Efficiency for Gene Silencing Induction in Nicotiana Species by a Viral Satellite DNA Vector

Virus-induced gene silencing （VIGS） is a useful technique for rapid plant gene function analysis. We recently reported a new VIGS vector modified from Tomato yellow leaf curl China virus （TYLCCNV） DNAβ （DNAm β）. In this study we compared in detail DNAmβ-induced gene silencing in four Nicotiana species including N. benthamiana, N. glutinosa, N. tabacum and N. paniculata. We found that DNAmβ-induced gene silencing in the four species was distinct in developing dynamics, tissue specificity, efficiency, and constancy in the plant life span. It was most efficient in N. benthamiana, where development of VIGS was most rapid, without tissue specificity and nearly 100% efficient. DNAmβ-induced gene silencing in N. glutinosa was also efficient despite being slightly less than in N. benthamiana. It initially occurred in veins, later was scattered to mesophyll, finally led to complete silencing in whole leaves. In both species, VIGS constantly expressed until the plants died. However, DNAmβ-mediated VIGS in the other two Nicotiana species, N. tabacum and N. paniculata, was significantly less efficient. It was strictly limited within the veins of the silenced leaves, and constantly occurred only over 3-4 weeks. The upper leaves that emerged later stopped showing the silencing phenotype. DNAmβ-induced gene silencing in N. benthamiana and N. glutinosa was not significantly influenced by the growth stage when the plants were agro-inoculated, and was not sensitive to high growth temperature up to 32℃. Our results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in some Nicotiana species.     

Biochemical and serological characterization of b-proteins fromNicotiana species

Summary Proteins associated with the hypersensitive response (b-proteins) were purified from variousNicotiana species and compared biochemically and serologically. The method developed to purify proteins b₁, b₂ and b₃ ofN. tabacum cv. Xanthi-nc was used to purify b-proteins present inN. sylvestris (b₀, b₁ and b₃) andN. tomentosiformis (b₂), the parental species ofN. tabacum, and b₁″ from bothN. glutinosa andN. debneyi. Ultracentrifugation and amino acid analysis of some of these proteins has shown that they are very similar and that they are all monomers in their native form (mol wt = 15 700 for b₀, b₁, b₂ and b₃; mol wt = 13 800 for b₁″). Based on their reactions to an antiserum produced against protein b₁ ofN. tabacum cv. Xanthi-nc, 3 serological groups can be recognized which are independent of the source species (I) b₀ and b₁, (II) b₁″ and b₂, (III) b₃. Thus, proteins in the same serological group but from different species are more closely related than the b-proteins in different serological groups but present in the same species. The implication of this site on the possible phylogeny of b-proteins is discussed. Serological tests confirmed the b-protein present as a constitutive component in the virus resistant interspecific hybrids ofN. glutinosa ×N. debneyi as protein b₁″.     

Seven of eight species in Nicotiana section Suaveolentes have common factors leading to hybrid lethality in crosses with Nicotiana tabacum

Background and Aims

Reproductive isolation is a mechanism that separates species, and is classified into two types: prezygotic and postzygotic. Inviability of hybrids, or hybrid lethality, is a type of postzygotic isolation and is observed in some plant species, including Nicotiana species. Previous work has shown that the Q chromosome, which belongs to the S subgenome of N. tabacum, encodes one or more genes leading to hybrid lethality in some crosses.

Methods

Interspecific crosses of eight wild species were conducted in section Suaveolentes (which consists of species restricted to Australasia and Africa) with the cultivated species Nicotiana tabacum. Hybrid seedlings were cultivated at 28, 34 or 36 °C, and PCR and chromosome analysis were performed.

Results and Conclusions

Seven of eight wild species produced inviable hybrids after crossing. Hybrid lethality, which was observed in all crosses at 28 °C, was Type II lethality, with the characteristic symptoms of browning of hypocotyl and roots; lethality was suppressed at elevated temperatures (34 or 36 °C). Furthermore, one or more genes on the Q chromosome of N. tabacum were absolutely responsible for hybrid lethality, suggesting that many species of section Suaveolentes share the same factor that triggers hybrid lethality by interaction with the genes on the Q chromosome. Exceptionally, only one wild species, N. fragrans, produced 100 % viable hybrids after crossing with N. tabacum, suggesting that N. fragrans has no factor triggering hybrid lethality.     

Random chloroplast segregation and frequent mtDNA rearrangements in fertile somatic hybrids between Nicotiana tabacum L. and N. glutinosa L.

Patterns of organelle inheritance were examined among fertile somatic hybrids between allotetraploid Nicotiana tabacum L. (2n=4x=48) and a diploid wild relative N. glutinosa L. (2n=2x=24). Seventy somatic hybrids resistant to methotrexate and kanamycin were recovered following fusion of leaf mesophyll protoplasts of transgenic methotrexate-resistant N. tabacum and kanamycin-resistant N. glutinosa. Evidence for hybridization of nuclear genomes was obtained by analysis of glutamate oxaloacetate transaminase and peroxidase isoenzymes and by restriction fragment length polymorphism (RFLP) analysis using a heterologous nuclear ribosomal DNA probe. Analysis of chloroplast genomes in a population of 41 hybrids revealed a random segregation of chloroplasts since 25 possessed N. glutinosa chloroplasts and 16 possessed N. tabacum chloroplasts. This contrasts with the markedly non-random segregation of plastids in N. tabacum (+)N. rustica and N. tabacum (+) N. debneyi somatic hybrids which we described previously and which were recovered using the same conditions for fusion and selection. The organization of the mitochondrial DNA (mtDNA) in 40 individuals was examined by RFLP analysis with a heterologous cytochrome B gene. Thirty-eight somatic hybrids possessed mitochondrial genomes which were rearranged with respect to the parental genomes, two carried mtDNA similar to N. tabacum, while none had mtDNA identical to N. glutinosa. The somatic hybrids were self-fertile and fertile in backcrosses with the tobacco parent.Contribution No. 1487 Plant Research Centre     

Compensation for a Mutated Auxin Biosynthesis Gene of Agrobacterium Ti Plasmid A66 in Nicotiana glutinosa Does Not Result from Increased Auxin Accumulation

Nicotiana glutinosa compensated for a mutated tumor-morphology-shooty (tms) (auxin biosynthesis) locus of Agrobacterlum tumefaciens strain A66 and showed the same virulent tumor response to infection by strain A66 or the wild-type strain A6. Cloned cell lines transformed by strains A6 or A66 were fully hormone independent in culture and grew rapidly as friable, unorganized tissues on hormone-free growth medium. Growth of N. glutinosa tumor cells was inhibited by addition of α-naphthaleneacetic acid to the growth medium, and A6- and A66-transformed cells showed similar dose responses to this auxin. On the other hand, A6-transformed cells contained much higher levels of indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) than A66-transformed cells. Differences in IAA and ACC levels in N. glutinosa tumor lines were consistent with the expected activity of the tms locus and were quantitatively similar to results obtained previously with A6- and A66-transformed cells of Nicotiana tabacum, which does not compensate for mutated tms genes. Thus, compensation for mutated tms genes in N. glutinosa did not result from increased auxin accumulation and did not appear to be related to the capacity of this host for auxin biosynthesis.     

Production of a hybrid of Nicotiana repanda willd.× N. tabacum L. by ovule culture

Nicotiana repanda (n = 24) × N. tabacum (n = 24) was obtained by ovule culture following regeneration from the callus. The hybrid showed an aneuploid chromosome number (44) and morphological characteristics intermediate between those of N. repanda and N. tabacum. The resistance of N. repanda to tobacco mosaic virus was incorporated into the hybrid.     

过量表达水稻细胞质型OsAPXs基因提高转基因烟草的耐盐性

为了验证水稻(Oryza sativa L.)细胞质型APXs与细胞耐盐性的关系,实验分别将OsAPXa和OsAPXb（基因登录号：D45423、AB053297）转化到烟草(Nictiana tabacum,N.plum)植株中。Southern结果表明,二基因分别整合到烟草的基因组;Northern分析表明,外源基因在转基因烟草中得到高效表达;在碳酸盐逆境下,T2代转基因植株与野生型对照相比,其APX活性呈现显著的提高,T₂代品系的H₂O₂含量和叶片受害程度显著低于野生型;T₂代品系分别在含有10 mmol·L^-1 NaHCO₃、5 mmol·L^-1 Na₂CO₃的MS培养基上生长,根的生长受到抑制,叶片产生黄化;野生型烟草则难以存活。水稻细胞质型OsAPXs基因的过量表达提高了转基因烟草的耐盐性,揭示出OsAPXa和OsAPXb在碳酸盐逆境应答过程中发挥着重要的作用。     

A Nicotiana Gametosomatic Hybrid and its Progenies

A Nicotiana gametosomatic hybrid between N. tabacum and N. glutinosawas studied. It was shown to have a stable somatic chromosomecomplement of 2n=5x=60. Karyotypes and measures of DNA contentshow that there have been no major changes in the parental chromosomenumbers or morphology accompanying hybridization. Forty-eightchromosomes are derived from N. tabacum (2n=4x=48); 12 fromN. glutinosa (2n=2x=24). Homologous pairs of N. tabacum chromosomesusually pair normally at meiosis. Trivalents incorporating aglutinosa chromosome do occur but usually these remain as univalents. The fertility of the hybrid is low but some products of self-pollinationwere obtained. These carry the complete N. tabacum genome witha few glutinosa chromosomes, some of which form supplementarybivalents at meiosis. All derivatives studied were mixoploidbut progressive selfing reduces the extent of abnormality ofmitotic divisions. The study of morphological and developmentaltraits indicates that the addition of even a few chromosomesof N. glutinosa to the N. tabacum complement can modify thetabacum phenotype substantially. There is considerable variationamong the derivatives and scope for fixing desirable qualitiesthrough selection. The presence of only a haploid set of glutinosachromosomes in the original hybrid makes the return to a desirablegenotype more efficient than that achieved through more conventionalbreeding methods. Key words: Nicotiana, gametosomatic hybrid, selfed progeny, cytology     

Characterization of nuclear and cytoplasmic information in the progeny of a somatic hybrid between male sterile Nicotiana tabacum and N. glutinosa

Summary Several nuclear and cytoplasmic characters of the back-crossed progeny of a somatic hybrid between male sterile Nicotiana tabacum (N. debneyi cytoplasm) and N. glutinosa have been analysed. Progeny were obtained by repeated back-crossing of a somatic hybrid with pollen from either N. tabacum or N. glutinosa. Nuclear ribosomal RNA genes (rDNA) were found to be a reliable marker to determine the constitution of nuclear genomes in the progeny. The progeny obtained by back-crossing with N. tabacum pollen maintained uniformity in leaf morphology. On the other hand, variation in leaf morphology was observed in the second back-cross population obtained with N. glutinosa pollen. This may be due to a variable contribution of N. tabacum chromosomes. Segregation of rDNA was also found in individuals of the same back-crossed progeny, but was not related to the chromosome number. The stable inheritance of chloroplast DNA in the back-crossed generation was confirmed regardless of the type of pollen donor. Male sterility was consistently maintained throughout several generations, suggesting that the nuclear genome of either N. tabacum or N. glutinosa does not influence the expression of cytoplasmic male sterility.     

Cellular markers indicative of ozone stress on bioindicator plants growing in a tropical environment

Ozone (O₃) is an oxidative pollutant that causes visible foliar symptoms in sensitive plants. Importantly, cellular markers induced by physiological alterations, including oxidative burst (OB), hypersensitive response-like (HR-like) reactions, and accelerated cell senescence (ACS), precede these symptoms. Because little is known about the effects of ozone in tropical environments, we aimed to identify these cellular markers in Nicotiana tabacum, Ipomoea nil and Psidium guajava growing in an urban area of tropical Sao Paulo, Brazil, and describe the specific physiological alterations that trigger the development of visible symptoms. Cell wall wart-like protrusions, an OB cellular marker, and swelling of thylakoids and mitochondrial membranes, as well as accumulation of plastoglobuli, all markers of ACS, were cell markers common to all species studied. Cellular markers indicating HR-like reaction, as observed in the asymptomatic leaves of N. tabacum and I. nil, are precursors of future development of necrotic areas, featuring the visible symptoms. On the other hand, the main cause of visible symptoms in P. guajava appeared to be the accumulation of phenolic compounds in the mesophyll tissue, an ACS marker. Based on this line of evidence, these bioindicator plants growing in a tropical environment show the damaging effects of O₃ pollution, even before visible symptoms develop. Moreover, this damage can be detected through a distinct group of cellular markers.     

Genes in S and T subgenomes are responsible for hybrid lethality in interspecific hybrids between Nicotiana tabacum and Nicotiana occidentalis

Background

Many species of Nicotiana section Suaveolentes produce inviable F₁ hybrids after crossing with Nicotiana tabacum (genome constitution SSTT), a phenomenon that is often called hybrid lethality. Through crosses with monosomic lines of N. tabacum lacking a Q chromosome, we previously determined that hybrid lethality is caused by interaction between gene(s) on the Q chromosome belonging to the S subgenome of N. tabacum and gene(s) in Suaveolentes species. Here, we examined if hybrid seedlings from the cross N. occidentalis (section Suaveolentes)×N. tabacum are inviable despite a lack of the Q chromosome.

Methodology/Principal Findings

Hybrid lethality in the cross of N. occidentalis×N. tabacum was characterized by shoots with fading color. This symptom differed from what has been previously observed in lethal crosses between many species in section Suaveolentes and N. tabacum. In crosses of monosomic N. tabacum plants lacking the Q chromosome with N. occidentalis, hybrid lethality was observed in hybrid seedlings either lacking or possessing the Q chromosome. N. occidentalis was then crossed with two progenitors of N. tabacum, N. sylvestris (SS) and N. tomentosiformis (TT), to reveal which subgenome of N. tabacum contains gene(s) responsible for hybrid lethality. Hybrid seedlings from the crosses N. occidentalis×N. tomentosiformis and N. occidentalis×N. sylvestris were inviable.

Conclusions/Significance

Although the specific symptoms of hybrid lethality in the cross N. occidentalis×N. tabacum were similar to those appearing in hybrids from the cross N. occidentalis×N. tomentosiformis, genes in both the S and T subgenomes of N. tabacum appear responsible for hybrid lethality in crosses with N. occidentalis.     

‘Hertwig Effect’ in plants: induced parthenogenesis through the use of irradiated pollen

Summary Studies of four combinations of Nicotiana involving four species, N. langsdorffii (N. l.), N. alata (N. a.), N. glutinosa (N. g.) and N. tabacum (N. t.), have shown that parthenogenetic haploid and diploid maternal individuals may arise with the use of male gametes (pollen) treated with high doses of ionising radiation in plants, similar to that found in animals (Hertwig Effect). At lower doses (10–20 Kr) rapidly diminishing numbers of seedlings were produced and many of these died soon after germination or before reaching maturity. In the intraspecific combination N. l. × N. l., viable seeds were produced only at the lower doses of 10 and 15 Kr. In the interspecific combination N. l. × N. a., at lower doses, all plants that came to bloom showed variable hybrid morphology. There were no plants resembling the female parent. In the combination N.t. × N.a., at lower doses there were rare surviving plants which were maternal dihaploid (1 plant out of 4 at 15 Kr) or tetraploid N. tabacum (all 5 plants at 20 Kr). All surviving plants at higher doses (50 and 100 Kr) were maternal tetraploids. In the combination N.t.×N.g., plants produced at lower than 20 Kr were almost all either aneuploid or triploid hybrids. Dihaploid, maternal N. tabacum plants appeared at 20 Kr and higher doses. After 50 Kr the large proportion of plants produced were maternal dihaploid or tetraploid N. tabacum.     

Host range and symptom differences between isolates of turnip mosaic virus obtained fromSisymbrium loeselii

Twenty-four isolates of turnip mosaic virus (TuMV) from spontaneously infectedSisymbriutn loeselii plants were collected in Bohemian localities. Single lesions on leaves ofNicotiana tabacum cv. Samsun served for inoculating petunia plants used as infection sources for twelve host species. In no case were two identical isolates obtained. 15 isolates could be transmitted toVicia faba, a new TuMV host. Almost all isolates infectedPhaseolus vulgaris cv. Prince locally andTrifolium incarnatum systemically. Seven isolates were transmissible toPisum sativum. No substantial differences between isolates were observed with infectedAn aranthus caudatus, Chenopodium quinoa, Datura innoxia andSinapis alba plants. Several isolates could not infectNicotiana glutinosa at all, other isolates caused in it latent systemic infection and some isolates only local infection contrary to normal local and systemic infections ofN. glutinosa. Attempts to transmit one isolate to cereals failed.     

A protein in the oxygen-evolving complex in the chloroplast is associated with symptom expression on tobacco leaves infected with cucumber mosaic virus strain Y

To elucidate the molecular basis of symptom expression in virus-infected plants, the changes in proteins between tobacco, Nicotiana tabacum cv. Ky57, leaves inoculated with cucumber mosaic virus strain Y [CMV(Y)] and strain O [CMV(O)], were compared by 2-dimensional (2-D) gel electrophoresis. The appearance of chlorotic spots in CMV(Y)-inoculated tobacco leaves accompanied an increase of 3 polypeptides and a decrease in 6 polypeptides, as compared with those in the CMV(O)-inoculated tobacco which showed no clear symptoms. The decrease in the amounts of two polypeptides of 22 and 23 kDa was particularly significant: these two polypeptides were compared with a 24 kDa polypeptide, which co-migrated with them in 2-D gel electrophoresis but did not clearly decrease at an early stage of infection, as well as major other proteins of CMV(Y)-inoculated tobacco leaves. However, the 22, 23 and 24 kDa polypeptides showed the same peptide mapping pattern. Furthermore, the 12 amino acid residues at N-termini of the three polypeptides match those of the extrinsic 23 kDa polypeptide of an oxygen-evolving complex from spinach. A comparative analysis of the 22, 23 and 24 kDa polypeptides in N. tabacum and its ancestral parents, N. sylvestris and N. tomentosiformis, revealed that the 22 kDa polypeptide derives from N. sylvestris and the 23 kDa polypeptide from N. tomentosiformis; the 24 kDa polypeptide derives from both ancestral Nicotiana species. The results indicate that the polypeptides whose amounts differentially decrease with the progress of symptom expression in N. tabacum inoculated with CMV(Y) are one component of the oxygen-evolving complex in photosystem II.     

Molecular cytogenetic analyses and phylogenetic studies in the Nicotiana section Tomentosae

Phylogenetic schemes based on changing DNA sequence have made a major impact on our understanding of evolutionary relationships and significantly built on knowledge gained by morphological and anatomical studies. Here we present another approach to phylogeny, using fluorescent in situ hybridisation. The phylogenetic scheme presented is likely to be robust since it is derived from the chromosomal distribution of ten repetitive sequences with different functions and evolutionary constraints [GRS, HRS60, NTRS, the Arabidopsis-type telomere repeat (TTTAGGG)_n, 18S-5.8S-26S ribosomal DNA (rDNA), 5S rDNA, and four classes of geminiviral-related DNA (GRD)]. The basic karyotypes of all the plant species investigated Nicotiana tomentosiformis, N. kawakamii, N. tomentosa, N. otophora, N. setchellii, N. glutinosa (all section Tomentosae), and N. tabacum (tobacco, section Genuinae) are similar (x=12) but the distribution of genic and non-genic repeats is quite variable, making the karyotypes distinct. We found sequence dispersal, and locus gain, amplification and loss, all within the regular framework of the basic genomic structure. We predict that the GRD classes of sequence integrated into an ancestral genome only once in the evolution of section Tomentosae and thereafter spread by vertical transmission and speciation into four species. Since GRD is similar to a transgenic construct that was inserted into the N. tabacum genome, its fate over evolutionary time is interesting in the context of the debate on genetically modified organisms and the escape of genes into the wild. Nicotiana tabacum is thought to be an allotetraploid between presumed progenitors of N. sylvestris (maternal, S-genome donor) and a member of section Tomentosae (T-genome donor). Of section Tomentosae, N. tomentosiformis has the most similar genome to the T genome of tobacco and is therefore the most likely paternal genome donor. It is known for N. tabacum that gene conversion has converted most 18S-5.8S-26S rDNA units of N. sylvestris origin into units of an N. tomentosiformis type. Clearly if such a phenomenon were widespread across the genome, genomic in situ hybridisation (GISH) to distinguish the S and T genomes would probably not work since conversion would tend to homogenise the genomes. The fact that GISH does work suggests a limited role for gene conversion in the evolution of N. tabacum. Received: 8 November 1999; in revised form: 23 February 2000 / Accepted: 1 March 2000     

Accumulation of scopoletin is associated with the high disease resistance of the hybrid Nicotiana glutinosa x Nicotiana debneyi

The high disease resistance of the amphidiploid hybrid of Nicotiana glutinosa x Nicotiana debneyi is associated with high constitutive levels of two phenolic compounds as analysed by high-performance liquid chromatography. The structures of these two compounds were elucidated by means of gas chromatography-tandem mass spectrometry, fluorescence- and light-spectrophotometry to be those of scopolin and scopoletin. They reached levels of 4 nmol·(g FW)^?1 and 35 nmol·(g FW)^?1, respectively, in leaf tissues of the hybrid, about 10–50 times the amount found in the parental species. Scopoletin showed a direct antimicrobial activity against Cercospora nicotianae, Phytophthora parasitica var. nicotianae, Pseudomonas syringae pvs. tabaci and syringae and tobacco mosaic virus when added to synthetic growth media, mixed with the inoculum or sprayed onto tobacco plants prior to inoculation. We postulate that the high amount of toxic phenolics in the leaves of the hybrid N. glutinosa x N. debneyi contributes to its high disease resistance.     

Expression of nuclear and chloroplastic genes coding for fraction-1 protein in somatic hybrids of Nicotiana tabacum + rustica

In the sexual interspecific cross, Nicotiana rustica L.xN. tabacum L., N. rustica can serve as the female but not as the male parent. By fusion of protoplasts, the barrier to fertilization was overcome and somatic hybrids containing N. tabacum cytoplasm were produced as shown by isoelectric focusing of the Fraction-1 protein (F-1-protein). All somatic hybrids displayed polypeptides of the large subunit of F-1 protein (which is coded by the chloroplast genome) characteristic of only one or the other parental species. Two hybrids had large subunits of the N. tabacum type and two hybrids had those of the N. rustica type. Three hybrids contained three smallsubunit polypeptides (coded by the nuclear genome), one being characteristic of N. rustica, one characteristic of N. tabacum, and one with an isoelectric point common to both species. A fourth hybrid contained only two small-subunit polypeptides of the N. tabacum type but in a F-1 protein macromolecule whose large subunits were of the N. rustica type. One somatic hybrid was self-fertile and its F₂ progeny contained large- and small-subunit polypeptides indistinguishable in their isoelectric points from those in the parent F₁ hybrid. All somatic hybrids showed an aneuploid chromosome number and morphological characteristics intermediate between those of N. rustica and N. tabacum.     

Ultrastructure of Necrotic Lesions in Nicotiana glutinosa Leaves Locally Infected with a Variant of Cucumber mosaic virus

CMV(Y/GM2)tr is a variant of Cucumber mosaic virus strain Y [CMV(Y)] which infects Nicotiana species, including N. glutinosa, to induce necrotic local lesions (NLLs) in inoculated leaves, although all other CMV strains including CMV(Y) systemically infect Nicotiana species. To investigate the morphological features of this unique host response in N. glutinosa leaves infected with CMV(Y/GM2)tr, the ultrastructure of cells surrounding completely collapsed NLLs in virus‐inoculated N. glutinosa leaves was compared with that of normal cells of mock‐inoculated N. glutinosa leaves. The changes, which have been reported in other several virus–host plant systems showing the hypersensitive response (HR), were frequently observed in cells surrounding the NLLs. Furthermore, clumping of the nuclear matrix within the nuclei, which is a feature of programmed cell death, also occurred in these cells. These results indicated that the HR‐like host response occurred at the fine structural level in the cells of N. glutinosa plants infected with CMV(Y/GM2)tr.     

Species Origin of Genomic Factors in Nicotiana nudicaulis Watson Controlling Hybrid Lethality in Interspecific Hybrids between N. nudicaulis Watson and N. tabacum L

Hybrid lethality is expressed at 28°C in the cross Nicotiana nudicaulis×N. tabacum. The S subgenome of N. tabacum has been identified as controlling this hybrid lethality. To clarify the responsible genomic factor(s) of N. nudicaulis, we crossed N. trigonophylla (paternal progenitor of N. nudicaulis) with N. tabacum, because hybrids between N. sylvestris (maternal progenitor of N. nudicaulis) and N. tabacum are viable when grown in a greenhouse. In the cross N. trigonophylla×N. tabacum, approximately 50% of hybrids were vitrified, 20% were viable, and 20% were nonviable at 28°C. To reveal which subgenome of N. tabacum was responsible for these phenotypes, we crossed N. trigonophylla with two progenitors of N. tabacum, N. sylvestris (SS) and N. tomentosiformis (TT). In the cross N. sylvestris×N. trigonophylla, we confirmed that over half of hybrids of N. sylvestris×N. trigonophylla were vitrified, and none of the hybrids of N. trigonophylla×N. tomentosiformis were. The results imply that the S subgenome, encoding a gene or genes inducing hybrid lethality in the cross between N. nudicaulis and N. tabacum, has one or more genomic factors that induce vitrification. Furthermore, in vitrified hybrids of N. trigonophylla×N. tabacum and N. sylvestris×N. trigonophylla, we found that nuclear fragmentation, which progresses during expression of hybrid lethality, was accompanied by vitrification. This observation suggests that vitrification has a relationship to hybrid lethality. Based on these results, we speculate that when N. nudicaulis was formed approximately 5 million years ago, several causative genomic factors determining phenotypes of hybrid seedlings were inherited from N. trigonophylla. Subsequently, genome downsizing and various recombination-based processes took place. Some of the causative genomic factors were lost and some became genomic factor(s) controlling hybrid lethality in extant N. nudicaulis.