Plant Molecular Biology 12: 329-337, 1989 © 1989 Kluwer Academic Publishers. Printed in Belgium

329

Transformation of homozygous diploid potato with an Agrobacterium tumefaciens binary vector system by adventitious shoot regeneration on leaf and stem segments R.G.F. Visser, 1.3 E. Jacobsen, L 3 A. Hesseling-Meinders, ~ M.J. Schans, ~ B. Witholt 2 and W.J. Feenstra ~

1Department of Genetics, University of Groningen, Kerklaan 30, 9751 NN Haren, Netherlands (*author for correspondence); 2Department of Biochemistry, University of Groningen, Nijenborgh 16, 9747 AG Groningen, Netherlands; 3present address: Department of Plant Breeding, Agricultural University, P.O. Box 386, 6700 AJ Wageningen, Netherlands Received 5 July 1988; accepted in revised form 20 December 1988

Key words: Agrobacterium tumefaciens, adventitious shoot regeneration, transformation, homozygous potato Abstract

Transformed potato (Solanum tuberosum) plants were obtained from homozygous diploid potato by using a transformation procedure in combination with an adventitious shoot regeneration method. Leaf and stem explants were inoculated with an Agrobacterium tumefaciens strain which contained a binary vector (pVU 1011) carrying the neomycin phosphotransferase gene. Shoot regeneration most effectively on stem explants, occurred within six weeks directly from the explants without introducing a callus phase. A strong seasonal influence on transformation efficiencies was observed. Analysis of a number of randomly selected regenerated shoots for their ability to root and form shoots on kanamycin-containing medium shows that over 90~o of the regenerated shoots obtained are transformed. In a number of shoots transformation was confirmed by a test for the presence and expression of the NPT-II gene.

Introduction

A number of procedures have been developed to transform plants or plant cells with specific D N A (reviewed by e.g. Fraley et al. [10]). The most widely used technique to introduce foreign D N A into plant cells is the one that uses Agrobacterium as a mediating system. Solanum tuberosum, an agronomically important crop, is also susceptible to Agrobacterium infection. Transformation of potato has been achieved with wild-type strains of Agrobacterium tumefaciens [22], Agrobacterium rhizogenes [32, 23] and even Agrobacterium rubi

[20]. Recently, it was also shown that transformation of potato can be achieved by using a binary T-DNA-vector system [1, 24, 27, 29]. The existing transformation procedures for potato have mostly been developed for tuber slices of tetraploid cultivars [27, 29]. This means that genotypes which give little or no tuber formation, such as most monoploids or homozygous diploids, cannot be transformed using these procedures. Transformation of potato stem or leaf explants has also been reported [ 1, 24]. The main problem here is the fairly long period of time needed to

330 regenerate transformed plants and, due to the long callus phase, an introduction of gross morphological and cytological changes. Here we describe the use of a transformation procedure in combination with the earlier described adventitious shoot regeneration procedure on leaf explants of the monoploid potato Mn 79.7322 [15]. The use of this transformation system resulted in an efficient regeneration of transgenic shoots within six weeks.

Introduction of the plasmids from E. coli into Agrobacterium tumefaciens LBA4404 was in the presence ofE. coli HB 101 [4] containing plasmid pRK2013 [7] by triparental mating. A. tumefaciens was grown on LB medium [ 17] or minimal medium A [5] with rifampicin (100 mg/1) at 29 ° C. The integrity of the vectors in Agrobacterium was verified by preparing DNA from Agrobacterium by the boiling method [13].

Transformation Materials and methods

Plant material Shoot cultures of S. tuberosum Mn 79.7322 (homozygous disploid 2n = 2x = 24, obtained by doubling the original monophaploid Mn 79.7322, denoted 7322) and amylose-free mutant 86.040 (amf-1 diploid form, 2n = 2 x = 24 [14] were grown in vitro on basal MS medium [19] supplemented with 10 or 30 g/1 sucrose (MS 10 or MS 30 respectively) at 25 °C in the light. They were propagated by subculturing stem segments every three to four weeks. Leaves and stem explants free of axillary buds of these genotypes were used in adventitious shoot regeneration and in transformation experiments.

Bacterial strains and plasmids Inoculations were made with the avirulent A. tumefaciens strain LBA4404 [21] containing the disarmed plasmid pVU 1011 (kindly provided by Dr P. van den Elzen) or derivatives thereof. pVU1011 is a derivative of pROK-1 [2] which is an expression cassette derivative of BIN19 [3]. The Escherichia coli hygromycin phosphotransferase (HPT) gene [8] was fused to the CaMV 35S RNA promotor and the nopaline synthase terminator. This vector allows the selection for kanamycin- and/or hygromycin-resistant transgenic plants.

Stem and leaf explants of approximately 5 mm x 3 mm were prepared from in vitro grown plants. The explants were floated overnight on liquid medium M 387 (MS supplemented with 80 mg/1 NHaNO3, 14.7 mg/1 CaC12, 10 mg/l NAA, 10 mg/1 BAP [15]. The next day the explants were soaked in an overnight culture of A. tumefaciens for 15 minutes and blotted dry on sterile filter paper. The treated explants were place on solidified (1.8~ agar) M 379 medium (callus induction medium, MS medium supplemented with l g/1 sucrose, 4g/1 mannitol, 2.25 mg/l BAP, 0.175mg/1 IAA [15] in sealed Petri dishes. After two days of cocultivation the explants were placed on M 379 plates with either cefotaxim (200 mg/1) and kanamycin (50 mg/1) for seven days (Procedure I) or cefotaxim (200 mg/1) alone for five to seven days (Procedure II). Consecutively the explants were transferred onto medium M 384 (shoot induction medium, MS supplemented with 15 g/1 sucrose, 2.25 mg/1BAP, 5 mg/1 GA 3 [15] with 200 mg/1 cefotaxim and 50 mg/1 kanamycin. All cultures were incubated at 25 °C, with a 16-h photoperiod. Every three to four weeks the explants, from both procedures, were transferred to fresh M 384 medium with 200 mg/1 cefotaxim and 50 mg/1 kanamycin. After varying times (from 6 weeks on) shoots were removed from the explants and placed onto MS 30 medium containing 200 mg/l cefotaxim. As a control uninoculated explants were treated according to both procedures.

331 Neomycin phosphotransferase-H (NPT-I1) assay

Shoot material (100mg fresh weight) was macerated in an Eppendorf tube with 100 #1 extraction buffer containing 62.5 mM Tris pH 6.8, 10~o glycerol, 5 ~ 2-mercaptoethanol, 0.2~o SDS, 0.025~o bromophenol blue and 0.025% xylencyanol. After centrifugation for 10 min at 4 °C in a microfuge the supernatant was subject to electrophoresis through a 10~o polyacrylamide gel at 55 V overnight in a cold room and assayed for N P T - I I activity in situ as described by Reiss et aL [26]. The running gel was overlayed with an agarose slab containing 33.5 mM Tris-maleate, 21 mM MgC12, 200mM NH4C1, 2~o agarose, 40 #g/ml kanamycin sulphate and 50 #Ci [ 7 - 32p] ATP, incubated for 60 min at room temperature and blotted onto Whatman P81 phosphocellulose paper for 4 hours. Excess background radioactivity was removed by a single wash in a solution of 1~ SDS and 1 mg/1 proteinase K for 30 min at 60 °C as described by White and Greenwood [31] and four washes in 1 mM phosphate buffer at 80 °C. The filter was dried at 80 °C and subsequently exposed to X-ray film overnight. Growth assays

Growth properties of 7322 and 86.040 shoots in the presence of kanamycin were estimated by measuring their rooting ability on MS 30 medium containing different kanamycin concentrations. Adventitious shoot regeneration on explants was according to the three-step procedure of Hovenkamp-Hermelink et al. [ 15]. To test the adventitious shoot regeneration ability on kanamycin leaf strips were transferred, after floating, on callus induction medium M 379 followed by shoot regeneration on M 384 medium containing cefotaxim and kanamycin. Cytological determinations

Determination of chromosome number in root tip cells was according to Pijnacker and Ferwerda

[25] and estimation of ploidy level by counting chloroplasts in stomatal guard cells was according to Frandsen [ 11 ].

DNA isolation and analysis

Total DNA was isolated from 500 mg of plant material according to Dellaporta etal. [6]. Agarose gel electrophoresis, Southern blotting, hybridization and plasmid isolation were done essentially according to Visser etal. [30]. Restricted DNA was probed for the kanamycin resistance gene using a fragment encompassing the neomycin phosphotransferase gene. DNA was labelled using the random primed labelling technique [9].

Results

Adventitious shoot regeneration on leaf and stem explants

When leaf explants of 7322 and 86.040 were transferred to M 384 medium formation of callus and shoots was visible from two weeks on. Within 30 days 70% of the leaf explants showed shoot regeneration, and this increased to 85 ~o after six weeks (Fig. 1). When the same procedure was used for shoot regeneration on stem explants wound sites started 100

xJ

Lu

Z

t.u Q: 50

20

30

4O DA Y5

Fig. 1. Adventitious shoot regeneration on 7322 potato leaf

(x-x) and stem (o-o) explants. % Regeneration is given as the number of explants developing one or more shoots.

332

Table1. Comparisonof shooting response and transformationefficiencybetween transformationprocedures I and II. Transformation procedure

I II

Number of experiments

7 3

Total number of infected segments

Number of explants with shoots on kana5°*

Transformation (~o)I

leaf

stem

leaf

stem

leaf

stem

1390 600

1390 520

4 14

12 42

0.3 2.3

0.9 8.1

* Kana5° indicates medium supplementedwith 50 mg/1kanamycin. Determined as number of explants producingkanamycin-resistantshoots divided by the total number of infected explants.

to swell within one week and a thin layer of callus proliferated. A comparison with leaf explants showed that shoot regeneration on stem segments was delayed, shoot buds appearing on both sides of the stem explant from three weeks on and only 2 0 ~ of the explants showing shoot regeneration within 30 days (Fig. 1). However, after six weeks the percentages of stem and leaf explants with shoot formation were equal. The addition of cefotaxim to any medium did not affect adventitious shoot regeneration on either leaf or stem explants.

Conditions for transformation of explants with binary T-DNA vectors Stem and leaf explants infected with binary strains of A. tumefaciens and cultured on cefotaxim containing media did not show a different pattern of adventitious shoot regeneration when

compared to uninfected explants. Untreated leaf or stem explants were never able to produce adventitious shoots on media with 10 mg/1 kanamycin or more. However, stem explants occasionally callused at this concentration.

Induction and selection of transgenic plants When inoculated explants were directly transferred to medium with kanamycin (Procedure I), callus was formed on stem explants, but hardly ever on leafexplants. Regenerating explants could hardly be obtained and the very few shoots that appeared did so only after nine weeks and longer. The calli themselves stopped growing and eventually died. When inoculated explants were transferred to M 384 medium containing kanamycin after 5 to 7 days (Procedure II), leaf and stem explants started to form a small layer of callus at their wound sites and shoots started to appear

Table2. Seasonalinfluenceon the transformationefficiencyin potato. Period

Transformation procedure

Number of experiments

Total number of inocculated explants2

Transformation ( ~o)1

Autumn

I II I II

4 1 3 2

2000 220 780 900

0.3 _3 1.2 6.2

Spring

1 See Table 1. 2 Leaf and stem explants. 3 Only one experimentwas performed, no explants with kanamycin-resistantshoots obtained.

333 within six weeks. Table 1 summarizes the results of several experiments performed with either transformation procedure. It is obvious that transformation procedure II is far more efficient (9 times) than procedure I and that with both procedures stem explants yielded more kanamycinresistant shoots than did leaf explants. It was found that, irrespective of the procedure followed, experiments that had been started in autumn or winter hardly produced any shoots, whereas those started in spring gave much better results (Table 2). Control explants did not show this strong seasonal influence, they formed adventitious shoots efficiently in all seasons.

able to form roots or adventitious shoots on these media. Twenty-eight of the 31 shoots, which were able to root on medium containing 50 mg/1 kanamycin, formed also roots on medium with 100 mg/l kanamycin. As one might expect multiple shoots isolated from one explant often showed different reactions to kanamycin, for every shoot might be the resuk of an independent transformation. When leaf explants from 7 shoots

Characterization of transgenic shoots From 56 regenerating explants obtained with procedure II, 145 plantlets were obtained, as all explants showed multiple shoot formation. 52 out of the 145 shoots were tested irrespective whether they were transgenic or not. Two criteria were taken into account: 1) the ability of adventitious shoot regeneration on leaf explants to occur in the presence of 10 mg/1 kanamycin; 2) the ability of root formation on MS 30 to occur with 50 rag/1 kanamycin. Table 3 contains the combined results and shows that 92 ~o of the shoots either form roots or adventitious shoots, or both, on kanamycin-containing media. Control (untransformed) shoots were unTable 3. Adventitious shoot formation on 10 mg/1 kanamycin and root formation on 50 mg/l kanamycin by transgenic (n = 52) and control (n = 15) shoots.

Root formation ~

Adventitious shoot formation

T

27

4

C

0

0

T C

17 0

4 15

T, transgenic shoots; C, control shoots.

Fig. 2. Characterization of transgenic shoots. A. The effect of kanamycin on root formation of untransformed (C) and transformed (PTi-53 and PTi-80) shoots. The average weight of roots from two independent experiments (4 shoots per experiment) after a culture period of 20 days is illustrated against the kanamycin concentration. The weight of the roots at 0 mg/1 has been taken as 100%. B. Autoradiograph showing NPT-II activity of extracts from kanamycin-resistant shoots. Lanes 1-5, transformants: lane 1, PTi-9; lane 2, PTi53; lane 3, PTi-150; lane 4, PTi-80; lane 5, PTi-144; lane 6, nontransformed control. The position of bacterial NPT-II as control is indicated in the right margin as Km-P (kanamycin phosphate).

334 (originally obtained from different explants) were put through a second adventitious shoot regeneration cycle, resulting shoots all showed the same reaction to the two transformation criteria as before, thus showing stable phenotypes and indicating that chimerism, which may be found among regenerated shoots [ 14] is not likely to be a cause of different reactions by shoots which had been obtained from the same explant. Some of the 52 regenerants were analysed in more detail for their resistance to kanamycin and the expression and presence of the NPT-II gene. Some typical results are illustrated in Figs. 2 and 3. The resistance of two transformants (PTi-53

Fig. 3. Southern blot analysis of DNA from potato transormants. A. Total DNA was digested with restriction endonuclease Hind III. The blot was probed with a radiolabeled NPT-II gene fragment. Lane 1 is a digest of nontransformed DNA and lanes 2-8 are digests of transformed DNA. Lane 2, PTi-9; lane3, PTi-53; lane4, PTi-150; lane5, PTi-177; lane 6, PTi-80; lane 7, PTi-138; lane 8, PTi-144. The sizes of the molecular weight markers are indicated. B. Diagram of the T-DNA region of pVU1011. Flags indicate the border fragments, region drawn in black denotes the probe. Abbreviations: PNOS, nopaline synthase promoter; NPT-II, neomycin phosphotransferase gene; TNOS, nopaline synthase terminator; PCaMV, cauliflower mosaic promotor; HPT, hygromycin phosphotransferase gene; E, Eco RI; H, Hind !II.

and PTi-80) to kanamycin was determined by comparing root growth on MS 30 medium with 0, 50, 100 and 150 mg/l kanamycin (Fig. 2A). It is evident from these results that both transformants were less sensitive to kanamycin than was the control, PTi-53 being more resistant than PTi-80. NPT II enzyme activity in extracts from 16 transformants was assayed after electrophoresis on a non-denaturing polyacrylamide gel. The activity was present in extracts from transformants (Fig. 2B, lanes 1-5 and data not shown), but absent in extracts from nontransformed control material (lane 6). Furthermore, these results show that pTi-53 (lane 2) contains more NPT-II enzyme activity than pTi-80 (lane 4) which accounts for its higher resistance to kanamycin. Molecular proof for the presence of the NPT-II gene was obtained by Southern blot analysis of DNA from 12 transformants. Hind III-digested DNA was hybridized to a NPT-II fragment thus revealing homologous sequences not found in untransformed plant material (Fig. 3). If only one copy of intact T-DNA was transformed the probe should hydribize to one Hind III fragment, of which one end is joined to plant DNA, with a minimal size of 1.6 kb (Fig. 3B). Three transformants revealed two to five bands, indicating the presence of multiple copies of the T-DNA. No relation was found suggesting that transformants which contain more copies (integrations) of the NPT-II gene have also a higher NPT-II enzyme activity. Transformant PTi-80, which contains only one independent integration (copy), has indeed a low NPT-II enzyme activity (Fig. 2B, lane 4), but for transformant PTi-9 (Fig. 2B, lane 1) with 2 integrtions the NPT-II activity is even less than for PTi-80, whereas for transformant PTi-144 with 2 integrations the NPT-II enzyme activity is at least equal to that of transformant PTi-150 with 5integrations (Fig. 2B, lanes 5 and 3). As some of the transformed shoots contain pVU1011 T-DNA, which also confers hygromycin resistance, they were tested for resistance on medium containing 10 mg/1 hygromycin, a concentration lethal for normal potato shoots. All 10 shoots stayed healthy-looking and dit not bleach

335 out like the control shoots did within two weeks. Only two shoots were able to form roots which were abnormal in appearance (date not shown). Forty shoots were micropropagated and transferred to soil. The morphology of 35 transgenic plants was identical to that of the untransformed control di- and tetraploid 86.040 and 7322 potato plants. Five abnormal plants were obtained, four of which differed only in their leaf shape. All plants, including the phenotypical abnormal ones, flowered well. All shoots tested gave in vitro tuberization on kanamycin-containing medium (50 mg/1).

Cytological analysis

The ploidy level from 20 transformed plants was estimated by counting chloroplasts in stomatal guard cells. Both diploid and tetraploid plants were present among the transformed plants. Of 13 of these plants the chromosome number in root tip cells was determined. Four plants contained the diploid number of chromosomes (2n = 2x = 24) and seven the doubled number of chromosomes (2n = 4x = 48). Two plants with an aneuploid number ' of chromosomes (2n = 2x = 45 and 47) were found among the tested plants. Only the plant with 45 chromosomes had an abnormal phenotype.

Discussion

The transformation procedure for potato described in this paper uses leaf and stem explants. This approach, in analogy to the transformation procedures described for petunia, tobacco, tomato and Brassica napus [ 16, 18, 12], yields transformants within six weeks. As can be seen from Table 1, transformation procedure II, involving a period without selective pressure, is more efficient and yields shoots faster than procedure I. The higher efficiency of procedure II might be caused by multiplication of transformed cells in the 5 to 7 days without selective

pressure. A larger cluster of transformed cells should be able to cope better with kanamycin stress than one single cell. The transformation efficiency obtained with procedure II (6-8%) is comparable to the transformation efficiencies obtained with tomato and Brassica napus: 7-15 % [ 18, 12]. Transformation frequencies reported for tuber slices of the potato cultivar Dtsir~e vary from 1% [29] to 20% [27]. Although the adventitious shoot regeneration is initially better with leaf explants (Fig. 1), about 75 % of the explants producing kanamycin-resistant shoots were stem explants. This is in contrast with the results of An et al. [ 1 ] who reported that stem explants were less amenable to transformation than leaf explants. The reason for higher yield of kanamycin-resistant shoots from stem explants could lie in the initial slow response to shoot regeneration in general. The seasonal influence on the efficiency of transformation was very large (Table 2). Experiments performed in the spring were with both transformation procedures much better than experiments performed in autumn. This effect is most probably due to the physiological state of the plant material in combination with the kanamycin stress. Control experiments on kanamycin-free medium with inoculated or untreated explants produced always numerous shoots in any time of the year. The 52 putative transgenic shoots which were further analyzed for their ability to regenerate roots and shoots in the presence of kanamycin could be divided in four classes (Table 3). Although most shoots were positive for one or both criteria, 8 % were negative. This class could be formed by the so called 'escapes' or by shoots that had a lower expression of their resistance gene or had completely lost it. The two classes which are positive for only one criterion are no doubt transformed as control shoots never showed a positive reaction to either one of the criteria. This is also evident from the fact that some shoots which did not form roots on 50 mg/1 kanamycin were able to root on a lower (25 mg/1) kanamycin concentration (results not shown). One of the shoots not able to produce adven-

336 titious shoots on kanamycin containing medium (PTi-9) possessed NPT-II enzyme activity (Fig. 2B, lane 1) and contained NPT-II sequences (Fig. 3A, lane 2). Results from the DNA analysis on 7 plants (Fig. 3) showed that four plants acquired more than one copy of the TDNA which is in good agreement with previous results obtained with binary vectors [3, 28]. In this respect it was observed that the presence of multiple T-DNA copies does not necessarily mean a high degree of resistance to kanamycin or a strong NPT-II reaction (confirm the results in Fig. 2B, lanes 1 and 4, with those in Fig. 3A, lanes 2 and 6). Regenerants from potato explants show a large degree of somaclonal variation [24]. Five plants out of a total of 40 of our plants were abnormal, but only one plant was grossly deformed. Cytological analysis showed that only two of these five morphological abnormal plants contained an aneuploid number of chromosomes. The fact that the kanamycin resistance trait is still present after prolonged culturing on kanamycinfree medium shows that the trait is stably integrated into the genome.

Acknowledgements We like to thank Dr P. van den Elzen, Mogen International, for providing plasmid pVU1011, D. la F6ber and B. Hermelink for technical assistance, and H. Mulder for preparing the photographs. This work was supported by a grant from ISP (Integraal Structuur Plan Noorden des Lands).

References 1. An G, Watson BD, ChiangCC: Transformation of tobacco, tomato, potato and Arabidopsis thaliana using a binary Ti vector system. Plant Physiol 81:301-305 (1986). 2. Baulcombe DC, Saunders GR, Bevan MW, Mayo MA, Harrison BD: Expression of biological active viral satellite RNA from the nuclear genome of transformed plants. Nature 321:446-449 (1986).

3. Bevan M: Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12:8711-8721 (1984). 4. Boyer HW, Roulland-Dussoix D: A complementation analysis of the restriction and modification of DNA in Escherichia coil J Mol Biol 41:459-472 (1969). 5. Davis BD, Mingioli ES: Mutants of Escherichia coli requiring methionine or vitamin B~2. J Bact 60:17-28 (1959). 6. Dellaporta SL, Wood J, Hicks JB: A plant DNA minipreparation: Version II. Plant Mol Biol Rep 1:19-21 (1983). 7. DittaG, Stanfieid S, Corbin D, Helinski DR: Broad host range DNA cloning system for gram negative bacteria; construction of a gene bank of Rhizobium melilote. Proc Natl Acad Sci USA 77:7374-7351 (1980). 8. van den Elzen PJM, Townsend J, Lee KY, Bedbrook JR: A chimeric hygromycin resistance gene as a selectable marker in plant cells. Plant Mol Biol 5:299-302 (1985). 9. FeinbergAP, VogelsteinB: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 (1983). 10. Fraley RT, Rogers SG, Horsch RB: Genetic transformation in higher plants. CRC Crit Rev Plant Sci 4:1-46 (1986). 11. Frandsen NO: Die Plastidenzahl als Merkmal bei der Kartoffel. Theor Appl Genet 38:153-167 (1968). 12. Fry J, BarnasonA, HorschRB: Transformation of Brassica napus with Agrobacterium tumefaciens based vectors. Plant Cell Rep 6:321-324 (1987). 13. Holmes DS, Quickly M: A rapid boiling method for the preparation of bactrial plasmids. Anal Biochem 114: 193-201 (1981). 14. Hovenkamp-Hermelink JHM, Jacobsen E, Ponstein AS, Visser RGF, Vos-Scheperkeuter GH, Bijmolt EW, Vries JN de, Witholt B, Feenstra WJ: Isolation of an amylose-free starch mutant of the potato (Solanum tuberosum L.). Theor Appl Genet 75:217-221 (1987). 15. Hovenkamp-Hermelink JHM, Jacobsen E, Pijnacker LP, de Vries JN, Witholt B, Feenstra WJ: Cytological studies on adventitious shoots and minitubers of a monoploid potato clone. Euphytica (in press). 16. Horsch RB, Fry JE, Hoffmann NL, Wallroth M, Eichholtz D, Rogers SG, Fraley RT: A simple and general method for transferring genes into plants. Science 227: 1229-1231 (1985). 17. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). 18. McCormick, Niedermeyer J, Fry J, Barnason A, Horsch R, Fraley R: Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens. Plant Cell Rep 5:81-84 (1986). 19. Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473-497 (1962)

337 20. Ondrej M, MatousekJ, VlasakJ: Differentiation of transformed plants from tumors induced by Agrobacterium rubi ATCC 13335. J Plant Physiol 126:397-407 (1987). 21. Ooms G, Hooykaas PJJ, van Veen RJM, van Bellen P, Regensburg-Tuink TJG, Schilperoort RA: Octopine Tiplasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T-region. Plasmid 7:15-29 (1982). 22. Ooms G, Karp A, Roberts J: From tumour to tuber: tumour cell characteristics and chromosome numbers of crown gall-derived tetraploid potato plants (Solanum tuberosum cv. Maris Bard). Theor Appl Genet 66:169-172 (1983). 23. Ooms G, Karp A, Burrell MM, Twell D, Roberts J: Genetic modification of potato development using RiTDNA. Theor Appl Genet 70:440-446 (1985). 24. Ooms G, Burrell MM, Karp A, Bevan MW, Hille J: Genetic transformation in two potato cultivars with TDNA from disarmed Agrobacterium. Theor Appl Genet 73:744-750 (1987). 25. Pijnacker LP, FerwerdaMA: Giemsa C-banding of potato chromosomes. Can J Genet Cytol 26:415-419 (1984). 26. Reiss B, Sprengel R, Willi M, Schaller H: A new, sensi-

27.

28.

29.

30.

31.

32.

tive method for qualitative and quantitative assay of the neomycin phosphotransferase in crude cell extracts. Gene 30:211-218 (t984). SheermanS, BevanMW: A rapid transformation method for Solanum tuberosurn using binary Agrobacterium tumefaciens vectors. Plant Cell Rep 7:13-16 (1988). Spielman A, Simpson RB: T-DNA structure in transgenic tobacco plants with multiple independent integration sites. Mol Gen Genet 205:34-41 (1986). Stiekema WJ, Heidekamp F, Louwerse JD, Verhoeven HA, Dijkhuis P: Introduction of foreign genes into potato cultivars Bintje and Desiree using an Agrobacterium tumefaciens binary vector. Plant Cell Rep 7:47-50 (1988). Visser RGF, Hoekstra R. van der Leij FR, Pijnacker LP, Witholt B, Feenstra WJ: In situ hybridization to somatic metaphase chromosomes of potato. Theor Appl Genet 76:420-424 (1988). White DWR, Greenwood D: Transformation of the forage legume Trifolium repens L. using binary Agrobacterium vectors. Plant Mol Biol 8:461-469 (1987). Willmitzer L, Sanchez-Serrano J, Buschfeld E, Schell J: DNA from Agrobacterium rhizogenes is transferred to and expressed in axenic hairy root plant tissues. Mol Gen Genet 186:16-22 (1982).