AOBPreview originally published online on June 26, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Annals of Botany 92: 281-288, 2003
© 2003 Annals of Botany Company
New CMS-associated Phenotypes in Cybrids Nicotiana tabacum L. (+Hyoscyamus niger L.)
1 International Institute of Cell Biology, Zabolotnogo Str. 148, 252143 Kiev-143, Ukraine and 2 Institut für Pflanzengenetik und Kulturpflanzenforschung Gatersleben, IPK Corrensstrasse 3, 06466 Gatersleben, Germany
* For correspondence. E-mail mikhajlo.zubko{at}man.ac.uk
Received: 28 February 2003; Returned for revision: 11 April 2003; Accepted: 6 May 2003 Published electronically: 26 June 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Morphological characteristics were studied in cytoplasmic male sterile (CMS) cybrids possessing the tobacco nuclear genome, Hyoscyamus niger plastome and recombinant mitochondria. After backcrosses with tobacco, new flower modifications were found, including: conversions of stamens into branched filamentous structures; alterations in the shape of petals and the corolla limb; and high degrees of reduction in most flower organs. Vegetative alterations (leaf elongation and stem branching) occurred in some cybrids. Results confirmed that a protoplast fusion-based alloplasmic cytoplasm transfer, followed by conventional backcrosses, is a useful tool for generating alternative CMS sources with novel nucleo-cytoplasmic compositions. These alterations in the genetic status were accompanied by modified floral and vegetative phenotypes.
Key words: Nicotiana, Hyoscyamus, cybrids, CMS flowers, leaf development, nucleo-cytoplasmic incompatibility.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Cytoplasmic male sterility (CMS), a maternally inherited failure in reproductive ability of male gametophytes, can often be a desirable genetic trait because it increases the efficiency of hybrid seed production in plant breeding (Kaul, 1988). CMS usually results from a genetically altered mitochondrial genome, owing to mutations or substitution of cytoplasm by sexual backcrosses (Hanson, 1991; Vedel et al., 1994; Elkonin and Tyrnov, 2000). Genetic manipulation with CMS using multiple backcrosses is time-consuming. Somatic hybridization was shown to be a feasible method for one-step transfer of existing CMS types within species (Belliard et al., 1978, 1979; Davey and Kumar, 1983; Gleba and Sytnik, 1984; Kumar and Cocking, 1987; Vedel et al., 1994; Elkonin and Tyrnov, 2000). Generation of CMS de novo is possible via interspecific cytoplasm transfer by protoplast fusions, but results are variable, with efficiency reported to be low (Aviv et al., 1984; Kumashiro et al., 1988) or high (Atanassov et al., 1998). Intergeneric cytoplasm transfer appears to be an effective method for producing a high frequency of CMS phenotypes and creating novel nucleo-cytoplasmic combinations from sexually incompatible, distant species (Melchers et al., 1992; Zubko et al., 1992, 1996; Dragoeva et al., 1998).
We have previously described a novel morphological type of alloplasmic CMS designated the ‘green flowers’ trait (Zubko et al., 1996). This extreme ‘homeotic’ character generated de novo in cybrids Nicotiana tabacum (+Hyoscyamus niger) and N. tabacum (+Scopolia carniolica) is characterized by the complete depletion of stamens and petals in flowers. Here, we report on new varieties of CMS phenotypes derived after parasexual transfer of the cytoplasm from a cybrid with ‘green flowers’ to tobacco, followed by sexual backcrosses. Also, we further characterize CMS flower and leaf morphologies in the cybrids N. tabacum (+Hyoscyamus niger).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Plant material
Cybrids Nicotiana tabacum L. (+Hyoscyamus niger L.) possessing the nuclear genome of N. tabacum and the cytoplasm of H. niger were constructed by fusions of protoplasts from these parental species (Zubko et al., 1996). Since N. tabacum was a plastome albino mutant, and protoplast-derived green colonies of H. niger had distinct morphological properties, the cybrids were selected as green colonies with the tobacco morphology.
Cybrid plants were grown either in vitro on basal MS medium (Murashige and Skoog, 1962) with 20 g l–1 sucrose, or in soil. Plants were grown at 24–26 °C, with a 14/10 h light/dark regime and a light intensity of 80–100 µmol m–2 s–1 (in vitro) or 150–180 µmol m–2 s–1 (in soil).
Backcrosses
Flowering cybrid plants were manually pollinated using pollen from wild-type N. tabacum ‘Wisconsin 38’, provided kindly by Hermine Gelin-Kowallis (Berlin, Germany). Emasculation and isolation of pollinated flowers were not necessary owing to complete male sterility of the cybrids. Seed from these backcrosses was germinated either in vitro or in soil to obtain progeny for analyses and further backcrosses. Seedlings germinated in vitro were transferred into soil when they had well-developed roots.
Scanning electron microscopy
For scanning electron microscopy, ‘green flowers’ with extremely reduced generative organs were collected from soil-grown cybrid plants, fixed, and prepared by slow-speed freezing, as described previously (Adler et al., 1996). Images were taken using a Digital Image Scanning System (DISS; point electronic GmbH, Halle/Saale, Germany) on a field emission scanning electron microscope (HITACHI S4100) equipped with a cryostage CT1500 (Oxford Instruments, Oxford, UK). Images were processed by computer using PhotoFinish 3.0 and Word 6.0 software.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Construction of cybrids by substitution of cytoplasm
Cybrids Nicotiana tabacum (+Hyoscyamus niger) were generated in protoplast fusion experiments by a one-step cytoplasm transfer from a wild-type H. niger to a plastome albino mutant of N. tabacum (Zubko et al., 1996). Three cybrid lines (Rhn1, Rhn2 and Rhn3) were regenerated after protoplast fusions. In addition, several repeated cybrids (Drhn lines) were generated by a re-transfer of the cytoplasm from Rhn1 cybrid to an albino plastome mutant DSR A15 (Svab and Maliga, 1986). Chloroplast genomes in all primary and repeated cybrids were inherited from H. niger, and the mitochondrial genomes were recombined to different extents (Zubko et al., 1996).
All cybrids manifested different types of CMS according to their flower morphology. The flower phenotypes of all cybrids are shown in Figs 1 and 2 and are described in Table 1.
|
|
|
Flower phenotypes in Rhn plants and their progeny
In contrast to wild-type tobacco flowers, which possess bell-like pink corollas surrounding stamens and pistil (Fig. 1A and B), cybrid line Rhn1 had large and complex ‘green flowers’, which lacked a corolla and stamens (Fig. 1D). These flowers usually contained one large pistil, sometimes with an increased number of stigma segments. Instead of stamens, several internal ‘green flowers’ of smaller size were located around the pistil (Fig. 1E). These internal ‘green flowers’ very often had their own smaller pistils. Backcrosses of Rhn1 with wild-type tobacco gave progeny with ‘green flowers’. The capacity for seed formation after backcrossing was significantly reduced in Rhn1. Only the central flower within these complex ‘green flowers’ formed capsules with seeds (Fig. 1F). Another line, Rhn2, possessed small ‘green flowers’, usually with one pistil of different size (Fig. 1G). Very often these flowers had extremely reduced generative organs (1–2 mm in size) that became brown and died within 1–2 weeks (Fig. 1H and I). Only flowers with longer pistils in the line Rhn2 were able to produce seed capsules after backcrosses with wild-type tobacco, but viable seed was not obtained. Flowers of Rhn3 cybrid (Fig. 1C) were similar in their general morphology to wild-type tobacco flowers (Fig. 1A and B) but they were cytoplasmically male sterile owing to the presence of limited amounts of unviable pollen in the anthers. The flower phenotype of Rhn3 plants was maternally inherited in backcrosses with wild-type tobacco. No alterations of this CMS phenotype were found among three to six sexual generations of the plants subsequently backcrossed with wild-type tobacco.
Flower phenotypes in Drhn cybrids
In most of the repeated cybrids (Drhn2, Drhn5, Drhn6, Drhn8 and Drhn9), flowers were similar to those of the Rhn2 cybrid (Fig. 1G–I; Table 1). More detailed morphological analysis of this extreme phenotype showed the conversion of stamens into very small leaf-like structures (Fig. 2A). These were covered with abundant trichomes, which were of an unusual shape for tobacco (Fig. 2B). The trichomes often consisted of four to six cells, with the upper cells tending to have horizontal cell divisions (Fig. 2C). Larger flowers of this type that occurred in the Rhn2 line and in the Drnh-lines possessed longer pistils and easily detectable ovaries. Within 2–3 weeks after backcrosses with tobacco, seed-buds with an unusual accumulation of anthocyanins (Fig. 1J and K) were formed in these flowers, in contrast to the colourless seed-buds of tobacco (Fig. 1L and M). These cybrid flowers never developed viable seeds. The pink seed-buds were more elongated (Fig. 1K) than seed-buds of tobacco (Fig. 1M).
Among the Drhn-cybrids, one line (Drnh3) developed flowers with petals (Fig. 1N). The symmetry of these flowers and the morphology of their floral organs were irregular: corollas were often split; stamens displayed different degrees of petalloidal conversion; some sepals were partially petalloid; and stigmas possessed two to four segments. In backcrosses with tobacco, Drhn3-flowers were efficient seed producers, and seeds generally developed into plants with regular CMS phenotypes (Fig. 1O–S), denoted as Drhn3 x W-1/25 (from a typical individual line). A different phenotype, denoted as line Drhn3 x W-2/25, was found in the same backcross generation (Fig. 1T–X). Both cybrid lines, Drhn3 x W-1/25 and Drhn3 x W-2/25, had funnel-like limbs of corollas (Fig. 1P and U), in contrast to the bell-like limb of the corolla in wild-type tobacco (Fig. 1A) and cybrid line Rhn3 (Fig. 1C). Flowers of Drhn3 x W-1/25 line were characterized by crimped petals (Fig. 1P and S), in contrast to the pointed petals of Drhn3 x W-2/25 (Fig. 1X). Stamens of Drhn3 x W-1/25 were converted into branched yellow-green filamentous structures (Fig. 1Q and R), whereas stamens of Drhn3 x W-2/25 were transformed into leaf-like structures that were whitish-pink in colour (Fig. 1V and W). The edges of these structures were often converted into green stigmoidal bumps (Fig. 1W). Corollas in flowers of Drhn3 x W-1/25 line were ‘ruptured’ by the relatively early formation of pistils (Fig. 1O), while the top edges of developing corollas in flowers of the Drhn3 x W-1/25 line were tightly closed and much longer (Fig. 1T), as in tobacco flowers.
Vegetative morphology of the cybrids
Apart from dramatic alterations in flower morphology, plants Rhn1 and Rhn2 had unusually elongated leaves, and there was a noticeable degree of variation in this trait in different vegetative subclones of the plants (Figs 3 and 4). For quantitative characterization of this character, we calculated the leaf elongation factor (LEF), based on the ratio of leaf length to leaf width (Fig. 4). Values of LEF for two distinct subclones of cybrid Rhn1 (LEF = 2·57 and 3·2) and cybrid Rhn2 (LEF = 2·29) are significantly higher than those for tobacco (LEF = 1·62 and 1·75). The value for LEF in cybrid Rhn3, which possessed a tobacco-type corolla, was close to that of tobacco.
|
|
Following transmission of cytoplasm from Rhn1 to tobacco, the resultant Drhn-lines had elongated leaves, similar to those of Rhn1 plants (data not shown). At least in some of the plants derived from backcrosses of repeated cybrids Drhn3 and Drhn9 with wild-type tobacco, leaves were elongated. Two of these plants, Drhn3 x W38-2/25 and Drhn9 x W38, are shown in Fig. 3C and D. Progeny from the second backcross of line Drhn3 x W38-2/25 also had elongated leaves (data not shown).
Plants Rhn1 and most of the repeated Drhn-cybrids displayed a pronounced degree of stem branching (Fig. 3). This feature was also characteristic of backcross-derived progeny of line Drhn9 (Fig. 3C).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
We have described the patterns of floral and vegetative morphology that emerged de novo in cell-engineered alloplasmic cybrids N. tabacum (+H. niger). These phenotypic traits are associated with genetically novel types of CMS. The plants possess a nucleus of tobacco, and novel combinations of plastid and mitochondrial genomes derived from H. niger, a species that is sexually incompatible with Nicotiana. The transferred H. niger plastid genome was stably inherited in all examined progeny of the cybrids derived after backcrosses over several years. Initial cybrids and their backcross-derived progeny showed several patterns of rearrangement in mitochondrial (mt) DNA (Zubko et al., 1996, 2001). The extensive recombination of mtDNA detected in the cybrids is a common phenomenon in cytoplasmic hybrids (Belliard et al., 1979; Gleba and Sytnik, 1984; Kofer et al., 1991). Establishing CMS de novo in somatic hybrids/cybrids is usually associated with highly recombined mitochondrial genomes (Aviv et al., 1984; Melchers et al., 1992; Zubko et al., 1992, 1996; Dragoeva et al., 1998). The recombinant mtDNA molecules might reflect optimization of mitochondrial functions into a newly formed nucleo-plastid-mitochondrial genetic background of a particular cybrid (Wolters et al., 1993). Such adaptive mtDNA recombination does not seem to be strongly determined by taxonomic distances between parental mitochondrial genomes, since some taxonomically distant cybrids are self-fertile and show limited mDNA rearrangements (Zubko et al., 2002).
The new morphological variants of CMS flowers appeared in the cybrids after repeated parasexual transfer of the cybrid cytoplasm to tobacco followed by sexual backcrosses. The corolla-less N. tabacum (+Hyoscyamus niger) cybrid Rhn1 with CMS ‘green flowers’ and recombined mitochondria was used as a donor of cytoplasm for tobacco in a second round of protoplast fusions, with subsequent selection for green chloroplasts of H. niger (Zubko et al., 1996, 2001). The incidence of ‘green flowers’, even if differently modified, in most of the recovered repeated cybrids of the Drhn group revealed the possibility of genetic transmission of cytoplasmically controlled flower morphology in protoplast fusion experiments. The ‘green flowers’ character is the most dramatic alteration of tobacco flowers observed as a result of manipulations involving alien cytoplasm.
A few types of ‘green flowers’ have been discussed previously in terms of the genetic interactions between altered mitochondrial genomes and intact nuclear homeotic genes which reflect an altered mode of phenotypic expression of homeotic genes during flower organ formation (Zubko et al., 1996, 2001). Here, we have described another two features that illustrate the diversity of this cytoplasmic homeosis: (1) the partial conversion of stamens into pistilloid structures that occurred in corolla-containing flowers of the line Drhn3 x W-2/25; and (2) the structurally reduced ‘green flowers’, described here for Rhn2 and most Drhn cybrids, manifested anthocyanin biosynthesis in ovules. This latter characteristic might be interpreted as a new example of altered nucleo-cytoplasmic control affecting the spatial tobacco anthocyanin accumulation strictly associated with petals (Weiss, 2000). In the case of Drhn cybrids, the pigment synthesis is shifted to its accumulation in ovules of petal-less flowers.
The phenotype of cybrid Drhn3, displaying corolla-type flowers with petalloid anthers, is obviously a result of less dramatic flower alterations which are quite common for interspecific nucleo-mitochondrial incompatibilities found during sexual (Gerstel, 1980; Nikova et al., 1991, 1997; Spangenberg et al., 1992) and parasexual (Aviv et al., 1984; Kofer et al., 1991; Vedel et al., 1994; Atanassov et al., 1998) transmission of cytoplasm. Plant Drhn3 with a perturbed floral morphology segregated two divergent floral phenotypes (Drhn3 x W38-1/25 and Drhn3 x W38-2/25). The segregation of the repeated cybrid into two phenotypes after backcrossing might result from heterogeneous mtDNA within the Drhn3 cybrid and sorting of this cytoplasm during the subsequent sexual cross.
Backcrosses were used as a tool to normalize the irregular phenotype and to generate phenotypic variability of flowers. Variations in flower morphology associated with CMS could be beneficial to the ornamental plant industry. These variations are based on altered nucleo-mitochondrial interactions and, therefore, are alternatives to those based on nuclear modifications by mutagenesis and genetic transformation. Strictly maternal inheritance of CMS types provides a basis for the production of stable CMS lines with a certain flower morphology, allowing these characteristics to be maintained after backcrosses with a number of desirable genotypes.
Altered morphological characteristics of floral organs, similar to some of those described here, were not only found in alloplasmic tobacco plants but also in transgenic tobacco lines over-expressing nuclear homeotic genes (Mandel et al., 1992). Since nucleo-cytoplasmic interactions were suggested to modulate the expression of homeotic genes (Zubko et al., 2001), more new flower varieties can be expected when using homeotic mutants and transgenics on floral homeotic genes for backcrossing CMS cybrids altered in flower morphology.
Leaf elongation and stem branching are striking features that were heritable after parasexual re-transmission of cytoplasm and backcrossing the repeated cybrids with wild-type tobacco. Mutations of different nuclear genes are known to influence leaf shape implying multiple nuclear control of leaf morphology (Hofer et al., 2001). In our experiments, repeated cybridization followed by backcrosses provided the nuclear background of wild-type tobacco in the cybrids. On this basis, we have shown for the first time that heritable transmission of leaf morphology could be under cytoplasmic control in the tobacco nuclear background. This is the first evidence that leaf morphology and stem branching could be subject to genetic manipulations by direct transmission of a genetically reconstructed cytoplasm via protoplast fusions, and that these traits could be transmitted to the progeny of sexual backcrosses. These two characters do not seem to be linked because in at least one line (Drhn3 x W38-2/25) leaf elongation is manifested alone, independently of stem branching. The association of these phenotypic characteristics with the altered cytoplasm suggests that cytoplasm is involved in specifying some aspects of both leaf and stem morphology. This implies a possible role for cytoplasmic genetic determinants in the evolution of morphogenesis. Restriction patterns of plastid DNA were identical and stably inherited for all the initial cybrids and their progeny tested to date, while the patterns of mtDNA were highly variable (Zubko et al., 1996, 2001). These facts imply that the leaf elongation and stem branching described are probably under the control of rearranged mitochondrial genomes. Previous genetic studies have suggested the involvement of cytoplasm in determining leaf shape in Epilobium (Michaelis, 1954) and Antirrhinum (Bergbusch, 2002). However, these studies did not attempt to attribute the control of leaf morphology to a particular cytoplasmic genome.
Two issues should be taken into account when considering the impact of alloplasmic genes on plant development and metabolism. First, any conclusion based on the correspondence between a particular phenotype and certain alterations in cytoplasmic genetic status formally meets difficulties owing to the presence of other (mitochondrial or plastidic) genetic compartments that might affect the phenotype as a result of organelle–organelle interactions, in addition to nucleo-cytoplasmic interactions (Zubko et al., 2001). Secondly, it is unlikely that the genetic effects of cytoplasmic genes are direct; they are probably mediated metabolically/energetically. Consistent with this hypothesis is the report of decreased ATP : ADP ratios in floral buds, indicating the reduced energy availability in mitochondria of alloplasmic CMS plants with morphological flower alterations (Bergman et al., 2000).
|
ACKNOWLEDGEMENTS |
|---|
We thank Dr S. Misera for his help in this work, Dr D. A. N. Edlin for reading the manuscript and Mrs Heike Ernst for photography. This work was supported by Körber-Stiftung (Hamburg, Germany) and a visiting fellow grant (M.K.Z.) from IPK (Gatersleben).
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
-
Adler K, Kruse J, Kunze G. 1996. Slow speed freezing of chemically unfixed biological tissues and long term storage of frozen samples for cryo-scanning electron microscopy. Microscopy Research and Technique 33: 262–265.[CrossRef][Web of Science][Medline]
Atanassov II, Atanassova SA, Dragoeva AI, Atanassov AI. 1998. A new CMS source in Nicotiana developed via somatic cybridization between N. tabacum and N. alata. Theoretical and Applied Genetics 97: 982–985.[CrossRef]
Aviv D, Bleichman S, Arzee-Gonen P, Galun E. 1984. Intersectional cytoplasmic hybrids in Nicotiana. Theoretical and Applied Genetics 67: 499–504.[CrossRef]
Belliard G, Vedel F, Pelletier G. 1979. Mitochondrial recombination in cytoplasmic hybrids of Nicotiana tabacum by protoplast fusion. Nature 281: 401–403.[CrossRef][Web of Science]
Belliard G, Pelletier G, Vedel F, Quétier F. 1978. Morphological characteristics and chloroplast DNA distribution in different parasexual hybrids of Nicotiana tabacum. Molecular and General Genetics 165: 231–237.[CrossRef]
Bergbusch VL. 2002. Evidence for cytoplasmic inheritance of a developmental organizer affecting growth habit and leaf shape in Antirrhinum majus. Heredity 89: 44–55.[CrossRef][Web of Science][Medline]
Bergman P, Edqvist J, Farbos I, Glimelius K. 2000. Male-sterile tobacco displays abnormal mitochondrial atp1 transcript accumulation and reduced floral ATP/ADP ratio. Plant Molecular Biology 42: 531–544.[CrossRef][Web of Science][Medline]
Davey MR, Kumar A. 1983. Higher plant protoplasts – retrospect and prospect. In: Giles KL, ed. Plant protoplasts. New York: Academic Press, 219–299.
Dragoeva A, Atanassov I, Atanassov A. 1998. CMS due to tapetal failure in cybrids between Nicotiana tabacum and Petunia hybrida. Plant Cell Tissue and Organ Culture 55: 67–70.[CrossRef]
Elkonin LA, Tyrnov VS. 2000. Studying genetic control of cytoplasmic male sterility in plants: state of the problem and current approaches. Russian Journal of Genetics 36: 347–358.
Gerstel DU. 1980. Cytoplasmic male sterility in Nicotiana. Technical Bulletin of the North Carolina Agricultural Research Series 263: 1–31.
Gleba Yu, Sytnik K. 1984. Protoplast fusion: Genetic engineering in higher plants. In: Schoerman R, ed. Monographs on theoretical and applied genetics. Berlin: Springer-Verlag, 115–161.
Hanson MR. 1991. Plant mitochondrial mutations and male sterility. Annual Review of Genetics 25: 461–486.[CrossRef][Web of Science][Medline]
Hofer JMI, Gourlay CW, Ellis THN. 2001. Genetic control of leaf morphology: a partial view. Annals of Botany 88: 1129–1139.
Kaul MLH. 1988. Male sterility in higher plants. In: Frankel R, Grossman M, Maliga P and Riley R, eds. Monographs of Theoretical Applied Genetics 10. Heidelberg: Springer-Verlag, 775–797.
Kofer W, Glimelius K, Bonnett HT. 1991. Modifications of mitochondrial DNA cause changes in floral development in homeotic-like mutants of tobacco. Plant Cell 3: 759–769.
Kumar A, Cocking EC. 1987. Protoplast fusion: a novel approach to organelle genetics in higher plants. American Journal of Botany 74: 1289–1303.[CrossRef]
Kumashiro T, Asahi T, Komari T. 1988. A new source of cytoplasmic male sterile tobacco obtained by fusion between Nicotiana tabacum and X-irradiated N. africana protoplasts. Plant Science 55: 247–254.[CrossRef]
Mandel MA, Bowman JL, Kempin SA, Ma H, Meyerowitz EM, Yanofsky MF. 1992. Manipulation of flower structure in transgenic tobacco. Cell 71: 133–143.[CrossRef][Web of Science][Medline]
Melchers G, Mohri Y, Watanabe K, Wakabayashi S, Harada K. 1992. One-step generation of cytoplasmic male sterility by fusion of mitochondrial-inactivated tomato protoplasts with nuclear-inactiv ated Solanum protoplasts. Proceedings of the National Academy of Sciences of the USA 89: 6832–6836.
Michaelis P. 1954. Cytoplasmic inheritance in Epilobium and its theoretical significance. Advances in Genetics VI: 287–401.
Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473–497.[CrossRef]
Nikova VM, Zagorska NA, Pundeva RS. 1991. Development of four tobacco cytoplasmic male sterile sources using in vitro techniques. Plant Cell, Tissue and Organ Culture 27: 289–295.[CrossRef]
Nikova V, Vladova R, Pundeva R, Shabanov D. 1997. Cytoplasmic male sterility in Nicotiana tabacum L. obtained through interspecific hybridization. Euphytica 94: 375–378.[CrossRef]
Spangenberg G, Pérez Vicente R, Oliveira MM, Osusky M, Nagel J, Pais MS, Potrykus I. 1992. CMS system in Nicotiana: flower development, patterns of mitochondrial DNA and mitochondrial gene expression. Sexual Plant Reproduction 5: 13–26.
Svab Z, Maliga P. 1986. Nicotiana tabacum mutants with chloroplast encoded streptomycin resistance and pigment deficiency. Theoretical and Applied Genetics 72: 637–643.[CrossRef]
Vedel F, Pla M, Vitart V, Gutierres S, Chétrit P, De Paepe R. 1994. Molecular basis of nuclear and cytoplasmic male sterility in higher plants. Plant Physiology and Biochemistry 32: 601–618.
Weiss D. 2000. Regulation of flower pigmentation and growth: multiple signalling pathways control anthocyanin synthesis in expanding petals. Physiologia Plantarum 110: 152–157.[CrossRef]
Wolters AMA, Koornneef M, Gilissen LJW. 1993. The chloroplast and mitochondrial DNA type are correlated with the nuclear composition of somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia. Current Genetics 24: 260–267.[CrossRef][Web of Science][Medline]
Zubko MK, Zubko EI, Gleba YuYu. 2002. Self-fertile cybrids Nicotiana tabacum(+Hyoscyamus aureus) with a nucleo-plastome incompati bility. Theoretical and Applied Genetics 105: 822–828.[CrossRef][Web of Science][Medline]
Zubko MK, Zubko EI, Patskovsky YV, Khvedynich OA, Gleba YY. 1992. New type of CMS in somatic hybrids Nicotiana+Scopolia and Nicotiana+Hyoscyamus reconstructed on cytoplasm. Doclady Akademii Nauk 325: 1062–1066.
Zubko MK, Zubko EI, Patskovsky YuV, Khvedynich OA, Fisahn J, Gleba YuYu, Schieder O. 1996. Novel ‘homeotic’ CMS patterns generated in Nicotiana via cybridization with Hyoscyamus and Scopolia. Journal of Experimental Botany 47: 1101–1110.
Zubko MK, Zubko EI, Ruban AV, Adler K, Mock H-P, Misera S, Gleba YuYu, Grimm B. 2001. Extensive developmental and metabolic alterations in cybrids Nicotiana tabacum(+Hyoscyamus niger) are caused by complex nucleo-cytoplasmic incompatibility. The Plant Journal 26: 1–14.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. R. Hanson and S. Bentolila Interactions of Mitochondrial and Nuclear Genes That Affect Male Gametophyte Development PLANT CELL, June 1, 2004; 16(suppl_1): S154 - S169. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||