar pollen phenotypes are seen in the Columbia ecotype but sidecar pollen is a gametophytic lethal in the Landsberg erecta ecotype. Thus this allele of sidecar pollen shows differential gametophytic penetrance and variable expressivity in different genetic backgrounds. The extra cell has the cell identity of a vegetative cell and is produced prior to any asymmetric microspore mitosis. Pollen tetrad analysis directly demonstrates that SIDECAR POLLEN is indeed expressed in male gametophytes. To our knowledge, scp is the first male gametophytic mutation to be described in Arabidopsis.

Abstract: Prior to this report, heat treatment (32.5°C, 24 h) was the method used to induce embryogenesis fromBrassica napus microspores. Continuous culture at 25°C results in pollen development. This study shows that colchicine alone, at the non-inductive temperature of 25°C, can induce embryogenesis, thus demonstrating that heat shock is not required for embryogenic induction inB. napus cv. Topas. Embryogenic frequencies of over 15% were obtained by culturing isolated microspores with 25 μM colchicine for 42 h at 25°C. The microspore developmental stages responsive to colchicine were unicellular vacuolate and late unicellular, somewhat earlier stages than the population responsive to heat induction. Other groups have reported that heat-shock proteins are essential to the induction of embryogenesis. The present study offers a method of embryogenic induction without the use of heat which will allow discrimination between the factors associated with response to heat shock and those involved with changing cell development.

Abstract: 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.

Abstract: 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.

Abstract: The structural pattern of tissue and meristem formation during zygotic and microspore embryo development in canola was examined. A defined sequence of cell divisions could be found during zygotic e...

Abstract: Male-sterile mutants are being studied to deepen our understanding of the complex processes of microsporogenesis and microgametogenesis. Due to difficulties associated with isolating the mutated gene, there is currently very little molecular information on the defects responsible for male sterility. As a first step in utilizing male-sterile mutants to better understand the bio-chemical and molecular processes that control pollen development, we have characterized a number of Arabidopsis thaliana lines that were generated by seed transformation and exhibit male sterility. We report here the identification and characterization of three male-sterile A. thaliana lines, all of which are tagged with T-DNA and show aberrant meiosis. A detailed cytochemical study was conducted on these lines to better understand the timing and nature of each mutation and to investigate how these mutations affect subsequent steps of pollen development. All three mutants undergo apparently normal morphogenesis until the onset of meiosis. In one line (6492) the mutation is most notable at the tetrad stage when up to eight microspores can be seen in each callose-encased tetrad. The resulting mutant microspores are of variable sizes and contain different amounts of DNA. Two other mutants (7219 and 7593) possess many common features, including variable developmental pathways, failure to produce callose, production of vacuolate, coenocytic (multi-nucleate) cells that are surrounded by persistent microsporocyte walls, and asynchronous patterns of development. Unlike the situation in wild-type plants, where developmental stages are correlated with bud length, such correlations are almost impossible with these two mutants. The sporogenous tissue within all three of these mutant lines collapses prior to anthesis.

Abstract: This report describes a very high genome doubling efficiency of Brassica napus cv. Topas plants, derived from microspores induced to undergo embryogenesis with a colchicine treatment, without the use of a heat treatment. The plants showed normal growth and development, and 90% were fertile. In contrast, only 6% of the plants derived from heat-induced embryos were fertile diploids. All cytological analysis of the progeny of fertile plants showed 2n=38 chromosomes. These results show that colchicine can simultaneously induce microspore embryogenesis and double the ploidy level to produce doubled haploid plants.

Abstract: Summary A large, heterogeneous, highly expressed gene family encoding oleosin-like proteins is described in the Brassicaceae. ieven related cDNA sequences were isolated from Brassica napus anther mRNA using RACE-PCR and compared with other recently described anther-specific oleosin-like genes from B. napus. The expression patterns of four representative members of this diverse gene family were analyzed by Northern blotting and in situ hybridization. In all cases, the genes were expressed specifically in the tapetum of 3–5 mm B. napus buds, which contained microspores at the late-vacuolate and bicellular stages of development. The predicted protein products are ordered into subclasses, each of which has a characteristic C-terminal domain, containing different amino acid motifs or repeated residues. Tryphine (pollen coat) fractions from mature B. napus pollen were found to be particularly enriched in polypeptides of apparent molecular weights 32–38 kDa, plus numerous less abundant polypeptides of less than 15 kDa. The N-terminal 15–20 residues of three of these polypeptides (12, 32 and 38 kDa) were found by microsequencing to be identical to parts of the predicted amino acid sequences of three of the tapetal-expressed oleosin-like genes. This indicates the possibility of post-translational modification of these proteins resulting in a cleavage of the primary translation products in order to generate the mature tryphine polypeptides. These data imply that a large and diverse group of oleosin-like proteins is synthesized in the tapeturn of B. napus anthers and that following tapetal degradation, these proteins, possibly in modified form, then relocate to the developing microspores where they eventually constitute some of the major components of the extracellular tryphine of mature pollen grains. These proteins share a conserved 70 amino acid residue hydrophobic domain and are related structurally to the seed-specific intracellular oleosins, although their biological function may be different.

Abstract: A clone for an embryoid-abundant, early cysteine-labeled metallothionein (EcMt) gene has been isolated from a wheat pollen embryoid cDNA library. The transcript of this gene was only expressed in embryogenic microspores, pollen embryoids, and developing zygotic embryos of wheat. Accumulation of the EcMt mRNA showed a direct and positive correlation with an increase of the plant hormone, abscisic acid (ABA) in developing pollen embryoids. Treating cultures with an inhibitor of ABA biosynthesis, fluridone, suppressed not only ABA accumulation but also the appearance of the EcMt gene transcript and the ability of microspores to form embryoids. These results suggest that the EcMt gene may act as a molecular marker for pollen embryogenesis because ABA biosynthesis is accompanied by the increased expression of the EcMt transcript that coincides with the differentiation of pollen embryoids in wheat anther cultures.

Abstract: Morphogenic calli were obtained efficiently from ab initio cultures of isolated microspores in eggplant. Initial culture of freshly isolated microspores in sucrose-free medium at high temperature (35°C) for 3 d was a prerequisite for callus induction. The microspores were re-cultured in modified NLN medium containing 2% sucrose and phytohormones (NAA 0.5 mg l(-1), BA 0.5 mg l(-1)) in the dark. After 4 weeks of re-culture, small calli derived from microspores were transferred to MS medium containing 4 mg l(-1) zeatin and 0.2 mg l(-1) IAA for shoot regeneration. The ploidy of 12 randomly selected regenerants was assessed by chromosome counts in root tips. Only one of the regenerants was haploid, 7 were diploid, 3 were triploid and one was tetraploid. The diploids set seeds after self-pollination and showed no segregation for morphological traits in the progeny, suggesting that they were spontaneously doubled haploids.

Abstract: Haploid embryos can be produced at high frequency by anther and microspore culture of many Brassica species and commercial cultivars (Keller et al.,1984, 1987; Lichter, 1989; Swanson, 1990; Arnison and Keller, 1990; Duijs et al., 1992; Palmer et al.,1994). The procedures for such production are routine although there are still significant genetic limitations to recovery of haploids from some genotypes. The development of procedures for efficient recovery of haploids was driven principally by their potential usefulness in the development of homozygous lines for breeding programs (Wenzel et al.,1977). The high regenerative potential of haploid microspores, especially when cultured in isolation from the anther tissues, provides an ideal system for mutagenesis and the recovery of mutants. In addition, the recovery of doubled haploids through chromosome doubling techniques ensures that mutant genes are fixed in the homozygous state.

Abstract: A male-sterile mutant, mei-1, of Arabidopsis thaliana is described. In this mutant, instead of a tetrad of four microspores being formed after meiosis, a "tet- rad" consisting of from five to eight microspores is formed. The microspores show a wide range of sizes and of DNA contents. The mutant is female-fertile. This mu- tant was produced by seed transformation with Agrobac- terium and appears to be T-DNA tagged.

Abstract: A number of factors influencing embryogenesis from isolated microspores of radish (Raphanus sativus) were examined Of 11 genotypes evaluated, six produced embryos ranging from 83 embryos per 10(5) microspores for 'Chugoku-ao' to 02 for 'Tenshun', but five genotypes were not responsive An initial culture period at elevated temperature before incubation at 25°C was essential for induction of microspore embryogenesis However, the optimum period of the treatment varied among genotypes and/or experiments Bud size also influenced microspore embryogenesis Though optimum bud size was different between genotypes, the microspore populations represented in these buds contained uninucleate and binucleate microspores Selection of embryogenic microspores using percoll density gradient resulted in up to 13-fold increase of embryo yield Though almost all embryos failed to develop directly into plantlets, plants were obtained by multiple subcultures The regenerated plants had hyperploid chromosome numbers

Abstract: Microspore culture is a very important and useful tool in plant breeding for haploid production and has been developed for many yearsBrassica campestris (Brassica rapa L sspoleifera) is an important oilseed crop, but it is relatively recalcitrant in tissue culture including microspore culture The microspore culture in our laboratory is based on the Canadian protocol Thirty genotypes ofB campestris were included in this study; twenty produced embryos The highest yield was 5930 embryos per 100 buds from Canadian genotype Cv-2, this result was one of the best that had been reported in microspore culture inB campestris The buds measuring 20 mm to 39 mm in length responded best to produce embryos, the optimum timing for microspore culture was confirmed to be during the mid-late to very-late uninucleate stage The buds could be removed from either the main raceme or lateral racemes Activated charcoal (150 mg l-1) was added to the liquid NLN medium, it promoted embryogenesis significantly; embryo development was faster and the embryo yield was significantly higher than those cultures without activated charcoal The donor plant condition was considered an important factor influencing embryogenesis; older donor plants (older than five weeks) and a cold treatment are recommended

Abstract: Microspores of several genotypes of Brassica campestris ssp. parachinensis have been cultured in vitro and induced to undergo embryogenesis and plant formation. Conditions favourable for embryogenesis in this species include a bud size of 2-2.9 mm, NLN-13 culture medium (Nitsch and Nitsch 1967; Lichter 1981, 1982; Swanson 1990), and an induction through exposure to 32°C for a period of 48 h. Longer periods of an elevated temperature for induction of embryogenesis resulted in embryo abortion at early developmental stages. With the protocol developed here, microspores of 60-80% of donor plants could be induced to produce embryos, although embryo yields were low, i.e. 2-5 embryos per 10 buds. Some genotypes responded to culture conditions with high numbers of embryo formation (100-150 embryos per 10 buds) but most of these subsequently failed to mature. The pattern of cell division and morphological changes of the microspores in culture were studied using various microscopic techniques.

Abstract: The life cycle of higher plants alternates between a diploid sporophytic phase and a haploid gametophytic phase. The gametophytic generation begins after meiosis with two types of haploid gametophytes or spores: microspores, which develop into pollen grains within the anther (male gametogenesis), and megaspores, which form the embryo sac within the ovule (female gametogenesis). During male gametogenesis microspores undergo a precisely defined and often synchronous sequence of cell divisions that lead to the formation of highly specialized cells: a large vegetative cell and two sperm cells. Although this tightly controlled developmental pathway consists of only two cell divisions, under certain experimental conditions, immature male gametophytes can be induced to undergo an altered development leading to the production of haploid embryos. During this process, known as androgenesis or microspore embryogenesis, the male gametophyte develops directly into an embryo, without an intervening fertilization. In this review, several recent findings on microspore embryogenesis will be described. A central issue will be what triggers microspores or pollen to change their developmental fate to produce sporophytes. The emphasis will be on molecular events that occur during the initial stages of microspore embryogenesis. Two main questions that still remain to be answered are: what is the genetic basis for embryogenic potential, and how is embryogenic development induced?

Abstract: The invention relates to transformed, embryogenic microspores and progeny thereof characterized by being transformed by Agrobacterium tumefaciens, capable of leading to non-chimeric transformed haploid or doubled haploid embryos that develop into fertile homozygous plants within one generation and containing stably integrated into their genome a foreign DNA, said DNA being characterized in that it comprises at least one gene of interest and at least base pairs within the right border sequence of Agrobacterium T-DNA. The invention furthermore relates to a method for the incorporation of foreign DNA into chromosomes of microspores comprising the following steps: a) infecting of embryogenic microspores with Agrobacteria, which contain plasmids carrying a gene of interest under regulatory control of initiation and termination regions bordered by at least one T-DNA border, b) washing out and killing the Agrobacteria after co-cultivation.

Abstract: The potential uses of doubled haploids in plant breeding and genetic studies have been well documented in the past two decades (Chen, 1986; De Buyser et al., 1987; Graner et al., 1991). There are at least three doubled haploid systems. They are anther or isolated microspore culture (Charmet and Bernard, 1984; Dunwell et al., 1987; Zhou and Konzak, 1989; Ziauddin et al., 1990), ovary culture (Wang and Kuang, 1981; Castillo and Cistue, 1993), and embryo rescue following interspecific crosses, such as the Hordeum bulbosum system (Kasha and Kao, 1970) and the wheat × maize hybridization method (Amrani et al., 1993). The anther culture system has been a favorable choice by many researchers because of the availability of a large number of microspores within each anther that could potentially produce doubled haploid plants. For many plant species, anther culture is the most efficient means to obtain ample haploid plants for selecting desirable F1 combinations in plant breeding.

Abstract: Several pollen-specific genes from different species have been isolated and characterized at the molecular level, but the precise role of most of them is unknown. Mutant analysis represents a direct approach to uncovering gene function, but the paucity of available mutants affecting pollen development and/or function and the poor characterization of the known mutants have so far limited the exploitation of this approach. Here we present the cytological characterization ofgametophytic male sterile-1 (gaMS-1), a maize mutant that we identified in a program of transposon insertion mutagenesis for the production of mutations in gametophytically acting genes involved in microsporogenesis.gaMS-1 is expressed during or immediately after the first microspore division and leads to the production of immature, nonfunctional pollen grains. The mutation appears to affect the events leading to the developmental switch that follows the first microspore mitosis.

Abstract: Twelve accessions of tronchuda cabbage landraces were tested for their ability to produce embryos through microspore culture in NLN-13 medium. A sample from all the isolations was stained with DAPI for determination of microspore developmental stage. The relationship between the microspore developmental stage and the production of microspore-derived embryos was evaluated. Embryos were obtained from all the accessions. Considerable variation was observed between isolations with different developmental stage of microspores, accessions and plants within the same accession. The best embryogenic responses occurred in microspore populations with approximately 10 to 89% of binucleate pollen. The highest embryo yield was obtained with one of the ‘Couve de Valhascos’ accessions (0.41 embryos / 1 000 microspores) and the lowest yields with the accessions of ‘Couve Portuguesa’ (less than 0.07 embryos / 1 000 microspores).

Abstract: In cultured microspores from Brassica napus, embryogenesis can be synchronously and irreversibly induced by elevating the culture temperature to 32°C for a minimum of 8 h. Culture at 18°C allows gametophytic development to continue, and results in the formation of pollen in vitro. This allows us to study the temperature controlled switch in developmental fate from gametophytic development to embryogenic development by molecular means. Analysis of protein synthetic patterns by [35S]-methionine incorporation and 2-dimensional gel electrophoresis, revealed that 25 proteins were differentially synthesized during the induction of microspore embryogenesis. Most of these proteins (17) appeared to belong to the class of heat shock proteins (HSPs). Four of these HSPs have been identified by Western blotting using antibodies raised against HSP17, HSP68 and HSP70. One protein that was only synthesized under embryogenic culture conditions, and did not belong to the heat inducible HSPs, is a candidate marker for early embryogenic development.

Abstract: Section 1. The Discovery of Anther Culture Technique for Production of Haploids - A Personal Reflection S. C. Maheshwari. Haploids in Plant Breeding G. S. Khush, S. S. Virmani. Reflections on Doubled Haploids in Plant Breeding P. S. Baenziger. Cytogenetics and Potentials of Haploidy in Forest Tree Genetics and Improvement S. Baldursson, M. R. Ahuja. Haploidy and Mutation Techniques M. Maluszynski et al. Cytological and Biochemical Aspects of in vitro Androgenesis in Higher Plants R. S. Sangwan, B. S. Sangwan-Norreel. Molecular and Biochemical Events During the Induction of Microspore Embryogenesis J. H. G. Cordewener et al. The Utility of Doubled Haploid Population for Studying the Genetic Control of Traits Determined by Recessive Alleles K. P. Pauls. Section 2. Anther Culture S. K. Sopory, M. Munshi. Influence of Ethylene in Microspore Embryogenesis T. Tiainen. Effect of Gelling Agents on Anther Culture E. K. Callenberg, L. B. Johnsson. Microspore Culture J. M. Dunwell. Ovary and Ovule Culture for Haploid Production E. R. J. Keller, L. Korzun. In Vitro Pollination and Fertilization S. S. Bhojwani, A. Raste. Irradiated Pollen for Haploid Production S. Sestila. Polyhaploid Production in the Triticeae by Sexual Hybridization A. Kazi-Mujeeb, O. Riera-Lizarazu. Conditional Lethal Markers: Spontaneous Haploid Selection in Plants C. Harlow et al. Methods to Double Haploid Chromosome Numbers P. S. Rao, P. Suprasanna.