Planta
Planta (1986) 168:2%35
9 Springer-Verlag 1986
Transplantation of isolated nuclei into plant protoplasts A novel technique for introducing foreign D N A into plant cells
P.K. Saxena 1, M. Mii 1, W.L. Crosby 2, L.C. Fowke 1 and J. King 1 i Department of Biology, University of Saskatchewan, Saskatoon, Sask. S7N 0W0, and 2 Plant Biotechnology Institute, National Research Council, Saskatoon, Sask. S7N 0W9, Canada
Abstract. The uptake of isolated nuclei from Vicia hajastana Grossh. cells into protoplasts of an auxotrophic cell line of Datura innoxia P. Mill. was induced under the influence of polyethylene glycol and Ca 2+ at pH 6.8. The frequency of nuclear uptake varied from 0.8 to 2.3% and that of the recovery of prototrophic clones from 10 -5 to 6" 10 -4. The prototrophic nuclear fusion products following nuclear uptake could be rescued by initial culture of the protoplasts in non-selective conditions and by the subsequent use of feeder cell layers to support the growth of surviving colonies on a selective medium. The presence of Vicia genomic DNA in some prototrophic clones was confirmed by dot-blot hybridization using Datura and Vicia DNA probes. In certain transformed clones, the recovery of prototrophy was accompanied by the restoration of morphogenetic potential. Welldeveloped shoots typical of wild-type Datura could be regenerated employing an appropriate regeneration medium. Key words: Auxotrophy - D a t u r a - DNA transfer - Nuclear transplantation - Protoplast - Prototrophy - Vicia (nuclear transplantation).
Introduction
Isolated plant protoplasts are valuable vehicles for the transfer of desired genetic information across related or relatively unrelated species (for review see Davey and Kumar 1983). The absence of cell walls around protoplasts provides an opportunity for genetic modification by inducing, for instance, Abbrevkttions: MS-Murashige and Skoog (1962); P E G = polyethylene glycol
the uptake of foreign organelle genome in the form of chloroplasts, mitochondria, nuclei and even whole single-cell organisms (for a review, see Fowke and Gamborg 1980). We are currently interested in evaluating the possibility of causing genetic transformation of plant cells by the incorporation of isolated, foreign nuclei. The regulation of various developmental and biochemical traits as a result of the transfer of genetic information by nuclear transplantation is well documented in animal cells (Briggs and King 1952; Kondorosi and Duda 1980). However, the success with plant systems has been very limited. In the first attempt at nuclear transplantation in higher plants, Potrykus and Hoffmann (1973) induced the uptake of isolated nuclei from Petunia hybrida into the protoplasts of several species by employing alternating-layer centrifugation. L6rz and Potrykus (1978) improved the procedure by using polyethylene glycol (PEG) and Ca 2 § to accomplish nuclear uptake and obtained higher uptake frequencies. However, although the experiment was well designed, based on the genetic complementation of recessive nuclear mutants of Nicotiana tabacum, proof of the transfer and biological expression of the transferred genome was not obtained. Complementation of the same mutants was, however, possible through protoplast fusion (L6rz and Potrykus 1978). In this communication, we report the successful transplantation of isolated nuclei into plant protoplasts and provide evidence of the transfer and function of a foreign genome following nuclear uptake. The selection system used to detect transformation was based on the correction of auxotrophy in a pantothenate-requiring cell line (Pnl) of Datura innoxia P. Mill. (Solanaceae) using nuclei isolated from a legume, Vicia hajastana Grossh. The
30
P.K. Saxena et al. : Transplantation nuclei - protoplasts: Introduction of D N A
auxotroph is blocked between o~-ketoisovalerate and pantoic acid in the metabolic pathway leading to coenzyme A (King et al. 1980). Pnl has an absolute requirement for pantothenate for growth (Savage et al. 1979) and has never produced revertants although tested for reversion at 14-d-intervals over the seven years since the auxotroph was first isolated. These properties make Pnl a very suitable experimental material for genetic transformation.
5 m M CaC12"2H20) and centrifuged at 120.g for 5 rain. The pellet was suspended on top of a 20% sucrose solution, centrifuged at 120.g for 5 min, and the floating protoplasts collected with a Pasteur pipette. Nuclei not adhering to the protoplasts together with the protoplasts broken by the Ca 2+-PEG treatment were pelleted. The purified protoplasts were again washed with the Ca-mannitol solution and subsequently cultured in 35-mm-diameter Petri dishes, at a density of 105 per ml, in MS liquid medium (2 ml) enriched with 0.5 rag-1-1 Ca-D-pantothenate, 0.2 M mannitol and 3% sucrose. The culture dishes were incubated in darkness at 25 + 1~ C for one week and later transferred to continuous light (32 ~tmol.m 2-s ~, 25_+ 1~ C).
Materials and methods
Rescue ofprototrophic clones. After 4-5 d of culture, the original culture medium was diluted with fresh medium devoid of pantothenate and mannitol. Fifteen-day-old colonies were collected in a centrifuge tube and the culture medium removed with a Pasteur pipette. The colonies were then resuspended in the fresh MS minimal medium, i.e., without pantothenate and mannitot, and transferred to feeder plates prepared as described in Horsch and King (1984). Cells from 3-d-old wild-type Datura innoxia cultures were filtered through a 500-gm Teflon screen (Albert Godde Bedin Sales, Elmsford, N.Y., USA), collected on Miracloth discs, and resuspended in fresh medium to a final density of 0.2 g F W . ml-1. Aliquots, 1 ml each, of this suspension were pipetted onto agar (0.8%, w/v)-solidified MS medium (25 ml) 'dispensed in plastic Petri dishes (10 cm diameter). The cell suspension was then spread over the surface of medium by careful tilting and swirling of the plates. The plates were kept at room temperature (23 + 2 ~ C) for 3-5 h to allow the agar to absorb excess liquid. On top of this cell layer was placed a sterile, snugly-fitting, black filter paper disc (No. 4740-C10; EatonDikeman, Mt. Holly Springs, Pa., USA) and a second disc, 7 cm in diameter, on top of the first. Black filter paper was used for easy observations of white cell colonies against a black background. The feeder plates were used 24-36 h after preparation. The surviving colonies were further transferred, after three to four weeks, to MS agar plates without a feeder layer. Such prototrophic clones were maintained as callus or cell suspension cultures in MS medium.
Cell Cultures. Both Vicia hajastana Grossh. and the auxotrophic cell lines of Datura innoxia P. Mill. were maintained in MS (Murashige and Skoog 1962) liquid medium containing 1 mg.1-1 of 2,4-dichlorophenoxyacetic acid (2,4-D). For P n l , the medium was supplemented with 0.5 mg.1 1 Ca-D-pantothenate. The cells were subcultured at 3-d-intervals by transferring 5 ml of cell suspension to 50 ml of fresh medium. The cell cultures were maintained at 28 ~ in continuous light (10 gmol photons, m - 2 s - 1 f r o m " cool-white" fluorescent-lamps; Westinghouse, Pittsburg, Pa., USA) on a gyratory shaker (Model G-10 rotatory shaker, 2.5 cm eccentricity; New Brunswick Scientific Co., Edison, N.J., USA) at 150 rpm. Isolation ofprotopIasts and nuclei. Protoplasts of P n l were isolated by modifying an earlier method (Mii et al. 1985). Cells were digested for 4 h with an enzyme solution (pH 5.6) containing 1% cellulase Onozuka " R - 1 0 " , 1% cellulase " R S " , 0.5% macerozyme (all from Kinki Yakult Pharmaceutical Industry, Nishinomiya, Japan), 0.5% each of driselase (Sigma Chemical Co., St. Louis, MO., USA) and rhozyme HP-150 (Genencor, New York, N.Y., USA), 5 m M CaC12" 2 H 2 0 and 0.5 M mannitol, at 25 ~ C. Before use, the enzyme solution was centrifuged at 3 000 .g to remove the carriers and then filter-sterilized using a Nalgene filterware (Nalgene Co., Rochester, N.Y., USA). A b o u t 1.5 2 g cells were incubated per 10 ml enzyme solution in a Petri dish (10 cm diameter) placed on a horizontal shaker (50 rpm). The resulting suspensions were passed through an 85-gm screen, centrifuged at 120.g and the protoplast pellet purified by flotation on a 20% sucrose solution (Saxena et al. 1985a). Isolation of Vicia hajastana protoplasts and nuclei was carried out as described in Saxena et al. (1985a, b). Briefly, the purified protoplasts were lysed in pre-sterilized 10 m M 2-(Nmorpholino)ethanesulphonic acid (Mes) buffer (pH 5.2) supplemented with 10 m M each of NaC1 and KC1, 2.5 m M ethylenediaminetetraacetic acid (EDTA), 2.5 m M dithiothreitol (DTT), 0.1 m M spermine, 0.2 M sucrose and 0.02% Triton X-100. The cytoplasmic debris was removed by filtration through two layers of Miracloth (Chicopee Mills, New York, N.Y., USA) followed by polycarbonate filters (Nucleopore, Pleasanton, Cal., USA) of 12- and 10-gm pore size. The nuclei were isolated and maintained at 4 ~ C until used for uptake experiments.
Uptake of nuclei and culture of protoplasts. P n l protoplasts and Vicia nuclei were mixed in at least a 1 : 25 ratio in a total volume of 2.5 ml and spread over the bottom of a 35-ram-diameter Petri dish. To this mixture was added 1 ml Ca a § solution containing 40% (w/v) P E G ( M W 8000) prepared in a solution of 0.1 M 4-(2-hydroxyethyl)-l-piperazine-ethanesulphonic acid (Hepes) buffer (pH 6.8) containing 0.1 M CaC12' 2H20. At the end of the incubation (15-20 min), the mixture was gradually diluted with 12 ml Ca-mannitol solution (0.5 M mannitol with
Chromosome counting. Cells from 3-d-old suspension cultures were transferred to fresh medium supplemented with 0.1% colchicine for 6 h, collected, and treated with 5 N HC1 (0.5 g cells in 10 nil) for 20 rain. Thereafter, the cells were washed three times with distilled water and stained with toluidine blue. The average chromosome number of each of the eight prototrophic clones analysed was determined from chromosome counts of 30 mitotic cells. Dot-blot hybridization. For the preparation of genomic DNA, 10 g (DW) of P n l cells were resuspended in 50 ml of a solution of 25% Sucrose, 50 m M 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)-HC1 (pH 8.0), 50 m M E D T A (pH 8.0), and homogenized three times for 2 s at 0 ~ C using a Brinkman " P o l y t r o n " homogenizer (Kinematica G.m.b.H., Kriens, Luzern, Switzerland) with medium probe_ The homogenate was filtered through three layers of sterile cheesecloth, and the filtrate centrifuged at 17000-g for 30 min at 4 ~ C. The supernatant was discarded and the pellet gently resuspended in 20 ml of a solution containing 50 m M Tris-HCl (pH 8.0), 20 m M E D T A (pH 8,0) and 1% sarkosyl. After 15 min of incubation at 70 ~ C, solid CsC1 was added at 0.9 g per 1 g of lysed mixture, dissolved, and the preparation incubated at 65 ~ C for 10 rain. Flocculant material was centrifuged to the meniscus at 12000-g for 10 rain at 4 ~ C. To the clarified CsC1 suspension, 0.5 ml of ethidium bromide (10 mg. m I - 1) was added per gram of orig-
P.K. Saxena et al. : Transplantation nuclei - protoplasts: Introduction of D N A inal lysate, mixed, and the mixture centrifuged to equilibrium. Genomic D N A was recovered by side puncture and extracted free of ethidium bromide using water-saturated isoamyl alcohol. The D N A was ethanol-precipitated, resuspended to approx. 200p, g.ml ~ in sterile 1 0 m M Tris-HC1 containing 0.1 m M EDTA, and stored frozen. For dot-blot hybridization, 1%tg aliquots of D N A were denatured in 100 gl of 0.5 N N a O H at 65~ for 10 min, cooled on ice, and neutralized by addition of 100 gl of 3 M Na-acetate, pH 5.6, at room temperature. Aliquots of 100 gl each were dot-blotted in triplicate, using a " h y b r i d - d o t " - t y p e apparatus (Bio-Rad Laboratories, Mississauga, Ont., Canada), to nitrocellulose sheets (0.45 p,m-pore size; Schleicher & Schuell, Keene, N.H., USA), prewetted 5 rain each in water and 2 x SSC (sodium chloride + sodium citrate). Filters were air-dried and baked in vacuo for 30 min at 80 ~ C before prehybridization for 4 h at 65~ in a mixture of 5 x SSPE (sodium chloride + sodium phosphate + EDTA), 1% sarkosyl, and 50 g g . l - 1 denatured herring sperm DNA. The solutions of SSC and SSPE were prepared as described in Maniatis et al. (1982). Probes to each genomic D N A were derived by nick translation in the presence of [3zP]dATP to specific activities of about 1.6.10 6 Bq per gg. Filters were probed in 2 ml (1.6-10 s Bq total) of 2 x S S P E , 1% sarkosyl at 65 ~ C for 13 h. Filters were subsequently washed 4 x 15 rain in 250 ml each of 2 x SSPE, 1% sarkosyl at 65 ~ C, and blotted to dampness prior to exposure to K o d a k XAR-5 autoradiography film (Eastman-Kodak, Rochester, N.Y., USA).
Regeneration of shoots. Small calli, 2-4 m m in diameter, rescued as above, were subcultured on MS medium without 2,4-D but supplemented with benzyladenine (1, 2.5, 5 r a g . l - I ) . Shoot buds, which developed in about two weeks, were transferred to MS medium, where they grew into well developed shoots.
Fine chemicals were purchased from Sigma Chemical Co., St. Louis, M., USA, unless otherwise indicated.
Results
Nuclear uptake. Initially, conditions were optimized for efficient nuclear uptake. The use of Ca 2 +-PEG treatment as described in the Materials and methods minimized damage to the protoplasts and produced a 0.8-2.3% uptake frequency with 60-78% viability of the protoplasts after treatment. The quality of the nuclear preparations was an important factor for the agglutination (Fig. 1 A) and subsequent entry (Fig. 1 B) of nuclei into the protoplasts (Fig. 1 C). In our experiments the maximal uptake frequency (2.3%) was achieved only if the nuclear preparations were free of cytoplasmic contamination and nuclear clumping. The method of nuclear isolation (Saxena et al. 1985a, b) employed in this study gives high yields of pure nuclei without any contamination by live protoplasts. Further, isolated nuclei of Vicia were cultured with Pnl protoplasts to check for survival of any intact protoplasts. No cell colony was seen in such cultures. After nuclear uptake, Pnl protoplasts were cultured in nonselective conditions, i.e., in MS medi-
31
um supplemented with pantothenate, to allow initial divisions. The level of pantothenate in the protoplast culture medium was diluted gradually by adding selective medium (1 ml, MS medium without pantothenate) at 3-d-intervals, Plating of twoweek-old protoplast-derived colonies onto the feeder plates resulted in necrosis of all regenerants except for the putative transformed clones (Fig. 1 D). The clones surviving on selection plates (Fig. 1 D) were picked up and maintained on the selective medium (MS medium minus pantothenate). Within a month such prototrophic clones attained a growth rate comparable to that of Pnl on agar-solidified or in liquid medium. Figure 1 E - H shows a comparison of the growth of Pnl and a representative prototrophic clone derived by nuclear transfer under selective and nonselective conditions. The Pnl cells were able to grow on a pantothenate-supplemented medium (Fig. 1 E) but turned black (Fig. 1 F) and underwent necrosis on plain MS medium. By contrast, the prototrophic clone sustained normal growth both in the presence (Fig. 1G) and absence of pantothenate (Fig. i H). Although the Pnl cell line has not reverted since its isolation, to rule out any possibility of reversion as a result of the Ca 2+-PEG treatment, control experiments were carried out at the same time. The controls used included the culture of (i) Pnl protoplasts alone before Ca 2 § treatment and (ii) Pnl protoplasts alone after CaZ+-PEG treatment. None of the control cultures produced a growing cell colony in any experiment.
Transfer and expression of foreign genome. The term "prototrophic clone" (PnV) was used to designate the calli recovered in nuclear transplantation experiments using the protocol described above. The PnV clones tested for the presence of foreign D N A at the molecular level were referred to as transformed (T) clones. So far, seventeen PnV clones have been isolated in seven different experiments (Table 1). All the clones have been maintained in MS minimal medium for approximately two years. In cytological analyses of eight PnV clones, it was not possible to pinpoint a Vicia chromosome with certainty, primarily because of the large number (96-t-11) of chromosomes present in Pnl but also because of the five pairs of Vicia chromosomes, four are indistinguishable from those of Pnl. The average chromosome number of the PnV clones was 94 q- 9. The incorporation of portions of Vicia genome into prototrophic clones was shown by dot-blot
32
P.K. Saxena et al. :Transplantation nuclei- protoplasts: Introduction of D N A
P.K. Saxena et al. : Transplantation nuclei - protoplasts : Introduction of D N A
33
Table 1. Recovery of prototrophic clones by the incorporation of isolated nuclei of Vicia hajastana into the protoplasts of an auxotrophic cell line of Datura innoxia Expt. No.
Ratio of protoplasts and nuclei
No. of acceptor protoplasts
Prototrophic clones recovered
Morphogenetic response"
1 2
1 : 100 1 : 100
6.105 6.105
PnV1 PnV2-10
+ + 0 (6)
+ (2) + + (2) 3 4 5 6
1:30 1:40 1:25 1:50
6-105 4.5-105 1.105 2.106
PnVIl,12 PnV13 PnV14 PnV15,16
7
1:40
7" 105
PnV17
0 (2) + + 0 + (1) o (1) 0
a Morphogenetic potential of the PnV clones is denoted by: 0, pale white; + , clones turning green; + + , clones turning green and producing shoots. In ( ) are the numbers of clones showing the particular morphogenetic response
hybridization. Three prototrophic clones tested in this experiment have been designated as T], T2 and T3 (Fig. 2). Dot-blot analysis was performed using genomic D N A from T1, T2 and T3 and nicktranslated Vicia and Datura (Pnl) total genomic probes. The degree of hybridization between the probes themselves was very low. However, both Vicia and Pnl probes strongly hybridized with genomic D N A of T2 and T3 clones, confirming the presence of Vicia repetitive D N A in a background of Pnl (Datura) sequences (Fig. 2). A relatively weak hybridization of the genomic D N A of T1 with Vicia probe indicated that this clone contained substantial Pnl genome and little Vicia DNA.
Restoration of regenerability. Some of the prototrophic clones, in addition to the elimination of auxotrophy, regained their morphogenetic capacity. Both the parents, Pnl and Vicia hajastana, grow as pale white calli or cell suspension cultures and have lost their organogenetic capacity. However, of the seventeen PnV clones, five turned green and four out of these developed numerous shoots (Table 1) on MS medium supplemented with 2.5 rag.
Fig. 2. Dot-blot hybridization of D N A from three clones with nick-translated Datura (Pnl) and Vicia probes. DP, Datura innoxia ( P n l ) p r o b e ; VP, Vicia hajastana probe; T1, T2, T3, the prototrophic clones (PnV) of Pnl, tested for transfer of Vicia D N A
1-1 benzyladenine (Fig. 1 I). However, the regenerated shoots failed to produce roots on several media which effectively induce rooting of wild-type shoots, e.g., MS medium with 0.1 rag.1-1 indole-3acetic acid. Discussion The Pnl cell line of Datura innoxia used in this investigation is a true auxotrophic mutant with an absolute requirement for pantothenate (Savage et al. 1979). In our experiments, isolated nuclei of Vicia hajastana were used as the source of foreign genetic material to correct the auxotrophy of Pnl protoplasts. The correction of auxotrophy served as a selection system and also provided the biological proof for the expression of the foreign genome in Pnl. The attainment of prototrophy also resulted in restoration of morphogenetic capacity in some of the transformed clones. So far, all the attempts to induce organogenesis in Pnl itself have been unsuccessful. However, the extent of the relationship between the correction of auxotrophy and the ability of the transformed prototrophic clones
Fig. 1 A-I. Selection of prototrophic clones of Datura innoxia following the uptake of isolated nuclei of Vicia hajastana by auxotrophic protoplasts. A Aggregation of nuclei with protoplasts after CaZ+-PEG treatment. B Isolated nucleus (arrow) in the process of entering the protoplast. C The foreign nucleus (arrow) inside the host protoplasts after 2 h of uptake. D Prototrophic clones surviving on the selective feeder plates. Note that both the survivors and the dead auxotrophic protoplasts could be clearly discerned on a black filter paper. E Auxotrophic cells (Pnl) growing on supplemented medium (MS with pantothenate). F Necrosis of Pnl cells on MS minimal medium, i.e., without pantothenate. G Growth of a prototrophic clone on supplemented medium. H Growth of the same clone on minimal medium. I Regeneration of shoots from prototrophic clones on MS medium enriched with benzyladenine (2.5 mg. 1-1)
34
P.K. Saxena et al. : Transplantation nuclei - protoplasts: Introduction of DNA
to turn green and differentiate shoots varied. Only four of the prototrophic clones (PnV) showed chlorophyll synthesis and organogenesis (Table 1). Karyotype analyses of PnV clones was not helpful in identifying Vicia chromosomes(s), if any, transferred following nuclear transplantation. The use of the chromosome-sorting technique (De Laat and Blass 1984) might help ascertain their presence. Relevant in this context is the example of a somatic hybrid between carrot and Aegopodium podagraria (Dudits et al. 1979) which had only chromosomes of carrot but showed selectable markers of A. podagraria. In animal cells, gene transfer by cell fusion or nuclear transplantation has also been shown to occur without the appearance of cytologically detectable whole or parts of chromosomes (Rodgers 1979; Kondorosi and Duda 1980). Dot-blot hybridization of D N A from prototrophic clones with D N A of Datura and Vicia confirmed that foreign nuclear D N A was indeed transferred in our experiments. The observation of a low degree of hybridization between Datura and Vicia D N A facilitated the detection of Vicia D N A in the transformed clones. Sala et al. (1985) have also noted little hybridization between distantlyrelated genomes, namely Nicotiana, Daucus and Oryza. In our further transformation experiments, we plan to use nuclei with defined markers, for example from genetically engineered plants having resistance to kanamycin (Fraley et al. 1984). The initial culture of Pnl protoplasts in the supplemented medium, after nuclear uptake, was found necessary since the minimal medium did not support sustained mitotic divisions and the whole population ultimately died. The rescuing of the putative prototrophic clones required a similar precaution. After the removal of pantothenate, the transfer ofprotoplast-derived colonies to the selective medium was done on feeder plates to prevent the death of survivors because of low density. Like protoplasts, plant cells also fail to survive at a low density and the use of a feeder layer is essential (see Thomas and Davey 1975). The usefulness of feeder plates in the rescuing of transformants, after infection of Petunia protoplasts with Agrobacterium tumefaciens (Fraley et al. 1984), has been recently emphasized. The frequency of transformation in our experiments varied between 6- 10- 4 and 10- 5. However, it should be noted that these values were derived from the initial number of Pnl protoplasts before the Ca2+-PEG treatment. The CaZ+-PEG treatment and the subsequent washing and purification steps preceding the culture of protoplasts resulted
in the loss of approx. 50% of the protoplast population. In conclusion, our experiments demonstrate the feasibility of nuclear mediated-transformation of higher-plant cells and provide a system to study nucleo-cytoplasmic interactions. They also serve to emphasize the usefulness of auxotrophic mutants over many others, such as light-sensitive mutants, for genetic modification experiments, as already pointed out by L6rz and Potrykus (1978). In this respect, the availability of the mutants with definite nuclear markers is highly desirable for an efficient selection in nuclear transfer experiments. Also unanswered are certain basic questions associated with the process of nuclear uptake. For example, the mechanism of entry of the nuclei into protoplasts - whether by endocytosis or direct fusion - is not known. We are carrying out ultrastructural studies to establish the method of uptake of nuclei into protoplasts. We are grateful to Krystyna Czechowicz for excellent technical help. Photographic and typing assistance from Dennis Dyck and Evelyn Peters are acknowledged with thanks.
References
Briggs, R., King, T.J. (1952) Transplantation of living nuctei from blastula cells into enucleated frogs' eggs. Proc. Natl. Acad. Sci. USA 38, 455-463 Davey, M.R., Kumar, A. (1983) Higher plant protoplasts Retrospect and prospect. Int. Rev. Cytot. [Suppl.] 16, 220-300 DeLaat, A.M.M., Blass, J. (1984) Flow-cytometric characterization and sorting of plant chromosomes. Theor. Appl. Genet. 67, 463467 Dudits, D., Hadlaczky, G., Koncz, C., Lfizfir, G., Horvath, G. (1979) Plant regeneration from intergeneric cell hybrids. Plant Sci. Lett. 15, 101-112 Fowke, L.C., Gamborg, O.L. (1980) Application of protoplasts to the study of plant cells. Int. Rev. Cytol. 68, 9 51 Fraley, R.T., Horsch, R.B., Matzke, A., Chilton, M.D., Chilton, W.S., Sanders, P.R. (1984) In vitro transformation of Petunia cells by an improved method of co-cultivation with A. tumefaciens strains. Plant Molec. Biol. 3, 371 378 Horsch, R.B., King, J. (1984) The isolation of auxotrophs from Datura innoxia Mill. cell cultures following recovery of arsenate-treated cells on feeder plates. Planta 160, 168-173 King, J., Horsch, R.B., Savage, A.D. (1980) Partial characterization of two stable auxotrophic cell strains of Datura innoxia Mill. Planta 149, 480-484 Kondorosi, E., Duda, E. (1980) Introduction of foreign genetic material into cultured mammalian cells by liposomes loaded with isolated nuclei. FEBS Lett. 120, 37-40 L6rz, H , Potrykus, I. (1978) Investigations on the transfer of isolated nuclei into plant protoplasts. Theor. Appl. Genet. 53, 251-256 Maniatis, T., Fritsch, E.F., Sambrook, J. (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Publications, New York, N.Y., USA Mii, M., Seeni, S., Fowke, L.C., King, J. (1985) The isolation and cultivation of protoplasts from cell suspensions of a
P.K. Saxena et al. : Transplantation nuclei - protoplasts: Introduction of DNA a pantothenate-requiring auxotroph of Datura. Can. J. Bot. 63, 779-783 Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473-497 Potrykus, I., Hoffmann, F. (1973) Transplantation of nuclei into protoplasts of higher plants. Z. Pflanzenphysiol. 69, 287-289 Rodgers, A. (1979) Detection of small amounts of human DNA in human-rodent hybrids. J. Cell Sci. 38, 391-403 Sala, C., Biasini, M.G., Morandi, C., Nielsen, E., Parisi, B., Sala, F. (1985) Selection and nuclear DNA analysis of cell hybrids between Daucus carota and Oryza sativa. J. Plant Physiol. 118, 409-419
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Savage, A.D., King, J., Gamborg, O.L. (1979) Recovery of a pantothenate auxotroph from a cell suspension culture of Datura innoxia Mill. Plant Sci. Lett. 16, 367-376 Saxena, P.K., Fowke, L.C., King, J. (1985a) An efficient procedure for isolation of nuclei from plant protoplasts. Protoplasma 128, 184-189 Saxena, P.K., Liu, Y., Mii, M., Fowke, L.C., King, J. (1985b) High nuclear yield from protoplasts of several plants. J. Plant Physiol. 121, 193-197 Thomas, E., Davey, M.R. (1975) From single cells to plants. Wykham Publications, New York, N.Y., USA Received 2 January; accepted 14 February 1986
Planta (1986) 168:2%35
9 Springer-Verlag 1986
Transplantation of isolated nuclei into plant protoplasts A novel technique for introducing foreign D N A into plant cells
P.K. Saxena 1, M. Mii 1, W.L. Crosby 2, L.C. Fowke 1 and J. King 1 i Department of Biology, University of Saskatchewan, Saskatoon, Sask. S7N 0W0, and 2 Plant Biotechnology Institute, National Research Council, Saskatoon, Sask. S7N 0W9, Canada
Abstract. The uptake of isolated nuclei from Vicia hajastana Grossh. cells into protoplasts of an auxotrophic cell line of Datura innoxia P. Mill. was induced under the influence of polyethylene glycol and Ca 2+ at pH 6.8. The frequency of nuclear uptake varied from 0.8 to 2.3% and that of the recovery of prototrophic clones from 10 -5 to 6" 10 -4. The prototrophic nuclear fusion products following nuclear uptake could be rescued by initial culture of the protoplasts in non-selective conditions and by the subsequent use of feeder cell layers to support the growth of surviving colonies on a selective medium. The presence of Vicia genomic DNA in some prototrophic clones was confirmed by dot-blot hybridization using Datura and Vicia DNA probes. In certain transformed clones, the recovery of prototrophy was accompanied by the restoration of morphogenetic potential. Welldeveloped shoots typical of wild-type Datura could be regenerated employing an appropriate regeneration medium. Key words: Auxotrophy - D a t u r a - DNA transfer - Nuclear transplantation - Protoplast - Prototrophy - Vicia (nuclear transplantation).
Introduction
Isolated plant protoplasts are valuable vehicles for the transfer of desired genetic information across related or relatively unrelated species (for review see Davey and Kumar 1983). The absence of cell walls around protoplasts provides an opportunity for genetic modification by inducing, for instance, Abbrevkttions: MS-Murashige and Skoog (1962); P E G = polyethylene glycol
the uptake of foreign organelle genome in the form of chloroplasts, mitochondria, nuclei and even whole single-cell organisms (for a review, see Fowke and Gamborg 1980). We are currently interested in evaluating the possibility of causing genetic transformation of plant cells by the incorporation of isolated, foreign nuclei. The regulation of various developmental and biochemical traits as a result of the transfer of genetic information by nuclear transplantation is well documented in animal cells (Briggs and King 1952; Kondorosi and Duda 1980). However, the success with plant systems has been very limited. In the first attempt at nuclear transplantation in higher plants, Potrykus and Hoffmann (1973) induced the uptake of isolated nuclei from Petunia hybrida into the protoplasts of several species by employing alternating-layer centrifugation. L6rz and Potrykus (1978) improved the procedure by using polyethylene glycol (PEG) and Ca 2 § to accomplish nuclear uptake and obtained higher uptake frequencies. However, although the experiment was well designed, based on the genetic complementation of recessive nuclear mutants of Nicotiana tabacum, proof of the transfer and biological expression of the transferred genome was not obtained. Complementation of the same mutants was, however, possible through protoplast fusion (L6rz and Potrykus 1978). In this communication, we report the successful transplantation of isolated nuclei into plant protoplasts and provide evidence of the transfer and function of a foreign genome following nuclear uptake. The selection system used to detect transformation was based on the correction of auxotrophy in a pantothenate-requiring cell line (Pnl) of Datura innoxia P. Mill. (Solanaceae) using nuclei isolated from a legume, Vicia hajastana Grossh. The
30
P.K. Saxena et al. : Transplantation nuclei - protoplasts: Introduction of D N A
auxotroph is blocked between o~-ketoisovalerate and pantoic acid in the metabolic pathway leading to coenzyme A (King et al. 1980). Pnl has an absolute requirement for pantothenate for growth (Savage et al. 1979) and has never produced revertants although tested for reversion at 14-d-intervals over the seven years since the auxotroph was first isolated. These properties make Pnl a very suitable experimental material for genetic transformation.
5 m M CaC12"2H20) and centrifuged at 120.g for 5 rain. The pellet was suspended on top of a 20% sucrose solution, centrifuged at 120.g for 5 min, and the floating protoplasts collected with a Pasteur pipette. Nuclei not adhering to the protoplasts together with the protoplasts broken by the Ca 2+-PEG treatment were pelleted. The purified protoplasts were again washed with the Ca-mannitol solution and subsequently cultured in 35-mm-diameter Petri dishes, at a density of 105 per ml, in MS liquid medium (2 ml) enriched with 0.5 rag-1-1 Ca-D-pantothenate, 0.2 M mannitol and 3% sucrose. The culture dishes were incubated in darkness at 25 + 1~ C for one week and later transferred to continuous light (32 ~tmol.m 2-s ~, 25_+ 1~ C).
Materials and methods
Rescue ofprototrophic clones. After 4-5 d of culture, the original culture medium was diluted with fresh medium devoid of pantothenate and mannitol. Fifteen-day-old colonies were collected in a centrifuge tube and the culture medium removed with a Pasteur pipette. The colonies were then resuspended in the fresh MS minimal medium, i.e., without pantothenate and mannitot, and transferred to feeder plates prepared as described in Horsch and King (1984). Cells from 3-d-old wild-type Datura innoxia cultures were filtered through a 500-gm Teflon screen (Albert Godde Bedin Sales, Elmsford, N.Y., USA), collected on Miracloth discs, and resuspended in fresh medium to a final density of 0.2 g F W . ml-1. Aliquots, 1 ml each, of this suspension were pipetted onto agar (0.8%, w/v)-solidified MS medium (25 ml) 'dispensed in plastic Petri dishes (10 cm diameter). The cell suspension was then spread over the surface of medium by careful tilting and swirling of the plates. The plates were kept at room temperature (23 + 2 ~ C) for 3-5 h to allow the agar to absorb excess liquid. On top of this cell layer was placed a sterile, snugly-fitting, black filter paper disc (No. 4740-C10; EatonDikeman, Mt. Holly Springs, Pa., USA) and a second disc, 7 cm in diameter, on top of the first. Black filter paper was used for easy observations of white cell colonies against a black background. The feeder plates were used 24-36 h after preparation. The surviving colonies were further transferred, after three to four weeks, to MS agar plates without a feeder layer. Such prototrophic clones were maintained as callus or cell suspension cultures in MS medium.
Cell Cultures. Both Vicia hajastana Grossh. and the auxotrophic cell lines of Datura innoxia P. Mill. were maintained in MS (Murashige and Skoog 1962) liquid medium containing 1 mg.1-1 of 2,4-dichlorophenoxyacetic acid (2,4-D). For P n l , the medium was supplemented with 0.5 mg.1 1 Ca-D-pantothenate. The cells were subcultured at 3-d-intervals by transferring 5 ml of cell suspension to 50 ml of fresh medium. The cell cultures were maintained at 28 ~ in continuous light (10 gmol photons, m - 2 s - 1 f r o m " cool-white" fluorescent-lamps; Westinghouse, Pittsburg, Pa., USA) on a gyratory shaker (Model G-10 rotatory shaker, 2.5 cm eccentricity; New Brunswick Scientific Co., Edison, N.J., USA) at 150 rpm. Isolation ofprotopIasts and nuclei. Protoplasts of P n l were isolated by modifying an earlier method (Mii et al. 1985). Cells were digested for 4 h with an enzyme solution (pH 5.6) containing 1% cellulase Onozuka " R - 1 0 " , 1% cellulase " R S " , 0.5% macerozyme (all from Kinki Yakult Pharmaceutical Industry, Nishinomiya, Japan), 0.5% each of driselase (Sigma Chemical Co., St. Louis, MO., USA) and rhozyme HP-150 (Genencor, New York, N.Y., USA), 5 m M CaC12" 2 H 2 0 and 0.5 M mannitol, at 25 ~ C. Before use, the enzyme solution was centrifuged at 3 000 .g to remove the carriers and then filter-sterilized using a Nalgene filterware (Nalgene Co., Rochester, N.Y., USA). A b o u t 1.5 2 g cells were incubated per 10 ml enzyme solution in a Petri dish (10 cm diameter) placed on a horizontal shaker (50 rpm). The resulting suspensions were passed through an 85-gm screen, centrifuged at 120.g and the protoplast pellet purified by flotation on a 20% sucrose solution (Saxena et al. 1985a). Isolation of Vicia hajastana protoplasts and nuclei was carried out as described in Saxena et al. (1985a, b). Briefly, the purified protoplasts were lysed in pre-sterilized 10 m M 2-(Nmorpholino)ethanesulphonic acid (Mes) buffer (pH 5.2) supplemented with 10 m M each of NaC1 and KC1, 2.5 m M ethylenediaminetetraacetic acid (EDTA), 2.5 m M dithiothreitol (DTT), 0.1 m M spermine, 0.2 M sucrose and 0.02% Triton X-100. The cytoplasmic debris was removed by filtration through two layers of Miracloth (Chicopee Mills, New York, N.Y., USA) followed by polycarbonate filters (Nucleopore, Pleasanton, Cal., USA) of 12- and 10-gm pore size. The nuclei were isolated and maintained at 4 ~ C until used for uptake experiments.
Uptake of nuclei and culture of protoplasts. P n l protoplasts and Vicia nuclei were mixed in at least a 1 : 25 ratio in a total volume of 2.5 ml and spread over the bottom of a 35-ram-diameter Petri dish. To this mixture was added 1 ml Ca a § solution containing 40% (w/v) P E G ( M W 8000) prepared in a solution of 0.1 M 4-(2-hydroxyethyl)-l-piperazine-ethanesulphonic acid (Hepes) buffer (pH 6.8) containing 0.1 M CaC12' 2H20. At the end of the incubation (15-20 min), the mixture was gradually diluted with 12 ml Ca-mannitol solution (0.5 M mannitol with
Chromosome counting. Cells from 3-d-old suspension cultures were transferred to fresh medium supplemented with 0.1% colchicine for 6 h, collected, and treated with 5 N HC1 (0.5 g cells in 10 nil) for 20 rain. Thereafter, the cells were washed three times with distilled water and stained with toluidine blue. The average chromosome number of each of the eight prototrophic clones analysed was determined from chromosome counts of 30 mitotic cells. Dot-blot hybridization. For the preparation of genomic DNA, 10 g (DW) of P n l cells were resuspended in 50 ml of a solution of 25% Sucrose, 50 m M 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)-HC1 (pH 8.0), 50 m M E D T A (pH 8.0), and homogenized three times for 2 s at 0 ~ C using a Brinkman " P o l y t r o n " homogenizer (Kinematica G.m.b.H., Kriens, Luzern, Switzerland) with medium probe_ The homogenate was filtered through three layers of sterile cheesecloth, and the filtrate centrifuged at 17000-g for 30 min at 4 ~ C. The supernatant was discarded and the pellet gently resuspended in 20 ml of a solution containing 50 m M Tris-HCl (pH 8.0), 20 m M E D T A (pH 8,0) and 1% sarkosyl. After 15 min of incubation at 70 ~ C, solid CsC1 was added at 0.9 g per 1 g of lysed mixture, dissolved, and the preparation incubated at 65 ~ C for 10 rain. Flocculant material was centrifuged to the meniscus at 12000-g for 10 rain at 4 ~ C. To the clarified CsC1 suspension, 0.5 ml of ethidium bromide (10 mg. m I - 1) was added per gram of orig-
P.K. Saxena et al. : Transplantation nuclei - protoplasts: Introduction of D N A inal lysate, mixed, and the mixture centrifuged to equilibrium. Genomic D N A was recovered by side puncture and extracted free of ethidium bromide using water-saturated isoamyl alcohol. The D N A was ethanol-precipitated, resuspended to approx. 200p, g.ml ~ in sterile 1 0 m M Tris-HC1 containing 0.1 m M EDTA, and stored frozen. For dot-blot hybridization, 1%tg aliquots of D N A were denatured in 100 gl of 0.5 N N a O H at 65~ for 10 min, cooled on ice, and neutralized by addition of 100 gl of 3 M Na-acetate, pH 5.6, at room temperature. Aliquots of 100 gl each were dot-blotted in triplicate, using a " h y b r i d - d o t " - t y p e apparatus (Bio-Rad Laboratories, Mississauga, Ont., Canada), to nitrocellulose sheets (0.45 p,m-pore size; Schleicher & Schuell, Keene, N.H., USA), prewetted 5 rain each in water and 2 x SSC (sodium chloride + sodium citrate). Filters were air-dried and baked in vacuo for 30 min at 80 ~ C before prehybridization for 4 h at 65~ in a mixture of 5 x SSPE (sodium chloride + sodium phosphate + EDTA), 1% sarkosyl, and 50 g g . l - 1 denatured herring sperm DNA. The solutions of SSC and SSPE were prepared as described in Maniatis et al. (1982). Probes to each genomic D N A were derived by nick translation in the presence of [3zP]dATP to specific activities of about 1.6.10 6 Bq per gg. Filters were probed in 2 ml (1.6-10 s Bq total) of 2 x S S P E , 1% sarkosyl at 65 ~ C for 13 h. Filters were subsequently washed 4 x 15 rain in 250 ml each of 2 x SSPE, 1% sarkosyl at 65 ~ C, and blotted to dampness prior to exposure to K o d a k XAR-5 autoradiography film (Eastman-Kodak, Rochester, N.Y., USA).
Regeneration of shoots. Small calli, 2-4 m m in diameter, rescued as above, were subcultured on MS medium without 2,4-D but supplemented with benzyladenine (1, 2.5, 5 r a g . l - I ) . Shoot buds, which developed in about two weeks, were transferred to MS medium, where they grew into well developed shoots.
Fine chemicals were purchased from Sigma Chemical Co., St. Louis, M., USA, unless otherwise indicated.
Results
Nuclear uptake. Initially, conditions were optimized for efficient nuclear uptake. The use of Ca 2 +-PEG treatment as described in the Materials and methods minimized damage to the protoplasts and produced a 0.8-2.3% uptake frequency with 60-78% viability of the protoplasts after treatment. The quality of the nuclear preparations was an important factor for the agglutination (Fig. 1 A) and subsequent entry (Fig. 1 B) of nuclei into the protoplasts (Fig. 1 C). In our experiments the maximal uptake frequency (2.3%) was achieved only if the nuclear preparations were free of cytoplasmic contamination and nuclear clumping. The method of nuclear isolation (Saxena et al. 1985a, b) employed in this study gives high yields of pure nuclei without any contamination by live protoplasts. Further, isolated nuclei of Vicia were cultured with Pnl protoplasts to check for survival of any intact protoplasts. No cell colony was seen in such cultures. After nuclear uptake, Pnl protoplasts were cultured in nonselective conditions, i.e., in MS medi-
31
um supplemented with pantothenate, to allow initial divisions. The level of pantothenate in the protoplast culture medium was diluted gradually by adding selective medium (1 ml, MS medium without pantothenate) at 3-d-intervals, Plating of twoweek-old protoplast-derived colonies onto the feeder plates resulted in necrosis of all regenerants except for the putative transformed clones (Fig. 1 D). The clones surviving on selection plates (Fig. 1 D) were picked up and maintained on the selective medium (MS medium minus pantothenate). Within a month such prototrophic clones attained a growth rate comparable to that of Pnl on agar-solidified or in liquid medium. Figure 1 E - H shows a comparison of the growth of Pnl and a representative prototrophic clone derived by nuclear transfer under selective and nonselective conditions. The Pnl cells were able to grow on a pantothenate-supplemented medium (Fig. 1 E) but turned black (Fig. 1 F) and underwent necrosis on plain MS medium. By contrast, the prototrophic clone sustained normal growth both in the presence (Fig. 1G) and absence of pantothenate (Fig. i H). Although the Pnl cell line has not reverted since its isolation, to rule out any possibility of reversion as a result of the Ca 2+-PEG treatment, control experiments were carried out at the same time. The controls used included the culture of (i) Pnl protoplasts alone before Ca 2 § treatment and (ii) Pnl protoplasts alone after CaZ+-PEG treatment. None of the control cultures produced a growing cell colony in any experiment.
Transfer and expression of foreign genome. The term "prototrophic clone" (PnV) was used to designate the calli recovered in nuclear transplantation experiments using the protocol described above. The PnV clones tested for the presence of foreign D N A at the molecular level were referred to as transformed (T) clones. So far, seventeen PnV clones have been isolated in seven different experiments (Table 1). All the clones have been maintained in MS minimal medium for approximately two years. In cytological analyses of eight PnV clones, it was not possible to pinpoint a Vicia chromosome with certainty, primarily because of the large number (96-t-11) of chromosomes present in Pnl but also because of the five pairs of Vicia chromosomes, four are indistinguishable from those of Pnl. The average chromosome number of the PnV clones was 94 q- 9. The incorporation of portions of Vicia genome into prototrophic clones was shown by dot-blot
32
P.K. Saxena et al. :Transplantation nuclei- protoplasts: Introduction of D N A
P.K. Saxena et al. : Transplantation nuclei - protoplasts : Introduction of D N A
33
Table 1. Recovery of prototrophic clones by the incorporation of isolated nuclei of Vicia hajastana into the protoplasts of an auxotrophic cell line of Datura innoxia Expt. No.
Ratio of protoplasts and nuclei
No. of acceptor protoplasts
Prototrophic clones recovered
Morphogenetic response"
1 2
1 : 100 1 : 100
6.105 6.105
PnV1 PnV2-10
+ + 0 (6)
+ (2) + + (2) 3 4 5 6
1:30 1:40 1:25 1:50
6-105 4.5-105 1.105 2.106
PnVIl,12 PnV13 PnV14 PnV15,16
7
1:40
7" 105
PnV17
0 (2) + + 0 + (1) o (1) 0
a Morphogenetic potential of the PnV clones is denoted by: 0, pale white; + , clones turning green; + + , clones turning green and producing shoots. In ( ) are the numbers of clones showing the particular morphogenetic response
hybridization. Three prototrophic clones tested in this experiment have been designated as T], T2 and T3 (Fig. 2). Dot-blot analysis was performed using genomic D N A from T1, T2 and T3 and nicktranslated Vicia and Datura (Pnl) total genomic probes. The degree of hybridization between the probes themselves was very low. However, both Vicia and Pnl probes strongly hybridized with genomic D N A of T2 and T3 clones, confirming the presence of Vicia repetitive D N A in a background of Pnl (Datura) sequences (Fig. 2). A relatively weak hybridization of the genomic D N A of T1 with Vicia probe indicated that this clone contained substantial Pnl genome and little Vicia DNA.
Restoration of regenerability. Some of the prototrophic clones, in addition to the elimination of auxotrophy, regained their morphogenetic capacity. Both the parents, Pnl and Vicia hajastana, grow as pale white calli or cell suspension cultures and have lost their organogenetic capacity. However, of the seventeen PnV clones, five turned green and four out of these developed numerous shoots (Table 1) on MS medium supplemented with 2.5 rag.
Fig. 2. Dot-blot hybridization of D N A from three clones with nick-translated Datura (Pnl) and Vicia probes. DP, Datura innoxia ( P n l ) p r o b e ; VP, Vicia hajastana probe; T1, T2, T3, the prototrophic clones (PnV) of Pnl, tested for transfer of Vicia D N A
1-1 benzyladenine (Fig. 1 I). However, the regenerated shoots failed to produce roots on several media which effectively induce rooting of wild-type shoots, e.g., MS medium with 0.1 rag.1-1 indole-3acetic acid. Discussion The Pnl cell line of Datura innoxia used in this investigation is a true auxotrophic mutant with an absolute requirement for pantothenate (Savage et al. 1979). In our experiments, isolated nuclei of Vicia hajastana were used as the source of foreign genetic material to correct the auxotrophy of Pnl protoplasts. The correction of auxotrophy served as a selection system and also provided the biological proof for the expression of the foreign genome in Pnl. The attainment of prototrophy also resulted in restoration of morphogenetic capacity in some of the transformed clones. So far, all the attempts to induce organogenesis in Pnl itself have been unsuccessful. However, the extent of the relationship between the correction of auxotrophy and the ability of the transformed prototrophic clones
Fig. 1 A-I. Selection of prototrophic clones of Datura innoxia following the uptake of isolated nuclei of Vicia hajastana by auxotrophic protoplasts. A Aggregation of nuclei with protoplasts after CaZ+-PEG treatment. B Isolated nucleus (arrow) in the process of entering the protoplast. C The foreign nucleus (arrow) inside the host protoplasts after 2 h of uptake. D Prototrophic clones surviving on the selective feeder plates. Note that both the survivors and the dead auxotrophic protoplasts could be clearly discerned on a black filter paper. E Auxotrophic cells (Pnl) growing on supplemented medium (MS with pantothenate). F Necrosis of Pnl cells on MS minimal medium, i.e., without pantothenate. G Growth of a prototrophic clone on supplemented medium. H Growth of the same clone on minimal medium. I Regeneration of shoots from prototrophic clones on MS medium enriched with benzyladenine (2.5 mg. 1-1)
34
P.K. Saxena et al. : Transplantation nuclei - protoplasts: Introduction of DNA
to turn green and differentiate shoots varied. Only four of the prototrophic clones (PnV) showed chlorophyll synthesis and organogenesis (Table 1). Karyotype analyses of PnV clones was not helpful in identifying Vicia chromosomes(s), if any, transferred following nuclear transplantation. The use of the chromosome-sorting technique (De Laat and Blass 1984) might help ascertain their presence. Relevant in this context is the example of a somatic hybrid between carrot and Aegopodium podagraria (Dudits et al. 1979) which had only chromosomes of carrot but showed selectable markers of A. podagraria. In animal cells, gene transfer by cell fusion or nuclear transplantation has also been shown to occur without the appearance of cytologically detectable whole or parts of chromosomes (Rodgers 1979; Kondorosi and Duda 1980). Dot-blot hybridization of D N A from prototrophic clones with D N A of Datura and Vicia confirmed that foreign nuclear D N A was indeed transferred in our experiments. The observation of a low degree of hybridization between Datura and Vicia D N A facilitated the detection of Vicia D N A in the transformed clones. Sala et al. (1985) have also noted little hybridization between distantlyrelated genomes, namely Nicotiana, Daucus and Oryza. In our further transformation experiments, we plan to use nuclei with defined markers, for example from genetically engineered plants having resistance to kanamycin (Fraley et al. 1984). The initial culture of Pnl protoplasts in the supplemented medium, after nuclear uptake, was found necessary since the minimal medium did not support sustained mitotic divisions and the whole population ultimately died. The rescuing of the putative prototrophic clones required a similar precaution. After the removal of pantothenate, the transfer ofprotoplast-derived colonies to the selective medium was done on feeder plates to prevent the death of survivors because of low density. Like protoplasts, plant cells also fail to survive at a low density and the use of a feeder layer is essential (see Thomas and Davey 1975). The usefulness of feeder plates in the rescuing of transformants, after infection of Petunia protoplasts with Agrobacterium tumefaciens (Fraley et al. 1984), has been recently emphasized. The frequency of transformation in our experiments varied between 6- 10- 4 and 10- 5. However, it should be noted that these values were derived from the initial number of Pnl protoplasts before the Ca2+-PEG treatment. The CaZ+-PEG treatment and the subsequent washing and purification steps preceding the culture of protoplasts resulted
in the loss of approx. 50% of the protoplast population. In conclusion, our experiments demonstrate the feasibility of nuclear mediated-transformation of higher-plant cells and provide a system to study nucleo-cytoplasmic interactions. They also serve to emphasize the usefulness of auxotrophic mutants over many others, such as light-sensitive mutants, for genetic modification experiments, as already pointed out by L6rz and Potrykus (1978). In this respect, the availability of the mutants with definite nuclear markers is highly desirable for an efficient selection in nuclear transfer experiments. Also unanswered are certain basic questions associated with the process of nuclear uptake. For example, the mechanism of entry of the nuclei into protoplasts - whether by endocytosis or direct fusion - is not known. We are carrying out ultrastructural studies to establish the method of uptake of nuclei into protoplasts. We are grateful to Krystyna Czechowicz for excellent technical help. Photographic and typing assistance from Dennis Dyck and Evelyn Peters are acknowledged with thanks.
References
Briggs, R., King, T.J. (1952) Transplantation of living nuctei from blastula cells into enucleated frogs' eggs. Proc. Natl. Acad. Sci. USA 38, 455-463 Davey, M.R., Kumar, A. (1983) Higher plant protoplasts Retrospect and prospect. Int. Rev. Cytot. [Suppl.] 16, 220-300 DeLaat, A.M.M., Blass, J. (1984) Flow-cytometric characterization and sorting of plant chromosomes. Theor. Appl. Genet. 67, 463467 Dudits, D., Hadlaczky, G., Koncz, C., Lfizfir, G., Horvath, G. (1979) Plant regeneration from intergeneric cell hybrids. Plant Sci. Lett. 15, 101-112 Fowke, L.C., Gamborg, O.L. (1980) Application of protoplasts to the study of plant cells. Int. Rev. Cytol. 68, 9 51 Fraley, R.T., Horsch, R.B., Matzke, A., Chilton, M.D., Chilton, W.S., Sanders, P.R. (1984) In vitro transformation of Petunia cells by an improved method of co-cultivation with A. tumefaciens strains. Plant Molec. Biol. 3, 371 378 Horsch, R.B., King, J. (1984) The isolation of auxotrophs from Datura innoxia Mill. cell cultures following recovery of arsenate-treated cells on feeder plates. Planta 160, 168-173 King, J., Horsch, R.B., Savage, A.D. (1980) Partial characterization of two stable auxotrophic cell strains of Datura innoxia Mill. Planta 149, 480-484 Kondorosi, E., Duda, E. (1980) Introduction of foreign genetic material into cultured mammalian cells by liposomes loaded with isolated nuclei. FEBS Lett. 120, 37-40 L6rz, H , Potrykus, I. (1978) Investigations on the transfer of isolated nuclei into plant protoplasts. Theor. Appl. Genet. 53, 251-256 Maniatis, T., Fritsch, E.F., Sambrook, J. (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Publications, New York, N.Y., USA Mii, M., Seeni, S., Fowke, L.C., King, J. (1985) The isolation and cultivation of protoplasts from cell suspensions of a
P.K. Saxena et al. : Transplantation nuclei - protoplasts: Introduction of DNA a pantothenate-requiring auxotroph of Datura. Can. J. Bot. 63, 779-783 Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473-497 Potrykus, I., Hoffmann, F. (1973) Transplantation of nuclei into protoplasts of higher plants. Z. Pflanzenphysiol. 69, 287-289 Rodgers, A. (1979) Detection of small amounts of human DNA in human-rodent hybrids. J. Cell Sci. 38, 391-403 Sala, C., Biasini, M.G., Morandi, C., Nielsen, E., Parisi, B., Sala, F. (1985) Selection and nuclear DNA analysis of cell hybrids between Daucus carota and Oryza sativa. J. Plant Physiol. 118, 409-419
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Savage, A.D., King, J., Gamborg, O.L. (1979) Recovery of a pantothenate auxotroph from a cell suspension culture of Datura innoxia Mill. Plant Sci. Lett. 16, 367-376 Saxena, P.K., Fowke, L.C., King, J. (1985a) An efficient procedure for isolation of nuclei from plant protoplasts. Protoplasma 128, 184-189 Saxena, P.K., Liu, Y., Mii, M., Fowke, L.C., King, J. (1985b) High nuclear yield from protoplasts of several plants. J. Plant Physiol. 121, 193-197 Thomas, E., Davey, M.R. (1975) From single cells to plants. Wykham Publications, New York, N.Y., USA Received 2 January; accepted 14 February 1986
