Plant Cell Reports

Plant Cell Reports (1995) 14:550-554

9 Springer-Verlag1995

Transformation of grapevine rootstocks with the coat protein gene of grapevine fanleaf nepovirus S. Krastanova 1.,, M. Perrin 1, p. Barbier 1, G. Demangeat 1, p. Cornuet L. Pinck 2, and B. Walter 1

1 N.

Bardonnet 2, L. Otten 3,

INRA 28, rue de Herrlisheim, BP 507, F-68021 Colmar, France 2 CNRS IBMP Virologie, 3 Phytopathologie mol6culaire, 12, rue du G6n6ral Zimmer, F-67084 Strasbourg, France * Present address: Universit6 de Neuch~tel, 18 chemin de Chantemerle, CH-2000 Neuchgttel, Switzerland Received 5 July 1994/Revised version received 31 October 1994 - Communicated by H. L6rz

Summary. C o n t r o l of fanleaf disease i n d u c e d by the G r a p e v i n e Fanleaf Nepovirus (GFLV) today is based o n s a n i t a r y s e l e c t i o n a n d soil d i s i n f e c t i o n w i t h n e m a t i c i d e s . This way of c o n t r o l is n o t always efficient a n d n e m a t i c i d e s c a n b e d a n g e r o u s p o l l u t a n t s . Coat p r o t e i n (CP) m e d i a t e d p r o t e c t i o n could be a n attractive alternative. We h a v e t r a n s f e r r e d a chimeric CP gene of GFLV-F13 via A g r o b a c t e r i u m rumefaciens LBA4404 into two rootstock varieties : Vitis r u p e s t r i s a n d 110 Richter (V. r u p e s t r i s X V. Berlandieri). T r a n s f o r m a t i o n was p e r f o r m e d on e m b r y o g e n i c callus obtained from a n t h e r s a n d on hypocotyl fragments from mature embryos. Success of the t r a n s f o r m a t i o n was assessed by polymerase chain reaction and Southern analyses. T r a n s f o r m a n t s with a n u m b e r of copies of the CP gene v a r y i n g from o n e to five were obtained. Enzyme-linked i m m u n o s o r b e n t assay with virus-specific a n t i b o d i e s revealed various levels of expression of the coat p r o t e i n in the different t r a n s f o r m a n t s . Abbreviations: 2,4D: 2,4 dichlorophenoxyacetic acid; BAP: 6-benzylaminopurine;CP: coat protein; EDTA:ethylene diamine tetraacetic acid; ELISA: enzyme-linked immunosorbent assay; GFLV: grapevine fanleaf virus; GUS: glueuronidase; IBA: indole3-butyric acid; NAA: 1-naphthaleneacetic acid; NOA: 6naphthoxyacetic acid; NOS: nopaline synthase; NPTII: neomycin phosphotransferase II; PCR: polymerase chain reaction.

et al. 1991). Only in the last two r e p o r t s whole plantlets expressing a m a r k e r g e n e c o u l d b e r e g e n e r a t e d from a n t h e r - d e r i v e d somatic e m b r y o s . Besides, i n all these e x p e r i m e n t s , t r a n s f o r m a t i o n e f f i c i e n c y was assessed based o n the activity of the g l u c u r o n i d a s e (GUS) gene, which has been shown i n some cases to be expressed in c o n t a m i n a t i n g a g r o b a c t e r i a d e s p i t e t h e u s e of e u k a r y o t i c p r o m o t e r s ( V a n c a n n e y t et al. 1990), thus possibly leading to false positives. Efficiency of selection a n d r e g e n e r a t i o n of t r a n s f o r m e d cells r e m a i n two major p r o b l e m s for g r a p e v i n e t r a n s f o r m a t i o n . Since the first r e p o r t of successful somatic e m b r y o g e n e s i s (Mullins a n d S r i n i v a s a n 1976), p l a n t l e t s f r o m d i f f e r e n t Vitis c u l t i v a r s a n d h y b r i d s h a v e b e e n r e g e n e r a t e d from somatic e m b r y o s . In l i q u i d m e d i u m , it is possible to grow a large q u a n t i t y of e m b r y o s , b u t i n m a n y cases, the r e g e n e r a t i o n rate r e m a i n s low a n d most e m b r y o s look a b n o r m a l (Mullins 1986 ; C o u t o s - T h 6 v e n o t et al. 1992). In t h e w o r k r e p o r t e d h e r e , we p r e s e n t a n i m p r o v e d p r o t o c o l for t r a n s f o r m a t i o n of s o m a t i c e m b r y o s from a n t h e r s of two rootstock varieties, Vitis r u p e s t r i s d u Lot ( V. r u p e s t r i s ) a n d V. Berlandieri X V. r u p e s t r i s 110 Richter ( l l 0 R ) , a v o i d i n g a l i q u i d m e d i u m p h a s e , e x c e p t for t h e i n o c u l a t i o n with Agrobacterium. These explants were t r a n s f o r m e d using c o n s t r u c t s with the CP g e n e of GFLV (Bardonnet et al. 1994) a n d a n i n t r o n - c o n t a i n i n g GUS r e p o r t e r gene to p r e v e n t its e x p r e s s i o n i n b a c t e r i a ( V a n c a n n e y t et al. 1990).

INTRODUCTION MATERIALS AND METHODS G r a p e v i n e Fanleaf Virus is t r a n s m i t t e d by the n e m a t o d e Xiphinema i n d e x a n d i n d u c e s o n e of the m o s t w i d e s p r e a d a n d d a m a g i n g diseases of g r a p e v i n e (Bovey et al. 1980). T h e use of v i n e s resistant to GFLV o b t a i n e d b y g e n e t i c e n g i n e e r i n g c o u l d a d v a n t a g e o u s l y replace soil d i s i n f e c t i o n with n e m a t i c i d e s which c o n s t i t u t e d a n g e r o u s pollutants. Coat-protein m e d i a t e d protection (Wilson 1993) a g a i n s t GFLV has b e e n d e m o n s t r a t e d to occur i n t r a n s g e n i c Nicotiana benthamiana (Bardonnet et al. 1994). T r a n s f o r m a t i o n of v a r i o u s g r a p e v i n e tissues has b e e n r e p o r t e d (Guellec et al. 1990; Baribault et al. 1 9 8 9 ; B a r i b a u l t e t al. 1 9 9 0 ; C o l b y e t al. 1991; Berres et al. 1992; Mullins et al. 1990; Mauro

Correspondence to: B. Walter

Plant material: The rootstock clones IIOR and V. rupestris used belong to the INRAcollection kept in Colmar. Somatic embryogenesis from anthers: Floral buds were harvested before flowering, from greenhouse or field-grown plants. Buds were disinfected during 20 rain in 6.7% (w/v) calcium hypochlorite containing 0.1% (v/v) Tween-20, and rinsed three times with sterile water. Anthers were isolated in sterile conditions and cultured at 28~ in the dark, at a density of 10 floral buds per Petri dish (diameter:5.5 cm), following the method described by Rajasekaran and Mullins (1979). The base medium was a Murashige and Skoog MS medium (1962) containing half-strength macroelements and the vitamins of

551 Morel and Martin (1952), 36.7 mg/l Fe-EDTA, 30 g/1 sucrose, 7 g / l DIFCO bacto-agar, pH 5.8. Three auxins were compared (2,4D, NAA or NOA at 1 mg/L) in combination with BAP at 0.25 mg/L. Embryogenic calluses developing from the anthers were regularly t r a n s f e r r e d at 20 day-intervals on fresh base m e d i u m for callus induction. Petri dishes were kept in the dark at 28 ~ III

PNOS

TNOS

P35S

R i n d III ~

TNOS TNOS

P35S

I

IKb

I

HGURE 1. T-DNA map of the binary plasmid p1660. The line under the GFLV CP gene represents the location of the probe used for southern hydridization.The position of the two primers used in PCR reaction is represented by two black triangles.

Transformation: Plasmid p1660, a pROKI b i n a r y vector derivative, is the construction pRCPI described in Bardonnet et al. (1994), into w h i c h the p35S/GUS-intron/tNOS gene of Vancanneyt et al. (1990) was inserted, between the p35S/GFLVCP/tNOS a n d the pNOS/NPTII/tNOS cassettes (Fig. 1). This plasmid was mobilized to A. turaefaciens strain LBA4404. Two types o f explants were t r a n s f o r m e d : e m b r y o g e n i c callus at d i f f e r e n t stages of d e v e l o p m e n t , or fragments of e m b r y o hypocotyls. Bacterial colonies were inoculated in 20 mL YEB, i n c u b a t e d o v e r n i g h t at 28~ and centrifuged. The bacterial pellet was r e s u s p e n d e d in 2 mL basal MS m e d i u m for embryogenic calluses or 2 mL liquid NN medium (Nitsch and Nitsch 1962) for hypocotyls. The explants were immersed in the bacterial suspension during 5 to 75 min for the embryogenic calluses and 30 s for the hypocotyl fragments. For co-cultivation (2 days), e m b r y o g e n i c calluses were t r a n s f e r r e d o n t o solid MS m e d i u m with 1 mg/L NOA a n d 0.25 m g / L BAP. T h e n t h e e m b r y o g e n i c calluses w e r e t r a n s f e r r e d o n t o MS m e d i u m c o n t a i n i n g 1 mg/L NOA, 0.25 mg/L BAP, 25 or 50 mg/L kanamycin a n d cefotaxime at a c o n c e n t r a t i o n stepwise d e c r e a s e d f r o m 5OO to 100 mg/L at each transfer, a n d were cultured in the dark at 28~ When embryos h a d r e a c h e d the heart- or torpedo-shape, they were transferred to 9 cm Petri dishes containing basal MS medium without h o r m o n e s for differentiation and grown at 250C with 16 h light at 4000 lux. When further developed, the embryos were progressively t r a n s f e r r e d onto MS m e d i u m containing 1 mg/L BAP. For co-cultivation (2 days), the hypocotyl fragments were transferred to Petri dishes containing NN medium plus O.1 mg/L NAA a n d 2 mg/L BAP, o n a piece o f Whatman p a p e r as d e s c r i b e d b y Mullins et al. (1990). Then the hypocotyl f r a g m e n t s w e r e t r a n s f e r r e d onto semi-solid NN m e d i u m containing 3.5 g/L agar, O.1 mg/L NAA, 2 mg/L BAP, 10 mg/L k a n a m y c i n a n d carbenicillin at a c o n c e n t r a t i o n which was stepwise decreased from 500 to 100 mg/L at each transfer. After 8 weeks in the dark at 22~ the hypocotyl fragments were transferred onto the same medium, under 16 hours light (4000 lux, 26~ a n d 8 h o u r s dark (20~ for 12 weeks, and then placed in tubes containing MM m e d i u m (Morel and Martin 1952) s u p p l e m e n t e d with 1 mg/L IBA, 1 mg/L BAP, 10 mg/L kanamycin a n d 100 mg/L carbenicillin. Developed embryos or buds were transferred individually to tubes containing MM medium without kanamycin, cefotaxime or carbenicillin for rooting. Control experiments were done on the same media without Agrobacterium, kanamycin and carbenicillin. Analysis o f transform,ants : NPT II and GUS activities were first assayed o n callus, embryos or y o u n g / n vitro plantlets using the m e t h o d s described by Mc Donnell et al. (1987) and Jefferson (1987), respectively. Later on, leaves of regenerated plants were assayed for b o t h enzymes using the rapid method of Peng et al. (1993). The expression of the CP gene was detected by ELISA according to Walter a n d Etienne (1987) with an amplification step using IgG-biotin and streptavidin-phosphatase conjugates.

The detection of integrated transgenes was p e r f o r m e d by PCR and Southern hybridization. Total DNA was extracted from 0.5 g leaf tissue of in vitro grown or glasshouse-acclimated according to Guillemaut a n d Mar6chal-Drouard (1992). For PCR analyses, the two primers used corresponded to positions 30003017 and 3554-3572 of the GFLV RNA-2 sequence. These two p r i m e r s g e n e r a t e a f r a g m e n t o f a b o u t 5 7 0 nucleotides corresponding to the 3' e n d of the GFLV CP gene. PCR was performed in 50 gL reaction mixture, containing 0.05 to 0.1 gg grapevine DNA for 20 pmoles each primer, 1 unit of Taq DNA polymerase (Promega), 2 raM MgC12 a n d buffer provided by the manufacturer. Four ng of pCPM plasmid carring the CP gene (Serghini et al. 1991) were used as positive control. After 35 cycles in a Techne cycler (denaturation 1 rain at 92~ annealing 1 rain at 50~ extension 1 rain at 72~ 10 pL of the reaction m i x t u r e w e r e a n a l y s e d in a g a r o s e gel. For S o u t h e r n hybridization, approximately 4 pg total DNA digested with the restriction enzyme HindIII, were t r a n s f e r r e d to a Hybond-N nylon membrane. Blots were hybridized to a 32p-labeUed SalIEcoRI (1.1kb) DNA-fragment excised from plasmid pCPM.

RESULTS AND DISCUSSION 1 2 5 5 g r o u p s o f a n t h e r s o f V. r u p e s t r i s , a n d 2 3 3 1 groups of anthers of ll0R were cultured. Anthers c o n t a i n e d m o s t l y u n i n u c l e a t e d (GP1) a n d b i n u c l e a t e d (GP2) p o l l e n g r a i n s a s r e v e a l e d b y a c e t i c c a r m i n s t a i n i n g . T h e p e r c e n t a g e o f GP1 o r GP2 a n t h e r s t h a t d e v e l o p e d c a l l u s v a r i e d b e t w e e n 2 4 a n d 5 0 % f o r V. rupestris, o r 4 9 a n d 6 5 % f o r i 101L T h e p e r c e n t a g e o f e m b r y o g e n i c c a l l u s (Fig 2a) r e l a t i v e t o t h e n u m b e r o f c u l t u r e d a n t h e r g r o u p s w e r e 5 - 2 1 % f o r V. rupestris a n d 4 . 5 - 1 3 % f o r l l 0 R . No s i g n i f i c a n t d i f f e r e n c e w a s f o u n d between hormone treatments, although the appearance of the calli varied with the auxin used. Three e m b r y o g e n i c c a l l u s e s o f V. r u p e s t H s a n d t w o o f l l 0 R were multiplied for transformation.

V. rupestris

110 R

Duration of Kanamycin inoculation concentration 30 min 25mg/L 50m~/L 60 min 25mg/L 50mg/L

E

GUS

E

GUS

23/240 11/130 8/155 4/170

13/20 3/4 4/6 2/2

12/150 5/140 5/110 2/120

4/12 3/3

4/5 2/2

Table 1: Rate of t r a n s f o r m a t i o n a n d selection of somatic embryos inoculated for 30 or 60 min with Agrobacterium and with two c o n c e n t r a t i o n s o f k a n a m y c i n . E : n u m b e r o f e m b r y o s / n u m b e r o f embryogenic calli. GUS:number of blue e m b r y o s / n u m b e r of tested e m b r y o s .

Transformation We first defined the o p t i m a l i n c u b a t i o n time of the embryogenic callus with A. t u m e f a c i e n s h a r b o u r i n g plasmid p1660, as well as the conditions of kanamycin selection. Embryogenic calluses were grown in the p r e s e n c e o f 25 rag/1 o r 5 0 m g / 1 k a n a m y c i n a f t e r a n i n c u b a t i o n p e r i o d o f 30 o r 6 0 m i n w i t h A. tumefaciens. Results a r e p r e s e n t e d i n T a b l e 1. A n i n c u b a t i o n o f 30 to 60 m i n e n a b l e d u s to o b t a i n t r a n s f o r m e d t i s s u e s (embryos at various stages of development, hypocotyls, leaflets) a s j u d g e d b y a b l u e GUS c o l o r a t i o n (Fig 2 b,c) For a n i n c u b a t i o n t i m e o f less t h a n 30 rain, t h e transformation efficiency was very poor and for more t h a n 60 min, t h e r e g e n e r a t i o n r a t e was c o n s i d e r a b l y d e c r e a s e d . T h e p e r c e n t a g e o f e m b r y o s g r o w i n g to plantlets was highly reduced by the presence of the

552 bacteria, f r o m 45% (controls w i t h o u t bacteria) to 2596 for 110R, a n d f r o m 60% to 35% for V. rupestris ( d a t a n o t s h o w n ) . In t h e a b s e n c e of k a n a m y c i n , tissues assayed for GUS activity after cocultivation exhibited a p a t c h y d i s t r i b u t i o n of b l u e zones i n d i c a t i n g that o n l y some cells were expressing the GUS gene. In most cases twice as m a n y e m b r y o s d e v e l o p e d o n 25 m g / L k a n a m y c i n t h a n o n 50 m g / L (Table 1). Some plantlets d e v e l o p e d o n 25 m g / L k a n a m y c i n a n d all p l a n t l e t s d e v e l o p e d o n 50 m g / L k a n a m y c i n were u n i f o r m l y blue. T h e o t h e r p l a n t l e t s d e v e l o p e d o n 25 m g / L were p a r t i a l l y b l u e . GUS assays showed that t r a n s f o r m e d t i s s u e s o b t a i n e d w i t h o t h e r m e t h o d s such as cell s u s p e n s i o n s ( B a r i b a u l t et al. 1990), p e t i o l e s t u b s (Mullins et al. 1990), leaf petioles (Colby et al. 1991), stem f r a g m e n t s o r leaf e x p l a n t s (Berres et al. 1992) were always p a t c h i l y d i s t r i b u t e d , suggesting chimeric t r a n s f o r m a t i o n a n d inefficient selection. Regeneratively c o m p e t e n t p r o m e r i s t e m s from leaf petioles m a y n o t be easily accessible to A. tumefaciens leading to chimeras (Colby et al. 1991). In a d d i t i o n , inefficient selection could result from the d e t r i m e n t a l effect of the antibiotic on untransformed tissue involved in nutrient t r a n s l o c a t i o n to the t r a n s f o r m e d p r o m e r i s t e m s (Colby et al. 1 9 9 1 ) . The r e s u l t s we h a v e o b t a i n e d are c o m p a r a b l e to those y i e l d e d b y t h e i m p r o v e d m e t h o d for somatic e m b r y o g e n e s i s of the r o o t s t o c k 41B of Mauro et al. ( 1 9 9 1 ) . All 110R p l a n t l e t s selected o n k a n a m y c i n s h o w e d a p o s i t i v e r e s p o n s e in t h e NPTII a n d GUS analyses. Leaflets h a r v e s t e d from these plantlets grown in vitro, n e a r l y all r e a c t e d positively in ELISA ( 1 4 / 1 7 p l a n t l e t s ) . T a b l e 2 shows ELISA r e s u l t s of t r a n s g e n i c 110R p l a n t s exhibiting different levels of expression of the CP gene. Similar expression levels were observed i n t h e i r r e s p e c t i v e cuttings. All 9 V. rupestris a n a l y s e d scored positive i n NPTII a n d GUS tests.

non transformedcontrol

Cuttings from transformed plants Plant ELISA number OD405 um (lh) 1.1 0.392 1.2 0.385 1.3 0.520 II.1 0.840 II.2 0.961 11.3 1.335 0.056

buffer-control

0.018

Transformed plants from embr~o[~e~ic callus Plant ELISA number OD 405 nm (lh) I 0.391

II

0.951

Table2 : ELISAvaluesobtainedwith two transformed110Rplants and their respective cuttings. T h e s e c o n d t y p e of tissue i n o c u l a t e d was h y p o c o t y l f r a g m e n t s f r o m m a t u r e e m b r y o s , as d e s c r i b e d b y M u l l i n s et al. (1990). A d v e n t i t i o u s b u d s d e v e l o p e d d u r i n g the two m o n t h s growth of hypocotyl fragments i n the dark. After t r a n s f e r to the light, the size of the hypocotyl fragments rapidly increased and buds d i f f e r e n t i a t e d i n t o p h o t o s y n t h e t i c organs. After 4 m o n t h s , 75% of the f r a g m e n t s of V. rupestris y i e l d e d b u d s o n k a n a m y c i n , 89% of which were GUS-positive. In

the case of l l0R, 56% of the f r a g m e n t s y i e l d e d buds, 51% of which were GUS-positive (Table 3). After 6 m o n t h s , e l o n g a t e d b u d s were t r a n s f e r r e d to tubes on h o r m o n e - f r e e rooting m e d i u m a n d grew i n t o plantlets, part of which were a n a l y s e d i n a GUS assay according to Jefferson (1987): 7 / 2 0 p l a n t l e t s of l l 0 R a n d 15/15 V. rupestris tested showed u n i f o r m l y t r a n s f o r m e d blue tissues. 4 months

rupestris

110R

6 months

H

GUS +

B

GUS+

LBA 4404 (p1660) Control

217/286

63/71

180/217

15/15

LBA 4404 (p1660) Control

108/192

41/41

23/31

40/41 33/64

84/108

7/20

18/26

Table 3 : Transformation of hypocotyls of somatic embryos. The hypocotyls were observed 4 and 6 months after the inoculation with Agrobacterium. It:number of hypocotyl fragments with buds/number of cultured fragments. ]]:number of growing buds/number of hypocotyl fragments with buds. GUS:number of blue buds/number of analysed fragments or buds. We f i n a l l y o b t a i n e d 99 110R p l a n t l e t s a n d 190 V. rupestris plantlets. From the 110R p l a n t l e t s , 63 were a n a l y s e d for GUS a n d NPTII activities: 17 were GUS+ a n d NPTII+ a n d 46 GUS- a n d NPTII-. From the V. rupestris plantlets, 160 were a n a l y s e d : 54 were GUS+ NPTII+ a n d 70 GUS- NPTII-; 7 GUS+ NPTII- a n d 29 GUSNPTII+ were also found. GUS- NPTII- p l a n t l e t s p r o b a b l y escaped k a n a m y c i n selection ( d o n e at 10 mg/L). GUS+ NPTII- a n d GUS- NPTII+ p l a n t s m a y arise from defective i n t e g r a t i o n of the T-DNA, o r from defective expression of t h e s e genes. M o r e o v e r , w h e n o n e p l a n t l e t was p r o p a g a t e d in vitro b y cuttings, there was n o t always a c o n s i s t e n t c o r r e l a t i o n b e t w e e n the e x p r e s s i o n of the three genes transferred. For example, i n o n e group of 6 cuttings d e r i v e d from a GUS+ NPTII+ ELISA+ plantlet, one c u t t i n g was NPTII+ a n d GUS-, the o t h e r 5 being NPTII+ a n d GUS+, with o n l y 1 o u t of 6 EHSA positive. This could indicate chimeric t r a n s f o r m a t i o n . In conclusion, the somatic e m b r y o g e n e s i s m e t h o d is more efficient t h a n c a u l o g e n e s i s for obtaining t r a n s g e n i c g r a p e v i n e . This m a y be d u e to a better contact of t h e antibiotic d u r i n g selection of transformed cells ( l e a d i n g to less escapes), as well as a b e t t e r accessibility of r e g e n e r a t i v e cells to the t r a n s f o r m i n g agrobacteria (higher n u m b e r of t r a n s f o r m e d cells). This difference p r o b a b l y also explains why A. tumefaciens strain LBA4404 which in o u r h a n d s was poorly efficient for t r a n s f o r m a t i o n of stem f r a g m e n t s a n d leaf explants (Berres et al. 1992), efficiently t r a n s f o r m e d somatic e m b r y o s . However, the l i m i t i n g factor of the overall t r a n s f o r m a t i o n p r o c e s s is t h e low e f f i c i e n c y of e m b r y o g e n e s i s . A c c o r d i n g to o u r r e s u l t s , t h e t r a n s f o r m a t i o n of e m b r y o g e n i c callus a p p e a r e d more reliable t h a n that of h y p o c o t y l f r a g m e n t s p r o v i d e d that a p p r o p r i a t e k a n a m y c i n selection is exerted.

553 FIGURE 2. a: e m b r y o g e n i c callus with n u m e r o u s e m b r y o s , b: somatic embryos showing blue coloration after transformation with Agrobacterium LBA 4 4 0 4 harboring the plasmid p 1 6 6 0 c: Blue coloration of leaflets regenerated from transformed somatic embryos.

FIGURE 3. Analysis of PCR products obtained with grapevine DNA from 110R (lane a to 1), R u p e s t r i s (lane m) and an untransformed plant (lane p). Lane n c o r r e s p o n d s to the positive control. L a m b d a DNA digested with HindIII/EcoRI was used as a size marker. The arrow o n t h e l e f t s h o w s the p o s i t i o n of the e x p e c t e d PCR product.

lane

FIGURE 4. S o u t h e r n Blot analysis. (a) DNA d i g e s t e d w i t h EcoRI + HindIII (size marker revealed by an i n d e p e n d e n t hybridization, the p o s i t i o n o f the f r a g m e n t s is indicated on the left); (b) DNA of u n t r a n s f o r m e d 110R; (c to i) DNA of 7 transgenic 110R.

554

A n a l y s i s o f t r a n s f o r m a n ts In o r d e r to g a i n f u r t h e r e v i d e n c e for t r a n s f o r m a t i o n , the i n t e g r a t i o n of T-DNA b o r n e transgenes was p r o b e d b y two methods. Fig 3 show s the analysis o n agarose gel of PCR p r o d u c t s o b t a i n e d with g r a p e v i n e DNA isolated f r o m t r a n s f o r m e d 110R (lanes a to 1) a n d V. rupestris ( l a n e m). A f r a g m e n t of the expected size (cf Fig 1) was a m p l i f i e d f r o m DNA i s o l a t e d f r o m all p u t a t i v e t r a n s g e n i c p l a n t s tested. This f r a g m e n t was n o t detected with DNA f r o m u n t r a n s f o r m e d 110R (lane p) a n d all a m p l i f i e d p r o d u c t s c o m i g r a t e d with t h e f r a g m e n t a m p l i f i e d from the pCPM p l a s m i d (lane n). Additional n o n - s p e c i f i c a m p l i f i c a t i o n p r o d u c t s were also detected in PCR r e a c t i o n s d, f, g, i. I n f o r m a t i o n a b o u t the n u m b e r of i n t e g r a t e d CP gene was o b t a i n e d by Southern blot analysis. A CP g e n e - i n t e r n a l p r o b e (cf Fig 1) was c h o s e n so t h a t it w o u l d r e v e a l a single f r a g m e n t , c o m p o s e d o f t h e left e n d of the T-DNA a n d the n e i g h b o u r i n g p l a n t DNA, for each i n t e g r a t i o n of a TDNA copy, The blot s h o w n i n Fig 4 shows examples of 7 t r a n s f o r m a n t s with v a r i o u s n u m b e r s of copies (up to 5 i n l a n e f). T h e p r o b e h y b r i d i z e d w e a k l y to u n t r a n s f o r m e d g r a p e v i n e DNA (lane b), a p r o b e from t h e r i g h t b o r d e r d o e s n o t ( d a t a n o t shown). Most t r a n s f o r m a n t s a n a l y z e d h a d several copies of the CP gene. Multiple i n t e g r a t i o n s m a y h a v e b e e n favored by the necessity of a strong k a n a m y c i n selection. We often f o u n d that p l a n t l e t s r e g e n e r a t e d from o n e t r a n s f o r m e d callus were i n fact d e r i v e d from the same t r a n s f o r m e d cell. Such a case is shown in lanes d a n d e, the p a t t e r n o f w h i c h a r e i d e n t i c a l . We i d e n t i f i e d so far 15 i n d e p e n d e n t l l 0 R t r a n s f o r m a n t s a n d 4 V, rupestris. By S o u t h e r n h y b r i d i z a t i o n , we could also check that the CP gene i n t e g r a t i o n is stable i n the c u t t i n g s d e r i v e d from o n e t r a n s f o r m a n t b y vegetative multiplication, i.e. that t h e s e t r a n s f o r m a n t s were n o t c h i m e r a s ( d a t a n o t shown). T h e r e s u l t s p r e s e n t e d i n this p a p e r c o m p l e t e o u r p r e v i o u s r e p o r t o n the successful t r a n s f e r of the CP g e n e of GFLV i n t o g r a p e v i n e r o o t s t o c k v a r i e t i e s (Krastanova et al. 1993). The expression of the GFLV CP g e n e in the t r a n s f o r m a n t s has b e e n d e m o n s t r a t e d by ELtSA. The stability of the i n s e r t i o n s has b e e n p r o v e n , for some of the t r a n s f o r m a n t s , b y analysis of plantlets o b t a i n e d a f t e r m i c r o p r o p a g a t i o n of the t r a n s g e n i c p l a n t s i n the a b s e n c e of k a n a m y c i n selection. No c o n t a m i n a t i n g a g r o b a c t e r i a were d e t e c t e d d u r i n g the in vitro p r o p a g a t i o n of t h e t r a n s f o r m a n t s o n c a r b e n i c i l l i n - a n d cefotaxime-free m e d i u m . Resistance of t r a n s g e n i c g r a p e v i n e s to GFLV is c u r r e n t l y u n d e r i n v e s t i g a t i o n b y using the n a t u r a l way of virus infection through nematodes.

Acknowledgments

This work was supported by ANVAR (Association Nationale pour la Valorisation de la Recherche). K.S. benefited from a grant from the French Ministry for Research. The gift of the GUS gene from Dr. Willmitzer is greatly acknowledged. References

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Transformation of grapevine rootstocks with the coat protein gene of grapevine fanleaf nepovirus.

Control of fanleaf disease induced by the Grapevine Fanleaf Nepovirus (GFLV) today is based on sanitary selection and soil disinfection with nematicid...
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