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Histological and Ultrastructural Analysis of A. rhizogenes-mediated Root Formation in Walnut Cuttings
Plant Genetic Engineering: Towards the Third Millennium A.D. Arencibia (Editors) 9 2000 Elsevier Science B.V. All rights reserved.
100
Histological and Ultrastructural Analysis of A. rhizogenes-mediated Root Formation in Walnut Cuttings M M Altamura ~*, G Falasca ~, M Reverberi ~, A De Stradis2
1. Dipartimento di Biologia Vegetale, Universita di Roma "La Sapienza"; P.le Aldo Moro 5; 00185 Rome, Italy. 2. Centro di Servizi Interdipartimentale di Microscopia, Universita degli Studi della Basilicata; 85100 Potenza, Italy. * Author for correspondence
Introduction
The induction of adventitious roots is crucial for vegetative propagation in plant or micropropagation in vitro of many woody species, yet many woody species are totally recalcitrant to rooting (Altamura 1996). This recalcitrance may be associated with various histologically detectable factors, such as the inability of the explant cells, after stimulation, to organize root meristemoids (Altamura 1996). In English walnut (J. regia L.), the propagation by grafting on seedling rootstocks is an expensive process, yet the alternative practice of grafting on black walnut seedling rootstocks makes trees vulnerable to the lethal blackline disease. In vitro, root formation may be a response to wounding per se or it may be associated with the presence of root inducers, such as auxin (Altamura, 1996). However, for recalcitrant woody species, auxin may not induce root formation. As an alternative or complementary strategy, localized infection with Agrobacterium rhizogenes may be used and has recently been adopted for walnut microcuttings (Caboni et al., 1996). The potential of A. rhizogenes to induce rooting has been attributed to the insertion and stable integration of a portion (T-DNA) of the bacterium's root-inducing (Ri) plasmid into the plant genome. Four loci involved in root formation have been identified ("rol A, B, C and D") (Vilaine et al., 1987). In particular, rolB seems to play an important role in inducing meristemoid formation, at least in herbaceous model systems (Altamura, 1996). The present study investigated histological and ultrastructural changes occurring in the agroinfected cuttings of a recalcitrant walnut genotype cultured under conditions inductive for rooting, with the aim of determining how agrobacteria trigger root formation in the stem of a recalcitrant woody cutting, their migration in the explant, and the cytological events resulting from the combined presence of infection and exogenous auxin. Materials and Methods
In vitro cultures of a seedling of J.regia L. (cv. Sorrento) were multiplied as suggested by Caboni et al. (1996). Microcuttings (2 cm in length, consisting of the 3 most apical internodes and the apex) were isolated from the shoot clusters at the end of the multiplication phase and used for the rooting experiments. The rooting medium contained the same salts and organics of the multiplication medium (Caboni et al., 1996) and was kept for 30 days under 16 h light per day either in hormone-free conditions (HF treatment) or with 10 laM IBA (IBA treatment) or 10 ~tM IAA (IAA treatment). The growth, inoculation, and co-cultivation of A.rhizogenes Conn. (wild type 1855 NCPPB) were carried out according to Caboni et al. (1996). The
101 infected microcuttings were then transferred onto the same rooting medium used for noninfected explants, containing 0.52 mM cefotaxime to inhibit further bacterial growth, and either 10 gM IBA (IBA Ar treatment), 10 gM IAA (IAA Ar treatment), or no hormones (HF Ar treatment), for 30 days, under 16h light per day. At 0, 10, 15, 20 and 30 days, the stem portion (15 mm in length starting from the basal end) of five microcuttings per hormonal treatment was fixed in FAA. (90 ml ethanol, 5 ml acetic acid, 5 ml formaldehyde), dehydrated, embedded in paraffin, sectioned at 10 gm and stained with safranine-fast green (Jensen, 1962) or toluidine blue-0 (O'Brien, 1965) for the histochemical detection of polyphenols.The presence of bacteria in the vessels showing polyphenol deposition was also detected in sections stained with safranine-fast green observed with a Zeiss-LSM3 Confocal Scanning Laser Microscope. Microcuttings taken on the same sampling days were alternatively post-fixed in 1 % osmium tetroxide and then embedded in Spurr's low viscosity resin at 70~ for 8h. Ultrathin sections (80 nm thick) of the samples were stained with citric acid lead (II) salt trihydrate and then observed with a Zeiss EM 10/C transmission electron microscope. Results and Discussion
At culture end (day 30), rooted explants were sporadic in the absence of infection, whereas in the presence of infection a conspicuous response was obtained even for HF treatment (about 40 % of the cultured explants showed roots). In the presence of IBA, a very high response was obtained (about 60 %), whereas in the presence of IAA the response was very low (about 20 %). In comparison with day 0 (Figure 1a), an increase in xylem development occurred in all the cuttings starting from day 10, and especially in those of IAA Ar treatment (Table 1). Polyphenols, absent at day 0, were present at day 10 in both the primary and secondary xylem (Figure 1b), as well as in the secretor cavities of the cortex. Parenchyma cells, flanking some vessels, produced these compounds, which were then secreted into the vessels (Figure 1b). Polyphenol deposition increased during the culture, and mainly in the combined presence of infection and exogenous IAA (Table 1). Table 1. Histological events occurring in infected (Ar) and non-infected walnut cuttings throughout the culture period. (+, low; ++, medium' +++, high; and ++++, very high occurrence of the event. -, event not observed). Xylem development
Treatm~ nt
Polyphenol deposition
10
15 days
HF Ar
4-
4-
HF
4-
4-
4-4-
4-4-
4-4-4-
IAA
4-
4-4-
4-4-
IBA Ar
4-
4-
4-
IBA
4-
4-
4-
IAA Ar
20
Root primordia
Root meristemoids
10
15 days
20
10
4-
4-
4-4-
4-4-
4-
4-4-4-
4-
4-
4-
4-
4-
4-4-4-
4-
4-
++4-
15 days
20
10
15 20 days
4-4-+4-
4-
4-
30 4-4-4-
4-4-
4-4-
4-
4-
4-
++
4-
4-
4-4-4-4-
4-
4-
4-
4-
4-
4-44-4-
4-
-
4-4-4-
4-4-4-4-
-
4-
Figure. 1. Histological events of adventitious root formamation in walnut microcuttings.[Transection under light microscopy. Bars= 300~m (a,d); 50~m (b); 10~m (c)]. a. Stem showing the secondary vascular structure at culture onset (day 0). b. Polyphenol deposition in primary and secondary xylem (IAA Ar-treated explant, day 15). c. Meristemoid in close proximity to the cambium (direct genesis) (IBA Ar-treated explant, day 10). d. Dome of a root primordium form in the cortical callus (indirect genesis) (IBA Ar-treated explant, day 20). In the infected cuttings, the bacteria were localized in the secretor cavities of the cortex and in the nearby cells (Figure 2a), and especially in the tracheary elements of the xylem.
103
:~: i ;i !i~ii i~Ji:.
: i=}?:! :i !ii ~i!;!i~?~ii~!ii ~a:i!~!i~i i ~:=:!;i i ~i'.i i~ii;i':~i~121 !;2il~i==~=~a~ .......
I!
'~ ~!~ ::.
i
..~i,:-.::::~!!iii:;ii
'
Figure. 2. Presence of A.rhizogenes in the infected cuttings after 10 days of culture in rooting media. [Thin longitudinal sections observed under TEM. Bars= 250nm (a); 260nm (b); l~m (c)]. a. Detail of a degenerating cortical cell showing irregularly shaped and plasmolysed bacterioids (IBA Ar-treated cutting), b. Detail of a xylem parenchyma cell showing bacterioids immersed in a fibril material (HF Ar-treated cutting). c. Wavy walls of cortical parenchyma cells (IAA Ar-treated cutting).
104 The bacteria were frequently plasmolysed and irregular in shape (Figure 2a-b). Bacteria were present not only in the vessels but also in the xylem parenchyma cells surrounding them (Figure 2b) and, less frequently, in the pith ray cells and the cambial cells. Agrobacteria were also observed in the intercellular spaces and between the plasmamembrane and the wall. The parenchyma cells lost their structural integrity when they became filled with bacteria (Figure 2a-b). Although these cells continued to show a quite regular wall texture (Figure 2a), their walls frequently became wavy (Figure 2c), and fibril material accumulated in the protoplast, surrounding the bacteria (Figure 2b). This material greatly differed from the material observed in the vessels colonized by the bacteria and located at the borders of the secondary wall thickenings. In the cells containing high amounts of polyphenols, a conspicuous presence of bacteria was detected. Neither bacteria nor fibril materials were present in the non-infected cuttings. Another event observed starting from day 10 in infected explants of all treatments, and in the non-infected explants treated with IBA only, was the formation of meristemoids (Table 1), with a preferential location in close proximity to the cambium (Figure l c) (direct genesis, Altamura, 1996). In the treatments with IBA, meristemoids were also present in the cortical callus (indirect genesis; Altamura, 1996). At day 15, a very high number of meristemoids was observed in the infected cuttings treated with IBA, and, to a lesser extent, in those of the HF treatment, whereas the quantity of meristemoids did not significantly increase in the IAA Ar treatment in comparison with day 10 (Table 1). Furthermore, in the cuttings of the IBA Ar treatment, the first root primordia were also present and showed either indirect or direct genesis. At day 20, the quantity of meristemoids (all with a strictly basal location in the stem) greatly increased in the HF Ar treatment, whereas it decreased in the IBA Ar treatment, in which there was instead an increase in root primordia formation (Table 1). In general, the number of root primordia progressively increased up to day 30 (Figure 1d, Table 1). Starting from day 15, in the IAA-treated explants, especially in the infected cuttings, there was significant xylem development (Table 1). At day 20, the radial extension of xylem was significantly greater (by about 33%) in the infected cuttings, compared to the non-infected ones treated with the same hormone. Furthermore, in the infected cuttings, vessels showing high polyphenol deposition (Table 1), frequently associated with bacteria were observed and were extended to the entire radius of the xylem. This study demonstrates that in a recalcitrant walnut, a specific IBA treatment and the infection with A. rhizogenes synergistically induce a very precocious and abundant formation of direct and indirect meristemoids, resulting in a high macroscopic rooting response. In fact, infection per se is also able to trigger a rather consistent rooting response, yet meristemoids are produced later and in lower quantities than those obtained when IBA is added to the medium, and their location in the stem is strictly basal. It is known that endogenous auxin positively affects rooting, and that it accumulates at the stem base because of basipetal transport (Lomax et al., 1995). Thus, infection might trigger the rooting process in cells with a sufficiently high auxin content. The observation that in the infected cuttings treated with exogenous IBA the meristemoids are present along an extensive part of the stem supports the hypothesis that IBA treatment increases the population of cells with the endogenous auxin content necessary for rooting. It is not clear which aspect of infection (contamination or transformation) triggers rhizogenesis in walnut cuttings. It cannot be excluded that
105 contaminated cells, before their death, may produce diffusible signals for other, healthy cells, which become capable of initiating the rooting process. However, it is probable that, more than bacterial contamination, the transformation of the plant genome with the T-DNA genes of the root-inducing plasmid of A. rhizogenes plays the pivotal role in triggering rhizogenesis. The ultrastructural analysis of the present paper shows that A. rhizogenes is present in the cutting up to the end of culture and that the bacterial loci are mainly located in the conducting elements of the xylem. A. tumefaciens has been reported to form microcolonies in the vascular tissue of in vitro shoot cultures of tobacco (Matzk et al., 1996), and A. radiobacter has been shown in damaged cells of the cortex and the pith and in the vessels of Kalancho~ daigremontiana (Bogers, 1972). Furthermore, two different types of fibril material are produced as the consequence of contamination. The fibril material near the pit cavities of the vessels resembles the adhesion fibril material, composed of polysaccharides, produced by other bacteria during various pathogeneses (Mount & Lacy, 1982). The other type of fibril material resembles that produced by the rice cells in the leaf blight disease incited by Xanthomonas oryzae (Horino, 1976). The waviness of the walls of the host cells may be interpreted as a stress-induced response to intracellular bacterial colonization. In fact, a similar wall alteration has been observed in tobacco cells subjected to the stress of polyamine biosynthesis inhibition (Berta et al., 1997). In walnut microcuttings, IAA has a specific effect on the differentiation of the secondary xylem and not on rooting. It is possible that IAA triggers the sensitivity of the potential provascular cells of the cutting before activating the sensitivity of potential pre-rhizogenic cells, thus irreversibly conditioning the explant towards the xylary response. Differently from rhizogenesis, high levels of auxin are necessary for both the induction and maturation of the xylem (Fukuda, 1996). Our investigations demonstrate that polyphenol deposition is higher in the infected explants of the IAA treatment, and that when the bacteria are present in the vessels, they are preferentially located in those showing depositions of polyphenolic compounds. Assuming that polyphenol deposition is stimulated by bacteria, the "protective" role of such compounds on IAA (Lee et al., 1982) might contribute to prolonging xylem formation. Thus, in the infected cuttings treated with IAA, the positive interaction between xylem differentiation and polyphenol deposition might explain the failure of the rooting response.
Acknowledgements The work was supported by funds of Universit5 La Sapienza (Rome, Italy), Progetti Ateneo, to M.MIA. The Authors wish to thank "The New Phytologist" for granting permission to reproduce parts of the text of this work.
References Altamura MM. Histological events in adventitious rooting. Agronomic 1996; 16: 589-602. Berta G, Altamura MM, Fusconi A, Cerruti F, Capitani F, Bagni N. The plant cell wall is altered by inhibition of polyamine biosynthesis. New Phytol 1997; 137: 569-577. Bogers RJ. On the interaction ofAgrobacterium tumefaciens with cells of Kalancho~ daigremontiana. Proceedings of the Third International Conference on Plant Pathogenic Bacteria. In: Maas Geesteranus HP (ed.) Netherlands: Zuid-Nederlandsche Drukkerij, NV's-Hertogenbosch, 1972. p. 239-250.
106 Caboni E, Lauri P, Tonelli MG, Falasca G, Damiano C. Root induction by A.rhizogenes in walnut. Plant Science 1996; 118: 203-208. Fukuda H. Xylogenesis: initiation, progression, and cell death. Annu Rev Plant Phys Plant Mol Biol 1996; 47: 299-325. Horino O. Induction of bacterial leaf blight resistance by incompatible strains of XanthomGnas oryzae in rice. Biochemistry and Cytology of Plant-Parasite Interaction. In: Tomiyama K, Daly JM, Uritani I, Oku H, Oughi S (eds.). Yokyo:Kodansha Ltd. 1976. p. 43-55. Jensen WA. Botanical histochemistry. Principles and practices. San Francisco: Freeman, 1962. p. 7090, 250-251. Lee TT, Starrat AN, Jeknikar JJ. Regulation of enzymic oxidation of indole-3-acetic acid by phenols: structure-activity relationship. Phytochemistry 1982; 21:517-523. Lomax TL, Muday GK, Rubery PH. Auxin transport. Plant Hormones: Physiology, Biochemistry and Molecular Biology, 2 "a ed, In: Davies PJ (ed.). Dordrecht, The Netherlands: Kluwer Academic Publisher. 1995. p. 509-530. Matzk A, Mantell S, Schiemann J. Localization of persisting agrobacteria in transgenic tobacco plants. MPMI 1996; 9 (5): 373-381. Mount MS, Lacy GH. Phytopathogenic Prokaryotes. New York: Academic Press. 1982. O'Brien TP, Feder N, McCuily ME. Polichromatic staining of plant cell walls by toluidine blue O. Protoplasma 1965; 59: 367-373. Villaine F, Charbonnier C, Casse-Delbart F. Further insight concerning the TL region of the Ri plasmid A.rhizogenes strain A4 : transfer of a 1.9 kb fragment is sufficient to induce transformed roots on tobacco leaf fragments. Mol Gen Genet 1987; 210:111-115.
100
Histological and Ultrastructural Analysis of A. rhizogenes-mediated Root Formation in Walnut Cuttings M M Altamura ~*, G Falasca ~, M Reverberi ~, A De Stradis2
1. Dipartimento di Biologia Vegetale, Universita di Roma "La Sapienza"; P.le Aldo Moro 5; 00185 Rome, Italy. 2. Centro di Servizi Interdipartimentale di Microscopia, Universita degli Studi della Basilicata; 85100 Potenza, Italy. * Author for correspondence
Introduction
The induction of adventitious roots is crucial for vegetative propagation in plant or micropropagation in vitro of many woody species, yet many woody species are totally recalcitrant to rooting (Altamura 1996). This recalcitrance may be associated with various histologically detectable factors, such as the inability of the explant cells, after stimulation, to organize root meristemoids (Altamura 1996). In English walnut (J. regia L.), the propagation by grafting on seedling rootstocks is an expensive process, yet the alternative practice of grafting on black walnut seedling rootstocks makes trees vulnerable to the lethal blackline disease. In vitro, root formation may be a response to wounding per se or it may be associated with the presence of root inducers, such as auxin (Altamura, 1996). However, for recalcitrant woody species, auxin may not induce root formation. As an alternative or complementary strategy, localized infection with Agrobacterium rhizogenes may be used and has recently been adopted for walnut microcuttings (Caboni et al., 1996). The potential of A. rhizogenes to induce rooting has been attributed to the insertion and stable integration of a portion (T-DNA) of the bacterium's root-inducing (Ri) plasmid into the plant genome. Four loci involved in root formation have been identified ("rol A, B, C and D") (Vilaine et al., 1987). In particular, rolB seems to play an important role in inducing meristemoid formation, at least in herbaceous model systems (Altamura, 1996). The present study investigated histological and ultrastructural changes occurring in the agroinfected cuttings of a recalcitrant walnut genotype cultured under conditions inductive for rooting, with the aim of determining how agrobacteria trigger root formation in the stem of a recalcitrant woody cutting, their migration in the explant, and the cytological events resulting from the combined presence of infection and exogenous auxin. Materials and Methods
In vitro cultures of a seedling of J.regia L. (cv. Sorrento) were multiplied as suggested by Caboni et al. (1996). Microcuttings (2 cm in length, consisting of the 3 most apical internodes and the apex) were isolated from the shoot clusters at the end of the multiplication phase and used for the rooting experiments. The rooting medium contained the same salts and organics of the multiplication medium (Caboni et al., 1996) and was kept for 30 days under 16 h light per day either in hormone-free conditions (HF treatment) or with 10 laM IBA (IBA treatment) or 10 ~tM IAA (IAA treatment). The growth, inoculation, and co-cultivation of A.rhizogenes Conn. (wild type 1855 NCPPB) were carried out according to Caboni et al. (1996). The
101 infected microcuttings were then transferred onto the same rooting medium used for noninfected explants, containing 0.52 mM cefotaxime to inhibit further bacterial growth, and either 10 gM IBA (IBA Ar treatment), 10 gM IAA (IAA Ar treatment), or no hormones (HF Ar treatment), for 30 days, under 16h light per day. At 0, 10, 15, 20 and 30 days, the stem portion (15 mm in length starting from the basal end) of five microcuttings per hormonal treatment was fixed in FAA. (90 ml ethanol, 5 ml acetic acid, 5 ml formaldehyde), dehydrated, embedded in paraffin, sectioned at 10 gm and stained with safranine-fast green (Jensen, 1962) or toluidine blue-0 (O'Brien, 1965) for the histochemical detection of polyphenols.The presence of bacteria in the vessels showing polyphenol deposition was also detected in sections stained with safranine-fast green observed with a Zeiss-LSM3 Confocal Scanning Laser Microscope. Microcuttings taken on the same sampling days were alternatively post-fixed in 1 % osmium tetroxide and then embedded in Spurr's low viscosity resin at 70~ for 8h. Ultrathin sections (80 nm thick) of the samples were stained with citric acid lead (II) salt trihydrate and then observed with a Zeiss EM 10/C transmission electron microscope. Results and Discussion
At culture end (day 30), rooted explants were sporadic in the absence of infection, whereas in the presence of infection a conspicuous response was obtained even for HF treatment (about 40 % of the cultured explants showed roots). In the presence of IBA, a very high response was obtained (about 60 %), whereas in the presence of IAA the response was very low (about 20 %). In comparison with day 0 (Figure 1a), an increase in xylem development occurred in all the cuttings starting from day 10, and especially in those of IAA Ar treatment (Table 1). Polyphenols, absent at day 0, were present at day 10 in both the primary and secondary xylem (Figure 1b), as well as in the secretor cavities of the cortex. Parenchyma cells, flanking some vessels, produced these compounds, which were then secreted into the vessels (Figure 1b). Polyphenol deposition increased during the culture, and mainly in the combined presence of infection and exogenous IAA (Table 1). Table 1. Histological events occurring in infected (Ar) and non-infected walnut cuttings throughout the culture period. (+, low; ++, medium' +++, high; and ++++, very high occurrence of the event. -, event not observed). Xylem development
Treatm~ nt
Polyphenol deposition
10
15 days
HF Ar
4-
4-
HF
4-
4-
4-4-
4-4-
4-4-4-
IAA
4-
4-4-
4-4-
IBA Ar
4-
4-
4-
IBA
4-
4-
4-
IAA Ar
20
Root primordia
Root meristemoids
10
15 days
20
10
4-
4-
4-4-
4-4-
4-
4-4-4-
4-
4-
4-
4-
4-
4-4-4-
4-
4-
++4-
15 days
20
10
15 20 days
4-4-+4-
4-
4-
30 4-4-4-
4-4-
4-4-
4-
4-
4-
++
4-
4-
4-4-4-4-
4-
4-
4-
4-
4-
4-44-4-
4-
-
4-4-4-
4-4-4-4-
-
4-
Figure. 1. Histological events of adventitious root formamation in walnut microcuttings.[Transection under light microscopy. Bars= 300~m (a,d); 50~m (b); 10~m (c)]. a. Stem showing the secondary vascular structure at culture onset (day 0). b. Polyphenol deposition in primary and secondary xylem (IAA Ar-treated explant, day 15). c. Meristemoid in close proximity to the cambium (direct genesis) (IBA Ar-treated explant, day 10). d. Dome of a root primordium form in the cortical callus (indirect genesis) (IBA Ar-treated explant, day 20). In the infected cuttings, the bacteria were localized in the secretor cavities of the cortex and in the nearby cells (Figure 2a), and especially in the tracheary elements of the xylem.
103
:~: i ;i !i~ii i~Ji:.
: i=}?:! :i !ii ~i!;!i~?~ii~!ii ~a:i!~!i~i i ~:=:!;i i ~i'.i i~ii;i':~i~121 !;2il~i==~=~a~ .......
I!
'~ ~!~ ::.
i
..~i,:-.::::~!!iii:;ii
'
Figure. 2. Presence of A.rhizogenes in the infected cuttings after 10 days of culture in rooting media. [Thin longitudinal sections observed under TEM. Bars= 250nm (a); 260nm (b); l~m (c)]. a. Detail of a degenerating cortical cell showing irregularly shaped and plasmolysed bacterioids (IBA Ar-treated cutting), b. Detail of a xylem parenchyma cell showing bacterioids immersed in a fibril material (HF Ar-treated cutting). c. Wavy walls of cortical parenchyma cells (IAA Ar-treated cutting).
104 The bacteria were frequently plasmolysed and irregular in shape (Figure 2a-b). Bacteria were present not only in the vessels but also in the xylem parenchyma cells surrounding them (Figure 2b) and, less frequently, in the pith ray cells and the cambial cells. Agrobacteria were also observed in the intercellular spaces and between the plasmamembrane and the wall. The parenchyma cells lost their structural integrity when they became filled with bacteria (Figure 2a-b). Although these cells continued to show a quite regular wall texture (Figure 2a), their walls frequently became wavy (Figure 2c), and fibril material accumulated in the protoplast, surrounding the bacteria (Figure 2b). This material greatly differed from the material observed in the vessels colonized by the bacteria and located at the borders of the secondary wall thickenings. In the cells containing high amounts of polyphenols, a conspicuous presence of bacteria was detected. Neither bacteria nor fibril materials were present in the non-infected cuttings. Another event observed starting from day 10 in infected explants of all treatments, and in the non-infected explants treated with IBA only, was the formation of meristemoids (Table 1), with a preferential location in close proximity to the cambium (Figure l c) (direct genesis, Altamura, 1996). In the treatments with IBA, meristemoids were also present in the cortical callus (indirect genesis; Altamura, 1996). At day 15, a very high number of meristemoids was observed in the infected cuttings treated with IBA, and, to a lesser extent, in those of the HF treatment, whereas the quantity of meristemoids did not significantly increase in the IAA Ar treatment in comparison with day 10 (Table 1). Furthermore, in the cuttings of the IBA Ar treatment, the first root primordia were also present and showed either indirect or direct genesis. At day 20, the quantity of meristemoids (all with a strictly basal location in the stem) greatly increased in the HF Ar treatment, whereas it decreased in the IBA Ar treatment, in which there was instead an increase in root primordia formation (Table 1). In general, the number of root primordia progressively increased up to day 30 (Figure 1d, Table 1). Starting from day 15, in the IAA-treated explants, especially in the infected cuttings, there was significant xylem development (Table 1). At day 20, the radial extension of xylem was significantly greater (by about 33%) in the infected cuttings, compared to the non-infected ones treated with the same hormone. Furthermore, in the infected cuttings, vessels showing high polyphenol deposition (Table 1), frequently associated with bacteria were observed and were extended to the entire radius of the xylem. This study demonstrates that in a recalcitrant walnut, a specific IBA treatment and the infection with A. rhizogenes synergistically induce a very precocious and abundant formation of direct and indirect meristemoids, resulting in a high macroscopic rooting response. In fact, infection per se is also able to trigger a rather consistent rooting response, yet meristemoids are produced later and in lower quantities than those obtained when IBA is added to the medium, and their location in the stem is strictly basal. It is known that endogenous auxin positively affects rooting, and that it accumulates at the stem base because of basipetal transport (Lomax et al., 1995). Thus, infection might trigger the rooting process in cells with a sufficiently high auxin content. The observation that in the infected cuttings treated with exogenous IBA the meristemoids are present along an extensive part of the stem supports the hypothesis that IBA treatment increases the population of cells with the endogenous auxin content necessary for rooting. It is not clear which aspect of infection (contamination or transformation) triggers rhizogenesis in walnut cuttings. It cannot be excluded that
105 contaminated cells, before their death, may produce diffusible signals for other, healthy cells, which become capable of initiating the rooting process. However, it is probable that, more than bacterial contamination, the transformation of the plant genome with the T-DNA genes of the root-inducing plasmid of A. rhizogenes plays the pivotal role in triggering rhizogenesis. The ultrastructural analysis of the present paper shows that A. rhizogenes is present in the cutting up to the end of culture and that the bacterial loci are mainly located in the conducting elements of the xylem. A. tumefaciens has been reported to form microcolonies in the vascular tissue of in vitro shoot cultures of tobacco (Matzk et al., 1996), and A. radiobacter has been shown in damaged cells of the cortex and the pith and in the vessels of Kalancho~ daigremontiana (Bogers, 1972). Furthermore, two different types of fibril material are produced as the consequence of contamination. The fibril material near the pit cavities of the vessels resembles the adhesion fibril material, composed of polysaccharides, produced by other bacteria during various pathogeneses (Mount & Lacy, 1982). The other type of fibril material resembles that produced by the rice cells in the leaf blight disease incited by Xanthomonas oryzae (Horino, 1976). The waviness of the walls of the host cells may be interpreted as a stress-induced response to intracellular bacterial colonization. In fact, a similar wall alteration has been observed in tobacco cells subjected to the stress of polyamine biosynthesis inhibition (Berta et al., 1997). In walnut microcuttings, IAA has a specific effect on the differentiation of the secondary xylem and not on rooting. It is possible that IAA triggers the sensitivity of the potential provascular cells of the cutting before activating the sensitivity of potential pre-rhizogenic cells, thus irreversibly conditioning the explant towards the xylary response. Differently from rhizogenesis, high levels of auxin are necessary for both the induction and maturation of the xylem (Fukuda, 1996). Our investigations demonstrate that polyphenol deposition is higher in the infected explants of the IAA treatment, and that when the bacteria are present in the vessels, they are preferentially located in those showing depositions of polyphenolic compounds. Assuming that polyphenol deposition is stimulated by bacteria, the "protective" role of such compounds on IAA (Lee et al., 1982) might contribute to prolonging xylem formation. Thus, in the infected cuttings treated with IAA, the positive interaction between xylem differentiation and polyphenol deposition might explain the failure of the rooting response.
Acknowledgements The work was supported by funds of Universit5 La Sapienza (Rome, Italy), Progetti Ateneo, to M.MIA. The Authors wish to thank "The New Phytologist" for granting permission to reproduce parts of the text of this work.
References Altamura MM. Histological events in adventitious rooting. Agronomic 1996; 16: 589-602. Berta G, Altamura MM, Fusconi A, Cerruti F, Capitani F, Bagni N. The plant cell wall is altered by inhibition of polyamine biosynthesis. New Phytol 1997; 137: 569-577. Bogers RJ. On the interaction ofAgrobacterium tumefaciens with cells of Kalancho~ daigremontiana. Proceedings of the Third International Conference on Plant Pathogenic Bacteria. In: Maas Geesteranus HP (ed.) Netherlands: Zuid-Nederlandsche Drukkerij, NV's-Hertogenbosch, 1972. p. 239-250.
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