PlantCeU Reports
Plant Cell Reports (1992) 11:192-195
9 Springer-Verlag 1992
Transformation of vitis tissue by different strains of Agrobacterium tumefaciens containing the T-6b gene R. Berres 2, L. Otten
2,
B. Tinland 2, E. Malgarini-Clog 1, and B. Walter 1
1 Institut National de Recherche Agronomique, Station Vigne et Vin, Laboratoire de Pathologic V6g&ale, 28, rue de Herrlisheim, 68021 Colmar Cedex, France z Institut de Biologie Mol6culaire des Plantes, Laboratoire de Phytopathologie Mol6culaire, 12, rue du G6n6ral Zimmer, 67084 Strasbourg Cedex, France Received July i, 1991/Revised version received November 20, 1991 - Communicated by H. L6rz
Summary. Stem pieces and leaf disks of Vitis spp. were cocultured with Agrobacterium tumefaciens strains carrying the UidA (fl-glucuronidase = GUS) gene. The transformation efficiency was highly increased by using a modified T-6b gene (a gene from pTiTm4) which interferes with normal growth and allows regeneration of normal Nicotiana rustica plants (Tinland 1990). The strains first tested on stem segments were subsequently tested in a leaf explant system. On leaves the transformation efficiency of the strains was much lower than with stems. Both the T6b gene and the hsp 70-T-6b gene (a modified T-6b gene under the control of a heat shock promoter) allowed the initiation of GUS-positive buds.
Materials and methods Plant material. Vitis vinifera Chardonnay and Gewurztraminer, V. vin. X V. Berlandieri 41 B, V. Berl. X V. riparia Kober 5BB and SO-4 were used. Leaves (4-6 cm in diameter) and stem pieces (2-3 cm long) were harvested from plants grown in growth chambers at 32~ or from 6 weeks old plantlets grown on a modified Murashige and Skoog medium (MM) + 6 g/l Difco Agar (Martin etal..1987). Mobilisation of binary vectors into Agrobacterium. The binary vector pBI121.2 was mobilized from E. coli into A g r o b a c t e r i u m GV3101(pTiTm4) or GV3101 by triple mating using an E. coli strain carrying the pRK2013 helper plasmid. Exconjagant strains GV3101(pTiTm4, pBI121.2) and GV3101(pBI121.2) were selected on minimal medium with 400 I.tg/1 neomycin and 100 ~tg/1 rifampiein (GV3101 marker, Leemans et al. 1983). Plasmid content of exconjugants was verified by electrophoresis of bacterial lysates on agarose (Rosenberg et al. 1981)
Table 1 : Bacterial strains and plasmids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Key-words : Grapevine -Agrobacterium tumefacienstransformation Abbreviations : GUS = 13-glucuronidase ; BAP = benzylaminopurine ; X-gluc = 5-bromo-4-chloro-3-indolyl glucuronide. Introduction Genetic engeneering seems to be suited to introduce new properties into grapevine cultivars and rootstocks. Reports on Agrobacterium-mediated transformation of woody perennials are still scarce. There are only few publications on grapevine transformation. Hemstad and Reisch (1985) reported on in vitro gall production by A. tumefaciens and A. rhizogenes on Vitis L. hybrids. Genin and Walter (unpublished results) succeeded in transforming stem tissue of rootstocks by means of a disarmed vector containing the GUS gene. Guellec et al. (1990) infected whole plants of Vitis vinifera cv. Grenache with A. rhizogenes and obtained transformed roots. Baribault et al. (1990) succeeded in the regeneration of shoots expressing 13glucuronidase by cocultivation of shoot apex cultures of Vitis vinifera L. cv. Cabernet Sauvignon with A. tumefaciens. Mullins et al. (1990) obtained transformed plantlets of Vitis rupestris Scheele and transformed buds of Vitis vinifera cv. Chardonnay and Cabernet Sauvignon. In this report, we present a rapid and reproducible method for the transformation of grapevine by using a growthstimulating gene from the grapevine-specific Agrobacterium strain Tm4. Correspondence to." B. Walter
Bacterial strain
Characteristics
Marker of transformed plant cell
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
LBA4404(pAL4404, pBI121.2)
GUS gene on pBI121.2 helper plasmid pAL4404 Ach5 background
GUS
(1)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GV3101(pBII21.2)
GUS gene on pBI121.2 no helper plasmid C58 background
(2)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GV3101(pTiTm4, pB1121.2)
GUS gene on pBI121.2 helper plasmid pTiTm4 C58 background
GUS (2)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GV3101(pPM6000)
ocs gene on disarmed pTiAch5
OCS
(3)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GV3101(pPM6001)
UidA (GUS) gene on disarmed pTiAch 5
GUS
(4)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GV3 I01(pPM6000:: pPM 37)
T-6b gene in disarmed GUS vector
T -6b tumors OCS (5)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GV3101(pPM6001:: pPM37)
T-6b gene in disarmed GUS vector
T-6b tumors GUS (4)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GV3101(pPM6001:: pPMS1)
heat shock inducible T-6b gene in disarmed GUS vector
T-6b tumors GUS (6)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(1) Jefferson 1987 (2) Otten unpublished (3) Bonnard etal. 1990 (4) Tinland etaL. 1990 (5) Tinland etal. 1989 (6) Tinland 1990. Cocultivation. After the leaf veins were cut with a sterile scalpel, the leaves were cocuhivated for one hour in Petri dishes containing 106-107 bacteria/ml from the various strains (Table 1). The leaves were dried and placed in Petri dishes with liquid MM medium + BAP 2 mg/1 + Cefotaxim (R) 500 mg/1 (Roussel Uclaf).
193 After about three weeks the developingbuds were transferred to tubes with solid MS medium (Murashige and Skoog 1962). Stem pieces were cocultivated in the same way, and were planted upside down in solid MM + BAP 2 mg/l. Explantswere subculturedin fresh mediumweekly. GUS test. The presence of the UidA gene was revealed using the method of Jefferson (1987). The percentage of GUS-positivecells was estimated on sections of treated material, by comparing the surface of blue-colouredsectionsto the surface of the newly growntissues.
Results Transformation of stem fragments. Results obtained with different bacterial strains are presented in Table 2. The control strain GV3101(pBI121.2) which contains the UidA (GUS) gene, but lacks the virulence genes required for T -DNA transfer did not lead to GUS-positive cells. The commonly used binary vector strain LBA 4404 (pAl_A404, pBI121.2) yields only very few blue cells. The pTiTm4, pBI121.2 combination yielded tumors of which 30-50% of the cells were blue, indicating a high transformation efficiency (Figure 1). The plasmid pTiTm4 transfers a complex set of tumor genes to the plant cells (Huss et al. 1990). One of these genes, T-6b, stimulates division of cells and has been shown to allow regeneration of normal Nieotiana rustica plants when placed under the control of a heat shock promoter (Tinland et al. 1990). The effect of the T-6b gene on transformation was tested with the following constructs : GV3101(,pPM6001), which transfers the UidA (GUS) gene, GV3101(pPM6001::pPM37), a cointegrate between pPM6001 and an intermediate vector containing the T-6b gene, - GV3101(pPM6001::pPM51), a cointegrate between pPM6001 and an intermediate vector containing the hsp 70 T6b gene. On rootstock stems (41 B, Kober 5BB, SO-4) GV3101(pPM6001::pPM37) (T-6b) induced calluses of the same size as the Tm4-GUS strain (Figure 2a). Sixty-two out of 63 calluses were GUS-positive, about 20% of the cells being blue. This represents a notable increase with respect to GV3101(pPM6001), and shows that the T-6b gene enhances the ratio of transformed versus untransformed cells. GV3101(pPM6001::pPM51) (hsp70-T-6b) induced calluses of the same size as those induced by the disarmed GV3101 (pPM6001) strain : they showed blue zones in 42 out of 49 infections and contained 80% blue cells. Table 2. Transformation of stem tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Vitis variety
Bacterial strain
GUS + * % blue calluses cells
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
LBA 4404 (pAL~04,pBI121.2)
Gewurztraminer 41 B
14/57 6/6
Plant Cell Reports (1992) 11:192-195
9 Springer-Verlag 1992
Transformation of vitis tissue by different strains of Agrobacterium tumefaciens containing the T-6b gene R. Berres 2, L. Otten
2,
B. Tinland 2, E. Malgarini-Clog 1, and B. Walter 1
1 Institut National de Recherche Agronomique, Station Vigne et Vin, Laboratoire de Pathologic V6g&ale, 28, rue de Herrlisheim, 68021 Colmar Cedex, France z Institut de Biologie Mol6culaire des Plantes, Laboratoire de Phytopathologie Mol6culaire, 12, rue du G6n6ral Zimmer, 67084 Strasbourg Cedex, France Received July i, 1991/Revised version received November 20, 1991 - Communicated by H. L6rz
Summary. Stem pieces and leaf disks of Vitis spp. were cocultured with Agrobacterium tumefaciens strains carrying the UidA (fl-glucuronidase = GUS) gene. The transformation efficiency was highly increased by using a modified T-6b gene (a gene from pTiTm4) which interferes with normal growth and allows regeneration of normal Nicotiana rustica plants (Tinland 1990). The strains first tested on stem segments were subsequently tested in a leaf explant system. On leaves the transformation efficiency of the strains was much lower than with stems. Both the T6b gene and the hsp 70-T-6b gene (a modified T-6b gene under the control of a heat shock promoter) allowed the initiation of GUS-positive buds.
Materials and methods Plant material. Vitis vinifera Chardonnay and Gewurztraminer, V. vin. X V. Berlandieri 41 B, V. Berl. X V. riparia Kober 5BB and SO-4 were used. Leaves (4-6 cm in diameter) and stem pieces (2-3 cm long) were harvested from plants grown in growth chambers at 32~ or from 6 weeks old plantlets grown on a modified Murashige and Skoog medium (MM) + 6 g/l Difco Agar (Martin etal..1987). Mobilisation of binary vectors into Agrobacterium. The binary vector pBI121.2 was mobilized from E. coli into A g r o b a c t e r i u m GV3101(pTiTm4) or GV3101 by triple mating using an E. coli strain carrying the pRK2013 helper plasmid. Exconjagant strains GV3101(pTiTm4, pBI121.2) and GV3101(pBI121.2) were selected on minimal medium with 400 I.tg/1 neomycin and 100 ~tg/1 rifampiein (GV3101 marker, Leemans et al. 1983). Plasmid content of exconjugants was verified by electrophoresis of bacterial lysates on agarose (Rosenberg et al. 1981)
Table 1 : Bacterial strains and plasmids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Key-words : Grapevine -Agrobacterium tumefacienstransformation Abbreviations : GUS = 13-glucuronidase ; BAP = benzylaminopurine ; X-gluc = 5-bromo-4-chloro-3-indolyl glucuronide. Introduction Genetic engeneering seems to be suited to introduce new properties into grapevine cultivars and rootstocks. Reports on Agrobacterium-mediated transformation of woody perennials are still scarce. There are only few publications on grapevine transformation. Hemstad and Reisch (1985) reported on in vitro gall production by A. tumefaciens and A. rhizogenes on Vitis L. hybrids. Genin and Walter (unpublished results) succeeded in transforming stem tissue of rootstocks by means of a disarmed vector containing the GUS gene. Guellec et al. (1990) infected whole plants of Vitis vinifera cv. Grenache with A. rhizogenes and obtained transformed roots. Baribault et al. (1990) succeeded in the regeneration of shoots expressing 13glucuronidase by cocultivation of shoot apex cultures of Vitis vinifera L. cv. Cabernet Sauvignon with A. tumefaciens. Mullins et al. (1990) obtained transformed plantlets of Vitis rupestris Scheele and transformed buds of Vitis vinifera cv. Chardonnay and Cabernet Sauvignon. In this report, we present a rapid and reproducible method for the transformation of grapevine by using a growthstimulating gene from the grapevine-specific Agrobacterium strain Tm4. Correspondence to." B. Walter
Bacterial strain
Characteristics
Marker of transformed plant cell
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
LBA4404(pAL4404, pBI121.2)
GUS gene on pBI121.2 helper plasmid pAL4404 Ach5 background
GUS
(1)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GV3101(pBII21.2)
GUS gene on pBI121.2 no helper plasmid C58 background
(2)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GV3101(pTiTm4, pB1121.2)
GUS gene on pBI121.2 helper plasmid pTiTm4 C58 background
GUS (2)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GV3101(pPM6000)
ocs gene on disarmed pTiAch5
OCS
(3)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GV3101(pPM6001)
UidA (GUS) gene on disarmed pTiAch 5
GUS
(4)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GV3 I01(pPM6000:: pPM 37)
T-6b gene in disarmed GUS vector
T -6b tumors OCS (5)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GV3101(pPM6001:: pPM37)
T-6b gene in disarmed GUS vector
T-6b tumors GUS (4)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GV3101(pPM6001:: pPMS1)
heat shock inducible T-6b gene in disarmed GUS vector
T-6b tumors GUS (6)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(1) Jefferson 1987 (2) Otten unpublished (3) Bonnard etal. 1990 (4) Tinland etaL. 1990 (5) Tinland etal. 1989 (6) Tinland 1990. Cocultivation. After the leaf veins were cut with a sterile scalpel, the leaves were cocuhivated for one hour in Petri dishes containing 106-107 bacteria/ml from the various strains (Table 1). The leaves were dried and placed in Petri dishes with liquid MM medium + BAP 2 mg/1 + Cefotaxim (R) 500 mg/1 (Roussel Uclaf).
193 After about three weeks the developingbuds were transferred to tubes with solid MS medium (Murashige and Skoog 1962). Stem pieces were cocultivated in the same way, and were planted upside down in solid MM + BAP 2 mg/l. Explantswere subculturedin fresh mediumweekly. GUS test. The presence of the UidA gene was revealed using the method of Jefferson (1987). The percentage of GUS-positivecells was estimated on sections of treated material, by comparing the surface of blue-colouredsectionsto the surface of the newly growntissues.
Results Transformation of stem fragments. Results obtained with different bacterial strains are presented in Table 2. The control strain GV3101(pBI121.2) which contains the UidA (GUS) gene, but lacks the virulence genes required for T -DNA transfer did not lead to GUS-positive cells. The commonly used binary vector strain LBA 4404 (pAl_A404, pBI121.2) yields only very few blue cells. The pTiTm4, pBI121.2 combination yielded tumors of which 30-50% of the cells were blue, indicating a high transformation efficiency (Figure 1). The plasmid pTiTm4 transfers a complex set of tumor genes to the plant cells (Huss et al. 1990). One of these genes, T-6b, stimulates division of cells and has been shown to allow regeneration of normal Nieotiana rustica plants when placed under the control of a heat shock promoter (Tinland et al. 1990). The effect of the T-6b gene on transformation was tested with the following constructs : GV3101(,pPM6001), which transfers the UidA (GUS) gene, GV3101(pPM6001::pPM37), a cointegrate between pPM6001 and an intermediate vector containing the T-6b gene, - GV3101(pPM6001::pPM51), a cointegrate between pPM6001 and an intermediate vector containing the hsp 70 T6b gene. On rootstock stems (41 B, Kober 5BB, SO-4) GV3101(pPM6001::pPM37) (T-6b) induced calluses of the same size as the Tm4-GUS strain (Figure 2a). Sixty-two out of 63 calluses were GUS-positive, about 20% of the cells being blue. This represents a notable increase with respect to GV3101(pPM6001), and shows that the T-6b gene enhances the ratio of transformed versus untransformed cells. GV3101(pPM6001::pPM51) (hsp70-T-6b) induced calluses of the same size as those induced by the disarmed GV3101 (pPM6001) strain : they showed blue zones in 42 out of 49 infections and contained 80% blue cells. Table 2. Transformation of stem tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Vitis variety
Bacterial strain
GUS + * % blue calluses cells
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
LBA 4404 (pAL~04,pBI121.2)
Gewurztraminer 41 B
14/57 6/6
