Selected Hybrid Clones Research Articles

Variation in susceptibility of Eucalyptus grandis and selected hybrid clones to two termite species Macrotermes bellicosus and M. subhyalinus in Uganda

The maximum productivity of plantation forestry and its role in climate change mitigation, adaptation and resilience cannot be met without proper management. Termites in the genus Macrotermes have been reported as a major challenge to Eucalyptus plantation forestry establishment. The current study evaluated the susceptibility of four Eucalyptus hybrid clones: GU 7, GC 796, GC 550 and GC 796/2, and E. grandis Hill ex Maid. to the most damaging Macrotermes bellicosus (Smeathman) and Macrotermes subhyalinus (Rambur) to identify tolerant material that can be planted in high incidence areas. The study involved exposure of moisture dry pieces of wood from E. grandis and the four hybrid clones to damage by M. bellicosus and M. subhyalinus. Results confirmed that M. bellicosus is the most aggressive. Results further revealed that E. grandis and GC 550 are the most susceptible whereas GC 796 is the most tolerant clone. The findings from the study will contribute to improved management of termites by planting tolerant material in high risk areas.

STMS marker assay based diversity in selected hybrid clones of sugarcane (Saccharum officinarumL.)

Molecular genetic diversity among 35 selected clones of sugarcane assayed using 30 sugarcane specific sequence tagged microsatellite site (STMS) primers. Present investigation revealed that the markers analyzed were highly informative by virtue of their PIC (polymorphic information content) values. Jaccard's similarity coefficient value estimated was ranged from 0.37 to 0.89 with an average of 0.63 indicating the existence of appreciable genetic diversity among the clones studied which opened the avenue for further improvement of the clones through hybridization. UPGMA clusters analysis detected six clusters of which, Cluster III was largest with 26 genotypes. There was poor correspondence between pedigree and molecular markers as the two genotypes evolved from crosses involving different parentages were also clustered together in addition to clustering together of clones with at least one common parent and the clones evolved from the same cross showed more diversity suggesting that involvement of genotypes derived from same parental combination need not always lead to close breeding.

Reprogramming somatic cells by fusion with embryonic stem cells does not cause silencing of theDlk1-Dio3region in mice

To examine the imprinted Dlk1-Dio3 locus in pluripotent embryonic stem (ES) cell/fibroblast hybrid cells. Gtl2, Rian, and Mirg mRNA expression in mouse pluripotent ES cell/fibroblast hybrid cells was examined by real-time reverse transcription-polymerase chain reaction. Pyrosequencing and bisulfate sequencing were used to determine the DNA methylation level of the Dlk1-Dio3 locus imprinting control region. The selected hybrid clones had a near-tetraploid karyotype and were highly pluripotent judging from their capacity to generate chimeric embryos and adult chimeras. Our data clearly demonstrate that Gtl2, Rian, and Mirg, which are imprinted genes within the Dlk1-Dio3 locus, are active in all examined ES cell/fibroblast hybrid clones. In spite of interclonal variability, the expression of the imprinted genes is comparable to that of ES cells and fibroblasts. Quantitative analysis of the DNA methylation status of the intergenic differentially methylated region (IG DMR) within the Dlk1-Dio3 locus by pyrosequencing and bisulfite sequencing clearly showed that the DNA methylation status of the imprinted region in the tested hybrid clones was comparable to that of both ES cells and fibroblasts. Reprogramming process in a hybrid cell system is achieved without marked alteration of the imprinted Dlk1-Dio3 locus.

Interspecific hybridization of Eucalyptus: key issues for breeders and geneticists

Eucalypt hybrids are significant in forestry, particularly in sub-tropic and tropical regions, where cost efficient, clonal propagation is the key to their exploitation. However, the outstanding success of selected hybrid clones has given a biased impression of the vigor of eucalypt hybrids and the strength of reproductive barriers in the genus. When full account is made of losses through the life cycle, a picture of high incompatibility and inviability often emerges. Hybrid inviability tends to increase with increasing taxonomic distance between parents, but there are exceptions. Hybrids also seem more susceptible to pests than their pure species. Intense selection may still result in elite hybrid clones, but such inviability and susceptibility adds a significant cost to their development. Breeders must carefully evaluate the costs of hybrid development and deployment compared with pure species options. A key to hybrid selection is the rapid development and testing of large populations and application of high selection intensities. However, eventually this approach must be linked with more formal breeding strategies. As most traits are intermediate in F1 hybrids there is increasing interest in advanced generation hybrids to provide desirable trait combinations. In such cases, there is a clear role for marker assisted selection to speed introgression.

Transfer of amiprophosmethyl resistance from a Nicotiana plumbaginifolia mutant by somatic hybridisation

Transfer of resistance to the phosphorothioamidate herbicide, amiprophosmethyl (APM), from the β-tubulin mutant of Nicotiana plumbaginifolia to the interspecific N. plumbaginifolia (+)N. sylvestris and to the intertribal N. plumbaginifolia (+) Atropa belladonna somatic hybrids has been demonstrated. Transfer to the recipient species was accomplished by: (1) symmetric hybridisationand (2) asymmetric hybridisation using γ-irradiation of donor protoplasts. Cytogenetic analysis confirmed the hybrid origin of the hybrids obtained. It was established that most of them typically inherited no more than three donor chromosomes, although it was possible to obtain symmetric hybrids in the case of symmetric fusion. Immunofluorescent microscopy analysis has shown that protoplasts of the mutant, and of the N. plumbaginifolia (+) N. sylvestris and N. plumbaginifolia (+) A. belladonna hybrids, retained the normal structure of interphase microtubule (MT) arrays and mitotic figures after treatment with 5 µM APM, whereas MTs of protoplasts of the recipients were destroyed under these conditions. It was also shown that hybrid clones contained an altered β-tubulin isoform originating from the N. plumbaginifolia mutant. The selected hybrid clones were characterised by cross-resistance to trifluralin, a dinitroaniline herbicide with the same mode of anti-MT action. Some of the somatic hybrids which could flower were fertile. It was established that seeds of some fertile hybrids were able to germinate in the presence of 5 µM APM. The results obtained thus support the conclusion that the technique of somatic hybridisation, especially asymmetric fusion, can be used to transfer APM resistance from the N. plumbaginifolia mutant to different (related and remote) plant species of the Solanaceae, including important crops.

Telomerase repressor sequences on chromosome 3 and induction of permanent growth arrest in human breast cancer cells.

Activation of the enzyme telomerase, which has been associated with cellular immortality, may constitute a key step in the development of human cancer. Telomerase is repressed in most normal human somatic cells. This study was conducted, using a genetic complementation approach, with the aim of identifying and mapping the genes responsible for repressing telomerase and, simultaneously, to establish the effect of experimentally induced telomerase repression on human tumor cell growth. Individual human chromosomes isolated from normal diploid cells and tagged with bacterial antibiotic resistance genes (for later selection) were introduced into cells of the human breast carcinoma cell line 21NT by means of microcell transfer. Selected hybrid clones were screened for telomerase activity by use of the polymerase chain reaction-based telomere repeat amplification protocol (TRAP) assay, and the proliferative fate of the hybrid clones was determined. Regions of the introduced chromosomes associated with telomerase repression were mapped using segregant hybrids and a deletion analysis that employed microsatellite DNA markers. Strong repression of telomerase was observed following transfer of human chromosome 3 into 21NT cells but not after transfer of chromosomes 8, 12, or 20. The vast majority of hybrid clones with repressed telomerase entered permanent growth arrest after 10-18 population doublings. Deletion analysis of nonrepressed segregant monochromosome 3 hybrids indicated two regions on the short arm of chromosome 3 (3p21.3-p22 and 3p12-21.1) where telomerase regulator genes may be located. Telomerase in human breast cancer cells is efficiently repressed by a gene or genes on normal human chromosome 3p, and this repression is associated with permanent growth arrest of the tumor cells.

Quantitative genetics of yield breeding for Populus short rotation culture. I. Dynamics of genetic control and selection model of yield traits

Variation patterns and selection efficiencies for the three growth traits height, diameter, and volume were studied in two selected interspecific hybrid populations of eastern cottonwood (Populusdeltoides Bartr.): P. deltoides × Populussimonii Carr. and P. deltoides × Populusnigra L. Growth variables differed quite significantly (P < 0.01) among clones in each of the two hybrid populations, and they were more dominantly controlled by genetic factors than by environmental regimes. There were higher broad-sense heritablity levels for growth traits in this study than in other related studies. Genetic variances and broad-sense heritabilities for all traits markedly increased with stand development in the two hybrid populations; but the increasing rates with age of these two genetic parameters for diameter and volume were more rapid for P. deltoides × P. simonii hybridization than for P. deltoides × P. nigra hybridization. Selection of the best 5% of the clones from P. deltoides × P. simonii and the best 10% from P. deltoides × P. nigra produced mean 6th-year volume growth increases of 51 and 37%, respectively. Relative to P. deltoides, their high parent, the ratio of clonal heterosis of these selected hybrid clones was 5–23% for 6th-year volume. With different selection intensities, early selection of juvenile growth traits for the two populations, P. deltoides × P. simonii and P. deltoides × P. nigra, can also produce substantial indirect genetic gains for short rotation harvest traits. Further studies of Populus yield breeding and selection models were suggested.

Characterization of morphological variation and cold resistance in interspecific somatic hybrids between potato (Solanum tuberosum L.) and S. brevidens Phil.

Somatic hybrids between Solanum tuberosum L. cv. Gracia (2n=4x=48) and Solanum brevidens Phil. (2n=2x=24) were produced via fusion of mesophyll protoplasts. Selection of the protoplast derived putative hybrid calli was based on their vigorous growth. Additive isozyme patterns and chromosome numbers as well as the expression of parental morphological characters have proved the hybrid origin of the selected regenerants. Extensive chromosome loss during the regeneration process resulted in aneuploid hybrids with high frequency. Genomic instability could not be detected in these plants during the period of vegetative propagation. Regenerants from hybrid tissues exhibited wide morphological variation especially in tuber formation. The detailed morphological analysis based on the use of multivariate method (principal component analysis, PCA) enabled to identify morphological groups among the hybrid clones. The positioning of hybrid clones in the PCA space could not be correlated with chromosome numbers. The genomic ratio represented by the tetraploid and diploid parents influenced the morphology of somatic hybrid population according to the applied analytical system. Two selected hybrid clones have exhibited an intermediate degree of frost tolerance compared to the parents, based on the recovery of plants from lower buds after freezing of potted plants.

Localization of the gene for the vitamin B12 binding protein, transcobalamin II, near the centromere on mouse chromosome 11, linked with the hemoglobin alpha-chain locus.

Somatic cell hybrids, recombinant inbred (RI) mouse strains, and backcross breeding experiments were used to locate the gene of transcobalamin II (Tcn-2), the vitamin B12 binding protein in mouse serum. TCN-2 was found to be useful genetic marker in the somatic cell hybrids. Selected hybrid clones were derived from fusions between GR mouse cells and the Chinese hamster cell line E36. Analysis of mouse specific chromosomal enzyme markers in relationship to TCN-2 secretion, in the hybrid clones, provided provisional evidence for assignment of the Tcn-2 locus to chromosome 11. The strain distribution pattern of the TCN-2 variants S and F in the RI series CXS, constructed from the cross of BALB/cHeA (TCN-2S) with STS/A (TCN-2F), implied a close linkage with the hemoglobin alpha-chain locus (Hba) on chromosome 11. Backcross breeding using inbred strains confirmed these findings and located the Tcn-2 gene closest to the centromere, linked with waved 2 (wa-2) and Hba with recombination frequencies of 6.9 and 19.2% each. The linkage group Tcn-2/wa-2/Hba was established.

Localization of a gene which complements branched-chain amino acid transaminase deficiency to the short arm of human chromosome 12.

A human X Chinese hamster somatic cell hybrid (J1) containing only one human chromosome (number 11) does not express branched-chain amino acid transaminase and, therefore, cannot grow if the branched-alpha-keto acids are substituted for the branched-chain amino acids in the growth medium. J1 cells, which are glycine auxotrophs (Gly-A), were fused with normal human lymphocytes and the resulting hybrids selectively isolated in glycine-free medium. A total of 16 primary and 46 secondary clones were analyzed for isozymic and nutritional markers. Cytogenetic analysis with chromosome banding was also performed on selected hybrid clones. The results provide evidence that branched-chain amino acid transaminase is syntenic with lactic dehydrogenase B and is located on the short arm of human chromosome 12.

Construction and testing of mouse--human heteromyelomas for human monoclonal antibody production.

FU-266, a mutant human myeloma cell line sensitive to hypoxanthine/aminopterin/thymidine (HAT), was transfected by protoplast fusion with DNA of the recombinant plasmid vector pSV2-neoR, thus acquiring a dominant marker conferring resistance to the antibiotic G-418. One of the resultant neoR clones, E-1, was fused to irradiated (500 rads) or unirradiated cells of the HAT-sensitive, G-418-sensitive, nonproducer mouse myeloma line X63-Ag8.653. Hybrid clones were selected in G-418 plus ouabain, thus preserving their HAT sensitivity. Small numbers of human chromosomes were retained in all such hybrids, but most of them ceased secreting human myeloma (IgE(lambda). Selected hybrid clones were then tested as malignant fusion partners in a series of fusions with polyclonally activated human B lymphocytes and with antigen-primed human B lymphocytes, in some instances after transformation of the latter with Epstein-Barr virus. The yield of viable chimeric hybridomas has been consistently high, as has the proportion of hybridomas secreting human immunoglobulin molecules unpermuted with mouse or human myeloma heavy or light chains. Secretion by many subcloned hybridomas has been stable for over 6 months, and several antigen-specific human monoclonal antibodies have been generated. Thus these heteromyeloma cell lines appear to have significant advantages for human monoclonal antibody production.

Confirmational, provisional, and/or regional assignment of 15 enzyme loci onto Chinese hamster autosomes 1, 2, and 7.

PEG-mediated fusion between mouse Cl1d cells and primary Chinese hamster spleen cells produced interspecific hybrids which slowly and nonrandomly segregated Chinese hamster chromosomes. Cytogenetic and isozyme analysis (31 loci) of HAT and BrdU selected hybrid clones and subclones and of members of a hybrid clone panel retaining different combinations of Chinese hamster chromosomes enabled provisional assignment of the following enzyme loci on Chinese hamster chromosomes: thymidine kinase, galactokinase, and acid phosphatase-1 to chromosome 7; galactose-1-phosphate uridyltransferase to chromosome 2; and adenosine kinase, esterase D, glutathione reductase, glyoxalase, nucleoside phosphorylase, peptidases B and S, and phosphoglucomutase (PGM) 2 to chromosome 1. Assignments of PGM1, 6-phosphogluconate dehydrogenase, and enolase 1 to chromosome 2 were confirmed, and a chromosome 2 deletion (q23-q33) enabled the provisional assignment of PGM1 to that region. The assignments provide markers for the study of the genetic consequences of chromosomal rearrangements in Chinese hamster cell lines and support the concept of conservation of mammalian autosomal linkage groups.

Hybrid clones of rat liver and hamster ovary cells as targets for carcinogen screening.

Many short-term carcinogen-screening assays use transformed cells which require exogenous metabolizing systems to activate carcinogens. We have sought untransformed target cell-lines with a high mitotic index and the intrinsic ability to metabolize carcinogens. Hybrid cell clones were established by fusing an untransformed rat liver cell-line (with a normal diploid karyotype, epithelial morphology and carcinogen-metabolizing capacity) and a Chinese hamster ovary cell-line (with a high mitotic index, ability to grow in soft agar, a characteristic of transformed cells, and which cannot metabolize most carcinogens). A number of such clones were characterized with respect to their morphology, karyology, growth properties and the ability to metabolize 7,12-dimethylbenz[a]anthracene. Selected hybrid clones were found to respond to several types of carcinogen, using the induction of sister chromatid exchange as the criterion. A significant increase in the induction was observed in cloned hybrid cell-lines compared with the hamster ovary cell line. Moreover, the same hybrid clones can be used repeatedly without an exogeneous activating system, which makes this test system very practical.

I. The breeding of resistant varieties of potatoes

1. An introductory historical survey of the breeding for late blight resistance is given. 2. The results of the investigation on specialization problems and on methods of selection for resistance for the interval of 1936–1945 (Muncheberg/ Mark) and 1947–1948 (Voldagsen/Hann.) are discussed. 3. The assortment of commercial varieties must be divided into two groups: (I) Pure tuberosum varieties, which are highly susceptible with the exception of very few varieties with some degree of resistance. (II) “Hybrid varieties” with different degrees of resistance, which may be used as differential varieties in specialization researches. 4. Some more wild species could be classified with regard to their behavior to late blight. Their genetics of resistance and their suitability for hybridization is discussed. A report is given concerning the results of the testing of selected hybrid clones for the interval 1947–1948.