Plant Cell Reports

Plant Cell Reports (1988) 7: 13-16

© Springer-Verlag1988

A rapid transformation method for Solanum tuberosum using binary A grobacterium tumefaciens vectors S. Sheerlnan and M. W. Bevan Molecular Genetics Department, Institute of Plant Science Research (Cambridge Laboratory), Marls Lane, Trumpington, Cambridge CB2 2LQ, UK Received September 14, 1987 / Revised version received December 14, 1987 - Communicated by J. Schell

ABSTRACT

improvement in this important plant.

A tuber disc transformation and regeneration system was devised for potato (Solanum tuberosum). Tuber discs were found to be the most morphogenetic organ on a medium previously optimised for tomato regeneration. Shoot regeneration from tuber discs was rapid and prolific, and a large proportion of these were transformed as shown by nopaline assays and Southern blot analysis. The ease and speed of the tuber disc method will allow for the increased use of this commercially important plant in transformation studies. Abbreviations: MS, Murashige and Skoog medium; indole 3-acetic acid; BAP, benzylaminopurine; naphthalene acetic acid, ZR, zeatin riboside.

IAA, NAA,

HATERL%LS AND METHODS Plant gr~th media Shoot regeneration medium (3C5ZR) contained the salts of Murashige and Skoog (1962), obtained from Flow Laboratories or Gibco. This was supplemented with 1 mg/l Thiamine HCI, 0.5 mg/l nicotinic acid and 0.5 mg/l pyridoxine HCI (R3 vitamins); 3% sucrose, 5 ~M zeatin riboside (Calbiochem) and 3 GM IAA aspartic acid (Research Organics), pH 5.9. The medium was solidified with 0.8% Difco agar. Transformed shoots were selected on shoot regeneration medium containing i00 ~g/ml kanamycin and 500 Gg/ml carbenicillin. Explanted shoots were rooted in MS medium containing R3 vitamins and sucrose with I00 ~g/ml kanamycin and 200 ~g/ml carbenicillin.

INTRODUCTION

Potato (Solanum tuberosum) is a major crop plant in many regions of the world. Due to its economic importance, well established tissue culture procedures have been developed for somatic hybridisation (Shepard et al., 1980) and the production of variants by somaclonal variation (Gunn and Day, 1986). However, corresponding advances in direct gene transfer by Agr0bacterium-mediated transformation have been slow. Although potato is able to be transformed by wild-type Agrobacterium strains (De Cleene and De Ley, 1976), problems have been encountered in the rapid regeneration of morphologically normal transformed shoots. For example, Ooms et al. (1987) have developed a mixed inoculation method using disarmed and shootinducing strains of Agrobacterium from which a small number of morphologically normal transformed shoots can be obtained after 8-16 weeks. An et al. (1986) also describe the transformation of potato with a kanamycin-resistanee binary vector using a method of shoot regeneration from callus from which shoots were obtained 5 months after co-cultivation. We aimed to develop a more efficient and rapid method of gene transfer based on the direct selection and shoot induction method devised by Horsch et al. (1985). Such direct shoot regeneration avoids the need for a callus stage which is a source of somaclonal variation (Karp et al., 1982; Thomas et al., 1982). The method we have developed allows for the rapid recovery of many transformed potato shoots directly from Agrobacterium-infected tuber tissue. This method will encourage the study of gene regulation and crop

Offprint requests to: S. Sheerman

Plant material Virus-free tubers of S. tuberosum var. Pentland Dell were obtained from SCRI; Desiree, Maris Piper, Maris Bard and Golden Wonder were from the PBI. Only fresh tubers (less than 6 months old and stored at 4-6°C) were used for transformation experiments. Bacterial strains Agrobacterium tumefaciens LBA 4404 containing the disarmed kanamycin resistance vector pBin6 (Bevan, 1984) was used for all infections. The strain was grown in i0 mls of LB at 30°C containing 50 ~g/ml kanamycin for 24 hours before use, at which time it was centrifuged and resuspended in 20 mls of MS medium before plant transformation. Co-cultivatlon of A. tumefaeiens and potato tissues Tubers were peeled, washed briefly in distilled water and surface-sterilised for 15 minutes in a 10% solution of sodium hypochlorite containing a few drops of 'Tween 20'. They were then washed six times in sterile distilled water, and finally immersed in liquid MS medium. Samples were taken using a sterile 1 cm diameter cork borer, and columnar sections were sliced into thin discs (approximately 1-2 mm thick) using a scalpel. The tuber discs were floated in 20 mls of MS medium containing A~robacterium and thoroughly wetted by gently shaking the petri dish. After 20 minutes the tuber discs were transferred to tobacco feeder plates (Horsch et al., 1985) containing 3C5ZR medium. After 48 hours, infected discs were replated on

]4 identical medium lacking the feeder layer containing carbenicillin (500 ~g/ml) for Agrobacterium elimination, and kanamycin (i00 ~g/ml) for selection of transformed tissue. The plates were sealed with parafilm and incubated at 25=C under a 16-hour daylength. Tuber discs were subcultured onto fresh regeneration medium at 3 weekly intervals, lowering the carbenicillin concentration from 500 ~g/ml to 200 ~g/ml after 4 weeks in culture. Developing shoots were removed and replated in large test tubes containing MS salts and R3 vitamins plus carbenicillin (200 ~g/ml -I) and kanamycin (i00 ~g/ml -i) for root induction.

Nopaline assays Nopaline was extracted from approximately i monthold shoots and analysed according to Otten and Schilperoort (1978). DNA analysis DNA was extracted from stem and leaf tissue of axenic plants as described (Bevan, 1984), digested w i t h BamHI and Southern b l o t t e d (Maniatis et al., 1982). The blot was probed with an oligomer primed DNA fragment containing part of the nopaline synthase gene. RESULTS Regeneration and transformation Preliminary experiments showed that 3C5ZR medium was markedly superior for shoot regeneration from tissue explants. Other media containing the growth reglators NAA, IAA, kinetin and benzylaminopurine were tried with little success. This medium was initially developed for tomato regeneration (Tatchell and Binns, 1986), and contains conjugates of IAA and zeatin. The effectiveness of conjugates of plant growth regulators in shoot regeneration is thought to be due to the slow and steady release of free growth regulator from the medium (Hangarter et al., 1980). We noted consistent differences in shoot regeneration ability between different eultivars. Desiree was more efficient than the other cultivars, although Pentland Dell gave lower but acceptable shoot formation. Large variations in the efficiency of shoot formation between cultivars of commercial potatoes has been noted previously (Foulger and Jones, 1986). Next we tested the response of different organs of the cultivars Desiree, Marls Piper, Naris Bard, P~it~lauu Dell ana Golaen Wonder to 3CbZR shoot induction medium. In all varieties, thin slices of tuber tissue gave the most uniform and rapid response compared to leaf and stem explants. In addition, varietal differences in shoot formation persisted, as shown in Table i. T a b l e I.

Variety

Shooting response on 3C5ZR (shoots/disc)

Transformation efficiency

Marls Piper

o/zoo

O/lOO

Marls Bard

o/too

O/lOO

80"/80

60*/300

Golden Wonder

n.t.

28/90 (after 3 months)

Pentland Dell

110"/120

8"/150

Deslree

* multiple shoots

Desiree and Pentland Dell tuber slices showed the most prolific shoot formation with shoots developing within 4 weeks from multiple points on the tuber disc. It was observed that tuber age was a critical parameter in these experiments; young firm tubers gave the best results, while older, softer tubers with sprouts formed shoots only sporadically on 3C5ZR medium. The variation in shoot formation between cultivars noted in Table i may have been due, in part, to the age of the tubers used, although to our knowledge all tubers were of approximately the same age. The most consistent results have been obtained from tubers less than 6 months old stored at 4-6°C. Tuber tissue is also a very convenient source of material for transformation experiments. Transformation The binary vector Bin 6 (Bevan, 1984) in the octopine-type helper strain LBA 4404 (Ooms et al., 1982) was used in transformation experiments with tuber discs from the varieties of potatoes mentioned previously. Tuber discs were mixed with overnight cultures of Agrobacterium harbouring pBin6 and transformants were selected for kanamycin as described in Materials and Methods. Discs were scored for shoot formation 4 weeks after transformation, and representative results are shown in Fig. 2, panels a and b. The varieties Desiree, Pentland Dell, and Golden Wonder responded with between 6-20% of discs giving rise to shoots after 4 weeks. At least half of the Desiree discs gave rise to multiple independent shoots (see Table

I). When shoots were excised and placed on rooting medium containing carbenicillin and kanamycin, prolific root formation occurred in about 80% of the shoots, mostly from the nodes, with a few from the cut surface (Fig. 2, panel c). This is unlike tobacco in which roots form most profusely at the cut base (Hererra Estrella et al., 1983). Untransformed shoots did not form roots on I00 ~g/ml kanamycin. Rooted transformants were transferred to soil in a glasshouse. We have analysed over 200 independent transformants as part of a separate study and have found that only one in a hundred is morphologically distinct from the parental Desiree plants. Fig. 2, panel d shows representative transformants from a single transformation experiment.

Aualysls of transformauts Nopaline assays (Otten and Schilperoort, 1978) were performed on 8 shoots from a Desiree transformation with LBA 4404/pBin6 that had rooted on kanamycincontaining medium. All 8 shoots contained nopaline (data not shown) as pBin6 contains an intact nopaline synthase gene. DNA was then prepared from five of these putative transformants and analysed by Southern blotting. Digestions with BamHl (Fig. i) revealed that the five nopaline positive Desiree plants contained T-DNA, as shown by the presence of an internal band of 2.6 kb in all transformants. All transformants except that in lane 3 appeared to contain a single copy of T-DNA when compared to a single copy reconstruction (Fig. i, lane Ic). The transformant in lane 3 contained several copies of the internal fragment and presumed junction fragments. We do not yet understand the DNA arrangement of this transformant.

DISCUSSION The method of potato transformation described here has several advantages compared to methods currently used. The first advantage is that a medium has been found that induces the formation of morphologically normal shoots without an intervening callus stage, so

15 lowering the frequency of somaclonal variation in potato tissue culture (Karp et al., 1982; Thomas et al., 1982). The second advantage lies in using tuber discs for transformation. These tissues are easy to store so providing a constant large source of material for transformation, and they have a greater morphogenetic potential than leaf pieces. Finally, the combination of 3C5ZR medium and tuber disc transformation leads to the relatively rapid and reproducible formation of transformed shoots. We routinely obtain many independently transformed shoots within 4-6 weeks from transformation, whereas previous methods (e.g. Ooms et al., 1987) took at least 8-16 weeks to obtain shoots. Current experiments studying the regulation of chimaeric patatin genes in transformed potatoes have involved the analysis of over 200 independent transformants. We believe that the methods described in this paper will be of general use for potato transformation.

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Acknowledgements

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We thank Sara Tatchell and Andy Binns for advice on the use of IAA conjugates. S.S. thanks the Rockefeller Foundation and the Agricultural Genetics Company for support.

3 REFERENCES

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An,

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G., Watson, B.D., Chiang, C.C. (1986) Plant Physiol. 81:301-305. Bevan, M.W. (1984) Nucl. Acids Res. 12:8711-8721. De Cleene, M., De Ley, J. (1976) Botanical Review 42:389-466. Foulger, D., Jones, M.G.K. (1986) Plant Cell Reports 5:72-77. Gunn, R.E., Day, P.R. (1986) In: Plant Tissue Culture and its Biotechnological Applications. Butterworths, London, pp 313-316. Hangarter, R.P., Peterson, M.D., Good, N.E. (1980) Plant Physiol. 65:761-767. Herrera Estrella, L., De Block, M., Messens, E., Hernalsteens, J.P., Van Montagu, M., Schell, J. (1983) EMBO J. 2:987-995. Horsch, R.B., Fry, S.E., Hoffmann, N.L., Eicholtz, D., Rogers, S.G., Fraley, R.T. (1985) Science 277:1229-1231. Karp, A., Nelson, R.S., Thomas, E., Bright, S.W.J. (1982) Theor. Appl. Genet. 63:265-272. Maniatis, T., Fritsch, E.F., Sambrook, J. (1982) Molecular Cloning: A laboratory manual. Cold Spring Harbor Press, New York. Murashige, T., Skoog, F. (1962) Physiol. Plant. 15:473-497. Ooms, G., Hooykaas, P.J.J., Van Veen, R.J.M., Van Beelten, P., Regensburg, T.J.G., Schilperoort, R.A. (1982) Plasmid 7:15-29. Ooms, G., Burrell, M.M., Karp, A., Bevan, M., Hille, J. (1987) Theor. Appl. Genet. 73:744-750. Otten, L.A.B.M., Schilperoort, R.A. (1978) Biochim. Biophys. Acta 527:494:500. Shepard, J.F., Bidney, D., Shahin, E. (1980) Science 208:17-24. Tatchell, S., Binns, A.N. (1986) Tomato Genetics Co-op. Newsletter 36:35-36. Thomas, E., Bright, S.W.J., Franklin, J., Lancaster, V.A., Miflin, B.J., Gibson, R. (1982) Theor. Appl. Genet. 72:65-68.

FIG. I.

SOUTHERN BLOT ANALYSIS OF TRANSFORMANTS.

The autoradiograph shows hybridisation of a radioactively labelled DNA fragment homologous to the 3' end of the nopaline synthase gene to a BamHI digest of DNA isolated from nopaline positive transformants (lanes i, 3, 4, 5, 6). Lane 2 contains DNA from a normal Desiree plant. Ic and lOc refer to 1 and I0 copies per haploid genome of pBin6 digested with BamHI. The arrow indicates the position of the internal 2.6 kb BamHI fragment. Higher M.W. bands are presumed to be border or fusion fragments. The sizes of M.W. markers are shown on the left in kilobase pairs.

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(a) FIG. 2.

VARIOUS STAGES DURING POTATO TRANSFORMATION.

(a)

Tuber disc 3 weeks Agrobacterium, plated Slight callusing is formation occurs.

after incubation with on selective medium. evident before shoot

(b)

Tuber disc 6 weeks after incubation with Agrobacterium, plated on selective medium. Several independent shoots have grown from the perimeter and surface of the disc, with minimal callus formation.

(c)

(b) (c)

Shoots exclsed from disc as in (b) and rooted. Prolific root formation on selective medium can be ~een. Rapid shoot elongation also occurs at this stage.

(d)

Mature transformants in a glasshouse. The plants are vigorous, phenotypically normal and produce a good crop of tubers.

A rapid transformation method for Solanum tuberosum using binary Agrobacterium tumefaciens vectors.

A tuber disc transformation and regeneration system was devised for potato (Solanum tuberosum). Tuber discs were found to be the most morphogenetic or...
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