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Root induction by Agrobacterium rhizogenes in walnut
WCiENCE AN
ELSEVIER
Plant Science I I8 (1996) 203-208
Root induction by Agrobacterium rhizogenes in walnut Emilia Caboni*, Paola Law-i, Mariagrazia Tonelli, Giuseppina Falasca’, Carmine Damiano Istituto Sperimentale per la Frutticoltura, OW40 Ciampino Aeroporto, Rome, Italy Received 18 December 1995; revised 3 April 1996;accepted 17 May I996
AbStTWt Agrobacterium rhizogenes (wild-type, strain 1855), when applied to the basal part of microcuttings of walnut (J. regia L.), produced numerous adventitious roots in vitro: 58.6% of rooting was induced in microcuttings in hormone free medium and 62.9% and abundant callus formation in the presence of IBA. A. rhizogenes did not induce rooting when IAA was present in the rooting medium. No explants were induced to root by the treatments with IBA, IAA or in hormone free medium without the presence of A. rhizogenes. Root vascular elements were connected to the microcutting vascular system in the treatment with A. rhizogenes in HFM and were successfully transferred to soil. However, microcuttings treated with A. rhizogenes + IBA showed vascular connections not correctly formed. Southern blots performed using the fragment containing rol genes, as probe, coming from pBIN19::&015, provided molecular evidence of the transgenic nature. of the roots induced by A. rhizogenes.
Keywordr: In vitro rooting; J. regia L.; Localized infection
1. Introdlletioo Walnut is an important species for the production of nuts as well as wood, but its propagation
by traditional grafting and layering is diflicult [l]. Rooting of walnut has been improved in vitro in recent years, yet, there remain problems in this
Abbreviations: CX, cefotaxime; DKW, Driver & Kuniyuki medium; HFM, hormone free medium; IAA, indole-3-acetic acid, IBA, indole-3-butyric acid; YMB, yeast extract mannitol broth. l Corresponding author. ’ Present add-: Universiti “La Sapienza” mento di Biologia Vegetale, Rome, Italy.
Diparti-
species mainly related with the genotype specific response [2,3]. The soil bacterium Agrobacterium rhizogenes induces abundant adventitious root formation at the site of infection [4]. This morphogenic event is due to the transfer of genetic information of a portion (T-DNA) of the Ri (Root Inducing) plasmid from bacteria to the plant genome [5-71. Four of the loci involved in the root formation, corresponding to open reading frames (ORFs) 10, 11, 12a, 15 of the T,_-DNA, have been identified and called root loci @of) A, B, C and D [6,8]. In particular, rol B has been shown to be the only Ri T-DNA gene capable alone of inducing root differentiation Ki9,lOl.
0168-9452/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SOl68-9452(96)04449-4
204
E. Caboni et al. /Plant
The inoculation with A. rhizogenes was reported to be a means of inducing adventitious root formation in cuttings of some woody species [I l-141. The objective of this work was to determine the effect of localized infection with A. rhizogenes, by itself or combined with different auxin treatments, in inducing adventitious root formation in a difficult-to-root walnut genotype. 2. Materials ad methods 2.1. Plant material Microcuttings (2.0-2.5 cm in length) of a difficult-to-root seedling of walnut (Jugfans regia, cv. Sorrento) grown for 4 years on DKW medium [ 15) (termed DKWl) containing 30 g 1-l sucrose, 6.5 g 1-i agar (Riedel de Haen) and with the pH adjusted to 5.6 prior to autoclaving were used. IBA, 0.05 PM, and 2.2 PM BA were also added. The explants were.maintained in a cool white light (Osram L40 white fluorescent), 45 PM me2 s-‘, under a 16-h photoperiod, at 21 f 1°C. 2.2. Bacterial suspension and inoculation procedure A. rhizogenes (wild-type, strain 1855 NCPPB) was grown in Y MB medium [ 161at 28°C to the optical density of 0.6, centrifuged at 2500 x g for 10 min at 4°C and resuspended in the same amount of liquid DKWl medium. Localized infection was done through the immersion of the microcutting basal part (lo- 15 mm) into bacterial suspension and gently shaken on a rotary shaker for 24 h in the dark. Excess bacteria were removed by using sterile filter paper and microcuttings were transferred to DKWl medium with 10 PM IAA, 10 PM IBA or without auxin, in dark at 22”C, for a co-cultivation period of 24 h. As a control, non-infected explants were dipped into liquid DKWl medium without bacteria and then cultured on the same condition as infected explants with and without antibiotic in the medium. Infected microcuttings were then transferred on the same media used for the co-cultivation and containing 250 mg 1-l filter-sterilized CX (Roussel Laboratories, Uxbridge, UK), added after autoclaving, to inhibit further growth of bacteria. Con-
Science 118 (19%)
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trol explants were cultured in the same media with and without CX. All microcuttings were maintained for 12 days in darkness and then transferred to light. The culture room conditions were the same as those reported for the multiplication phase. Number of rooted explants and number of roots per explant were recorded 40 days after the beginning of the rooting treatment. For each experiment 30 microcuttings per treatment were used and results are the mean of 3 separate experiments f SE. (percentage scores of explants forming shoots were previously transformed with the formula arcsin J % + 1). 2.3. Molecular analysis Total DNA was isolated from 0.5 g of roots and from the same amount of tissue of the basal part of unrooted microcuttings using the procedure described by Dellaporta et al. [17]; this method was slightly modified adding 14 mM diethyldithiocarbamic acid (Sigma) and 40% w/w polyvinylpolypyrrolidone (Sigma), to avoid polysaccharides or polyphenols interfering in DNA purification [ 181. DNA was restricted with EcoRI (Boehringer) and fractionated in 1% agarose (Sigma) gel electrophoresis in Tris-borate-EDTA buffer (Sigma) [19] containing 1 pg ml-’ of ethidium bromide (Sigma). The run was performed at room temperature for 16 h at 1.5 V cm-‘. DNA was then transferred to a nylon membrane (Zeta-Probe Biorad) and hybridized with fragments of Ri plasmid random priming labelled with [32P]d-CTP (Amersham), using a U.S.B. kit and hybridizations were performed in 30% formamide (Sigma) for 16 h at 42°C. At the end of hybridization periods, filters were washed 30 min twice in 2 x SSC + 0.1% SDS at room temperature and 30 min twice in 0.1 SSC + 0.1% SDS [ 181 at 60°C. Filters were then exposed on X-ray films (Hyperfilm Amersham) at -70°C for 3 days. EcoRI-EcoRI fragment (4374 bp), containing rol genes [20] and coming from pBIN19::&015, was used to detect the presence of Tr_-DNA in the EcoRI digest of total DNA extracted from explants. To be sure that there was no bacterial DNA contamination in the DNA extracted from
E. Caboni et al. / PIam Science 118 (1996) 203-208
infected microcuttings, an EcoRI-EcoRI fragment of pMP162 plasmid, containing vir genes [21], was used as control. 2.4. Histological analysis The basal region, about 5-6 mm, of a sample of five microcuttings per each treatment inducing rooting was fmed in FAA (formalinglacial acetic acid:70% ethanol = 1:1:18, Merck), embedded in paraffin (melting point: 51-53”C, Merck), after dehydration, and sectioned at 10 pm with a standard rotary microtome, slides were stained either with eosin and Carazzi’s haemalum [22] or safranine-fast green [23].
3. Rewlts and disawion Infected microcuttings with A. rhizogenes maintained in the rooting medium without auxin induced 58.6% of explants to root and only a slight callus formation. In the presence of IBA a satisfactory rooting percentage (62.9%) was obtained (Table l), but an abundant callus formation was evident (Fig. 1). A. rhizogenes localized infection
Table I Effect of localixed infection with Agrobacterium rhizogenes, auxins (IAA and IBA) and cefotaxime on rooting of walnut microcuttings (data collected 40 days after transferring to the rooting medium) Rooting (%)
No. roots/explant
58.6 l 4.2 62.9 f 4.7 0
2.6 zt 0.1 2.4 f 0.1
Infected explants
HFM+CX IBA + CX IAA+CX Non-infected explants
HFM + CX HFM - CX IBA + CX IBA - CX IAA+CX IAA-cx
0 0 0 0 0 0
-
HFM, hormone free medium; CX, cefotaxime. Results are the mean of 3 separate experiments (n = 30) f S.E. (percentage scores of explants forming shoots were previously transformed with the formula arcsin 4 % + 1).
205
did not induce rooting when IAA was present in the rooting medium and only a small callus formation was induced (Fig. 1). No explants were induced to root by the IBA, IAA treatments or in HFM without bacterium infection complemented with or without cefotaxime (Table 1, Fig. 1). Rooted explants obtained by treatment with A. rhizogenes in the absence of auxin were successfully transferred to the soil. The hybridization between genomic DNA extracted from roots or basal parts of microcuttings and the fragment containing rol genes, coming from pBIN 19::Eco 15, provided molecular evidence of the transgenic nature of the roots and of the callw induced by IAA + A. rhizogenes; the absence of hybridization with the fragment containing vir genes, coming from pMP162, showed that there was no A. rhizogenes contamination in the explants used for DNA extraction (Fig. 2). Histological analysis showed good connection of roots to the vascular system in microcuttings induced to root by A. rhizogenes without auxin (Fig. 3a). Cuttings treated with A. rhizogenes + IBA, which formed abundant callus, showed connection to the stem vascular system only in some of the microcuttings, but some others had vascular elements not completely connected (Fig. 3b). Our results showed that localized infection with A. rhizogenes was critical to obtain rooting in microcuttings of a difficult-to-root walnut genotype. In fact, localized infection of microcuttings induced root formation and small callus production in the HFM. It was particularly effective also combined with the IBA treatment, but in this case abundant callus formation was also induced. Thus, these findings suggest that A. rhizogenes may be a useful method to improve rooting in walnut as reported in other woody species like apple, almond, olive and pistachio [lo-131. It has been reported that T,-DNA of the Ri plasmid of A. rhizogenes makes cells sensitive to auxin addressing them towards rhizogenic process [24,25] and that rol B (ORF 1l), among the 18 ORFs whose TL-DNA is formed, is the genetic determinant responsible for rooting [6,9,10]. Estruch et al. [26] showed that the protein encoded by the rol B releases indoles from indoxyl-B-
206
E. Caboni et al. /Plant Science 118 (19%) 203-208
Fig. I. In vitro rooting (40 days after transferring rhizogenes and/or auxins (IAA, IBA).
to the rooting
ABCDEFG N
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4361
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medium)
of walnut
microcuttings
treated
with Agrobacferium
glucosides and propose that the release of free IAA from its glucoside conjugates by the enzymatic activity of the rol B-encoded protein leads to the alteration of plant physiological process. However, a substantial free IAA homeostasis in both rol B transgenic and control plants has been recently demonstrated [27]. Whatever the rol B mechanism is, rooting induced by A. rhizogenes seems to be precisely regulated in terms of hormonal balance and/or hormone sensitivity with strong
Fig, 2. Southern blot of DNA of roots or basal parts of microcuttings treated with Agrobacrerium rhizogenes using the 4374 bp fragment containing rol genes, as probe, coming from pBINW:EcoIS (lines C, E, G in presence of IAA, IBA or in HFM, respectively) or the EcoRI-EcoRI fragment of pMPI62 plasmid (lines 9, D, F, in presence of IAA, IBA or in HFM, respectively). Line A is the untransformed control.
E. Caboni et al. /Plant
Science 118 (19%)
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207
Fig. 3. Transverse section of basal part of walnut microcuttings treated with A. rhizogenes in hormone free medium showing a root connected to the central vascular system (A) or in presence of IBA showing vascular elements not completely connected (B). (A) bar = 0.8 mm; (B) bar = 0.6 mm.
relation with the kind of auxin. In fact, when the treatment with A. rhizogenes without auxin was applied in our experiment, the cellular hormone content must have been correct to obtain a satisfactory rooting response and no callus formation. The addressed hormonal change caused by TDNA by itself induced root formation, but exogenous auxins produced modification of that
precise balance inducing rooting but also abundant callus formation in the treatment with IBA and only small callus in the presence of IAA. An evident strong reduction of rooting, which could be explained with excess of endogenous IAA-level, was also shown when A. rhizogenes infection was applied combined with exogenous IAA to almond microcuttings in previous experiments [ 121.
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E. Caboni et al. /Plant
In both the treatments with IBA and IAA either roots and calli were shown to be transgenic; thus neither IBA nor IAA inhibited infection ability of the bacterium. Anyway, the different effect of IBA and IAA is not clear in terms of auxin balance. Unfortunately, no data are available on catabolism of exogenous auxin in Juglans regia and only new studies on the metabolism of exogenous IBA and IAA during in vitro rooting process will allow an understanding of the rooting response. Acknowledgements
The authors thank Mrs. Loreta Marina&o for technical assistance in the micropropagation of the material. References
[II H.T. Hartmann, D.E. Kester and F.D. Davies, Propagation methods and rootstock for the important fruit and nut species, in: H.D. Hartmann, D.E. Kester and F.D. Davies (Eds.), Plant propagators, Principles and practices, 5th edn., Prentice Hall Inc., Englewood, NJ, 1990, pp. 527-565. PI R. Gruselle and F. Boxus, Walnut propagation. Acta Hart., 284 (1990) 45-52. [31 G. McGranaham, J.A. Driver and W. Tulecke, Tissue culture of Juglans, in: J.M. Bonga and D.J. Dmzan (Eds.), Cell Tissue Culture and Forestry Vol. 3, Martinus Nijhoff, Boston, 1987, pp. 261-271. [41 C. Elliot, Manual of Bacterial Plant Pathogens, 2nd edn., Chronica Botanica, Waltham, MA, 1951. PI L. Moore, G. Warren and G. Strobel, Involvement of a plasmid in the hairy root disease of plants caused by Agrobacterim rhizogenes. Plasmid, 2 (1979) 617-626. I61 F.F. White, G. Ghidossi, M.P. Gordon and E.W. Nester, Tumour induction by Agrobucterium rhizogenes involves the transfer of plasmid DNA to the plant genome. Proc. Nati. Acad. Sci. USA, 79 (1982) 3193-3197. I71 M.D. Chilton, D.A. Tepfer, A. Petit, C. David, F. CasseDelbart and J. Tempe, Agrobacterium rhizogenes inserts T-DNA into the genome of the host plant root cells. Nature, 295 (1982) 432-434. PI F. Vilaine, C. Charbonnier and F. Casse-Delbart, Further insight concerning the TL region of the Ri plasmid of Agrobucterium rhizogenes strain A4: transfer of a 19 kb fragment is suflicient to induce transformed roots on tobacco leaf fragments. Mol. Gen. Genet., 210 (1987) 111-115. PI I. Capone, L. Span& M. Cardarelli, D. Bellincampi, A. Petit and P. Costantino, Induction and growth properties of carrot roots with different complements of Agrobacterium rhizogenes T-DNA. Plant Mol. Biol., 13 (1989) 43-52. WI A. Spena, T. Schmulling, C. Koncz and J.S. Shell, lndependent and synergistic activity of rot A, B and C loci in
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stimulating abnormal growth in plants. EMBO J., 6 (1987) 3891-3899. L. Patena, E.G. Sutter and A.M. Dandekar, Root induction by Agrobacterium rhizogenes in a difficult-to-root woody species. Acta Hort., 227 (1988) 324-329. WI C. Damiano, T. Archilletti, E. Caboni, P. Lauri, G. Falasca, D. Mariotti and G. Ferraiolo, Agrobacterium mediated transformation of almond: in vitro rooting through localized infection of A. rhizogenes w.t. Acta Hort., 392 (1994) 161-169. 1131 C. Lambert and D. Tepfer, Utilisation de la transformation g&tique par Agrobacterium rhizogenes pour amkliorer la rhiiogenbse d’arbres fruitiers. Colloque Arboriculture fruit&, (1988) 1- 12. u41 E. Rugini and D. Mariotti, Agrobacterium rhizogenes TDNA genes and rooting in woody species. Acta Hart., 300 (1992) 301-308. 1151 J.A. Driver and A.H. Kuniyuki, In vitro propagation of Paradox walnut rootstock. Hort. Sci., 19 (1984) 507-509. [1’51 P.J.J. Hooykaas, P.M. Klapwjik, M.P. Nuti, R.A. Schilperoort and A. Rorsch, Transfer of the A. fwnefaciens ti plasmid to avirulent Agrobacteria and Rhizobium ex planta. J. Gen. Microbial., 98 (1977) 477-484. (171 S.L. Dellaporta, J. Wood and J.B. Hicks, A plant DNA minipreparation version II. Plant Mol. Biol. Rep., 1 (1983) 19-21. WI T. Archilletti, P. Lauri and C. Damiano, Agrobacteriummediated transformation of almond leaf pieces. Plant Cell Rep., 14 (1995) 267-272. 1191 J. Sambrook, E.F. Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press, NY, 1989. WI M. Cardarelli, D. Mariotti, M. Pomponi, L. Span& 1. Capone and P. Costa&no, Agrobacterium rhizogenes TDNA gene capable of inducing hairy root phenotype. Mol. Gen. Genet., 209 (1987) 475-480. WI M. Pomponi, L. Span& M.G. Sabbadini and P. Costantino, Restriction endonuclease mapping of the rootinducing plasmid of Agrobacrerium rhizogenes 1855. Plasmid, 10 (1983) 119-129. di tecniche istologiche ed WI V. Mazzi, Man&e istochimiche, Piccin (Padova), 1977, p. 127. ~31 W.A. Jensen, Botanical histochemistry. Principles and Practice, Freeman, San Francisco, 1962, pp. 250-251. 1241 L. Span& D. Mariotti, M. Cardarelli, C. Branca and P. Costantino, Morphogenesis and auxin sensitivity of transgenic tobacco with different complements of R, TDNA. Plant Physiol., 87 (1988) 479-483. ~51 H. Barbier-Brygoo, C. Maurel, W.H. Shen, G. Ephritikhine, A. Delbarre and J. Guem, Use of mutants and transformed plants to study the action of auxins, in: J.A. Roberts, C. Kirk and M. Venis (Eds.), Hormone perception and signal transduction in animals and plants, Company of Biologists, Cambridge, 1990. WI J.J. Estruch, J. Schell and A. Spena, The protein encoded by the rol B plant oncogene hydrolyses indole-giucosides. EMBO J., 10 (1991) 3125-3128. [271 0. Nilsson, A. Crozien, T. S&mulling, G. Studberg and 0. Olsson. Indole-3-acetic acid homeostasis in transgenic tobacco plants expressing the Agrobacterium rhizogenes rol B gene. Plant J., 3 (1993) 681-689.
ELSEVIER
Plant Science I I8 (1996) 203-208
Root induction by Agrobacterium rhizogenes in walnut Emilia Caboni*, Paola Law-i, Mariagrazia Tonelli, Giuseppina Falasca’, Carmine Damiano Istituto Sperimentale per la Frutticoltura, OW40 Ciampino Aeroporto, Rome, Italy Received 18 December 1995; revised 3 April 1996;accepted 17 May I996
AbStTWt Agrobacterium rhizogenes (wild-type, strain 1855), when applied to the basal part of microcuttings of walnut (J. regia L.), produced numerous adventitious roots in vitro: 58.6% of rooting was induced in microcuttings in hormone free medium and 62.9% and abundant callus formation in the presence of IBA. A. rhizogenes did not induce rooting when IAA was present in the rooting medium. No explants were induced to root by the treatments with IBA, IAA or in hormone free medium without the presence of A. rhizogenes. Root vascular elements were connected to the microcutting vascular system in the treatment with A. rhizogenes in HFM and were successfully transferred to soil. However, microcuttings treated with A. rhizogenes + IBA showed vascular connections not correctly formed. Southern blots performed using the fragment containing rol genes, as probe, coming from pBIN19::&015, provided molecular evidence of the transgenic nature. of the roots induced by A. rhizogenes.
Keywordr: In vitro rooting; J. regia L.; Localized infection
1. Introdlletioo Walnut is an important species for the production of nuts as well as wood, but its propagation
by traditional grafting and layering is diflicult [l]. Rooting of walnut has been improved in vitro in recent years, yet, there remain problems in this
Abbreviations: CX, cefotaxime; DKW, Driver & Kuniyuki medium; HFM, hormone free medium; IAA, indole-3-acetic acid, IBA, indole-3-butyric acid; YMB, yeast extract mannitol broth. l Corresponding author. ’ Present add-: Universiti “La Sapienza” mento di Biologia Vegetale, Rome, Italy.
Diparti-
species mainly related with the genotype specific response [2,3]. The soil bacterium Agrobacterium rhizogenes induces abundant adventitious root formation at the site of infection [4]. This morphogenic event is due to the transfer of genetic information of a portion (T-DNA) of the Ri (Root Inducing) plasmid from bacteria to the plant genome [5-71. Four of the loci involved in the root formation, corresponding to open reading frames (ORFs) 10, 11, 12a, 15 of the T,_-DNA, have been identified and called root loci @of) A, B, C and D [6,8]. In particular, rol B has been shown to be the only Ri T-DNA gene capable alone of inducing root differentiation Ki9,lOl.
0168-9452/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SOl68-9452(96)04449-4
204
E. Caboni et al. /Plant
The inoculation with A. rhizogenes was reported to be a means of inducing adventitious root formation in cuttings of some woody species [I l-141. The objective of this work was to determine the effect of localized infection with A. rhizogenes, by itself or combined with different auxin treatments, in inducing adventitious root formation in a difficult-to-root walnut genotype. 2. Materials ad methods 2.1. Plant material Microcuttings (2.0-2.5 cm in length) of a difficult-to-root seedling of walnut (Jugfans regia, cv. Sorrento) grown for 4 years on DKW medium [ 15) (termed DKWl) containing 30 g 1-l sucrose, 6.5 g 1-i agar (Riedel de Haen) and with the pH adjusted to 5.6 prior to autoclaving were used. IBA, 0.05 PM, and 2.2 PM BA were also added. The explants were.maintained in a cool white light (Osram L40 white fluorescent), 45 PM me2 s-‘, under a 16-h photoperiod, at 21 f 1°C. 2.2. Bacterial suspension and inoculation procedure A. rhizogenes (wild-type, strain 1855 NCPPB) was grown in Y MB medium [ 161at 28°C to the optical density of 0.6, centrifuged at 2500 x g for 10 min at 4°C and resuspended in the same amount of liquid DKWl medium. Localized infection was done through the immersion of the microcutting basal part (lo- 15 mm) into bacterial suspension and gently shaken on a rotary shaker for 24 h in the dark. Excess bacteria were removed by using sterile filter paper and microcuttings were transferred to DKWl medium with 10 PM IAA, 10 PM IBA or without auxin, in dark at 22”C, for a co-cultivation period of 24 h. As a control, non-infected explants were dipped into liquid DKWl medium without bacteria and then cultured on the same condition as infected explants with and without antibiotic in the medium. Infected microcuttings were then transferred on the same media used for the co-cultivation and containing 250 mg 1-l filter-sterilized CX (Roussel Laboratories, Uxbridge, UK), added after autoclaving, to inhibit further growth of bacteria. Con-
Science 118 (19%)
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trol explants were cultured in the same media with and without CX. All microcuttings were maintained for 12 days in darkness and then transferred to light. The culture room conditions were the same as those reported for the multiplication phase. Number of rooted explants and number of roots per explant were recorded 40 days after the beginning of the rooting treatment. For each experiment 30 microcuttings per treatment were used and results are the mean of 3 separate experiments f SE. (percentage scores of explants forming shoots were previously transformed with the formula arcsin J % + 1). 2.3. Molecular analysis Total DNA was isolated from 0.5 g of roots and from the same amount of tissue of the basal part of unrooted microcuttings using the procedure described by Dellaporta et al. [17]; this method was slightly modified adding 14 mM diethyldithiocarbamic acid (Sigma) and 40% w/w polyvinylpolypyrrolidone (Sigma), to avoid polysaccharides or polyphenols interfering in DNA purification [ 181. DNA was restricted with EcoRI (Boehringer) and fractionated in 1% agarose (Sigma) gel electrophoresis in Tris-borate-EDTA buffer (Sigma) [19] containing 1 pg ml-’ of ethidium bromide (Sigma). The run was performed at room temperature for 16 h at 1.5 V cm-‘. DNA was then transferred to a nylon membrane (Zeta-Probe Biorad) and hybridized with fragments of Ri plasmid random priming labelled with [32P]d-CTP (Amersham), using a U.S.B. kit and hybridizations were performed in 30% formamide (Sigma) for 16 h at 42°C. At the end of hybridization periods, filters were washed 30 min twice in 2 x SSC + 0.1% SDS at room temperature and 30 min twice in 0.1 SSC + 0.1% SDS [ 181 at 60°C. Filters were then exposed on X-ray films (Hyperfilm Amersham) at -70°C for 3 days. EcoRI-EcoRI fragment (4374 bp), containing rol genes [20] and coming from pBIN19::&015, was used to detect the presence of Tr_-DNA in the EcoRI digest of total DNA extracted from explants. To be sure that there was no bacterial DNA contamination in the DNA extracted from
E. Caboni et al. / PIam Science 118 (1996) 203-208
infected microcuttings, an EcoRI-EcoRI fragment of pMP162 plasmid, containing vir genes [21], was used as control. 2.4. Histological analysis The basal region, about 5-6 mm, of a sample of five microcuttings per each treatment inducing rooting was fmed in FAA (formalinglacial acetic acid:70% ethanol = 1:1:18, Merck), embedded in paraffin (melting point: 51-53”C, Merck), after dehydration, and sectioned at 10 pm with a standard rotary microtome, slides were stained either with eosin and Carazzi’s haemalum [22] or safranine-fast green [23].
3. Rewlts and disawion Infected microcuttings with A. rhizogenes maintained in the rooting medium without auxin induced 58.6% of explants to root and only a slight callus formation. In the presence of IBA a satisfactory rooting percentage (62.9%) was obtained (Table l), but an abundant callus formation was evident (Fig. 1). A. rhizogenes localized infection
Table I Effect of localixed infection with Agrobacterium rhizogenes, auxins (IAA and IBA) and cefotaxime on rooting of walnut microcuttings (data collected 40 days after transferring to the rooting medium) Rooting (%)
No. roots/explant
58.6 l 4.2 62.9 f 4.7 0
2.6 zt 0.1 2.4 f 0.1
Infected explants
HFM+CX IBA + CX IAA+CX Non-infected explants
HFM + CX HFM - CX IBA + CX IBA - CX IAA+CX IAA-cx
0 0 0 0 0 0
-
HFM, hormone free medium; CX, cefotaxime. Results are the mean of 3 separate experiments (n = 30) f S.E. (percentage scores of explants forming shoots were previously transformed with the formula arcsin 4 % + 1).
205
did not induce rooting when IAA was present in the rooting medium and only a small callus formation was induced (Fig. 1). No explants were induced to root by the IBA, IAA treatments or in HFM without bacterium infection complemented with or without cefotaxime (Table 1, Fig. 1). Rooted explants obtained by treatment with A. rhizogenes in the absence of auxin were successfully transferred to the soil. The hybridization between genomic DNA extracted from roots or basal parts of microcuttings and the fragment containing rol genes, coming from pBIN 19::Eco 15, provided molecular evidence of the transgenic nature of the roots and of the callw induced by IAA + A. rhizogenes; the absence of hybridization with the fragment containing vir genes, coming from pMP162, showed that there was no A. rhizogenes contamination in the explants used for DNA extraction (Fig. 2). Histological analysis showed good connection of roots to the vascular system in microcuttings induced to root by A. rhizogenes without auxin (Fig. 3a). Cuttings treated with A. rhizogenes + IBA, which formed abundant callus, showed connection to the stem vascular system only in some of the microcuttings, but some others had vascular elements not completely connected (Fig. 3b). Our results showed that localized infection with A. rhizogenes was critical to obtain rooting in microcuttings of a difficult-to-root walnut genotype. In fact, localized infection of microcuttings induced root formation and small callus production in the HFM. It was particularly effective also combined with the IBA treatment, but in this case abundant callus formation was also induced. Thus, these findings suggest that A. rhizogenes may be a useful method to improve rooting in walnut as reported in other woody species like apple, almond, olive and pistachio [lo-131. It has been reported that T,-DNA of the Ri plasmid of A. rhizogenes makes cells sensitive to auxin addressing them towards rhizogenic process [24,25] and that rol B (ORF 1l), among the 18 ORFs whose TL-DNA is formed, is the genetic determinant responsible for rooting [6,9,10]. Estruch et al. [26] showed that the protein encoded by the rol B releases indoles from indoxyl-B-
206
E. Caboni et al. /Plant Science 118 (19%) 203-208
Fig. I. In vitro rooting (40 days after transferring rhizogenes and/or auxins (IAA, IBA).
to the rooting
ABCDEFG N
23130
-9416 76557
21’
-
4361
-
2322
4
2027
medium)
of walnut
microcuttings
treated
with Agrobacferium
glucosides and propose that the release of free IAA from its glucoside conjugates by the enzymatic activity of the rol B-encoded protein leads to the alteration of plant physiological process. However, a substantial free IAA homeostasis in both rol B transgenic and control plants has been recently demonstrated [27]. Whatever the rol B mechanism is, rooting induced by A. rhizogenes seems to be precisely regulated in terms of hormonal balance and/or hormone sensitivity with strong
Fig, 2. Southern blot of DNA of roots or basal parts of microcuttings treated with Agrobacrerium rhizogenes using the 4374 bp fragment containing rol genes, as probe, coming from pBINW:EcoIS (lines C, E, G in presence of IAA, IBA or in HFM, respectively) or the EcoRI-EcoRI fragment of pMPI62 plasmid (lines 9, D, F, in presence of IAA, IBA or in HFM, respectively). Line A is the untransformed control.
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207
Fig. 3. Transverse section of basal part of walnut microcuttings treated with A. rhizogenes in hormone free medium showing a root connected to the central vascular system (A) or in presence of IBA showing vascular elements not completely connected (B). (A) bar = 0.8 mm; (B) bar = 0.6 mm.
relation with the kind of auxin. In fact, when the treatment with A. rhizogenes without auxin was applied in our experiment, the cellular hormone content must have been correct to obtain a satisfactory rooting response and no callus formation. The addressed hormonal change caused by TDNA by itself induced root formation, but exogenous auxins produced modification of that
precise balance inducing rooting but also abundant callus formation in the treatment with IBA and only small callus in the presence of IAA. An evident strong reduction of rooting, which could be explained with excess of endogenous IAA-level, was also shown when A. rhizogenes infection was applied combined with exogenous IAA to almond microcuttings in previous experiments [ 121.
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In both the treatments with IBA and IAA either roots and calli were shown to be transgenic; thus neither IBA nor IAA inhibited infection ability of the bacterium. Anyway, the different effect of IBA and IAA is not clear in terms of auxin balance. Unfortunately, no data are available on catabolism of exogenous auxin in Juglans regia and only new studies on the metabolism of exogenous IBA and IAA during in vitro rooting process will allow an understanding of the rooting response. Acknowledgements
The authors thank Mrs. Loreta Marina&o for technical assistance in the micropropagation of the material. References
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