Mechanism Of 2n Pollen Formation Research Articles

High frequency 2n pollen formation in black poplar (Populus nigra L.) induced by colchicine

Populus nigra L. is one of the most important male genetic donors in populus genetic improvement and tree breeding over the world. Many excellent Populus nigra clones are identified as triploids potentially obtaining by hybridization of 2n pollen and normal oogamete. This study revealed the cytological mechanism of 2n pollen formation in Populus nigra L. for the first time and concluded the best treated combination of colchicine treatment, which obtained 2n pollen with the highest rate reaching at 87.11% (even 100% 2n pollen in some floral buds) which made the polyploid hybridization utilizing 2n pollen in section Aigeiros possible.
 
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Cytological mechanism of 2n pollen formation in Chinese jujube (Ziziphus jujuba Mill. ‘Linglingzao’)

In order to elucidate the cytological mechanism of 2n pollen formation in Chinese jujube, a cultivar named ‘Linglingzao’ (2n = 2x = 24) which produces relative more 2n pollens naturally was employed for microsporogenesis analysis. Chromosomes paired in 12 bivalents at diakinesis and the first meiotic division was normal, whereas, the second division was characterized by frequent abnormal spindle orientation (parallel and tripolar spindles). Perpendicular, tripolar, and parallel spindles at metaphase II accounted for 72.93, 17.22, and 9.85% respectively. Perpendicular and tripolar spindles led to tetrads and triads formation respectively. Two types of parallel spindles were observed and a significant association correlation (r = 0.84, P < 0.05) between parallel spindle II (fused spindles) and dyads was found which meant only fused spindles can form dyads, while parallel spindle I with a long distance between two sets of chromosomes seemed to lead tetrad formation. At tetrad stage, the observed frequencies of tetrads, triads and dyads were 80.99, 16.40, and 2.61% respectively. By the rule that each tetrad can form four n pollens, each triad can form one 2n pollen and two n pollens, each dyad can form two 2n pollens, the frequency of 2n pollen estimated was 5.71%. Based on the pollen diameter difference between n and 2n pollens, frequency of 2n pollen observed was 6.15%. No significant difference was observed between frequency of 2n pollen estimated and that of observed. The results indicated that, tripolar spindles and fused spindles at metaphase II followed by more triads and few dyads formation at tetrad stage were responsible for the production of 2n pollen in Chinese jujube. 2n gametes observed correspond to first division restitution. Research on the cytological mechanism of unreduced pollen will provide a platform for unreduced pollen induction and polyploidy breeding in Chinese jujube.

Occurrence and cytological mechanism of 2n pollen formation in Chinese Diospyros spp. (Ebenaceae) staminate germplasm

SummarySome Diospyros spp. (Ebenaceae) staminate germplasm (2n = 6x = 90) recently found in Hubei Province, China, was of potential value for breeding. This study evaluated the natural rate of occurrence and cytological mechanism responsible for the formation of 2n pollen in this staminate germplasm. The frequencies of giant pollen, dyads, and triads detected in all accessions were 0 – 2.3%, 0.8 – 2.2%, and 2.3 – 4.3%, respectively. The correlation coefficient (r = 0.98**; P < 0.01) between the frequency of giant pollen and those of dyads and triads was highly significant, which strongly suggests that the giant pollen was 2n pollen. Cytological analysis of pollen mother cell meiosis showed that the main cytological mechanisms responsible for the formation of 2n pollen were tripolar spindles and fused spindles, instead of parallel spindles and vertical spindles. In addition, an abnormal disintegration of sister chromatids, which also produced 2n pollen in the end, was occasionally found at metaphase II. Diploid (2n) pollen produced by tripolar spindles and fused spindles was equivalent to first division restitution (FDR) gametes. Therefore, this Diospyros spp. staminate germplasm may have value for sexual polyploidisation in breeding programmes of Japanese persimmon.

Cytological mechanisms of 2n pollen formation in the wild potato Solanum okadae and pollen-pistil relations with the cultivated potato, Solanum tuberosum

Solanum okadae Hawkes et Hjert is a wild diploid potato species endemic to Argentina and Bolivia, of potential breeding value. However, no genetic studies have been carried out with this species and its crossability relations with the common tetraploid potato, S. tuberosum L. ssp. tuberosum, are unknown. Furthermore, accessions from both countries differed in their morphological phenotypes. To ascertain the feasibility of incorporating this wild germplasm into cultivated potato, 2n pollen screening was carried out in 10 accessions and families derived from crosses between accessions; also, pollen-pistil compatibility relations were studied in reciprocal interspecific crosses. Plants of four of the 10 accessions produced 2n pollen (0.1 to 5.0%) and 4n pollen (0.0 to 3.0%). Parallel and tripolar spindles at Anaphase II were the cytological mechanisms involved in 2n pollen formation; lack of chromosome migration in both meiotic divisions originated the 4n pollen. Both full compatibility and incompatibility at various sites along the pistil were observed in the S. tuberosum × S. okadae combinations; most reciprocal combinations were incompatible. Compatible genotypes produced 2n pollen. However, only a few seeds were obtained and chromosome counts could not be carried out in the hybrid progeny because seedlings died at an early stage. The pollen-pistil barriers are incomplete and can be circumvented by the appropiate choice of parents. The identification of the post-zygotic barriers will be the focus of further studies.

Creating triploid germplasm via induced 2n pollen in Capsicum annuum L.

Summary2n pollen of diploid chili (Capsicum annuum L. ‘Xianjiao’) was induced by heat shock (35.5˚C, 4.h) or low temperature treatment (118C, 48.h). The percentages of 2n pollen were increased to 8.3% and 10.5% respectively. The cytogenetic mechanisms of 2n pollen formation were first division restitution, second division restitution and abnormal cytokinesis. Diploid 2n pollen was less competent than n pollen in fertilization. Only one triploid plant arose from 78 progenies after fertilization by 2n pollen. The four offspring plants of the triploid were diploid. The morphological characters of the triploid and microsporogenesis were also evaluated.

Occurrence and cytological mechanism of 2n pollen formation in a tetraploid accession of Ipomoea batatas (sweet potato).

We evaluated a 4x accession of Ipomoea batatas (L.) Lam. and four 2x accessions of Ipomoea triloba for 2n pollen production. Approximately 90% of the genotypes of accession 81.2 (I. batatas, 4x) produced 2n pollen with different frequencies. In contrast, none of the genotypes of I. triloba produced 2n pollen. The diameter of the 2n pollen was approximately 30% ((3) sqrt 2) larger than that of the n pollen, making it easy to identify, measure, and quantify. The correlation (r = 0.93**) between the frequency of giant pollen and the frequency of dyads and triads was highly significant, strongly suggesting that the giant pollen grains were 2n pollen. The 2n pollen producers presented either a parallel or tripolar spindle arrangement (Y shaped) at anaphase II instead of the normal 60 degrees crossed spindle orientation. These two abnormal spindle configurations produced dyads and triads, with different frequencies (13-67%), instead of tetrads. Occasionally a metaphase II spindle variation was found with a single fused spindle, which also forms a dyad. The correlation (r = 0.89**) between the frequency of 2n pollen and the frequency of parallel, fused, and tripolar spindle arrangements was also highly significant, suggesting that these abnormal spindle configurations are involved in the production of 2n pollen in I. batatas. When we evaluated the efficiency of 2n pollen in polyploidization using 4x x 4x (2n) crosses, all progenies were 4x, suggesting the existence of barriers to crossability between 4x genotypes and their 2n pollen-producer counterparts.

Homoeologous recombination in 2n-gametes producing interspecific hybrids ofLilium(Liliaceae) studied by genomic in situ hybridization (GISH)

Interspecific hybridization of Lilium longiflorum (L) with Asiatic (A) lily hybrids results in so-called LA-hybrids. Some of these hybrids produce 2n-pollen, which were used to perform crosses on Asiatic and Oriental (O) hybrids, resulting in ALA- and OLA-hybrids. Recombination between homoeologous chromosomes (introgression) and the mechanism of 2n-pollen formation in these hybrids were studied using genomic in situ hybridization (GISH). A clear differentiation between the chromosomes of L. longiflorum, Asiatic, and Oriental hybrids was observed in four ALA- and one OLA-hybrid using GISH. Two ALA-hybrids showed 3 and 5 recombinant chromosomes with a total of 5 and 10 crossover sites per hybrid, respectively. These occurred at random positions on the chromosomes. The number and the location of the rDNA-sites were determined using in situ hybridization and provided a tool, the FISH-marker, for identifying the NOR-bearing chromosomes in the lily hybrids. Evidence for the occurrence of the FDR-mechanism (first division restitution) of 2n-pollen formation in the LA-hybrids was obtained on the basis of absence of homologous chromosomes of L. longiflorum in the ALA- and OLA-hybrids.Key words: Lilium longiflorum, introgression, FDR, interspecific hybridization, FISH.

RFLP Detection of 2n Pollen Formation by First and Second Division Restitution in Perennial Ryegrass

Unreduced gametes can be used to transfer desirable traits from parent to polyploid offspring. Codominant restriction fragment length polymorphism (RFLP) markers were applied to determine the mechanism of 2n pollen formation in diploid (2n = 2x = 14) Lolium perenne L. Sixty heterologous probes were randomly chosen from a Festuca arundinacea Schreb. PstI‐genomic DNA library. A total of 30 and 33 heterozygous allelic loci were detected in two genotypes of parental L. perenne, respectively, based on segregation of RFLP loci in 13 triploid (3x) and two tetraploid (4x) hybrids, produced in 1993, between diploid L. perenne and tetraploid F. mairei St. Yves. Approximately 30 and 83% of the heterozygosity was transmitted by 2n pollen from L. perenne into two tetraploid hybrids, respectively. Both meiotic first division restitution (FDR) and second division restitution (SDR) occurred in 2n pollen formation in L. perenne. Omission of the second meiotic division (OS) and fusion of adjacent polar nuclei (FA) were found during microsporogenesis. These two mechanisms were supposedly responsible for forming 2n gametes by SDR and FDR, respectively. Formation of 2n pollen in diploid L. perenne could facilitate efficient transfer of germplasm from diploid to higher ploidy levels in Lolium and Festuca.

Cytological basis of 2n pollen formation in a wide range of 2x, 4x, and 6x taxa from tuber-bearing Solanum species

Cytological mechanisms of 2n pollen formation were investigated in microsporogenesis and sporad development in 2x, 4x, and 6x taxa of tuber-bearing Solanum species that were originally collected from Mexico, and Central and South America. Parallel spindles and tripoles were seen at anaphase II in the majority of genotypes observed. The occurrence of parallel spindles and tripoles were well correlated with the occurrence of dyads and triads at the sporad stage. Further, the frequency of 2n pollen predicted by frequencies of parallel spindles and tripoles and by frequencies of dyads and triads were also well correlated with observed 2n pollen frequencies. Premature cytokinesis at telophase I to prophase II was seen only in 2x and 4x species from series Conicibaccata. The frequency of premature cytokinesis corresponded well with the frequency of dyads leading to 2n pollen formation. It was concluded that the major mechanisms of 2n pollen formation in tuber-bearing Solanum species is parallel spindles.

Cytology of 2n Pollen Formation in Diploid Crested Wheatgrass, Agropyron cristatum

Hybrids between induced tetraploids of diploid crested wheatgrass, Agropyron cristatum (L.) Gaertn. (2n = 2x = 14), and tetraploid crested wheatgrass, A. desertorum (Fisch. ex Link) Schult. (2n = 4x = 28), have provided improved germplasm with unique agronomic traits. Introgression from diploids to tetraploids would be simplified if diploids that produce 2n gametes (gametes with the sporophytic chromosome number) could be identified. This study was initiated to determine if 2n pollen is produced in diploid crested wheatgrass and to determine the cytological mechanisms of 2n pollen formation. Pollen diameters were investigated by measuring 60 pollen grains from each of 600 diploid and 100 tetraploid genotypes. Pollen diameters averaged 37.9 μm (SE = 0.17) and 41.1 μm (SE = 0.33) for diploid and tetraploid genotypes, respectively, with a range of 35.3 to 41.3 μm among the diploids, and 39.7 to 46.8 μm among the tetraploids. Approximately 16% of the diploid genotypes produced some pollen grains with twice the mean volume of n pollen. Cytological analyses of 17 individuals that produced variable‐sized pollen indicated that 2n pollen was produced via restitution at the second meiotic division. Meiotic abnormalities that could give rise to 2n microspores include i) arrest at metaphase II, ii) lack of sister chromatid movement in anaphase II, iii) movement of all or most sister chromatids to a single pole in anaphase II, and iv) abnormal cytokinesis after telophase II. The formation of 2n pollen in diploid crested wheatgrass should facilitate efficient transfer of germplasm from diploids to tetraploids within the genus Agropyon.

Some Aspects of Inbreeding in Derived Tetraploids of Potatoes

Diploid (2n = 2x = 24) potato (Solanum) species that produce 2n gametes, by either first division or second division restitution mechanisms, are being widely used in potato breeding programs to enhance the genetic diversity of the tetraploid potato (S. tuberosum L.). initial biometrical modeling of tetraploid X diploid hybridizations in potatoes has revealed complex genetic relationships between diploid parents and their tetraploid offspring. Three breeding schemes are presently used by potato breeders to transfer genetic material from diploid parents to their tetraploid offspring: 2n gamete-producing diploids may be crossed directly to the tetraploids; haploids (2n = 2x = 24) may be extracted from the tetraploids and crossed to the diploid species parent to form a haploid-species hybrid that is then crossed to the tetraploid; or 2n gamete-producing diploids may be crossed to each other. I have determined the inbreeding coefficient for tetraploids derived by any of these three breeding strategies. These inbreeding coefficients are complex functions of the coancestry of the parents, the inbreeding of the parents, the coefficient of double reduction in the tetraploid parent, and the frequency of single exchange tetrads in the diploid parent(s), and they depend on the mechanism of 2n pollen formation.

Estimation of preferential pairing in tetraploid x diploid hybridizations

Crosses made between tetraploid and diploid, 2n pollen-producing species directly transfer from one-half to the entire diploid genome from the diploid to the tetraploid level, depending on the mechanism of 2n pollen formation and the amount of crossing-over that occurs. Tetraploid plants that result from tetraploid x diploid hybridizations can be further utilized in a breeding program. It is postulated that preferential pairing between homologous chromosomes derived from the original tetraploid or diploid parent occurs in the tetraploid x diploid hybrid. Depending on the genetic divergence of the species involved, preferential pairing of homologous chromosomes may range from zero to one. Theoretical estimates of the amount of preferential pairing and the standard errors of these estimates are derived for cases where the diploid parent produces 2n gametes by either a first division or a second division restitution mechanism.

Introgression of Solarium acaule germ plasm from the endosperm balance number 2 gene pool into the cultivated endosperm balance number 4 potato gene pool via triplandroids

Triploid plants from interspecific crosses between selected clones of Solanum acaule and a 2n pollen producing, cultivated diploid expressed an average of 7.7% to 2n pollen, called triplandroids. Percent stainable pollen of these triploids ranged from 1.8 to 22.1%. The cytological mechanism of 2n pollen formation was parallel orientation of spindles in the second division of meiosis. Three triplandroid-producing triploids were crossed to several tetraploid potato cultivars and breeding clones. Of these 4x−3x pollinations, 49% produced berries, containing an average of 15.2 seeds. Progeny from 4x−3x crosses were 67% pentaploid, 2n = 60, and 33% aneuploids 2n = 56–59, mostly 2n = 59. Thus, functional microspores from triploids were predominantly triplandroids, 2n = 3x = 36, with low frequencies of aneuploidy. This use of a triplandroid bridge is a rapid means of introducing S. acaule germ plasm to the cultivated tetraploid gene pool.Key words: interspecific hybrids, parallel spindles, pollen restitution, triploidy, 2n pollen.

Occurrence of 2n pollen and ps gene frequencies in cultivated groups and their related wild species in tuber-bearing Solanums

The gene frequency for parallel spindles (ps) was estimated from the frequency of plants producing 2n pollen in three cultivated groups: 2x Phureja (phu), 2x Stenotomum (stn), and 4x Andigena (adg), as well as in four related wild taxa: 2x Solanum brevicaule (brc), 2x S. sparsipilum (spl), 4x S. gourlayi (grl) and 4x S. gourlayi-S. infundibuliforme hybrids (grl-ifd). Plants with more than 1% large pollen were considered as 2n pollen producers. Observations of meiosis in a sample of 2n pollen-producing plants indicated that parallel spindles is the mechanism of 2n pollen formation. The number of plants with 2n pollen among the total examined was 228 plants (15.5%) of 1,473 in 2x spl, 31 (26.7%) of 116 in 2x brc, 92 (17.4%) of 528 in 2x stn, 665 (22.1%) of 3,008 in 2x phu, 731 (51.4%) of 1,421 in 4x adg, 591 (41.2%) of 1,436 in 4x grl, and 36 (64.3%) out of 56 in 4x grl-ifd. The ps gene frequencies assuming Hardy-Weinberg equilibrium were: 0.393 for 2x spl, 0.462 for 2x brc, 0.417 for 2x stn, 0.470 for 2x phu, 0.847 for 4x adg, 0.801 for 4x grl, and 0.895 for 4x grl-ifd. Twenty-five adg clones were randomly selected from a large population and were crossed with 2x clone W5295.7, which produces 2n pollen by parallel spindles (ps). The 4x progenies from 4x×2x crosses were used to determine the genotypes at the ps locus by screening 10-20 plants in each family for 2n pollen. Based on chromosome segregation at the ps locus, 9, 14, 1, and 1 clones were nulliplex, simplex, simplex or duplex, and duplex, respectively. The frequency of the ps gene in the adg population was estimated to be 0.825 and 0.815 for chromosome and chromatid segregation, respectively. The high frequencies of 2n pollen and the ps gene in cultivated 2x and 4x groups, and in wild taxa closely related to them, provide evidence for sexual polyploidization in the tuber-bearing Solanums.

Cytological mechanism of 2n pollen formation and unilateral sexual polyploidization in Lolium

Plants of Lolium perenne, L. multiflorum and L. hybridum (331, 51 and 18, respectively) were screened for 2n pollen production. The screening was based on the size differences that are expected to be found between n and 2n pollen of a plant. It was found that 28 plants of perennial ryegrass-belonging to 13 cultivars-produced produced pollen grains of heterogenous size (big and small). The estimated frequency of big pollen grain production was higher than 10% in ten out of 28 plants and in three of them it reached a value of 100%. Eight plants obtained from two 4x × 2x crosses, in which the male parent had been previously identified as producing pollen grains of heterogenous size, was tetraploid. The cytological mechanism of big pollen grain formation observed in three plants consists in the lack of spindle formation and, consequently, of chromosome migration in anaphase II. From these breeding and cytological results, it was concluded that the big pollen grains observed were viable 2n pollen grains. The nuclei of the 2n pollen grains produced by this mechanism are genetically equivalent to those formed by the restitution of the second meiotic division. The value of these gametophytes in a breeding program of Lolium is discussed.

The mechanism of 2n pollen formation in diploids of Costus speciosus (Koenig) J.E. Smith and role of sexual polyploidization in the origin of intraspecific chromosomal races.

Microsporogenesis in the two diploid clones of C. speciosus was studied in detail. The absence of cytokinesis detected in some of the dyads at the close of the second meiotic division was the mechanism producing 2n pollen in both the diploids. The distinct variation in n and 2n pollen size indicated the possibility of its use as a reliable criterion for estimating the frequency of 2n pollen in large populations. In addition to the normally expected diploids, certain triploid and tetraploid genotypes were identified in the selfed progenies of both the diploids which confirmed the occurrence of functional 2n gametes in their parental diploid clones. The present study suggested that sexual polyploidization involving the function of 2n gametes might have played an important role in the origin of naturally occurring intraspecific chromosomal races of C. speciosus.

The occurrence and frequency of 2n pollen in three diploid solanums from Northwest Argentina

A group of wild, tuber-bearing species from Northwest Argentina, belonging to the series Tuberosa, Solarium spegazzini Bitt. (spg, 2n=2x=24), S. gourlayi Hawkes (grl, 2n=2x=24 and 2n=4x=48) and S. oplocense Hawkes (opl, 2n=6x=72), and Cuneolata, S. infundibuliforme Phil (ifd, 2n=2x=24), is being used to investigate the mode of origin of polyploids in the genus Solanum. 2n gametes have been detected in the diploid species ifd and spg and in a diploid race of grl, using cytological and breeding approaches. Twenty-two introductions of spg, 8 of grl and 26 of ifd have been tested for 2n pollen; 59%, 63% and 54% of them, respectively, had at least one 2n pollen producing plant. These introductions comprised 238, 76 and 235 plant respectively, of which 20, 16, and 32 plant produced 5% or more 2n pollen. The mechanism of 2n pollen formation was determined in several plant of 2x spg, 2x grl and 2x ifd. All of them were found to form diplandroids via parallel spindles. This mechanism, which gives meiotic products genetically equivalent to first division restitution gametes, is under control of the Mendelian recessive ps. The results suggest that the allele ps is widely distributed in natural populations of the three diploids, and that its frequency is very high. These species are seen as valuable material for population genetic studies, and for the eventual incorporation into a breeding scheme involving sexual polyploidization via 2n gametes.

CYTOLOGY OF 2n POLLEN FORMATION IN DIPLOID ALFALFA, MEDICAGO SATIVA

Four diploid (2x) clones of alfalfa, Medicago sativa L., which produced good seed set when used as male parents in 4x-2x crosses were selected for study. The 2x clones descended from 2x haploids of cultivated 4x alfalfa. Fertility in the 4x-2x cross was due to the production of pollen with the unreduced chromosome number (2n pollen) from the 2x parent. The cytological mechanism of 2n pollen formation was found to be disorientation of spindles at metaphase II in up to 38% of the pollen mother cells. Thus, both n and 2n pollen were produced by all four diploids examined. Normal spindles at metaphase II were oriented such that they defined the poles of a tetrahedron and resulted in normal tetrads in a tetrahedral arrangement. Disoriented spindles were basically parallel to each other and resulted in formation of dyads and occasionally a triad. Dyads developed into two 2n pollen grains; triads developed into one 2n and two n pollen grains. Since both n and 2n pollen grains are produced by the diploids, they can be maintained as diploids or they can be used as male parents in crosses to tetraploids. The genetic constitution of 2n pollen resulting from parallel spindles is similar to that expected after first division restitution of meiosis and much of the heterozygosity of the diploid parent is conserved in the gametes. The 2n gamete mechanism has potential application in germplasm transfer and in maximizing heterozygosity in tetraploid hybrids.

THREE MECHANISMS OF 2nPOLLEN FORMATION IN DIPLOID POTATOES

Three mechanisms of 2n pollen formation observed in diploid hybrids between Solatium tuberosum Group Phureja (2x) and haploids of S. tuberosum Group Tuberosum (4x) are termed parallel spindles (ps), premature cytokinesis 1 (pc 1) and premature cytokinesis 2 (pc 2). A II spindles are parallel to each other in many cells of clones producing 2n pollen by ps. A single cleavage furrow follows and forms a dyad which generates two 2n microspores. The other two mechanisms, pc 1 and pc 2, produce 2n pollen by omission of the second meiotic division. Premature cytokinesis 1 (pc 1) involves asynchronized movement of bivalents to and from the M I plate and falling apart of chromatids at T I. A cleavage furrow follows the first meiotic division, no second division occurs and products of meiosis are dyads. Premature cytokinesis 2 (pc 2) exhibits no abnormalities at the first meiotic division. However, cytokinesis occurs prematurely at P II, no second division occurs, and only dyads are formed. Genetic data suggested that these three mechanisms may be controlled by single loci, and are inherited independently as simple recessives.