Partial Genome Transfer Research Articles

A review on cybrids : An approach for plant improvement

Somatic hybridization is a mechanism for gene transfer from related species to cultivated crops, especially when wide hybridization is not applicable. Protoplast fusion resulting in the development of cybrids is known as cybridization. Interaction of plastome and genome contributes to the formation of cybrids (cytoplasmic hybrids). Cybridization has been successfully used to transfer CMS in rice and maize. It is very promising allows partial genome transfer better tolerated than whole-genome transfer. It enables transfer of genetic materials between distantly-related and sexually-incompatible species with great potential for improvement of crops. It demands less common skills and expertise, but ranks as a method yielded good results. But introgression and stable inheritance of the alien DNA carrying the desired genes have to take place. Moreover, an important factor to investigate in the future is the mechanism involved in the incorporation of donor DNA into recipient chromosomes.

Symmetric and Asymmetric Somatic Hybridization in Citrus: Review

Somatic hybridization by nuclear or cytoplasmic fusions leads to three distinct possible outcomes -[a] “symmetric” hybrid: fusion of whole nuclei, [b] “asymmetric” hybrid: fusion of parental nuclei, one or both deficient for one or more chromosome(s), and [c] “cybrid”: fusion without a nuclear contribution by one parent, i.e., an extremely asymmetric hybrid. Somatic hybridization in citrus has been a powerful tool for the production of novel allotetraploid somatic genotypes that combine desirable characteristics of two parents. These hybrids open new opportunities for sexual hybridization at the tetraploid level, and for interploid crosses for the production of potentially seedless triploids, among other uses. Somatic symmetric hybrids have great potential for rootstock improvement and are excellent as breeding parents, but they may have limited direct application as scion cultivars because they typically contain the entire genomes of both fusion parents, and are likely to exhibit the desirable and undesirable traits of both parents. Asymmetric hybridization is very promising as it allows partial genome transfer and may be better tolerated than a whole-genome transfer. A brief review about symmetric and asymmetric somatic hybridization and cybridization in citrus is presented. Somatic hybridization seems to be a great tool for citrus breeding. However, the release of new improved cultivars produced by this technology is what is going to prove its effectiveness.

Isolation and characterization of gametic microprotoplasts from developing microspores of Lilium longiflorum for partial genome transfer in the Liliaceous ornamentals

We have established a system for isolating gametic microprotoplasts from developing microspores of Lilium longiflorum 'Hinomoto' (2n =2× =24) as a first step toward the production of intergeneric hybrid plants with one or a few alien chromosomes via microprotoplast fusion in the Liliaceous ornamentals. Anthers of this cultivar containing microsporocytes at mainly diakinesis to metaphase I were cultured for 3–4 days in a medium containing half-strength MS salts, double-strength MS vitamins, 1 g l–1 casamino acids, 100 g l–1 sucrose, and one of four spindle toxins: amiprophos-methyl, isopropyl N-(3-chlorophenyl)carbamate (CIPC), colchicine or propyzamide. Of the four spindle toxins examined, CIPC at concentrations of 5 or 10 µM efficiently induced micronucleation with the mean number of nuclei per meiocyte being 7.5. CIPC treatment also efficiently induced micronucleation in the other five Lilium genotypes evaluated (L. regale, L. longiflorum 'Georgia', L. speciosum 'Uchida', the Asiatic hybrid lily 'Connecticut King' and the Aurelian hybrid lily 'Golden Splendor') with the mean number of nuclei per meiocyte falling within the range 5.4–11.7. In 'Hinomoto', each meiocyte nucleus formed a microcell 4–5 days after initiation of CIPC treatment. Following isolation of such meiocytes from the anthers and their incubation in a cell-wall-digesting enzyme solution, gametic (micro)protoplasts of less than 10 µm, 10–20 µm and 20–50 µm in diameter were obtained with yields of 5.5×104, 6.6×104 and 4.9×104 per anther, respectively. Smaller microprotoplasts with DNA content below the 2C level, as indicated by flow cytometric analysis, were enriched by sequential filtration through nylon sieves of decreasing pore size (50, 20 and 10 µm). The relative fluorescence intensity in some of their nuclei corresponded to that of one or only a few chromosomes.

Preparation of Microprotoplasts for Partial Genome Transfer via Microprotoplast Fusion in Liliaceous Ornamental Plants

We aimed to produce intergeneric hybrid plants with only one or a few alien chromosomes via microprotoplast fusion for genetic improvement and chromosome studies in Liliaceous ornamental plants. In order to apply this technique, it is essential to establish an efficient system for mass-preparation of microprotoplasts. We have established 2 different systems for isolating microprotoplasts, one from partially synchronized cell suspension cultures of Hemerocallis hybrida and the other from developing microspores of Lilium longiflorum. Here, the induction of micronucleated cells, isolation of microprotoplasts, and enrichment of smaller microprotoplasts containing one or a few chromosomes are described for both systems.

Characterization and molecular analysis of transgenic plants obtained by microprotoplast fusion in sunflower

Asymmetric somatic hybrid (ASH) plants were obtained by PEG-mediated mass fusion of microprotoplasts from perennial Helianthus species and hypocotyl protoplasts of Helianthus annuus. The formation of micronuclei in perennial sunflower cell cultures was induced, at early log phase, by addition of the herbicides amiprophos-methyl or oryzalin. Sub-diploid microprotoplasts were isolated by high-speed centrifugation and the smallest enriched by sequential filtration through nylon sieves of decreasing pore size. Fusion products were cultured and the regenerated plants phenotypically, genetically and cytologically characterized. DNA analysis using RAPD markers revealed that 28 out of 53 regenerated plants were asymmetric hybrids. Subsequent nuclear-DNA flow cytometric analysis showed that these plants had a higher DNA content than the receptor H. annuus, suggesting that they represented addition lines. Cytological investigation of the metaphase cells of 16 hybrids revealed an addition of 2–8 extra chromosomes in these plants. The phenotype of most ASH plants resembled H. annuus. These results indicate that micronuclear induction and asymmetric somatic hybridization represent a potent tool for partial genome transfer aimed at the specific transfer of economically important traits in breeding programs.

Efeito de herbicidas sobre a polimerização dos microtúbulos e indução de micronúcleos em protoplastos de Helianthus maximiliani

Com o objetivo de estudar a cinética da despolimerização dos microtúbulos e a dinâmica da indução de micronúcleos, usaram-se dois herbicidas com ação antimitótica, amiprofós-metil (APM) e oryzalin (ORY), em células de Helianthus maximiliani. A aplicação de ambos os herbicidas em células com ativo crescimento resultou num elevado número de células com micronúcleos, em virtude das modificações mitóticas causadas pela ausência de microtúbulos. Primeiramente, cromossomos isolados ou grupos de 2 ou mais cromossomos espalharam-se pelo citoplasma. Duas a três horas após a aplicação dos herbicidas, os microtúbulos, estavam despolimerizados e os cromossomos metafásicos condensaram-se, formando diretamente os micronúcleos sem ter havido a divisão do centrômero e a separação das cromátides. A concentração mais eficiente de ORY foi 20 µM, ao passo que para APM foi 60 µM, para um período de incubação de 36 e 48 h respectivamente. O prolongamento do período de tratamento elevou a freqüência de micronúcleos deformados, bem como aumentou a morte celular. A adição de citocalasina-B (CB) (20 µM), após 24 h da aplicação dos herbicidas, aumentou significativamente a micronucleação e estabilização dos micronúcleos nas células. O controle dos microtúbulos de células em suspensão torna-se uma ferramenta eficiente para a indução de micronúcleos, bem como representa um método elegante para a transferência genômica parcial.

Factors influencing the utility of gametic microprotoplasts for partial genome transfer in potato

The potential of gametic microprotoplasts as a tool for partial genome transfer in potato (Solanum tuberosum L.) was investigated and a number of technical findings are reported. The phosphoric amide herbicide amiprophos-methyl was effective for microcell induction when applied at a concentration of 25 μm to excised anther material; however, neither whole buds nor isolated microspores responded to treatment. One selected clone responded well to spindle toxin treatment, and microcells were observed in 9% of tetrads, for which 4% (wt/vol) cellulase Onozuka R10 and 0.25% (wt/vol) driselase was found to be optimal for the release of protoplasts from the tetrads with conversion rates exceeding 40%. Technical problems identified at a number of crucial stages that may preclude the more widespread application of this technology are discussed.

Cremart: A new chemical for efficient induction of micronuclei in cells and protoplasts for partial genome transfer

Results on efficient induction of micronuclei by Cremart in suspension cells and protoplasts of potato are reported. Cremart is a highly effective phosphoric amide herbicide, which acts on the mitotic spindle, and induces micronuclei through modification of mitosis. After treatment with Cremart, metaphase chromosomes changed directly into micronuclei without centromere division and chromatid separation. When suspension cells were treated with Cremart (3.7-15.0 μM) for 48h, and subsequently incubated in a mixture of cell wall-digesting enzymes in the presence of cytochalasin-B and Cremart for 18h, the frequency of micronucleation in the cell/protoplast mixture increased significantly, as compared to that obtained after treatment of suspension cells with Cremart (3.7-15.0 μM) for 48 h. Sieving after enzyme incubation resulted in the recovery of protoplasts, showing mass induction of micronuclei. Also synchronized suspension cells of Nicotiana plumbaginifolia responded with high frequency of micronucleation after Cremart (3.7 μM) treatment. The application of this procedure for partial genome transfer is discussed.

Somatic hybridization as a tool for tomato breeding

Protoplast fusion can be used to produce somatic hybrids of species that cannot be obtained by sexual hybridization. The possibility to introgress genes from Solanum species into the cultivated tomato species Lycopersicon esculentum, and to obtain novel cytoplasm-nucleus combinations (cybrids) was considered as an important strategy to extend the genetic variation available for tomato breeding. Somatic hybrids between L. esculentum and other Lycopersicon species, as well as between L. esculentum and Solanum or Nicotiana species, have been produced. Specific mutants, genotypes with antibiotic resistances, and metabolic inhibition by iodoacetate or iodoacetamide and irradiation were used for the selection of hybrids. In addition, the improvement of protoplast culture techniques and the use of the favourable tissue culture traits derived from species such as L. peruvianum, which have been introduced into tomato by classical breeding, allowed the efficient recovery of somatic hybrids. However, the occurrence of somatic incongruity in fusion combinations of L. esculentum and Solanum and even more in L. esculentum and Nicotiana, did not allow the production of true cybrids and/or fertile hybrids, indicating the importance of both cytoplasm-nucleus and nucleus-nucleus interactions in somatic incongruity. Another problem with fusions between distantly related species is the strongly reduced fertility of the hybrids and the very limited homoeologous recombination between chromosomes of the parental species. Partial genome transfer from donor to recipient through microprotoplast (+) protoplast fusion, and the production of monosomic or disomic chromosome addition lines, light overcome some of these problems. In symmetric somatic hybrids between L. esculentum and S. tuberosum the occurrence of limited somatic and meiotic recombination was demonstrated. Fertile progeny plants could be obtained, though at a low frequency, when embryo rescue was performed on a large scale after backcrossing hexaploid somatic tomato (+) potato hybrids with a tetraploid potato genotype. The potential value of genomic in situ hybridization (GISH) and RFLPs for the analysis of the genome/chromosome composition of the hybrids has been demonstrated for intergeneric somatic hybrids between Lycopersicon and Solanum.

Isolation of sub-diploid microprotoplasts for partial genome transfer in plants: Enhancement of micronucleation and enrichment of microprotoplasts with one or a few chromosomes

Results on the enhancement of the frequency of protoplasts with micronuclei, and on the isolation and enrichment of smaller sub-diploid microprotoplasts in transformed Nicotiana plumbaginifolia Viv. are reported. Suspension cells were treated with the spindle toxin amiprophos-methyl (APM) for 48 h, and subsequently incubated in a mixture of cell-wall-digesting enzymes in the presence of APM and cytochalasin-B. During enzyme incubation, the frequency of micronucleated protoplasts increased by a factor of 2–6. A shorter period (3 h) of incubation with a higher concentration of enzymes as well as a longer period (16 h) of incubation with a lower concentration of enzymes gave similar frequencies of micronucleated protoplasts and yields of micronuclei. Further, synchronization by sequential treatment with the DNA-synthesis inhibitor hydroxy urea, or aphidicolin, followed by APM and enzyme incubation, significantly increased the frequency of micronucleated protoplasts and the number of micronuclei. The suspension of protoplasts (mono- and micronucleated) obtained after enzyme incubation was fractionated throuh a continuous iso-osmotic gradient of Percoll, using high-speed centrifugation. This resulted in one large and a few small bands, which contained a heterogeneous population of microprotoplasts, protoplasts and cytoplasts. In contrast to the large band, the small bands contained a relatively higher frequency of small sub-diploid microprotoplasts. To separate the small sub-diploid microprotoplasts from the large microprotoplasts and protoplasts of the bands, discontinuous Percoll gradients and sequential filtration through nylon sieves of decreasing pore size (48-20-15-10-5 μm) were investigated. Compared with the former method, the latter gave a highly enriched fraction containing predominantly (≈80%) small subdiploid microprotoplasts with DNA contents equivalent to that of one to four chromosomes, as revealed by microdensitometric and flow-cytometric analyses. The application of this technique for partial genome and limited gene transfer is discussed.

Microprotoplast isolation, enrichment and fusion for partial genome transfer in plants

Somatic hybridization using protoplasts with whole genomes has often resulted in complex hybrids with many unwanted chromosomes or genes. Several researchers have attempted to reduce the number of undesired chromosomes through irradiation of the donor protoplasts, but so far without much success. Alternatively, micropro‐toplasts containing one or a few chromosomes can be used for partial genome transfer, as has been demonstrated in human and other mammalian cell systems using microcells. Recently, we have optimized the ‘microprotoplast system’ for several donor cell lines (potato, Nicotiana, sugar beet) carrying various genetic markers, such as kanamycin resistance, β‐glucuronidase, nitrate reductase deficiency, hormone autotrophy, opines, etc. Protocols were developed to obtain higher yields of micro‐protoplasts as well as to enrich sub‐diploid microprotoplasts containing one or a few chromosomes. These microprotoplasts were fused with whole mesophyll protoplasts of recipient lines using polyethylene glycol. Various requirements for lines used as donor and recipient partners in microprotoplast‐protoplast fusions are described. The results are discussed in the context of partial genome transfer.

Microprotoplast isolation, enrichment and fusion for partial genome transfer in plants

Microprotoplast isolation, enrichment and fusion for partial genome transfer in plants

Partial genome transfer through micronuclei in plants

Acta Botanica NeerlandicaVolume 40, Issue 2 p. 97-113 Review Partial genome transfer through micronuclei in plants H. A. Verhoeven, H. A. Verhoeven Department of Cell Biology, Centre for Plant Breeding and Reproduction Research CPRO, P.O. Box 16, 6700 AA Wageningen, The NetherlandsSearch for more papers by this authorK. Sree Ramulu, K. Sree Ramulu Department of Cell Biology, Centre for Plant Breeding and Reproduction Research CPRO, P.O. Box 16, 6700 AA Wageningen, The NetherlandsSearch for more papers by this authorL. J. W. Gilissen, L. J. W. Gilissen Department of Cell Biology, Centre for Plant Breeding and Reproduction Research CPRO, P.O. Box 16, 6700 AA Wageningen, The NetherlandsSearch for more papers by this authorI. Famelaer, I. Famelaer Department of Cell Biology, Centre for Plant Breeding and Reproduction Research CPRO, P.O. Box 16, 6700 AA Wageningen, The NetherlandsSearch for more papers by this authorP. Dijkhuis, P. Dijkhuis Department of Cell Biology, Centre for Plant Breeding and Reproduction Research CPRO, P.O. Box 16, 6700 AA Wageningen, The NetherlandsSearch for more papers by this authorJ. Blaas, J. Blaas Department of Cell Biology, Centre for Plant Breeding and Reproduction Research CPRO, P.O. Box 16, 6700 AA Wageningen, The NetherlandsSearch for more papers by this author H. A. Verhoeven, H. A. Verhoeven Department of Cell Biology, Centre for Plant Breeding and Reproduction Research CPRO, P.O. Box 16, 6700 AA Wageningen, The NetherlandsSearch for more papers by this authorK. Sree Ramulu, K. Sree Ramulu Department of Cell Biology, Centre for Plant Breeding and Reproduction Research CPRO, P.O. Box 16, 6700 AA Wageningen, The NetherlandsSearch for more papers by this authorL. J. W. Gilissen, L. J. W. Gilissen Department of Cell Biology, Centre for Plant Breeding and Reproduction Research CPRO, P.O. Box 16, 6700 AA Wageningen, The NetherlandsSearch for more papers by this authorI. Famelaer, I. Famelaer Department of Cell Biology, Centre for Plant Breeding and Reproduction Research CPRO, P.O. Box 16, 6700 AA Wageningen, The NetherlandsSearch for more papers by this authorP. Dijkhuis, P. Dijkhuis Department of Cell Biology, Centre for Plant Breeding and Reproduction Research CPRO, P.O. Box 16, 6700 AA Wageningen, The NetherlandsSearch for more papers by this authorJ. Blaas, J. Blaas Department of Cell Biology, Centre for Plant Breeding and Reproduction Research CPRO, P.O. Box 16, 6700 AA Wageningen, The NetherlandsSearch for more papers by this author First published: June 1991 https://doi.org/10.1111/j.1438-8677.1991.tb01518.xCitations: 11AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume40, Issue2June 1991Pages 97-113 RelatedInformation

Chromosome behaviour and formation of micronuclei after treatment of cell suspension cultures with amiprophos-methyl in various plant species

Chromosome behaviour and the formation of micronuclei after treatment of cell suspensions of Solanum tuberosum, Daucus carota and Haplopappus gracilis with the microtubule depolymerizing herbicide amiprophos-methyl (APM) are reported. The addition of APM at 32 and 64 μM to suspension cells resulted in a high degree of metaphase arrest, and single as well as groups of two or more chromosomes were scattered throughout the cell offering good prospects for cytology and metaphase chromosome isolation. The course of mitosis was strikingly different from normal. Metaphase chromosomes showed no centromere division or separation of chromatids (devoid of anaphase). The single as well as grouped metaphase chromosomes started decondensation after a 3-h application of APM and within 10 h they developed nuclear membranes and formed micronuclei. The frequency of APM-induced micronucleation varied according to the species, cell growth and mitotic activity. Fast-growing cell cultures responded with higher frequency of micronucleate cells than the relatively slow-growing cultures. After removal of APM, chromosome grouping and micronucleation disappeared and cells showed normal mitosis and growth. Various factors related to cell cycle progression, mitotic index and cell growth which are important for increasing micronucleation, are discussed. The application of micronuclei for partial genome transfer and gene mapping in plants is suggested.

Efficient induction by amiprophos-methyl and flow-cytometric sorting of micronuclei in Nicotiana plumbaginifolia

Amiprophos-methyl (APM) is a potential herbicide which acts at the level of microtubules. By exposure of suspension cells of Nicotiana plumbaginifolia to this agent, a high degree of metaphase arrest was observed and single as well as groups of chromosomes were scattered throughout the cell, offering good prospects for application in cytology and chromosome isolation. After prolonged exposure to the drug, the chromosomes decondensed and micronuclei were formed. Based on their DNA content, the micronuclei were sorted by flow cytometry. Prospects for application of isolated micronuclei for partial genome transfer and gene mapping are discussed.