Plant tissue culture is recognized as an important tool to generate useful genetic variability for crop improvement. Regenerated plants from ... the utilization of somaclonal variation made it possi- ..... Therefore, a possible approach is to use.
Euphytica 80:5-11, 1994. © 1994KluwerAcademicPublishers. Printedin the Netherlands.
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S o m a c l o n a l v a r i a t i o n for r e s i s t a n c e to Verticillium dahliae in p o t a t o (Solanum tuberosum L.) p l a n t s r e g e n e r a t e d f r o m callus L. Sebastiani 1, A. Lenzi 2, C. Pugliesi 2 & M. Fambrini 2 1 Scuola Superiore di Studi Universitari e di Perfezionamento S. Anna, Via Carducci 40, 56124 Pisa, Italy; 2 Dipartimento di Biologia delle Piante Agrarie Sezione di Genetica, Universitft di Pisa, Via Matteotti l/B, 56124 Pisa, Italy
Received20 January 1994;accepted25 June 1994 Key words: disease resistance, filtrate selection, Solanum tuberosum (cvs D6sir6e, Kondor), somaclonal variation, Verticillium dahliae
Summary Plant tissue culture is recognized as an important tool to generate useful genetic variability for crop improvement. Regenerated plants from callus induced from stem explants of Solanum tuberosum cv D6sir6e were assessed by in vitro selection, for resistance to Verticillium dahliae. This fungus is the causal agent of Verticillium wilt, a serious vascular wilt disease both in crops and wild species. The rate of in vitro multiplication by single node cuttings was used as a parameter of screening in two selection cycles with different concentrations of V. dahliae filtrate. One resistant clone was selected and then evaluated by inoculation in the growth chamber. Induced damage, and morphological traits (dry weight, leaf area and tuber production) were estimated. The selected clone was comparable to the resistant control, cv Kondor. The results suggest that genetic variation induced in tissue culture could be utilized to generate disease resistance.
Introduction Verticillium wilt is caused by two soilborne fungi, Verticillium dahliae Kleb and V. albo-atrum Renke and Berthold (Rowe et al., 1987). This disease is a limiting factor for production of all the commonly grown varieties of potato especially in dry climates (Susnoschi et al., 1976). It effects the quality of potato tubers, causing browning of cortical and parenchymal cells and cellular collapse around the xylem. V. dahliae invades the vascular elements of host plant mainly through wounds in roots, induces collapse of vessels and fills them with mycelia and gums leading to plant death (Isaac & Harrison, 1968). Although it has no specific hosts, V. dahliae infects a wide range of crops (tomato, alfalfa, strawberry, eggplant, watermelon), different genera (Chenopodium, Taraxacum, Papaver, Capsella), and even many species of trees (Rich, 1983). The genetics of resistance is unclear but polygenic control in eggplant, potato, alfalfa and Capsicum is reported (Alconero et al., 1988; Goth & Webb, 1981;
Latunde-Dada & Lucas, 1983; Palloix et al., 1990; Martin et al., 1993), while in tomato, resistance is controlled by a single dominant gene (Schaible et al., 1951). The genus Solanum is characterized by broad genetic variability and resistance to V. dahliae exists within the wild species S. chacoense, S. sparsipilliurn, S. microdontum and S. torvum. That resistance could be transferred to S. tuberosum but for the necessity to preserve allelic and non allelic genetic interaction in cultivated varieties and the occurrence of sexual incompatibility that hinders the interspecific hybridization. New genetic variability induced by in vitro tissue culture has been reported for many crops (for review see, e.g., Scowcroft et al., 1987; Karp, 1991). A wide range of plant characteristics can be altered as a result of regeneration from cell and tissue culture, including agronomically important traits such as disease resistance (Van den Bulk, 1991). Particularly in potato, the utilization of somaclonal variation made it possi-
ble to obtain resistance to several pathogens such as Phytophthora infestans (Matern et al., 1978; Behnke, 1979, 1980a), Alternaria solani (Shepard et al., 1980), Fusarium oxysporum (Behnke, 1980b), Streptomyces, PLRV, PVY (Thomson et al., 1986) and PVX (Wenzel et al., 1987). Resistance to Verticillium wilt induced by in vitro tissue culture has been attained in Medicago sativa (Latunde Dada & Lucas, 1983, 1987, 1988; Frame et al., 1991), while in potato breeding, in vitro techniques have been exploited to transfer this trait from Solanum torvum S.W. into Solanum tuberosum by protoplast electrofusion (Jadari et al., 1992). The purpose of the present work was to screen regenerated plants from callus culture of potato stem explants of cv D6sir6e, for resistance to V. dahliae. Disease symptoms and morphological data after inoculation of a selected resistant clone were determined.
Callus cultures and plant regeneration Stem explants of cv D6sir6e (0.5 cm long) from multiplied plantlets were placed in Petri dishes (100 x 15 mm) on callus induction medium composed of MS basal medium supplemented with 2.25 mg/1 6benzylamino purine (BAP) and 0.186 mg/1 NAA (Webb et al., 1983). Cultures were incubated in growth chamber in the same conditions previously described. After 30 days the induced calli were transferred to the regeneration medium (MS basal medium plus 10 mg/1 gibberelic acid (GA3) and 2.25 mg/l BAP) (Webb et al., 1983) and subcultured on fresh medium every three or four weeks. The developed shoots (2 cm long) were excised and placed on solidified (0.8% Bactoagar) MS basal medium without growth regulators for rooting. Verticillium dahliae filtrate
Materials and methods
Plant materials Potato tubers (Solanum tuberosum L.) cv D6sir6e (tolerant to V. dahliae; Susnoschi et al., 1976) and the control cultivar Kondor (resistant to V. dahliae; Buchner et al., 1989), supplied by Department of Agricultural Plant Biology, Horticultural and Floriculture Section, University of Pisa, were maintained with dark condition at 25 + 1o C in growth chamber until sprouting.
Conidial suspensions of V. dahliae, strain PAl (supplied by Department of Plant Pathology, University of Bologna) were added to 250 ml of liquid medium according to Nachmias et al. (1985) in 1,000 ml Erlenmeyer flasks. These were placed on a rotary shaker (80 rpm) at 25 + 1° C in the dark for three weeks. Mycelia and conidia were removed by filtration through Whatman N ° 1 filter paper and following centrifugation for 10 min at 3,000 rpm (Durrands & Cooper, 1988). Fungal filtrate pH were adjusted to 5.7 with 1 M KOH before filter sterilization through Millipore membrane (0.22 pm).
In vitro multiplication In vitro selection Shoots (5 cm long) were cut from tubers and the wounds covered with liquid paraffin. Successively they were surface sterilized for 10 min in 10% ACE (sodium hypochlorite) and then washed three times for 10 rain with sterilized distilled water. After sterilization they were aseptically placed on 20 ml solidified (0.8% Bactoagar) medium in 150 ml Erlenmeyer flasks and incubated in growth chamber at 25 + 1° C under a 16/8 h day/night regime (60 #E/m2/sec fluorescent light). Cultural medium was composed of MS basal medium (Murashige & Skoog, 1962) containing 0.5 mg/1 of a-naphthaleneacetic acid (NAA) and 0.5 rag/1 Zeatin riboside (ZR) (modified from Hu & Wang, 1983). Rooted shoots were then multiplied by single node cuttings on MS medium without growth regulators (Hussey & Stacey, 1981).
Nodes from cvs D6sir6e and Kondor were grown for two weeks on 20 ml MS liquid medium ammended with three concentrations (12.5%, 25% and 50%) of V. dahliae filtrate, in the same conditions described in 'In vitro multiplication'. Each treatment was replicated 50 times and all experiments were repeated at least three times. Vitality was calculated as percentage of well grown shoots obtained from the total nodal explants cultured. Discrimination between resistant (Kondor) and tolerant (D6sir6e) cultivars was possible at 12.5% and 25% fungal filtrate concentration (Table 1). Nodal explants from regenerated shoots of cv D6sir6e were selected (first selection cycle) in 25% fungal filtrate concentration. Developed, non deformed shoots were propagated through the system described in 'In vitro multiplication' and successively submitted
Table 1. Effect of different VerticiUium dahliae filtrate concentrations on in vitro multiplication by single node cuttings expressed as percentageof obtainedviable shoots
Cultivar
V dahliae filtrate concentration 0% 12.5% 25% 50% Percentage of viable shoots1
DEsir6e Kondor
90.1 a 93.3 a
26.6 a 80.1 b
16.5 a 34.1 b
2.5 a 7.5 a
I Means values within a vertical column followed by the same letter were not significantlydifferent (p = 0.05) using a X2-test of independence.
150
STEM EXPLANTS (cvD~sir~) I
In Vitro culture (60days)(Webb etal.,1983)
32$ REGENERATEDSHOOTS
325 NODALEXPLANTS Selection (14 days) on 25% V. dah//ae filtrate (FIRSTSELECTIONCYCLE) 1J SURVIVEDSHOOTS
weeks. Resulting suspension was adjusted to 106 conidia/ml by count with haemocytometer and dilution. Plantlets of cvs Ddsir6e and Kondor and of the selected clone R3-90, obtained by single node cutting propagation, were inoculated as described for Medicago sativa (Latunde-Dada & Lucas, 1988). Roots, gently cut and washed with sterilized water, were soaked for 20 min in conidial suspensions. Roots of control plants were cut and washed as well, and then soaked for 20 min in sterilized water. All inoculated plantlets were transferred to pots containing a 2:1:1 (v/v) mixture of sterilized peat, sand and loam and grown in controlled conditions at 25 41° C, 60% relative humidity, under a 16/8 h day/night regime (180/zE/m2/sec fluorescent light). After four weeks plants were analyzed for disease severity. The external symptoms o f Verticillium wilt were assessed using a subjective scoring system previously described in the aifalfa/Verticillium interaction (Latunde-Dada & Lucas, 1988). Average dry weight, tuber production and leaf area measured with a DT area meter MK2 (Delta-T Devices), were determined in control and infected plants of all genotypes. The treatment was replicated 30 times and repeated three or four times. Morphological characters o f 20 random non infected plants from three replicates of the selected resistant clone and cv Ddsir6e, were also analyzed.
Singlenode cuttings(Hussey & Stacey,1981) Statistical analyses
15 CLONES Selection (14 days) on 50% V. dah//ae filtrate (SECONDSI::/FL-'TIONCYCLE) R3-90 RESISTANTCLONE Fig. 1. Origin of R3-90 clone.
to a second selection cycle in 50% filtrate concentration (Fig. 1). Selected clone R3-90 was multiplied in vitro by single node cuttings and infected in vivo. Its behaviour was compared with that o f c v s Ddsir6e and Kondor. Inoculation
Inoculation cultures were prepared by washing V. dahliae mycelium grown on PDA medium (Potato Dextrose Agar, Difco) at 25 -4- 1o C, in the dark for two
The Chi-squared test for independence was used to determine significant differences (p = 0.05) among the responses of single node cuttings to V. dahliae filtrate. Statistical significance of differences in morphological traits of inoculated plants was examined by analysis of variance and the means separated using Fisher's Least Significant Differences (LSD) test at p = 0.01.
Results Influence o f V. dahliae filtrate on in vitro multiplication
Data in Table 1 show the effect of different V. dahliae filtrate concentrations on in vitro multiplication by single node cuttings in resistant (Kondor) and tolerant (Ddsir6e) cultivars. The response was evaluated as percentage of viable shoots of the total cultured nodes. This technique, that enables an early in vitro selec-
Table 2. Effect of Verticillium dahliae filtrate (25%) on in vitro multiplicationby single node cuttingsof different genotypes
Genotype
Control V. dahliae filtrate Percentage of viable shoots1
Dtsirte R3-90 Kondor
86.6 a 92.3 ab 99.8 b
14.2 a 35.1 b 32.5 b
Table 3. Effect of V.dahliae inoculationon dry weight (g) of in vitro multipliedplantlets
Genotype
Mean dry weight(g) Reduction ControlI Infected respect plantsI to control(%)
Dtsirte R3-90 Kondor
77.1 a 69.2 a 73.5 a
38.5 b 67.1 a 60.5 a
50.1 3.2 17.7
I Means values within a verticalcolunmfollowed by the same letter(s) were not significantlydifferent (p = 0.05) using a x2-test of independence.
1 Valueswere comparedby analysisof variance and the means within a row separated by LSD test at 1% level. Valuesfollowedby the same letter were not significantlydifferent.
tion, has already been utilized in potato/Phytophthora infestans interaction (Crin6 et al., 1989). The discrimination between tolerant and resistant genotypes was possible at 12.5% and 25% while the highest concentration (50%) was too drastic.
Table 4. Effect of V. dahliae inoculation on leaf area (mm2) of in vitro multipliedplantlets
Selection o f a somaclonal variant
Nodal explants from 325 regenerated plantlets (cv D t s i r t e ) were submitted to in vitro selection with fungal filtrate (Fig. 1). After a first selection cycle (25% fungal filtrate in cultural medium) the most vigorous material (15 plantlets) were micropropagated by single node cuttings. Successively the 15 clones were transferred to higher filtrate concentration (50%) and R3-90 clone was isolated (Fig. 1). R3-90 resistance was first assessed comparing its sensibility to culture filtrate with that of resistant and tolerant cultivars. Data in Table 2 indicate that the selected clone behaves as the resistant cultivar Kondor.
Genotype
Mean leaf area (mm2) Control I Infected plantsI
Reduction respect to control (%)
Dtsirte R3-90 Kondor
1737 a 1633 a 2035 a
50.4 13.3 15.3
861 b 1415 a 1724a
t Valueswere compared by analysisof variance and the means withina row separatedby LSD test at 1% level. Valuesfollowedby the same letter were not significantlydifferent.
Morphological analysis of in vivo growing plants shows no difference in phenotypical characters between R3-90 clone and the parental cultivar D6sirte (Table 6).
Confirmation o f resistance
Discussion
Resistance was then confirmed by the analysis of dry weight and leaf area reduction observed after inoculation (Tables 3 and 4). Both characters showed a reduction in R3-90 clone similar to that of the resistant cultivar. Similarly for tuber production, a negligible decrease was observed in infected plants both of R3-90 clone and cv Kondor while cv D t s i r t e tuber production lowered to 50% (Table 5). Foliar symptoms assessed with a subjective scoring system, revealed that damage in infected R3-90 clone was three times smaller than in cv D t s i r t e (Fig. 2).
The results of the present investigation suggest the possibility to obtain plants resistant to fungal diseases from callus cultures of potato stem explants. In the last several years genetic variability induced by in vitro tissue culture, termed somaclonal variation (Larkin & Scowcroft, 1981), has been exploited in breeding many crops. In vegetatively propagated plants such genetic modifications can be directly incorporated into new varieties. The source of somaclonal variation is not completely known but it is thought to be due to a c o m -
9
o
o
m 2
t~ o ,--4 o 4J o
1
r~
0 K. control
K. infected
D. control
D. infected
R3-90 control R3-90 infected
Fig. 2. Symptomatology mean score of inoculated potato plants of tolerant (D6sir6e: D.) and resistant (Kondor: K.) cultivars and of the selected clone R3-90, in comparison with respective controls. Bars represent the standard error of the mean.
Table 5. Effect of V. dahliae inoculation on tuber production (g) in cvs D6sir6e and Kondor and in R3-90 resistant clone
Genotype
D6sir6e R3-90 Kondor
Table 6. Observed traits for in vivo morphological comparison between R3-90 clone and parental cultivar D6sirde
Mean tuber production (g)
Reduction
Traits
Control I
Infected plants 1
respect to control (%)
Leaf colour I
253.4 a 237.8 a 417.2 a
130.1 b 234.5 a 405.4 a
48.7 1.4 2.0
1 Values were compared by analysis of variance and the means within a row separated by LSD test at 1% level. Values followed by the same letter were not significantly different.
bination of factors. One of these surely consists of preexisting mutations in the cells of the explants material ( D ' A m a t o , 1985). N e v e r t h e l e s s n u c l e a r i r r e g u l a r i t i e s o c c u r r i n g in t i s s u e - c u l t u r e c e l l s a r e k n o w n to f r e q u e n t l y c a u s e p h e n o t y p i c a b n o r m a l i t i e s in r e g e n e r a t e d p l a n t s ( K a r p , 1991).
Variegation Leaf lamina fusion 6 th
leaf length
Main stem length Stem number per plant Vigour2 Anthocyanins Tuber production per plant (g) Tuber number per plant Tuber average weight (g)
R3-90
D6sir6e
2
2
0 0 15.2 (0.53) 27.9 (1.18) 4.4 (0.30) 1.8 (0.11) yes 227.7 (11.7) 6.9 (0.40) 33.0 (2.60)
0 0 15.7 24.0 2.8 1.6 yes 234.9 8.8 26.7
(0.57) (0.99) (0.26) (0.11) (10.0) (0.60) (2.20)
1 Pale green: 1; normal: 2; dark green: 3. 2 1-4 (weak-greatly vigorous). Numbers in brackets are standard errors of the means of three experiments.
Genetic background of plants can affect the amount o f v a r i a b i l i t y t h a t o c c u r s ( L o r z , 1984). I n t h e i r s t u d i e s
10 on disease resistance in tobacco somaclones, Daub & Jenns (1989) concluded that the observed variability depended on the disease as well as on the genotype of the parents. Therefore, a possible approach is to use source material that already shows some resistance as was suggested by Daub (1986). In fact, Wright & Lacy (1988) observed a much higher frequency of highly resistant plants to F u s a r i u m in a population of celery regenerants obtained from a moderately resistant cultivar than in a population derived from highly susceptible source material. In our study plants were regenerated from callus cultures induced from stem explants of a cultivar tolerant to V. dahliae (D6sir6e). Selection of disease resistant variants at the plant level has been reported for many crops (Van den Bulk, 1991). This approach is necessary when the pathogenetic mechanism is not clear or fungal disease is expressed only in organized tissues (Daub, 1986). V. dahliae toxin does not diffuse in potato tissues and cytoimmunofluorescence studies in tubers and stems infected by the fungus demonstrated that the toxin was localized on the walls of the xylem vessels (Nachmias et al., 1985; Buchner et al., 1989). A recent report on the reaction of potato tissue cultures to V. dahliae Vd toxin asserted that calli were unaffected, except for the cells in direct contact with the toxic medium (Nachmias et al., 1990). That can be explained by the lack of xilem bundles in callus cultures which hinder the exposure of the inner cells to the toxin. As reported by Daub (1986) frequencies of somaclonal variants selected at the plant level for a particular trait are estimated to range from 0.2 to almost 3%. In our experiment among 325 regenerated plants we have isolated one variant not sensitive to fungal filtrate (Fig. 1 and Table 2) and resistant to inoculation (Fig. 2; Tables 3, 4 and 5). A major problem associated with the evaluation of somaclonal variation for disease resistance is the availability of efficient, reliable screening methods. Verticillium culture filtrate contains toxic metabolites (Buchner et al., 1982; Nachmias et al., 1982, 1985, 1987) which may be useful to select Verticillium-resistant plants (Nachmias et al., 1990). Our results demonstrate that the fungal filtrate assayed on cultured single node explants enables a discrimination between tolerant (D6sir6e) and resistant (Kondor) cultivars (Table 1). Analogous results have been obtained with this selection method in potato/Phytophthora infestans interaction (Crin6 et al., 1989). Verticillium wilt causes lower yields; the host plants are stunted with shorter internodes, leaf growth reduc-
tion and lack of root branching (Isaac & Harrison, 1968; Harrison & Isaac, 1968; Susnoshi et al., 1976). Data in Tables 3, 4 and 5 attest that fungal infection induces a reduction of plant dry weight, leaf area and tuber production in the tolerant cultivar (D6sir6e). The results obtained in infected plants of R3-90 clone demonstrate its resistant behaviour (Tables 3, 4 and 5). In some cases plants selected for disease resistance show changes in other, mostly unwanted traits as well (Shahin & Spivey, 1987). In our case morphological analysis indicates the lack of variability in R3-90 clone whose plants are characterized by a parental phenotype (Table 6).
Acknowledgements The authors are greatly indebted to Prof. G. Vannacci for his advice and to Mr M. Rocca for very efficient technical assistance.
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