Skip to main content
SpringerLink
Log in

The culture of manually isolated heterokaryons ofNicotiana tabacum andNicotiana rustica

  • Published:
Proceedings / Indian Academy of Sciences

Abstract

Protoplasts derived from leaf mesophyll tissue ofNicotiana tabacum were fused with cell suspension-derived protoplasts ofN. rustica. Heterokaryons were isolated using a micromanipulator and were cultured. Nuclear fusion was observed to occur in many of these heterokaryons after culturing them for a few days. Hybrid cell division was also observed. Fluorescein isothiocyanate (FITC) staining ofN. rustica protoplasts prior to fusion did not interfere with subsequent hybrid cell division, and theFITC fluorescence was observed to persist beyond the first division stage of hybrid cells. From a total of thirty heterokaryons which were placed in a nurse culture of protoplasts of albinoPetunia hybrida, thirteen green colonies were subsequently obtained of which six have regenerated somatic hybrid plants. Plants were characterised for their hybrid nature by analysis of vegetative and floral morphology, isoelectric focusing pattern of leaf esterases and Fraction 1 protein. All the six plants are nuclear hybrids. Chloroplast segregation appears to have occurred in these plants, with five having the Fraction 1 protein large subunit ofN. rustica, and one having the large subunit ofN. tabacum. Some of the plants possess sexual fertility.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Ahuja P S, Lu D Y, Cocking E C and Davey M R 1983 An assessment of the cultural capabilities ofTrifolium repens L. (White Clover) andOnobrychis vicifolia Scop. (Sainfoin) mesophyll protoplasts;Plant Cell Rep. 2 269–272

    Article  Google Scholar 

  • Binding H 1975 Reproducibly high plating efficiencies of isolated protoplasts from shoot cultures of tobacco;Physiol. Plant 35 225–227

    Article  Google Scholar 

  • Cammerts D and Jacobs K 1980 A simple electrophoretic procedure for the determination of the polypeptide composition of Fraction 1 Protein;Anal. Biochem. 109 317–320

    Article  Google Scholar 

  • Chen K, Wildman S G and Smith H H 1977 ChloroplastDNA distribution in parasexual hybrids as shown by polypeptide composition of Fraction 1 protein;Proc. Natl. Acad. Sci. (USA) 74 5109–5112

    Article  CAS  Google Scholar 

  • Cocking E C, Davey M-R, Pental D and Power J B 1981 Aspects of plant genetic manipulation;Nature (London) 293 265–270

    Article  CAS  Google Scholar 

  • Constabel F, Dudits D, Gamborg O L and Kao K N 1975 Nuclear fusion in intergeneric heterokaryons: A note;Can J. Bot. 53 2092–2095

    Article  Google Scholar 

  • Davey M R and Kumar A 1983 Higher Plant Protoplasts—Retrospect and Prospect;Int. Rev. Cytol. Suppl 16 219–299

    CAS  Google Scholar 

  • Douglas G C, Keller W A and Setterfield G 1981a Somatic hybridization betweenNicotiana rustica andN. tabacum III. Biochemical, morphological and cytological analysis of somatic hybrids;Can. J. Bot. 59 228–237

    CAS  Google Scholar 

  • Douglas G C, Wetter L R, Keller W A and Setterfield G 1981b Somatic hybridization betweenNicotiana rustica andN. tabacum IV. Analysis of nuclear and chloroplast genome expression in somatic hybrids;Can. J. Bot. 59 1509–1513

    Article  CAS  Google Scholar 

  • Dudits D, Kao K N, Constabel F and Gamborg O L 1976 Fusion of carrot and barley protoplasts and division of heterokaryocytes;Can. J. Genet. Cytol. 18 263–269

    Google Scholar 

  • Evans D A, Flick C E and Jensen R A 1981 Disease resistance: Incorporation into sexually incompatible somatic hybrids of the genusNicotiana;Science 213 907–909

    Article  PubMed  Google Scholar 

  • Evans D A 1983 Agricultural applications of plant protoplast fusion;Biotechnology 1 253–261

    Article  Google Scholar 

  • Fowke L C, Bech-Hansen C W, Gamborg O L and Constabel F 1975 Electron-microscope observations of mitosis and cytokinesis in multinucleate protoplasts of soybean;J. Cell Sci. 18 491–507

    PubMed  CAS  Google Scholar 

  • Frearson E M, Power J B, and Cocking E C 1973 The isolation, culture and regeneration ofPetunia leaf protoplasts;Dev. Biol. 33 130–137

    Article  PubMed  CAS  Google Scholar 

  • Gleba Y Y and Hoffman F 1978 Hybrid cell linesArabidopsis thaliana +Brassica campestris. No evidence for specific chromosome elimination;Mol. Gen. Genet. 165 257–264

    Article  CAS  Google Scholar 

  • Gleba Y Y, Memot V P, Cherep N N and Sharzynshaya M V 1982 Inter-tribal hybrid cell lines ofAtropa belladona (×)Nicotiana chinensis obtained by cloning individual protoplast fusion products;Theor. Appl. Genet. 62 75–79

    Google Scholar 

  • Hamill J D 1983Studies on somatic hybridization in the genus Nicotiana. Ph.D. Thesis; University of Nottingham, Nottingham NG7 2RD, England

    Google Scholar 

  • Harms C T 1983 Somatic incompatibility in the development of higher plant somatic hybrids;Q. Rev. Biol. 58 325–353

    Article  Google Scholar 

  • Harris H 1974Nucleus and cytoplasm (Oxford: Clarendon Press)

    Google Scholar 

  • Hein T, Przewózyny T and Schieder O 1983 Culture and selection of somatic hybrids using an auxotrophic cell line;Theor. Appl. Genet. 64 119–122

    Article  Google Scholar 

  • Iwai S, Nago T, Nakata K, Kawashima N and Matsukyma S 1980 Expression of nuclear and chloroplastic genes coding for Fraction 1 protein in somatic hybrids ofNicotiana tabacum + N. rustica;Planta 147 414–417

    Article  CAS  Google Scholar 

  • Jensen W A 1964 Observations on the fusion of nuclei in plants;J. Cell. Biol. 23 669–672

    Article  PubMed  CAS  Google Scholar 

  • Jinks J L, Caligari P D S and Ingram N R 1981 Gene transfer inNicotiana rustica using irradiated pollen;Nature (London) 291 586–588

    Article  Google Scholar 

  • Kao K N 1975 A nuclear staining method for plant protoplasts. inPlant tissue culture methods (eds) O L Gamborg and L R Wetter (Ottawa: NRC Publications)

    Google Scholar 

  • Kao K N 1977 Chromosomal behaviour in somatic hybrids of soybean—Nicotiana glauca;Mol. Gen. Gen. 150 225–230

    Article  Google Scholar 

  • Keller W A and Melchers G 1973 The effect of high pH and calcium on tobacco leaf protoplast fusion;Z. Naturforsch. 28C 737–741

    Google Scholar 

  • Lowry O H, Rosebrough N J, Farr A L and Randall R J 1951 Protein measurement with the folin phenol reagent;J. Biol. Chem. 193 265–275

    PubMed  CAS  Google Scholar 

  • Lu D Y, Pental D and Cocking E C 1982 Plant regeneration from seedling cotyledon protoplasts;Z. Pflanzenphysiol. 107 59–63

    Google Scholar 

  • Menczel L, Lázár and Maliga P 1978 Isolation of somatic hybrids by cloningNicotiana heterokaryons in nurse cultures;Planta 143 29–32

    Article  Google Scholar 

  • Musashige T and Skoog F A 1962 A revised medium for rapid growth and bio-assay with tobacco tissue cultures;Physiol. Plant. 15 473–497

    Article  Google Scholar 

  • Patnaik G, Wilson D and Cocking E C 1981 Importance of enzyme purification from single protoplasts ofPetunia parodii;Z. Pflanzenphysiol. 102 199–205

    CAS  Google Scholar 

  • Patnaik G, Cocking E C, Hamill J and Pental D 1982 A simple procedure for the manual isolation and identification of plant heterokaryons;Plant Sci. Lett. 24 105–110

    Article  Google Scholar 

  • Pental D, Cooper-Bland S, Harding K, Cocking E C and Müller A J 1982 Cultural studies on nitrate reductase deficientNicotiana tabacum mutant protoplasts;Z. Pflanzenphysiol. 105 219–227

    CAS  Google Scholar 

  • Redenbaugh K, Ruzin S, Bartholomew J and Bassham J A 1982 Characterization and separation of plant protoplasts via flow cytometry and cell sorting;Z. Pflanzenphysiol. 107 65–80

    Google Scholar 

  • Scowcroft W R and Larkin P J 1981 ChloroplastDNA assorts randomly in intraspecific somatic hybrids ofNicotiana debneyii;Theor. Appl. Genet. 60 179–184

    Article  CAS  Google Scholar 

  • Smith H H, Hamill D E, Waver E A and Thompson K H 1970 Multiple molecular forms of peroxidases and esterases amongNicotiana species and amphiploids;J. Hered. 61 203–212

    CAS  Google Scholar 

  • Uchimiya H and Murashige T 1974 Evaluation of parameters in the isolation of viable protoplasts from cultured tobacco cells;Plant Physiol. 54 939–944

    Article  Google Scholar 

  • Vasil I K and Vasil V 1980 Isolation and culture of protoplasts;Int. Rev. Cytol. Suppl. B11 1–19

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, NG7 2RD, Nottingham, England

    J D Hamill, G Patnaik, D Pental & E C Cocking

Authors
  1. J D Hamill
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. G Patnaik
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D Pental
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. E C Cocking
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Since deceased

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hamill, J.D., Patnaik, G., Pental, D. et al. The culture of manually isolated heterokaryons ofNicotiana tabacum andNicotiana rustica . Proc. Indian Acad. Sci. 93, 317–327 (1984). https://doi.org/10.1007/BF03053085

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF03053085

Keywords

Search

Navigation