Effect of Radiation Spectral Composition on Nicotiana spp. Seedlings Grown in vitro

The aim of this work was to assess the responses of seedlings of five species of Nicotiana genus to red and far red radiation. N. acuminata exhibits positive photoblastic behaviour and germination was completely inhibited under far red and darkness. In N. glauca germination was reduced under far red and darkness, but the other species showed neutral behaviour. The hypocotyl elongation was inhibited in N. glauca and N. tabacum under white and far red radiation. In N. langsdorffii and N. debneyi hypocotyl was elongated under far red radiation. Only in N. acuminata red radiation promote greater hypocotyl elongation than dark condition. On the phylogenetic tree obtained from restriction analysis N. glauca and N. acuminata are grouped in one branch, while the other species, N. langsdorffii, N. debneyi and N. tabacum, are grouped in the other branch cluster. Moreover, the N. debneyi behaviours under different radiation treatments were similar to those of N. tabacum. These two species are allopolyploid members of the genus Nicotiana, as also was confirmed by this study. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tumor formation and morphogenesis on different Nicotiana sp and hybrids induced by Agrobacterium tumefaciens T-DNA mutants

A series of experiments are presented that have been performed to observe the interactions between Agrobacterium tumefaciens strains mutated in the T-DNA genes involved in indoleacetic acid and cytokinin biosynthesis and several Nicotiana species and hybrids. Infections were induced on leaf cuttings of Nicotiana debneyi, N. knightiana, N. clevelandii, N. bigelovii var bigelovii, N. bigelovii var quadrivalvis, N. glauca, N. langsdorffii, the amphidiploid tumorous hybrid N. glauca × N. langsdorffii, and a nontumorous mutant of it. The effect of deletions of the Ti plasmid varied according to plant genotype. Insertion mutants in iaaM and iaaH suppressed tumor formation in N. langsdorffii, reduced it in N. bigeloviivar quadrivalvis, had no effect in N. glauca and the two amphidiploid hybrids, and promoted tumorigenesis when compared to the wild-type Agrobacterium strain B6S3 in N. bigelovii N. debneyi, and N. knightiana. The same mutations induced shoot formation in N. glauca, increased it in N. debneyi, and suppressed root formation in N. knightiana. On the other hand, an insertion mutation of the isopentenyl transferase gene (ipt-) had no effect in N. bigelovii var quadrivalvis, N. debneyi, the tumorous hybrid, suppressed tumor formation in N. langsdorffii, and inhibited it in N. glauca, the nontumorous hybrid, N. bigelovii var bigelovii, and N. knightiana. Insertion in ipt suppressed shoot formation in the nontumorous hybrid and inhibited it in the nontumorous amphidiploid and N. debneyi, while promoting root formation in N. glauca and N. debneyi. The suggestion of the existence of specific hormone equilibria necessary for the shift to each morphogenetic pattern was supported by experiments with exogenous hormone treatments of three genotypes (N. glauca, N. langsdorffii, and the nontumorous N. glauca × N. langsdorffii).     

Possible involvement of genes on the Q chromosome of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> in expression of hybrid lethality and programmed cell death during interspecific hybridization to <Emphasis Type="Italic">Nicotiana debneyi</Emphasis>

Hybrid seedlings from the cross between Nicotiana tabacum, an allotetraploid composed of S and T subgenomes, and N. debneyi die at the cotyledonary stage. This lethality involves programmed cell death (PCD). We carried out reciprocal crosses between the two progenitors of N. tabacum, N. sylvestris and N. tomentosiformis, and N. debneyi to reveal whether only the S subgenome in N. tabacum is related to hybrid lethality. Hybrid seedlings from reciprocal crosses between N. sylvestris and N. debneyi showed lethal characteristics identical to those from the cross between N. tabacum and N. debneyi. Conversely, hybrid seedlings from reciprocal crosses between N. tomentosiformis and N. debneyi were viable. Furthermore, hallmarks of PCD were observed in hybrid seedlings from the cross N. debneyi × N. sylvestris, but not in hybrid seedlings from the cross N. debneyi × N. tomentosiformis. We also carried out crosses between monosomic lines of N. tabacum lacking the Q chromosome and N. debneyi. Using Q-chromosome-specific DNA markers, hybrid seedlings were divided into two groups, hybrids possessing the Q chromosome and hybrids lacking the Q chromosome. Hybrids possessing the Q chromosome died with characteristics of PCD. However, hybrids lacking the Q chromosome were viable and PCD did not occur. From these results, we concluded that the Q chromosome belonging to the S subgenome of N. tabacum encodes gene(s) leading to hybrid lethality in the cross N. tabacum × N. debneyi.     

Restoration of shooty morphology of a nontumorous mutant of Nicotiana glauca x N. langsdorffii by cytokinin and the isopentenyltransferase gene

The shooty morphology of a nontumorous amphidiploid mutant of Nicotiana glauca Grah. x N. langsdorffii Weinm. was restored by cytokinins, whether exogenously applied or endogenously produced by transformation of the mutant with a transfer DNA (T-DNA) cytokinin-biosynthesis gene (isopentenyltransferase; ipt). Auxins alone did not confer this effect. Similar transformation was not achieved for the parental species. In the case of transformation with the ipt gene, selection of the transformed tissues was based on its hormone-independent growth in the presence of the antibiotic kanamycin. Transformed tissues exhibited a shooty morphology, indistinguishable from that of wildtype genetic tumors N. glauca x N. langsdorffii. This altered phenotype was caused by the presence and constitutive expression of the ipt gene. The insertion and expression of this gene in transformed tissues was confirmed by using the polymerase chain reaction (PCR) technique as well as conventional molecular hybridization analysis. Expression of the ipt gene led to an elevated level of cytokinin in the transformed mutant tissues. This evidence supports the notion that genetic tumors are caused, at least in part, by elevated levels of cytokinin in interspecific hybrids.     

Somatic hybrid plants of Nicotiana glauca and Nicotiana tabacum obtained by protoplast fusion

Somatic hybrid plants were produced by fusion of protoplasts from cell cultures of the Nicotiana tabacum L. sulfur mutant Su/Su and from leaf mesophyll of Nicotiana glauca Graham. After fusion the N. glauca protoplasts failed to survive under the selected culture condition. From the hybrid cells light green shoots were produced. The hybrid plants exhibited intermediate characters between parental species with respect to leaf morphology, trichome density, floral structure and flower color. The chromosome number of 25 hybrid plants was 2n = 72 and both N. glauca and N. tabacum chromosomes were identified in the hybrids. Results of isoenzyme analysis showed bands of both parents and a specific (hybrid) band for aspartate amino-transferase. Small subunit fraction-1-protein of somatic hybrids also consisted of the sum of N. glauca and N. tabacum bands. Leaf spot formation associated with the Su locus of N. tabacum was observed in somatic hybrids.     

Morphological and genetic characteristics of genetic tumor in Nicotiana glauca

It is well understood that genetic tumors develop in certain interspecific Nicotiana hybrids. Nicotiana species are divided into “plus” and “minus” groups and crosses between “plus” and “minus” species give rise to tumorous hybrids. However, it has been proposed that parents and hybrids derived from crosses among members within the same group do not produce tumors. In this study, genetic tumors were only obtained in Nicotiana glauca, which exhibited tumor features similar to those of N. glauca × N. langsdorffii. Our results suggest that genetic factors may control tumor formation independent of tumor induction dependent on the specific interspecific cross. Genetic tumor formation exhibited high B-type and D-type cyclin expression levels, indicating tumor cells are characterized by an uncontrolled cell cycle.     

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.     

Change in the solubility of crystalline Fraction I proteins correlated with change in the composition of the small subunit

Fraction I protein crystals were obtained by a simple methodfrom four additional species in addition to seven species ofNicotiana previously reported and from Solanum melongena. Crystalswere obtained neither from several other genera of the Solanacealnor from N. debneyi, but ¹⁴C protein from the latter co-crystallizedwith N. tabacum Fraction I protein. Co-crystallization did notoccur with ¹⁴C proteins from species of Tagetes, Allium, Beta,Brassica and Hyocyamus whose Fraction I proteins were evidentlytoo different in their quaternary structures to occupy the samecrystal lattice with N. tabacum protein. Fraction I proteinsfrom N. gossei and N. excelsior differed in solubility as afunction of the NaCl concentration. The two proteins were alikein the isoelectric point of the three primary peptides composingthe large subunit, but differed in the isoelectric point ofone out of four primary peptides of the small subunit; thisdifference was also consistent with a difference in trypticpeptide fingerprints. Proteins from N. tabacum and N. glaucadiffered both in the composition of their large and small subunitsbut did not differ in solubility. However, by changing the compositionof the small subunit without changing the large subunit, thesolubility of each protein was changed. The change in smallsubunit composition could be achieved by isolating proteinsfrom the reciprocal F₁ hybrids of N. tabacum ? N. glauca wherethe maternal inheritance regulates the composition of the largesubunit, whereas both maternal and paternal genes regulate thecomposition of the small subunit. ¹Present address: Department of Physiology, Hyogo College ofMedicine, Nishinomiya, Hyogo, Japan. (Received March 20, 1974; )     

Physiological Comparisons of Pith Callus With Crown-Gall and Genetic Tumors of Nicotiana glauca, N. langsdorffii, and N. glauca-langsdorffii Grown in Vitro. II. Nutritional Physiology

Explants of genetic tumors, tumors initiated by Agrobacterium tumefaciens strains B-6 and T-37, and excised pith plugs from Nicotiana glauca, N. langsdorffii, and N. glauca-langsdorffii were cultured on Murashige and Skoog medium. All cultures, pith callus and tumors with the exception of N. langsdorffii pith grew on this medium. Addition of glutamine to the medium resulted in highly organoid growth in N. langsdorffii pith. In order to have material comparable to other pith cultures, N. langsdorffii was initiated on 2,4-dichlorophenoxyacetic acid medium, after which it grows on complete medium as amorphous pith callus. Except for the initiation of N. langsdorffii (and N. glauca) pith, the 2,4-dichlorophenoxyacetic acid medium, caused bleaching in cultures of T-37 induced tumors and death of B-6 induced tumors. Tumor cultures, except for the seedling tumor, grew well on a minimal medium lacking kinetin, indoleacetic acid, vitamins, glycine, and inositol. Glycine was necessary only in the growth of N. langsdorffii pith callus. A tissue culture model is presented which permits comparison of the various tissue types.     

Cytogenetics of flower modification of a cytoplasmic male-sterile tobacco

Plants combining the cytoplasm of Nicotiana debneyi and the 48 chromosomes from N. tabacum are male sterile. Early backcross generations of the amphidiploid hybrid to male N. tabacum produced a great variety of plants from which a series of phenotypes with characteristic flower forms and transmission rates have been isolated. Type 1A possesses completely feminized stamens and deeply split corollas, breeds true when backcrossed to normal males and carries 48 N. tabacum chromosomes. Other phenotypes, 2C, 3E and 4H, range toward normal morphology of corollas and stamens. Like 1A, 2C forms no anther tissue and has 48 chromosomes. This type is transmitted to 36.3% of the backcross progeny, the remainder being of type 1A; presumably 2C carries a chromosome segment from N. debneyi that is responsible for the partial restoration of flower structure. In contrast, both 3E and 4H produce anthers and possess an extra chromosome. The extra chromosomes are transmitted to only 19.9% and 7.1% of the progeny, respectively. Significantly, the extra chromosomes found in the anther-forming types are nucleolus organizing and carry a satellite from N. debneyi. On the basis of these observations, we surmise that differentiation of anthers in plants with N. debneyi cytoplasm may depend on the presence of a nucleolus-organizing chromosome from that species. This chromosome is unstable; unaltered, it conditions a highly restored phenotype (4H), but when structurally modified, it may control different phenotypic expressions. Other examples of satellited restorer chromosomes had been reported for different cytoplasmically male-sterile combinations; therefore, the phenomenon may have more general significance.     

Physical mapping of plastid DNA variation among eleven Nicotiana species

Summary Plastid DNA of seven American and four Australian species of the genus Nicotiana was examined by restriction endonuclease analysis using the enzymes Sal I, Bgl I, Pst I, Kpn I, Xho I, Pvu II and Eco RI. These endonucleases collectively distinguish more than 120 sites on N. tabacum plastid DNA. The DNAs of all ten species exhibited restriction patterns distinguishable from those of N. tabacum for at least one of the enzymes used. All distinctive sites were physically mapped taking advantage of the restriction cleavage site map available for plastid DNA from Nicotiana tabacum (Seyer et al. 1981). This map was extended for the restriction endonucleases Pst I and Kpn I. In spite of variation in detail, the overall fragment order was found to be the same for plastid DNA from the eleven Nicotiana species. Most of the DNA changes resulted from small insertions/deletions and, possibly, inversions. They are located within seven regions scattered along the plastid chromosome. The divergence pattern of the Nicotiana plastid chromosomes was strikingly similar to that found in the genus Oenothera subsection Euoenothera (Gordon et al. 1982). The possible role of replication as a factor in the evolution of divergence patterns is discussed. The restriction patterns of plastid DNA from species within a continent resembled each other with one exception in each instance. The American species N. repanda showed patterns similar to those of most Australian species, and those of the Australian species N. debneyi resembled those of most American species.Abbreviations ims isonuclear male sterile - ptDNA plastid chloroplast DNA - Rubisco ribulosebisphosphate carboxylase/oxygenase - kbp kilobase pairs - LSU large subunit of Rubisco     

T-DNA analysis of plants regenerated from hairy root tumors

Summary Plants regenerated from hairy root tumors induced on Nicotiana glauca and Nicotiana tabacum by Agrobacterium rhizogenes strain A4 were examined for the presence of T-DNA. Regenerated N. tabacum plants contained intact copies of both TL-DNA and TR-DNA. However, plants regenerated from N. glauca tumors did not contain the TR-DNA region corresponding to the tms (auxin synthesis) genes. Some of the regenerants exhibited an abnormal phenotype which is characterized by severe leaf wrinkling. This phenotype is correlated with the presence of TL-DNA, but not TR-DNA.     

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.     

烟草大小孢子发生和雌雄配子体发育研究

采用卡宝品红染色压片和石蜡切片法对迪勃纳氏烟草(Nicotiana debneyi)大小孢子发生和雌雄配子体发育过程进行了研究.结果发现:(1)迪勃纳氏烟草为两性花,其小孢子发生和雄配子体发育早于大孢子.(2)迪勃纳氏烟草小孢子发生和发育过程基本正常,减数分裂过程有少数细胞出现滞后染色体、染色体断片和染色体桥等异常现象;其胞质分裂为同时型,四分体为十字交叉型和正四面体型,成熟的花粉粒为2细胞型.(3)迪勃纳氏烟草为2室子房,中轴胎座,倒生胚珠,胚珠多数,胚囊发育为蓼型,大孢子发生和发育过程未观察到异常现象.     

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.     

Morphological,cytological and molecular characterization of a novel symmetric somatic hybrid between N. tabacum and N. glauca

Abstract

By using the protoplast fusion technique, we have obtained 44 regenerated plants, phenotypically different and distinct from their parents, among which we have identified a fertile symmetric somatic hybrid, designated as TG-32, between N. tabacum var. Gexin No.1 and N. glauca. The morphology, fertility, chromosome number and nuclear constitution of the somatic hybrid have been studied in detail. Unlike other asymmetric interspecific somatic hybrids, the chromosome number of the symmetric somatic hybrid is 72, equal to the sum of chromosomes of both parents. The TG-32 plant has flowers similar to those of N. tabacum, but with petals similar to those of N. glauca. Interestingly the offspring of TG-32 vary in seed production ability with temperature, and produce more seeds under a relatively low temperature. Two SCAR markers were used to evaluate genetic variability and structure. The hybrid amplified the expected fragment, but the parents showed only one of two markers. This experimental result supports the hypothesis of the co-existence of two parental genomes in the somatic hybrid.     

Isolation and characterization of Tnd-1, a retrotransposon marker linked to black root rot resistance in tobacco

 In Nicotiana debneyi, resistance to a wide range of black root rot (Chalara elegans) isolates is conferred by a single dominant gene. This gene has been transferred to cultivated tobacco (Nicotiana tabacum) and was recently discovered to be linked in coupling to a 1050-bp random amplified polymorphic DNA (RAPD) marker generated with the UBC 418 primer. We have cloned and sequenced the UBC418₁₀₅₀ marker and found it to be part of a retrotransposon. This retrotransposon is a remnant of the N. debneyi genome and is the first to be isolated from this species. Transposon N. debneyi (Tnd)-1 is present in the tobacco genome as two directly repeated copies, and in multiple copies in the donor species N. debneyi and in a number of related Nicotiana species. The retrotransposon appears to have been introduced into the Nicotiana genome after the development of the Suavolentes progenitors. The gene associated with black root rot resistance co-segregates with the retrotransposon in tobacco and is thought to be contained within the introgressed fragment marked by Tnd-1. The retrotransposon will therefore be a useful species-specific landmark that can be used for future cloning of the resistance gene. Received: 3 March 1998 / Accepted: 18 August 1998     

ORIGIN OF ADVENTITIOUS SHOOTS REGENERATED FROM CULTURED TOBACCO LEAF TISSUE

Leaf discs from Nicotiana tabacum L., N. glauca Grahm., and a series of interspecific periclinal chimeras were cultured on Murashige and Skoog (MS) medium containing either 2.5 mg/1 6-benzylaminopurine(BA) or 3.0 mg/16-furfurylaminopurine (kinetin). Most shoots regenerated from chimeral leaf discs were non-chimeral but 51 of 658 shoots were chimeral. The histogenic composition of plants regenerated from leaf discs of periclinal chimeras indicated that any cell layer in a leaf can be the ultimate source of shoots, and that shoots can be multicellular in origin. Certain periclinal arrangements were recovered more frequently and their chimeral nature was more stable during subsequent shoot growth. While N. tabacum leaf discs regenerated shoots on MS medium supplemented with either BA or with kinetin, N. glauca leaf discs did not form shoots on the medium containing kinetin. However, chimeras which possessed cells of both species arose on medium containing BA or kinetin, indicating that the morphogenetically competent (i.e., able to produce shoots in culture), N. tabacum cells either interacted with N. glauca cells leading to their competence or incorporated non-competent N. glauca cells into nascent shoot apical meristems.     

Transmission genetics of the somatic hybridization process in Nicotiana

Summary Callus protoplasts of a Nicotiana tabacum chlorophyll-deficient mutant were fused with mesophyll protoplasts from one of following five sources: 4 cmsanalogs of tobacco bearing the cytoplasms of N. plumbaginifolia, N. suaveolens, N. repanda, and N. undulata, respectively, as well as wild species N. glauca. In another series of experiments, callus protoplasts from the chlorophyll-deficient genome Su/Su mutant of tobacco were fused with mesophyll protoplasts of the wild species N. glauca and those of a plastome chlorophyll-deficient tobacco mutant. The screening of hybrids consisted of visual identification followed by mechanical isolation and cloning of heteroplasmic fusion products in microdroplets of nutrient medium. Studies of regenerated plants included the analyses of gross morphology of plants, leaf and flower morphology, analysis of chromosome size and morphology and chromosome numbers, studies of multiple molecular forms of esterase and amylase, analysis of chloroplast DNA restriction patterns and analyses of chlorophyll-deficiency controlled by Su and P ^– genes. The study of progeny of 41 clones representing all species' combinations demonstrated that regenarants of most (63%) clones from intraspecific (for nuclear genes) combinations were cybrid forms, whereas in the case of the fusion N. tabacum + N. glauca, the true nuclear hybrids prevailed and the proportion of cybrids did not exceed 26%. Clones regenerating both hybrid and cybrid plants from the same fusion product were also found.