Plant Cell Reports
Plant Cell Reports (1991) 10:354-357
9 Springer-Verlag1991
Gene transfer into peanut (Arachis hypogaea L.) by Agrobacterium tumefaciens C. Lacorte a, E. Mansur 2, B. Timmerman a, and A.R. Cordeiro a 1 Department of Genetics, Federal University of Rio de Janeiro, Caixa Postal 68011, CEP 21944- Rio de Janeiro, Brazil z Department of Cellular Biology and Genetics, University of the State of Rio de Janeiro, Brazil Received January 14, 1991/Revised version received June 13, 1991 - Communicated by I.K. Vasil
ABSTRACT. Introduction of foreign genes into plant tissues via AQrobacterium tumefaciens based vectors requires specific knowledge of Agrobacterium-host compatibility. Therefore, to develop a transformation protocol for peanut (Arachis hypogaea L . ) , five Brazilian cultivars were screened with four wild-type A.tumefaciens strains. Successful transformation was dependent on specific bacterial strain-plant cultivar interactions and strain A281 was the most effective for tumor induction. Tumors displayed hormone autonomous growth, were opine positive and contained DNA that was homologous to the T-DNA of the inciting strain. Tumors induced on seed and seedling explants by A281 (pTD02) also expressed the reporter genes gus and npt-II contained in the binary vector. These results show that peanut is a permissive host for the acceptance of genes from specific A.tumefaciens gene vectors. Abbreviations: GUS: B-glucuronidase (EC 3.2.1.31) NPTII: neomycin phosphotransferase II (EC 2.7.1.95), EDTA: ethylene-diamine-tetracetic acid.
INTRODUCTION
Gene transfer to plants via Agrobacterium tumefaciens is an efficient way to introduce desirable traits into crop plants. Transformation by Agrobacterium was demonstrated in the family Leguminosae in soybean (Hinchee et al. 1988), pea (Puonti-Kaerlas et al. 1989) and forage legumes (Deak et al. 1 9 8 6 ; Manners 1987). Susceptibility to Agrobacterium varies among related species and cultivars of legumes (Byrne et al. 1987; Manners 1987; Hinchee et al. 1988; Owens and Cress 1985), indicating that the transformation process depends on the combined action of bacterial and plant genotypes. Therefore, evaluation of strain-cultivar compatibility
Offprint requests to: A.R. Cordeiro
is an important step in the establishment of a transformation protocol. In this report, the susceptibility of five Brazilian cultivars of peanut to four wild strains of A.tumefaciens was evaluated by determining tumor induction frequencies. Variations in strain-cultivar compatibility were determined and the most efficient host-pathogen interaction was chosen for the study of tumor induction in explants The transformation process was confirmed by opine and T-DNA integration analysis. The expression of chimeric genes in transformed tissues was demonstrated by using a binary vector (pTP02) containing npt-II and gus genes.
MATERIALS AND METHODS
Plant material and inoculation conditions Seeds of Arachis hypogaea L. (Cvs Tatu, Tatuf, Tatu branco, T u p ~ and Pen&polis) were obtained from I n s t i t u t o Agron6mico, Campinas, B r a z i l and planted in a s o i l : s a n d mixture ( 3 : 1 ) . Four-week-old plants were inoculated with w i l d - t y p e s t r a i n s of A.tumefaciens by wounding stem internodes with a hypodermic needle and applying 10 #l of an overnight culture. I n o c u l a t i o n s i t e s were covered with p l a s t i c f i l m for one week to prevent d e s s i c a t i o n . Two p l a n t s of each c u l t i v a r were inoculated with each s t r a i n of bacterium (Table I ) , at s i x s i t e s per p l a n t . Frequency of tumor formation and sizes were scored f i v e weeks a f t e r inoculation. As a control for virulence, tumors were induced on Kallanchoe tubiflora stem internodes with the same procedure used for peanut plants. In order to obtain in vitro grown plants, seeds were surface-sterilized in 0.1% (w/v) HgCl2, plus a few drops of tween 80 for 15 minutes with constant agitation, rinsed six times with sterile distilled water and soaked for one minute in 5mM EDTA after the third wash. Seeds were germinated in jars containing cotton wool wetted with 1/2 strength MS salts (Murashige and Skoog 1962), (pH 5.8) supplemented with 3% sucrose. Ten-day-old seedlings were cut above the cotyledonary node and placed in MS medium solidified with 0.8% agar. Rooted plants were inoculated as
355 described f o r greenhouse p l a n t s , except that p l a s t i c f i l m was not used t o cover wound s i t e s . In a d d i t i o n , cefotaxime at a final concentration of 500 #g/ml was added to the culture medium 24 hours after inoculation to prevent bacterial growth. Seed explants (embryonic axes and cotyledon slices) were excised from surface-sterilized seeds. Leaf and petiole explants were obtained from seven-day-old in vitro-grown plantlets. Tissue fragments were infected with 10 ~l of an overnight culture, blot-dried on sterile filter paper and cultured on solid MS salts and vitamins supplemented with 3% sucrose for 24 hours. After this period the explants were rinsed once in liquid MS medium, blot-dried and transferred to solid MS medium plus 500 #g/ml cefotaxime. Tumor appearance was recorded after 5 weeks. Tumors induced by the tested strains on tobacco leaf explants and plantlets were cultured and used as positive controls for opine and NPT-II assays. B a c t e r i a l s t r a i n s and plasmids A.tumefaciens s t r a i n s T37, A281, Bo542 and A136 ( a v i r u l e n t ) were obtained from U n i v e r s i t y of Gent, Belgium. Strains A208 and LE392 (pEHBI07) were provided by Dr. Elizabeth Hood, Utah U n i v e r s i t y , Logan, USA. Plasmid pTD02 was a g i f t of Dr. D. E. O l i v e i r a and contains n p t - I I and gus genes, the l a t e r under the control of ATC1 promotor ( A l l i o t e et a l . 19891. S t r a i n A281 (pTD02) was constructed by t r i p a r e n t a l mating ( D i t t a et a l . 19801. Relevant phenotypes of A.tumefaciens s t r a i n s used are l i s t e d in Table I .
Table I: A.tumefaciens wild type strains used STRAIN
PLASMID
A281 Bo542 A208 T37 A136
pTiBo542 pTiBo542 pTiT37 pTiT37
CHROMOSOMAL BACKGROUND C58 Bo542 C58 T37 C58
OPINE(a)
REF
AGR, L,L.SAP, LOP AGR, L,L.SAP, LOP NOP, AGC NOP, AGC
(I) (2) (2) (2) (2)
(a) AGR, agropine; L,L,SAP, L,L,succinamopine; LOP, leucinopine; NOP, nopaline; AGC, agrocinopine. ( I ) Hood et al. 1986a. (2) Sciaky et al. 1978. Wild-type strains were grown in YEB medium (bacto beef extract 5g/l, bacto yeast extract Ig/l, peptone 5g/l, sucrose 5g/l, MgSO 4 240 mg/l, pH 7.2) with 100 #g/ml rifampicin. Strain A281 (pTD02) was grown in YEB medium containing 300 #g/ml streptomycin and 100 #g/ml spectinomycin. Prior to inoculation, bacteria were grown overnight in Minimum Medium A (K2HPO4 10.5g/l, KH2PO4 4.5g/I, (NH4)2SO 4 1.0g/l, sodium citrate 2H20 0.5 g/l, MgSO4.7H20 200 mg/l, glucose 2 g/l).
28oc. Agropine assays were performed as described by Reynaerts et at. (1989), and nopaline detection by the method of Otten and Schilperoort (1978). GUS and NPT-II assays. In vitro induced tumors were used for NPT-II and histochemical GUS assays, after being checked for sterility. GUS activity was assayed as described by Jefferson et al. (1987) and NPT-II activity was demonstrated by the dot-blot assay (McDonnell et al. 1987). Southern hybridization analysis. DNA was isolated from in vitro cultured, axenic tumors according to the procedure of Dellaporta et at. (1983) and analysed by Southern hybridiztion (Scott 1988) with probes prepared using a nick translation kit of Bethesda Research Laboratories. The DNA restriction fragment used as probe for A281 induced tumors was Bam HI fragment 10 of T-DNA region of pTiBo542 (Hood et al. 1986b), isolated from pEHBI07. The probe for DNA from tumors induced by A281 (pTD02) was the purified fragment of Sat I restriction of pTD02 containing npt-II gene. RESULTS AND DISCUSSION
Tumor induction in green-house plants All peanut cultivars tested may be considered as AQrobacterium permissive hosts, because tumors developed in response to infection with all virulent strains tested, (Fig. I) although strain T37 did not induce tumors in Cvs Tatu branco and Tatuf. In general, sensitivity of Cv Tatu branco was lower considering both tumor size and induction frequency (Table If), except when inoculated with strain A281. Tatu a n d Tup~ were most sensitive to all strains. Other legumes, including soybean, show genotypic variability of response upon infection with Agrobacterium (Byrne et al. 1987; Manners 1987; Hinchee et al. 1988; Owens and Cress 1985).
Culture of tumors. Tumors obtained from greenhouse plants were excised, and the internal part was surface sterilized in 2% (w/v) NaClO for 15 min, rinsed 6 times in sterile distilled water and placed in liquid MS medium, with 500 #g/ml cefotaxime. After two days, tumors were transferred to solid MS medium (agar 0.8%), with 250 #g/ml cefotaxime. Tumors of in vitro-grown plants and explants were excised and directly transferred to the culture medium plus antibiotic. Tumors incited by A281 (pTD02) were cut into approximately 2 rnm cubes and plated in solid MS medium containing 50 - 300 ~g/ml kanamycin. Opine detection. Putative tumors were tested for sterility by incubation of segments on YEB medium for 48 hours at
Fig. I - Tumors induced in green-house plants of Cv Tatu by different strains of A.tumefaciens. A- strain A281; B- strain 13o542, C- strain A208; D- strain T37; E- strain A136. S t r a i n A281 induced the l a r g e s t and most r a p i d l y appearing tumors, which could be observed w i t h i n two weeks (Table I I ) . This s t r a i n has been described as h i g h l y v i r u l e n t in other p l a n t s i n c l u d i n g soybean, alfalfa (Hood et al. 1986a; Jin et al. 1987;
356 Table If: Response of peanut cuttivars to wild type Agrobacterium tumefaciens strains Peanut genotype Tatu Tup~ Tatu Branco Tatuf Pen~polis
Botanical Type Valencia Valencia Spanish Spanish Virginia
A.tumefaciens strains and plant response A281 Bo542 A208 T37 A136 1.0a++++b 0.8 +++ 0.8 ++ 0.3 + 0 0.9++++ 0.9 +++ 0.7+++ 0.5 + 0 1.0 +++ 0.2 ++ 0.5 + 0 0 0.7 ++ 0.5 +++ 0.8 +++ 0 0 0.5 +++ 0.5 ++ 0.7 ++ 0.3 ++ 0
a: Tumor formation frequencies calculated as the number of tumors in 2 plants divided by the number of inoculation sites (6 per plant). b: Tumor sizes: + = < I mm; ++ = I--~ 1,5 mm; +++ = 1 , 5 - - ~ 2 ram; ++++ =_> 2 mm. Hood e t a[. 1987) and pea (Puonti-Kaerlas e t a_!. 1989). Susceptibility is also related to plant age, since twomonth-old plants inoculated with A281 showed reduced frequency or absence of tumor induction (data not shown). Although strains A208 and T37 contain the same plasmid, T37 was significantly less virulent in all cultivars tested. This fact might reflect the effect of chromosomal genes involved in the infection process. Tumor induction in vitro.
on hormone free medium, and were agropine positive. Approximately 50% of segments from A281 (pTD02) incited tumors grew in the presence of kanamycin, in concentrations up to 300 #g/ml while tumors induced by A281 had reduced growth in 50 #g/mr. Concentrations of kanamycin greater than 50 #g/mr resulted in cessation of growth and necrosis after six weeks in culture. NPT-II activity in the transformed tissues was demonstrated by dot-blot assays (Fig. 3).
Peanut tissues with potential for plant regeneration are mainly restricted to seed and seedling explants (McKently eta[. 1990) Therefore, testing the susceptibility of these tissues to Agrobacterium is essential for the establishment of a transformation protocol. To meet this objective, cotyledon segments, embryonic axes, petioles and leaves were inoculated with strain A281(pTD02). Undifferentiated tumors were formed on explants of the five cultivars except cotyledon tumors, which developed roots. Explant browning in hormone-free medium may have affected tumor development frequencies and, thus, the reproducibility of the data. Strains A281 and Bo542 when inoculated on in vitrogrown plantlets produced tumors that were hard, yellowish and appeared two weeks and one month, respectively, after infection. Strains T37 and A208 incited hard, dark green tumors after one month (data not shown). Excised tumors cultured in solid MS medium lacking growth regulators grew as large, friable calli, from which cell suspension cultures could be established.
Fig. 2 - Agropine assay of tumors induced by strain A281. A- tumor of A.hypogaea Cv Tatu; B- untransformed control callus; C o tumor of N.tabacum Cv. Wisconsin 38; D- untransformed tobacco callus. Starting from the top: agropine, mannopinic acid/mannopine and neutral sugars.
Opine d e t e c t i o n . Tumors i n c i t e d by s t r a i n s A281 and 80542 produce mainly agropine, and those by s t r a i n s A208 and T37 produce nopaline (Table I ) . A l l in v i t r o and in vivo induced tumors t e s t e d were opine p o s i t i v e . Agropine assays produced r e s u l t s s i m i l a r to those shown in Fig. 2. Expression of NPT-II and GUS in tumors Expression of screenable (gus) and selectabte (npt-II) markers was studied in tumors i n c i t e d on explants by A281 (pTD02) in a l l c u l t i v a r s . Axenic tumors obtained by i n f e c t i o n with t h i s s t r a i n also grew
Fig. 3 - Dot b l o t assay f o r d e t e c t i o n of NPT-II a c t i v i t y in tumors induced by A281 (pTD02). A) E.coti HBIOI::Tn5 (1) and HBI01 ( 2 ) ; B) A.hypoQaea A281 (pTD02) induced tumor (1) and c o n t r o l c a l l u s (2); C) N.tabacum A281 (pTD02) induced tumor (1) and control callus (2).
357 Histochemical assays for GUS activity demonstrated the presence of positive tumor sectors (unpublished results). This irregular distribution is expected, as tumor cells might support the growth of non-transformed cells.
Southern hybridization analysis Southern blot hybridization (Fig. 4) of DNA from axenic tumors induced by A281 and A281 (pTD02) confirms the integration of T-DNA into the genome of tumor cells. Hind III digested DNA of A281 induced tumors showed bands of 2.6 and 2.9 Kb, representing the two major Hind Ill fragments of pTiBo542 (Hood et al. 1986b). Sal I restricted DNA from tumors induced by A281(pTD02), probed with Sal I fragment of pTD02 also produced a band of the expected size (2.7 Kb).
Fig. 4 - Southern hybridization analysis of peanut tumor DNA. (A) Hind Ill digested DNA of an A281 induced tumor probed with Bam HI fragment 10 of T-DNA region of pTiBo542: lane I- DNA from A281 induced tumor; lane 2- DNA from untransformed tissue (B) Sal I digested DNA of A281 (pTD02) induced tumor probed with Sat I fragment of pTD02 containing npt-II: lane I- DNA from A281 (pTD02) induced tumor; lane 2- DNA from untransformed tissue.
We have demonstrated the integration and expression of foreign genes in A.hypogaea. Although peanut has been described as an A.rhizogenes and A.tumefaciens host (Mugnier 1988, Dang et at. 1990), no evidence of T-DNA integration has been presented previously. Optimization of parameters which influence transformation can result in a model system that, combined with efficient regeneration conditions, may lead to the production of transgenic plants.
AKNOWLEDGMENTS The authors are indebted to Drs. W. Krul and G. Engler for critical reading of the manuscript and to Drs. D.E.
de Oliveira and M.V. Montagu for helpful suggestions. We aknowledge Drs. E. Hood, C. Genetello and D.E. Oliveira for providing bacterial strains, Dr. J.l. Godoy and Fundaq~o Maria Julieta Drummond de Andrade for supplying peanut seeds and Dr. O. Machado for assistance with opine detection. We also thank L. Frade for technical assistance and J. Damasceno for typing the manuscript. This research was suported by Financiadora de Estudos e Projetos (FINEP) and Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq).
REFERENCES
Alliotte T, Tir~ C, Engler G, Peleman J, Caplan A, Montagu MV Inz~ D. 1989. Plant Physiol., 89:743752. Byrne MC, McDonnell RE, Wright M, Carnes MG. 1987. Plant Cell, Tissue Organ Cult., 8:3-15. Deak M, Kiss G, Koncz C, Dudits D. 1986. Plant Cell Rep., 5:97-100. Dellaporta SL, Wood J, Hicks JB. 1983. Plant Mol. Biol. Rep., I(4):19-21. Ditta G, Stanfield S, Corbin D, Helinski DR. 1980. Proc. Natl. Acad. Sci. USA, 77:7347-7351. Dang JD, Bi YP, Xia LS, Sun SM, Song ZH, Guo B, Jiang XC, Shao QQ. 1990. Acta Genet. Sin, 17(I):1316. Hinchee MAW, Conno~-Ward DV, Newel[ CA, McDonnell RE, Sato SJ, Gasser CS, Fischhoff DA, Re DB, Fraley RT, Horsch RB. 1988. Bio/technology, 6:915-922. Hood EE, Chilton M-D, Fraley RT. 1986a. J. Bacterial. 168:1283-1290. Hood EE, Fraley RT, Chilton M-D. 1987. Plant. Physiol. 83:529-534. Hood EE, Helmer GL, Fraley RT, Chilton M-D. 1986b. J. Bacterial., 168::1291-1301. Jefferson RA, Kavanagh TA, Bevan MW. 1987. Embo J., 13:3901-3907. Jin S, Komari T, Gordon M, Nester EW. 1987. J. Bacterial. 169:4417-4425. Manners JM. 1987. Plant Cell Rep., 6:204-207. McDonnell RE, Clark RD, Smith WA, Hinchee MA. 1987. Plant Mol. Biol. Rep., 5:380-386. McKently AH, Moore GA, Gardner FP. 1990. Crop. Sci., 30:192-196. Mugnier J. 1988. Plant Cell Rep., 7:9-12. Murashige T, Skoog F. 1962. Physiol. Plant., 15:473497. Otten LABM, Schilperoort RA. 1978. Biochem. Biophys. Acta, 527:497-500. Owens LD, Cress DE. 1985. Plant Physiol., 77:87-94. Puonti-Kaerlas J, Stabel P, Eriksson T. 1989. Plant Cell Rep., 8:321-324. Reynaerts A, De Block M, HernaIsteens J-P, Van Montagu M. 1989. In: Gelvin SB, Schilperoort RA, (eds.), Plant Molecular Biology Manual, Kluwer Academic Publishers, The Netherlands., pp. A9 1-15. Sciaky D, Montoya AL, Chilton M-D. 1978. Plasmid, 1:238-253. Scott R. 1988. In: Draper J, Scott R, Armitage P, (eds.), Plant Genetic Transformation, A Laboratory Manual, Blackwell Scientific Publications, pp. 237261.
Plant Cell Reports (1991) 10:354-357
9 Springer-Verlag1991
Gene transfer into peanut (Arachis hypogaea L.) by Agrobacterium tumefaciens C. Lacorte a, E. Mansur 2, B. Timmerman a, and A.R. Cordeiro a 1 Department of Genetics, Federal University of Rio de Janeiro, Caixa Postal 68011, CEP 21944- Rio de Janeiro, Brazil z Department of Cellular Biology and Genetics, University of the State of Rio de Janeiro, Brazil Received January 14, 1991/Revised version received June 13, 1991 - Communicated by I.K. Vasil
ABSTRACT. Introduction of foreign genes into plant tissues via AQrobacterium tumefaciens based vectors requires specific knowledge of Agrobacterium-host compatibility. Therefore, to develop a transformation protocol for peanut (Arachis hypogaea L . ) , five Brazilian cultivars were screened with four wild-type A.tumefaciens strains. Successful transformation was dependent on specific bacterial strain-plant cultivar interactions and strain A281 was the most effective for tumor induction. Tumors displayed hormone autonomous growth, were opine positive and contained DNA that was homologous to the T-DNA of the inciting strain. Tumors induced on seed and seedling explants by A281 (pTD02) also expressed the reporter genes gus and npt-II contained in the binary vector. These results show that peanut is a permissive host for the acceptance of genes from specific A.tumefaciens gene vectors. Abbreviations: GUS: B-glucuronidase (EC 3.2.1.31) NPTII: neomycin phosphotransferase II (EC 2.7.1.95), EDTA: ethylene-diamine-tetracetic acid.
INTRODUCTION
Gene transfer to plants via Agrobacterium tumefaciens is an efficient way to introduce desirable traits into crop plants. Transformation by Agrobacterium was demonstrated in the family Leguminosae in soybean (Hinchee et al. 1988), pea (Puonti-Kaerlas et al. 1989) and forage legumes (Deak et al. 1 9 8 6 ; Manners 1987). Susceptibility to Agrobacterium varies among related species and cultivars of legumes (Byrne et al. 1987; Manners 1987; Hinchee et al. 1988; Owens and Cress 1985), indicating that the transformation process depends on the combined action of bacterial and plant genotypes. Therefore, evaluation of strain-cultivar compatibility
Offprint requests to: A.R. Cordeiro
is an important step in the establishment of a transformation protocol. In this report, the susceptibility of five Brazilian cultivars of peanut to four wild strains of A.tumefaciens was evaluated by determining tumor induction frequencies. Variations in strain-cultivar compatibility were determined and the most efficient host-pathogen interaction was chosen for the study of tumor induction in explants The transformation process was confirmed by opine and T-DNA integration analysis. The expression of chimeric genes in transformed tissues was demonstrated by using a binary vector (pTP02) containing npt-II and gus genes.
MATERIALS AND METHODS
Plant material and inoculation conditions Seeds of Arachis hypogaea L. (Cvs Tatu, Tatuf, Tatu branco, T u p ~ and Pen&polis) were obtained from I n s t i t u t o Agron6mico, Campinas, B r a z i l and planted in a s o i l : s a n d mixture ( 3 : 1 ) . Four-week-old plants were inoculated with w i l d - t y p e s t r a i n s of A.tumefaciens by wounding stem internodes with a hypodermic needle and applying 10 #l of an overnight culture. I n o c u l a t i o n s i t e s were covered with p l a s t i c f i l m for one week to prevent d e s s i c a t i o n . Two p l a n t s of each c u l t i v a r were inoculated with each s t r a i n of bacterium (Table I ) , at s i x s i t e s per p l a n t . Frequency of tumor formation and sizes were scored f i v e weeks a f t e r inoculation. As a control for virulence, tumors were induced on Kallanchoe tubiflora stem internodes with the same procedure used for peanut plants. In order to obtain in vitro grown plants, seeds were surface-sterilized in 0.1% (w/v) HgCl2, plus a few drops of tween 80 for 15 minutes with constant agitation, rinsed six times with sterile distilled water and soaked for one minute in 5mM EDTA after the third wash. Seeds were germinated in jars containing cotton wool wetted with 1/2 strength MS salts (Murashige and Skoog 1962), (pH 5.8) supplemented with 3% sucrose. Ten-day-old seedlings were cut above the cotyledonary node and placed in MS medium solidified with 0.8% agar. Rooted plants were inoculated as
355 described f o r greenhouse p l a n t s , except that p l a s t i c f i l m was not used t o cover wound s i t e s . In a d d i t i o n , cefotaxime at a final concentration of 500 #g/ml was added to the culture medium 24 hours after inoculation to prevent bacterial growth. Seed explants (embryonic axes and cotyledon slices) were excised from surface-sterilized seeds. Leaf and petiole explants were obtained from seven-day-old in vitro-grown plantlets. Tissue fragments were infected with 10 ~l of an overnight culture, blot-dried on sterile filter paper and cultured on solid MS salts and vitamins supplemented with 3% sucrose for 24 hours. After this period the explants were rinsed once in liquid MS medium, blot-dried and transferred to solid MS medium plus 500 #g/ml cefotaxime. Tumor appearance was recorded after 5 weeks. Tumors induced by the tested strains on tobacco leaf explants and plantlets were cultured and used as positive controls for opine and NPT-II assays. B a c t e r i a l s t r a i n s and plasmids A.tumefaciens s t r a i n s T37, A281, Bo542 and A136 ( a v i r u l e n t ) were obtained from U n i v e r s i t y of Gent, Belgium. Strains A208 and LE392 (pEHBI07) were provided by Dr. Elizabeth Hood, Utah U n i v e r s i t y , Logan, USA. Plasmid pTD02 was a g i f t of Dr. D. E. O l i v e i r a and contains n p t - I I and gus genes, the l a t e r under the control of ATC1 promotor ( A l l i o t e et a l . 19891. S t r a i n A281 (pTD02) was constructed by t r i p a r e n t a l mating ( D i t t a et a l . 19801. Relevant phenotypes of A.tumefaciens s t r a i n s used are l i s t e d in Table I .
Table I: A.tumefaciens wild type strains used STRAIN
PLASMID
A281 Bo542 A208 T37 A136
pTiBo542 pTiBo542 pTiT37 pTiT37
CHROMOSOMAL BACKGROUND C58 Bo542 C58 T37 C58
OPINE(a)
REF
AGR, L,L.SAP, LOP AGR, L,L.SAP, LOP NOP, AGC NOP, AGC
(I) (2) (2) (2) (2)
(a) AGR, agropine; L,L,SAP, L,L,succinamopine; LOP, leucinopine; NOP, nopaline; AGC, agrocinopine. ( I ) Hood et al. 1986a. (2) Sciaky et al. 1978. Wild-type strains were grown in YEB medium (bacto beef extract 5g/l, bacto yeast extract Ig/l, peptone 5g/l, sucrose 5g/l, MgSO 4 240 mg/l, pH 7.2) with 100 #g/ml rifampicin. Strain A281 (pTD02) was grown in YEB medium containing 300 #g/ml streptomycin and 100 #g/ml spectinomycin. Prior to inoculation, bacteria were grown overnight in Minimum Medium A (K2HPO4 10.5g/l, KH2PO4 4.5g/I, (NH4)2SO 4 1.0g/l, sodium citrate 2H20 0.5 g/l, MgSO4.7H20 200 mg/l, glucose 2 g/l).
28oc. Agropine assays were performed as described by Reynaerts et at. (1989), and nopaline detection by the method of Otten and Schilperoort (1978). GUS and NPT-II assays. In vitro induced tumors were used for NPT-II and histochemical GUS assays, after being checked for sterility. GUS activity was assayed as described by Jefferson et al. (1987) and NPT-II activity was demonstrated by the dot-blot assay (McDonnell et al. 1987). Southern hybridization analysis. DNA was isolated from in vitro cultured, axenic tumors according to the procedure of Dellaporta et at. (1983) and analysed by Southern hybridiztion (Scott 1988) with probes prepared using a nick translation kit of Bethesda Research Laboratories. The DNA restriction fragment used as probe for A281 induced tumors was Bam HI fragment 10 of T-DNA region of pTiBo542 (Hood et al. 1986b), isolated from pEHBI07. The probe for DNA from tumors induced by A281 (pTD02) was the purified fragment of Sat I restriction of pTD02 containing npt-II gene. RESULTS AND DISCUSSION
Tumor induction in green-house plants All peanut cultivars tested may be considered as AQrobacterium permissive hosts, because tumors developed in response to infection with all virulent strains tested, (Fig. I) although strain T37 did not induce tumors in Cvs Tatu branco and Tatuf. In general, sensitivity of Cv Tatu branco was lower considering both tumor size and induction frequency (Table If), except when inoculated with strain A281. Tatu a n d Tup~ were most sensitive to all strains. Other legumes, including soybean, show genotypic variability of response upon infection with Agrobacterium (Byrne et al. 1987; Manners 1987; Hinchee et al. 1988; Owens and Cress 1985).
Culture of tumors. Tumors obtained from greenhouse plants were excised, and the internal part was surface sterilized in 2% (w/v) NaClO for 15 min, rinsed 6 times in sterile distilled water and placed in liquid MS medium, with 500 #g/ml cefotaxime. After two days, tumors were transferred to solid MS medium (agar 0.8%), with 250 #g/ml cefotaxime. Tumors of in vitro-grown plants and explants were excised and directly transferred to the culture medium plus antibiotic. Tumors incited by A281 (pTD02) were cut into approximately 2 rnm cubes and plated in solid MS medium containing 50 - 300 ~g/ml kanamycin. Opine detection. Putative tumors were tested for sterility by incubation of segments on YEB medium for 48 hours at
Fig. I - Tumors induced in green-house plants of Cv Tatu by different strains of A.tumefaciens. A- strain A281; B- strain 13o542, C- strain A208; D- strain T37; E- strain A136. S t r a i n A281 induced the l a r g e s t and most r a p i d l y appearing tumors, which could be observed w i t h i n two weeks (Table I I ) . This s t r a i n has been described as h i g h l y v i r u l e n t in other p l a n t s i n c l u d i n g soybean, alfalfa (Hood et al. 1986a; Jin et al. 1987;
356 Table If: Response of peanut cuttivars to wild type Agrobacterium tumefaciens strains Peanut genotype Tatu Tup~ Tatu Branco Tatuf Pen~polis
Botanical Type Valencia Valencia Spanish Spanish Virginia
A.tumefaciens strains and plant response A281 Bo542 A208 T37 A136 1.0a++++b 0.8 +++ 0.8 ++ 0.3 + 0 0.9++++ 0.9 +++ 0.7+++ 0.5 + 0 1.0 +++ 0.2 ++ 0.5 + 0 0 0.7 ++ 0.5 +++ 0.8 +++ 0 0 0.5 +++ 0.5 ++ 0.7 ++ 0.3 ++ 0
a: Tumor formation frequencies calculated as the number of tumors in 2 plants divided by the number of inoculation sites (6 per plant). b: Tumor sizes: + = < I mm; ++ = I--~ 1,5 mm; +++ = 1 , 5 - - ~ 2 ram; ++++ =_> 2 mm. Hood e t a[. 1987) and pea (Puonti-Kaerlas e t a_!. 1989). Susceptibility is also related to plant age, since twomonth-old plants inoculated with A281 showed reduced frequency or absence of tumor induction (data not shown). Although strains A208 and T37 contain the same plasmid, T37 was significantly less virulent in all cultivars tested. This fact might reflect the effect of chromosomal genes involved in the infection process. Tumor induction in vitro.
on hormone free medium, and were agropine positive. Approximately 50% of segments from A281 (pTD02) incited tumors grew in the presence of kanamycin, in concentrations up to 300 #g/ml while tumors induced by A281 had reduced growth in 50 #g/mr. Concentrations of kanamycin greater than 50 #g/mr resulted in cessation of growth and necrosis after six weeks in culture. NPT-II activity in the transformed tissues was demonstrated by dot-blot assays (Fig. 3).
Peanut tissues with potential for plant regeneration are mainly restricted to seed and seedling explants (McKently eta[. 1990) Therefore, testing the susceptibility of these tissues to Agrobacterium is essential for the establishment of a transformation protocol. To meet this objective, cotyledon segments, embryonic axes, petioles and leaves were inoculated with strain A281(pTD02). Undifferentiated tumors were formed on explants of the five cultivars except cotyledon tumors, which developed roots. Explant browning in hormone-free medium may have affected tumor development frequencies and, thus, the reproducibility of the data. Strains A281 and Bo542 when inoculated on in vitrogrown plantlets produced tumors that were hard, yellowish and appeared two weeks and one month, respectively, after infection. Strains T37 and A208 incited hard, dark green tumors after one month (data not shown). Excised tumors cultured in solid MS medium lacking growth regulators grew as large, friable calli, from which cell suspension cultures could be established.
Fig. 2 - Agropine assay of tumors induced by strain A281. A- tumor of A.hypogaea Cv Tatu; B- untransformed control callus; C o tumor of N.tabacum Cv. Wisconsin 38; D- untransformed tobacco callus. Starting from the top: agropine, mannopinic acid/mannopine and neutral sugars.
Opine d e t e c t i o n . Tumors i n c i t e d by s t r a i n s A281 and 80542 produce mainly agropine, and those by s t r a i n s A208 and T37 produce nopaline (Table I ) . A l l in v i t r o and in vivo induced tumors t e s t e d were opine p o s i t i v e . Agropine assays produced r e s u l t s s i m i l a r to those shown in Fig. 2. Expression of NPT-II and GUS in tumors Expression of screenable (gus) and selectabte (npt-II) markers was studied in tumors i n c i t e d on explants by A281 (pTD02) in a l l c u l t i v a r s . Axenic tumors obtained by i n f e c t i o n with t h i s s t r a i n also grew
Fig. 3 - Dot b l o t assay f o r d e t e c t i o n of NPT-II a c t i v i t y in tumors induced by A281 (pTD02). A) E.coti HBIOI::Tn5 (1) and HBI01 ( 2 ) ; B) A.hypoQaea A281 (pTD02) induced tumor (1) and c o n t r o l c a l l u s (2); C) N.tabacum A281 (pTD02) induced tumor (1) and control callus (2).
357 Histochemical assays for GUS activity demonstrated the presence of positive tumor sectors (unpublished results). This irregular distribution is expected, as tumor cells might support the growth of non-transformed cells.
Southern hybridization analysis Southern blot hybridization (Fig. 4) of DNA from axenic tumors induced by A281 and A281 (pTD02) confirms the integration of T-DNA into the genome of tumor cells. Hind III digested DNA of A281 induced tumors showed bands of 2.6 and 2.9 Kb, representing the two major Hind Ill fragments of pTiBo542 (Hood et al. 1986b). Sal I restricted DNA from tumors induced by A281(pTD02), probed with Sal I fragment of pTD02 also produced a band of the expected size (2.7 Kb).
Fig. 4 - Southern hybridization analysis of peanut tumor DNA. (A) Hind Ill digested DNA of an A281 induced tumor probed with Bam HI fragment 10 of T-DNA region of pTiBo542: lane I- DNA from A281 induced tumor; lane 2- DNA from untransformed tissue (B) Sal I digested DNA of A281 (pTD02) induced tumor probed with Sat I fragment of pTD02 containing npt-II: lane I- DNA from A281 (pTD02) induced tumor; lane 2- DNA from untransformed tissue.
We have demonstrated the integration and expression of foreign genes in A.hypogaea. Although peanut has been described as an A.rhizogenes and A.tumefaciens host (Mugnier 1988, Dang et at. 1990), no evidence of T-DNA integration has been presented previously. Optimization of parameters which influence transformation can result in a model system that, combined with efficient regeneration conditions, may lead to the production of transgenic plants.
AKNOWLEDGMENTS The authors are indebted to Drs. W. Krul and G. Engler for critical reading of the manuscript and to Drs. D.E.
de Oliveira and M.V. Montagu for helpful suggestions. We aknowledge Drs. E. Hood, C. Genetello and D.E. Oliveira for providing bacterial strains, Dr. J.l. Godoy and Fundaq~o Maria Julieta Drummond de Andrade for supplying peanut seeds and Dr. O. Machado for assistance with opine detection. We also thank L. Frade for technical assistance and J. Damasceno for typing the manuscript. This research was suported by Financiadora de Estudos e Projetos (FINEP) and Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq).
REFERENCES
Alliotte T, Tir~ C, Engler G, Peleman J, Caplan A, Montagu MV Inz~ D. 1989. Plant Physiol., 89:743752. Byrne MC, McDonnell RE, Wright M, Carnes MG. 1987. Plant Cell, Tissue Organ Cult., 8:3-15. Deak M, Kiss G, Koncz C, Dudits D. 1986. Plant Cell Rep., 5:97-100. Dellaporta SL, Wood J, Hicks JB. 1983. Plant Mol. Biol. Rep., I(4):19-21. Ditta G, Stanfield S, Corbin D, Helinski DR. 1980. Proc. Natl. Acad. Sci. USA, 77:7347-7351. Dang JD, Bi YP, Xia LS, Sun SM, Song ZH, Guo B, Jiang XC, Shao QQ. 1990. Acta Genet. Sin, 17(I):1316. Hinchee MAW, Conno~-Ward DV, Newel[ CA, McDonnell RE, Sato SJ, Gasser CS, Fischhoff DA, Re DB, Fraley RT, Horsch RB. 1988. Bio/technology, 6:915-922. Hood EE, Chilton M-D, Fraley RT. 1986a. J. Bacterial. 168:1283-1290. Hood EE, Fraley RT, Chilton M-D. 1987. Plant. Physiol. 83:529-534. Hood EE, Helmer GL, Fraley RT, Chilton M-D. 1986b. J. Bacterial., 168::1291-1301. Jefferson RA, Kavanagh TA, Bevan MW. 1987. Embo J., 13:3901-3907. Jin S, Komari T, Gordon M, Nester EW. 1987. J. Bacterial. 169:4417-4425. Manners JM. 1987. Plant Cell Rep., 6:204-207. McDonnell RE, Clark RD, Smith WA, Hinchee MA. 1987. Plant Mol. Biol. Rep., 5:380-386. McKently AH, Moore GA, Gardner FP. 1990. Crop. Sci., 30:192-196. Mugnier J. 1988. Plant Cell Rep., 7:9-12. Murashige T, Skoog F. 1962. Physiol. Plant., 15:473497. Otten LABM, Schilperoort RA. 1978. Biochem. Biophys. Acta, 527:497-500. Owens LD, Cress DE. 1985. Plant Physiol., 77:87-94. Puonti-Kaerlas J, Stabel P, Eriksson T. 1989. Plant Cell Rep., 8:321-324. Reynaerts A, De Block M, HernaIsteens J-P, Van Montagu M. 1989. In: Gelvin SB, Schilperoort RA, (eds.), Plant Molecular Biology Manual, Kluwer Academic Publishers, The Netherlands., pp. A9 1-15. Sciaky D, Montoya AL, Chilton M-D. 1978. Plasmid, 1:238-253. Scott R. 1988. In: Draper J, Scott R, Armitage P, (eds.), Plant Genetic Transformation, A Laboratory Manual, Blackwell Scientific Publications, pp. 237261.
