Intergeneric somatic hybridization and its application to crop genetic improvement

Related or distant species of cultivated cs are a large pool of many desirable genes. Gene transfer from these species through conventional breeding is difficult owing to post- and pre-zygotic sexual incompatibilities. Somatic hybridization via protoplast fusion is a possible alternative for gene transfer from these species to cultivated crops. Since the early days of somatic hybridization many intergeneric somatic hybrids have been developed through symmetric fusion, asymmetric fusion and microfusion. Somatic hybrids are mainly selected by using markers such as specific media or fusion parents with special features, biochemical mutants, antibiotic resistance and complementation strategy. The hybridity of the regenerants is determined based on morphological, cytological and molecular analysis. The inheritance patterns of nuclear and cytoplasmic genomes in the somatic hybrids are diverse. Nuclear DNA from both fusion parents co-exists congruously in some hybrids with translocation and rearrangement of chromosomes, but spontaneous elimination of chromosomes from either or both fusion parents has been observed very often. In asymmetric fusion, chromosome elimination is an important issue that is a complicated process influenced by many factors, such as irradiation dose, phylogenetic relatedness, ploidy level of fusion parent and regenerants. As for chloroplast genome, uniparental segregation is mainly detected, though co-existence is also reported in some cases. The mitochondrial genome, in contrast to chloroplast, undergoes recombination and very frequent rearrangements. Somatic cell fusion has potential applications for crop genetic improvement by overcoming sexual incompatibility or reproductive barriers, and by realizing novel combinations of nuclear and/or cytoplasmic genomes.     

Protoplast fusion for production of tetraploids and triploids: applications for scion and rootstock breeding in citrus

Protoplast fusion technology has been utilized in many crops to generate allotetraploid somatic hybrids, and sometimes autotetraploids as a byproduct of the process. A brief history of this technology development is provided, along with a simple protocol developed for citrus, which can be easily adapted to other plants. Protoplast fusion has become a significant tool in ploidy manipulation that can be applied in various cultivar improvement schemes. In rare cases, a new somatic hybrid may have direct utility as an improved cultivar; however, the most important application of somatic hybridization is the building of novel germplasm as a source of elite breeding parents for various types of conventional crosses for both scion and rootstock improvement. Somatic hybridization is generating superior allotetraploid breeding parents for use in interploid crosses to generate seedless triploids. Seedlessness is a primary breeding objective for new fresh fruit citrus varieties, and several thousand triploid hybrids have been produced using somatic hybrids as the tetraploid parent. Protoplast fusion is also being utilized to produce somatic hybrids that combine complementary diploid rootstocks, which have shown good potential for tree size control. Tree size control has gained importance as a means of reducing harvesting costs, maximizing the efficiency of modern cold protection methodology, and facilitating the adaptation of new fruit production systems. Successful somatic hybridization in citrus rootstock improvement has enabled rootstock breeding at the tetraploid level via sexual hybridization, which can yield maximum genetic diversity in zygotic progeny upon which to impose selection for the many traits required in improved rootstock cultivars, including disease and insect resistance, broad adaptation, tree size control, and the ability to consistently produce high yields of quality fruit. Recent progress and successful examples of these applications are discussed. Finally, a discussion of the genetic potential of somatic hybrids as breeding parents, including meiotic behavior and inheritance is provided.     

Genotyping of somatic hybrids between <Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb. and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration.     

Biotechnology and agricultural improvement

A range of emerging technologies are expected to play a significant role in agricultural improvement in the next 20 years. Some are only now being explored, but others have already produced significant results. Recent progress in the tissue culture and genetic engineering of crop plants has opened the door to: (1) large scale and rapid propagation of genetically uniform plants from elite materials; (2) the selection of novel and improved varieties using somaclonal variation technology; (3) the development of new hybrids between different cultivars and species by means of protoplast fusion and (4) the use of recombinant DNA to introduce new genetic material into plant cells. It is expected that, by the year 2000, a wide range of crops will be affected by these advances in biotechnology.     

Characterization of a Wheat Breeders’ Array suitable for high‐throughput SNP genotyping of global accessions of hexaploid bread wheat (Triticum aestivum)

Targeted selection and inbreeding have resulted in a lack of genetic diversity in elite hexaploid bread wheat accessions. Reduced diversity can be a limiting factor in the breeding of high yielding varieties and crucially can mean reduced resilience in the face of changing climate and resource pressures. Recent technological advances have enabled the development of molecular markers for use in the assessment and utilization of genetic diversity in hexaploid wheat. Starting with a large collection of 819 571 previously characterized wheat markers, here we describe the identification of 35 143 single nucleotide polymorphism‐based markers, which are highly suited to the genotyping of elite hexaploid wheat accessions. To assess their suitability, the markers have been validated using a commercial high‐density Affymetrix Axiom^® genotyping array (the Wheat Breeders’ Array), in a high‐throughput 384 microplate configuration, to characterize a diverse global collection of wheat accessions including landraces and elite lines derived from commercial breeding communities. We demonstrate that the Wheat Breeders’ Array is also suitable for generating high‐density genetic maps of previously uncharacterized populations and for characterizing novel genetic diversity produced by mutagenesis. To facilitate the use of the array by the wheat community, the markers, the associated sequence and the genotype information have been made available through the interactive web site ‘CerealsDB’.     

TISSUE CULTURE IN THE PRODUCTION OF NOVEL DISEASE-RESISTANT CROP PLANTS

1. Plant tissue cultures form the basis of a number of techniques which have been developed to effect genetic changes in plants. Progress is being made in the application of these techniques in breeding new, disease-resistant cultivars. 2. It is possible to induce and select for mutants among populations of cultured plant cells. Novel disease-resistant plants of a small number of species have been regenerated from cells selected in culture for their resistance to toxins produced by pathogens, both with and without prior exposure to mutagens. It is not known whether such procedures are widely applicable, and the nature of the genetic changes involved has not yet been determined. 3. The tissues of plant species which are propagated vegetatively are normally genetic mosaics with regard to many characteristics, including resistance to disease. Thus, some of the plants regenerated from cultured cells of such species are more resistant to pathogens than the parent plants. Novel plants produced in this way are already being used in some breeding programmes. 4. Many attempts have been made to modify the genomes of cultured plant cells by means of exogenous nucleic acids. The evidence for integration and replication of this genetic material is equivocal. The technique, therefore, offers no immediate prospects for the development of novel disease-resistant plants, but may be important in the long term as methods are perfected for using plasmids and other agents as carriers of useful genes. 5. Steady advances are being made in producing somatic hybrids of crop plants by fusion of isolated protoplasts. In the long term it may be possible to use protoplast fusion to transfer desirable disease-resistance traits between related species which cannot be hybridized by conventional breeding methods. 6. The culture of excised embryos may be used to grow interspecific and inter-generic hybrid plants in cases where incompatibility occurs after normal fertilization. The technique is already being used by breeders in the production of disease-resistant hybrids of crop species. 7. It is concluded that tissue culture has a limited but useful role to play in the development of novel disease-resistant crop plants.     

Molecular characterisation of inter and intra-specific somatic hybrids of potato using randomly amplified polymorphic DNA (RAPD) markers

Summary Protoplast fusion allows the transfer of both mono- and polygenic traits between species that are sexually incompatible. This approach has particular relevance for potato, and somatic hybridisation has been used to introduce a range of disease resistance genes from sexually incompatible wild species into the cultivated potato gene pool. In addition, protoplast fusion allows the resynthesis of tetraploid genotypes from pre-selected diploid or dihaploid donor parents. A limiting factor for the efficient exploitation of this technology in potato breeding is the difficulty of unequivocally identifying nuclear hybrids (heterokaryons). In order to facilitate the identification of hybrids at an early stage following fusion, Randomly Amplified Polymorphic DNA markers (RAPDs) have been used to characterise molecularly both inter- and intra-specific somatic hybrids of potato. RAPD markers detect naturally occurring polymorphism in the donor genotypes and utilise short oligonucleotide primers of arbitrary nucleotide sequence in combination with the polymerase chain reaction (PCR). The exploitation of RAPDs in the characterisation of both somatic and sexual hybrids is discussed.     

Progress of chromosome engineering mediated by asymmetric somatic hybridization

Plant somatic hybridization has progressed steadily over the past 35 years. Many hybrid plants have been generated from fusion combinations of different phylogenetic species, some of which have been utilized in crop breeding programs. Among them, asymmetric hybrid, which usually contains a fraction of alien genome, has received more attention because of its importance in crop improvement. However, few studies have dealt with the heredity of the genome of somatic hybrid for a long time, which has limited the progress of this approach. Over recent ten years, along with the development of an effective cytogenetical tool ＂in situ hybridization （ISH）＂, asymmetric fusion of common wheat （Triticum aestivum L.） with different grasses or cereals has been greatly developed. Genetics, genomes, functional genes and agricultt, ral traits of wheat asymmetric hybrids have been subject to systematic investigations using gene cloning, genomic in situ hybridization （GISH） and molecular makers. The future goal is to fully elucidate the functional relationships among improved agronomic traits, the genes and underlying molecular mechanisms, and the genome dynamics of somatic introgression lines. This will accelerate the development of elite germplasms via somatic hybridization and the application of these materials in the molecular improvement of crop plants.     

Organelle segregation following plant protoplast fusion

Organelle segregation and genome recombination can lead to the production of novel nuclear—cytoplasmic genome combinations following plant protoplast fusion. These unique hybrids are potentially useful in crop improvement.     

植物细胞质雄性不育系育种的反向核置换技术分析

通过不育细胞质为选择背景,在田间事先鉴定出杂种后代的优异完全不育株,用花药培养或诱导孤雌生殖使其纯合,测定其配合力,可以筛选到优良的目标不育系。以下3种方法则可能通过目标不育系而获得其同型保持系：一是通过体细胞变异(花药培养)产生;二是在不育系孕穗期高温或低温处理使其转换成可育,选择仍具有不育保持能力的作为保持系,或作为轮回亲本,将其细胞核换到可育细胞质中;三是用原生质体融合的方式向不育系导入已杀死细胞核的可育细胞质而获得配套保持系。它可以使杂种优势利用变得有预见性,可能提高现有杂种优势水平。在创造雄性不育新质源,排除微效可育基因,进行不育系的定向改造,选育高配合力不育系,以及加速育种进程等方面具有重要价值。     

Protoplast fusion as a means of producing new industrial yeast strains

The ability of the yeast Saccharomyces cerevisiae to produce ethanol and carbon dioxide from carbohydrates has been exploited by man for thousands of years. During its brief existence protoplast fusion has already become an invaluable tool for investigating the molecular genetics of yeast, as well as an important part of the arsenal of genetic manipulations available to develop new strains. In the case of industrial strains, a mating reaction is usually lacking. Protoplast fusion overcomes this barrier and allows for the genetic analysis of commercially valuable traits. A major block toward broader applicability of fusion is that hybrids becomes more unstable as the genetic backgrounds of the parents diverge. As greater progress in overcoming this problem is made, fusion, by itself and in conjunction with classical hybridization, will become increasingly important in the development of new strains. The incorporation of cytoplasmic elements into yeast protoplasts has the potential to vastly expand the array of biochemical reactions performed by yeasts, thereby increasing the importance of this microbe to mankind.     

Somatic hybridization in citrus: An effective tool to facilitate variety improvement

Summary Citrus somatic hybridization and cybridization via protoplast fusion has become an integral part of citrus variety improvement programs worldwide. Citrus somatic hybrid plants have been regenerated from more than 200 parental combinations, and several cybrid combinations have also been produced. Applications of somatic hybridization to citrus scion improvement include the production of quality tetraploid breeding parents that can be used in interploid crosses to generate seedless triploids, and the direct production of triploids by haploid + diploid fusion. Applications of somatic hybridization to citrus rootstock improvement include the production of allotetraploid hybrids that combine complementary diploid rootstocks, and to combine citrus with sexually incompatible or difficult to hybridize genera that possess traits of interest for germplasm expansion. A few somatic hybrid tetraploid breeding parents have flowered, are fertile, and are being used as pollen parents to generate triploids. Several allotetraploid somatic hybrid rootstocks are performing well in commercial field trials, and show great promise for tree size control. Seed trees of most of these somatic hybrid rootstocks are producing adequate nucellar seed for standard propagation. Somatic hybridization is expected to have a positive impact on citrus cultivar improvement efforts.     

Synthetic hexaploid wheat enhances variation and adaptive evolution of bread wheat in breeding processes

Synthetic hexaploid wheat (SHW) that combines novel and elite genes from the tetraploid wheat Triticum turgidum L. and wild ancestor Aegilops tauschii Coss., has been used to genetically improve hexaploid common wheat. The abundant genetic diversity in SHW can effectively make breakthroughs in wheat genetic improvement through the inclusion of increased variation. In this paper, we reviewed the current advances in research and utilization of the primary SHW lines and SHW-derived wheat varieties that have enhanced evolution of modern wheat under conditions of natural and artificial selection in southwestern China. Using primary SHW lines, four high-yielding wheat varieties have been developed. In addition, using the SHW-derived varieties as breeding parents, 12 new wheat varieties were also developed. Results of genotype–phenotype and fingerprint analysis showed that the introgressed alleles from SHW lines have contributed a great number of elite characters to the new wheat varieties, and these elite characters include disease resistance, more spikes per plant, more grains per spike, larger grains, and higher grain-yield potential. We found that the primary SHW lines and SHW-derived varieties have identifiable effects to enhance genetic variation and adaptive evolution of modern hexaploid wheat, which significantly increased the grain yields of hexaploid wheat in recent years. These findings have significant implications in the breeding of high-yielding wheat varieties resistant to biotic and abiotic stresses using SHW as genetic resources.     

植物细胞质雄性不育系育种的反向核置换技术分析

通过不育细胞质为选择背景,在田间事先鉴定出杂种后代的优异完全不育株,用花药培养或诱导孤雌生殖使其纯合,测定其配合力,可以筛选到优良的目标不育系.以下3种方法则可能通过目标不育系而获得其同型保持系:一是通过体细胞变异(花药培养)产生;二是在不育系孕穗期高温或低温处理使其转换成可育,选择仍具有不育保持能力的作为保持系,或作为轮回亲本,将其细胞核换到可育细胞质中;三是用原生质体融合的方式向不育系导入已杀死细胞核的可育细胞质而获得配套保持系.它可以使杂种优势利用变得有预见性,可能提高现有杂种优势水平.在创造雄性不育新质源,排除微效可育基因,进行不育系的定向改造,选育高配合力不育系,以及加速育种进程等方面具有重要价值.     

Cryopreservation of the Norway spruce tissue culture line able to produce extracellular lignin

Somatic hybridization has been used in potato to overcome the sexual barriers between the cultivated (Solanum tuberosum L.) and wild species. To date hundreds of inter/intra-specific somatic hybrids have been produced via protoplast fusions using 23 Solanum species and characterized for multiple traits such as agronomic, disease/pest resistance, salinity, frost and others. With increasing success in recovery of fusion products, somatic hybrids have been exploited in potato genetics, breeding and genomics studies. Here, we report on progress in somatic hybridization research in potato during the past 40 years.     

Hybrid wheat: quantitative genetic parameters and consequences for the design of breeding programs

Key message

Commercial heterosis for grain yield is present in hybrid wheat but long-term competiveness of hybrid versus line breeding depends on the development of heterotic groups to improve hybrid prediction.

Abstract

Detailed knowledge of the amount of heterosis and quantitative genetic parameters are of paramount importance to assess the potential of hybrid breeding. Our objectives were to (1) examine the extent of midparent, better-parent and commercial heterosis in a vast population of 1,604 wheat (Triticum aestivum L.) hybrids and their parental elite inbred lines and (2) discuss the consequences of relevant quantitative parameters for the design of hybrid wheat breeding programs. Fifteen male lines were crossed in a factorial mating design with 120 female lines, resulting in 1,604 of the 1,800 potential single-cross hybrid combinations. The hybrids, their parents, and ten commercial wheat varieties were evaluated in multi-location field experiments for grain yield, plant height, heading time and susceptibility to frost, lodging, septoria tritici blotch, yellow rust, leaf rust, and powdery mildew at up to five locations. We observed that hybrids were superior to the mean of their parents for grain yield (10.7 %) and susceptibility to frost (?7.2 %), leaf rust (?8.4 %) and septoria tritici blotch (?9.3 %). Moreover, 69 hybrids significantly (P < 0.05) outyielded the best commercial inbred line variety underlining the potential of hybrid wheat breeding. The estimated quantitative genetic parameters suggest that the establishment of reciprocal recurrent selection programs is pivotal for a successful long-term hybrid wheat breeding.     

Somatic hybridization in higher plants.

Somatic hybridization in higher plants has come into focus since methods have been established for protoplast fusion and uptake of foreign DNA and organelles by protoplasts. Polyethylene glycol (PEG) was an effective agent for inducing fusion. Treatment of protoplasts with PEG resulted in 5 to 30% heterospecific fusion products. Protoplasts of different species, genera and even families were compatible when fused. A number of protoplast combinations (soybean + corn, soybean + pea, soybean + tobacco, carrot + barley, etc.) provided fusion products which underwent cell division and callus formation. Fusion products initially were heterokaryocytes. In dividing heterokaryocytes, random distribution of mitotic nuclei was observed to be accompanied by multiple wall formation and to result in chimeral callus. Juxtaposition of mitotic nuclei suggested nuclear fusion and hybrid formation. Fusion of heterospecific interphase nuclei was demonstrated in soybean + pea and carrot + barley heterokaryons. Provided parental protoplasts carry suitable markers, the fusion products can be recognized. For the isolation and cloning of hybrid cells, fusion experiments must be supplemented with a selective system. Complementation of two non-allelic genes that prevent or inhibit growth under special culture conditions appears as the principle on which to base the selection of somatic hybrids. As protoplasts of some species have been induced to regenerate entire plants, the development of hybrid plants from protoplast fusion products is feasible and has already been demonstrated for tobacco.     

Trends in genetic diversity among European maize cultivars and their parental components during the past 50 years

It has been claimed that the system that delivers the products of plant breeding reduces the diversity of cultivated varieties leading to an increased genetic vulnerability. The main goal of our study was to monitor the temporal trends in genetic diversity over the past five decades among maize cultivars with the largest acreage in Central Europe. Our objectives were to (1) investigate how much of the genetic diversity present in important adapted open-pollinated varieties (OPVs) has been captured in the elite flint germplasm pool, (2) examine changes in the genetic diversity among the most important commercial hybrids as well as in their dent and flint parents, (3) analyze temporal changes in allele frequencies between the dent and flint parental inbreds, and (4) investigate linkage disequilibrium (LD) trends between pairs of loci within the set of parental dent and flint lines. We examined 30 individuals of five prominent OPVs from Central Europe, 85 maize hybrids of economic importance, and their dent and flint parental components with 55 SSRs. LD was significant at probability level P=0.01 for 20.2% of the SSR marker pairs in the 82 dent lines and for 17.2% in the 66 flint lines. The dent and flint heterotic groups were clearly separated already at the beginning of hybrid breeding in Central Europe. Furthermore, the genetic variation within and among varieties decreased significantly during the five decades. The five OPVs contain numerous unique alleles that were absent in the elite flint pool. Consequently, OPVs could present useful sources for broadening the genetic base of elite maize breeding germplasm.     

Organelle assortment and mitochondrial DNA rearrangements in Brassica somatic hybrids and cybrids

Protoplast fusion permits manipulations of organelle genomes not readily achieved by other in vitro procedures or sexual crosses. Although considerable information is now available about the fate of chloroplasts and mitochondria in fusion products of various genera, many additional questions about factors affecting organelles after fusion remain to be answered. Brassica species are particularly favorable materials for such studies. Organelle assortment, mitochondrial DNA (mtDNA) recombination, and plant phenotypes observed after fusion of protoplasts from cytoplasmic male sterile B. oleracea with protoplasts from B. campestris, B. oleracea or B. napus are described. The somatic hybrids and cybrids obtained at Cornell have been used for detailed studies of recombinant mtDNA, including correlation of a specific mtDNA region with the ogura type of cytoplasmic male sterility, and have provided plant materials for possible use in hybrid breeding programs.     

Can spelt wheat be used as heterotic group for hybrid wheat breeding?

Key message

Spelt wheat is a distinct genetic group to elite bread wheat, but heterosis for yield and protein quality is too low for spelt to be recommended as heterotic group for hybrid breeding in wheat.

Abstract

The feasibility to switch from line to hybrid breeding is currently a hot topic in the wheat community. One limitation seems to be the lack of divergent heterotic groups within wheat adapted to a certain region. Spelt wheat is a hexaploid wheat that can easily be crossed with bread wheat and that forms a divergent genetic group when compared to elite bread wheat. The aim of this study was to investigate the potential of Central European spelt as a heterotic group for Central European bread wheat. We performed two large experimental field studies comprising in total 43 spelt lines, 14 wheat lines, and 273 wheat–spelt hybrids, and determined yield, heading time, plant height, resistance against yellow rust, leaf rust, and powdery mildew, as well as protein content and sedimentation volume. Heterosis of yield was found to be lower than that of hybrids made between elite wheat lines. Moreover, heterosis of the quality trait sedimentation volume was negative. Consequently, spelt wheat does not appear suited to be used as heterotic group in hybrid wheat breeding. Nevertheless, high combining abilities of a few spelt lines with elite bread wheat lines make them interesting resources for pre-breeding in bread wheat. Thereby, the low correlation between line per se performance and combining ability of these spelt lines shows the potential to unravel the breeding value of genetic resources by crossing them to an elite tester.