Plant Cell Reports
Plant Cell Reports (1991) 10:291-295
Increase of rooting ability in the woody species kiwi (Actinidia deliciosa A. Chev.) by transformation with Agrobacterium rhizogenes rol genes
9 Springer-Verlag1991
Eddo Rugini 1, Alessandro Pellegrinesehi 1, Massimo Mencueeini 2, and Domenico Mariotti 3 1 Istituto di Ortofloroarboricoltura,Facoltfi di Agraria, Universit/tdella Tuscia, Via S. Camillo De Lellis, 01100 Viterbo, Italy z CNR Istituto per l'Olivicoltura, loc. Madonna Alta, 06100 Perugia, Italy 3 Istituto di Radiobiochimicaed Ecofisiologiavegetali, Via Salaria km. 29.300, 00016 Monterotondo Scalo, Roma, Italy Received January 15, 1991/Revisedversion receivedApril 17, 1991 - Communicatedby J. Schell
Summary. The woody species kiwi (Actinidia deliciosa A. Chev.), a male and late flowering clone of the cv Hayward, has been transformed by a T-DNA fragment encompassing rol A, B, C genes of A. rhizogenes. Transgenic plants, regenerated from leaf disc callus, showed the typical "hairy root" phenotype as described in herbaceous species. Explants from these plants (both leaf discs or 3 to 4 node leafy microcuttings) showed an increased ability to produce roots. Since root formation is one of the limiting factors in the vegetative propagation of woody species, the results have been discussed in relation to the use of A. rhizogenes rol genes in improving root morphogenesis in trees.
Introduction Transgenic plants with a normal morphological appearance have been recently produced in a number of species including woody plants, e.g. poplar (Fillatti 1987), apple (James et al. 1989), walnut (Dandekar et al. 1988; Mc Granahan et al. 1988) by using disarmed vectors (Bevan et al. 1984). The achievement of transgenic fruit cultivars with "useful" genes has not been reported yet, mainly because of the difficulties encountered in the in vitro manipulation of these species. Agronomically valuable cultivars or rootstocks often show a very poor rooting efficiency which, up to date, has to be regarded as the most limiting factor in the vegetative spreading of basic genotypes. This makes the improvement of rooting ability one of the major objectives in plant micropropagation. The scarce basic knowledge concerning plant morphogenetic processes encourages new approaches to this problem. Dicotyledonous plants infected by Agrobacteriurn rhizogenes show the development of roots (hairy roots) at Offprint requests to: E. Rugini
the inoculation site (Elliot 1951). This morphogenetic process is due to the mobilization, stable integration and expression in the plant host cell of a segment (T-DNA) of the large Ri plasmid harboured by the bacterium (Birot et al. 1987). The use of wild type A. rhizogenes as root inducer, by simple inoculation of the stems, has produced a transformed root system in almond and olive woody plants (Rugini 1984; Strobel and Nachmias 1985; Rugini 1986). Since this strategy was laborious and time consuming, studying rooting ability of T-DNA transformed woody plants could be of interest. Herbaceous plants, transgenic for the whole T-DNA, show the so-called "hairy root" phenotype (Tepfer 1984) characterized by the ability to profusely regenerate roots from leaf explants cultured on hormone-free media (Spanb and Costantino 1982). Four T-DNA genetic loci (rol A,B,C,D) were found to be involved in the rooting process promoted by this microorganism in dicots (White et al. 1985). More recently, the number of genes capable of inducing rhizogenesis has been reduced to ORFs (Open Reading Frames) 10, 11, 12 (Slightom et al. 1986), corresponding to rol A,B,C, which altogether have been identified as responsible for the induction and maintenance of the "hairy root" phenotype (Cardarelli et al. 1987a; Spena et al. 1987; Vilaine et al. 1987). Further results have finally proved that the stimulation of root formation is substantially due to ORF 11 (rol B) action, rol A and C being mainly involved in modifying the morphology and growth rate of the induced roots (Cardarelli et al. 1987a; Schmulling et al. 1988; Capone et al. 1989a). Since the ability to form roots in herbaceous transgenic plants containing the whole T-DNA or T-DNA segments containing rol B is increased with respect to the controls (Tepfer 1984; Spanb et al. 1987), it is interesting to see whether the same effects can be obtained in woody species where self-rooted cuttings are highly desirable for propagation especially when the rootstock is not required.
292 The aim of this work was to investigate whether possible positive alterations in rooting ability could be obtained by transforming trees with these "bacterial" genes, by using the woody species kiwi which easily regenerates from callus even after a long culture period.
Materials and Methods Plant tissue culture. Kiwi (Actinidia deliciosa A. Chev.), a male and late flowering clone of the cultivar Hayward (kindly supplied by Vitrocoop, Cesena) multiplied in vitro with monthly subcultures on the proliferation agarized (0.5% w/v agar, Biolife) MS medium (Murashige and Skoog 1962) supplemented with 1 mg/1 BAP (6benzylaminopurine), was the source of the leaf material. Callusing of leaf explants was obtained on the following induction medium: MS basal salts, 3% w/v sucrose, 5 mg/1 NAA (alpha-naphthaleneacetic acid), 0.1 rag/1 BAP (6-benzylaminopurine). Adventitious shoots from callus were obtained in the regeneration B5 medium (Gamborg et al. 1968), with 1 mg/l BAP, 3% sucrose and 0.5% w/v agar. The pH of the various media was adjusted to 5.8 before autoclaving at 121~ for 15 rain. Callus induction and growth was carried out in 25 hole multiwells containing one leaf disc or about 1 g of callus on 3 ml medium per hole, while shoot regeneration was obtained in 9 cm diameter Petri dishes containing 2 to 3 g callus/plate. The cultures were incubated in a growth room at 23 ~ in the dark except for the regeneration phase which was done under a 15 h photoperiod (fluorescent lamps at 40 ~ E m -2 s-l). Bacterial strains and media. Cloning of the TL-DNA Eco 15 fragment encompassing ORFs 10, 11 and 12 has been extensively reported elsewhere (Cardarelli et al. 1987b). The restriction fragment EcoR1 15, extracted from a suitable pRi 1855 pBR322 clone (Pomponi et al. 1983), cloned in Bin 19 (Bevan et al. 1984) and transferred into Agrobacterium strain LBA 4404 (Ooms et al. 1982) was used to transform kiwi explants. Bacteria were grown at 28 ~ in solidified YMB medium (Hooykaas et al. 1977) supplemented with 100 mg/l kanamycin for 48 h and then transferred for the co-cultivation procedure to MS liquid medium (approximatively 108 cells/ml). Transformation and selection procedure. Leaf discs (1 cm in diameter) were cut with a cork borer, immersed in bacterial suspension and gently scratched on both surfaces by pressing them on carborundum granules with a copper wire brush. The discs remained in contact with bacterial suspension for about 1 h before being placed singularly, after blotting, onto the callus induction medium in the adaxial side up position. Three days later carbenicillin (200 mg/l) was added and explants were maintained in the same medium plus antibiotic for another 15 days. Selection was carried out by adding an increasing level of kanamycin (from 50 to 200 mg/l) during the subsequent subcultures. In this concentration the callus was subcultured twice before being transferred to the regeneration medium. Control plates, lacking kanamycin, but with carbenicillin, were also used to obtain callus and to regenerate control shoots. Kanamycin resistance. To assess the level of resistance of untransformed callus and tissue to kanamycin, kiwi callus and apical shoots (3-4 cm in length) were cultured on callus induction and
proliferation media, respectively, with kanamycin at the levels 0, 20, 30, 50, 100, 200 and 400 mg/l.
Micropropagation of clones. Shoots regenerated from kanamycin resistant callus were proliferated by axillary bud stimulation in proliferation medium containing 200 mg/I carbenieyllin and 50 rag/1 kanamycin for 2-3 subcultures before omitting the last antibiotic. To assess the absence of bacteria or other contaminants in the plant cultures, small explants were cultivated in media suitable for microorganism growth (YMB). Axenic shoots were then used for rooting, NPTII and Southern tests. Shoots regenerated from untransformed callus were also proliferated. Rooting test. Rooting tests were carried out on half strength MS medium, without hormones, except for the case in which it was supplemented with 0.2 mg/l IBA (indol-3-butyrie acid), by using both leaf discs or 3 to 4 node leafy explants under the above described environmental conditions. The percentage of rooted microcuttings or leaf discs and the number of roots per disc or microcutting were observed at various intervals. Biochemical tests. NPT II (neomycin phosphotransferase II) tests were performed according to Tavazza et al. (1988). Plant DNA extraction was done as described by Della Porta et al. (1983). Southern blot hybridization was carried out as reported by Span6 et al. (1982).
Results
Ideal candidates for testing possible beneficial effects on root formation induced by rol genes in woody species would be those fruit cultivars totally incapable of producing roots from cuttings. On the other hand, in vitro plant regeneration is preliminary to gene transfer experiments and, to our knowledge, the above-mentioned genotypes are extremely difficult to manipulate in vitro. Even if Actinidia deliciosa is to some extent capable of rooting in vitro, it represents a valuable substitute as a model plant, since it is responsive to callus induction and meristematic shoot neoformation from dedifferentiated cells. MS medium supplemented with a high level of NAA (5 mg/l) and BAP(0.1 mg/l) proved to be ideal for callus induction and for supporting its vigorous growth. Leaf discs produced profuse callus both around the cut surface and from injuries inflicted by carborundum, which stimulated dedifferentiation along the midrib and on the whole disc surface. The regeneration medium (B5 containing 1 mg/l BAP) induced abundant meristematic shoot production on the callus; the earliest regenerative events took place two weeks after the subculture but the morphogenetic process continued for months. Preliminary to gene transfer experiments, callus and shoots were tested for kanamycin resistance on various concentrations of this selective antibiotic. Callus proliferation was completely inhibited at 20 mg/1 and explant death was observed at the same concentration
293 when apical shoots were used. No visible effects were evoked by carbenicillin.
plants of the genes responsible for the "hairy root" phenotype in herbaceous species, and second, because rol A and C have been reported to positively affect rooting, in terms of growth rate, when they are operating together with rol B (Schmulling et al. 1988). Gene transfer was carried out via leaf disc transformation; to select transformed cell lines, the leaf disc-derived callus obtained in the absence of kanamycin was subcultured on selection medium. Only a few colonies of the numerous inocula survived and continued to proliferate up to 200 mg/l kanamycin. Despite the high concentration of NAA (5 mg/l), usually prohibitive for any form of morphogenesis, a few roots emerged from the white callus. This result has to be put in relation to the strong disposition of rol B gene in directing all the transformed cells towards root morphogenesis.
Fig. 1.-A) Southern blot analysis of plant DNA from thekanamycin resistant, NPT II positive clone 9. Lane l=restriction profile of the pMP 66 T-DNA fragment which covers more than 9 0 % of pRI 1855 TL-DNA region; Lane 2 = Autoradiogram of clone 9 DNA probed by the Eco RI fragment 15 encompassing rol A, B, C genes; Lane 3 = Autoradiogram of a control plant DNA probed with the same T-DNA fragment. B) EcoRI restriction map of pRi 1855 TL-region covered by pMP 66 containing the fragment 15 encompassing rol A, B, C genes
Fig. 2. -Neomycin phosphotransferase II assay in kanamycirrresistant clone 9 kiwi plant. Lane A = Extract from clone 9; Lane B = Extract from untransformed kiwi (negative control); Lane C = Extract from a transformed potato (positive control)
The role of ORF 11 (rol B), with the upstream regulatory region of about 1000 bp, has been dearly established in the induction of hairy roots (Schmulling et al. 1988; Capone et al. 1989a). We have initially chosen, instead of the sole rol B, a construct containing the Eco 15 T-DNA fragment in Bin 19 (Fig. 1 B) encompassing rol A, B, C, first, in order to define the effects on woody
Fig. 3.- A) Rooting of leaf discs in hormone-free MS medium: transgenic explants (left) and control.- B) Transgenic kiwi plant after potting: evidence of the reversion from "hairy root" to "normal" phenotype. Arrow shows "hairy root" leaf phenotype.
Putatively transformed callus regenerated as many shoots as untransformed callus when transferred to regeneration medium containing 1 mg/l BAP. For each callus, 5 to 10 shoots were regenerated, numbered and micropropagated. The transformed state of the shoots proliferating in the selection medium was tested by
294 analysing NPT II enzyme activity and by Southern blot hybridization. NPT II was positive for all the randomly chosen plantlets (see an example in Fig. 2). The presence of the transferred T-DNA segment in the kiwi clone no. 9 DNA was checked by hybridizing a 32 p _ labelled EcoRI fragment against a Southern blot of EcoRI - digested clone 9 total DNA (Fig. 1 A). The morphology of kanamycin resistant kiwi shoots, compared to normal ones, during the in vitro growth, was characterized by the typical "hairy root" alterations described in various species after transformation with rol A,B,C genes from A. rhizogenes T-DNA. The slower Table 1. Percentage of rooted leaf discs of various transformed kiwi clones on hormone-free m e d i u m after 30 and 60 days and average n u m b e r of roots p e r disc (_+ SE) evaluated at 60 days. Thirty explants
A remarkable variability in regard to the number of roots radiating from the leaf discs was observed only among the transgenic clones. The typical "hairy root" phenotype characterized the transgenic roots. To study the behaviour of transformed plantlets in root production two experiments were conducted by evaluating first the rooting ability in 1/2 MS without hormones, and second the influence of rol genes on the timing of root production in 1/2 MS supplemented with 0.2 mg/l IBA, a growth regulator largely used to stimulate root morphogenesis woody species in vitro propagated. Table 2 shows that after 40 days all the transgenic microcuttings had produced roots (with some variability among the clones) in hormone-free MS medium, while only 36% of the controls showed roots (in general, with a lower number per plant).
p e r each clone were used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table 2. Percentage of rooted microcuttings of randomly chosen
Clone
% of rooted
roots
transformed kiwi clones and average n u m b e r of roots p e r cutting (_+
number
leaf discs
p e r disc
SE) in hormone-free medium at 40 days. Thirty explants per clone were
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
30 days .
CONTROL
.
.
.
.
.
60 days .
.
.
.
.
.
.
.
60 days .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Clone
% of rooted
roots per
number
microcuttings
microcutting
0
33
3.0 _+ 0.8
9
66
66
15.0 -+ 4.3
14
100
100
3.6 -+ 1.0
15
100
100
4.0 _+ 1.2
9
100
6.0 _+ 1.2
16
100
100
3.3 _+ 0.7
14
100
4.0 -+ 0.8
20
100
100
20.0 +_ 5.1
16
100
2.6 _+ 0A
24
83
90
3.6 -+ 1.3
20
100
3.0 _+ 1.4
27
100
100
12.5 -+ 3.2
24
100
2.0 + 0.2
28
100
100
4.3 + 1.8
27
100
4.0 -+ 1.0
33
100
100
7.0 -+ 1.5
28
100
3.0 _+ 1.2
37
100
100
7.5 -+ 0.7
33
100
1.6 -- 0.3
39
100
100
16.0 -+ 4.1
37
100
6.5 -+ 1.8
initial growth, shorter internodes, the dark green wrinkled leaves and the high rooting ability proved that woody plants behaved, during this phase, like herbaceous ones. After potting, transgenic somaclones (10 to 15 cm high) were inclined, in most cases, to lose the aereal "hairy root" characteristics (Fig. 3B). It has been reported for various herbaceous species that leaf discs from plants transformed by T-DNA fragments containing rol B with an upstream regulatory region of about 1000 bp possess the ability to produce profuse rhizogenesis when cultured on hormone-free media, where controls, as a rule, fail (SchmuUing et al. 1988; Spanb and Costantino 1982). Table 1 shows the rooting test in leaf discs of transgenic kiwi plants on hormonefree MS medium (see also Fig.3A). Almost all the discs of the randomly chosen transformed clones produced roots after 4 weeks, while the controls did not show any morphogenetic event. One month later, in only one third of the control discs, a few roots appeared with an average number of roots per disc lower than the transgenic ones.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CONTROL
36
2.0 -+ 0.3
Table 3. Percentage of rooted cuttings of randomly chosen transformed kiwi clones and average n u m b e r of roots per cutting (-+ SE) in MS medium plus 0.2 rag/1 IBA at 15 days. (Thirty explants per each clone were used) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Clone
% of rooted
roots per
number
microcuttings
microcutting
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CONTROL
40
2.2 + 0,2
4
100
3.0 + 0,3
9
100
2.0 + 0.1
14
100
2.0 + 0,8
16
100
2.5 + 0,3
18
100
2.3 +_ 0,4
20
100
2.5 -+ 0.4
24
100
1.0 ___0.2
26
100
2.0 _+ 0.4
28
100
3.5 -+ 1.0
295 In the absence of hormones, rooting starts after about two weeks while I B A supply usually reduces this period. Transgenic plantlets cultured in the presence of IBA behaved as expected with regard to rooting ability; in fact, after only 15 days all of them were rooted but only 40% of the controls showed rooting formation (Table 3). In both cases transgenic roots showed the "hairy root" appearance. Experiments are in progress to evaluate their behaviour in soil.
Discussion The results presented in this paper indicate that: I) it is possible to transform the woody plant Actinidia deliciosa by means of Agrobacterium derived vectors; lI) rol A,B,C genes from A. rhizogenes can be used to enhance the rooting ability of this woody species. This is the first report concerned with the effects of rol genes in trees. Transgenic kiwi plants obtained by transferring rol A, B and C genes show, as described in herbaceous plants, the typical "hairy root" phenotype. However, we observed that after potting several transgenic plants became similar, from a morphological point of view, to the mother plants or those regenerated from untransformed callus. Hairy roots, induced byAgrobacterium rhizogenes wild type are, in general, m o r e branched and lose gravitropic response, while other functions remain unaffected. To date, however, some contradictory findings have been reported about the effect of the various combinations of rol genes on root morphology and tropism; for example, different species transformed by the same T - D N A constructs seem to behave differently (Cardarelli et al. 1987a; Spanb et al. 1987; Schmulling et al. 1988; Capone et al. 1989b). Data from this lab obtained by a large experiment with three categories of transgenic tobaccos ( L D R = w i t h the full TD N A complement; E 15 = with rol A,B,C genes; SHp15 = with O R F 11 (rol B) plus the upstream region of 1000 bp and S R I = control), dearly show that roots radiating from SHp 15 microcuttings are morphologically and gravitropic similar to the SR1 control, while E 15 roots are similar to those of L D R which are m o r e branched and definitively less gravitropic. These results support the idea that the loss of geotropism is due to genes other than rol B, and the preliminary data reported in this paper should lead to more extensive use of the "root inducing" gene rol B. Even if additional work has to be done, this paper first demonstrates that our approach is worthy of further investigation. Moreover, the use of these morphogenetic "bacterial" genes should also help us in a better understanding of root differentiation in trees.
Acknowledgements. This work was supported by the "Piano Nazionale di rieerca: Sviluppo di tecnologie avanzate applicate alle piante" of the Italian Ministry of Agriculture and Forestry.
References Bevan M (1984) NucleicAcid Res 12:8711-8721 Birot AM, Bouchez D, Casse-Delbart F, Durant Tardif M, Jouanin L, Pautot V, Robaglia C, Tepfer D, Tepfer M, Tourneur J, Vilaine F (1987) Plant Physiol Biochem 25:323-335 Capone I, Cardarelli M, Trovato M, Costantino P (1989a) Mol Gen Genet 216:239-244 Capone I, Spanb L, Cardarelli M, Petit A, Costantino P (1989b) Plant Mol Biol 13:43-52 Cardarelli M, Mariotti D, Pomponi M, Span6 L, Capone I, Costantino P (1987a) Mol Gen Genet 209:475-480 Cardarelli M, Span6 L~ Mariotti D, Mauro ML, Van Sluys MA, Costantino P, (1987b) Mol Gen Genet 208:457-463 Dandekar AM, Martin LA, Macgranahan G (1988) J Am Soc Hort Sci 113:945-949 Della Porta SL, Wood J, Hicks JB (1983) Plant Mol Biol Rept 1:19-27 Elliot C (1951) Manual of bacterial plant pathogens, 2nd rev Edn Chronica Botanica, Waltham, Mass Fillatti JJ, Sellmer J, McCown B, Haissing B, Comai L (1987) Mol Gen Genet 206:192-199 Gamborg OL, Miller RA, Ojima K (1968) Exp Cell Res 50:151-158 Hooykaas PJJ, Klapwijck P, Nuti MP, Schilperoort RA, Rorsch A (1977) J Gen Microbiol 98:477-484 James DJ, Passey AJ, Barbara DJ, Bevan M (1989) Plant Cell Reports 7:658-661 McGranahan GH, Leslie CA, Uratsu SL, Martin LA, Dandecar AM (1988) Bio-Technology6:800-804 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Ooms G, Hooykaas PJJ, Van Veen RJM, Van Beelen P, RegensburgTuink AJG, Schilperoort RA (1982) Plasmid 7:15-25 Pomponi M, Spanb L, Sabbatini MG, Costantino P (1983) Plasmid 10:119-120 Rugini E (1984) Progress in Studies on in vitro Culture of Almonds. Proc. 41X Conf on Plant Tissue Culture and its Agricultural Applications, Nottingham, 17-21 Sept, p 73 Rugini E (1986) Olive In: Bajaj YPS (ed) Biotechnologyin Agriculture and Forestry Trees. Springer,Verlag, Berlin, Heidelberg, New York, Tokio, pp 253-267 Schmulling T, Schell J, Spena A (1988) Embo J 7:2621-2629 Slightom JL, Durant Tardif M, Jouanin L, Tepfer D (1986) J Biol Chem 261:108-121 Span6 L, Costantino P (1982) Z Pflanzenphysio1106:87-92 Span6 L, Mariotti D, Pezzotti M, Damiani F (1987) Theor AppI Genet 73:523-530 Span6 L, Pomponi M, Costantino P, Van Slogteren GMS, Tempe J (1982) Plant Mol Biol 1:291-300 Spena A, SchmullingT, Koncz L, Schell J (1987) Embo J 6:3891-3899 Strobel AG, NachmiasA (1985) J Gen Microbiol 131:1245-1249 Tavazza R, Ordas RJ, Tavazza M, Ancora G, Benvenuto E (1988) J Plant Physiol 133:640-644 Tepfer D (1984) Cell 37:959-967 Vilaine F, Charbonnier C, Casse Delbart F (1987) Mol Gen Genet 210:111-115 White FF, Taylor BH, Huffmann GA, Gordon MP, Nester EW (1985) J Bacteriol 164:33-44
Plant Cell Reports (1991) 10:291-295
Increase of rooting ability in the woody species kiwi (Actinidia deliciosa A. Chev.) by transformation with Agrobacterium rhizogenes rol genes
9 Springer-Verlag1991
Eddo Rugini 1, Alessandro Pellegrinesehi 1, Massimo Mencueeini 2, and Domenico Mariotti 3 1 Istituto di Ortofloroarboricoltura,Facoltfi di Agraria, Universit/tdella Tuscia, Via S. Camillo De Lellis, 01100 Viterbo, Italy z CNR Istituto per l'Olivicoltura, loc. Madonna Alta, 06100 Perugia, Italy 3 Istituto di Radiobiochimicaed Ecofisiologiavegetali, Via Salaria km. 29.300, 00016 Monterotondo Scalo, Roma, Italy Received January 15, 1991/Revisedversion receivedApril 17, 1991 - Communicatedby J. Schell
Summary. The woody species kiwi (Actinidia deliciosa A. Chev.), a male and late flowering clone of the cv Hayward, has been transformed by a T-DNA fragment encompassing rol A, B, C genes of A. rhizogenes. Transgenic plants, regenerated from leaf disc callus, showed the typical "hairy root" phenotype as described in herbaceous species. Explants from these plants (both leaf discs or 3 to 4 node leafy microcuttings) showed an increased ability to produce roots. Since root formation is one of the limiting factors in the vegetative propagation of woody species, the results have been discussed in relation to the use of A. rhizogenes rol genes in improving root morphogenesis in trees.
Introduction Transgenic plants with a normal morphological appearance have been recently produced in a number of species including woody plants, e.g. poplar (Fillatti 1987), apple (James et al. 1989), walnut (Dandekar et al. 1988; Mc Granahan et al. 1988) by using disarmed vectors (Bevan et al. 1984). The achievement of transgenic fruit cultivars with "useful" genes has not been reported yet, mainly because of the difficulties encountered in the in vitro manipulation of these species. Agronomically valuable cultivars or rootstocks often show a very poor rooting efficiency which, up to date, has to be regarded as the most limiting factor in the vegetative spreading of basic genotypes. This makes the improvement of rooting ability one of the major objectives in plant micropropagation. The scarce basic knowledge concerning plant morphogenetic processes encourages new approaches to this problem. Dicotyledonous plants infected by Agrobacteriurn rhizogenes show the development of roots (hairy roots) at Offprint requests to: E. Rugini
the inoculation site (Elliot 1951). This morphogenetic process is due to the mobilization, stable integration and expression in the plant host cell of a segment (T-DNA) of the large Ri plasmid harboured by the bacterium (Birot et al. 1987). The use of wild type A. rhizogenes as root inducer, by simple inoculation of the stems, has produced a transformed root system in almond and olive woody plants (Rugini 1984; Strobel and Nachmias 1985; Rugini 1986). Since this strategy was laborious and time consuming, studying rooting ability of T-DNA transformed woody plants could be of interest. Herbaceous plants, transgenic for the whole T-DNA, show the so-called "hairy root" phenotype (Tepfer 1984) characterized by the ability to profusely regenerate roots from leaf explants cultured on hormone-free media (Spanb and Costantino 1982). Four T-DNA genetic loci (rol A,B,C,D) were found to be involved in the rooting process promoted by this microorganism in dicots (White et al. 1985). More recently, the number of genes capable of inducing rhizogenesis has been reduced to ORFs (Open Reading Frames) 10, 11, 12 (Slightom et al. 1986), corresponding to rol A,B,C, which altogether have been identified as responsible for the induction and maintenance of the "hairy root" phenotype (Cardarelli et al. 1987a; Spena et al. 1987; Vilaine et al. 1987). Further results have finally proved that the stimulation of root formation is substantially due to ORF 11 (rol B) action, rol A and C being mainly involved in modifying the morphology and growth rate of the induced roots (Cardarelli et al. 1987a; Schmulling et al. 1988; Capone et al. 1989a). Since the ability to form roots in herbaceous transgenic plants containing the whole T-DNA or T-DNA segments containing rol B is increased with respect to the controls (Tepfer 1984; Spanb et al. 1987), it is interesting to see whether the same effects can be obtained in woody species where self-rooted cuttings are highly desirable for propagation especially when the rootstock is not required.
292 The aim of this work was to investigate whether possible positive alterations in rooting ability could be obtained by transforming trees with these "bacterial" genes, by using the woody species kiwi which easily regenerates from callus even after a long culture period.
Materials and Methods Plant tissue culture. Kiwi (Actinidia deliciosa A. Chev.), a male and late flowering clone of the cultivar Hayward (kindly supplied by Vitrocoop, Cesena) multiplied in vitro with monthly subcultures on the proliferation agarized (0.5% w/v agar, Biolife) MS medium (Murashige and Skoog 1962) supplemented with 1 mg/1 BAP (6benzylaminopurine), was the source of the leaf material. Callusing of leaf explants was obtained on the following induction medium: MS basal salts, 3% w/v sucrose, 5 mg/1 NAA (alpha-naphthaleneacetic acid), 0.1 rag/1 BAP (6-benzylaminopurine). Adventitious shoots from callus were obtained in the regeneration B5 medium (Gamborg et al. 1968), with 1 mg/l BAP, 3% sucrose and 0.5% w/v agar. The pH of the various media was adjusted to 5.8 before autoclaving at 121~ for 15 rain. Callus induction and growth was carried out in 25 hole multiwells containing one leaf disc or about 1 g of callus on 3 ml medium per hole, while shoot regeneration was obtained in 9 cm diameter Petri dishes containing 2 to 3 g callus/plate. The cultures were incubated in a growth room at 23 ~ in the dark except for the regeneration phase which was done under a 15 h photoperiod (fluorescent lamps at 40 ~ E m -2 s-l). Bacterial strains and media. Cloning of the TL-DNA Eco 15 fragment encompassing ORFs 10, 11 and 12 has been extensively reported elsewhere (Cardarelli et al. 1987b). The restriction fragment EcoR1 15, extracted from a suitable pRi 1855 pBR322 clone (Pomponi et al. 1983), cloned in Bin 19 (Bevan et al. 1984) and transferred into Agrobacterium strain LBA 4404 (Ooms et al. 1982) was used to transform kiwi explants. Bacteria were grown at 28 ~ in solidified YMB medium (Hooykaas et al. 1977) supplemented with 100 mg/l kanamycin for 48 h and then transferred for the co-cultivation procedure to MS liquid medium (approximatively 108 cells/ml). Transformation and selection procedure. Leaf discs (1 cm in diameter) were cut with a cork borer, immersed in bacterial suspension and gently scratched on both surfaces by pressing them on carborundum granules with a copper wire brush. The discs remained in contact with bacterial suspension for about 1 h before being placed singularly, after blotting, onto the callus induction medium in the adaxial side up position. Three days later carbenicillin (200 mg/l) was added and explants were maintained in the same medium plus antibiotic for another 15 days. Selection was carried out by adding an increasing level of kanamycin (from 50 to 200 mg/l) during the subsequent subcultures. In this concentration the callus was subcultured twice before being transferred to the regeneration medium. Control plates, lacking kanamycin, but with carbenicillin, were also used to obtain callus and to regenerate control shoots. Kanamycin resistance. To assess the level of resistance of untransformed callus and tissue to kanamycin, kiwi callus and apical shoots (3-4 cm in length) were cultured on callus induction and
proliferation media, respectively, with kanamycin at the levels 0, 20, 30, 50, 100, 200 and 400 mg/l.
Micropropagation of clones. Shoots regenerated from kanamycin resistant callus were proliferated by axillary bud stimulation in proliferation medium containing 200 mg/I carbenieyllin and 50 rag/1 kanamycin for 2-3 subcultures before omitting the last antibiotic. To assess the absence of bacteria or other contaminants in the plant cultures, small explants were cultivated in media suitable for microorganism growth (YMB). Axenic shoots were then used for rooting, NPTII and Southern tests. Shoots regenerated from untransformed callus were also proliferated. Rooting test. Rooting tests were carried out on half strength MS medium, without hormones, except for the case in which it was supplemented with 0.2 mg/l IBA (indol-3-butyrie acid), by using both leaf discs or 3 to 4 node leafy explants under the above described environmental conditions. The percentage of rooted microcuttings or leaf discs and the number of roots per disc or microcutting were observed at various intervals. Biochemical tests. NPT II (neomycin phosphotransferase II) tests were performed according to Tavazza et al. (1988). Plant DNA extraction was done as described by Della Porta et al. (1983). Southern blot hybridization was carried out as reported by Span6 et al. (1982).
Results
Ideal candidates for testing possible beneficial effects on root formation induced by rol genes in woody species would be those fruit cultivars totally incapable of producing roots from cuttings. On the other hand, in vitro plant regeneration is preliminary to gene transfer experiments and, to our knowledge, the above-mentioned genotypes are extremely difficult to manipulate in vitro. Even if Actinidia deliciosa is to some extent capable of rooting in vitro, it represents a valuable substitute as a model plant, since it is responsive to callus induction and meristematic shoot neoformation from dedifferentiated cells. MS medium supplemented with a high level of NAA (5 mg/l) and BAP(0.1 mg/l) proved to be ideal for callus induction and for supporting its vigorous growth. Leaf discs produced profuse callus both around the cut surface and from injuries inflicted by carborundum, which stimulated dedifferentiation along the midrib and on the whole disc surface. The regeneration medium (B5 containing 1 mg/l BAP) induced abundant meristematic shoot production on the callus; the earliest regenerative events took place two weeks after the subculture but the morphogenetic process continued for months. Preliminary to gene transfer experiments, callus and shoots were tested for kanamycin resistance on various concentrations of this selective antibiotic. Callus proliferation was completely inhibited at 20 mg/1 and explant death was observed at the same concentration
293 when apical shoots were used. No visible effects were evoked by carbenicillin.
plants of the genes responsible for the "hairy root" phenotype in herbaceous species, and second, because rol A and C have been reported to positively affect rooting, in terms of growth rate, when they are operating together with rol B (Schmulling et al. 1988). Gene transfer was carried out via leaf disc transformation; to select transformed cell lines, the leaf disc-derived callus obtained in the absence of kanamycin was subcultured on selection medium. Only a few colonies of the numerous inocula survived and continued to proliferate up to 200 mg/l kanamycin. Despite the high concentration of NAA (5 mg/l), usually prohibitive for any form of morphogenesis, a few roots emerged from the white callus. This result has to be put in relation to the strong disposition of rol B gene in directing all the transformed cells towards root morphogenesis.
Fig. 1.-A) Southern blot analysis of plant DNA from thekanamycin resistant, NPT II positive clone 9. Lane l=restriction profile of the pMP 66 T-DNA fragment which covers more than 9 0 % of pRI 1855 TL-DNA region; Lane 2 = Autoradiogram of clone 9 DNA probed by the Eco RI fragment 15 encompassing rol A, B, C genes; Lane 3 = Autoradiogram of a control plant DNA probed with the same T-DNA fragment. B) EcoRI restriction map of pRi 1855 TL-region covered by pMP 66 containing the fragment 15 encompassing rol A, B, C genes
Fig. 2. -Neomycin phosphotransferase II assay in kanamycirrresistant clone 9 kiwi plant. Lane A = Extract from clone 9; Lane B = Extract from untransformed kiwi (negative control); Lane C = Extract from a transformed potato (positive control)
The role of ORF 11 (rol B), with the upstream regulatory region of about 1000 bp, has been dearly established in the induction of hairy roots (Schmulling et al. 1988; Capone et al. 1989a). We have initially chosen, instead of the sole rol B, a construct containing the Eco 15 T-DNA fragment in Bin 19 (Fig. 1 B) encompassing rol A, B, C, first, in order to define the effects on woody
Fig. 3.- A) Rooting of leaf discs in hormone-free MS medium: transgenic explants (left) and control.- B) Transgenic kiwi plant after potting: evidence of the reversion from "hairy root" to "normal" phenotype. Arrow shows "hairy root" leaf phenotype.
Putatively transformed callus regenerated as many shoots as untransformed callus when transferred to regeneration medium containing 1 mg/l BAP. For each callus, 5 to 10 shoots were regenerated, numbered and micropropagated. The transformed state of the shoots proliferating in the selection medium was tested by
294 analysing NPT II enzyme activity and by Southern blot hybridization. NPT II was positive for all the randomly chosen plantlets (see an example in Fig. 2). The presence of the transferred T-DNA segment in the kiwi clone no. 9 DNA was checked by hybridizing a 32 p _ labelled EcoRI fragment against a Southern blot of EcoRI - digested clone 9 total DNA (Fig. 1 A). The morphology of kanamycin resistant kiwi shoots, compared to normal ones, during the in vitro growth, was characterized by the typical "hairy root" alterations described in various species after transformation with rol A,B,C genes from A. rhizogenes T-DNA. The slower Table 1. Percentage of rooted leaf discs of various transformed kiwi clones on hormone-free m e d i u m after 30 and 60 days and average n u m b e r of roots p e r disc (_+ SE) evaluated at 60 days. Thirty explants
A remarkable variability in regard to the number of roots radiating from the leaf discs was observed only among the transgenic clones. The typical "hairy root" phenotype characterized the transgenic roots. To study the behaviour of transformed plantlets in root production two experiments were conducted by evaluating first the rooting ability in 1/2 MS without hormones, and second the influence of rol genes on the timing of root production in 1/2 MS supplemented with 0.2 mg/l IBA, a growth regulator largely used to stimulate root morphogenesis woody species in vitro propagated. Table 2 shows that after 40 days all the transgenic microcuttings had produced roots (with some variability among the clones) in hormone-free MS medium, while only 36% of the controls showed roots (in general, with a lower number per plant).
p e r each clone were used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table 2. Percentage of rooted microcuttings of randomly chosen
Clone
% of rooted
roots
transformed kiwi clones and average n u m b e r of roots p e r cutting (_+
number
leaf discs
p e r disc
SE) in hormone-free medium at 40 days. Thirty explants per clone were
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
30 days .
CONTROL
.
.
.
.
.
60 days .
.
.
.
.
.
.
.
60 days .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Clone
% of rooted
roots per
number
microcuttings
microcutting
0
33
3.0 _+ 0.8
9
66
66
15.0 -+ 4.3
14
100
100
3.6 -+ 1.0
15
100
100
4.0 _+ 1.2
9
100
6.0 _+ 1.2
16
100
100
3.3 _+ 0.7
14
100
4.0 -+ 0.8
20
100
100
20.0 +_ 5.1
16
100
2.6 _+ 0A
24
83
90
3.6 -+ 1.3
20
100
3.0 _+ 1.4
27
100
100
12.5 -+ 3.2
24
100
2.0 + 0.2
28
100
100
4.3 + 1.8
27
100
4.0 -+ 1.0
33
100
100
7.0 -+ 1.5
28
100
3.0 _+ 1.2
37
100
100
7.5 -+ 0.7
33
100
1.6 -- 0.3
39
100
100
16.0 -+ 4.1
37
100
6.5 -+ 1.8
initial growth, shorter internodes, the dark green wrinkled leaves and the high rooting ability proved that woody plants behaved, during this phase, like herbaceous ones. After potting, transgenic somaclones (10 to 15 cm high) were inclined, in most cases, to lose the aereal "hairy root" characteristics (Fig. 3B). It has been reported for various herbaceous species that leaf discs from plants transformed by T-DNA fragments containing rol B with an upstream regulatory region of about 1000 bp possess the ability to produce profuse rhizogenesis when cultured on hormone-free media, where controls, as a rule, fail (SchmuUing et al. 1988; Spanb and Costantino 1982). Table 1 shows the rooting test in leaf discs of transgenic kiwi plants on hormonefree MS medium (see also Fig.3A). Almost all the discs of the randomly chosen transformed clones produced roots after 4 weeks, while the controls did not show any morphogenetic event. One month later, in only one third of the control discs, a few roots appeared with an average number of roots per disc lower than the transgenic ones.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CONTROL
36
2.0 -+ 0.3
Table 3. Percentage of rooted cuttings of randomly chosen transformed kiwi clones and average n u m b e r of roots per cutting (-+ SE) in MS medium plus 0.2 rag/1 IBA at 15 days. (Thirty explants per each clone were used) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Clone
% of rooted
roots per
number
microcuttings
microcutting
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CONTROL
40
2.2 + 0,2
4
100
3.0 + 0,3
9
100
2.0 + 0.1
14
100
2.0 + 0,8
16
100
2.5 + 0,3
18
100
2.3 +_ 0,4
20
100
2.5 -+ 0.4
24
100
1.0 ___0.2
26
100
2.0 _+ 0.4
28
100
3.5 -+ 1.0
295 In the absence of hormones, rooting starts after about two weeks while I B A supply usually reduces this period. Transgenic plantlets cultured in the presence of IBA behaved as expected with regard to rooting ability; in fact, after only 15 days all of them were rooted but only 40% of the controls showed rooting formation (Table 3). In both cases transgenic roots showed the "hairy root" appearance. Experiments are in progress to evaluate their behaviour in soil.
Discussion The results presented in this paper indicate that: I) it is possible to transform the woody plant Actinidia deliciosa by means of Agrobacterium derived vectors; lI) rol A,B,C genes from A. rhizogenes can be used to enhance the rooting ability of this woody species. This is the first report concerned with the effects of rol genes in trees. Transgenic kiwi plants obtained by transferring rol A, B and C genes show, as described in herbaceous plants, the typical "hairy root" phenotype. However, we observed that after potting several transgenic plants became similar, from a morphological point of view, to the mother plants or those regenerated from untransformed callus. Hairy roots, induced byAgrobacterium rhizogenes wild type are, in general, m o r e branched and lose gravitropic response, while other functions remain unaffected. To date, however, some contradictory findings have been reported about the effect of the various combinations of rol genes on root morphology and tropism; for example, different species transformed by the same T - D N A constructs seem to behave differently (Cardarelli et al. 1987a; Spanb et al. 1987; Schmulling et al. 1988; Capone et al. 1989b). Data from this lab obtained by a large experiment with three categories of transgenic tobaccos ( L D R = w i t h the full TD N A complement; E 15 = with rol A,B,C genes; SHp15 = with O R F 11 (rol B) plus the upstream region of 1000 bp and S R I = control), dearly show that roots radiating from SHp 15 microcuttings are morphologically and gravitropic similar to the SR1 control, while E 15 roots are similar to those of L D R which are m o r e branched and definitively less gravitropic. These results support the idea that the loss of geotropism is due to genes other than rol B, and the preliminary data reported in this paper should lead to more extensive use of the "root inducing" gene rol B. Even if additional work has to be done, this paper first demonstrates that our approach is worthy of further investigation. Moreover, the use of these morphogenetic "bacterial" genes should also help us in a better understanding of root differentiation in trees.
Acknowledgements. This work was supported by the "Piano Nazionale di rieerca: Sviluppo di tecnologie avanzate applicate alle piante" of the Italian Ministry of Agriculture and Forestry.
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