Haploid Level Research Articles

Stable haploid poplar callus lines from immature pollen culture.

Embryogenesis and plant regeneration have been obtained from isolated immature pollen of two poplar hybrids (Populus nigra L. x hybrid 'Aue1' and 'Aue2'). In total, 1487 calli or embryos, respectively, larger than 1 mm were generated in a 2-year study. By using a cytokinin containing induction medium, on average 19 calli per responsive immature catkin were formed. Additional supplementation with auxin in 2002 increased the frequency to 72 calli per catkin. Microsatellite marker analyses confirmed haploid origin in most regenerants studied. So far six out of eight obtained regenerative callus lines have maintained their haploid level up to 24 months of development. A number of haploid and doubled haploid plants of different lines are available and have been transferred to soil.

Powdery mildew (Sphaerotheca fuliginea) resistance in melon is selectable at the haploid level.

The major cause of powdery mildew in melons (Cucumis melo L.) is the fungus Sphaerotheca fuliginea. There are several cultivar- and season-specific races of this fungus. In order to control powdery mildew, it is important to introduce resistance to fungal infection into new cultivars during melon breeding. Haploid breeding is a powerful tool for the production of pure lines. In this study, it was investigated whether powdery mildew resistance could be manifested at the haploid level from two disease-resistant melon lines, PMR 45 and WMR 29. the effects of various races of S. fuliginea on diploid and haploid plants of PMR 45 and WMR 29 and of a disease-susceptible line, Fuyu 3 were measured. The responses of haploid and diploid plants to powdery mildew were identical. In addition, haploids that were generated from hybrids between Fuyu 3 and disease-resistant lines were examined. Seven out of 13 haploids from a Fuyu 3xPMR 45 cross and 10 out of 12 haploids from a Fuyu 3xWMR 29 cross were classified as resistant plants because they showed the same responses as their disease-resistant diploid parents to the various fungal races. These results indicate that resistance in PMR 45 and WMR 29 is selectable at the haploid level. All of the plant responses were observed by microscopy. A possible mechanism for generating powdery mildew resistance in two different melon lines is discussed.

Karyotype analysis reveals interspecific differentiation in the genus Cedrus despite genome size and base composition constancy

The DNA content and GC% of the four true cedar (Cedrus) species, C. atlantica, C. brevifolia, C. deodara and C. libani, were assessed. Genome size was homogeneous among representative populations of the four species with an average of 32.6±0.6 pg per 2 C or 15.7×109 base pairs per 1 C. The composition in GC was calculated to be 40.7%. A simple monosomatic haploid level was found in the megagametophyte, as compared to the diploid level of the corresponding embryo. Cytogenetic studies showed a diploid chromosome number of 2n=2x=24 in 11 populations sampled over the four species. The chromosome complements have similar morphology and symmetry. However, fluorochromes revealed specific banding patterns in each of the four cedar species. Eight GC-rich chromomycin A3 bands were observed in Cedrus deodara chromosomes, six in both Cedrus libani and Cedrus brevifolia, and four bands in Cedrus atlantica chromosomes. Moreover, Hoechst 33258 fluorochrome revealed AT-rich sequences specifically located in the centromeric regions while the GC-rich sequences appeared negatively stained. These investigations provide a systematic characterisation of the Cedrus genus and should contribute towards clarification of the phylogenetic relationships among the four species.

Is maize B chromosome preferential fertilization controlled by a single gene?

In previous work, genotypes for high and low B chromosome transmission rate were selected from a native race of maize. It was demonstrated that the B transmission is genetically controlled. The present work reports the fourth and fifth generations of selection and the F1 hybrids between the lines. The native B is characterized by a constant behaviour, with normal meiosis and nondisjunction in 100% of postmeiotic mitosis. It is concluded that genetic variation for B transmission between the selected lines is due to the preferential fertilization process. The F1 hybrids show intermediate B transmission rate between the lines. They are uniform, the variance of the selected character being one order of magnitude lower than that of the native population. In addition, 0B x 2B and 2B x 2B crosses were made to study the effect of the presence of B chromosomes in the female parent, resulting in non-significant differences. Several crosses were made both in Buenos Aires and in Madrid to compare the possible environmental effect, but significant differences were not found. Our results are consistent with the hypothesis of a single major gene controlling B transmission rate in maize, which acts in the egg cell at the haploid level during fertilization. It is also hypothesized that maize Bs use the normal maize fertilization process to promote their own transmission.

Gynogenetic haploids of Citrus after in vitro pollination with triploid pollen grains

This study reports haploid plantlet regeneration through gynogenesis in Citrus clementina Hort. ex Tan., cv. Nules, induced by in vitro pollination with pollen grains of Oroblanco, a triploid cultivar of grapefruit. It indicates that parthenogenesis induced in vitro by triploid pollen can be an alternative method to obtain haploids in monoembryonic cultivars of Citrus. Actually, despite considerable efforts, androgenesis has not been yet successful in many genotypes of Citrus. Pollination and mature stage of pistils was necessary for gynogenic embryo regeneration. Fourteen haploid gynogenic embryos of Nules clementine were obtained. Embryo conversion was high (85.7%) and embryos vigorously germinated producing twelve plantlets. Chromosome counting, performed in the roots of obtained embryos, showed the haploid level (n=x=9). Isozyme analyses confirmed the expected homozygous state of embryos and plantlets.

Long term maintenance of an anther-derived haploid callus line of the Asiatic hybrid lily ‘Connecticut King’

A haploid callus line from anther cultures of the Asiatic hybrid lily ‘Connecticut King’ was maintained for a long term. The survival and growth of the haploid calluses were affected by auxins of picloram, α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) and temperatures of 25, 15 and 7 °C during culture. Picloram was more suitable for maintenance of the haploid calluses, whereas NAA and 2,4-D led to root and shoot formation from the haploid calluses. The best temperature for maintenance was 25 °C. About 90% of cells in calluses were maintained in haploid level during 60 weeks of subculture, and about 80% of cells were haploid in the calluses maintained over 2 years with the MS medium containing 4 μM picloram in the dark at 25 °C.

Studies on morphology and metabolism of prothalli during GA3-induced formation of antheridia in Anemia phyllitidis

In Schizaeaceae ferns, including Anemia phyllitidis, formation of antheridia is known to be induced by exogenously applied gibberellic acid. Also present studies show that GA3 (10−5 mol·dm−3) modifies the development of gametophytes of Anemia phyllitidis. Simultaneously with formation of antheridia, they exhibit lower number of cells but only slightly lowered profile areas and lengths of prothalli. Growth in size of individual cells compensates for lowered division frequency. Cytophotometric measurements reveal no essential changes in the DNA content in vegetative cells of the control and GA3-stimulated gametophytes. It remains at haploid level and therefore it is assumed that cell cycle is blocked at G1 phase. Application of GA3 increases the total amount of proteins. CZE (Capillary Zone Electrophoresis) separation of peptides extracted from control and GA3-treated prothalli indicates the differences in the ratio of their particular forms. In GA3-treated gametophytes the activities of acid and basic phosphatases, contents of carbohydrates (glucose, starch), chlorophyll, the number of chloroplasts and dry mass of prothalli are increased. GA3-intensified metabolism, evidenced in gametophytes of A. phyllitidis, may be interpreted as a stimulatory mechanism which influences metabolic pathways involved in forming, developing and maturing of male sex organs.

Haploid maternal genome derived from early diplotene oocytes can substitute for the female pronucleus in preimplantation mouse development

We describe the preimplantation development of mouse embryos that have received the haploid maternal genome derived from early diplotene nuclei of primordial oocytes (PO). Two generations of recipient egg-cells were used. Induction of two meiotic divisions of the PO nucleus and the reduction of the number of chromosomes to the haploid level were achieved in preovulatory oocytes (primary recipients). The developmental potential of the obtained haploid genome was examined in zygotes (secondary recipients). The nuclei of PO obtained from newborn mice were transferred by cell electrofusion to in vitro maturing (IVM) and enucleated preovulatory mouse oocytes. The reconstructed oocytes which had completed maturation, i.e., reached metaphase II, were artificially activated (8% ethanol + CHX). Activated oocytes were used as donors of haploid pronuclei of PO origin which were transferred (by karyoplast fusion) to partially enucleated zygotes containing only the male pronucleus. Thus, reconstituted zygotes were transplanted to the ligated oviducts of the cycling mice and 27% of them developed to the blastocyst stage. Our experiments demonstrate that 1) the nucleus of PO can be induced to premature meiotic divisions in an IVM enucleated preovulatory oocyte; 2) in the presence of a normal male pronucleus, the haploid pronucleus of PO origin can substitute for a female pronucleus during preimplantation development.

Haploid maternal genome derived from early diplotene oocytes can substitute for the female pronucleus in preimplantation mouse development

We describe the preimplantation development of mouse embryos that have received the haploid maternal genome derived from early diplotene nuclei of primordial oocytes (PO). Two generations of recipient egg-cells were used. Induction of two meiotic divisions of the PO nucleus and the reduction of the number of chromosomes to the haploid level were achieved in preovulatory oocytes (primary recipients). The developmental potential of the obtained haploid genome was examined in zygotes (secondary recipients). The nuclei of PO obtained from newborn mice were transferred by cell electrofusion to in vitro maturing (IVM) and enucleated preovulatory mouse oocytes. The reconstructed oocytes which had completed maturation, i.e., reached metaphase II, were artificially activated (8% ethanol + CHX). Activated oocytes were used as donors of haploid pronuclei of PO origin which were transferred (by karyoplast fusion) to partially enucleated zygotes containing only the male pronucleus. Thus, reconstituted zygotes were transplanted to the ligated oviducts of the cycling mice and 27% of them developed to the blastocyst stage. Our experiments demonstrate that 1) the nucleus of PO can be induced to premature meiotic divisions in an IVM enucleated preovulatory oocyte; 2) in the presence of a normal male pronucleus, the haploid pronucleus of PO origin can substitute for a female pronucleus during preimplantation development. Mol. Reprod. Dev. 48:488–495, 1997. © 1997 Wiley-Liss, Inc.

Chromatid exchanges may be induced by damage in sites of transcriptional activity.

A conditional expression system has allowed us to vary the expression level of the xeroderma pigmentosum group A (XPA) photoproduct-specific DNA-binding protein in human cells and so control the response of cells to damage by UV light. Using a form of XPA that contains a single missense mutation (R207G) enabled us to study a lower range of function than that obtained with the wild-type sequence. This form of XPA has been previously shown to stimulate pyrimidine dimer excision preferentially in actively transcribed genes. We found that UV resistance increased as a linear function of XPA expression levels. Excision of (6-4) pyrimidine-pyrimidone photoproducts in the whole genome increased to a maximum at about the haploid level of XPA expression, but there was little pyrimidine dimer excision from the whole genome. SCE frequency induced by UV light was high in cells with no SPA expression and fell rapidly with increasing levels of SPA expression within 0-50% of the haploid level of expression. No further reduction in SCE frequency was produced at the highest levels of XPA expression, when repair replication extended to the overall genome. We speculate that a low level of repair, especially that occurring in actively transcribed genes, may selectively eliminate photoproducts that are particularly important in causing cell killing and SCEs.

On the occurrence of somatic meiosis in embryogenic carrot cell cultures.

During the establishment of an embryogenic cell line from a carrot hypocotyl explant, processes closely resembling meiotic divisions are seen. A microdensitometric analysis revealed that the amount of cellular DNA diminished in the majority of cells to the haploid level. However, the diploid level was re-established in a matter of a few days. The genetic consequences of this segregation were studied by analyzing restriction fragment length polymorphisms (RFLP) and randomly amplified polymorphic DNAs (RAPD). The results showed that the great majority of embryos regenerated from segregants and that different segregants had different genetic constitutions.

Aneuhaploids and tetrasomics in rice (Oryza sativaL.) derived from anther culture of trisomics

Eight types of aneuhaploids (Aneuhaplo 4, 5, 6, 8, 9, 10, 11, and 12) and eight types of tetrasomics (Tetraplo 4, 5, 6, 7, 8, 9, 10, and 12) of rice have been obtained from anther culture of trisomics. This paper reports the plant morphology of these aneuploids and their chromosome behavior at metaphase I. Aneuhaploids for different chromosomes are distinguishable from each other and are morphologically similar to the parental trisomics, suggesting that the extra chromosome has similar genetic effects on plant morphology at the haploid level as at the diploid level. Similarly, tetrasomics with different extra chromosomes are distinguishable from each other and are similar morphologically to the parental trisomic. However, stronger changes in morphological characters were observed in tetrasomics compared with trisomics having the same extra chromosome, as a result of a dosage effect of the extra chromosomes. Comparing plant size between aneuhaploid, tetrasomic, and trisomic with the same extra chromosome, it was shown that the trisomic was the largest, the tetrasomic was of medium size, and the aneuhaploid was the smallest, except for those plants with an extra chromosome 8 in which plant size is dramatically decreased in both the aneuhaploid and the tetrasomic. At metaphase I, aneuhaploids showed chromosome configurations of 1 II + 11 I and 13 I. The frequency of the 1 II + 11 I configuration is higher than 70%, indicating that homologous chromosomes in aneuhaploids tend to stay associated in meiosis. Intragenome chromosome pairing (2 II + 9 I), so called secondary association, was observed in the aneuhaploid for chromosome 5. Tetrasomic plants showed 5 kinds of chromosome configurations: 1 IV + 11 II, 1 III + 11 II + 1 I, 13 II, 12 II + 2 I, and 11 II + 4 I. A chromosome configuration of 13 II was often observed in tetrasomics with shorter extra chromosomes and a chromosome configuration of 1 IV + 11 II was often observed in tetrasomics with longer extra chromosomes. Aneuhaploids had complete seed sterility. Tetrasomics showed very poor pollen fertility and complete seed sterility, except for a few shriveled seeds that were observed in Tetraplo 6 and 9. This is the first report in rice where many aneuhaploids and tetrasomics have been characterized. This information will help to further unravel rice aneuploidy and cytogenetics. The aneuploids obtained here will be very useful tools for the study of genetics and breeding in rice.

Genome reorganization from polyteny to polyploidy in the nurse cells found in onion fly (Delia antiqua) and cabbage root fly (Delia radicum) ovaries (Diptera, Anthomyiidae)

The material required to ensure successful embryogenesis in the onion fly (Delia antiqua) and the cabbage root fly (Delia radicum) (Diptera, Anthomyiidae) is supplied by 15 nurse cells, while the oocyte chromosomes enter a quiescent stage during prophase I of meiosis. This level of transcription is achieved by the polyploidization of the nurse cell DNA. Elongate polytene chromosomes form in both species, but lack the banding and conspicuous puffing commonly seen in other dipteran tissues. The polytene chromosomes contract until they finally appear as small, densely staining spheres. These fragment into large numbers of endochromosomes that are much smaller than their mitotic counterparts, which then despiralize, resulting in the flocculate appearance of the nurse cell nucleus. Photodensitometry revealed a gradient of DNA values between nurse cells near the oocytes and those further away. Final DNA values 1000 times the haploid level were recorded in the nurse cell nearest to the oocyte compared with 336 times the C-value in the most distal cell. At lower temperatures (< 10 degrees C), the polytene chromosomes become banded and longer. None of the onion flies kept in these conditions produced viable eggs, though there was some reproductive success among the cabbage root flies.

Development of Anther‐Derived Dihaploids to Combine Disease Resistance in Maryland Tobacco

Anther culture can reduce the amount of time required to develop completely homozygous lines of tobacco (Nicotiana tabacum L.). The objectives of this study were to determine the value of anther culture in combining resistance to three diseases in dihaploid lines of Maryland tobacco and to evaluate the agronomic performance of these lines relative to the parent cultivars. ‘MD 609’, which is resistant to black shank (Phytophthora parasitica var. nicotianae), was crossed with ‘MD 341’, which is resistant to tobacco mosaic virus (TMV) and wildfire (Pseudomonas syringae pv. tabaci). Anthers from the F1 were cultured to produce approximately 2000 haploid plantlets. The haploids were screened for resistance to TMV and wildfire, and midveins from 315 resistant plants were cultured to generate 133 dihaploids. These 133 dihaploids were then screened for black shank resistance, and 16 (12%) had levels of resistance similar to MD 609. In a 2‐yr field study involving 24 dihaploids, the average dihaploid was inferior to the midparent mean for price and quality index, had longer internodes, and flowered 6% later. These results indicate that whenever possible, screening for desired traits should be conducted at the haploid level to maximize efficiency. Although reduced yields were frequently observed with the dihaploids, certain lines with desirable levels of disease resistance equaled, and occasionally exceeded, the midparent value for yield and other agronomically important traits.

Karyotype analysis inPeperomiaRuiz and Pav.

SUMMARYKaryotype analysis conducted in 5 species of Peperomia, reveals that there are 2 lines of evolution, one with basic number n = 11 and the other with n = 12. Peperomia clusiaefolia (Jacq.) Hook. (2n = 22), P. rotundifolia (L.) HBK (2n = 22), P. sandersii C.DC. (2n = 24) and P. verschaffeltii Hem. (2n = 24) are diploids. Where as P. bicolor has 2n = 36 chromosomes, evidently a triploid. Karyotype is symmetrical in all the investigated species, eventhough there is a steady diminution in chromosome length. Species of Peperomia differ in minute details of karyotype even at haploid level, there by suggesting the role of minute structural alterations in speciation.

The effect of linkage on sample size determination for multiple trait selection

Sufficient sample sizes are needed in breeding programs to be confident, with a specified probability α, of obtaining a specified number of plants of a desired genotype in segregating populations. We develop a method of determining the minimum sample size needed to produce, with specified probability α, at least m individuals of a desired genotype. This method takes into consideration factors affecting differential selection of gametes, segregation at a single locus, and linkage among the loci of interest. We first consider the effects in the gametophyte (haploid level) of fitness and linkage on the frequencies of alleles at two linked loci, then at three or more linked loci. The probability of obtaining at least m successes, or occurrences of the desired allele, among n gametes is given by a formula based on the binomial distribution. This probability is affected by fitness and linkage through their impact on the probability that a single randomly chosen gamete is of the desired type. Using an extension of this approach, we examine the effects of the altered allelic frequencies on the likelihood of obtaining the desired genotype from a randomly chosen pair of gametes in the sporophyte (diploid level). A table and a figure show the sample size required to produce, with probability 0.95, m individuals of the desired g enotype or phenotype, as a function of m and the probability that a randomly selected individual is of the desired type.

Potato haploid performance in 2x × 4x crosses

Nineteen haploids (2n = 2x = 24) extracted from four tetraploids ofSolanum tuberosum (2n = 4x = 48) with high 2n egg frequencies and known mode of 2n egg formation were evaluated via 4x progeny from 2x × 4x crosses (haploid × cultivar) for total tuber yield, tuber appearance and tuber set. Ninety-eight, 4x families (15-20 genotypes each) were planted in the randomized complete block design with two replications at two locations. Analyses of variance were conducted for all haploids over three common testers, and each of the four groups of haploids individually over a variable number of testers. The performance of haploids, based on general combining ability effects, was consistent for all traits, when tested with either three or more testers. Diploid clones can be evaluated in 2x × 4x crosses using three unrelated adapted 4x testers. Nonorthogonal comparisons indicated no significant differences for all traits among four groups of haploids, and between modes of 2n egg formation. Significant variation within each group of haploids indicates that selection at the haploid level should be carried out among haploids from either the same or different cultivars. Superior haploids can be utilized in 2x × 4x and 2x × 2x breeding schemes to generate 4x clones.

Characterization of Escherichia coli ATP synthase .beta.-subunit mutations using a chromosomal delection strain

(1) We constructed Escherichia coli strain JP17 with a deletion in the ATP synthase beta-subunit gene. JP17 is completely deficient in ATP synthase activity and expresses no beta-subunit. Expression of normal beta-subunit from a plasmid restores haploid levels of ATP synthase in membranes. JP17 was shown to be efficacious for studies of beta-subunit mutations. Site-directed mutants were studied directly in JP17. Randomly generated chromosomal mutants were identified by PCR and DNA sequencing, cloned, and expressed in JP17. (2) Eight novel mutations occurring within the putative catalytic nucleotide-binding domain were characterized with respect to their effects on catalysis and structure. The mutations beta C137S, beta G152D, beta G152R, beta E161Q, beta E161R, and beta G251D each impaired catalysis without affecting enzyme assembly or oligomeric structure and are of interest for future studies of catalytic mechanism. The mutations beta D301V and beta D302V, involving strongly conserved carboxyl residues, caused oligomeric instability of F1. However, growth characteristics of these mutants suggested that neither carboxyl side chain is critical for catalysis. (3) The mutations beta R398C and beta R398W rendered ATP synthase resistant to aurovertin, giving strong support to the view that beta R398 is a key residue in the aurovertin-binding site. Neither beta R398C or beta R398W impaired catalysis significantly.

The Giemsa banding pattern of canine chromosomes, using a cell synchronization technique

Cytogenetic investigations of the domestic dog, Canis familiaris, were performed on the Doberman pinscher and two Boxer dogs. Conventional homogeneously stained and G-banded metaphases from peripheral blood lymphocyte cultures synchronized with amethopterin and bromodeoxyuridine were studied. These procedures permitted the unequivocal identification of all canine chromosomes. A canine chromosome idiogram was constructed on the basis of the G-banding pattern at the haploid 327-band resolution level. The secondary constrictions and tapering of the telomeric regions characteristic of several canine chromosomes are described. Q-, C-, and NOR-banding were also performed and the salient features are described. This karyotype should enhance the value of the canine species in cytogenetic investigations.

Isolation and characterization of S49 mouse lymphoma cell mutants deficient in adenosine deaminase.

Adenosine deaminase-deficient mutants of a mouse lymphoma cell line S49 have been isolated by a two-step selection process. In the first step, we derived mutant lines containing haploid levels of adenosine deaminase activity from wild-type cells. The selective medium contained tritiated deoxyadenosine, deoxycytidine, and deoxycoformycin. Wild-type cells were killed, presumably because of suicidal incorporation of tritiated deoxyadenosine via the adenosine deaminase pathway. The second step was to derive, from the partially deficient mutants, sublines that were virtually lacking adenosine deaminase, using tritiated deoxyadenosine and deoxycytidine. Four mutant clones were found to contain less than 5% of the enzyme activity of wild-type cells and virtually no immunoreactive adenosine deaminase protein. Northern blot analysis showed that the levels of adenosine deaminase mRNA were drastically reduced. Back-selection for adenosine deaminase-positive revertants can be accomplished by using a medium containing deoxyadenosine (as a sole source of purine), aminopterin, and thymidine or, alternatively, by using deoxyadenosine alone in a serum-free medium.