A study of morphology,cytology and sterility in interspecific hybrids and amphidiploids of Nicotiana knightiana X N. umbratica

Summary Interspecific hybrids and amphidiploids of Nicotiana knightiana Goodspeed (n= 12)x N. umbratica Burbidge (n = 23) resembled either parent in some characters and were intermediate in other characters. The F₁ hybrids (2n = 35) showed mostly univalents during meiosis, while the amphidiploids (2n = 70) formed bivalents almost regularly. The former were completely sterile and the latter fully male fertile but predominantly female sterile. This female sterility was due to disintegration of the embryo sacs leading to collapsed ovules. The few fertile ovules, however, showed normal development of embryo sac and embryo. The occurrence of fertile and sterile ovules was believed to be due to segregation of the genes governing sterility.     

Nucleotide polymorphism in chloroplast dna of Nicotiana debneyi

Summary EcoR1 restriction endonuclease analysis of chloroplast DNA isolated from several distinct populations of Nicotiana debneyi has revealed a naturally occurring polymorphism. The chloroplast DNA of seven of the nine populations analysed possessed an additional EcoRl site. The origin of the additional restriction endonuclease fragments was confirmed by hybridisation of [³² P]-cRNA to fractionated EcoRl restricted chloroplast-DNA fragments adsorbed to nitrocellulose filters. Reciprocal f₁ hybrids between plants carrying the variant chloroplast-DNA's confirmed maternal inheritance of chloroplast-DNA.Communicated by G. Melchers and D. von Wettstein     

Chloroplast DNA assorts randomly in intraspecific somatic hybrids of Nicotiana debneyi

Summary Plants were regenerated following intraspecific fusion of leaf protoplasts from two naturally occurring genotypes of Nicotiana debneyi. The two genotypes differed in the EcoRl fragmentation pattern of chloroplast DNA and in the nuclear-coded phosphoglucomutase (Pgm) isozymes. There was no conscious selection for hybrid genotypes during protoplast culture or plant regeneration. Among 225 plants screened for Pgm, six were identified as nuclear hybrids. Restriction endonuclease and filter hybridisation analysis revealed that the cytoplasms of the hybrids contained one or other but never both parental chloroplast DNAs. The sorting out of chloroplasts was random and complete; the limit of detecting a rare chloroplast-DNA type in a mixture was 0.1%.     

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.     

Fertile somatic hybrids between transgenicNicotiana tabacum and transgenicN. debneyi selected by dual-antibiotic resistance

Summary A simple, yet effective selection system was used to produce fertile somatic hybrids betweenNicotiana tabacum andN. debneyi. This approach utilized transgenic antibiotic-resistantN. tabacum andN. Debneyi as donor plants for mesophyll protoplast fusions. Thirteen somatic hybrid plants were regenerated from calli capable of growth on medium containing both antibiotics. The majority of the hybrids displayed a range of leaf and floral morphologies and growth habits that were intermediate to those of the parental species, and had chromosome numbers varying from amphidiploid (2n = 96) to hypoaneuploid (2n = 60). Isoenzyme and RFLP analysis demonstrated the presence and expression of nuclear genes from both parents in all of the hybrids. Most plants are fully fertile. Thus, these plants differ from the malesterile tobacco cybrids and alloplasmic lines produced by transferring theN. debneyi cytoplasm to tobacco. A nonrandom pattern of cytoplasmic segregation in the fusion products occurred with a bias towards the presence ofN. debneyi cp and mtDNA. Evidence for the presence of rearranged or recombinant cp and mtDNA in some of the hybrids was obtained. The somatic hybrids were successfully backcrossed to theN. tabacum parent and are now being tested for immunity to black root rot, a trait specific toN. debneyi, but not existent in theN. tabacum parental line.     

The diversity of interspecific pollen-pistil incongruity in Nicotiana

Nicotiana tabacum was used as a pistillate parent and crossed with three self-compatible species, N. rustica, N. repanda and N. trigonophylla, which were previously reported to have pollen tubes unilaterally inhibited by N. tabacum pistil. Temporal and morphological observations revealed distinct differences of pollen tube behavior among these incongruous crosses. Pollen tubes of N. repanda were arrested in stigma and those of N. rustica in the middle of the style. On the other hand, pollen tubes of N. trigonophylla continued growing at a slow rate. Tubes of N. repanda and N. rustica showed morphological abnormalities such as swelling, thick wall, and irregular callose deposition. In addition, tubes of N. rustica often elongated in reverse direction and wound about in the middle of the style. Although the tubes of N. trigonophylla were apparently normal in morphology, they were distributed throughout the transmitting tissue, differing from the self-pollination of N. tabacum in which they were confined to the peripheral region of it. The diversity of pollen tube behavior indicates that physiological causes of incongruity are different among the three crosses. Bud pollination enabled pollen tubes to reach the ovary in all crosses, indicating that the N. tabacum pistil acquired its ability to inhibit foreign pollen tube elongation with its development. When interspecific hybrids between N. tabacum and the other three species were pollinated by parental species, tubes reached the ovary in all crosses, but the elongation rate of tubes slowed down and morphology was abnormal.     

Mapping genetic factors controlling pollen viability in an interspecific cross in Helianthus sect. Helianthus

Segregation of 48 genetic markers, including one CMS restorer gene, one morphological character gene, six isozymes and 40 RAPD loci, was scored in a backcross progeny of an interspecific hybrid H. argophyllusxH. annuus cv RHA274. A linkage map was generated taking into account segregation distortions for 11 of the 48 loci in the frame of two different models considering locus-pair segregation in the context of either independent selection pressures or non-equilibrated parental classes. The map consists of nine linkage groups and nine isolated markers covering 390 cM. Approximately half of the plants of the BC1 were male fertile as expected for the segregation of one dominant male-fertility restorer gene; however, these displayed a large range of variation for pollen viability. About 80% of this variation was explained by three genomic regions located on linkage groups 1, 2 and 3. The observation of meiotic chromosomes revealed a significant rate of mispairing (rod bivalents and tetravalents) in tight correlation with pollen viability, indicating that chromosome rearrangements (translocations) are the preponderant factors reducing pollen viability in this progeny. Cytogenetic and mapping data suggest that the three genomic regions involved in pollen-viability variation are located close to translocation points which differentiate the parental-species karyotypes. Segregation distortion was observed for loci correlated with pollen-viability variation. These were most likely the result of two possible suggested mechanisms.     

Transformation of Nicotiana tabacum,N. debneyi,and N. rustica: Inheritance and protoplast expression of antibiotic resistance

Agrobacterium tumefaciens strains harbouring plasmid vectors pBCAT1, pVU1011 or pMON806 were used to transform leaf explants of Nicotiana tabacum cultivars Delgold and Candel, N. debneyi, and N. rustica var. NRT. Transgenic plants resistant to the selective agents kanamycin, hygromycin or methotrexate were regenerated and used as sources of leaf mesophyll protoplasts. Protoplasts divided and regenerated plants in the presence of selective agents at levels inhibitory to protoplasts of non-transformed plants. Cross-resistance of protoplasts to more than one selective agent was not observed in this study which suggests that this approach may lead to an efficient interspecific somatic hybrid selection system.     

Rare symmetric and asymmetric Nicotiana tabacum (+) N. megalosiphon somatic hybrids recovered by selection for nuclear-encoded resistance genes and in the absence of genome inactivation

Following protoplast fusion between Nicotiana tabacum (dhfr) and N. megalosiphon (nptII) somatic hybrids were selected on the basis of dual resistance to kanamycin and methotrexate. Despite strong selection for parental nuclear-encoded resistances, only nine N. tabacum (+) N. megalosiphon somatic hybrids were obtained. A preferential loss of the parental N. tabacum nuclear and organelle genome was apparent in some plants in spite of the lack of genomic inactivation by the irradiation or chemical treatment of the parental protoplasts. Only six of the nine hybrids recovered possessed both parental profiles of nuclear RFLPs and isoenzymes. The remaining three hybrids were highly asymmetric with two being identical to N. megalosiphon except for minor morphological differences and rearranged or recombined mitochondrial DNAs (mtDNA), while the other one was distinguishable only by the presence of a rearranged or recombined mtDNA, and was therefore possibly a cybrid. Overall, eight somatic hybrids possessed rearranged or recombined mtDNAs and chloroplast inheritance was non-random since eight possessed N. megalosiphon-type chloroplasts and only one had N. tabacum chloroplasts. In contrast, using the same selection approach, numerous morphologically similar symmetric somatic hybrids with nuclear RFLPs and isozymes of both the parental species were recovered from control fusions between N. tabacum and the more closely related N. sylvestris. In spite of the low frequency of recovery of symmetric N. tabacum (+) N. megalosiphon hybrids in this study, one of these hybrids displayed a significant degree of self-fertility allowing for back-crosses to transfer N. megalosiphon disease-resistance traits to N. tabacum. Plant Research Centre Contribution No. 1579     

Cloning and morphological properties of Nicgl;CYCD3;1 gene in genetic tumors from interspecific hybrid of N. langsdorffii and N. glauca

Plant genetic tumors represent neoplastic growths, which arise spontaneously in hybrid plants without apparent external induction. To understand the molecular nature of unregulated cell proliferation, a cyclin D cDNA clone encoding a cyclin D of 1104bp was isolated from a genetic tumor and designated Nicgl;CYCD3;1 gene. DNA gel blot analysis suggested that there are two copies of Nicgl;CYCD3;1 in the genetic tumors. Northern analysis showed that this gene had the highest expression level in genetic tumor compared to Nicotiana glauca, N. langsdorffii and hybrid plants. Plant morphology of hybrid plant was an intermediate between N. glauca and N. langsdorffii and was altered in the genetic tumors. The cell cycle distribution in N. glauca was G0/G1, 90.59; S, 0.60; G2/M, 8.81; in N. langsdorffii it was G 0/G1, 86.22; S, 6.90; G2/M, 6.88; in hybrid plants it was G 0/G1, 96.40; S, 1.79; G2/M, 1.81; and in genetic tumors G 0/G1, 74.70; S, 2.35; G2/M, 22.94. These data provide new insights into the molecular mechanisms underlying genetic tumor formation from interspecific hybrid between N. langsdorffii and N. glauca.     

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     

Identification of two RAPD markers tightly linked with the Nicotiana debneyi gene for resistance to black root rot of tobacco

Linkage of randomly amplified polymorphic DNA (RAPD) markers with a single dominant gene for resistance to black root rot (Chalara elegans Nag Raj and Kendrick; Syn. Thielaviopsis basicola [Berk. and Broome] Ferraris) of tobacco (Nicotiana tabacum L.), which was transferred from N. debneyi Domin, was investigated in this study. There were 2594 repeatable RAPD fragments generated by 441 primers on DNAs of Delgold tobacco, a BC₅F₈ near isogenic line (NIL) carrying the resistance gene in a Delgold background, and PB19, the donor parent of the resistance gene. Only 7 of these primers produced eight RAPD markers polymorphic between Delgold and PB19, indicating there are few RAPD polymorphisms between them despite relatively dissimilar pedigrees. Five of the eight RAPD markers were not polymorphic between Delgold and the NIL. All of these markers proved to be unlinked with the resistance gene in F₂ linkage tests. Of the remaining three RAPD markers polymorphic between Delgold and the NIL, two were shown to be strongly linked with the resistance gene; one in coupling and the other in repulsion. Application of the two RAPDs in the elimination of linkage drag associated with the N. debneyi resistance gene and marker-assisted selection for the breeding of new tobacco cultivars with the resistance gene is discussed.     

Analysis of fertility in somatic hybrids of Nicotiana rustica and N. tabacum and progeny over two sexual generations

Summary Somatic hybrid plants, produced between Nicotiana rustica and N. tabacum by heterokaryon isolation and culture and also by mutant complementation, were examined regarding their ability to set seed. From a total of seventeen independent somatic hybrids, three were found to be partially self-fertile while the others did not set seed. Differences regarding the methods of hybrid selection, parental varieties and chloroplast composition of hybrids did not appear to be significant regarding the ability of plants to set seed. Much variation in fertility was observed in subsequent generations and by recurrent selection of the most fertile, over two generations, it was possible to increase the level of self-fertility in some of the progeny. One R2 derivative possessed approximately a tenfold higher level of self-fertility than it's somatic hybrid parent. The presence of genetic markers from both parents were observed in all progeny indicating their hybrid nature.     

Morphology and cytology of Hordeum chilense X H. bulbosum hybrids

Summary An interspecific hybrid between Hordeum chilense and H. bulbosum was produced. The hybrid resembles the male parent, but some characters from H. chilense are also present. Transgressive inheritance for other characters has also been observed. Neither chromosome instability nor homoeologous pairing was found.     

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.     

Hybrid lethality of cultured cells of an interspecific F1 hybrid of Nicotiana gossei Domin and N. tabacum L.

Cultured cells were established from the hypocotyl of F₁ hybrid seedlings of Nicotiana gossei Domin and N. tabacum L. The cultured cells started to die at 26°C, but not at 37°C, which is similar to what occurred in cells of the original hybrid plants. An increase in the number of cells without cytoplasmic strands and acidification of the cytoplasm followed by decomposition of the mitochondria and chloroplasts indicated that vacuolar collapse plays a central role in the execution of cell death. Oxygen but not light was required for cell death. Cellular levels of the superoxide anion and hydrogen peroxide temporarily increased during the early phase at 26°C, while no such oxidative burst was observed at 37°C. The reactive oxygen intermediates are potentially involved in the death of the hybrid cells.     

Non-random chloroplast segregation inNicotiana tabacum (+)N. rustica somatic hybrids selected by dual nuclear-encoded resistance

Nicotiana tabacum (+)N. rustica interspecific somatic hybrids were produced by fusion of leaf mesophyll protoplasts of transgenic methotrexate-resistantNicotiana tabacum L. with leaf mesophyll protoplasts of transgenic kanamycin-resistantN. rustica L. Somatic hybrids were selected on the basis of resistance to both methotrexate and kanamycin. Evidence for nuclear hybridization was obtained for 21 hybrids by restriction-fragment-length-polymorphism (RFLP) analysis using a heterologous wheat nuclear ribosomal-DNA (rDNA) probe and by analysis of glutamate-oxaloacetate transaminase (GOT) isoenzymes. Chloroplasts segregated non-randomly as 20 of the somatic hybrids possessedN. rustica chloroplasts and only one hadN. tabacum chloroplasts. Patterns of mitochondrial inheritance were examined by hybridization of a heterologous wheat cytochrome oxidase subunit II (coxII) gene with genomic DNA of the somatic hybrids. Four somatic hybrids with hybridization patterns similar toN. rustica and 17 with hybridization patterns consistent with mitochondrial DNA (mtDNA) rearrangement or recombination were obtained. None of the somatic hybrids had patterns ofcoxll hybridization identical withN. tabacum. Male-fertility levels in the hybrids ranged from undetectable to 87% and only nine hybrids produced a limited amount of viable seed. There was no apparent correlation between the patterns of organelle inheritance in the somatic hybrids and the relative degree of fertility.Contribution No. 1439 Plant Research CentreCurrent address: Plant Biotechnology Institute, National Research Council, 110 Gymmasium Road, Saskatoon, Saskatchewan S7N OW9, Canada     

Prospects for interspecific hybridization of Lupinus atlanticus Gladst. with L. cosentinii Guss.

Summary Successful crossing is reported between L. atlanticus Gladst. (2n = 38) and L. cosentinii Guss. (2n = 32), using lines of both species selected for crossability followed by selection of relatively fertile progenies. In one cross, 82E75, from a single F₂ segregating plant, 22 F₃ seeds were obtained. Some other less crossable combinations were completely sterile in the f₁ or F₂. Backcrossing to both parent species was successful, but some crosses gave relatively more seed by using F₂ plants for backcrossing rather than F₂'s. It is concluded that potential exists for introgression of useful genes in both directions.     

An apomictic polyhaploid obtained from a pearl millet x Pennisetum squamulatum apomictic interspecific hybrid

Summary In a research program to transfer apomixis from Pennisetum squamulatum Fresen to pearl millet, P. americanum L. Leeke, a polyhaploid plant (2n=21) was discovered in the uniform open-pollinated progeny of an apomictic interspecific hybrid (2n = 41) between pearl millet and P. squamulatum. The polyhaploid was shorter, less vigorous and was smaller morphologically than its maternal parent. It probably originated by parthenogenetic development of a reduced gametophyte in the apomictic interspecific hybrid. The most common metaphase I chromosome association in the polyhaploid was 4 bivalents plus 13 univalents. Irregular chromosome distribution, tripolar spindles, bridges and fragments were observed at anaphase I and telophase I. The polyhaploid was male-sterile and partially female- fertile having multiple aposporous embryo sacs in 95% of the ovules. Seed set was 3% when open-pollinated and 33% when pollinated with pearl millet pollen. Low seed set was due to competition among multiple embryos developing in the same ovule. Seventeen progeny from seed produced under open-pollination on the polyhaploid each had 2n=21 chromosomes and were morphologically uniform and identical to the female parent. The expression of obligate apomixis in the polyhaploid conditioned by the P. squamulatum genome between the simplex and duplex condition indicates that apomictic reproduction is possible in nonpolyploid plants.     

Overcoming hybrid incompatibility between Nicotiana africana Merxm. and N. tabacum and development of cytoplasmically male sterile tobacco forms

The incompatibility between the wild species N. africana Merxm. and the cultivated species N. tabacum has been overcome by in vitro techniques. Underdeveloped F₀ seeds, placed on MS medium with supplements, produced plants which upon reaching the stage of anthesis proved to be completely sterile. Female sterility of F₁ hybrids was overcome by applying tissue culture methods. Explants of stem parenchyma were grown in vitro. In every passage investigations were made of their callus production, organogenesis and cell polyploidization. The regenerants showed a great diversity in their morphological and cytological characters. Pollination of the R₁ plants (N. africana × N. tabacum) with N. tabacum produced normally seeded capsules. BC₁ plants were male sterile. The male sterility of the first backcross generation was preserved in BC₂ and BC₃, proving its cytoplasmic origin.