Genomic factors controlling the lethality exhibited in the hybrid between Nicotiana suaveolens Lehm. and N. tabacum L.

Interspecific hybrid plants between Nicotiana suaveolens and N. tabacum exhibit lethal symptoms at the seedling stage and cannot grow to maturity. In this investigation, an attempt was made to clarify the genomic factors responsible for this lethality. N. suaveolens was crossed to N. sylvestris (genomic constitution: SS) and N. tomentosiformis (TT), these latter two species being the progenitors of N. tabacum (SSTT). From the cross N. suaveolens x N. tomentosiformis, many seedlings were obtained through ovule culture, and these subsequently grew to maturity without exhibiting any lethality. In the reciprocal crossing between N. sauvelons and N. sylvestris, only a few hybrid seedlings were obtained through ovlue culture and all died after unfolding their cotyledons when cultured at 28 °C. This lethality could be avoided by culturing the ovules at 36 °C. These features of hybrid lethality resembled those observed in the interspecific hybrid between N. suaveolens and N. tabacum. These findings suggest that the S genome in N. tabacum is responsible for the lethality exhibited in the hybrid between N. suaveolens and N. tabacum.     

Endogenous levels of auxin and tumorigenesis in a Nicotiana amphiploid

Young seedlings of the tumor-prone amphiploid Nicotiana suaveolens X N. langsdorffii were grown aseptically on nutrient medium in a controlled environment chamber. At regular intervals after the seeds had been sown, the incidence of tumor formation was scored and plants were harvested. Indole-3-acetic acid was extracted, purified, and assayed spectrofluorometrically. A close correlation in time between onset of tumor formation and decline in endogenous level of indole-3-acetic acid was demonstrated.     

ENDOGENOUS BETA IRRADIATION AND TUMOR PRODUCTION IN AN AMPHIPLOID NICOTIAN A HYBRID

The amphiploid Nicotiana suaveolens × N. langsdorffii, which is genetically constituted to produce tumors spontaneously late in development, can be induced to form them in the early seedling stage by treatment of the apical meristem with kinetin and indoleacetic acid. Application of H³-or C¹⁴-labeled plant-growth regulators resulted in most plants and notably with C¹⁴-labeled indoleacetic acid in a significant increase in the rate of tumor production over growth-regulator treatment alone. Endogenous radiation alone was shown to be tumefacient since radioactive nucleosides, tritiated water, and C¹⁴-sodium carbonate also enhance tumorization.     

Effect of kinetin on auxin uptake and distribution in normal and tumor-prone Nicotiana shoots

Stem segments of non-tumorous Nicotiana glauca and N. langsdorffiiplants and of their tumor-producing amphidiploid F₁ hybrid weretreated with 6-furfurylaminopurine (kinetin) prior to transporttests with applied labeled indoleacetic acid (IAA-2-¹⁴C). Kinetin-treatmentsincreased the uptake of IAA in non-tumorous shoots; the IAAuptake by N. langsdorffii segments was increased up to 3-fold.The auxin uptake in stem-segments of the tumor-forming hybrid,however, could not be increased significantly by kinetin. Theeven distribution of IAA-¹⁴C in segments of normal and tumorproneNicotiana shoots is stimulated by kinetin. Data are discussedin conjunction with previous results on auxin transport andtumorformation in Nicotiana. (Received August 8, 1972; )     

Hybrid lethality in interspecific hybrids between Nicotiana tabacum and N. suaveolens: evidence that the Q chromosome causes hybrid lethality based on Q-chromosome-specific DNA markers

Hybrid seedlings from the cross Nicotiana tabacum × N. suaveolens express lethality at 28°C. We carried out a cross between monosomic lines of N. tabacum lacking the Q chromosome and N. suaveolens by test-tube pollination and ovule culture at 28°C. To suppress hybrid lethality, hybrid seedlings obtained were transferred to 36°C immediately after germination and cultured. We determined whether Q-chromosome-specific DNA markers were detected among hybrid seedlings. When hybrid seedlings cultured at 36°C were transferred to 28°C, hybrid seedlings in which Q-chromosome-specific DNA markers were detected expressed hybrid lethality, while hybrid seedlings in which Q-chromosome-specific DNA markers were not detected did not express hybrid lethality. From these results, we concluded that the presence of the Q chromosome of N. tabacum is related to hybrid lethality observed in crosses between N. tabacum and N. suaveolens. This is the first report that clearly demonstrates the relationship between a certain chromosome and hybrid lethality in the genus Nicotiana using chromosome-specific DNA markers. Additionally, we confirmed that the Q chromosome belongs to the S subgenome because Q-chromosome-specific DNA markers were detected only in N. sylvestris.     

A CYTOCHEMICAL INVESTIGATION OF A CHLOROPLAST INCLUSION

The chemical composition of a membrane-bound plastid inclusion observed in genetic tumor cells of the amphiploid Nicotiana suaveolens X N. langsdorffii was studied by means of electron microscope cytochemistry. Treatment with the following enzymes had no effect on the ultrastructure of the inclusion: α-amylase, DNase, lipase, papain, pronase, protease, RNase, and trypsin. The only enzyme to alter the fine structure of the inclusion was pepsin, which decreased the electron density of the granular component of the structure. These results indicate that protein is the major chemical constituent of this membrane-bound plastid inclusion. This finding is discussed in relation to the possible role of the inclusion in plastid ontogeny.     

The possible role of cyclic AMP in the control of genetic tumor induction

Young seedlings of the tumor-prone amphiploid Nicotiana suaveolensx N. langsdorffii, grown aseptically on nutrient medium, weretreated with 1x10^–2 M cyclic adenosine 3': 5'-monophosphate(cyclic AMP). The incidence of tumor formation was scored atregular intervals subsequent to exposure. Cyclic AMP causeda significant reduction in the rate of tumor formation. In addition,untreated plants grown on nutrient medium were harvested atregular intervals after the seeds had been sown. Cyclic AMPwas extracted, partially purified, and assayed radioimmunologically.The endogenous level of cyclic AMP in stem tissue was highestin young seedlings, rapidly fell reaching a low point in 18day-old plants, and eventually leveled off. The presence ofindoleacetic acid (IAA) in the growth medium at a final concentrationof 2x10^–5 M prevented the decline in cyclic AMP that occurredin seedlings grown on unsupplemented medium. (Received May 21, 1976; )     

Paper chromatography of auxins and inhibitors in two Nicotiana species and their hybrid

Auxins and auxin inhibitors from tissue extracts of normal Nicotiana plants, Nicotiana glauca, N. langsdorffii and their hybrid (which spontaneously produces tumors) were separated by ascending paper chromatography with n-butanol-distilled water. An Avena curvature test was used for demonstrating growth-promoting and growth-inhibiting substances. IAA could be found in extracts of the parents and the hybrid (R_F 0.75). Hybrid tissue yielded the highest amount (37.1°), N. glauca tissue less (30.8°), and N. langsdorffii tissue the least amount (8.5°) of IAA. A second growth promoter (R_F 0.35) could be separated from the tissue extracts of the parents and the hybrid, but it showed only low activity in the Avena test. Three inhibitors were present in extracts from N. langsdorffii and the hybrid at R_F 0.25, 0.45, and 0.85, whereas N. glauca showed only two of them (R_F 0.25 and 0.85). The inhibitor with an R_F of 0.45 seemed to be identical with the acidic, benzene-insoluble “inhibitor β” of Bennet-Clark and Kefford (1953). The inhibitor (neutral, benzene-soluble) at R_F 0.85 could be found in some tissue extracts of the parents and the hybrid, but showed only little activity in the curvature tests. From neutral and from acidic plant extracts within a pH range of 4.4 to 5.8 a third inhibitor with an R_F of 0.25 could be separated. It seems that the high concentration of natural IAA in the hybrid is regulated by a variety of inhibitors with different specificities in the growth-regulating process. Nicotiana langsdorffii tissue has much less auxin but the same variety of inhibitors as the hybrid, whereas N. glauca tissue contains less auxin than the hybrid and only two of the three inhibitors found in N. langsdorffii and hybrid extracts.     

MORPHOLOGY AND HISTOLOGY OF TUMOR TYPES INDUCED BY X,GAMMA, AND BETA IRRADIATION OF A TOBACCO HYBRID

Hagen , George L. (The Institute for Cancer Research, Philadelphia, Pa.), James E. Gunckel , and Arnold H. Sparrow . Morphology and histology of tumor types induced by X, gamma and beta irradiation of a tobacco hybrid. Amer. Jour. Bot. 48(8): 691–699. Illus. 1961.—Radiation-induced tumors of an interspecific hybrid, Nicotiana glauca × N. langsdorffii, were of 6 morphological types: white-smooth; green-smooth; green-tomentose; purple-pigmented; green-leafy and stalked, although all intergrades could be found in 1 tumor mass. The morphological responses were essentially the same after treatment with acute X rays, chronic gamma or beta rays. Gamma rays were from an external cobalt-60 source and beta rays from internally deposited P³². The histology of the 6 morphological tumor types was not distinctive in terms of meristem, vascular and surface development, but rather followed an ontogenetic series from non-organoid to organoid. The results are discussed in terms of abnormal genome and physiological imbalances, and are compared to similar responses from infection with crown gall and wound-tumor virus.     

Somatic hybrids of plants by fusion of protoplasts

Summary Fusions of protoplasts from Nicotiana langsdorffii and Nicotiana glauca were induced using polyethylene glycol. Parasexual hybrid colonies were selected for their ability to grow without growth substances. Hybrid plants, regenerated after grafting, were all tumorous and exhibited morphological and chromosome number variations. Out of 48 colonies selected in vitro only 6 regenerated flowering plants. Two of these plants had 42 chromosomes and were morphologically identical to the sexual amphidiploid Nicotiana glaucaxlangsdorffii.     

Interspecific hybrid plant formation by electrofusion in Nicotiana

Summary We obtained complete hybrid plants by electrofusion of mesophyll protoplasts from Nicotiana glauca and N. langsdorffii. Electrofocusing analysis of Fraction I proteins isolated from the leaves of these plants confirmed their hybridity. Cytological analysis indicated that the chromosome number (2n) of these plants is between 60 to 66, suggesting that they are the products of triple fusion. All plants were fertile and set viable seeds after self pollination. As we did not use an AC field for electrofusion, the present results indicate that an AC field is not essential for obtaining hybrid plants with electrofusion.     

Somatic hybridization in Nicotiana: Segregation of organellar traits among hybrid and cybrid plants

Protoplasts from a nitrate reductase-deficient mutant of Nicotiana tabacum L. were fused with protoplasts from a stamen-less, cytoplasmically malesterile cultivar of tobacco containing the cytoplasm from N. suaveolens Lehm. Plants were regenerated from the fused protoplasts and characterized with respect to stamen development, chromosome number, and chloroplast composition. Of 29 regenerated plants, stamen production was restored in 26 plants and pollen production in 22. One plant was male sterile and two plants have never flowered. Analysis of the electrophoretic mobility of ribulose-1,5-bisphosphate carboxylase (RuBPcase) showed that 19 of the plants contained RuBPcase of the N. suaveolens type, six plants contained enzyme of the N. tabacum type, and four plants contained both types. Analysis of resistance to tentoxin in seedlings from 20 of the plants demonstrated that 14 had N. suaveolens-type chloroplasts, three had N. tabacum type, and three contained both types. Many of the plants which produced stamens and pollen still contained chloroplasts of the N. suaveolens type. Thus, the trait of cytoplasmic male sterility in tobacco is not an expression of the type of chloroplast genetic material.     

In vivo observations of nucleolar extrusion in cultured callus cells of a non tumorousNicotiana glauca ×N. langsdorffii hybrid

Summary In vivo observations ofNicotiana glauca ×Nicotiana langsdorffii hybrid callus cells cultured on hanging-drop slides show a continuous pulsating movement of nucleoli containing vesicles from the nucleus into the cytoplasm and the occasional release of the nucleoli into the cytoplasmic stream. The phenomenon is discussed in relation to similar findings in other plant materials, particularly in critical phases of development.Contribution No. 151 from the Laboratorio di Mutagenesi e Differenziamento, Consiglio Nazionale delle Ricerche (C.N.R.), Pisa, Italy. Investigation supported by Consiglio Nazionale delle Ricerche (C.N.R.) under the programme Biology of Reproduction.     

THE FINE STRUCTURE OF GENETIC TUMOR CELLS

Tissue from genetic tumors at an early stage of development on young seedlings of Nicotiana suaveolens x N. langsdorffii was examined with the electron microscope. Such tumors, which first appear on the stem immediately below the petioles of the first and second leaves, are composed essentially of three cell types. They are covered by a single layer of epidermal cells of which two specializations, guard cells and trichomes, were observed. The majority of cells in the tumors are large, irregularly shaped, highly vacuolated, parenchymal cells. Meristematic cells, which are found in clusters close to the surface of the tumor, are the third cell type. A membrane-bound inclusion was observed within the plastids of all of the cell types within the tumor. It consists of granular material which accumulates within an intrathylakoid space. There are no major differences in ultrastructure between parenchymal cells of genetic tumors and their normal counterparts from stems without any signs of tumor formation.     

THE EXTRACTABLE AND DIFFUSIBLE AUXIN-AUXIN INHIBITOR LEVEL IN NICOTIANA GLAUCA,NICOTIANA LANGSDORFFII AND THEIR AMPHIDIPLOID HYBRID

The auxin-auxin inhibitor (effective auxin content—EAC)² level of stems and leaves of normal Nicotiana plants (N. glauca, N. langsdorffii and of their spontaneously tumor-producing hybrid) was investigated by the use of the diffusion method into agar blocks and by the wet-ether and wet-alcohol extraction method. In all series, the EAC was tested by the Avena curvature test. Although the Avena curvature produced from extracts and diffusates in these tobacco plants is low, in general there is a consistently greater curvature in the parents (on an average for N. glauca, 6.7° in the diffusion and 6.2° in the extraction tests; for N. langsdorffii, 2.7° in the diffusion and 4.6° in the extraction tests) than in the hybrid (on an average of 0.7° in the diffusion and 2.6° in the extraction tests). This suggests that the question “Why do tumors form in the hybrid?” cannot be answered simply by suggesting that it is a function of excessive auxin. It is known that IAA must be added in high concentrations to aseptic cultures of N. langsdorffii and N. glauca whereas cultures of hybrid tissues do not require such an exogenous auxin supply for growth. This might indicate differences in the availability of IAA for growth of these tissues.     

Morphological and cytological characteristics of somatic hybrids of Nicotiana tabacum L. (+) N. megalosiphon Heurk. et Müll

Summary Somatic hybrid plants of Nicotiana tabacum (bar) (+) Nicotiana megalosiphon (npt II) were recovered after polyethylene glycol (PEG) mediated fusion. Hybrid calluses were selected on the basis of their dual resistance to bialaphos and kanamycin or UV inactivation of the donor species (Nicotiana megalosiphon) protoplasts. The hybrid nature of the individual clones obtained was confirmed by AFLP analysis. An array of plants were recovered including self-fertile hybrid plants with N. tabacum or N. megalosiphon phenotype, self-sterile plants with N. tabacum habit, leaf and intermediate flower morphology, self-sterile plants with N. megalosiphon habit, abnormal leaves and intermediate flowers, and self sterile plants of N. megalosiphon type with abnormal characters. Viable pollen was observed in hybrid plants from the third group. The hybrids possessed a nuclear DNA content near that of the diploid tobacco or N. megalosiphon, and also near that of the tetraploid genome size of N. megalosiphon. The results provide evidence for nonpreferential loss of one of the parental genomes and spontaneous asymmetrization of hybrid plants. The present study shows that by means of somatic hybridization a great genetic diversity in the hybrid clones can be achieved.     

Growth inhibition of tobacco tissue cultures with 6-azauracil, 6-azauridine and maleic hydrazide

Tissue cultures of Nicotiana tabacum, Nicotiana suaveolens and Nicotiana suaveolens × Nicotiana langsdorffii were cultured in the presence of the growth inhibitors maleic hydrazide and 6-azauracil as well as 6-azauridine. Inhibition of growth was complete at 10⁻⁴ molar concentrations in all 3 genotypes of cells. The inhibition due to 6-azauracil could be completely relieved with uridine and partially relieved with uracil. The effect with maleic hydrazide was irreversible, a fact which indicates a different mechanism or level of inhibition. This study supports the concept that derivatives of 6-azauracil inhibit the synthesis of uridine derivatives required for ribose nucleic acid synthesis and growth.     

Bound Form Indole-3-acetic Acid Synthesis in Tumorous and Nontumorous Species of Nicotiana

The synthesis of H₂O-soluble and NaOH-hydrolyzable bound forms of indole-3-acetic acid (IAA) in petiole slices of Nicotiana glauca, Nicotiana langsdorffii, and their tumorous and nontumorous hybrids in the presence of exogenous ¹⁴C-IAA was investigated. The synthesis of conjugates progressively increased during 6 hours of incubation in ¹⁴C-IAA. The results showed that the rate of synthesis of IAA conjugates was higher in tumorous hybrids supplied exogenous IAA than in the parental species similarly supplied, and the rate of synthesis was higher in amphidiploid tumor plants than in a nontumorous mutant. It was also found that after 10 to 12 hours of incubation, 45% of the IAA taken up by F1 hybrids was in conjugated form whereas only 10 to 25% of the IAA taken up by a nontumorous mutant, N. langsdorffii, or N. glauca was conjugated. An F1 hybrid and an amphidiploid hybrid were found equally efficient in conjugating exogenously supplied IAA. It is postulated on the basis of these and other findings that IAA conjugates play an important role in tumorigenesis in Nicotiana.     

Indole-3-acetic Acid Synthesis in Tumorous and Nontumorous Species of Nicotiana

The synthesis of indole-3-acetic acid (IAA) in the enzyme extracts of Nicotiana glauca, Nicotiana langsdorffii, their F1 hybrid, their amphidiploid hybrid, and the nontumorous mutant of the hybrid was investigated. Tryptamine, a possible precursor of IAA biosynthesis in Nicotiana tabacum, was not found in the callus tissue of N. glauca, N. langsdorffii, and their F1 hybrid.

In petiole slices, the synthesis of IAA progressively increased during 5 hours of incubation in [¹⁴C]tryptophan. The rate of synthesis was about equal in the hybrid and N. langsdorffii but lower in N. glauca on either a cell or fresh weight basis. It was also found that tryptophan was about 25 times more efficient than tryptamine in promoting synthesis of IAA in petiole slices.

It was found that indoleacetaldehyde oxidase, indoleacetaldehyde reductase, and tryptophan aminotransferase activities were present in all of the species examined; however, tryptophan decarboxylase activity was not found. The tryptophan aminotransferase activity in N. glauca, N. langsdorffii, and the nontumorous mutant required α-ketoglutaric acid and pyridoxal 5-phosphate whereas the addition of pyridoxal 5-phosphate seemed not to increase the enzyme activity in tumor plants.

The tryptophan aminotransferase in the amphidiploid hybrid was partially purified by acetone precipitation. The enzyme activity had a temperature optimum at 49 C and a pH optimum at 8.9. It is suggested that there is an indolepyruvic acid pathway in the synthesis of IAA in the Nicotiana species examined.