Plant Molecular Biology6:303-312, 1986 © Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands

Genetic transformation of Brassica campestris vat. rapa protoplasts with an engineered cauliflower mosaic virus genome Jerzy Paszkowski, Barbara Pisan, Raymond D. Shillito, Thomas Hohn, Barbara Hohn & Ingo Potrykus Friedrich Miescher Institut, P.O. Box 2543, CH-4002 Basel, Switzerland

Keywords: Brassica campestris var. rapa, engineered Cauliflower Mosaic virus DNA, infectivity in planta, protoplast transformation

Summary A hybrid Cauliflower Mosaic Virus (CaMV) genome containing a selectable marker gene was constructed by replacing the gene VI coding region with the aminoglycoside (neomycin) phosphotransferase type II [APH(3')II] gene from Tn5. This modified viral genome was tested for its infectivity both in planta and in a protoplast transformation system of Brassica campestris var. rapa. Stable, genetically transformed cell lines of B. campestris var. rapa were obtained after transformation. DNA of the hybrid CaMV genome was found to be integrated into high molecular weight plant genomic DNA. Transformation was achieved only when the hybrid genome was supplied together with wild type viral DNA. A possible complementation of the modified CaMV genome with the wild type viral DNA as a helper molecule in planta and in the protoplast system is discussed.

Introduction Several ways exist at present to deliver and express foreign genes in plant cells. The most widely used method has exploited the natural gene transfer system of the soil bacterium Agrobacterium tumefaciens, the causal agent of crown gall disease (1, 12). Foreign genes inserted into the part of the bacterial plasmid which is normally transferred to the plant genome (T-DNA) have been shown to be co-transferred and to function in plant cells (2, 5, 10, 16, 17, 30). Deletion of tumor inducing functions from the T-DNA gives the possibility of regenerating fertile plants and of transmission of introduced genes to the progeny (3, 18, 30). It has recently been shown that direct gene transfer, independent of the A. tumefaciens tumor inducing plasmid, is possible, and yields genetically engineered, morphologically normal plants regenerated from transformed protoplasts. The transformed gene is also in this case transmitted to the next and further sexual generations (31, 35). Use of a viral vector for expression of a foreign

gene in plants has recently been reported. Precise replacement of the dispensable part of Cauliflower Mosaic Virus (CaMV) - open reading frame (ORF) II - by the protein coding part of a bacterial dihydrofolate reductase gene gave rise to a functional, infective virus expressing the foreign gene during systemic infection (4). The tight organisation of the CaMV genome and our as yet incomplete understanding of the function of its genes limits its accessibility for modifications without loss of infectivity. ORF VI codes for the main protein of the viral inclusion bodies and its function is essential for viral infectivity (6, 41). The protein coding region of gene VI was replaced by the coding part of the bacterial gene for aminoglycoside phosphotransferase II [APH(3')II] which has been used before as a selectable marker gene in several plant transformation experiments (2, 10, 16, 17). Therefore, the APH(3')II hybrid gene was expected to be expressed under the control of gene VI expression signals. The replication and infectivity of this disarmed viral molecule and its possible functional com-

304 plementation by wild-type CaMV was studied in planta and in a protoplast transformation system. Protoplast derived, genetically transformed cell lines of turnip (Brassica campestris var. rapa) were recovered and analysed for the form, organisation and expression of the transforming viral DNA within the plant cells.

Material and methods

Construction of the hybrid CaMV genome 2 #g of EcoRV fragment of plasmid pBR327km ÷ (31) containing the hybrid gene was electroeluted from a 1070 agarose gel and cloned into the EcoRV fragment of plasmid pCa20-Ball (4). This was obtained by partial digestion of pCa20-Ball with EcoRV and isolation from the gel of the fragment lacking the EcoRV fragment covering CaMV gene VI. All manipulations were carried out as described by Maniatis et al. (29).

Plant inoculation Plants were inoculated as described in (19). 2 #g of Sal 1 restricted plasmid DNA (pCaMV6km, pCa20-Ball) was used in every inoculation and applied to a young turnip leaf of 5 weeks old plants.

Protoplast isolation Turnip plants Brassica campestris var. rapa c.v. Just Right were grown in a phytotron (12/12 hours day-night cycle at 27/20 °C, 5 000 lux cool fluorescence light). Leaves were sterilized [30 min in 0.507o Ca(OCI)2] and then washed thoroughly in sterile H20. Sterile leaves were cut into thin (1 mm) strips which were incubated in enzyme solution (1070 w/v Cellulase R-10 Onozuka, 0.1070 Macerozyme R-10 Onozuka, 0.3 M mannitol, 0.043 M CaCI2, pH 5.4, 500 mOs/kgH20) for 16 hours at 12°C. In order to separate the protoplasts from nondigested tissue, the digest was passed through a 100/~m stainless steel sieve. Protoplast were washed 3 times by centrifugation (5 min, 100xg) and resuspension in 0.175 M CaC12, 0.507o w/v MES [2-(N-morpholino)ethansulphonic acid] pH 5.7, 510 mOs/kgHzO.

Protoplast transformation and culture The method used for protoplast transformation was as originally developed by Krens et al. (22) and was used with modifications as described previously (31). 2x106 protoplasts were used per transformation treatment, each of which contained a total of 50/~g of DNA (10 #g of each plasmid molecule digested with Sal 1 in order to release the viral genome from the bacterial vector and 30-40/~g of calf thymus DNA as carrier) (Table 1). After transformation and washing of the DNA with F medium (22, 23) with a pH of 5.3 (adjusted with KOH after autoclaving) (31), the protoplasts were embedded in 1.5°70 agarose (2.5x105 protoplasts/ml) in modified KO-medium (26) containing 0.5 mg/1 NAA, 0.25 mg/1 2,4-D, 0.1 mg/l BAP, 520 mOs/kgH20 and cultured in an agarose bead type culture system (39).

Selection of transformed cell clones Selection was applied at day 4 of culture by the replacement of the culture medium surrounding the agarose beads with fresh medium containing antibiotic (50 mg/1 kanamycin or 10 mg/l G-418) (21). After 3 weeks of culture with replacement of the selective medium at 5 day intervals, the osmotic pressure of the culture medium was gradually reduced to 200 mOs/kgH20 by reducing the mannitol concentration. After 6 weeks visible resistant clones were picked up individually and were further Table 1. Turnip protoplast transformation experiments.

DNA mixtures used in transformation

Clones obtained Resistant to G-418 10 mg/l

pCaMV6km (10 #g) calf thymus (40/zg) pCaMV6km (10 pg) pCa20-Ball (10 #g) calf thymus (30/~g)

0

28

pCa20-Ball (10 #g) calf thymus (40/~g)

0

calf thymus (50 ~tg)

0

Resistant to kanamycin 50 mg/l

305 cultured on 0.8% w/v agar-solidified KO-medium supplemented with 50 mg/1 kanamycin sulphate or 20 mg/1 G-418. We have found that kanamycin was superior to G-418 for selection. G-418 rapidly killed wild type cell colonies as long as they were small, but was not effective for calli larger than 30-50 cells. Therefore the developmental stage of culture at the time of G-418 application was very critical, and G-418 had to be applied at an early stage of culture (first cell division) in order to avoid development of background colonies in the control treatments. G-418 was also not effective in maintaining constant selection pressure in later stages of the experiment when calli reached sizes of several thousand cells. We have observed that control turnip calli could proliferate at approximately the same rate on agar-solidified media containing 20 mg/l G-418 as on antibiotic free medium. However, 5 mg/l of G-418 was sufficient to inhibit growth of protoplast derived cell cultures at the 2 to 8 cell stage. Kanamycin sulphate (at a concentration of 50 mg/1) was effective over a longer period of colony development (up to several thousands cells). Larger cell colonies of non-transformed turnip calli were able to proliferate on kanamycin-containing media (50 mg/l), but at reduced rates compared to antibiotic free media (data not shown). We have observed that addition of antibiotic to the cell environment (to the liquid phase surrounding agarose beads) (39) after the first division allowed sensitive cell colonies to complete an additional round of mitosis in the presence of G-418 (10 mg/1) and 3 - 5 rounds in the presence of kanamycin sulphate (50 mg/1). Only transformed cell lines continued to proliferate further in kanamycin. Earlier addition of the antibiotics had a rapid killing effect on the total population of protoplasts or newly formed cells. The plating efficiencies in transformation experiments were estimated by counting the number of proliferating clones in protoplast derived cultures equivalent to transformed samples cultured without selection.

DNA isolation from plant material and Southern blot analysis The methods and conditions for DNA isolation and Southern blot hybridisation were as described previously (31).

APH(3 ')H activity assay APH(3')II activity was detected using the method described by Reiss et al. (37) and adapted to plant material as previously described (31).

Results

Replacement of CaMV gene VI by the APH(3 ')H gene The well characterised CaMV gene VI is transcribed as an abundant 19S mRNA from a typical eukaryotic promoter region preceding the protein coding part of the gene (13). An EcoRV fragment of plasmid pBR327Cakm + (31), containing the gene VI promoter region and 81 base pairs of the 5' coding region of gene VI linked in the correct reading flame to the protein coding part of a bacterial APH(3')II gene from Tn5, was used to replace the gene VI EcoRV fragment of cloned CaMV. The replacement restored the gene VI expression signals in the 5' region of the hybrid gene and placed the 3' end of the APH(3')II hybrid gene close to the gene VI transcription termination site. The gene product predicted from this construction [N terminal fusion of APH(3')II, 23 amino acid residues longer than bacterial gene] has been shown to be biologically active (31). Otherwise the CaMV genome of the resulting hybrid molecule (pCaMV6km) was not altered. Details of the construction are shown in Fig. 1. For a more detailed restriction map of CaMV genome see (7, 11).

Infectivity and complementation test of engineered CaMV genome CaMV DNA, amplified in E. coli by cloning on bacterial vector plasmids, is infectious on plants when cut out from the vector molecule and applied to wounded leaves (19, 24). Infectivity studies with CaMV are normally carried out with turnip (Brassica campestris var. rapa) as the model host plant. We therefore used B. campestris var. rapa in infectivity tests and in protoplast transformation experiments. Infectivity was determined by inoculation of young plants with Sal 1 restricted DNA of pCaMV6km or pCa20-Ball (Fig. l) (4). DNA's were

306

0 E c o R ~

v,//

y

SallBomH1~ ~ / ~ E c ° R V APH(Y)/

~

/ .......... +'~ EcoRV'~' ~ X

oCnMV6~m ~ H

-BomH1 X

EcoRV

I "~

~

V

/In

V

Soil Fig. 1A. Replacement of the CaMV gene VI coding region with the coding region of the APH(3')II gene. CaMV strain cloned as plasmid pCa20-Ball is described in ( 4 ) , plasmid pBR327Cakm + is described in (31). Open boxes - ORF's of CaMV, hatched box - APH(3')II gene coding region, thick lines - intergenic regions of CaMV, thin lines - bacterial vector plasmid.

7435

/,630 4833 5383 5708 58/,8 Bglll Sail P~tl EcoR~rHind~ 1 GENE V

I

73&0l

7670

EcoR-_V SgllT

GENE 17]"

TArAATG

TGA

GENE ?7 REGION OF CoMV

ATG

TGA

pCaMV6krn Fig. lB. CaMV gene VI modification. Numbers above restriction endonuclease sites indicate their position as described in (11). Arrows mark the start of the 35S RNA transcript at position 7435 on the CaMV map.

applied separately or as a hi mixture. After 3 weeks, plants were examined for the development of CaMV specific mosaic symptoms. At the same time, DNA from inoculated leaves (site of infection) and from secondary infected leaves (infected by systemic spread) was examined by Southern blot hybridistion (40) in order to follow the fate of the applied DNA (Fig. 2).

Fig. 2. Infectivity and complementation test In planta of viral genomes released from plasmids pCaMV6km and pCA20-Ball. 5 #g of the plant DNA was restricted with the enzyme EcoRV. (a) turnip DNA probed with APH(3')II coding region for detection of pCaMV6km. (b) turnip DNA probed with gene VI coding region for detection of pCa20-Ball. lanes h DNA of healthy turnip leaf. 2: DNA of leaf inoculated with Sail digested pCaMV6km. 3: DNA of secondary leaf of plant inoculated with pCaMV6km. 4" DNA of leaf inoculated with Sail digested pCaMV6km and pCa20-Ball. 5: DNA of secondary leaf of plant inoculated with pCaMV6km and pCA20-Ball. 6: DNA of leaf inoculated with Sail digested pCa20-Ball. 7: DNA of secondary leaf of plant inoculated with pCa20-Ball. The arrows point to the 1.2 kb and 1.6 kb EcoRV fragments of the pCa20-Ball and pCa6km respectively. (compare Fig. !.)

307 Application of pCaMV6km DNA alone never led to the development of mosaic symptoms. Infection with pCa20-Ball DNA, or With mixtures of both DNA's caused the mosaic symptoms typical of systemic infection by the wild-type virus. Southern blot analysis, using specific probes which discriminate between the two DNA's applied, revealed that pCaMV6km was not able to spread systemically through the plant. This was also true in the case where pCaMV6km was applied together with pCa20-Ball (Fig. 2). We have never found pCa6km specific hybridisation signals in the secondary leaves of inoculated plants. A weak signal specific to the APH(3')II coding region - (pCaMV6km detection) was visualised by hybridisation to the DNA isolated from inoculated (DNA rubbed) leaves 3 weeks after infection with pCaMV6km alone (Fig. 2, lane 2). This probably reflected residual DNA from the application, since the control bacterial plasmid pUC8 DNA could also be detected after the same time interval after infection (data not shown). A reproducibly stronger pCaMV6km specific signal was observed with DNA from inoculated leaves in cases where pCaMV6km was mixed with pCa20-Ball prior to the infection. This may indicate some form of helper virus effect of pCa20-Ball at the primary site of infection. We can conclude that replacement of CaMV gene VI by the foreign gene thus had produced a non viable CaMV hybrid genome which could neither spread systemically throughout the plant tissues nor could be complemented in trans for systemic spread by co-infection with the wild-type CaMV.

Transformation of isolated turnip mesophyll protoplasts and selection of antibiotic resistant cell lines In order to study the possibility of pCaMV6km replication at the cellular level in cultured plant cells turnip protoplast transformation experiments were undertaken. We have recently improved turnip mesophyll protoplast culture to c.a. 20°7o plating efficiency (34), a level sufficient to approach DNA transformation. For protoplast transformation a modification of a DNA uptake procedure previously developed by Krens et al. (22, 23) was used. Readjustment of the

pH of F medium (22) to 5.5, which falls to 4.8 after autoclaving, was a prerequisite for survival of turnip protoplasts during the DNA treatment. Even so, recovery of protoplasts after transformation was not satisfactory in the majority of experiments. In only 4 out of 15 equivalent experiments was the plating efficiency at a level (10-1507o) sufficient to carry out selection of transformants. Antibiotic resistant cell lines were recovered in 2 of these experiments (Table 1). We obtained 28 resistant clones from G-418 selection and 2 clones from kanamycin sulphate selection (tab. 1). These clones were grown further on agar-solidified media containing 20 mg/1 G-418 or 50 mg/1 kanamycin sulphate, respectively. As soon as the clones reached a size of approximately 1 g fresh weight of tissue, part of the material was subjected to Southern blot analysis and assayed for the presence of the APH(3')II gene product.

Form and arrangement of the foreign DNA in transformed G-418 and kanamycin resistant cell lines After 12 weeks of culture, DNA from 18 G-418 resistant cell lines was isolated, separated by electrophoresis in a 1°70agarose gel, blotted to nitrocellulose and probed for the presence of pCaMV6km specific sequences, pCaMV6km specific hybridisation to high molecular weight DNA (greater than 50 kb.) was detected in all but one of the 18 clones tested (data not shown). Transformed cell lines were grown further on G-418 containing medium for an additional 3 months and the Southern blot analyses were repeated. At this later time we were not able to detect either pCaMV6km or pCa20-Ball sequences in any of the previously tested 18 lines. As stated in Material and Methods section, G-418 does not maintain selective pressure on larger calli and it is possible that the transforming DNA was lost during the longer culture period under these nonselective conditions. DNA of the two clones selected and cultured on kanamycin-containing media (T12A and T12B) were analysed after 3, 6, 9, and 12 months of culture - the foreign DNA was stably maintained. When non restricted total cellular DNA was analysed, hybridisation with radioactively labelled pCaMV6km was detected only in the region of high

308 molecular weight D N A greater than 50 kilobases in length. There was no detectable hybridisation in the region of 7.1 or 7.5 kb, the expected sizes for free copies of viral like molecules derived from plasmids pCaMV6km and pCa20-Ball, respectively. Further analysis of nuclear D N A confirmed the supposition that the major part of the Sal 1 fragment of pCaMV6km containing the hybrid viral genome had been integrated, gene.rating junction fragments with plant D N A in both of the selected cell lines T12A and T12B. Reconstructions and mapping experiments suggested the presence of a single copy of the transforming D N A molecule per diploid genome of B. campestris var. rapa. The results of the Southern blot analyses (Fig. 3) suggest the following conclusions on the junctions of pCaMV6km D N A and plant DNA: a) The 1144 bp EcoRV fragment containing the hybrid marker gene is present in a non-rearranged form, which when correlated to tests for A P H ( 3 ' ) I I activity, and to previous results from tobacco protoplast transformation (31, 35), can be taken as evi-

dence for the integration of a functional copy of this gene into the plant genome. b) EcoRV, BstEII, and EcoRI restriction digests of nuclear D N A of T12A and T12B hybridised to probes specific for either the A P H ( 3 ' ) I I coding region or the 5' and 3' gene flanking sequences allowed for approximate mapping Of the regions of the integrated pCaMV6km Sail fragment (Fig. 4). The fragments of foreign DNA integrated in two transformed lines are different and are probably integrated at different sites in the genome (as judged from the sizes of the border fragments). There are, however, also c o m m o n features, in that both lines contain an unaltered (2297 bp long) EcoRI fragment of the linear molecule used for transformation and there is little rearrangement of the transforming D N A as compared to that found after transformation of tobacco (15, 31). Approximate maps of the pCaMV6km insert in the Brassica campestris var. rapa genome are shown on Fig. 4.

Fig. 3. Southern blot analysis of DNA of kanamycinresistant cell lines T12A and T12B. (a) Probed with pCaMV6km. (b) Probed with 5' flanking region of the hybrid gene (Sail - EcoRVfragment of pCaMV6km, Fig. 4, probe 1). (c) Probed with the region covering hybrid gene (EcoRV - EcoRVfragment of pCaMV6km, Fig. 4, probe 2). (d) Probed with 3' flankingregion of the hybrid gene [EcoRV - BstEH fragment of pCaMV6km, Fig. 4, probe 3). Lanes 1 and 2 on every panel; DNA digested with EcoRV. Lanes 3 and 4 on panel a DNA digested with BstEIl. Lanes 3 and 4 on panels b, c, d, DNA digested with EcoR1. Lanes 2 and 4 on panel a; T12A, lines I and 3; T12B. Lanes 1 and 3 on panels b, c, d; T12A. Lanes 2 and 4 on panels b, c, d; T12B. Numbers next to arrows represent DNA length in kilo bases.

309

EcoR1

&O T12A

~ I ~ ' ~ ~ 4 Sot1 EcoR1 EcoR5

"1"12

Ecol,5

01,26~8 , < BsfE2

2t4t,2t~7&283& "If f" r

EcoR1

2×EcoR1 EcoR5 "'

P~ob~3' r ~ ~ _ .

'~'Eco R1

t~833

I

Sol 1

Eco R'~5

Fig. 4. The approximate map of the pCa6km insert into the Brassica campestris var. rapa genome for transformants T2A and T2B. Wavy lines represent plant genomic DNA. Numbers above the restriction sites indicate their position on the CaMV map (11). Marked probes - these fragments were used for Southern blot analysis shown on Fig. 3.

APH(3 ')H activity test of transformed cell lines Kanamycin-specific phosphorylation, reflecting activity of APH(3')II, could be detected in crude plant protein extracts electrophoresed under non denaturing conditions in a 10% polyacrylamide gel (31, 37). Extracts of kanamycin and G-418 resistant cell lines were tested for the presence of the APH(3')II activity. In the G-418 resistant cell lines, assayed seven months after transformation (at the time when the presence of the transforming DNA could no longer be detected) no APH(3')II activity was found. Extracts of the kanamycin resistant cell lines (T12A and T12B) contained the expected APH(3')II specific activity, confirming the biological function of the integrated hybrid marker gene (Fig. 5). Moreover the banding pattern of the enzyme was identical to that found in tobacco tissues expressing the enzyme from the same fused gene (Fig. 5) (31).

Discussion

We have constructed a non-infectious viral vector molecule based on Cauliflower Mosaic Virus DNA by the replacement of the CaMV gene VI coding region with the APH(3')II marker gene. Gene VI product - the main component of viral inclusion bodies - is considered to be essential for assembly of viral particles (8). Complementation of the disarmed CaMV in trans with a helper strain was not sufficient to allow the engineered viral genome to spread systemically throughout the plant tissues al-

Fig. 5. APH(3')II activity test. Line 1: protein extract from T12A. Line 2: protein extract from T12B. Line 3. protein extract from control B. campestris var rapa callus. Line 4. protein extract from transformed tobacco plant T2-1 (31). The arrows show the position of the APH(3')II activity.

though there appears to be some helper function in the primary leaf. Since pCaMV6km was altered only in the region of open reading flame VI, these results indicate that the replaced region of CaMV may not only have a protein coding function but may carry other signals important in the CaMV life cycle (see below). We have previously observed that wild type CaMV can replicate in B. campestris var. rapa cell clones derived from protoplasts from infected plants, and that during viral propagation in tissue culture its plant infectivity can be lost. This seemed to be due to spontaneous deletion of parts of the CaMV genome (32). Therefore we expected that it might be possible to observe replication in tissue

310 culture of viral genomes which are defective for in planta replication due to deletions or gene replacement, such as pCaM6km. This was studied by using protoplast transformation experiments. Southern blot analysis indicated that the linear DNA used in transformation was probably directly integrated without prior circularization (Figs. 3 and 4). Since recircularisation of the CaMV DNA is a prerequisite for its replication (25), it is likely that we have observed direct foreign DNA integration which has not been preceeded by rounds of replication. The apparent lack of replication of pCaMV6km could also be due to an alteration of the promoter sequence for the main 35S CaMV transcript (28) which is proposed to be the viral replication intermediate (14, 20, 33). The 35 S promoter deletion of pCaMV6km might lower the promoter activity, although the long transcript should still be produced (28). Why then did the only transformants obtained come from treatments including the mixture of pCaMV6km and pCa20-Ball? The low transformation frequences and the low total number of transformants obtained and analysed do not allow a conclusive answer to be given. Since we have observed high rate of cotransformation of separate DNA molecules to plant protoplasts (38), it is likely that both molecules were present in the same cell. It may be that virus infected and therefore stressed cells are more accessible for uptake or integration of the foreign DNA. This possibility is currently being tested. The integration pattern of pCaMV6km into the turnip genome is simple. We have not observed major DNA rearrangements or integration of separate small fragments of the transforming molecule. This might reflect the low nuclease activity found in turnip protoplasts in comparison to other plant protoplasts (9). The protein product of the hybrid marker gene produced in turnip appears to be identical to that found in tobacco transformed by the same gene construction (31). It has been genetically proven for tobacco that genes introduced via direct gene transfer are integrated into the nuclear genome (31). In turnip, transforming DNA is most probably integrated into the nuclear genome since pCaMV6km copies were found in the nuclear DNA fraction at approximately a single copy per diploid genome of B. campestris var. rapa (as concluded from the presence of

only one kind of border fragments). Genetic transformation experiments published so far have been performed mainly within one plant family, the Solanaceae. The results presented are the first case of genetic transformation for a dicotyledonous plant species not related to Solanaceae. The Cruciferae, here represented by B. campestris var. rapa, include several important crop plants (e.g. rape, cabbage) and interesting model plants (e.g. Arabidopsis thaliana). We were able to integrate a foreign marker gene into the genome of B. campestris var. rapa and stably maintain it in tissue culture for over one year. Unfortunately, we could not develop conditions for plant regeneration from B. campestris var. rapa protoplasts in order to be able to study the genetics of the introduced trait. However, plant regeneration is now possible for other Brassica species (26) and therefore experiments continuing this research line are now in progress. The results suggest that direct DNA transformation to plant cells is not likely to be limited to a narrow range of competent plant species and should therefore become a useful technique in the genetic engineering of variety of plants. This supposition is further supported by recent results with genetic transformation of protoplasts of the Graminaceous monocots Lolium multiflorum (36) and Triticum monococcum (27).

Acknowledgements The authors thank M. Saul, I. Negrutiu and E. Balazs for helpful comments during the preparation of this manuscript and Mrs S. Wilhelm for photographic assistance.

References 1. Bevan MW, Chilton M-D: T-DNA of the Agrobacterium Ti and Ri plasmids. Annual Rev Genet 16:357 - 384, 1982. 2. Bevan MW, Flavell RB, Chilton M-D: A chimaeric antibiotic resistance gene as a selectable marker for plant cell transformation. Nature 304:184- 187, 1983. 3. De Block M, Herrera-Estrella L, Van Montagu M, Schell J, Zambyrski P: Expression of foreign genes in regenerated plants and their progeny. EMBO J 3:168I- 1689, 1984. 4. Brisson N, Paszkowski J, Penswick JR, Gronenborn B, Potrykus I, Hohn T: Expression of a bacterial gene in plants by using a viral vector. Nature 310:511 -514, 1984. 5. Broglie R, Coruzzi G, Fraley RT, Rogers SG, Horsch RB,

311 Niedermeyer JG, Fink CL, Flick JS, Chua N-H: Light regulated expression of a pea ribulose-l-,5-bisphosphate carboxylase small subunit gene in transformed plant cells. Science 224:838- 843, 1984. 6. Daubert S, Shephert R J, Gardner RC: Insertional mutagenesis of the cauliflower mosaic virus genome. Gene 25:201 - 208, 1983. 7. Dixon LK, Hohn T: Cloning and manipulating cauliflower mosaic virus, in Recombinant DNA Research and Viruses. ed Y. Becker, in press, 1985. 8. Favali MA, Bassi M, Conti GG: A quantitative autoradiographic study of intracellular sites for replication of Cauliflower Mosaic Virus. Virology 53:115- 119, 1973. 9. Fernandes SM, Lurquin PF, Kado CI: Incorporation and maintenance of recombinant-DNA plasmid vehicles pBR313 and pCR1 in plant protoplasts. FEBS Lett 87:277-282, 1978. 10. Fraley RT, Rogers SG, Horsch RB, Sanders PR, Flick JS, Adams SP, Bittner ML, Brand LA, Fink CL, Fry JS, Galluppi GR, Goldberg SB, Hoffmann NL, Woo SC: Expression of bacterial genes in plant cells. Proc Natl Acad Sci USA 80:4803-4807, 1983. 11. Gardner RC, Howarth A J, Hahn P, Brown-Luedi M, Shephert RD, Messing J: The complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by Ml3mp7 shotgun sequencing. Nucleic Acid Res 9:2871 - 2888, 1981. 12. Gheysen G, Dbaese P, Van Montague M, Shell J: DNA flux across genetic barriers: the crown gall phenomenon. In: Hohn B, Dennis E (eds) Plant Gene Research. Genetic flux in plants, pp 12- 47, 1985. 13. Guilley H, Dudley RG, Jonard G, Balazs E, Richards KE: Transcription of Cauliflower Mosaic Virus DNA: Detection of promoter sequences, and characterization of transcripts. Cell 30:763-773, 1982. 14. Guilley H, Richards KE, Jonard G: Observations concerning the discontinuous DNA of Cauliflower Mosaic Virus. EMBO J 2:277-282, 1983. 15. Hain R, Stabel P, Czernilofsky AP, Steinbiss HH, HerreraEstrella L, Schell J: Uptake, integration, expression and genetic transmission of a selectable chimeric gene by plant protoplasts. Mol Gen Genet 199:161 - 168, 1985. 16. Herrera-Estrella L, De Block M, Messens E, Hernalsteens J-P, Van Montagu M, Shell J: Chimeric genes as dominant selectable markers in plant cells. EMBO J 2:987-995, 1983. 17. Herrera-Estrella L, Depiker A, Van Montagu M, Shell J: Expression of chimeric genes transferred into plant cells using a Ti-plasmid-derived vector. Nature 303:209-213, 1983. 18. Horsch RB, Fraley RT, Rogers SG, Sanders PR, Loyd A, Hoffmann N: Inheritance of functional foreign genes in plants. Science 223:496- 498, 1984. 19. Howell SH, Walker LL, Dudley RK: Cloned cauliflower mosaic virus DNA infects turnips (Brassica rapa). Science 208:1265 - 1267, 1980. 20. Hull R, Covey SN: Does cauliflower mosaic virus replicate by reverse transcription. TIBS 8:119-121, 1983. 21. Jimenez A, Davies J: Expression of a transposable antibiotic resistance element in Saccharomyces. Nature 287:869- 871, 1980.

22. Krens FA, Molendijk L, Wullems G J, Schilperoort RA: In vitro transformation of plant protoplast with Tiplasmid DNA. Nature 296:72-74, 1982. 23. Krens FA, Schilperoort RA: Ti-plasmid DNA uptake and expression by protoplasts of Nicotiana tabacum. In: Vasil IK (ed) Cell Culture and Somatic Cell Genetics of Plants, Vol. 1, Laboratory Techniques. Academic Press, New York, 1984, pp 522-534. 24. Lebeurier G, Hirth L, Hohn T, Hohn B: Infectivities of native and cloned DNA of cauliflower mosaic virus. Gene 12:139- 146, 1980. 25. Lebeurier G, Hirth L, Hohn B, Hohn T: In vivo recombination of cauliflower mosaic virus DNA. Proc Natl Acad Sci USA 75:1428- 1432, 1982. 26. Liang-cai L, Kohlenbach W: Somatic embryogenesis in quite a direct way in cultures of mesophyll protoplasts of Brassica napus. Plant Cell Rep 1:209-211, 1982. 27. Lorz H, Baker B, Schell J: Gene transfer to cereal cells mediated by protoplast transformation. Mol Gen Genet 199:178- 182, 1985. 28. Odell JT, Nagy F, Chua N-H: Identification of DNA sequences required for activity of the cauliflower mosaic virus 35 S promoter. Nature 313:810-812, 1985. 29. Maniatis, Fritsch EF, Sambrook J: Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, New York, 1982. 30. Murai N, Sutton DW, Murray MG, Slighton JL, Merlo D J, Reichert NA, Senupta-Goplan C, Stock CA, Barker RF, Kemp JD, Hall TC: Phaseolin gene from bean is expressed after transfer to sunflower via tumor-inducing plasmid vectors. Science 222:476-482, 1983. 31. Paszkowski J, Shillito RD, Saul M, Mandak V, Hohn T, Hohn B, Potrykus I: Direct gene transfer to plants. EMBO J 3:2717-2722, 1984. 32. Paszkowski J, Shinshi H, Konig I, Lazar GB, Hobn T, Mandak V, Potrykus I: Proliferation of Cauliflower Mosaic Virus in protoplast-derived clones of turnip (Brassica rapa). In: Potrykus I e t al. (eds) protoplasts 1983, Poster Proceedings. Birkhauser, Basel, 1983, pp 138-139. 33. Pfeiffer P, Hohn T: Involvement of reverse transcription in the replication of Cauliflower Mosaic Virus: A detailed model and test of some aspects. Cell 33:781- 789, 1983. 34. Pisan B, Potrykus I, Paszkowski J: Optimalization of turnip (Brassica rapa) protoplast culture for Cauliflower Mosaic Virus transformation. In: Potrytus 1 et al..(eds) Protoplast 1983, Poster Proceedings. Birkhauser, Basel, 1983, pp 44 - 45. 35. Potrykus I, Paszkowski J, Saul MW, Petruska J, Shillito RD: Molecular and general genetics of a hybrid foreign gene introduced to tobacco by direct gene transfer. Mol Gen Genet 199:169- 177, 1985. 36. Potrykus I, Saul M, Petruska I, Paszkowski J, Shillito RD: Direct gene transfer to cells of a Graminaceous monocot. Mol Gen Genet 199:183 - 188, 1985. 37. Reiss B, Sprengel R, Will H, Schaller H: A new sensitive method for qualitative and quantitative assay of neomycin pbosphotransferase in crude cell extracts. Gene 30:211-218, 1984. 38. Schocber R J, Shillito RD, Saul MW, Paszkowski J, Potrykus I: Co-transformation of foreign genes to plants, submired.

312 39. Shillito RD, Paszkowski J, Potrykus I: Agarose plating and a bead type culture technique enable and stimulate development of protoplast-derived colonies in number of plant species. Plant Cell Rep 2:244-247, 1983. 40. Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503-517, 1975.

41. Xiong C, Muller S, Lebeurier G, Hirth L: Identification by immunoprecipitation of cauliflower mosaic virus in vitro major translation product with a specific serum against viroplasm protein. EMBO J 1:971-976, 1982. Received 23 September 1985; in revised form 9 January 1986; accepted 14 January 1986.