A re-examination of the mechanisms of 2n gamete formation in potato and its implications for breeding

Summary The claim that first division restitution gametes are produced through the unique meiotic abnormality of parallel spindle formation at anaphase II of microsporogenesis in Solanum species appears to be not justified. An examination of the phenomenon of meiotic nuclear restitution in fifty-six diploid Solanum clones revealed that the parallel orientation of spindles either at metaphase or anaphae II does not necessarily lead to dyad or unreduced microspore formation. However, the fused spindle formation at metaphase II of microsporogenesis invariably leads to dyad and unreduced microspore formation. The 2n gametes produced through fused spindles may be genetically equivalent to first division restitution gametes, and therefore, they may be useful in potato breeding. In addition to fused spindle formation, at least three other types of meiotic abnormalities that could lead to 2n gamete formation were also observed. Although any one, or two, of these mechanisms were predominantly observed in certain clones, it was by no means certain that other mechanisms did not operate in these clones at the same time, or at a later stage. Because of the great modifying influence of environment on the process of meiosis, and meiotic nuclear restitution, it might be erroneous to conclude that one or the other meiotic nuclear restitution mechanism alone operates at a time in a given clone. In order to elucidate the genetic basis of 2n gamete formation in potato, it is desirable (i) to answer whether there are exclusive genetic mechanisms for the formation of first or second division restitution gametes; (ii) to establish reliable cytological criteria for their detection; and (iii) to determine the effect of environmental factors on the occurrence of different mechanisms.     

Further cytological characteristics of a male-sterile mutant in soybean affecting cytokinesis and microspore development

A spontaneous male‐sterile mutation affecting microspore development has been found in BR97‐13509H line of the official Brazilian soybean breeding programme. Slightly abnormal and irregular chromosome segregation occurred in both meiotic divisions, albeit insufficient to explain total pollen sterility. The main cause of male sterility was the absence of cytokinesis after telophase II. Instead of the typical tetrads of microspores, four‐nucleate coenocytic microspores were formed. Coenocytic microspores resulting from the absence of cytokinesis have also been described in ms1 and ms4 male‐sterile soybean mutants, although with certain differential characteristics. In the BR97‐13509H mutant line, after interphase the four nuclei were rejoined in one or two metaphase plates and progressed through the first pollen mitosis. Pollen then underwent a progressive process of degeneration characterized by continuous cytoplasmic shrinkage. The second pollen mitosis failed to occur, and pollen sterility became total.     

Formation of unreduced gametes is impeded by homologous chromosome pairing in tetraploid <Emphasis Type="Italic">Triticum turgidum</Emphasis> × <Emphasis Type="Italic">Aegilops tauschii</Emphasis> hybrids

It is believed that unreduced gametes with somatic chromosome numbers play a predominant role in natural polyploidization. Allohexaploid bread wheat originated from spontaneous hybridization of Triticum turgidum L. with Aegilops tauschii Coss. Unreduced gametes originating via meiotic restitution, including first-division restitution (FDR) and single-division meiosis (SDM), are well documented in triploid F₁ hybrids of T. turgidum with diploid Ae. tauschii (genomic constitution ABD, usually with 21 univalents in meiotic metaphase I). In this study, two T. turgidum lines known to carry genes for meiotic restitution were crossed to tetraploid Ae. tauschii. The resulting F₁ hybrids (genomes ABDD), had seven pairs of homologous chromosomes and regularly formed 14 univalents and seven bivalents at metaphase I. Neither FDR nor SDM were observed. The distribution of chromosome numbers among progeny obtained by self pollination and a backcross to T. turgidum showed the absence of unreduced gametes. These results suggest that high homologous pairing interfered with meiotic restitution and the formation of unreduced gametes. This may be related to asynchronous movement during meiosis between paired and unpaired chromosomes or to uneven distribution of chromosomes in anaphases, resulting in nonviable gametes.     

Functional genomics of microspore embryogenesis

Isolated plant microspores, when stressed and cultured in vitro, can be diverted from their normal gametophytic pathway towards sporophytic development, with the formation of haploid embryos and ultimately doubled-haploid plants. This process is called androgenesis or microspore embryogenesis, and is widely used in plant breeding programmes to generate homozygous lines for breeding purposes. Protocols for the induction of microspore embryogenesis and the subsequent regeneration of doubled haploid (DH) plants have been successfully developed for more than 200 species. These practical advances stand in stark contrast to our knowledge of the underlying molecular genetic mechanism controlling this process. The majority of information regarding the genetic and molecular control of the developmental switch from gametophytic to sporophytic development has been garnered from four intensely studied (crop) plants comprising two dicotyledonous species, rapeseed (Brassica napus) and tobacco (Nicotiana tabacum), and two monocotyledonous species, wheat (Triticum aestivum) and barley (Hordeum vulgare). In these species the efficiency of microspore embryogenesis is very high and reproducible, making them suitable models for molecular studies. In the past, molecular studies on microspore embryogenesis have focussed mainly on the identification of genes that are differentially expressed during this developmental transition and/or early in embryo development, and have identified a number of genes whose expression marks or predicts the developmental fate of stressed microspores. More recently, functional genomics approaches have been used to obtain a broad overview of the molecular processes that take place during the establishment of microspore embryogenesis. In this review we summarise accumulated molecular data obtained in rapeseed, tobacco, wheat and barley on embryogenic induction of microspores and define common aspects involved in the androgenic switch.     

Abnormal pollen development of bread wheat-<Emphasis Type="Italic">Leymus mollis</Emphasis> partial amphiploid

Summary Octoploid Tritileymus was the partial amphiploid obtained from Triticum aestivum × Leymus mollis, its chromosome constitution was 14 Leymus chromosomes and 42 wheat chromosomes. In the pollen development of Tritileymus, some abnormalities were observed, such as the absence of tetrad formation, cytomixis of microspores, irregular shape of some microspores and multi-germination pores, symmetrical first mitosis, chromosomes or chromatids randomly dispersed in the cytoplasm of microspore.     

Pollen and coenocytic microspore germination in male-fertile and malesterilemsl soybean

Summary Light and fluorescence microscopy were used to study coenocytic microspore germination from male-sterile (ms1 ms1) soybean plants. Anther squashes from male-sterile plants revealed that a low frequency of natural coenocytic microspore germination occurred in male-sterile anthers of four independent lines; [ms1-North carolina (T260H),ms1-Urbana (T266H),ms1-Tonica (T267H), andms1-Ames (T268H)]. Abnormalities such as giant tubes, branched tubes, tubes with swollen areas, and multiple tubes were observed from coenocytic microspores from all four lines. The Urbana line, however, demonstrated a higher percentage of coenocytic microspore germination than did the other three lines. Flowers of the Urbana line from both malefertile and male-sterile plants, as well as gynoecia pollinated with coenocytic microspores from sterile plants, were used for in vivo studies. Pollen-tube growth appeared normal in male-fertile plants. In contrast, coenocytic microspore tubes rarely were observed in gynoecia from male-sterile plants or in gynoecia from malefertile plants that had been artificially cross-pollinated withms1 ms1 plants. Few tubes from coenocytic microspores were observed in the vicinity of the micropylar region. A low frequency of seed set was achieved in the greenhouse on Urbana male-sterile plants grown in the absence of male-fertile plants. Thus, we believe either that some gametes from coenocytic microspores are able to participate in fertilization at low frequency or that apomixis occurs inms1 ms1 plants.Joint contribution: Agricultural Research Service, U.S. Department of Agriculture, and Journal Paper No. J-12310 of the Iowa Agriculture and Home Economics Experiment Station, Ames IA, 50011 USA. Project 2471.     

Frequent occurrence of unreduced gametes in <Emphasis Type="Italic">Triticum turgidum</Emphasis>–<Emphasis Type="Italic">Aegilops tauschii</Emphasis> hybrids

Spontaneous chromosome doubling via union of unreduced (2n) gametes has been thought to be the way that common wheat (Triticum aestivum L.) was originated from the hybridization of T. turgidum L. with Ae. tauschii Cosson. Previous works have observed unreduced gametes in F₁ hybrids of Ae. tauschii with six of the eight T. turgidum subspecies. It is not clear, however, whether the formation of these unreduced gametes is a norm in the F₁ hybrids. In the present study, we tried to answer this question by assessing the occurrence frequency of unreduced gametes in 115 T. turgidum–Ae. tauschii hybrid combinations, involving 76 genotypes of seven T. turgdium subspecies and 24 Ae. tauschii accessions. Our data show that these hybrid combinations differed significantly (P ≤ 0.01, F = 11.40) in selfed seedset, an indicator for production of unreduced gametes. This study clearly showed that meiotic restitution genes are widely distributed within T. turgidum. However, significant differences were found between as well as within T. turgidum subspecies and in the interaction of the T. turgidum genotypes with those of Ae. taushii. The possible application of the meiotic restitution genes from T. turgidum in production of double haploids is also discussed.     

Chromosome number and microsporogenesis in a pentaploid accession of Brachiaria brizantha (Gramineae)

Chromosome number, microsporogenesis and mode of reproduction are described for an accession of Brachiaria brizantha, a grass of African origin. Cytological analysis revealed that the accession BRA005886 is pentaploid (2n = 5x = 45), with a base number of x = 9. Multivalent chromosome associations, from tri‐ to pentavalents, were recorded in diakinesis, and the further meiotic behaviour was highly irregular as well. Most abnormalities were related to irregular chromosome segregation commonly found in polyploids. Micronuclei were observed following telophases I and II. Some micronuclei near the cell wall were released as microcytes, whereas others remained in the tetrad. Other meiotic abnormalities, such as cell fusion and the absence of cytokinesis causing the formation of dyads and triads were also recorded. Binucleate microspores and 2n microspores resulting from nucleus restitution were observed among the normal ones. Limitations of this accession for use in hybridization programmes are discussed.     

Transformation studies in Hordeum vulgare using a highly regenerable microspore system

Summary A highly regenerable, isolated microspore system for barley, Hordeum vulgare L. cv. Igri, has been developed which is amenable to transformation studies using particle bombardment. The system allows DNA to be delivered to microspores at the single cell stage and both transient and stable transformation events have been demonstrated. The potential advantages of using isolated microspores as the target tissue in routine transformation systems are discussed.     

大麦的小孢子发生和雄配子体发育的细胞学研究

本文研究了大麦(Hordeum vulgare L.)的小孢子发生和雄配子体发育的细胞学过程,主要结论是:小孢子母细胞减数分裂胞质分裂为连续型,四分体排列为左右对称型.小孢子第一次有丝分裂形成生殖和营养细胞.生殖细胞再分裂一次,形成一对精子.所以,大麦的成熟花粉粒为三细胞型.绒毡层为分泌型.母细胞减数分裂时,绒毡层细胞内多数具双核.小孢子释放前,绒毡层开始退化,二核花粉粒时仅留残痕.     

Development of the male gametophyte of an infertile plant Armoracia rusticana Gaertn.

Horseradish, Armoracia rusticana , is propagated vegetatively through root cuttings. It is described as a male-sterile plant as it does not form seeds or only develops them in small quantities. This study discusses the anatomical structure of anthers and the events throughout meiosis up to the formation of a male gametophyte. Microscopic observations have shown that the anatomical structure of the anther wall of A. rusticana is typical of the whole family of Brassicaceae , and all its layers are properly developed and fully functional. Meiosis in the sporogenous tissue proceeds correctly and leads to the formation of microspores. Also the configurations of the cytoplasmic skeleton are typical of plants with simultaneous cytokinesis. Further development, however, exhibits many abnormalities. A majority of the cells formed in the sample studied were dysfunctional uninucleate microspores with apparent symptoms of degeneration of the cytoplasm and organelles. Binucleate pollen grains, in which mitosis did occur, constituted only 10% of the cells analysed. Anatomical and cytological observations of male sterility in A. rusticana indicated inhibition of development at the microspore, stage which was manifested by cytoplasm degeneration with a low number of membranes and a scarce amount of starch in proplastids. Usually, in the prolonged microspore stage and in the young pollen no mitosis occurs at the time of anthesis. Cytological disorders may, to a great extent, limit the number of functional bicellular male gametophytes. Both in microspores and in pollen grains, there is exceptionally little starch in plastids.     

2n Gamete formation in the genus Brachiaria (Poaceae: Paniceae)

Microsporogenesis of several Brachiaria species of the Brazilian collection at Embrapa Beef Cattle has been analyzed in detail. This paper reports abnormal cytokinesis in three accessions of three different species (Brachiaria humidicola, 2n = 4x = 36, Brachiaria decumbens, 2n = 4x = 36, and Brachiaria dura, 2n = 6x = 54). Chromosomes paired in bi-, tri-, and quadrivalents in these accessions, whereas chromosome segregation at meiosis I was characterized by exclusion of laggards as micronuclei. In a high number of meiocytes, the first sign of cytokinesis appeared only in metaphase II and did not divide the meiocyte into a dyad. Total absence of cytokinesis was also detected among meiocytes in the second division. Since in both cases the two metaphase plates were very close, they favored the rejoining of chromosome sets after anaphase II and formed a restitutional nucleus in telophase II. Second cytokinesis occurred after telophase II in most meiocytes. Monads, dyads, and triads with n or 2n nuclei were observed among meiotic products. The 2n gametes observed correspond to the first division restitution (FDR). The number of affected cells in each accession was variable, but the number of microspores with restitutional nucleus, including those scored in tetrads and the released ones, did not exceed 9%. Although polyploidy is common in the genus Brachiaria, its origin is still unclear. Current results suggest that 2n gametes may have contributed to the evolutionary history of the genus.     

Fertile maize lines obtained from isolated microspores

Microspore response of three- way cross maize hybrid genotype 3AL/95 (Zea mays L.) was studied under simplified isolation and culture conditions. Fertile plant production was achieved through abundant plant regeneration. As a total, microspores of 160 tassels were inoculated and five sustainable microspore derived callus cultures (SMC) were obtained. Hybrid seeds (ML SC), which were produced by crossing of regenerates from two SMCs, gave rise to subsequent vigorous and fertile progeny. The response of the 3AL/95 and ML SC microspores was studied in three liquid culture media in order to improve the early viability of microspores. Them N6M medium provided better survival of cultured microspores (p = 5%) than the ppN6M/89 and the YPM-G media. The pH 5.8 in mN6M medium revealed significant increase (p = 1%) in microspore viability as compared to pH 3.0. The ML SC microspores showed higher viability(30%) on the first day of culture in the mN₆M than those of the3AL/95 (19%) but without improved rate of callus formation and plant regeneration. This revised version was published online in August 2006 with corrections to the Cover Date.     

Matromorphy in Brassica oleracea L. VII. Research on products of microsporogenesis and gametogenesis from prickle pollinated plants

Summary A research has been carried out on the occurrence of 2n gametes in Brassica oleracea and on their possible way of origination. After microsporogenesis it appeared that unreduced PMC's, dyads and triads occurred. Giant pollen grains with two (diploid) and four (haploid) generative nuclei, respectively, were found, resulting from a deviating cytokinesis and karyokinesis. Diploid heterozygous matromorphic embryos may develop from such unreduced gametes formed after macrosporogenesis.     

Breeding for quantitative resistance to potato cyst nematode (Globodera pallida) in tetraploid potatoes using dihaploids and unreduced gametes

Summary Dihaploids were produced from tetraploids resistant to potato cyst nematode (Globodera pallida (Stone)). High levels of resistance were found in the dihaploids and three were used to produce tetraploid progenies by crossing them with susceptible tetraploid cultivars. One dihaploid, PDH505, produced more highly resistant offspring than the other two, PDHs 417 and 418. The latter gave progenies whose levels of resistance were similar to those obtained from susceptible dihaploids crossed with resistant tetraploids.The differences between the progenies of the resistant dihaploids were probably due to different modes of unreduced gamete formation (PDH505 producing gametes by first division restitution (FDR) and PDHs 417 and 418 by second division restitution (SDR)) although cytological studies would be necessary to confirm this. The methods by which dihaploids could be utilised in a tetraploid potato breeding programme are discussed in relation to the mode of unreduced gamete formation.     

Meiotic restitution mechanisms and 2n pollen formation in a Solanum tuberosum dihaploid and in dihaploid × wild species hybrids*

A Solatium tuberosum dihaploid that proved to be a 2n pollen producer, exhibited two types of meiotic abnormality involved in 2n pollen formation: premature cytokinesis at prophase II and parallel/tripolar spindles in meiosis II. For this reason such a dihaploid was preferentially used as a seed parent in crosses with the diploid species S. tarijense, S. sanctae-rosae, and S. phureja. Diploid interspecific hybrids were evaluated for male fertility, for 2n pollen production and stability over three years and for meiotic nuclear restitution mechanisms. Pollen stainability ranged from 6 to 85% and 2n pollen from 5 to 35% among hybrids. The meiotic mechanism generating 2n pollen was that of spindle abnormality in meiosis II and consequent dyad/triad formation. High correlation was found between fused/parallel spindles and dyads.     

Efficient Production of Doubled Haploid Plants through Chromosome Doubling of Isolated Microspores in Brassica napus

Three methods of chromosome doubling to produce doubled haploid plants from microspore cultures of Brassica napus were compared: colchicine treatment of microspore-derived plants, microspore-derived embryos, and isolated microspores. In the whole plant treatment, 53% of the treated plants set seed, but the treatment delayed plant growth and reduced seed set. When microspore-derived embryos were treated with colchicine, the doubling frequency was 32% (compared to 15% for spontaneous doubling). Direct colchicine treatment of isolated microspores resulted in a doubling efficiency of 70 % of the whole plants. This treatment also stimulated embryogenesis in microspore culture, leading to increased plant regeneration. Thus, direct chromosome doubling of isolated microspores is efficient and more than 10 000 doubled haploid plants have been produced in this manner in the past three years in order to accelerate the plant-breeding process.     

Structural observations during androgenic microspore culture of the 4c1 genotype of Zea mays L.

Summary The capacity of the maize genotype 4c1 to regenerate microcalli and embryos from cultured microspores has been examined by comparing various cold pretreatments and culture media, using microspores and pollen at different stages of development. Viability of cultured cells was tested with FDA and their development was traced with light and fluorescence microscopy using DAPI as a nuclear dye.It was found that a pre-incubation of dissected flowers floating in a liquid nutrient medium at 8°C during 10–14 days was most successful for the induction of cell division. Among the developmental stages tested only the microspores appeared to regenerate. Subculture at 25°C in the same liquid medium, supplemented with 0.1 mg/l TIBA, gave highest rates of microspore division, i.e. up to 70% at 4 to 6 days of culture.All pathways described earlier for maize androgenic embryogenesis were observed within the 4c1 genotype. Symmetric divisions occurred in cultured microspores but most frequently asymmetric divisions lead to the formation of microcalli within 12 days of culture. In at least 60% of all dividing microspores cells were derived from the generative nucleus. Microcalli further developed either into loose or compact calli. Compact calli formed embryo-like structures.Abbreviations DAPI 4,6-diamidino-2-phenylindole - Dicamba 3,6-dichloro-2-methoxy benzoic acid - 2,4D 2,4 dichlorophenoxyacetic acid - FDA fluorescein diacetate - PAA phenylacetic acid - TIBA 2,3,5-triiodobenzoic acid - YP medium Yu-Pei basal salt medium     

A cytological study on aluminium-treated wheat anther cultures resulting in plants with increased Al tolerance

The in vitro selection of microspores and microspore‐derived structures under Al stress is one way to improve the Al tolerance of crops. In our study, cytological alterations caused by Al were examined in anther cultures of a commercial wheat (Triticum aestivum L.) variety ‘Mv Pálma’, and the efficiency of in vitro selection was demonstrated. Although the anther walls retarded the appearance of toxicity symptoms, cytological changes similar to those observed in root cells (inhibition of cell division, intense vacuolisation, occurrence of micronuclei and cell wall thickening) were detected in the microspores. The severity of Al toxicity and the efficiency of selection depended on the Al concentration and the mode of treatment. Single Al treatments (0.6 and especially 1.6 mM) allowed DH lines with increased Al tolerance to be selected. Repeated Al treatment severely inhibited the cell division of the microspores and it was lethal even at a concentration as low as 0.6 mM. The results show that microspore embryogenesis can be exploited for studying the cytological effect of Al and for increasing the Al tolerance of wheat.     

Morphological and cytological study in a new type of cytoplasmic male-sterile line CMS-GIG2 in sunflower (Helianthus annuus)

Cytoplasmic male sterility (CMS) is essential for sunflower hybrid production. CMS-GIG2, a new sunflower CMS type, was further confirmed by crossing with the maintainer and restorer lines for the CMS-PET1, both of which maintain the male sterility of CMS-GIG2. Meiotic division in CMS-GIG2 was observed with 4'6-diamidino-2-phenylindole dihydrochloride staining, indicating that microspore formation was disrupted before the meiotic cytokinesis was completed. Light microscopy observation showed that both middle layer and tapetal cells expand radially rather than degrade over time, followed by failure to form tetrads and normal microspores. This morphological defect leading to male sterility in CMS-GIG2 differs from that observed in the PET1 CMS type. CMS-GIG2 will certainly provide additional genetic diversity for sunflower hybrid breeding programmes.