Journal of Experimental Botany, Vol. 45, No. 274, pp. 649-656, May 1994Journal ofExperimentalBotany

Regeneration of transgenic plants of grapevine(Vitis vinifera L.) via Agrobacterium rhizogenes-mediated transformation of embryogenic calli

M. Nakano1, Y. Hoshino and M. Mii2

Laboratory of Plant Cell Technology, Faculty of Horticulture, Chiba University, 648 Matsudo, Chiba 271, Japan

Received 25 October 1993; Accepted 26 January 1994

Abstract

Genetically transformed roots and calli were inducedfrom leaf segments of grapevine (Vitis vinifera L. cv.Koshusanjaku) after co-cultivation with wild-typeAgrobacterium rhizogenes strains, but plant regenera-tion from them was not achieved. On the other hand,transgenic grapevine plants were obtained via somaticembryogenesis after co-cultivation of embryogeniccalli with an engineered A. rhizogenes strain includingboth the neomycin phosphotransferase II (NPT II) andthe /7-glucuronidase (GUS) genes, followed by selectionof secondary embryos for kanamycin resistance. Allthese plants showed GUS gene expression revealedby histochemical assay. Southern blot analysisrevealed the stable integration of the GUS cordingregion in their genome. Transformants containing RiT-DNA exhibited various phenotypes: most of themshowed a typical Ri-transformed phenotype such aswrinkled leaves, while the others looked normal.

Key words: Agrobacterium rhizogenes, grapevine, trans-genic plants, Vitis vinifera.

Introduction

Grapes are the world's most widely-grown fruit crop.Breeding of this crop has been mainly based on intra-and interspecific hybridization. However, like manyother fruit crops, production of novel grapevine cultivarsby these conventional methods is difficult and time-consuming because of the long generation cycle and highlevels of heterozygosity. It is, therefore, considered thatgenetic transformation is one of the most attractive means

to improve this important crop because genes encodingagriculturally desirable traits can be directly introducedinto pre-existing and desirable genotypes (Alleweldt andPossingham, 1988; Scorza, 1991).

Production of transgenic plants by Agrobacterium-mediated transformation has already been reported insome fruit crops including apple (James et al, 1989;Maheswaran et al, 1992; Lambert and Tepfer, 1992),Rubus (Graham et al, 1990), Ribes (Graham andMcNicol, 1991), Citrus (Hidaka et al., 1990; Moore et al.,1992), kiwi fruit (Uematsu et al., 1991; Rugini et al.,1991) and papaya (Fitch et al., 1993). In grapevines,considerable effort has also been made to establish genetictransformation systems (Hemstad and Reisch, 1985;Baribault et al, 1989, 1990; Guellec et al, 1990; Mullinset al, 1990; Matsuta et al, 1991; Berres et al, 1992).However, only two reports have appeared on the regen-eration of transgenic plants of grapevines: V. rupestris, aroot stock species (Mullins et al, 1990), and V. vinifera(Matsuta et al, 1991). In both reports, transgenic plantswere produced by disarmed A. tumefaciens strains. In thispaper, we report the production of transgenic plants ofV. vinifera by co-cultivating embryogenic calli with agenetically-engineered virulent A. rhizogenes strain.

Materials and methods

Plant material

In vitro shoot cultures of grapevine (V. vinifera L. cv.Koshusanjaku) initiated from shoot tips were maintained onNitsch's medium (Nitsch and Nitsch, 1969) lacking plantgrowth regulators but with 30 g dm"3 sucrose, and solidifiedwith 2g dm"3 gellan gum (Gelrite; Kelco, Division of Merck& Co. Inc., San Diego, USA) (NHF medium). The cultures

1 Present address: Iwate Biotechnology Research Centre, 22-174-4 Narita, KJtakami, Iwate 024, Japan.2 To whom correspondence should be addressed: Fax: + 81 473 66 2234.

© Oxford University Press 1994

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were kept at 27 °C under continuous illuminationm~2s~') with fluorescent lamps. Subculture was performedonce every two months by transferring terminal and lateralcuttings.

Induction of embryogenic calli

Embryogenic calli of cv. Koshusanjaku were induced accordingto the method of Matsuta and Hirabayashi (1989) with severalmodifications. Fully expanded leaves of plantlets were collectedone month after subculture, cut into pieces and placed onNitsch's medium containing 5 fiM 2,4-dichlorophenoxyaceticacid (2,4-D; Sigma), 5 /xM JV-2-chloro-4-pyridyl-./V-phenylurea(4PU; Kyowa Hakko Kogyo Co., Japan) and 30g dm"3

sucrose, and solidified with 2 g dm"3 gellan gum. After 40 d ofculture in the dark, leaf-derived calli were transferred to Nitsch'smedium lacking vitamins, inositol and glycine but with 1 ^M2,4-D and 30 g dm"3 sucrose, and solidified with 2g dm"3

gellan gum (NEC medium). They were then cultured at 27 °Cunder continuous illumination (35 fimo\ m" 2 s" ' ) . Four to 5months after transfer, somatic embryos developed from theprimary callus were transferred to fresh NEC medium.Embryogenic calli derived from the somatic embryos duringone month after transfer were maintained by subculturingmonthly on to NEC medium.

For inducing somatic embryos from the embryogenic callus,the calli were transferred to NHF medium and incubated at27°C under continuous illumination (50^mol m"2s"1).Somatic embryos developed from the calli after one month onNHF medium were transferred individually on to the freshNHF medium for germination and further growth. Secondarysomatic embryos frequently developed from both somaticembryos and somatic embryo-derived plantlets on this medium.

Agrobacterium strains

Three mikimopine-type A. rhizogenes strains, A13 (MAFF02-10266), A5 and D6 (Daimon et al., 1990), one agropine-type strain 15834 (Petit et al., 1983), and one engineeredA. rhizogenes strain A13/pBI121 were used in this study. StrainA13/pBI121 harboured both a wild-type Ri plasmid and thebinary vector pBI121. The pBI121 contained the neomycinphosphotransferase II (NPT II) gene linked to the nopalinesynthase promoter and the jS-glucuronidase (GUS) gene linkedto the cauliflower mosaic virus 35S promoter (Jefferson et al.,1987). Prior to co-cultivation, YEB liquid medium (Herrera-Estrella and Simpson, 1988) containing 20 /*M acetosyringone,and the same medium containing 100 mg dm"3 kanamycin inaddition, were inoculated with strain A13 and A13/pBI121,respectively. The cultures were incubated overnight at 27 °Cwith rotary shaking (200 cycles min"1).

Co-cultivation of leaf explants

Leaves harvested from plantlets one month after subculturewere cut into pieces 1 cm square. These explants were soakedinto overnight-cultured bacterial suspensions (about 1 x 108

cells cm"3) and incubated with rotary shaking (100cycles min"1) for 10 min. Then, they were blotted on tosterilized filter papers and co-cultivated on NHF mediumcontaining 20 ̂ M acetosyringone at 27 °C in the dark for 3 d.After the co-cultivation period, explants were transferred toNHF medium containing 500mg dm"3 cefotaxime (Claforan;Hoechst AG). Adventitious roots and calli that developedwithin one month were transferred to the same medium, andAgrobacterium-ftee cultures were established after several suc-cessive subcultures.

Co-cultivation of embryogenic calli

About 0.5 g fresh weight of embryogenic calli were suspendedin 10cm" of overnight-cultured bacterial suspensions andincubated with rotary shaking (100 cycles min"1). After 10 min,calli were transferred on to NEC medium containing 20 jxMacetosyringone. After 3 d of co-cultivation at 27 °C in the dark,calli were washed with sterilized distilled water containing500 mg dm"3 cefotaxime, and were spread over NHF mediumcontaining 500 mg dm"3 cefotaxime. The cultures were main-tained at 27 °C under continuous illumination. Somatic embryosarose within one month. Secondary embryos developed fromthe primary embryos were successively transferred to the samemedium. When the calli were inoculated with the engineeredstrain, 50 mg dm"3 kanamycin (Sigma) was added tocefotaxime-containing NHF medium.

Opine assay

About 100 mg fresh weight of tissues were ground in amicrotube. After centrifugation at 6000 xg for 15 min, 10 mm3

of crude extract was subjected to high-voltage paper electrophor-esis (Petit et al., 1983). The detection of mikimopine wasperformed with Pauly reagent (Isogai et al., 1990). Agropineand mannopine detection was performed as described earlier(Petit et al., 1983).

Plant DNA isolation and Southern blot analysis

DNA isolation and Southern blot analysis were performedaccording to the method of Baribault et al. (1990). Ashybridization probes, a 3.0 kbp Hindlll-EcoRl fragment frompBI121 (Jefferson et al., 1987) (for the GUS cording region)and a 4.9 kbp EcoRl fragment from pLJl (Jouanin et al., 1984)(for Ri T-DNA) were used.

GUS histochemical assay

Localization of GUS enzyme activity was detected using thehistochemical GUS assay (Jefferson et al., 1987). Leaf, stemand root segments excised from in v;7ro-growing plantlets weresubjected to the assay.

Results

Co-cultivation of leaf explants with wild-type A. rhizogenesstrains

Our initial trials on the co-cultivation of leaf explantswith wild-type A. rhizogenes strains, A5, A13, D6, and15834, resulted in the development of roots and calli fromthe cut ends in all four strains. However, significantdifferences (/><0.05) in per cent explants developing rootsand calli were found for A. rhizogenes strains (Table 1).Among the four bacterial strains, A13 and D6 effectivelyinduced roots and calli (over 60%). These roots and callishowed rapid growth which was sustained after excisingfrom the original explants and transferring to NHFmedium. Calli cultured on this medium occasionallydeveloped roots. In the control cultures, although fewYEB medium-inoculated explants produced roots andcalli, they did not show further growth on the fresh NHFmedium.

To confirm transformation, roots and calli showing

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Table 1. Effect of wild-type A. rhizogenes strain on per cent ofleaf explant with roots and/or calli

A. rhizogenes Per cent of leaves with Per cent of roots and callistrain roots and/or calli' containing opine*

ControlA5A13D615834

7.4 a22.2 b65.0 c63.4 c36.6 b

0100100100100

The data were recorded one month after the co-cultivation."The values represent the mean of three independent experiments.

Means followed by the same letter are not significantly different(P<0.05; Duncan's New Multiple Range Test).

'Opine analyses were performed on roots and calli from at least10 leaves.

sustained growth were subjected to opine assay. All ofthem showed the presence of mikimopine (for strains A5,A13 and D6), or agropine and mannopine (for strain15834), indicating that they were transformed tissues(Table 1). On the other hand, roots and calli developedin the control culture, and roots excised from originalplantlets did not produce detectable amounts of opine.

After establishing Agrobacterium-free cultures, trans-formed tissues were transferred to media supplementedwith various concentrations and combinations of auxins[2,4-D, a-naphthaleneacetic acid (NAA; Sigma)] andcytokinins [benzyladenine (BA; Sigma), zeatin (Sigma)and 4PU] in order to induce morphogenesis. However,plant regeneration has not so far been achieved (datanot shown).

Co-cultivation of embryogenic calli with a wild-typeA. rhizogenes strain A13

The embryogenic cell line induced from somatic embryosof V. vinifera cv. Koshusanjaku retained a highembryogenic ability after maintaining for over two yearson NEC medium, and produced numerous somaticembryos by transferring to NHF medium. In addition,somatic embryos developed from embryogenic calli alsofrequently produced secondary embryos.

Embryogenic calli were initially co-cultivated with awild-type strain A13, with which transformed roots andcalli were most efficiently developed from leaf explants.After the co-cultivation, most calli turned brown within3 to 5 d of culture on cefotaxime-containing NHFmedium. However, some of them started to grow there-after, and a number of somatic embryos were con-sequently obtained after one month of culture. Someembryos developed roots vigorously, which has neverbeen observed in the control embryos derived from YEBmedium-inoculated embryogenic calli. About 10% of thesomatic embryos developed into plantlets, while the otherseither showed no further growth or only developed roots.Somatic embryo-derived plantlets were successively sub-

Regeneration of transgenic plants 651

cultured by terminal cuttings to cefotaxime-containingNHF medium in order to eliminate Agrobacterium.

To select transformed plantlets, opine assay of leafextracts was carried out. Six out of 10 plantlets analysedproduced mikimopine. However, opine production inthese plantlets was unstable, and opine was not detectedafter several successive subcultures in all six lines. Inaddition, Southern blot analysis of DNAs extracted fromthem indicated that the T-DNA region did not exist inthe genome of these six clones after successive subcultures(data not shown). Thus, production of transgenic plantsby a wild-type strain of A. rhizogenes was unsuccessful.The plantlets initially producing opine appeared to bechimeric and transformed cells might have been lostduring the successive subcultures.

Co-cultivation of embryogenic calli with an engineeredA. rhizogenes strain A13/pBI121

To obtain non-chimeric transgenic plants of V. vinifera,a selection method of transformants for kanamycin resist-ance was employed by using an engineered A. rhizogenesstrain A13/pBI121, which harboured the binary vectorpBI121 including the NPT II and GUS genes. Afterco-cultivation, calli were spread over cefotaxime-containing NHF medium further supplemented with50 mg dm"1 kanamycin. Our preliminary experimentsindicated that this level of kanamycin in NHF mediumwas sufficient to inhibit somatic embryo formation fromembryogenic calli and also to inhibit secondary embryoformation from developed somatic embryos, althoughembryogenic calli could sustain their growth on NECmedium supplemented with up to 100 mg dm"1 kanamy-cin. A total of 45 somatic embryo clones were obtainedfrom three independent co-cultivation experiments afterone month on the kanamycin-containing medium(Fig. 1). On the other hand, the control YEB medium-inoculated embryogenic calli never produced somaticembryos even after three months on the same kanamycin-containing medium.

Three to 10 embryos from each of the 45 primaryembryo clones were subcultured on to the kanamycin-containing medium, and newly formed embryos wereselected and further subcultured on the same medium.During a two month selection period, only newlydeveloped embryos were maintained and subclones whichdid not produce secondary embryos were discarded.Thus, 16 subclones continuously producing secondaryembryos were considered to be putative non-chimerictransformants. Plantlets were regenerated from 12 out of16 subclones, and were successively subcultured on tocefotaxime-containing medium to eliminate Agrobac-terium.

After successive subcultures, plantlets from these 12subclones were subjected to both opine and Southern blot

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Fig. 1. Effect of kanamycin selection (50 mg dm 3) on somatic embryo development from co-cultivated (right) and non-co-cultivated (left)embryogenic calli of V. vinifera cv. Koshusanjaku. The photograph was taken one month after the co-cultivation with A. rhizogenes A13/pBI121.

analyses to confirm transformation. Among them, 10subclones produced mikimopine in their roots and leaves(Fig. 2). In addition, Southern blot analysis of DNAsextracted from roots and leaves indicated that all 12subclones selected contained the GUS cording sequencein their genomes (Fig. 3a). Furthermore, 10 opine-positivesubclones also contained a wild-type Ri T-DNA (Fig. 3b).Transformation was also confirmed by GUS histochem-ical assay; Leaves, stems and roots of plantlets from all12 subclones showed GUS activity (Fig. 4).

Differences in phenotype of in Wrro-growing plantletswere observed among 12 transformed subclones (Fig. 5).Transgenic plantlets from four subclones which containeda wild-type Ri T-DNA exhibited a typical Ri-transformedphenotype such as wrinkled leaves and abundant rootsystem. On the other hand, those of the other eightsubclones looked normal, even though six contained awild-type Ri T-DNA. Most of these plantlets were suc-cessfully transferred to the greenhouse. However, thosefrom one subclone exhibiting a Ri-transformed phenotypewere severely dwarfed and their transfer to the greenhousehas so far been unsuccessful.

Discussion

Grapevines have been considered as recalcitrant crops forthe production of transgenic plants. Although manyworkers have already tried to produce transgenic grapev-ine plants mediated by Agrobacterium, only limited suc-cess has so far been obtained, although as suggested byMullins et al. (1990), it is easy to obtain transgenic cellsand tissues by Agrobacterium-mediated transformation.

Fig. 2. Mikimopine detection in leaves and roots of transgenic plantsof V vinifera cv. Koshusanjaku. Lane 1. hairy roots of tobaccotransformed with A. rhizogenes A13; lane 2, leaves of an untransformedplant; lane 3, roots of an untransformed plant; lane 4, leaves of atransgenic plant; lane 5, roots of a transgenic plant M, mikimopine.

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a

•3.0

1 2 3 4 5

Fig. 3. Southern blot analyses of transgenic plants of V. vinifera cv.Koshusanjaku. (a) Hindlll- and £coRI-digested DNA samples hybrid-ized with a 3.0 kbp //mdIII-£coRI fragment from pBI121. Lane 1,DNA from pBI 121 as a positive control; lane 2, leaves of anuntransfonned plant; lanes 3 and 4, leaves of different transgenicsubclones; lane 5, roots of the same traasgenic subclone as in lane 3.(b) £coRI-digested DNA samples hybridized with a 4.9 kbp EcoRlfragment from pLJl. Lane 1, DNA from pLJl as a positive control;lanes 2 to 5, leaves of different transgenic subclones; lane 6, leaves ofan untransfonned plant. Numbers indicate kbp.

Fig. 4. GUS activity in (a) leaf, (b) stem and (c) root samples of atransgenic plant of V. vinifera cv. Koshusanjaku.

In this study, transformed roots and calli were obtainedby all of the wild-type A. rhizogenes strains used, althoughdifferences in transformation frequency were observedamong the strains (Table 1).

Two main problems other than Agrobacterium infectiv-ity have so far been found to producing transgenic

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Fig. 5. Transgenic plantlets of V. vinifera cv. Koshusanjalm. Left, aplantlet with Ri T-DNA exhibiting a typical Ri-transformed phenotypesuch as abundant root system; right, a plantlet without Ri T-DNAlooking normal.

grapevine plants. Firstly, there have been few reliablesystems for regenerating plants from transformed tissuesin grapevines, especially in V. vinifera which is the mostimportant species. Guellec et al. (1990) failed to induceplant regeneration from transformed roots of V. viniferaobtained by A. rhizogenes-mediated transformation. Inaddition, although Mullins et al. (1990) obtained GUS-positive buds of V. vinifera transformed by a disarmedA. twnefaciens strain, their further development was notobserved. Plant regeneration from transformed roots andcalli was also unsuccessful in this study. The secondproblem is the generation of chimeras containing bothtransformed and untransformed cells. Baribault et al.(1990) and Berres et al. (1992) obtained chimeric trans-formed shoots of V. vinifera by A. tumefaciens-mediatedtransformation, and they demonstrated a multicellularorigin of these shoots.

In this study, these two problems were overcome bycombining the use of embryogenic calli and a selectionmethod. The usefulness of embryogenic cultures as atarget material for Agrobacterium-mediated transforma-

tion has already been reported in some recalcitrant speciesincluding walnut (McGranahan et al., 1988) and papaya(Fitch et al., 1993). Embryogenic cultures have beenshown to have a high plant regeneration ability in anumber of species and, furthermore, it has been generallyaccepted that somatic embryos arise from single cells(Ammirato, 1983; Krul and Worley, 1977). Theembryogenic cell line of V. vinifera used in this study,which was initially reported by Matsuta and Hirabayashi(1989), also had a high regeneration ability. Therefore,non-chimeric transgenic plants were initially expected tobe obtained efficiently by using this embryogenic cell line.However, possible chimeric regenerants were obtainedfrom this callus after co-cultivation with a wild-type strainA13. Since embryogenic calli used in this study containednumerous somatic embryos, the chimeric regenerantsseem to originate from those somatic embryos in whichthe transformation event occurred beyond the single cellstage. In the present study, however, the use of anengineered strain A13/pBI121 and successive selection ofsecondary embryos on kanamycin-containing mediumproved to overcome this unfavourable chimera formation.At the same time, this study may provide additionalevidence to the previous report demonstrating that sec-ondary embryos originate from single cells of the primarysomatic embryos (Polito et al., 1989).

To date, genetic transformation using virulentA. rhizogenes strains has been reported in a number ofplant species (Visser et al., 1989; Damiani and Arcioni,1991; Saito et al., 1992). In contrast with disarmedAgrobacterium strains, virulent A. rhizogenes is usable notonly as a transformation vector for introducing foreigngenes into the plant genome, but also as a genetic sourceof agriculturally interesting traits. A typical transformedphenotype including increased branching, dwarfness,wrinkled leaves, modified flowering and abundant rootsystem has shown to be induced by Ri T-DNA (Tepfer,1984; Oono et al., 1987; Spena et al., 1987). These genesare useful to create new genotypes and phenotypes especi-ally in fruit crops. In fact, improvement of rooting abilityby transformation with Ri T-DNA has been demonstratedin kiwi (Rugini et al., 1991) and apple (Lambert andTepfer, 1992). Although detailed phenotypic characteriza-tion of Ri-transformed grapevine plants obtained in thisstudy has not been performed at present, several morpho-logical changes, such as wrinkled leaves and an abundantroot system, were observed in some in v/fro-growingplantlets. On the other hand, transformants which didnot contain Ri T-DNA were also obtained. In thosetransformants no morphological abnormalities wereobserved. Therefore, the transformation system developedin this study is useful for the production of transgenicgrapevine plants with incorporation of only the desiredgenes since, in this case, regeneration of 'true-to-type'plants without vector T-DNA is desirable.

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In this study, we efficiently obtained transgenic plantsof V. vinifera by using embryogenic calli with a highregeneration ability. However, to date, the induction ofembryogenic cell line is restricted to V. vinifera cv.Koshusanjaku. Therefore, further experimentation shouldbe directed to induce the embryogenic cell line in otherimportant grapevine cultivars. Also, further characteriza-tion of transgenic plants obtained in this study should beperformed.

Acknowledgement

We wish to thank Dr T. Handa, Tsukuba University, Japan,for the gift of A. rhizogenes strain A13/pBI121.

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