Proc. Nati. Acad. Sci. USAVol. 84, pp. 5345-5349, August 1987Genetics

T-DNA organization in tumor cultures and transgenic plants of themonocotyledon Asparagus officinalis

(Agrobacterium tumefaciens/monocotyledon transformation/kanamycin resistance)

BENNY BYTEBIER*, FRANCINE DEBOECK*, HENRI DE GREVE*, MARC VAN MONTAGU*t,AND JEAN-PIERRE HERNALSTEENS**Laboratorium voor Genetische Virologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium; and tLaboratorium voorGenetica, Rijksuniversiteit Gent, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium

Contributed by Marc Van Montagu, April 6, 1987

ABSTRACT Asparagus officinalis was the first monocot-yledonous plant from which hormone-independent and opine-producing crown gall tissue could be isolated. We confirm byDNA hybridization that tumor lines obtained after infection ofthis plant by Agrobacterium strains harboring wild-type no-paline and octopine tumor-inducing (Ti) plasmids are stablytransformed and contain transferred DNA (T-DNA) segmentsidentical to the T-DNA found in dicotyledonous plants. Wehave also infected Asparagus with a nononcogenic T-DNAvector that carries a chimeric aminoglycoside phosphotrans-ferase [NOS-APH(3')IIJ gene and selected transformed tissueson kanamycin-containing medium. The transformed status ofthese tissues was then confirmed by DNA hybridization. Fromthese calli we regenerated kanamycin-resistant shoots that weresubsequently rooted. Thus we report the isolation of transgenicmonocotyledonous plants engineered via the Agrobacteriumvector system.

The soil bacterium, Agrobacterium tumefaciens, causescrown gall, a neoplastic transformation of the wounded tissueof a wide range of dicotyledonous plants. All oncogenicAgrobacterium strains have a large tumor-inducing (Ti)plasmid. Tumor induction is the result of the transfer andstable integration of a well-defined portion of the Ti plasmid,called the T-region, into the plant genome. The transferredDNA (T-DNA) encodes oncogenic functions involved inphytohormone biosynthesis, which cause tumorous prolifer-ation of the transformed tissue. The T-DNA also encodesenzymes that synthesize tumor-specific compounds calledopines. The Ti plasmids are classified according to the typeof opine they specify. Best studied are the nopaline andoctopine type Ti plasmids. For a recent review on themolecular biology of crown gall disease, see ref. 1.The organization of the T-DNA of both nopaline and

octopine tumors of dicotyledonous plants has been elucidat-ed by Southern transfer and hybridization experiments (2-4).The nopaline T-DNA is a continuous stretch of 23 kilobases(kb) (2). Octopine tumors, however, may contain two inde-pendently transferred T-DNAs (3, 4). The left-hand T-DNA(TL) of 13.2 kb is always present. It includes the genesresponsible for tumorous proliferation of the transformedtissue (5-7) in addition to the octopine synthase gene (5, 8).The right-hand T-DNA segment (TR) is 7.9 kb in length andoriginates from a part of the plasmid located to the right of theTL region. The TR-DNA encodes the genes for the synthesisof agropine and mannopine (9). It is often absent from crowngall tumor cultures (3, 4).Three other findings were of importance in our understand-

ing of the A. tumefaciens DNA transfer system: (i) The

T-DNA is bounded by essentially identical 25-base-pair (bp)direct repeats. These sequences define the T-DNA. Thejunction between plant and bacterial DNA occurs within oradjacent to the border sequence in a number of independenttumor lines (10-13). (ii) The oncogenicity genes that preventnormal regeneration of transformed tissue are not necessaryfor T-DNA transfer and integration (5, 7). (iii) Any DNAsequence inserted between the 25-bp repeats is cotransferredwith the T-DNA to the plant genome (14). Based on thesefindings a number of vector systems were developed thattoday are commonly used to transform dicotyledonousplants.

Historically monocotyledonous plants were thought insen-sitive to Agrobacterium infection, although it has beenreported that at least some monocotyledons are susceptibleto infection (for review, see ref. 15). Only recently, however,has the evidence for transformation extended beyond mor-phological changes in the infected tissue (16-18). Hernal-steens et al. (16) isolated tumor tissue that would grow onhormone-free medium after infecting stem fragments ofAsparagus officinalis, a member of the family Liliaceae, withthe wild-type A. tumefaciens strain C58. This tissue producedthe opines nopaline and agrocinopine. However, the physicalorganization of T-DNA in the monocotyledon genomne wasnot demonstrated at that time, and therefore it was not knownwhether normal T-DNA integration had occurred in thisuncommonly used host.We report on DNA hybridization data of this tissue and of

another tumor line induced by A. tumefaciens strain C58C1pTiB6S3 harboring an octopine type Ti plasmid. The analysisof both Asparagus tumor lines shows that T-DNA integrationis very similar to the pattern of integration found in dicoty-ledons.Because the oncogenicity genes prevent normal regener-

ation of transformed tissue, we then tested whether anononcogenic Ti plasmid-derived vector containing a domi-nant selectable marker for plant cells could be used toconstruct transgenic monocotyledonous plants. After infect-ing cells with a vector that carried a chimeric kanamycin-resistance gene, we selected transformed callus on kana-mycin-containing medium. DNA hybridization confirmedthat T-DNA integration had occurred normally. From thistissue we could regenerate transgenic Asparagus plants.

MATERIALS AND METHODS

Hybridization Analysis. DNA was prepared (19) fromaxenically cultured tumor tissue. Before the final DNAprecipitation step, S ,tl of a 2.5 mg of pancreatic RNase A perml solution was added, and the reaction mixture was incu-

Abbreviations: Ti, tumor inducing; TL, left-hand 13.2-kb transferredDNA of octopine tumors; TR, right-hand 7.9-kb transferred DNA ofoctopine tumors; T-DNA, transferred DNA.

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bated at 370C for 15 min. Ten micrograms of DNA wasdigested, electrophoresed through a 0.8% agarose gel, andtransferred onto GeneScreenPlus (New England Nuclear) orHybond N (Amersham) membranes according to the manu-facturers' instructions. 32P-labeled DNA probes were pre-pared by nick-translation (20) or by the procedure describedby Feinberg and Vogelstein (21, 22). Hybridization andwashing was done using standard procedures (23). The filterswere exposed to x-ray films using an intensifying screen at-700C.Transformation and Selection. Asparagus transformation

by A. tumefaciens strain C58C1 pTiB6S3 was done asdescribed by Hernalsteens et al. (16). The transformationprocedure used for A. tumefaciens strain C58C1 pGV-3850::1103neo(dim) (24) relied on the same protocol. Invivo-grown Asparagus spears were surface-sterilized and cutinto 3-cm-long pieces, which were inverted and planted 5 mmdeep in half-strength Murashige and Skoog (MS) medium(35). A. tumefaciens C58C1 pGV3850::1103neo(dim) wascultured on Luria-Bertani medium (23) with 25 mg of kan-amycin per liter and applied directly to the upper end of theAsparagus segment with a spatula. One month after infec-tion, a 2-mm-thick slice ofinfected tissue was incubated twiceon Linsmaier and Skoog (LS) medium (36) with 200 mg ofglutamine per liter, 1 mg of 6-benzylaminopurine per liter,and 1 mg of a-naphthaleneacetic acid per liter (25) for 1 mo.The tissue was then transferred to the same callus-inducingmedium now supplemented with 50 mg of kanamycin sulfateper liter.

Regeneration. Kanamycin-resistant callus was transferredto LS medium with 200 mg of glutamine per liter, 40 mg ofadenine per liter, 4 mg of 6-benzylaminopurine per liter, and1 mg of a-naphthaleneacetic acid per liter (25) on which itregenerated shoots. These shoots were then moved to LS

AProbe A Probe 8

~co c9

Probe C

am.E

medium with 0.1 mg of 6-benzylaminopurine per liter forshoot proliferation. Clumps of shoots were divided into partscontaining 3-10 spears each and put on LS medium with 0.5mg of indole-3-acetic acid (26) for rooting. Rooted plantswere transferred into glass jars containing a 3-cm layer ofsterile horticultural perlite (Sibli, Liege, Belgium) moistenedwith half-strength MS mineral medium, and covered withplastic film. Daily the plastic cover was punctured until theplants were completely exposed to their surroundings; theywere then transferred to pots filled with perlite and wateredwith fertilizer-containing solution (Peters 20+20+20; 200 mgper liter).

RESULTS AND DISCUSSION

Analysis of Asparagus Tumor DNA Transformed by A.tumefaciens Strain C58. Fig. 1 presents the nucleic acidhybridization data that shows the T-DNA in this tissue isintegrated into the Asparagus genome. Internal fragmentsspanning the T-DNA from EcoRI fragment 16 on the left-hand side to the BamHI fragment 10 on the right-hand sidecan be detected in the different lanes. These fragments coverthe T-DNA over a length of 23 kb, which means that nodetectable deletions resulted from transfer or integration.The BamHI, HindIlI, and Sal I-digested DNA fragmentswere hybridized with a probe homologous to the right-borderregion (HindIII fragment 23); each lane shows four compositefragments of different lengths (the BamHI lane contains afifth fragment of 5 kb, which is the internal BamHI fragment10). Therefore, we conclude that at least four copies of theT-DNA are integrated into the Asparagus genome. Becausethis tumorous tissue is not of clonal origin, we do not knowwhether these copies represent multiple T-DNA insertions

Probe D

_-49CF 44-4.1 =4.1

-2.2-1.9

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-6.3-4.6

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-^ -2.05

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-2.25CF--2.1 CF

C

Hind IllEcoR 1BamH ISal!Sac 11

| 6.7(10) | 4.55(15) | 4.6(14b) 4 (19) L71 3.45(22) 119(31 3.3(23) 1E)I 11.4 (2)4.1(15) 631 4.1(16) 11.902)22(2.8T 4.4(14) I 4x 1 14.85 (1) l

12(1) L61 10(3) I 1 5(10) I 11.1 (2)1.81 7.75(3) | 8.2(2) | 3.85(3) 1 2.051 22 I I 4 (10) 1 1.7 1 1.6 1

I 1 4.4 1 6.3(5) 1 101) 1 5.25 1 2 1.4 1.7

nop T-DNA

FIG. 1. Southern blot analysis ofDNA from A. officinalis tumor tissue transformed by A. tumefaciens C58. (A) Hybridization pattern. Thesize of the restriction fragments is given in kb. Fragments marked CF are composite fragments; all others are internal fragments. Thehybridization probes used were as follows: probe A, pGV0369 (HindIII fragment 10 of pTi C58 in pBR322; ref. 2); probe B, Sac 11 fragment5 of pTi C58; probe C, Sac 1I fragment 1 of pTi C58; probe D, pGV0422 (Hindilll fragment 23 of pTi C58 in pBR322; ref. 2). (B) Restrictionmap of the T-region pTi C58. Size of restriction fragments is indicated in kb; fragment number is within parentheses (27). nop, nopaline.

.Vf-.

* +12 CF

_ -9.0 CF=6.8 CF6.2CF7CF6.2 CF

_-4.5 CF

B

-3CF

D

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into one founder cell or single insertions into several inde-pendently transformed founder cells. In the EcoRI lanehybridized with a left-border probe (HindIII fragment 10) twobands of 4.1 and 4.9 kb are detected. The 4.1-kb bandcorresponds to the internal EcoRI fragment 16. The leftborder is situated -50 bp to the left of the junction betweenEcoRI fragment 35 and EcoRI fragment 16 (10, 11); conse-quently there is insufficient homology with EcoRI fragment35 to visualize composite fragments in an EcoRI digestion.The second band of4.9 kb in this EcoRI lane can be explainedby assuming that, in this case, the T-DNA ended withinEcoRI fragment 16. Integration around the left border isknown to be less precise: junction fragments between plantsequences and EcoRI fragment 16 have also been found inNicotiana (2, 10, 12). That this 4.9-kb band is due to a partialdigestion of the DNA cannot be excluded, however, becauseit does not appear in the EcoRI-digested DNA hybridizedwith the Sac II fragment 5 (probe B). To summarize we canstate that, as in dicotyledons (2), multiple contiguous T-DNAs of 23 kb can be transferred and integrated into theAsparagus genome.

Isolation and Analysis of Asparagus Tumor DNA Trans-formed by A. tumefaciens Strain C58C1 pTiB6S3. A similarexperiment in which Asparagus stem fragments were infect-ed with the octopine type A. tumefaciens strain C58C1pTiB6S3 was done. One of twenty infected fragments devel-oped a tumor that could be cultured axenically on hormone-free medium. This tumor contained octopine, mannopine,and agropine (data not shown), which indicated that both TLand TR were present.

A Probe A Probe B

Fig. 2 shows DNA hybridization data. The left border ofTLis located in Sma I fragment 17, and the right border lies tothe right of Hpa I fragment 13 in EcoRI fragment 19.Fragments between these two, spanning the whole TL region,can also be seen in different lanes. In the EcoRI lanehybridized with a TR-specific probe (BamHI fragment 2) wedetect the internal EcoRI fragment 12. In this tissue left andright borders of TR appear located in their normal positionson EcoRI fragments 19 and 20, respectively. That severalcomposite fragments are detected in both hybridization withTL- and TR-specific probes points to insertions ofT-DNA intomore than one locus of the Asparagus genome. However,because this is not clonal tissue, we do not know if themultiple insertions of T-DNA are present in one cell. Fromhybridization of the EcoRI lane with a probe that hasspecificity for the right border of TL and the left border of TR(HindIll fragment 1) we could not detect EcoRI fragment 19.This must mean that TL and TR integrate independently intothe Asparagus genome and not as one contiguous fragment ashas been noticed in Helianthus annuus (30).Transformation ofAsparagus Using a Nononcogenic T-DNA

Vector. Fragments ofAsparagus spears were infected with A.tumefaciens strain C58C1 pGV3850::1103neo(dim) (24). TheTi plasmid harbored by this strain is devoid of all oncogenicsequences and carries, between the T-DNA border se-quences, the nopaline synthase gene and a chimerickanamycin-resistance gene consisting of the nopalinesynthase promoter fused to the coding region of the amino-glycoside phosphotransferase [aph(3')II] gene of Tn5 fol-lowed by the polyadenylylation signal of the octopinesynthase gene. Callus was induced from the wound tissue on

, ,Probe9

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'4 TL No * TR - s1 3.2 (14) I 2.8 1 2i .11. 1 1 41.11) I 5,7(5)1 I

19.15(11 1 7.6(8 7(17) I 15.15(2 .95 14.65(17) 11.511.25 (3) 1.7 17.3(7] II Z.2191 if[J III1465 f3a) 12117)1 2.6 ) I W.5 I3b) II 9.7a (7) 1 I 5.5(1)

FIG. 2. Southern blot analysis of DNA from A. officinalis tumor tissue transformed by A. tumefaciens strain C58C1 pTiB6S3. (A)Hybridization pattern. Only internal T-DNA fragments are indicated; size is given in kb. The hybridization probes used were restrictionfragments of the T-region of pTiB6S3. Probe A, BamHI fragment 8; probe B, Hindlll fragment 1; and probe C, BamHI fragment 2. (B) Restrictionmap of the T-region of pTiB6S3. Size of the restriction fragment is indicated in kb; fragment number is within parentheses (28, 29).

-73

-1.0

.-0.7

B

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AH E E HE S:1'1'I-H HindIll -10 - AmpR

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digesthomology with

probe A +

probe B

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16 18 19

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FIG. 3. Southern blot analysis of DNA from calli and transgenic plants of A. officinalis transformed by A. tumefaciens strain C58C1pGV3850::1103neo(dim). (A) Schematic representation of the plasmid cointegrate pGV3850::1103neo(dim). The plasmid pGV3850::1103neo-(dim) (24) was generated by introducing (31) pLGV1103neo(dim) (24) into A. tumefaciens C58C1 containing pGV3850. The vector pGV3850 (32)is a disarmed nopaline Ti plasmid in which all internal T-DNA sequences between Hind1li fragment 10 (carrying the left-border sequence) andHindlil fragment 23 (carrying the right-border sequence and the nopaline synthase gene) are substituted by the cloning vector pBR322. Theintermediate vector pLGV1103neo(dim) is a tandem duplication of pLGV1103neo (33) except for the Sal I fragment from Tn9O3 that is presentonly once. This pBR322 derivative contains a kanamycin-resistance gene, KmR, from Tn9O3 allowing selection in bacteria and a chimericNOS-APH(3')Il kanamycin-resistance gene, KmE-R, as a dominant selectable marker for plants. This chimeric gene consists of the TnS aph(3')1lcoding region fused on its 5' side to the nopaline synthase gene promoter and on its 3' side to a fragment of the octopine synthase gene containingthe polyadenylylation signal. A single recombination event between the regions of homology of pGV3850 and pLGV1103neo(dim), provided bythe pBR322 sequences, results in the T-region structure indicated. E, H, and S indicate EcoRI, Hindlil, and Sal I respectively; wavy lines indicateleft and right T-DNA border. AmpR is the ampicillin gene of pBR322, nos is the nopaline synthase gene. (B) Homology between the plasmidpGV3850::1103neo(dim) and the probes used. Probe A, pGV0369 (Hind)ll fragment 10 in pBR322; ref. 2); probe B, pGV0422 (Hind111 fragment23 in pBR322; ref. 2); probe C, pLGV1103neo(dim). The 3.0-kb HindlIl/EcoRl fragment does not contain any pBR322 sequences; thereforeit has no homology with probe A. However, it does have a small (300-bp) region of homology with probe B, due to the nos promoter. Becauseof this small homologous region, indicated as ± in the homology table, a faint internal 3.0-kb band appears after hybridization with probe B(see Fig. 3C). For the same reason (i.e., nos promoter homology) probe C has a small region of homology with the right border fragment; however,we see no composite bands appearing in the hybridization pattern. (C) Hybridization pattern. DNA was doubly digested with Hind11) and EcoRI.The numbers 16, 18, and 19 indicate three transformed callus lines. The transgenic plants 19-1, 19-2, and 19-3 were regenerated from callus line19. Only internal fragments are indicated; size is in kb. Differences in intensity of bands equal in size are due to unequal amounts ofDNA havingbeen loaded.

nonselective medium and transferred on kanamycin-contain-ing medium after 2 months. Three oftwenty induced calli (no.16, 18, and 19) continued to grow. Large amounts of nopaline,as well as the APH(3')II enzyme, could be detected in crudeextracts from these calli (data not shown).The results of hybridization experiments with the total

DNA extracted from these cultures are presented in Fig. 3.The lanes hybridized with probes A and B show that allfragments between the right HindIII site of HindIII fragment10 and the left HindIII site ofHindIII fragment 23 are present.This was also confirmed by a HindIII digestion (data notshown). The lanes hybridized with probe B (HindIII fragment10 in pBR322) further show a band of 2.9 kb correspondingto the right EcoRI/HindIII fragment of HindIII fragment 10in strain pGV3850::1103neo(dim). Because we cannot detectany composite bands in this hybridization we conclude thatthe T-DNA must end just inside EcoRI fragment 35. Com-posite fragments would have appeared if there were sufficienthomology with EcoRI fragment 35. They do appear after

digestion with HindIII (data not shown). Hybridization witha right-border probe also shows several composite frag-ments-meaning that more than one copy of this T-DNAinserted into the Asparagus genome.

Regeneration of Transgenic Asparagus Plants. Two of threetransformed callus lines regenerated shoots. These shootscontain nopaline, and three of them (no. 19-1, 19-2, and 19-3)that originated from one callus line were further examined forDNA content. Fig. 3 shows that no major rearrangements ofthe T-DNA occurred during regeneration. Several of theseshoots developed roots when transferred to a root-inducingmedium. They were then transferred into perlite (Fig. 4).Their appearance is similar to untransformed young Aspar-agus plants.Our results clearly demonstrate that T-DNA integration in

Asparagus is comparable to that in dicotyledons. The isola-tion of transgenic Asparagus plants gives us confidence in theuse of Agrobacterium as a vector system for the transfor-mation of other monocotyledons like the Gramineae (18, 34).

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FIG. 4. Transgenic Asparagus plants, both expressing thenopline synthase and NOS-APH(3')II gene.

We thank Marc De Beuckeleer for helpful advice on the DNAhybridization experiments; Plant Genetic Systems for hospitality;Dr. Depicker and Dr. Caplan for critical reading of the manuscript;Dr. Gruselle for advice on Asparagus regeneration and HildeNeirynck, Stefaan Van Gijsegem, and Karel Spruyt for preparationof this manuscript and figures. B.B. is supported by a fellowship ofthe Instituut tot aanmoediging van het Wetenschappelijk Onderzoekin Nijverheid en Landbouw. J.-P.H. is a Research Associate of theBelgian National Fund for Scientific Research.

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