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Plant Cell Reports (1988) 7:88-91 PlantCeU Reports © Springer-Verlag1988 Genetic transformation of cauliflower (Brassica oleracea L. var. Botrytis) by A grobacterium rhizogenes Chantal David and Jacques Temp~ Groupe de Recherche sur les Interactions entre Microorganismes et Plantes, G~n6tique et Amelioration des Plantes, INRA et CNRS, Institut de Microbiologie, Facult6 des Sciences, Bat. 409, F-91405 Orsay C6dex 05, France Received September 29, 1987 / Revised version received December 21, 1987 - Communicated by J. Schell Abstract Cauliflower plantlets were inoculated with different Agrobacterium rhizogenes strains. Numerous hairy roots were induced on cauliflower hypocotyls by agropine-type strains. Fewer roots were obtained with mannopine-type strains. In vitro cultures were established both from normal and hairy roots. Plant regeneration occured spontaneously from normal and transformed roots. Regenerated plants contained the same opines (if present) as root cultures. Some mannopine-positive regenerants displayed a modified phenotype. Relationships between phenotype, opine content, T-DNA content and/or expression are discussed. Introduction The soil bacteria A. tumefaciens and A.rhizogenes are able, via a natural system of genetic transformation, to insert DNA sequences (T-DNA for Transferred DNA), originally borne on their pathogenic plasmids (called respectively Ti or Ri) into the genome of dicotyledonous plant cells (for a recent review, see Gheysen et aL 1985). The expression of T-DNA inserts in plant cells results in proliferative overgrowths (crown gall or hairy root). Homology between Ti and Ri plasmids is limited to some essential functions involved in the T-DNA transfer process (Huffman et aL 1984, Jouanin 1984), or opine synthesis and degradation. At the DNA level, Ti plasmids share homologous sequences known as "common T-DNA", which are responsible for tumor development. Common T-DNA essentally carries genes involved in cytokinin and auxin synthesis (Schroeder etal. 1984, Akiyoshi et al. 1984). Mutations in the cytokinin gene result in reoty tumors that, on some hosts, closely look like hairy roots (Garfinkel et al. 1981, Ooms et al. 1981, Leemans et aL 1982, Joos et al. 1983a). Agropine-type Ri plasmids (Petit et al. 1983) have T-DNA borne auxin genes (de Paolis et aL 1985, Huffman etaL 1984, Jouanin 1984) but these are accessory to the phenomenon. Hairy root rhizogenesis is due to the expression of T-DNA borne gene(s) (White et al. 1985) whose product(s) are still unidentified (Cardarelli et al. 1987, Spena et al. 1987, Vilaine and Casse-Delbart 1987). A major, and possibly fundamental, difference between hairy Offprint requests to: C. David root and crown gall is stressed by the following features : for plants known to regenerate, viable and fertiles plants can be obtained from hairy root cultures, with a full T-DNA complement (Costantino et al. 1984, David et aL 1984, Tepfer 1984, Jouanin et al. 1987, Birot et al. 1987). In contrast, it is very difficult to obtain regenerants from crown gall tumors incited by wild type A. tumefaciens strains with wild type T-DNA. These regenerants from crown gall tumors showed rearrangements in their T-DNA content (Wullems et al. 198i). In the genus Brassica, which includes species of agronomic importance, oilseed rape (Brassica napus ) (Guerche et al. 1987) and cabbage (Brassica oleracea var. capitata ) (Birot et al. 1987) have already been transformed using agropine-type A. rhizogenes strains. We report transformation of cauliflower (Brassica oleracea L. var.. botrytis ) by Ri T-DNA of different A. rhizogenes strains, regeneration of transformed plants, analysis of opine and T-DNA content of regenerants. Material and Methods Seeds of cauliflower from strain ES 5-1 were kindly supplied by B. Smets from graines Caillard, chemin de Pouill6, BP 30, 49130 Les Ponts-de-C~, France. A. rhiZogenes strains used were previously described (Petit et aL 1983). The physical map of the A. rhizogenes strain 8196 virulence plasmid (pRi 8196) has been published ; plasmids harbouring inserts from pRi 8196 were described previously (Koplow et al. 1984). A. rhizogenes strains were grown on solid rich medium (LB, Maniatis, 1982) at 28°C. E. colt strain LE 392, harbouring recombinant plasmid 218 (Fig. la) was grown at 37°C on solid LB medium with ampicillin (25 #g/ml) and cMoramphenicol (25 I~g/ml). Cauliflower seeds were disinfected with a solution of calcium hypochlorite (90g/I for 15 minutes) and washed with sterile water. Seeds were germinated in the dark in Petri dishes on Monnier's medium (Monnier, 1976). Hypocotyls from ten day-old etiolated plantlets were inoculated with bacteria after wounding. Inoculated seedlings were transferred on the same medium without sucrose and maintained at 25°C under a 16 h photoperiod.

Genetic transformation of cauliflower (Brassica oleracea L. var. Botrytis) by Agrobacterium rhizogenes

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Page 1: Genetic transformation of cauliflower (Brassica oleracea L. var. Botrytis) by Agrobacterium rhizogenes

Plant Cell Reports (1988) 7:88-91 PlantCeU Reports © Springer-Verlag 1988

Genetic transformation of cauliflower (Brassica oleracea L. var. Botrytis) by A grobacterium rhizogenes

Chantal David and Jacques Temp~

Groupe de Recherche sur les Interactions entre Microorganismes et Plantes, G~n6tique et Amelioration des Plantes, INRA et CNRS, Institut de Microbiologie, Facult6 des Sciences, Bat. 409, F-91405 Orsay C6dex 05, France

Received September 29, 1987 / Revised version received December 21, 1987 - Communicated by J. Schell

Abstract

Cauliflower plantlets were inoculated with different Agrobacterium rhizogenes strains. Numerous hairy roots were induced on cauliflower hypocotyls by agropine-type strains. Fewer roots were obtained with mannopine-type strains. In vitro cultures were established both from normal and hairy roots. Plant regeneration occured spontaneously from normal and transformed roots. Regenerated plants contained the same opines (if present) as root cultures. Some mannopine-positive regenerants displayed a modified phenotype. Relationships between phenotype, opine content, T-DNA content and/or expression are discussed.

Introduct ion

The soil bacteria A. tumefaciens and A.rhizogenes are able, via a natural system of genetic transformation, to insert DNA sequences (T-DNA for Transferred DNA), originally borne on their pathogenic plasmids (called respectively Ti or Ri) into the genome of dicotyledonous plant cells (for a recent review, see Gheysen et aL 1985). The expression of T-DNA inserts in plant cells results in proliferative overgrowths (crown gall or hairy root). Homology between Ti and Ri plasmids is limited to some essential functions involved in the T-DNA transfer process (Huffman et aL 1984, Jouanin 1984), or opine synthesis and degradation.

At the DNA level, Ti plasmids share homologous sequences known as "common T-DNA", which are responsible for tumor development. Common T-DNA essentally carries genes involved in cytokinin and auxin synthesis (Schroeder etal . 1984, Akiyoshi et al. 1984).

Mutations in the cytokinin gene result in reoty tumors that, on some hosts, closely look like hairy roots (Garfinkel et al. 1981, Ooms et al. 1981, Leemans et aL 1982, Joos et al. 1983a). Agropine-type Ri plasmids (Petit et al. 1983) have T-DNA borne auxin genes (de Paolis et aL 1985, Huffman etaL 1984, Jouanin 1984) but these are accessory to the phenomenon. Hairy root rhizogenesis is due to the expression of T-DNA borne gene(s) (White et al. 1985) whose product(s) are still unidentified (Cardarelli et al. 1987, Spena et al. 1987, Vilaine and Casse-Delbart 1987).

A major, and possibly fundamental, difference between hairy

Offprint requests to: C. David

root and crown gall is stressed by the following features : for plants known to regenerate, viable and fertiles plants can be obtained from hairy root cultures, with a full T-DNA complement (Costantino et al. 1984, David et aL 1984, Tepfer

1984, Jouanin et al. 1987, Birot et al. 1987). In contrast, it is very difficult to obtain regenerants from crown gall tumors incited by wild type A. tumefaciens strains with wild type T-DNA. These regenerants from crown gall tumors showed rearrangements in their T-DNA content (Wullems et al. 1 9 8 i ) .

In the genus Brassica, which includes species of agronomic importance, oilseed rape (Brassica napus ) (Guerche et al. 1987) and cabbage (Brassica oleracea var. capitata ) (Birot et al. 1987) have already been transformed using agropine-type A. rhizogenes strains.

We report transformation of cauliflower (Brassica oleracea L. var.. botrytis ) by Ri T-DNA of different A. rhizogenes strains, regeneration of transformed plants, analysis of opine and T-DNA content of regenerants.

Material and Methods

Seeds of cauliflower from strain ES 5-1 were kindly supplied by B. Smets from graines Caillard, chemin de Pouill6, BP 30, 49130 Les Ponts-de-C~, France.

A. rhiZogenes strains used were previously described (Petit et aL 1983). The physical map of the A. rhizogenes strain 8196 virulence plasmid (pRi 8196) has been published ; plasmids harbouring inserts from pRi 8196 were described previously (Koplow et al. 1984). A. rhizogenes strains were grown on solid rich medium (LB, Maniatis, 1982) at 28°C. E. colt strain LE 392, harbouring recombinant plasmid 218 (Fig. la) was grown at 37°C on solid LB medium with ampicillin (25 #g/ml) and cMoramphenicol (25 I~g/ml).

Cauliflower seeds were disinfected with a solution of calcium hypochlorite (90g/I for 15 minutes) and washed with sterile water. Seeds were germinated in the dark in Petri dishes on Monnier's medium (Monnier, 1976). Hypocotyls from ten day-old etiolated plantlets were inoculated with bacteria after wounding. Inoculated seedlings were transferred on the same medium without sucrose and maintained at 25°C under a 16 h photoperiod.

Page 2: Genetic transformation of cauliflower (Brassica oleracea L. var. Botrytis) by Agrobacterium rhizogenes

Roots developed in 8 to 24 days at the site of inoculation. Single root tips (ca. 2 cm in length) were transferred on solid Monnier's medium (25°C, 16 h photoperiod). In some cases, carbenicillin (50 pg/l) was added to the medium for the two or three first transfers. Absence of bacterial contamination was assessed by squashing root pieces and incubating them at 28°C on LB medium.

Plant extracts were analyzed for the presence of mannopine or mannopine and agropine as described previously (Petit et aL 1983).DNA was extracted from roots, leaves or curds using 30 to 40 g (fresh weight) for each extraction as previously described (Chilton et a l . 1982). Small scale preparation (less than 5 g fresh material) was performed by the method of Dellaporta et a l . (1983), followed by an additional purification by CsCI-Ethidium bromide gradient centrifugation. Plasmid DNA used as probe was prepared by the clear lysate method (Clewe]l and Helinski, 1969). Plant DNA was digested for 5 hours with restriction endonucleases (5 to 10 u/p.g DNA) according to recommendations of the supplier (Boehringer). Digested DNA (51~g/well) was electrophoresed (1-1.5 V/cm ; 15-24 hours) in Tris-borate horizontal agarose gels.

Agarose gels were prepared for blotting according to Southern (1975). Membranes (Gene Screen Plus, New England Nuclear) were prepared and handled as recommended by the manufacturer. The probe used was plasmid clone 218 (Fig. 1), labeled with 32p dCTP using a nick translation kit (Amersham) at a specific activity of ca. 5.107dpm/l~g. Hybridized blots were autoradiographed with Kodak X©mat AR films with one intensification screen.

Results

Pathoaenicity of A. rhizoqenes strains

As previously mentioned (Petit et al. 1983), agropine strains (A4, 15834) were the most virulent ones. Mannopine strains (8196, TR101, TR7) incited roots on hypocotyls, but these were fewer and developed later than after an inoculation with agropine strains.

Presence of silver nitrate-positive substances of the aarooine family

Roots incited at the wound site (primary roots) by strains 8196, TR7 and TR101 always contained mannopine. Only a portion of hairy roots incited by strains A4 and 15834 contained opines (3/30) (Petit et aL 1983). The same opines were found in primary roots in hairy root cultures derived from them and in the culture medium (Fig. lb). Lines incited by strain 8196 were found to be very stable with respect to opine synthesis, as previously observed (David et aL 1984). Mannopine was the most abundant of these (ca. 0.2% of the fresh weight of roots). In contrast, the opine content of hairy root cultures incited by agropine-type Ri plasmid varied from one culture to the other.

Phenotype of in vitro cultures

Normal cauliflower roots did not grow for more than one month when isolated and in vitro cultivated. Growth stopped and roots gave rise to neoformed buds (Fig. lc). Cauliflower hairy root lines did not display a pronounced hairy root phenotype (i. e. fast growth rate, high lateral branching and lack of geotropism ; David et al. 1984, Tepfer 1984). Nevertheless, they grew better and for a longer period than normal roots (Fig. ld ) .

89

Plant reaeneration

Regeneration occurred spontaneously for both normal and transformed cauliflower roots. Buds developed earlier on normal roots (40 days after root isolation) than on hairy roots (2 to 8 months according to the root lines studied). Neoformed buds appeared on roots, cultivated either on solid or in liquid medium (Fig. l e). Regenerated plantlets were first grown aseptically (Fig. le, f) before transfer to the greenhouse (Fig. l f ) .

Adventitious buds developed often from the first shoot. Both hairy root and normal root cultures re-isolated from regenerated plantlets had more pronounced tendency to bud neoformation than primary cultures.

Transformed plant 0henotype

Thirty plants were regenerated from ten different root lines incited by strains A4 and 15834. Both for opine-positive and opine-negative regenerants, the presence of TL-DNA was established, by dot-blot hybridization. The probe used was clone PMP 30 (Pomponi et al. 1983). They all contained TL-DNA (data not shown). Nevertheless, they did not exhibit the morphological traits associated with the presence of hairy root TL-DNA (Tepfer 1984, Ooms et al. 1985, Guerche et aL 1987). They closely looked like normal ES 5-1 plants. Regenerants from the mannoplne hairy root line ES 5-1 8196 were phenotypically more variable. Among six plants, two exhibited a typical hairy root phenotype, i. e. wrinkled leaves, plagiotropic roots, short internodes, first described on tobacco by Ackermann (1977), (Fig. 2a, left) while the others appeared normal when compared to plants regenerated from normal roots (Fig. 2a, right). Plants with an altered phenotype formed very wrinkled leaves and had smaller curds. They did not flower (even 5 months after transfer to the greenhouse) and finally died.

Ouine content of reaenerants

Extracts were prepared from leaves, roots, curds, floral tissues and siliquae from regenerants developed on the root lines incited by agropine-type Ri plasmids. When original root lines contained mannopine and agropine, all of the analyzed tissues contained these opines, whose amount, as estimated on high voltage paper electrophoresis, were as variable as observed in the hairy root lines (data not shown). Extracts were prepared from leaves and roots of the six plants regenerated from the line ES 5-1 8196. Mannopine content of leaves appeared very homogeneous and represented about 0.05% of the fresh weight of the leaves (Fig. 2b). In contrast, mannopine concentration in roots was more variable, ranging from less than 0.01% (Tz-IX) to more than 0.06% (Tl-I; Fig. 2b).

T-DNA content of mannopine-po~itive regenerants

DNA was extracted from roots, leaves and cauliflower curds. The best yield of extraction was obtained with curds, and DNA could be digested with restriction endonucleases in this case after ethanol precipitation without further purification. Root and leaf DNAs were analyzed for three T1 regenerants from the hairy root line ES 5-1 8196, two with normal morphology and one with a typical hairy root phenotype. Hybridization patterns were identical. Two internal fragments, corresponding to Barn HI restriction fragments 6 and 19, which belong to the core T-DNA (Byrne et a l . 1983 ) were present in the three regenerants analyzed (Fig. 2c). Only two plant DNA - T-DNA junction fragments were visible. This T-DNA structure is similar to that observed in some carrot lines (Byrne et al.

Page 3: Genetic transformation of cauliflower (Brassica oleracea L. var. Botrytis) by Agrobacterium rhizogenes

90

Fig. l a . : Barn HI restriction map of the T-region of pRi 8196 (Koplow et aL 1984). The length of insert of clone 218, used as a probe for T-DN& analysis, is shown.

Fig. l b : Electrophoresis analysis of extracts from cauliflower ES 5-1 hairy root line (B, D, F, H). Each spot corresponds to 1.4 mg (fresh weight) for roots and respectively to 100 I~1 (B), 20 ILl (D), 40111 (F, H) for culture media. For each line, root extracts (A, C, E, G) and used growth medium (B, D, F, H) have been analyzed side by side. Extracts were spotted on Whatmann 3 MM paper and electrophoresed in acetic acid/formic acid buffer (pH 1.9) at 100 V/cm for 15 min. Staining was performed with silver nitrate reagent. Hairy roots were incited by Agrobacterium rhizogenes strains 8196 (A to D) and A4 (E to H). The standard(s) contained : I, agropine ; II + III, mannopine and mannopinic acid (unresolved) ; IV, agropinic acid. N corresponds to neutral compounds (e.g. mannitol in standard).

Fig. l c : Normal cauliflower (ES 5-1) root culture, with neoformed shoots. Fill. l d : Cauliflower hairy root culture (ES 5-1 8196). Fig. l e : Neoformed cauliflower shoot on hairy root. Fig. I f : T1 regenerant from a cauliflower hairy root line incited by an agropine-type Ri

plasmid.

~ o k b ~ a o 15

core _T_DNA

Barn H !

lb Figure 1

218

Fig. 2a : Comparison between two T1 regenerants from the cauliflower hairy root line ES 5-1 8196. Left, TI-I with wrinkled leaves ; right, regenerant with normal leaves.

Fig. 2b : Electrophoretic analysis of extracts from leaves (a) and roots (b) of T1 plants regenerated from the hairy root line ES 5-1 8196. One microliter of extract corresponding to 2 mg of leaves (fresh weight) and to 10 mg of roots (fresh weight) wel:e spotted on Wathmann 1 MM paper and electrophoresed at 100 V/cm for 15 min. Staining was performed with silver nitrate reagent. I to A, T1 regenerants ; N, extracts from leaf and root of one normal ES 5-1 regenerant. II : mannopine, ---, origin of separation ; ~ , unknown silver nitrate-positive compound present in normal roots.

Fig 2c : Southern blot analysis of DNA prepared from roots (r) and leaves (1) of 3 regenerants from the hairy root line ES 5-1 8196. Lanes 1, 2, 3 : regenerants with ne rr~=~ leaves ; lanes 4 and 5 : regenerant TI-I with wrinkled leaves). Lane 6 : R, reconstru hybridization where Bam HI digested DNA from the clone 218 (6, 10, 13, 14, 19 : fragments ; v : vector) was loaded at a concentration equivalent to 5 copies of T-DNA / di cauliflower genome. ~ : junction fragments.

F igure 2

Page 4: Genetic transformation of cauliflower (Brassica oleracea L. var. Botrytis) by Agrobacterium rhizogenes

1983, David et al. 1987). However, the difference is that only one insert seems to be present in the cauliflower hairy root line studied.

Discussion

Regeneration of whole plants from hairy root cultures is readily obtained with many species (Tepfer 1984, Costantino et al., 1984, Petit et aL, 1986, Jouanin et al. 1987). Crucifers are known to regenerate from roots, either in vivo or in vitro (North 1953, Bajaj and Nietsch 1975, Grout and Crisp 1980, Lazzeri and Dunwell 1984) and this property is maintained in hairy root cultures, including cauliflower.

Cauliflower regenerants observed had generally a normal phenotype, and only two plants regenerated from the mannopine-containing line 81 96 exhibited a modified phenotype. An altered phenotype was described for several plant species like tobacco (Ackermann 1977, Tepfer 1984), potato (Ooms et aL 1985), oilseed rape (Guerche et aL 1987). This phenotype is more or less pronounced : leaf wrinkling is negligible in alfafa transformants (Spano et aL 1987) and not always visible on Lotus comiculatus regenerants (Petit et aL 1987). Since plants with normal and altered phenotype had similar T-DNA content, we suppose that the variability we observed is due to differential expression of T-DNA genes in regenerated plants. This is consistent with previous observations on tobacco regenerants from one hairy root line, incited by an agropine-type strain (Tepfer 1984), in which this variability could indeed be correlated with TL-DNA expression (Durand-Tardif etal . 1985).

Acknowledgements

This research was supported by grants from the CNRS (U.A. 136), INRA (Groupe de Recherche sur les Interactions Microorganismes-PIantes), EEC grant n°6B1-4-018F and BAP-0015-F and Minist~re de l'lndustrie et de la Recherche.

References

Ackermann C. (1977) Plant Sci. Lett. 8:23-30. Akiyoshi DE., Klee H., Amasino RM., Nester EW., Gordon MP.

(1984) Prec. Natl. Acad. Sci. USA 81:5994-5998. Byrne MC., Koplow J., David C., Temp~ J., Chilton MD.

(1983) J. Mol. Appl. Genet. 2:201-209. Bajaj YPS., Nietsch P. (1975) J. Exptl. Bet. 26:883-890. Birot AM., Bouchez D., Casse-Delbart F., Durand-Tardif M.,

Pautot V., Rebaglia C., Tepfer D., Tepfer M., Tourneur J., Viiaine F. 1987) Plant Physiol. and Biochem. 7:323-335.

Cardarelli M., Spano L., Mariotti D., Mauro ML., Van SLuys MA., Costantino P. (1987) Mol, Gen. Genet. in press.

Chilton MD., Tepfer DA., Petit A., David C., Casse-Delbart F., Temp~ J. (1982) Nature 295:432-435.

Clewell DB., Helinski DR. (1969) Prec. Natl. Acad. Sci. USA 62:1 156-1 166.

Costantino P., Spano L., Pomponi M., Benvenuto E., Ancora G. (1984) J. Mol. Appl. Genet. 2:465-470.

David C., Chilton MD., Temp~ J. (1984) Bio/technology 2:73-76.

9]

David C., Petit A., Temp~ J. (1987) PI. Ceil Reports, in press.

Dellaporta SL., Wood J., Hicks JB. (1983) Plant Mol. Biol. reporter 1:19-21.

De Paolis A., Mauro ML., Pomponi M., Cardarelli M., Spano L., Costantino P. (1985) Plasmid 13:1-7.

Durand-Tardif M., Broglie R., Slightom J., Tepfer D. (1985) J. Mol. Biol. 186:557-564.

Garfinkel DJ., Simpson RB., Ream LW., White FF., Gordon MP., Nester EW. (1981) Cell 27:143-153.

Gheysen G., Dhaese P., Van Montagu M., Schell J. (1985) in : Plant gene research : genetic flux in plants. Hohn B., Dennis ES. (Eds.) Springer Verlag, Wien, New York, pp. 11-47.

Guerche P., Jouanin L., Tepfer D., Pelletier G. (1987) Mol. Gen. Genet. 206:382-386.

Grout BWW., Crisp P. (1980) J. Hort, Sci. 55:65-70. Huffman GA., White FF., Gordon MP., Nester EW. (1984) J.

Bacteriol. 157:269-276. Joos H., Inze D., Caplan A., Sormann M., Van Montagu M.,

Schell J. (1983a) Cell 32:1057-1067. Jouanin L. (1984) Plasmid 12:91-102. Jouanin L., Guerche P., Pamboukdjian N., Tourneur C.,

Casse-Delbart F., Tourneur J. (1987) Mol. Gen. Genet. 206:387-392.

Koplow J., Byrne MC., Temp~ J., Chilton MD. (1984) Plasmid 11:17-27.

Lazzeri PA., Dunwell JM. (1984) Annals of Botany 54:341 -361.

Leemans J., Deblaere R., Willrnitzer L., De Greve H., Hernalsteens JP., Van Montagu M., Schell J. (1982) EMBO J. 1:147-152.

Maniatis T., Fritsch EF., Sambrook J. (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory.

Monnier M. (1976) Rev. Cytol. Biol. Veg. 39:1-120. North C. (1953) Ann. Appl. Biol. 40:250-261. Ooms G., Hooykas PJ., Moleman G., Schilperoort RA. (1981)

Gene 14:33-50. Corns G., Kamp A., Burrell MM., Roberts J. (1985) Theor.

Appl. Genet. 70:440-446. Petit A., David C., Dahl G., Ellis JG., Guyon P., Casse-Delbart

F., Temp~ J. (1983) Mol. Gen. Genet. 190:204-214. Petit A., Berkaloff A., Temp~ J. (1986) Mol. Gen. Genet.

202:388-393. Petit A., Stougaard J., K0hle A., Marcker KA., Temp~ J.

(1987) Mol. Gen. Genet. 207: 245-250. Pomponi M., Spano L., Sabbadini MG., Costantino P. (1983)

Plasmid 10:119-129. Schroeder G., Waffenschmidt S., Weiler EW., Schroeder J.

(1984) Eur. J. Biochem. 138:387-391. Southern E. (1975) J. Mol. Biol. 95:503-517. Spano L., Mariotti D., Pezzotti M., Damiani F., Arcioni S.

(1987) Theor. Appl. Genet. submitted. Spena A., Schm[Jlling T., Koncz C., Schell JS. (1987)

submitted. Tepfer D. (1984) Cell 37:959-967. Vilaine F., Casse-Delbart F. (1987) Gene 55:105-114. White FF., Taylor BH., Huffman GA., Gordon MP., Nester EW.

(1985) J. Bacteriol. 164:33-44. Wullems GJ., Molendijk L., Corns G., Schilperoort RA.

(1981) Cell 24:719-727.