- Email: [email protected]
[6] Interspecific transfer of partial nuclear genomic information by protoplast fusion
[6]
PROTOPLASTFUSION
45
70% o f the treated t o b a c c o mesophyll protoplasts at a multiplicity of 3 × 105 R N A molecules per protoplast. D N A - m e d i a t e d transformation o f tobacco mesophyll protoplasts could also be demonstrated using the recombinant plasmid p L G V 2 3 n e o encapsulated into similar liposomes. Transformation frequencies calculated from the n u m b e r o f kanamycin-resistant colonies were of the order of 4 x 10 -5 transformant per viable protoplast. Plants regenerated from these colonies did express the n e o gene as evidenced by the presence of APH(3')II activity and contained several integrated copies of this gene as shown by Southern blot analysis. Acknowledgments This work was supported in part by a grant from the State of Washington High Technology Center (P.F.L.).
[6] I n t e r s p e c i f i c T r a n s f e r o f P a r t i a l N u c l e a r G e n o m i c Information by Protoplast Fusion
By
OTTO
SCHIEDER
The technique o f protoplast fusion offers great potential to somatic cell genetics and crop improvement, because the genetic information of species which cannot be crossed by sexual methods can be combined. It has been shown that not only complete nuclear genomes o f different plant species can be combined by the fusion technique,~-3 but also the cytoplasmic genetic information can be transferred from one species into another one. 4,5 H o w e v e r , the transfer of cytoplasmic genetic information is of limited importance for crop improvement because most of the desirable genetic information is located in the nucleus. A transfer of limited nuclear 1C. T. Harms, Experientia, Suppl. 46, 69 (1983). 2 y. y. Gleba and K. M. Sytnik, "Protoplast Fusion." Springer-Verlag, Berlin and New York, 1984. 30. Schieder and H. Kohn, in "Cell Culture and Somatic Cell Genetics of Plants" (I. K. Vasil, ed.), Vol. 3, p. 569. Academic Press, New York, 1986. 4 E. Galun and D. Aviv, in "Handbook of Plant Cell Culture" (D. A. Evans, W. R. Sharp, P. V. Amirato, and Y. Yamada, eds.), p. 358. Macmillan, New York, 1983. 5 E. Galun and D. Aviv, in "Efficiency in Plant Breeding" (W. Lange, A. C. Teven, and N. G. Hagenboom, eds.), p. 228. Pudoc, Wageningen, 1984. METHODS IN ENZYMOLOGY, VOL. 148
Copyright © 1987 by Academic Press, Inc. All fights of reproduction in any form reserved.
46
CELLS, PROTOPLASTS, AND LIPOSOMES
[6]
genomic information is more important. Recently several transformation systems using cloned genes have been developed.6-9 However, the limitation of this method is the difficulty in isolating and cloning most of the desirable genes, e.g., genes coding for disease resistances. Even greater is the difficulty when the resistant phenotype is polygenically inherited. An alternative method for overcoming this problem might be the uptake of isolated chromosomes in protoplasts ~° or the production of asymmetric somatic hybrids possessing only a part of the nuclear genomic information of one of the two partners combined by protoplast fusion. This chapter will present some details about the production of asymmetric somatic hybrids induced through fusion of heavily irradiated "donor protoplasts" with nonirradiated "recipient protoplasts. ''11-16
Transfer of Nuclear Genomic Controlled Traits
The principal method for the construction of asymmetric somatic hybrid cell lines or plants is the use of protoplasts from a donor plant pretreated with X rays and subsequently fused with the protoplasts of a recipient plant. This principal method has been used successfully also for the transmission of organelle-controlled traits) 7
6 j. Schell, L. Herrera-Estrella, P. Zambryski, M. de Block, H. Joos, L. Willmitzer, P. Eckes, S. Rosahl, and M. Van Montagu, in "The Impact of Gene Transfer Techniques in Eukaryotic Cell Biology" (J. S. Schell and P. Starlinger, eds.), p. 73. Springer-Verlag, Berlin and New York, 1984. 7 j. Paszkowski, R. D. Shillito, M. Saul, V. Mandak, T. Hohn, B. Hohn, and I. Potrykus, EMBO J. 3, 2717 (1984). 8 I. Potrykus, M. W. Saul, J. Petruska, J. Paszkowski, and R. D. Shillito, Mol. Gen. Genet. 199, 183 (1985). 9 H. L6rz, B. Baker, and J. Schell, Mol. Gen. Genet. 199, 178 (1985). 10 D. Dudits and T. Praznovszky, in "Biotechnology in Plant Science: Relevance to Agriculture in the Eighties" (M. Zaitlin, P. Day, and A. Hollaender, eds.), p. 115. Academic Press, Orlando, 1985. n D. Dudits, O. Fej6r, G. Hadlaczky, C. Konc, G. L~izfir, and G. Horvfith, Mol. Gen. Genet. 179, 283 (1980). t2 p. p. Gupta, M. Gupta, and O. Schieder, Mol. Gen. Genet. 188, 378 (1982). 13 p. p. Gupta, O. Schieder, and M. Gupta, Mol. Gen. Genet. 197, 30 (1984). 14 K. Itho and Y. Futsuhara, Jpn. J. Genet. 58, 545 (1983). 15 O. Schieder, P. P. Gupta, G. Krumbiegel-Schroeren, T. Hein, and A. Steffen, in "Plant Tissue and Cell Culture: Application to Crop Improvement" (F. J. Novfik, L. Havel, and J. Dolezel, eds.), p. 371. Czech. Acad. Sci., Prague, 1984. ~60. Schieder, in "Biotechnology in Plant Science: Relevance to Agriculture in the Eighties" (M. Zaitlin, P. Day, and A. Hollaender, eds.), p. 77. Academic Press, Orlando, 1985. 17 A. Zelcer, D. Aviv, and E. Galun, Z. Pflanzenphysiol. 911, 397 (1978).
[6]
PROTOPLASTFUSION
47
Protoplast Source So far only a few experiments have been undertaken to transfer nuclear genomic traits from a donor into a recipient plant. In most of the experiments nuclear coded metabolic deficiencies have been used in order to correct them through a transfer via fusion with irradiated donor protoplasts. 11-13Protoplasts can be isolated from both the donor and the recipient either from mesophyll or from cell suspensions. A prerequisite is that at least the recipient protoplasts can be regenerated to callus or to plants. The isolation and preparation of the protoplasts can be undertaken as described in detail for Datura.18
Mitotic Inactivation of Donor Protoplasts In general, X rays have been used for the mitotic inactivation of the donor protoplasts. The principle is the fragmentation of the nuclear genome to avoid the fusion of complete genomes of both the donor and the recipient. Irradiation with y-rays might also be useful but this technique has not yet been used. Inactivation with chemicals such as iodoacetate, that are useful in transfer experiments of organelle genomes, 19might not be advantageous because with this treatment no fragmentation of the nuclear genome can be achieved. After isolation and preparation of the donor protoplasts they have to be suspended in 0.6 M mannitol and exposed to X rays in a shallow layer. The dose rate for total mitotic inactivation of the protoplasts can differ depending on the species used as donor. For Datura innoxia, for example, the application of 5 krad of X rays leads to a total mitotic inactivation of its mesophyll protoplasts, 12 whereas the mesophyll protoplasts of Physalis minima and the cell suspension protoplasts of Nicotiana paniculata require for total inactivation at least 15 krad. T M It is essential that before starting an experiment the dose rate necessary for a total mitotic inactivation of the donor protoplasts has been determined. Differences in sensitivity against irradiation in different plant species are well known.
Fusion of Protoplasts The fusion procedure of protoplasts can be carried out by chemical treatment, as in regular somatic hybridization experiments where symmetric somatic hybrids have been produced.~-3 However, the new fusion 18 O. Schieder, in "Cell Culture and Somatic Cell Genetics of Plants" (I. K. Vasil, ed.), Vol. 1, p. 350. Academic Press, New York, 1984. J9 V. A. Sidorov, L. Menczel, F. Nagy, and P. Maliga, Planta 152, 341 (1981).
48
CELLS, PROTOPLASTS, AND LIPOSOMES
[6]
technique using electrical pulse treatment may also be of practical value in future asymmetric hybridization experiments. 2°,21
Culture of Protoplasts After fusion the protoplasts have to be resuspended in a protoplast regeneration medium suitable for the respective recipient plant species. Numerous regeneration media have already been dev610ped and detailed descriptions for several plant species have been published. 22
Selection Procedures As already mentioned, in most of the experiments undertaken so far, metabolic deficiencies have been used in order to correct them via interspecific protoplast fusion. Selection of the asymmetric somatic hybrids was based on their wild-type phenotype. In two experiments the nuclearcoded albinism was corrected by fusion of wild-type protoplasts inactivated with X rays. The albinism in a carrot mutant could be corrected after fusion with inactivated wild-type protoplasts of petrosilium.11 A similar experiment using an albino mutant of Datura innoxia as the recipient and wild-type of Physalis minima as the donor was carded out by Gupta et al.13 Obviously, the selection was based on the green color of the asymmetric somatic hybrids. In other experiments the nitrate reductase deficiency in tobacco mutants was partially corrected after fusion with inactivated donor protoplasts of either Physalis minima and Datura innoxia or Hyoscyamus muticus. Selection here was based on the ability of the asymmetric somatic hybrids to develop on a medium containing only NO3- ions as the sole nitrogen source. 12,13Itho 14 fused X-ray-inactivated protoplasts of Nicotiana langsdorffii with protoplasts of Nicotiana glauca. Selection of asymmetric somatic hybrids is based on their hormone-independent growth behavior, a phenomenon well known in sexual and symmetric hybrids of these two species. When no markers are available which can serve for the selection of asymmetric somatic hybrids the t-DNA ofAgrobacterium tumefaciens can be incorporated into the donor plant material. 6 Selection of asymmetric somatic hybrids is then based on their hormone-independent growth behavior. With such a method asymmetric somatic hybrid cell lines have been produced between the nitrate reductase-deficient cell line cnx-68 of tobacco as the recipient and a tumorous cell line of Nicotiana paniculata.16 In these experiments besides 20 U. Zimmermann and P. Scheurich, Planta 151, 26 (1981). 21 H. Kohn, R. Schieder, and O. Schieder, Plant Sci. 38, 121 (1985).
22I. K. Vasil, ed., "Cell Culture and Somatic Cell Genetics of Plants," Vol. 1. Academic Press, New York, 1984.
[6]
PROTOPLASTFUSION
49
the T - D N A other nuclear genomic D N A of the donor was simultaneously transferred by this method. 23,24 F o r transformation of the donor plant material with A g r o b a c t e r i u m tumefaciens leaf disks or stem segments of aseptic grown shoots have to be incubated with 100 /~1 of the late log phase o f the bacteria suspension at 27o. 25 After 2 days of incubation wash the leaf disks or stem segments with the respective protoplast regeneration medium containing 0.05% of the antibiotic carbenicillin to avoid overgrowing the bacteria. Since tumors developing after incubation with A g r o b a c t e r i u m normally are mixed up with untransformed cells, a leaf disk or stem segment cloning 25 immediately after incubation with the bacteria via protoplasts is necessary for getting cell lines from which all the cells contain the T-DNA. F o r such a cloning, protoplast isolation, preparation, and culture can be carried out as described elsewhere. 18,22 H o w e v e r , the protoplast regeneration media must also contain carbenicillin (0.025%). 25 When the protoplasts have developed to macroscopic cell colonies transfer them onto agar medium lacking p h y t o h o r m o n e s but still containing carbenicillin (0.025%). Growing colonies can be picked and used for the establishment of a cell suspension 26 which can serve as the source for the d o n o r protoplasts. Mitotic inactivation of the donor protoplasts with X rays, their fusion with the recipient protoplasts, and culture can be carried out as already described. The selection of the asymmetric somatic hybrid cell lines can be undertaken on hormone-free agar media when the protoplasts have developed into visible colonies. ~6 H o w e v e r , the usage o f wild-type T - D N A o f Agrobacterium tumefaciens for the selection has the disadvantage that only tumorous asymmetric somatic cell lines can be obtained. F o r obtaining nontumorous asymmetric somatic hybrid cell lines and finally plants it has been proposed using TD N A constructions which are disarmed from all the tumor genes but contain the genetic information coding for the neomycin phosphotransferase (strain pGLVne02103). This would lead to cells resistant to neomycin or kanamycin. 27,28Figure 1 indicates how such a T - D N A construction can perhaps be used for asymmetric hybridization experiments where z~E. M~ller-Gensert and O. Schieder, in "Genetic Manipulation in Plant Breeding" (W. Horn, C. J. Jensen, W. Odenbach, and O. Schieder, eds.), p. 697. de Gruyter, Berlin, 1986. 24E. Mfiller-Gensert and O. Schieder, Mol. Gen. Genet., in press (1987). z5A. Steffen and O. Schieder, Theor. Appl. Genet. 72, 135 (1986). 26p. King, in "Cell Culture and Somatic Cell Genetics of Plants" (I. K. Vasil, ed.), Vol. I, p. 130. Academic Press, New York, 1984. 27O. Schieder, T. Hein, and H. Kohn, in "Genetic Manipulation in Plant Breeding" (W. Horn, C. J. Jensen, W. Odenbach, and O. Schieder, eds.), p. 641. de Gruyter, Berlin, 1986. R. Hain, P. Stabel, A. P. Czernilofsky, H. H. Steinbiss, L. Herrera-Estrella, and J. Schell, Mol. Gen. Genet. 199, 161 (1985).
50
CELLS, PROTOPLASTS,AND LIPOSOMES
[6]
Recipient plant without disease resistance
Donor plant with disease resistance
I.
Transformatlon with pGLVneo2103 Protoplasts Inacti with X
!
Protoplasts ~
~
~
Fusion
Culture
l 1 1
Selection of colonies resistant to kanamycin
Regeneration of selected colonies to plants
Selection of disease resistant plants FxG. 1. A scheme indicting how the s ~ n pGLVneo2103 of Agrobacterium tumefacwns, disarmed from all the tumor genes, can be used ~ r a simultaneous gans~r of nucle~ genomic information coding ~ r disease resistance. nuclear g e n o m i c information for disease resistances will be transferred f r o m a d o n o r into a recipient plant simultaneously with the T - D N A . Verification of Somatic A s y m m e t r y It was concluded in e x p e r i m e n t s to correct nitrate reductase deficiency in t o b a c c o through X-ray-inactivated wild-type protoplasts o f Phy-
[6]
PROTOPLAST FUSION
51
salis minima and Datura innoxia that a transfer of nuclear genomic DNA of the donor is responsible 12for this correction. No direct proof exists for this predication. Famelaer et al. 29 treated nitrate reductase-deficient protoplasts of Nicotiana plumbaginifolia with the total DNA of the wild type and the mutant itself and recovered corrected phenotypes from both treatments. They concluded that the mutagenic effects of the donor DNA is the most plausible mechanism. This mutagenic effect may also occur after fusion with inactivated donor protoplasts. Cytological observations that demonstrate the presence of only a few chromosomes of the donor in the selected asymmetric somatic hybrids 13 are better. The study of the electrophoretic pattern of proteins (isoenzymes) in the selected presumed asymmetric somatic hybrids in comparison to the recipient and the donor plant material can serve as an indication of the transfer of nuclear genomic information. 1~-14,3°However, the most convincing method for demonstrating an incomplete transfer of nuclear genomic DNA of the donor into a recipient is the usage of repetitive and species-specific nuclear DNA sequences of the donor in combination with Southern blotting) l This method, which worked for identifying symmetric somatic hybrids between Hyoscyamus muticus and Nicotiana tabacum, 32 is also very useful for confirming asymmetric somatic hybridity.Z3,24, 33
For isolation and cloning of repetitive and species-specific nuclear DNA sequences follow the method of Saul and Potrykus. 32 Total DNA of the donor and the recipient plant material as well as that of the presumed asymmetric somatic hybrids can be prepared either according to Bedbrook 34 or to Murray and Thompson. 35 After Southern blotting the differences in the hybridization strength between the restricted total DNA of the donor and the restricted total DNA of the presumed asymmetric somatic hybrids can be used to estimate the amount of transferred donor nuclear D N A . 23,33
29 y . Famelaer, M. Heindrickx, I. Negrutiu, and M. Jacobs, in "Genetic Engineering of Plants and Microorganisms Important for Agriculture" (E. Magnien and D. de Nettancourt, eds.), p. 172. Martinus Nijhoff/Dr. W. Junk Publishers, Dordrecht, Netherlands, 1985. 30 L. R. Wetter, in "Cell Culture and Somatic Cell Genetics of Plants" (I. K. Vasil, ed.), Vol. 1, p. 651. Academic Press, New York, 1984. 31 E. M. Southern, J. Mol. Biol. 98, 503 0975). 32 W. Saul and I. Potrykus, Plant Cell Rep. 3, 65 (1984). 33 j. Imamura, "Annual Report," p. 51. Friedrich-Miescher-Institute, Basel, 1984. 34 j. Bedbrook, Plant Mol. Biol. Newsl. 2, 24 (1981). 35 H. G. Murray and W. F. Thompson, Nucleic Acids Res. 8, 4321 (1980).
PROTOPLASTFUSION
45
70% o f the treated t o b a c c o mesophyll protoplasts at a multiplicity of 3 × 105 R N A molecules per protoplast. D N A - m e d i a t e d transformation o f tobacco mesophyll protoplasts could also be demonstrated using the recombinant plasmid p L G V 2 3 n e o encapsulated into similar liposomes. Transformation frequencies calculated from the n u m b e r o f kanamycin-resistant colonies were of the order of 4 x 10 -5 transformant per viable protoplast. Plants regenerated from these colonies did express the n e o gene as evidenced by the presence of APH(3')II activity and contained several integrated copies of this gene as shown by Southern blot analysis. Acknowledgments This work was supported in part by a grant from the State of Washington High Technology Center (P.F.L.).
[6] I n t e r s p e c i f i c T r a n s f e r o f P a r t i a l N u c l e a r G e n o m i c Information by Protoplast Fusion
By
OTTO
SCHIEDER
The technique o f protoplast fusion offers great potential to somatic cell genetics and crop improvement, because the genetic information of species which cannot be crossed by sexual methods can be combined. It has been shown that not only complete nuclear genomes o f different plant species can be combined by the fusion technique,~-3 but also the cytoplasmic genetic information can be transferred from one species into another one. 4,5 H o w e v e r , the transfer of cytoplasmic genetic information is of limited importance for crop improvement because most of the desirable genetic information is located in the nucleus. A transfer of limited nuclear 1C. T. Harms, Experientia, Suppl. 46, 69 (1983). 2 y. y. Gleba and K. M. Sytnik, "Protoplast Fusion." Springer-Verlag, Berlin and New York, 1984. 30. Schieder and H. Kohn, in "Cell Culture and Somatic Cell Genetics of Plants" (I. K. Vasil, ed.), Vol. 3, p. 569. Academic Press, New York, 1986. 4 E. Galun and D. Aviv, in "Handbook of Plant Cell Culture" (D. A. Evans, W. R. Sharp, P. V. Amirato, and Y. Yamada, eds.), p. 358. Macmillan, New York, 1983. 5 E. Galun and D. Aviv, in "Efficiency in Plant Breeding" (W. Lange, A. C. Teven, and N. G. Hagenboom, eds.), p. 228. Pudoc, Wageningen, 1984. METHODS IN ENZYMOLOGY, VOL. 148
Copyright © 1987 by Academic Press, Inc. All fights of reproduction in any form reserved.
46
CELLS, PROTOPLASTS, AND LIPOSOMES
[6]
genomic information is more important. Recently several transformation systems using cloned genes have been developed.6-9 However, the limitation of this method is the difficulty in isolating and cloning most of the desirable genes, e.g., genes coding for disease resistances. Even greater is the difficulty when the resistant phenotype is polygenically inherited. An alternative method for overcoming this problem might be the uptake of isolated chromosomes in protoplasts ~° or the production of asymmetric somatic hybrids possessing only a part of the nuclear genomic information of one of the two partners combined by protoplast fusion. This chapter will present some details about the production of asymmetric somatic hybrids induced through fusion of heavily irradiated "donor protoplasts" with nonirradiated "recipient protoplasts. ''11-16
Transfer of Nuclear Genomic Controlled Traits
The principal method for the construction of asymmetric somatic hybrid cell lines or plants is the use of protoplasts from a donor plant pretreated with X rays and subsequently fused with the protoplasts of a recipient plant. This principal method has been used successfully also for the transmission of organelle-controlled traits) 7
6 j. Schell, L. Herrera-Estrella, P. Zambryski, M. de Block, H. Joos, L. Willmitzer, P. Eckes, S. Rosahl, and M. Van Montagu, in "The Impact of Gene Transfer Techniques in Eukaryotic Cell Biology" (J. S. Schell and P. Starlinger, eds.), p. 73. Springer-Verlag, Berlin and New York, 1984. 7 j. Paszkowski, R. D. Shillito, M. Saul, V. Mandak, T. Hohn, B. Hohn, and I. Potrykus, EMBO J. 3, 2717 (1984). 8 I. Potrykus, M. W. Saul, J. Petruska, J. Paszkowski, and R. D. Shillito, Mol. Gen. Genet. 199, 183 (1985). 9 H. L6rz, B. Baker, and J. Schell, Mol. Gen. Genet. 199, 178 (1985). 10 D. Dudits and T. Praznovszky, in "Biotechnology in Plant Science: Relevance to Agriculture in the Eighties" (M. Zaitlin, P. Day, and A. Hollaender, eds.), p. 115. Academic Press, Orlando, 1985. n D. Dudits, O. Fej6r, G. Hadlaczky, C. Konc, G. L~izfir, and G. Horvfith, Mol. Gen. Genet. 179, 283 (1980). t2 p. p. Gupta, M. Gupta, and O. Schieder, Mol. Gen. Genet. 188, 378 (1982). 13 p. p. Gupta, O. Schieder, and M. Gupta, Mol. Gen. Genet. 197, 30 (1984). 14 K. Itho and Y. Futsuhara, Jpn. J. Genet. 58, 545 (1983). 15 O. Schieder, P. P. Gupta, G. Krumbiegel-Schroeren, T. Hein, and A. Steffen, in "Plant Tissue and Cell Culture: Application to Crop Improvement" (F. J. Novfik, L. Havel, and J. Dolezel, eds.), p. 371. Czech. Acad. Sci., Prague, 1984. ~60. Schieder, in "Biotechnology in Plant Science: Relevance to Agriculture in the Eighties" (M. Zaitlin, P. Day, and A. Hollaender, eds.), p. 77. Academic Press, Orlando, 1985. 17 A. Zelcer, D. Aviv, and E. Galun, Z. Pflanzenphysiol. 911, 397 (1978).
[6]
PROTOPLASTFUSION
47
Protoplast Source So far only a few experiments have been undertaken to transfer nuclear genomic traits from a donor into a recipient plant. In most of the experiments nuclear coded metabolic deficiencies have been used in order to correct them through a transfer via fusion with irradiated donor protoplasts. 11-13Protoplasts can be isolated from both the donor and the recipient either from mesophyll or from cell suspensions. A prerequisite is that at least the recipient protoplasts can be regenerated to callus or to plants. The isolation and preparation of the protoplasts can be undertaken as described in detail for Datura.18
Mitotic Inactivation of Donor Protoplasts In general, X rays have been used for the mitotic inactivation of the donor protoplasts. The principle is the fragmentation of the nuclear genome to avoid the fusion of complete genomes of both the donor and the recipient. Irradiation with y-rays might also be useful but this technique has not yet been used. Inactivation with chemicals such as iodoacetate, that are useful in transfer experiments of organelle genomes, 19might not be advantageous because with this treatment no fragmentation of the nuclear genome can be achieved. After isolation and preparation of the donor protoplasts they have to be suspended in 0.6 M mannitol and exposed to X rays in a shallow layer. The dose rate for total mitotic inactivation of the protoplasts can differ depending on the species used as donor. For Datura innoxia, for example, the application of 5 krad of X rays leads to a total mitotic inactivation of its mesophyll protoplasts, 12 whereas the mesophyll protoplasts of Physalis minima and the cell suspension protoplasts of Nicotiana paniculata require for total inactivation at least 15 krad. T M It is essential that before starting an experiment the dose rate necessary for a total mitotic inactivation of the donor protoplasts has been determined. Differences in sensitivity against irradiation in different plant species are well known.
Fusion of Protoplasts The fusion procedure of protoplasts can be carried out by chemical treatment, as in regular somatic hybridization experiments where symmetric somatic hybrids have been produced.~-3 However, the new fusion 18 O. Schieder, in "Cell Culture and Somatic Cell Genetics of Plants" (I. K. Vasil, ed.), Vol. 1, p. 350. Academic Press, New York, 1984. J9 V. A. Sidorov, L. Menczel, F. Nagy, and P. Maliga, Planta 152, 341 (1981).
48
CELLS, PROTOPLASTS, AND LIPOSOMES
[6]
technique using electrical pulse treatment may also be of practical value in future asymmetric hybridization experiments. 2°,21
Culture of Protoplasts After fusion the protoplasts have to be resuspended in a protoplast regeneration medium suitable for the respective recipient plant species. Numerous regeneration media have already been dev610ped and detailed descriptions for several plant species have been published. 22
Selection Procedures As already mentioned, in most of the experiments undertaken so far, metabolic deficiencies have been used in order to correct them via interspecific protoplast fusion. Selection of the asymmetric somatic hybrids was based on their wild-type phenotype. In two experiments the nuclearcoded albinism was corrected by fusion of wild-type protoplasts inactivated with X rays. The albinism in a carrot mutant could be corrected after fusion with inactivated wild-type protoplasts of petrosilium.11 A similar experiment using an albino mutant of Datura innoxia as the recipient and wild-type of Physalis minima as the donor was carded out by Gupta et al.13 Obviously, the selection was based on the green color of the asymmetric somatic hybrids. In other experiments the nitrate reductase deficiency in tobacco mutants was partially corrected after fusion with inactivated donor protoplasts of either Physalis minima and Datura innoxia or Hyoscyamus muticus. Selection here was based on the ability of the asymmetric somatic hybrids to develop on a medium containing only NO3- ions as the sole nitrogen source. 12,13Itho 14 fused X-ray-inactivated protoplasts of Nicotiana langsdorffii with protoplasts of Nicotiana glauca. Selection of asymmetric somatic hybrids is based on their hormone-independent growth behavior, a phenomenon well known in sexual and symmetric hybrids of these two species. When no markers are available which can serve for the selection of asymmetric somatic hybrids the t-DNA ofAgrobacterium tumefaciens can be incorporated into the donor plant material. 6 Selection of asymmetric somatic hybrids is then based on their hormone-independent growth behavior. With such a method asymmetric somatic hybrid cell lines have been produced between the nitrate reductase-deficient cell line cnx-68 of tobacco as the recipient and a tumorous cell line of Nicotiana paniculata.16 In these experiments besides 20 U. Zimmermann and P. Scheurich, Planta 151, 26 (1981). 21 H. Kohn, R. Schieder, and O. Schieder, Plant Sci. 38, 121 (1985).
22I. K. Vasil, ed., "Cell Culture and Somatic Cell Genetics of Plants," Vol. 1. Academic Press, New York, 1984.
[6]
PROTOPLASTFUSION
49
the T - D N A other nuclear genomic D N A of the donor was simultaneously transferred by this method. 23,24 F o r transformation of the donor plant material with A g r o b a c t e r i u m tumefaciens leaf disks or stem segments of aseptic grown shoots have to be incubated with 100 /~1 of the late log phase o f the bacteria suspension at 27o. 25 After 2 days of incubation wash the leaf disks or stem segments with the respective protoplast regeneration medium containing 0.05% of the antibiotic carbenicillin to avoid overgrowing the bacteria. Since tumors developing after incubation with A g r o b a c t e r i u m normally are mixed up with untransformed cells, a leaf disk or stem segment cloning 25 immediately after incubation with the bacteria via protoplasts is necessary for getting cell lines from which all the cells contain the T-DNA. F o r such a cloning, protoplast isolation, preparation, and culture can be carried out as described elsewhere. 18,22 H o w e v e r , the protoplast regeneration media must also contain carbenicillin (0.025%). 25 When the protoplasts have developed to macroscopic cell colonies transfer them onto agar medium lacking p h y t o h o r m o n e s but still containing carbenicillin (0.025%). Growing colonies can be picked and used for the establishment of a cell suspension 26 which can serve as the source for the d o n o r protoplasts. Mitotic inactivation of the donor protoplasts with X rays, their fusion with the recipient protoplasts, and culture can be carried out as already described. The selection of the asymmetric somatic hybrid cell lines can be undertaken on hormone-free agar media when the protoplasts have developed into visible colonies. ~6 H o w e v e r , the usage o f wild-type T - D N A o f Agrobacterium tumefaciens for the selection has the disadvantage that only tumorous asymmetric somatic cell lines can be obtained. F o r obtaining nontumorous asymmetric somatic hybrid cell lines and finally plants it has been proposed using TD N A constructions which are disarmed from all the tumor genes but contain the genetic information coding for the neomycin phosphotransferase (strain pGLVne02103). This would lead to cells resistant to neomycin or kanamycin. 27,28Figure 1 indicates how such a T - D N A construction can perhaps be used for asymmetric hybridization experiments where z~E. M~ller-Gensert and O. Schieder, in "Genetic Manipulation in Plant Breeding" (W. Horn, C. J. Jensen, W. Odenbach, and O. Schieder, eds.), p. 697. de Gruyter, Berlin, 1986. 24E. Mfiller-Gensert and O. Schieder, Mol. Gen. Genet., in press (1987). z5A. Steffen and O. Schieder, Theor. Appl. Genet. 72, 135 (1986). 26p. King, in "Cell Culture and Somatic Cell Genetics of Plants" (I. K. Vasil, ed.), Vol. I, p. 130. Academic Press, New York, 1984. 27O. Schieder, T. Hein, and H. Kohn, in "Genetic Manipulation in Plant Breeding" (W. Horn, C. J. Jensen, W. Odenbach, and O. Schieder, eds.), p. 641. de Gruyter, Berlin, 1986. R. Hain, P. Stabel, A. P. Czernilofsky, H. H. Steinbiss, L. Herrera-Estrella, and J. Schell, Mol. Gen. Genet. 199, 161 (1985).
50
CELLS, PROTOPLASTS,AND LIPOSOMES
[6]
Recipient plant without disease resistance
Donor plant with disease resistance
I.
Transformatlon with pGLVneo2103 Protoplasts Inacti with X
!
Protoplasts ~
~
~
Fusion
Culture
l 1 1
Selection of colonies resistant to kanamycin
Regeneration of selected colonies to plants
Selection of disease resistant plants FxG. 1. A scheme indicting how the s ~ n pGLVneo2103 of Agrobacterium tumefacwns, disarmed from all the tumor genes, can be used ~ r a simultaneous gans~r of nucle~ genomic information coding ~ r disease resistance. nuclear g e n o m i c information for disease resistances will be transferred f r o m a d o n o r into a recipient plant simultaneously with the T - D N A . Verification of Somatic A s y m m e t r y It was concluded in e x p e r i m e n t s to correct nitrate reductase deficiency in t o b a c c o through X-ray-inactivated wild-type protoplasts o f Phy-
[6]
PROTOPLAST FUSION
51
salis minima and Datura innoxia that a transfer of nuclear genomic DNA of the donor is responsible 12for this correction. No direct proof exists for this predication. Famelaer et al. 29 treated nitrate reductase-deficient protoplasts of Nicotiana plumbaginifolia with the total DNA of the wild type and the mutant itself and recovered corrected phenotypes from both treatments. They concluded that the mutagenic effects of the donor DNA is the most plausible mechanism. This mutagenic effect may also occur after fusion with inactivated donor protoplasts. Cytological observations that demonstrate the presence of only a few chromosomes of the donor in the selected asymmetric somatic hybrids 13 are better. The study of the electrophoretic pattern of proteins (isoenzymes) in the selected presumed asymmetric somatic hybrids in comparison to the recipient and the donor plant material can serve as an indication of the transfer of nuclear genomic information. 1~-14,3°However, the most convincing method for demonstrating an incomplete transfer of nuclear genomic DNA of the donor into a recipient is the usage of repetitive and species-specific nuclear DNA sequences of the donor in combination with Southern blotting) l This method, which worked for identifying symmetric somatic hybrids between Hyoscyamus muticus and Nicotiana tabacum, 32 is also very useful for confirming asymmetric somatic hybridity.Z3,24, 33
For isolation and cloning of repetitive and species-specific nuclear DNA sequences follow the method of Saul and Potrykus. 32 Total DNA of the donor and the recipient plant material as well as that of the presumed asymmetric somatic hybrids can be prepared either according to Bedbrook 34 or to Murray and Thompson. 35 After Southern blotting the differences in the hybridization strength between the restricted total DNA of the donor and the restricted total DNA of the presumed asymmetric somatic hybrids can be used to estimate the amount of transferred donor nuclear D N A . 23,33
29 y . Famelaer, M. Heindrickx, I. Negrutiu, and M. Jacobs, in "Genetic Engineering of Plants and Microorganisms Important for Agriculture" (E. Magnien and D. de Nettancourt, eds.), p. 172. Martinus Nijhoff/Dr. W. Junk Publishers, Dordrecht, Netherlands, 1985. 30 L. R. Wetter, in "Cell Culture and Somatic Cell Genetics of Plants" (I. K. Vasil, ed.), Vol. 1, p. 651. Academic Press, New York, 1984. 31 E. M. Southern, J. Mol. Biol. 98, 503 0975). 32 W. Saul and I. Potrykus, Plant Cell Rep. 3, 65 (1984). 33 j. Imamura, "Annual Report," p. 51. Friedrich-Miescher-Institute, Basel, 1984. 34 j. Bedbrook, Plant Mol. Biol. Newsl. 2, 24 (1981). 35 H. G. Murray and W. F. Thompson, Nucleic Acids Res. 8, 4321 (1980).