Plant Physiol. (1986) 81, 301-3050032-0889/86/8 1/0301/05/$0 1.00/0

Short Communication

Transformation of Tobacco, Tomato, Potato, andArabidopsis thaliana Using a Binary Ti Vector System'

Received for publication November 27, 1985 and in revised form February 10, 1986

GYNHEUNG AN*, BRIAN D. WATSON, AND CHIN C. CHIANGInstitute ofBiological Chemistry, Washington State University, Pullman, Washington 99164-6340

ABSTRACT

Using a binary tumor-inducing (Ti) plasmid vector system, severalplant species were transformed with a kanamycin resistance marker(neomycin phosphotransferase gene). Four Nicotiana species, seven to-mato cultivars, two potato cultivars, and Arabidopsis thaliana weretransformed by the binary vector transformation method. In this method,various plant organ pieces were co-cultivated with Agrobacterium tumc-faciens cells carrying the binary vector, pGA472, and a helper Ti plasmid.We have also demonstrated that a wild type Ti plasmid can be used as ahelper to obtain a transformed plant.

Several plant vectors have been developed from Ti2 or Riplasmids ofAgrobacterium (4, 7, 24). The most convenient vectorsystem in terms of molecular manipulations and plant transfor-mation is the binary vector system (15) in which the vectorcontains the cis-acting elements required for plant transforma-tion (2, 6). The other necessary functions are provided in transby several genes on either the helper Ti plasmid (15, 18) or thechromosome (12) of Agrobacterium. Although many higherplants are naturally susceptible to Agrobacterium infection (3),each plant species is sensitive to only certain strains ofAgrobac-terium (9). This host specificity is dependent both on the natureof the Ti or Ri plasmid harbored by the bacteria and thechromosomal background (9, 22). Therefore, the full host rangeofAgrobacterium can be utilized with binary vectors by choosingthe appropriate Ti (or Ri) plasmid and the bacterial strain.Tobacco and a few closely related species have been trans-

formed with nontumorigenic markers such as the kanamycinresistance gene (2, 5, 11, 14) using the protoplast co-cultivationmethods (20) based on Ti plasmid vector system of Agrobacte-rium tumefaciens. Tobacco and petunia protoplasts were trans-formed with the kanamycin resistance gene and plants expressingthe transferred gene were successfully regenerated (8, 16). Be-cause regeneration of protoplasts is difficult for many plantspecies (10) new transformation methods have been developed(1, 2, 17) in which leaf slices or cultured calli are directly co-cultivated (without protoplasting) with Agrobacterium and sub-

'Supported in part by grants from the National Science Foundation(DCB-8417721), the United States Department of Agriculture (85-CRCR-1-1746), and Rockefeller Foundation. This is Scientific PaperNo. 7339, College of Agriculture Research Center, Washington StateUniversity.

2Abbreviations: Ti, tumor inducing; Ri, root inducing; MS, Mura-shige-Skoog.

sequently selected for drug resistant transformants. In an earlierstudy, a binary vector system was developed which mediatedtransformation of tobacco at a high frequency (1, 2). Althoughthe host-range specificity of Agrobacterium is quite large, it wasnot clear whether a wide variety of plants could be successfullytransformed by this vector-transformation system. Here we re-port transformation of leaf or stem pieces from several plantspecies using the binary Ti plasmid system.

MATERIALS AND METHODSPlant Materials. Four species of tobacco plants (Nicotiana

glauca, N. plumbaginifolia, N. rustica, and N. tabacum cv Xan-thi) and two potato (Solanum tuberosum) cultivars (tetraploidline, Russet Burbank, and diploid line, ADX262-9) were main-tained on a MS agar medium (23) as sterile shoot cultures. Plantsof seven tomato (Lycopersicon esculentum) cultivars (Ace 55VF,Beefsteak, Bonny Best, Fireball, Roma, Rutgers, and L. esculen-tum x L. peruvianum habrid) and ofA. thaliana strain Columbiawere developed on a MS agar medium from sterile seeds. Allplants were grown under light (3,000 lux) at 28°C for 12 h andin dark at 24°C for 12 h.

Transformation of Plants. Leaves were cut to about 0.1 to 0.5cm2 and stems were cut to about 1 to 2 cm long. The plantsections were wounded with forceps and cultivated in 2 ml ofMS liquid medium in 60 mm Petri dishes with about 108 A.tumefaciens cells. After co-cultivation for 2 d, bacterial cells werewashed off and the plant slices were grown on a callus or shootinduction agar medium containing 200 mg/L of kanamycin and500 mg/L of carbenicillin. Transformants were selected on a MSagar medium with the following supplements: 2 mg/L of naph-thaleneacetic acid and 0.5 mg/L of BA for tobacco callus induc-tion; 0.5 mg/L of BA for tobacco shoot induction; 2 mg/L of2,4-D, 0.5 mg/L BA, and 15 mg/L glutamine for potato callus;0.5 mg/L BA and 15 mg/L glutamine for potato shoot induction;2 mg/L of 2,4-D for tomato callus; 0.5 mg/L, 2,4-D and 0.05mg/L of kinetin for A. thaliana (10).Neomycin Phosphotransferase Assay. Plant materials used for

the enzyme assay were from independently isolated tissues andfree of the Agrobacterium. Plant tissues were lysed by homoge-nization in 1 ml/g ofplant extraction buffer (0.5 M sucrose, 0.1%ascorbic acid, 0.1% cystine-HCl, and 0.1 M Tris-HCl [pH 7.5]).The lysate was centrifuged for 5 min in an Eppendorf centrifugeand supematant adjusted to 1 mg/ml of total protein. Twenty zlof the crude extract was used for the neomycin phosphotransfer-ase assay as described previously (2, 13).

RESULTS AND DISCUSSIONTobacco plants maintained as shoot cultures were sliced and

co-cultivated with A. tumefaciens strains containing the binary301

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vector pGA472 and a helper Ti plasmid pTiBo542 or pAL4404in MS liquid medium for 2 d without agitation. The binary Tiplasmid pGA472 (2) carries, in addition to the other cis-actingelements such as the T-DNA borders and a wide host rangereplicon, a chimeric fusion of the nopaline synthase promoterand the neomycin phosphotransferase gene that confers kana-mycin resistance to transformed plant cells. The helper Ti plas-mid Bo542 is supervirulent in nature (22) and transforms tobaccocells several-fold better than other Ti plasmids (1, 2). pAL4404is an avirulent derivative of the octopine Ti plasmid Ach5obtained by deletion of the entire T-DNA that carries the tumorgenes (15). During the co-cultivation period Agrobacterium cellscoaggregated and attached to plant tissues. Small pieces of to-bacco tissues were normally killed by the treatment. After co-cultivation, bacterial cells were removed by washing the plantswith a MS medium and the co-cultivated tobacco plant sliceswere placed on either callus or shoot induction medium contain-ing 200 mg/L of kanamycin. Transformed tobacco calli or shootsdeveloped from the co-cultivated leaf tissues within 3 to 4 weeks(Fig. 1). Using pAL4404 as a helper, several hundred transformedcalli or shoots can be routinely produced per Petri plate from allfour tobacco species. Since each callus may represent manytransformation events, this represents a minimum value for thenumber of plant cells transformed. Transformed tissues alsodeveloped from tobacco stems at a much lower frequency. Ahigher frequency of transformed calli was observed with Agro-bacterium containing Bo542 as a helper compared with pAL4404as observed previously (1,2). Since the helper Ti plasmid Bo542carries tumorigenic genes on its T-DNA, kanamycin resistanttransformants may alsocanry the tumor genes and these wouldinhibit normal regeneration. When regenerating tobacco proto-plasts were co-cultivated with Agrobacterium containing pGA472and pTiBo542, about 20% of the kanamycin resistant transform-ants were co-transferred with the tumor genes (2). Therefore,some of the transformed tobacco calli should be free of the tumorgenes and therefore be able to regenerate to mature plants. Infact, we were able to induce shoots and roots from some of thekanamycin resistant tobacco calli transformed with the wild typehelper Ti plasmid, pTiBo542 (Fig. 2A). Mature plants regener-ated from the transformed calli continued to carry and expressthe kanamycin resistance gene (TableI).Two lines of potato shoot cultures were also transformed with

the Agrobacterium strains containing the binary vector pGA472and a helper Ti plasmid (pTiBo542 or pAL4404) by the sameco-cultivation method described for tobacco plants. Both leafand stem were transformed with the kanamycin resistance gene(Fig. 3), although stems were less amenable to transformation bythis method. Several transformed calli were developed from eachleaf section as observed with tobacco transformation. The trans-formation frequency was similar to that of tobacco plants. Acouple of hundred kanamycin resistance calli can be obtainedper Petri plate. Transformed potato calli continue to grow onkanamycin medium and express the drug resistance gene (TableI). At a low frequency, kanamycin resistance shoots and rootswere regenerated from the transformed potato calli (Figs. 2B and3).We have examined seven tomato cultivars for the ability to be

transformed by the co-cultivation methods. Surface sterilizedseeds were germinated on a MS agar medium and sections ofcotyledons and hypocotyls were co-cultivated with the Agrobac-terium strain containing pGA472 and pTiBo542. Several trans-formed calli were developed from most of the treated cotyledonsof all seven tomato cultivars (Fig. 4). The transformation fre-quency was similar to that of tobacco and potato. At low fre-quency hypocotyls were also transformed by the methods.Expression of the kanamycin resistance gene in the calli furtherconfirmed that the resistant cells were genuine transformants

~~~~~~~~~~~~~L-

FIG. 1. Transformation of N. Tabacum cv Xanthi. Kanamycin re-sistant calli developed on a callus induction medium 4 weeks (A) or 6weeks (B) after co-cultivation. Kanamycin resistant shoots regeneratedon a shoot induction medium 5 weeks (C) or 6 weeks (D) after co-cultivation.

(Table I).A. thaliana plants grown on sterile agar medium for 4 to 5

weeks were cut and wounded as described in "Materials andMethods" and used for the co-cultivation experiments. Kana-mycin resistant calli were developed only on wounded stems,primarily at the axil (Fig. 5). The transformation frequency wassignificantly lower than that of tobacco. Less than 10% of stemsco-cultivated with pTiBo542 as a helper produced transformedcalli. No transformants were obtained with pAL4404. Expressionof the kanamycin resistance gene in the transformed calli wasdetected by measuring neomycin phosphotransferase activity(Table I).

302AN ET AL.

TRANSFORMATION OF PLANT SPECIES AND ARABIDOPSIS THALIANA

A

r,III

C

FIG. 2. Regenerated plants. N. Tabacum cv Xanthi (A) or S. tubero-sum cv ADX 262-9 (B) plant was regenerated on a MS medium contain-ing 50 mg/L kanamycin.

Table I. Neomycin Phosphotransferase Activity ofthe Transformed andUntransformed Cells

Plant Isolate Transformed UntransformedNo. cpm/20 gi ojfcrude extract

Tobacco 1 1450 1002 1210 90

Potato 1 930 1302 1700 110

Tomato 1 1160 902 1320 80

A. thaliana 1 1700 902 1340 110

B

D

FIG. 3. Transformation of S. tuberosum cv ADX 262-9. Kanamycinresistant calli developed on a leaf slice (A) or a stem section (B) 6 weeksafter co-cultivation. Shoot regeneration (C and D) from the kanamycinresistant calli 5 months after co-cultivation.

We observed that several factors influence transformationfrequencies. Addition of different amounts (106-l08 cells) ofAgrobacterium to the co-cultivation medium did not significantlyinfluence the transformation frequency. However, it may benecessary to use minimum amounts of bacteria for the transfor-mation of certain plants that are sensitive to Agrobacterium. Forsome plants, such as A. thaliana, efficient transformation wasobtained when the selection occurred in the dark, but no trans-formants have been obtained under light. However, both tobaccoand potato can be efficiently transformed under light and sinceshoot induction is delayed in the dark, we routinely select fortransformed shoots under light. Therefore, the level of lightintensity may have a pronounced effect on the transformationfrequency depending on the plant species. Frequently transfer-ring (every 2-3 weeks) of the co-cultivated tissues to a newselective medium enhanced development of transformed tissues,probably because untransformed tissues die on the kanamycinselection medium and release toxic compounds.The transformation method used in this study can be applied

to a variety of plant species by selecting the best helper strain.We have demonstrated that a wild type helper Ti plasmid con-taining the tumor inducing genes on the T-DNA can be used toobtain transformed plants. Therefore, it will not be necessary toconstruct avirulent helper Ti plasmids in which the tumor genesare deleted. Various species or cultivars of tobacco, potato, andtomato tested in this study were more or less equally transformedby the binary vector transformation system. The binary vectorsystem can conceivably be used for other plant species as well.Certain plants, however, may be refractive to transformation dueto the nature of the helper Ti plasmid. The utility of this binaryvector system permits the use of any wild type Ti plasmid.Therefore, by employing this binary vector with the appropriatehelper plasmid and Agrobacterium strain, the range ofplants thatcan be amenable to transformation technique may be quite large.

A. thaliana contains a small genome size (19) and thereforemuch attention has been recently directed to this cruciferousspecies as a model for the molecular analysis of gene expressionand regulation (21). We were able to transform only stem sections

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FIG. 4. Transformation of L. esculentum. Kanamycin resistant callideveloped on leaf (A, B, C, E, F) or stem (D) 8 weeks after co-cultivationof L. esculentum cultivar Fireball (A), Rutgers (B), Roma (C, D), Ace55VF (E), and Beefsteak (F).

of the mature plants. Therefore, careful selection of plant mate-rials is important for transformation of some plant species. Weare currently regenerating the transformed Arabidopsis calli.

Acknowledgments-We thank E. M. Meyerowitz, C. A. Ryan, and W. H.Loescher for the plant materials and T. W. Okida, C. Reeves, R. Thomburg, A.

Mitra, and P. R. Ebert for helpful discussion.

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