plant Cell
Plant Cell Reports (1986) 3:212-214
Reports © Springer-Verlag1986
Somatic hybrids between Solarium brevidens and Solarium tuberosum: Expression of a late blight resistance gene and potato leaf roll resistance J. P. Helgeson 1, G.J. Hunt 2, Geraldine T. Haberlach 1, and Sandra Austin 2 1 United States Department of Agriculture, University of Wisconsin, 1630 Linden Drive, Madison, WI 53706, USA 2 Department of Plant Pathology, ARS, University of Wisconsin, 1630 Linden Drive, Madison, WI 53706, USA Received December 31, 1985 / Revised version received February 22, 1986 - Communicated by J. M. Widholm
ABSTRACT
Hexaploid somatic hybrids resulting from mesophyll protoplast fusions between Solanum brevidens Phil., PI 218228, and Solanum tuherosum L., PI 203900 were tested for late blight resistance using two races of Phytophthora infestans Monte., de Bary. The S. tuherosum parent was a "late blight differential" possessing the R4 gene which confers resistance to race 0. The S. hrevidens parent is resistant to potato leaf roll virus. Inoculations with both compatible (race 1.3.4.5) and incompatible (race 0) races of P. infestans clearly demonstrated the expression of the late blight resistance gene in all of the hybrid progeny tested. Most of the hybrids tested were also resistant to potato leaf roll virus (PLRV), indicating that the S. brevidens genes for PLRV resistance were present and expressed. INTRODUCTION
Wild Solanum species have been important as sources of useful characteristics in the development of potato cultivars. Some potentially useful species, however, are sexually incompatible or cross with difficulty with Solanum tuberosum L. For example, Hermsen and Taylor (1979) pollinated 140 flowers of S. brevidens with pollen from a 2x (dihaplold) S. tuberosum cultivar and 89 flowers of S. tuberosum with S. brevidens pollen but obtained no seeds in either case. More success can be obtained by use of bridging species (e.g., S. plnnatisectum Dun. and S. fez-nandezianum Phil.) but, again, the introduction of the germplasm into S. tuberosum proves difficult, although not impossible (Hermsen 1983, Ehlenfeldt and Hanneman 1984). Somatic hybridization is one means by which genes of sexually incompatible plants may be combined directly with S. tuberosum (e.g., Melehers et al. 1978, Binding et al. 1982, Barsby et al. 1984, Austin et al. 1985). Fusion of protoplasts from wild species with those of S. tuberosum could result in the transfer of disease and pest resistances into potato. A previous study reported the incorporation of resistance to potato leaf roll virus ( P L R V ) f r o m S. brevidens into tetraploid somatic hybrids with a diploid S. tuberosum llne selected from a gp. Phureja-Stenotomum population (Austin et al. 1985). Protoplasts from a tetraploid S. tuberosum late blight differential line have also been fused with those of another accession of S. brevidens. The production and phenotypic characteristics of these
Offprint requests to: J. P. Hclgeson, Attn. S. Austin
hybrids have been described (Austin et al. 1986; in press). In the present study, these hybrids were tested for late blight resistance (derived from S. tuberosum PI 203900) and PLRV resistance (derived from S. hrevidens PI 218228). MATERIALS AND METHODS
Plants of the parental S. tuberosum PI 203900 and S. hrevidens PI 218228 clones, as well as multiple clonal copies of the somatic hybrids were maintained in sterile culture on modified MS medium (Prop) as given earlier (Haberlach et al. 1985). Clonal S. tuberosum cv. Russet Burbank plants were also grown in identical fashion for use as a reference standard. Six weeks prior to inoculation with Phytophthora infestans Monte., de Bary, plants were transferred to Jiffy-7 peat pellets I (Jiffy Products Ltd., Shippegan, Canada) and maintained in a growth chamber (24=C, 12 h photoperiod). Most plants were 20-30 cm tall when transferred to 6.3 cm dia plastic pots 10-12 days prior to inoculation. They were watered with Hoagland's solution as needed throughout the growth period. Hybrids, parental plants and Russet Burbank were arranged in two completely randomized groups and inoculated using a procedure based on that of Maleomson (1976) as modified by Stewart et al. (1983). All of the plants were sprayed, with the aid of a glass atomizer attached to an air line, to run-off with a suspension of zoospores and sporangia of P. infestans in sterile distilled water. One group was sprayed with 24,000 zoospores plus sporangia/ml of race 0 of P. infestans (incompatible with S. tuberosum PI 203900); the other group was inoculated with 30,000 zoospores plus sporangia/ml of race 1.3.4.5 (compatible with all plants tested). Immediately after inoculation, plants were placed in dew chambers for 24 h, in darkness at 20=C. Plants were then transferred to growth chambers maintained at 16°C, with 12 h days for symptom development. At least eight clonal copies of each somatic hybrid, parents, and Russet burbank were tested in each experiment.
I Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.
213 Five days after inoculation each plant was scored visually for percent necrotic tissue as illustrated by Cruickshank et al. (1982). Plants on which all the leaves were dead but having no stem lesions were rated as 70% necrotic. Plants were then returned to the dew chambers to promote sporulation and spread of lesions. Plants were examined as a group 48 h later for lesion spread and sporulation. Fusion hybrids and parents were tested for PLRV resistance as previously described (Austin et a l . 1985) following standard techniques (Casper 1977, Clark and Adams 1977). Some of the hybrids were clonal copies of those used for late blight tests. Five viruliferous aphids were confined on each plant for 5-6 days and feeding was observed. Plants were assayed for the presence of PLRV in leaf tissue by the ~ z y m e linked immunosorbent assay (ELISA) method using a PLRV test kit (Agdia, Inc., Mishiwaka, IN 46544) and following the instructions given. A known positive was included in each assay. Preliminary screening showed that uninoculated hybrids and parental lines gave uniform background readings (0.01-0.07) as would be expected. RESULTS AND DICSUSSION
In the late blight test, the appearances of leaves from susceptible and resistant plants were markedly different. All the fusion progeny inoculated with race 0 of P. infestans were significantly less necrotic (p
Plant Cell Reports (1986) 3:212-214
Reports © Springer-Verlag1986
Somatic hybrids between Solarium brevidens and Solarium tuberosum: Expression of a late blight resistance gene and potato leaf roll resistance J. P. Helgeson 1, G.J. Hunt 2, Geraldine T. Haberlach 1, and Sandra Austin 2 1 United States Department of Agriculture, University of Wisconsin, 1630 Linden Drive, Madison, WI 53706, USA 2 Department of Plant Pathology, ARS, University of Wisconsin, 1630 Linden Drive, Madison, WI 53706, USA Received December 31, 1985 / Revised version received February 22, 1986 - Communicated by J. M. Widholm
ABSTRACT
Hexaploid somatic hybrids resulting from mesophyll protoplast fusions between Solanum brevidens Phil., PI 218228, and Solanum tuherosum L., PI 203900 were tested for late blight resistance using two races of Phytophthora infestans Monte., de Bary. The S. tuherosum parent was a "late blight differential" possessing the R4 gene which confers resistance to race 0. The S. hrevidens parent is resistant to potato leaf roll virus. Inoculations with both compatible (race 1.3.4.5) and incompatible (race 0) races of P. infestans clearly demonstrated the expression of the late blight resistance gene in all of the hybrid progeny tested. Most of the hybrids tested were also resistant to potato leaf roll virus (PLRV), indicating that the S. brevidens genes for PLRV resistance were present and expressed. INTRODUCTION
Wild Solanum species have been important as sources of useful characteristics in the development of potato cultivars. Some potentially useful species, however, are sexually incompatible or cross with difficulty with Solanum tuberosum L. For example, Hermsen and Taylor (1979) pollinated 140 flowers of S. brevidens with pollen from a 2x (dihaplold) S. tuberosum cultivar and 89 flowers of S. tuberosum with S. brevidens pollen but obtained no seeds in either case. More success can be obtained by use of bridging species (e.g., S. plnnatisectum Dun. and S. fez-nandezianum Phil.) but, again, the introduction of the germplasm into S. tuberosum proves difficult, although not impossible (Hermsen 1983, Ehlenfeldt and Hanneman 1984). Somatic hybridization is one means by which genes of sexually incompatible plants may be combined directly with S. tuberosum (e.g., Melehers et al. 1978, Binding et al. 1982, Barsby et al. 1984, Austin et al. 1985). Fusion of protoplasts from wild species with those of S. tuberosum could result in the transfer of disease and pest resistances into potato. A previous study reported the incorporation of resistance to potato leaf roll virus ( P L R V ) f r o m S. brevidens into tetraploid somatic hybrids with a diploid S. tuberosum llne selected from a gp. Phureja-Stenotomum population (Austin et al. 1985). Protoplasts from a tetraploid S. tuberosum late blight differential line have also been fused with those of another accession of S. brevidens. The production and phenotypic characteristics of these
Offprint requests to: J. P. Hclgeson, Attn. S. Austin
hybrids have been described (Austin et al. 1986; in press). In the present study, these hybrids were tested for late blight resistance (derived from S. tuberosum PI 203900) and PLRV resistance (derived from S. hrevidens PI 218228). MATERIALS AND METHODS
Plants of the parental S. tuberosum PI 203900 and S. hrevidens PI 218228 clones, as well as multiple clonal copies of the somatic hybrids were maintained in sterile culture on modified MS medium (Prop) as given earlier (Haberlach et al. 1985). Clonal S. tuberosum cv. Russet Burbank plants were also grown in identical fashion for use as a reference standard. Six weeks prior to inoculation with Phytophthora infestans Monte., de Bary, plants were transferred to Jiffy-7 peat pellets I (Jiffy Products Ltd., Shippegan, Canada) and maintained in a growth chamber (24=C, 12 h photoperiod). Most plants were 20-30 cm tall when transferred to 6.3 cm dia plastic pots 10-12 days prior to inoculation. They were watered with Hoagland's solution as needed throughout the growth period. Hybrids, parental plants and Russet Burbank were arranged in two completely randomized groups and inoculated using a procedure based on that of Maleomson (1976) as modified by Stewart et al. (1983). All of the plants were sprayed, with the aid of a glass atomizer attached to an air line, to run-off with a suspension of zoospores and sporangia of P. infestans in sterile distilled water. One group was sprayed with 24,000 zoospores plus sporangia/ml of race 0 of P. infestans (incompatible with S. tuberosum PI 203900); the other group was inoculated with 30,000 zoospores plus sporangia/ml of race 1.3.4.5 (compatible with all plants tested). Immediately after inoculation, plants were placed in dew chambers for 24 h, in darkness at 20=C. Plants were then transferred to growth chambers maintained at 16°C, with 12 h days for symptom development. At least eight clonal copies of each somatic hybrid, parents, and Russet burbank were tested in each experiment.
I Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.
213 Five days after inoculation each plant was scored visually for percent necrotic tissue as illustrated by Cruickshank et al. (1982). Plants on which all the leaves were dead but having no stem lesions were rated as 70% necrotic. Plants were then returned to the dew chambers to promote sporulation and spread of lesions. Plants were examined as a group 48 h later for lesion spread and sporulation. Fusion hybrids and parents were tested for PLRV resistance as previously described (Austin et a l . 1985) following standard techniques (Casper 1977, Clark and Adams 1977). Some of the hybrids were clonal copies of those used for late blight tests. Five viruliferous aphids were confined on each plant for 5-6 days and feeding was observed. Plants were assayed for the presence of PLRV in leaf tissue by the ~ z y m e linked immunosorbent assay (ELISA) method using a PLRV test kit (Agdia, Inc., Mishiwaka, IN 46544) and following the instructions given. A known positive was included in each assay. Preliminary screening showed that uninoculated hybrids and parental lines gave uniform background readings (0.01-0.07) as would be expected. RESULTS AND DICSUSSION
In the late blight test, the appearances of leaves from susceptible and resistant plants were markedly different. All the fusion progeny inoculated with race 0 of P. infestans were significantly less necrotic (p
