Somatic hybrid plants of Nicotiana x sanderae (+) N. debneyi with fungal resistance to Peronospora tabacina

Background and Aims

The genus Nicotiana includes diploid and tetraploid species, with complementary ecological, agronomic and commercial characteristics. The species are of economic value for tobacco, as ornamentals, and for secondary plant-product biosynthesis. They show substantial differences in disease resistance because of their range of secondary products. In the last decade, sexual hybridization and transgenic technologies have tended to eclipse protoplast fusion for gene transfer. Somatic hybridization was exploited in the present investigation to generate a new hybrid combination involving two sexually incompatible tetraploid species. The somatic hybrid plants were characterized using molecular, molecular cytogenetic and phenotypic approaches.

Methods

Mesophyll protoplasts of the wild fungus-resistant species N. debneyi (2n = 4x = 48) were electrofused with those of the ornamental interspecific sexual hybrid N. × sanderae (2n = 2x = 18). From 1570 protoplast-derived cell colonies selected manually in five experiments, 580 tissues were sub-cultured to shoot regeneration medium. Regenerated plants were transferred to the glasshouse and screened for their morphology, chromosomal composition and disease resistance.

Key Results

Eighty-nine regenerated plants flowered; five were confirmed as somatic hybrids by their intermediate morphology compared with parental plants, cytological constitution and DNA-marker analysis. Somatic hybrid plants had chromosome complements of 60 or 62. Chromosomes were identified to parental genomes by genomic in situ hybridization and included all 18 chromosomes from N. × sanderae, and 42 or 44 chromosomes from N. debneyi. Four or six chromosomes of one ancestral genome of N. debneyi were eliminated during culture of electrofusion-treated protoplasts and plant regeneration. Both chloroplasts and mitochondria of the somatic hybrid plants were probably derived from N. debneyi. All somatic hybrid plants were fertile. In contrast to parental plants of N. × sanderae, the seed progeny of somatic hybrid plants were resistant to infection by Peronospora tabacina, a trait introgressed from the wild parent, N. debneyi.

Conclusions

Sexual incompatibility between N. × sanderae and N. debneyi was circumvented by somatic hybridization involving protoplast fusion. Asymmetrical nuclear hybridity was seen in the hybrids with loss of chromosomes, although importantly, somatic hybrids were fertile and stable. Expression of fungal resistance makes these somatic hybrids extremely valuable germplasm in future breeding programmes in ornamental tobacco.     

混合小麦亲本与新麦草不对称体细胞杂交体系的建立

以小麦品种济南177悬浮细胞系来源的原生质体与同品系胚性愈伤组织制备的原生质体混合后作为受体;以经过380μＷ/ｃｍ²紫外线照射1min、2min的新麦草原生质体分别作为供体,用PEG法诱导融合。组合Ⅰ(176+cha9+新麦草UV 1min)获得16个再生克隆。经过形态学、同工酶、染色体和RAPD分析,确定其全部为属间体细胞杂种。其中的5个克隆再生杂种植株。用7对小麦SSR引物对杂种克隆的叶绿体基因组进行了分析;组合Ⅱ(176+cha9+新麦草UV 2min)只获得3个克隆,且逐渐褐化死亡。表明以小麦济南177的两种培养细胞混合作受体的融合体系有利于杂种的获得及再生;紫外线对融合产物的生长发育有明显的剂量效应。     

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.     

An approach for quantitative assessment of fluorescence in situ hybridization (FISH) signals for applied human molecular cytogenetics.

A number of applied molecular cytogenetic studies require the quantitative assessment of fluorescence in situ hybridization (FISH) signals (for example, interphase FISH analysis of aneuploidy by chromosome enumeration DNA probes; analysis of somatic pairing of homologous chromosomes in interphase nuclei; identification of chromosomal heteromorphism after FISH with satellite DNA probes for differentiation of parental origin of homologous chromosome, etc.). We have performed a pilot study to develop a simple technique for quantitative assessment of FISH signals by means of the digital capturing of microscopic images and the intensity measuring of hybridization signals using Scion Image software, commonly used for quantification of electrophoresis gels. We have tested this approach by quantitative analysis of FISH signals after application of chromosome-specific DNA probes for aneuploidy scoring in interphase nuclei in cells of different human tissues. This approach allowed us to exclude or confirm a low-level mosaic form of aneuploidy by quantification of FISH signals (for example, discrimination of pseudo-monosomy and artifact signals due to over-position of hybridization signals). Quantification of FISH signals was also used for analysis of somatic pairing of homologous chromosomes in nuclei of postmortem brain tissues after FISH with "classical" satellite DNA probes for chromosomes 1, 9, and 16. This approach has shown a relatively high efficiency for the quantitative registration of chromosomal heteromorphism due to variations of centromeric alphoid DNA in homologous parental chromosomes. We propose this approach to be efficient and to be considered as a useful tool in addition to visual FISH signal analysis for applied molecular cytogenetic studies.     

Rearrangements in the mitochondrial genome of somatic hybrid cell lines of Pennisetum americanum (L.) K. Schum. + Panicum maximum Jacq.

Summary Mitochondrial DNA from three somatic hybrid cell lines of Pennisetum americanum + Panicum maximum was compared with mitochondrial DNA of the parents. Gel electrophoresis of BamHI-restricted mitochondrial DNA indicated that extensive rearrangements had occurred in each of the three hybrid lines. The hybrid restriction patterns showed a combination of some bands from each parent plus novel fragments not present in either parent. Additional evidence for rearrangements was obtained by hybridization of eight DNA probes, carrying sequences of known coding regions, to Southern blots. Each of the somatic hybrids exhibited a partial combination of the parental mitochondrial genomes. These data suggest recombination or amplification of the mitochondrial genomes in the somatic hybrids.     

Generation of high frequency of novel alleles of the high molecular weight glutenin in somatic hybridization between bread wheat and tall wheatgrass

Somatic hybridization between bread wheat and tall wheatgrass (Agropyron elongatum) has generated fertile introgression progenies with novel combinations of high molecular weight glutenin subunits (HMW-GS). Most of these novel HMW-GS alleles were stably inherited. Sixteen HMW-GS sequences were PCR amplified from three introgression progeny lines and sequenced. The alignment of these sequences indicated that five, probably derived from point mutations of the parental genes, whereas eight likely represent the product of replication slippage. Three Glu-1Ay sequences appear to have lost the transposon presented in the parental gene. Two subunits carry an additional cysteine residue, which may be favorable to the quality of end-use product. We demonstrate that novel HMW-GS alleles can be rapidly generated via asymmetric somatic hybridization. Heng Liu and Shuwei Liu have contributed equally to the work.     

Regeneration of asymmetric somatic hybrid plants from the fusion of two types of wheat with Russian wildrye

Two types of protoplasts of wheat (Triticum aestivum L. cv. Jinan 177) were used in fusion experiments—cha9, with a high division frequency, and 176, with a high regeneration frequency. The fusion combination of either cha9 or 176 protoplasts with Russian wildrye protoplasts failed to produce regenerated calli. When a mixture of cha9 and 176 protoplasts were fused with those of Russian wildrye, 14 fusion-derived calli were produced, of which seven differentiated into green plants and two differentiated into albinos. The morphology of all hybrid plants strongly resembled that of the parental wheat type. The hybrid nature of the cell lines was confirmed by cytological, isozyme, random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analyses. GISH analysis revealed that only chromosome fragments of Russian wildrye were transferred to the wheat chromosomes of hybrid calli and plants. Simple sequence repeat (SSR) analysis of the chloroplast genome of the hybrids with seven pairs of wheat-specific chloroplast microsatellite primers indicated that all of the cell lines had band patterns identical to wheat. Our results show that highly asymmetric somatic hybrid calli and plants can be produced via symmetric fusion in a triparental fusion system. The dominant effect of two wheat cell lines on the exclusion of Russian wildrye chromosomes is discussed.Abbreviations GISH Genome in situ hybridization - RAPD Random amplified polymorphic DNA - SCF Small chromosome fragment - SSR Simple sequence repeat     

Production and molecular characterization of two new Citrus somatic hybrids for scion improvement

Somatic hybridization by protoplast fusion from cell suspension cultures and leaf parent has been a well-established technique holding great potential for citrus variety improvement. In this study, somatic hybrid plants were regenerated from the following two fusion combinations: ‘Murcott’ tangor (Citrus reticulata Blanco × C. sinensis (L.) Osbeck) + Hirado Buntan Pink pummelo (HBP) (C. grandis (L.) Osbeck) and ‘Bingtang’ orange (C. sinensis (L.) Osbeck) + Calamondin (C. microcarpa Bunge). Somatic hybrids were selected at an early stage based on their higher capacity for embryogenesis comparing to non-hybrid cells. Flow cytometry analysis showed that all plants from pre-selected lines of the two combinations were tetraploid. SSR analysis confirmed their hybrid nature, with nuclear DNA from both fusion parents, and absence of parental specific bands was also detected. Cytoplasmic compositions of the recovered plants were further revealed by CAPS and cpSSR analysis. The allotetraploid somatic hybrids from the ‘Murcott’ tangor + HBP combination will be applied to develop triploid seedless cultivars by interploid crossing with diploid seedy citrus cultivars, and those from ‘Bingtang’ orange + Calamondin could be valuable for Asiatic citrus canker-tolerant and ornamental citrus breeding.     

2004 SIVB Congress Symposium Proceedings “Thinking Outside the Cell”: Applications of Somatic Hybridization and Cybridization in Crop Improvement,with Citrus as a Model

Summary Although somatic hybridization techniques are being ignored by variety improvement programs for most commodities, their contribution to citrus variety improvement continnes to expland and with increasing complexity. Citrus is, one of the few commodities where somatic hybridization is reaching its predicted potential, as somatic hybrids are now possible from most desirable parental combinations. Somatic hybrid citrus plants have been produced from more than 250 parental combinations, including more than 130 at the CREC. The CREC hybrids include 34 from sexually compatible intergeneric combinations, 16 from sexually incompatible combinations, and 81 interspecific combinations. The objective of this report is to demonstrate the impact of somatic hybridization on citrus improvement programs, and to discuss its potential with other commodities. For citrus scion improvement, several applications are aimed at the development of improved seedless fresh fruit varieties, and these include symmetric somatic hybridization, haploid+diploid fusion, targeted cybridization to transfer cytoplasmic male sterility (mtCMS) from Satsuma mandarin, and triploidy via interploid crosses using somatic hybrid allotetrapoid breeding parents. For rootstock improvement symmetric somatic hybridization provides an opportunity to hybridize complementary rootstocks without breaking up successful gene combinations. Rootstock somatic hybridization is providing opportunities for improving disease and inseet resistance, soil adaptation, and tree size control. Wide somatic hybridization provides an opportunity for gene transfer from related species, including some that are sexually incompatible. Extensive field research on citrus somatic hybrid rootstocks combined with emerging molecular analyses of citrus has allowed for the development of additional strategies for rootstock improvement. These include rootstock breeding and selection, at the tetraploid level using somatic hybrid parents, and the resynthesis of important rootstocks at the tetraploid level via fusion of selected superior parents. Ongoing examples of each strategy will be provided, along with ideas for extending the technology to other commodities.     

Citrus somatic hybridization with potential for improved blight and CTV resistance

Summary The production of five new somatic hybrids with potential for improved disease resistance is reported herein. Protoplast isolation, fusion, and plant regeneration was achieved from Caipira sweet orange (Citrus sinensis L. Osbeck) as an embryogenic parental source and Volkamer lemon (C. volkameriana Pasquale), Cleopatra mandarin (C. reticulata Blanco), and Rough lemon (C. jambhiri Lushington) as non-embryogenic parental sources. Fusion involving Cleopatra mandarin and Rangpur lime (C. limonia L. Osbeck) as embryogenic parental sources with Sour orange (C. aurantium L.) also resulted in somatic hybrid plants. Somatic hybridization was confirmed by leaf morphology evaluation, chromosome counting, and randomly amplified polymorphic DNA (RAPD) analyses. Somatic hybrids may combine complementary characteristics from both parental sources and have potential for tolerance to blight and citrus tristeza virus (CTV).     

Production of a highly asymmetric somatic hybrid between rice and Zizania latifolia (Griseb): evidence for inter-genomic exchange

 A highly asymmetric and fertile somatic hybrid plant was obtained via protoplast fusion in an intergenric combination. Gamma-ray-irradiated Zizania latifolia (Griseb). Turcz. ex Stapf mesophyll protoplasts were electrofused with idoacetamide-inactivated rice protoplasts derived from a 2-month-old suspension cell culture. Two of the six putative hybrid calli regenerated plants. Cytological observation showed that the somatic chromosome numbers of both plants were the same as the rice parent (2n=24). Nevertheless, the hybrid nature and inter-genomic exchange events of one of the plants, i.e. SH6 (SH for somatic hybrid), were confirmed by Southern analysis using both total genomic DNA and moderate-copy, Z. latifolia-abundant DNA sequences as probes; in both cases, parental specific and/or new intergenomic recombinant hybridization fragments were detected. In both plant and seed morphology, the hybrid (SH6) was distinct from its rice parental cultivar, as well as from the wild donor species, Z. latifolia. Received: 15 August 1998 / Accepted: 30 September 1998     

Male sterility systems in wheat and opportunities for hybrid wheat development

The common wheat (Triticum aestivum L.) is a poly(hexa)ploid, derived from an amphi-diploidization process involving the donor species—Triticum urartu, Aegilops speltoides, Triticum turgidum, and Aegilops tauschii. The genetic diversity of the autogamous wheat is narrow, which is a major reason for lesser rate of yield gain in wheat, in contrast to rice and maize. It is desirable to encourage hybrid breeding, i.e., combining different lines into genetically divergent heterotic pools. Thus, hybrid plants are a unique combination of desired alleles produced by crossing between genetically different parental lines. Hybrid seed production in a crop requires male-sterile female parents along with a reliable outcrossing system. The male-sterile female parent prevents pollen shedding and self-fertilization, maintaining the purity of hybrid seeds. An outcrossing system enhances hybrid seed production. This article emphasizes the biological relevance of crossbreeding and self-pollination in wheat, and reviews different male sterility systems which could be utilized for the development of hybrid wheat. Several biotechnological approaches and their practical utility in generating cross-compatible male-sterile female parent lines have been discussed.     

Homoploid hybrid speciation and genome evolution via chromosome sorting

Genomes of numerous diploid plant and animal species possess traces of interspecific crosses, and many researches consider them as support for homoploid hybrid speciation (HHS), a process by which a new reproductively isolated species arises through hybridization and combination of parts of the parental genomes, but without an increase in ploidy. However, convincing evidence for a creative role of hybridization in the origin of reproductive isolation between hybrid and parental forms is extremely limited. Here, through studying Agrodiaetus butterflies, we provide proof of a previously unknown mode of HHS based on the formation of post-zygotic reproductive isolation via hybridization of chromosomally divergent parental species and subsequent fixation of a novel combination of chromosome fusions/fissions in hybrid descendants. We show that meiotic segregation, operating in the hybrid lineage, resulted in the formation of a new diploid genome, drastically rearranged in terms of chromosome number. We also demonstrate that during the heterozygous stage of the hybrid species formation, recombination was limited between rearranged chromosomes of different parental origin, representing evidence that the reproductive isolation was a direct consequence of hybridization.     

落叶松树种的体细胞胚胎发生与规模化技术体系

植物体细胞胚胎发生技术是植物体细胞在人为可控条件下通过与合子胚胎发生类似的途径 ,发育出新个体的再生技术 ,自问世以来 ,已被广泛用于生命科学领域。对落叶松及其杂种胚性细胞系的大规模增殖培养、原胚发育状态、体细胞胚成苗和专用生物反应器技术体系的建立以及其应用、展望等方面进行了探讨 ,表明落叶松体细胞胚胎发生过程中专用生物反应器技术体系的完善与建立迫在眉睫 ,并将为现代林木育种与繁殖工程及生命科学相关学科的发展带来重要影响 。     

温室条件下条形柄锈菌体细胞重组的分子确证

为了获得温室条件下条形柄锈菌发生体细胞重组而导致毒性变异的直接证据,本研究选取7个美国条形柄锈菌小麦专化型菌系和2个美国条形柄锈菌大麦专化型菌系按照夏孢子颜色和专化型与毒性差异组成9对菌系组合,对于室内混合接种产生的子代菌系用具有不同抗性的小麦或大麦品种进行筛选,采用毒性分析及SSR分子标记技术对条形柄锈菌体细胞重组现象进行了研究。对获取的413个单孢子代菌系进行的毒性分析结果显示,有84个单孢子代菌系的毒性谱表现与亲本菌系不同,初步证明体细胞重组过程的存在。SSR标记分析结果显示,11对SSR引物中有6对引物在5对菌系组合的28个毒性谱不同的单孢子代菌系中,检测发现3个单孢菌系的扩增条带与其亲本菌系不同,且表现为亲本菌系扩增条带的重组,为体细胞重组菌系。这一结果从分子水平上证明了条形柄锈菌在室内接种条件下可以通过体细胞重组产生新小种而导致毒性变异。     

High-frequency generation and characterization of intergeneric hybrids and haploids from new wheat–barley crosses

Key message

Hybrid plants and a high frequency of maternal haploids were obtained using an efficient wheat–barley hybridization system (with new genotype combinations) and confirmed by several cytological and molecular tools.

Abstract

An efficient hybridization system between wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) is presented on the basis of three new genotype combinations. A particularly high, 14 % frequency of plant regeneration per florets was achieved in the wheat–barley genotype combination of ‘Sichuan’ × ‘Morex’. The genome composition in 42 of the 95 plants regenerated by embryo rescue was determined using ploidy analysis, genomic in situ hybridization and the application of chromosome arm-specific molecular markers (SSR and STS). A high overall frequency (76 %) of maternal (wheat) haploids was observed in all the tests for all three cross combinations. A major implication of this observation is that this new hybridization system represents a useful tool to study the mechanism of uniparental chromosome elimination in cereals.     

Mobilization of Stowaway-like MITEs in newly formed allohexaploid wheat species

Transposable elements (TEs) dominate the genetic capacity of most eukaryotes, especially plants, where they can account for up to 90?% of the genome, such as in wheat. The relationship between TEs and their hosts and the role of TEs in organismal biology are poorly understood. In this study, we have applied next generation sequencing, together with a transposon display technique in order to test whether a Stowaway-like MITE, termed Minos, transposes following allopolyploidization events in wheat. We have generated a 454-pyrosequencing database of Minos-specific amplicons (transposon display products) from a newly formed wheat allohexaploid and its parental lines and retrieved hundreds of novel MITE insertions in the allohexaploid. Clear mobilization of Minos was also seen by site-specific PCR analysis and sequence validation. In addition, using real-time qPCR analysis we observed an insignificant change in the relative quantity of Minos from the expected value of merging the two parental genomes, indicating that, despite its activation, no significant burst in Minos copy number can be seen in the newly formed allohexaploid. Interestingly, we found that CCGG sites surrounding Minos underwent massive hypermethylation following the allohexaploidization process. Our data suggest that MITEs have maintained their capacity for activity throughout the evolution of wheat and might be epigenetically deregulated in the first generations following allopolyploidization.     

Improved isolation of dihaploidsolanum tubersoum protoplasts and the production of somatic hybrids between dihaploidS. tuberosum andS. brevidens

Summary A modified protoplast isolation technique, applicable to a range of dihaploidSolanum tuberosum genotypes, has been developed. A combination of high calcium and high pH was used to fuse mesophyl protoplasts of dihaploidS. tuberosum (PDH40) and the diploid wild speciesS. brevidens. Large numbers of colonies were obtained after fusion and putative hybrids selected on the basis of phenotype from regenerated shoots. From these, 11 somatic hybrid plants have been identified by their isoenzyme patterns and morphologic characteristics. Four of these hybrids had the expected chromosome number of 48. The approach of mass culture after fusion followed by selection of hybrids from regenerated shoots and the application of somatic hybridization to potato breeding are discussed.     

A new α-gliadin gene family for wheat breeding: somatic introgression line II-12 derived from <Emphasis Type="Italic">Triticum aestivum</Emphasis> and <Emphasis Type="Italic">Agropyron elongatum</Emphasis>

Ninety-five α-gliadin open reading frames were cloned and sequenced from the somatic hybrid wheat introgression line II-12 and its parents Triticum aestivum cv. Jinan177 (JN177) and Agropyron elongatum. Novel α-gliadin genes were found to originate via point mutation, unequal crossover or slippage of a parental gene, demonstrating that new genes could be rapidly created through somatic hybridization in a manner similar to that previously shown for high-molecular-weight glutenin subunits (HMW-GS) genes. The data reveal the composition and origin of the α-gliadin gene in II-12, showing that: (1) most were homologous to those of JN177; (2) a few were derived direct from A. elongatum; and (3) some new genes were created de novo. A particular quality attribute of interest was the presence or absence of celiac disease (CD) epitopes, which were found to be four times more common among α-gliadin genes from the parent wheat JN177 than in those from A. elongatum. Although four types of CD epitopes were found in introgression line II-12, the number of genes encoded CD epitopes was lower than in JN177 due to the occurrence of pseudogenes. We discuss the benefit of these α-gliadins to wheat breeding. Fanguo Chen, Chunhui Xu, and Mengzhu Chen contributed equally to this work.