Plant Cell Reports
Plant Cell Reports (1992) 10:629-632
9 Springer-Verlag 1992
Molecular analysis of the nuclear organellar genotype of somatic hybrid plants between tomato (Lycopersicon esculentum) and Lycopersicon chilense Augusta B. Bonnema 1 and Mary A. O'Connell 2 1 Department of Agronomy and Horticulture, Plant Genetic Engineering Lab, New Mexico State University, Las Cruces, N M 88003, U S A 2 Present address: INRA, Centre de Recherches de Toulouse, L B M R P M , BP27, 31326 Castanet Tolosan, France Received August 21, 1991/Revised version received November 1, 1991
Summary. Somatic hybrid plants were recovered following fusion of leaf mesophyll protoplasts isolated from tomato (Lycopersicon esculentum) cultivar UC82 with protoplasts isolated from suspension cultured cells of L. chilense, L A 1959. Iodoacetate was used to select against the growth of unfused tomato protoplasts. Two somatic hybrids were recovered in a population of 16 regenerants. No tomato regenerants were recovered; all of the non-hybrid regenerants were L. chilense. The L. chilense protoplast regenerants were tetraploid. The hybrid nature of the plants was verified using species-specifc restriction fragment length polymorphisms for the nuclear, chloroplast and mitochondrial genomes. The somatic hybrids had inherited the chloroplast DNA of the tomato parent, and portions of the mitochondrial DNA of the L. chilense parent. The somatic hybrids formed flowers and developed seedless fruit.
Communicated by G. C. Phillips
products between tomato and L. chilense. L. chilense, a self-incompatible species, inhabits one of the most arid of the world's temperate deserts (Rick 1973). The adaptation ofL. chilense to these deserts depends on the foraging ability of its root system. This member of the Lycopersicon genus is most similar to L. peruvianurn (Rick 1979). While there are several reports on protoplast culture and fusion products for tomato-L, peruvianum (Kinsara et al. 1986; San et al. 1990; Wijbrandi et al. 1990), no reports on protoplast culture or fusion with L. chilense have been described. In order to determine if the protoplast fusion products were somatic hybrids, we used restriction fragment length polymorphisms (RFLPs) for five mapped nuclear loci (Mutschler et al. 1987; Zamir and Tanksley 1988). Materials and Methods
Key words: Protoplast fusion - RFLP - Mitochondrial DNA - Chloroplast DNA
Introduction
The application of protoplast fusion techniques to create novel plant genotypes has been in use for over 15 years and, in recent years, examples of the production of improved crop species using these techniques have been reported (Austin et al. 1985; Morgan and Maliga 1987; Kemble et aL 1988; Kyozuka et aL 1989; Sjodin and Glimelius 1989). We have applied protoplast fusion techniques to members of the related genera Lycopersicon and Solanum in attempts to understandnuclear-organellarinteractions, to construct useful novel genotypes, and to optimize methods for partial genome transfer (O'Connell and Hanson 1986; 1987; Bonnema et aL 1991; Melzer and O'Connell 1990; 1991). In this report we describe the construction and characterization of protoplast fusion Offprint requests to: M. A. O'Connell
Plant material. Seed of Lu chilense Dun., LA 1959, were obtained from Charles Rick, Tomato Genetics Stock Center, University of California, Davis; seed of L. esculentum Mill., cv. UC82, were provided by PetoSeed, Co. Suspension cell cultures of L. chilense were generated from hypocotyls of aseptically germinated seedlings. Callus was initiated on UM1A (O'Connell and Hanson 1985), and a suspension culture was subsequently established in the same medium. The suspension culture used for the isolation of protoplasts for fusions was 6-8 months old. UC82 plants to be used for the isolation of leaf mesophyll protoplasts were germinated in soil mix in a growth chamber, 16 h photoperiod, 25~ day, 18~ night, watered daily with a modified Nitsch salt solution (O'Connell and Hanson 1985). Plants were between 4 and 6 weeks old when leaves were collected for protoplast isolation. The plants received a cold pretreatment as described (Bonnema et al. 1991). Protoplast isolation~ fusion and culture. Protoplasts were isolated as described by Bonnema et al. (1991). The leaf mesophyll protoplasts were treated with 3 mM iodoacetate for 20 rain at 50C in W5 solution. The protoplasts were fused in a 1:1 ratio, final concentration of 6 x 10~/ml also as described by Bonnema et al. (1991). After fusion the protoplasts were plated at 2 x 1 ~ / m l in TraP. Approximately a 15% fusion frequency was observed based on doubly fluorescent ceils, red autofluorescence of chlorophyll in UC82 protoplasts and yellow-green fluorescence of fluorosceneisothiocyanate stained L. chilense protoplasts. The
630
Fig. 1. Comparisonof the leaf shape of several regenerants, 3D, 9A, 12A, 14, 42A, and 76, with the fusion parents,L. esculentum cv. UC82 (E), ..I~. chilense LA1959 (C).
protoplasts, microcalli and calli were cultured as descibed by Bonnema et al. (1991). Shoots were regenerated using these media: JSC-12 for greening of calli, TR-1 for shoot induction, and subsequently rooted on MS-0 supplemented with 50 t~M indole3-butyric acid (O'Connell and Hanson 1985). DNA isolation~ restriction digestion~and southern hybridization. Total DNA was isolated from leaf tissue of regenerated plants and the parental species as descibed by Doyle and Doyle(1989), with the addition of a CsC1 centrifugation step after the isopropanol precipitation. The DNA (10 ~ g) was digestedwith the indicated restriction endonucleases, electrophoresed on 0.8% agarose gels, and transferred to nylon membranes using standard procedures. Radiolabelled probes were generated usinga 3~P-dCrP and either random primer or'nick translation procedures. For hybridization with probes to detect nuclear RFLPs, hybridizationand washing of the blots was as described by Melzer and O'Connell (1991). For hybridization with probes to detect organellar RFLPs, hybridization and washing of the blots was as described by Bonnema et al. (1991). Results and Discussion
Recovery of regenerated plants Approximately six weeks following protoplast fusion, 100 microcalli were transferred from liquid protoplast culture medium, TmD, onto solid greening medium, JSC-12. Within a week these calli turned green, and after increasing in size they were transferred to a shoot inducing medium, TR-1. Most of the calli regenerated multiple shoots. The major difficulty encountered in recovering plants from this fusion construction was to induce roots. When shoots were transferred to MS-0 or to MS-0 plus charcoal, no roots formed and the shoots degenerated. However, when shoots were transferred to MS-0 plus 50 # M indole-3-butyric acid, roots were induced on most, approx. 75%, of the shoots within 10 days. Over 60 plants were established in soil, and of these, 16 were characterized extensively using molecular analyses. These 16 individuals were each from a unique callus and represented a regenerant from a single protoplast or protoplast fusion product. Some
examples of the leaf shapes of these regenerants are shown in Fig. 1. None of the regenerants resembled either parent sufficiently to suggest they were either tomato protoplast regenerants orL. chilense protoplast regenerants. A sexually derived hybrid between these two species was not available, so we were unable to predict the expected phenotype of a hybrid individual. The leaves of all of the regenerants were coarse, thick and brittle, and smaller than the leaves on tomato and L. chilense. Many of the plants produced flowers and often seedless fruit.
Molecular analysis of nuclear genotype of the regenerants In order to determine if the regenerants we recovered were somatic hybrids we needed to test the genotype of the plants at the molecular level. We were unable to predict which regenerants might be hybrid based on the morphology of the plants. Five isozyme activities were tested for useful polymorphisms between the two fusion parents. We also scored 66 individual regeuerants with these five activities. No useful assays were determined primarily because the L. chilense parent was hybrid for these activities. L. chilense is a self-incompatible species (Rick 1979) and there was a great deal of polymorphism among L. chilense individuals. We had not expected this degree of heterozygosity in one of our fusion parents. Our next strategy was to use RFLPs to determine the genotype of the regenerants. Using cDNA and total genomic clones mapped on the tomato nuclear genome (Mutschler et al. 1987; Zamir and Tanksley 1988), species-specifc RFLPs were determined for 5 loci: CD15 and CD24 on chromosome 1, CD35 on chromosome 2, TG18 on chromosome 9, and TG30 on chromosome 11. Total DNA was isolated from 16 unique individual regenerants and the genotype determined at some or all of these five loci. The results of the analysis for TG18 are shown in Fig. 2. Tomato contains a 2.9 kb ECoR1 fragment which
631
Fig. 2. RFLP analysis at locus TG18. Total DNA was isolated, restricted with EcoR1 and probed with TG18. Lanes 117 contain DNA from individual regenerants, lane E contains DNA from L. esculentum, cv. UC82 and lane C contains DNA from L chilense LA1959. The species-specific fragments are indicated in kb.
hybridizes to the probe for this locus, while L. chilense contains a 12.5 kb EcoR1 fragment. All of the individuals tested contained the L. chilense fragment, and two also contained the tomato fragment (lanes 2, 3, and 8). Lanes 2 and 3 in Fig. 2 contain DNA from the same individual, 3D. Since this regenerant was variegated, DNA was isolated from dark and light green leaves separately. No difference was ever observed between the two samples from individual 3D. Regenerants 3D and 37A (lane 8) were scored as hybrid at this locus. The intensity of the tomato fragment in lane 8 is just detectable. For both hybrids, the intensity of the tomato-specific hybridization with this probe is not equal to the L. chilense-specific hybridization. The somatic hybrids may not have retained both of the two tomato chromosome 9 homologues.
the suspension cell line carried the 18.0 and 12.5 kb HindIII fragments. From these results, two individualswere scored as hybrid, lane 1 and lane 5, which contain DNA isolated from individuals 3D and 37A respectively. These two regenerants were the only ones to demonstrate any tomato RFLP signal at the other nuclear loci tested. All of the other regenerants scored as L. chilense at the tested loci. Table 1 lists the loci tested and the genotype determined for these two individuals. Two individuals in a population of 16 tested, suggests a reasonable recovery rate of fusion products, estimated at 12%. We never recovered any individuals which scored as tomato. Selection against one parent, using iodoacetate inactivation, was sufficient to allow for the recovery of somatic hybrids.
Table 1. Description of regenerated plants following culture of protoplast fusion between L. chilense and L. esculentum. Nuclear loci or organelles were scored as L. chilense (C), L. esculentum (E), or hybrid (H) using RFLP results, nd indicates not determined; the number in parentheses indicates the chromosomal location of the locus.
Plant 3D 6C 1ZA 35A 37A
nuclear DNA CD15 CD24 CD35TG18TG30 (1) (1) (2) (9) (11) H nd nd nd H
H nd nd nd H
H C C C H
H C C C H
H C C C H
organellar DNA cp mt E C C nd E
C C C C C
The genotype of the regenerants was scored at four other loci and the results of the analysis for TG30 are shown in Fig. 3. Tomato contains a 4.3 kb HindlII fragment which hybridizes to the probe for this locus. In this case some of the individual polymorphisms described at the isozyme level between L. chilense individuals are apparent in this Southern blot. The individual L. chilense plant used for the isolation of DNA is not the same plant used for the initiation of the suspension cell line. These two plants are hybrid and polymorphic at the TG30 locus. The lane marked C displays two HindIII fragments which hybridize to the probe for this locus, 15.0 and 12.5 kb, while the regenerants contain 18.0 and 12.5 kb fragments. We presume that the L. chilense used for the intiation of
Fig. 3. RFLP analysis at locus TG30. Total DNA was isolated, restricted with HindIII and probed with TG30. Lanes 1-5 contain DNA from individual regenerants, lane E contains DNA fromL. esculentum, cv. UC82 and lane C contains DNA from L. chilense LA1959. The species-specific fragments are indicated in kb.
The chromosome number of several of the regenerants was determined from root tip squashes. All of the individuals counted contained 48 chromosomes. Somatic hybrid 37A had 48 chromosomes; we were unable to determine the chromosome number in somatic hybrid 3D, this plant was not very vigorous and has since died. We suspect that the non-parental appearance of the L. chilense protoplast regenerants, i.e., 12A is due to the doubling
632
Fig. 4. RFLP analysis of cpDNA. Total DNA was isolated, restricted with EcoR1 and probed with cpDNA isolated from L. esculentum, cv. UC82. Lanes 1-17 contain DNA from individual regenerants, lane E contains DNA from L. esculentum, cv. UC82, lane C contains DNA from L. chilense LA1959, and lane cpE contains purified cpDNA isolated from UC82. The species-specific fragments are indicated in kb. Only the upper portion of the Southern (> 6 kb), which contains the RFLPs is shown.
of their chromosome number. Several of the L. chilense regenerants and both of the somatic hybrids, 3D and 37A developed flowers and produced seedless fruit. This was quite different from the behavior of the L. chilense parent, which never developed fruit. Organellar genomes
The chloroplast genotype of 16 regenerants was determined based on an EcoR1 species-specific polymorphism. Tomato chloroplast DNA (cpDNA) contains a 9.2 kb fragment missing in L. chilense, and L. chilense contains a 6.4 kb fragment missing in tomato (Fig. 4). All but two of the regenerants contained the cpDNA of L. chilense, regenerants 3D (lanes 2 and 3, Fig. 4) and 37A (lane 8, Fig. 4), both contained the cpDNA of tomato. The mitochondrial genotype of the regenerants was also characterized, however, only one genetic locus was examined, the ribosomal RNA genes 18S + 5S. These genes are linked and in tomato mitochondrial DNA (mtDNA) three copies reside on three HindIII fragments, 15, 10.5 and 8.8 kb, while L. chilense contains only two copies, 15.0 and 8.8 kb HindlII fragments (Fig. 5). The hybridization signal in the 7.9 kb HindlII fragment is from cross hybridization with sequences in cpDNA (data not shown). All of the regenerants tested contained the 18S + 5S ribosomal genes from L. chilense.
Fig. 5. RFLP analysis of mtDNA. Total DNA was isolated, restricted with Hindlll and probed with the tomato mitochondrial 18S + 5S ribosomal RNA gene. Lanes I-5 contain DNA from individual regenerants, lane E contains DNA from L. esculentum, cv. UC82 and lane C contains DNA from L. chilense LA1959. The sizes of the species-specific fragments are indicated in kb.
Conclusions
Tomato-L. chilense somatic hybrids could be recovered following protoplast fusion, however, the morphology of the regenerated somatic hybrids was not sufficiently different from tetraploid L. chilense protoplast regenerants to allow identification. Nuclear RFLPs were used to allow identification of the somatic hybrid plants. The somatic hybrid plants had inherited the tomato cpDNA and at least portions of L. chilense mtDNA. Fertility is required in the hybrid regenerants if the material is to be used for a crop improvement. While no seed was recovered following selfing of the somatic hybrids, a more agressive approach using either L. chilense, or tomato as parents in reciprocal crosses might yield seed. Acknowledgements. We thank Mary Lucero and Suzanne Rozario for technical assistance, and Steve Tanksley for the tomato nuclear RFLP markers. This work was supported by the New Mexico AES and USDA Competitive Grant 87-CRCR-t-2291.
References Austin S, Baer MA, Helgeson JP (1985) Plant Sci 39:75-82 Bonnema AB, Melzer JM, O'Connell MA (1991) Theor Appl Genet 81:339-348 Doyle .IF, Doyle JL (1989) Focus 12:13-14 Kemble ILl, Barsby T L Yarrow SA (1988) Mol Gen Genet 213:202-205 Kinsara A, Patnaik SN, Cocking EC, Power JB (1986) Plant Physiol 125:225-234 Kyozuka J, Kaneda T, Shimamoto K (1989) Biotech 7:1171-1174 Melzer JM, O'ConneU MA (1990) Theor Appl Genet 79:193-200 Melzer JM, O'ConneU MA (1991) Theor Appl Genet (in press) Morgan A, Maliga P (1987) Mol Gen Genet 209:240-246 Mutschler MA, Tanksley SD, Rick CM (1987) Tomato Genet Coop Pep 37:5-34 O'Connell MA, Hanson MR (1985) Theor Appl Genet 70:1-12 O'Connell MA, Hanson MR (1986) Theor Appl Genet 72:59-65 O'ConneU MA, Hanson MR (1987) Theor Appl Genet 75:83-89 Rick CM (1973) In: Srb AM (ed) Genes, Enzymes and Populations. Plenum Press, New York, pp 255-269 Rick CM (1979) In: Hawkes JG, Lester RN, Skelding AD (eds) The Biology and Taxonomy of the Solanaceae. Academic Press, London, pp 667-678 San LH, Vedel F, Sihacbakr D, Remy R (1990) Mol Gen Genet 221:17-26 Sjodin C, Glimelius K (1989) Theor Appl Genet 78:513-520 Wijbrandi J, Zabel P, Koornneef M (1990) Mol Gen Genet 222:270-277 Zamir D, Tanksley SD (1988) Mol Gen Genet 213:254-261
Plant Cell Reports (1992) 10:629-632
9 Springer-Verlag 1992
Molecular analysis of the nuclear organellar genotype of somatic hybrid plants between tomato (Lycopersicon esculentum) and Lycopersicon chilense Augusta B. Bonnema 1 and Mary A. O'Connell 2 1 Department of Agronomy and Horticulture, Plant Genetic Engineering Lab, New Mexico State University, Las Cruces, N M 88003, U S A 2 Present address: INRA, Centre de Recherches de Toulouse, L B M R P M , BP27, 31326 Castanet Tolosan, France Received August 21, 1991/Revised version received November 1, 1991
Summary. Somatic hybrid plants were recovered following fusion of leaf mesophyll protoplasts isolated from tomato (Lycopersicon esculentum) cultivar UC82 with protoplasts isolated from suspension cultured cells of L. chilense, L A 1959. Iodoacetate was used to select against the growth of unfused tomato protoplasts. Two somatic hybrids were recovered in a population of 16 regenerants. No tomato regenerants were recovered; all of the non-hybrid regenerants were L. chilense. The L. chilense protoplast regenerants were tetraploid. The hybrid nature of the plants was verified using species-specifc restriction fragment length polymorphisms for the nuclear, chloroplast and mitochondrial genomes. The somatic hybrids had inherited the chloroplast DNA of the tomato parent, and portions of the mitochondrial DNA of the L. chilense parent. The somatic hybrids formed flowers and developed seedless fruit.
Communicated by G. C. Phillips
products between tomato and L. chilense. L. chilense, a self-incompatible species, inhabits one of the most arid of the world's temperate deserts (Rick 1973). The adaptation ofL. chilense to these deserts depends on the foraging ability of its root system. This member of the Lycopersicon genus is most similar to L. peruvianurn (Rick 1979). While there are several reports on protoplast culture and fusion products for tomato-L, peruvianum (Kinsara et al. 1986; San et al. 1990; Wijbrandi et al. 1990), no reports on protoplast culture or fusion with L. chilense have been described. In order to determine if the protoplast fusion products were somatic hybrids, we used restriction fragment length polymorphisms (RFLPs) for five mapped nuclear loci (Mutschler et al. 1987; Zamir and Tanksley 1988). Materials and Methods
Key words: Protoplast fusion - RFLP - Mitochondrial DNA - Chloroplast DNA
Introduction
The application of protoplast fusion techniques to create novel plant genotypes has been in use for over 15 years and, in recent years, examples of the production of improved crop species using these techniques have been reported (Austin et al. 1985; Morgan and Maliga 1987; Kemble et aL 1988; Kyozuka et aL 1989; Sjodin and Glimelius 1989). We have applied protoplast fusion techniques to members of the related genera Lycopersicon and Solanum in attempts to understandnuclear-organellarinteractions, to construct useful novel genotypes, and to optimize methods for partial genome transfer (O'Connell and Hanson 1986; 1987; Bonnema et aL 1991; Melzer and O'Connell 1990; 1991). In this report we describe the construction and characterization of protoplast fusion Offprint requests to: M. A. O'Connell
Plant material. Seed of Lu chilense Dun., LA 1959, were obtained from Charles Rick, Tomato Genetics Stock Center, University of California, Davis; seed of L. esculentum Mill., cv. UC82, were provided by PetoSeed, Co. Suspension cell cultures of L. chilense were generated from hypocotyls of aseptically germinated seedlings. Callus was initiated on UM1A (O'Connell and Hanson 1985), and a suspension culture was subsequently established in the same medium. The suspension culture used for the isolation of protoplasts for fusions was 6-8 months old. UC82 plants to be used for the isolation of leaf mesophyll protoplasts were germinated in soil mix in a growth chamber, 16 h photoperiod, 25~ day, 18~ night, watered daily with a modified Nitsch salt solution (O'Connell and Hanson 1985). Plants were between 4 and 6 weeks old when leaves were collected for protoplast isolation. The plants received a cold pretreatment as described (Bonnema et al. 1991). Protoplast isolation~ fusion and culture. Protoplasts were isolated as described by Bonnema et al. (1991). The leaf mesophyll protoplasts were treated with 3 mM iodoacetate for 20 rain at 50C in W5 solution. The protoplasts were fused in a 1:1 ratio, final concentration of 6 x 10~/ml also as described by Bonnema et al. (1991). After fusion the protoplasts were plated at 2 x 1 ~ / m l in TraP. Approximately a 15% fusion frequency was observed based on doubly fluorescent ceils, red autofluorescence of chlorophyll in UC82 protoplasts and yellow-green fluorescence of fluorosceneisothiocyanate stained L. chilense protoplasts. The
630
Fig. 1. Comparisonof the leaf shape of several regenerants, 3D, 9A, 12A, 14, 42A, and 76, with the fusion parents,L. esculentum cv. UC82 (E), ..I~. chilense LA1959 (C).
protoplasts, microcalli and calli were cultured as descibed by Bonnema et al. (1991). Shoots were regenerated using these media: JSC-12 for greening of calli, TR-1 for shoot induction, and subsequently rooted on MS-0 supplemented with 50 t~M indole3-butyric acid (O'Connell and Hanson 1985). DNA isolation~ restriction digestion~and southern hybridization. Total DNA was isolated from leaf tissue of regenerated plants and the parental species as descibed by Doyle and Doyle(1989), with the addition of a CsC1 centrifugation step after the isopropanol precipitation. The DNA (10 ~ g) was digestedwith the indicated restriction endonucleases, electrophoresed on 0.8% agarose gels, and transferred to nylon membranes using standard procedures. Radiolabelled probes were generated usinga 3~P-dCrP and either random primer or'nick translation procedures. For hybridization with probes to detect nuclear RFLPs, hybridizationand washing of the blots was as described by Melzer and O'Connell (1991). For hybridization with probes to detect organellar RFLPs, hybridization and washing of the blots was as described by Bonnema et al. (1991). Results and Discussion
Recovery of regenerated plants Approximately six weeks following protoplast fusion, 100 microcalli were transferred from liquid protoplast culture medium, TmD, onto solid greening medium, JSC-12. Within a week these calli turned green, and after increasing in size they were transferred to a shoot inducing medium, TR-1. Most of the calli regenerated multiple shoots. The major difficulty encountered in recovering plants from this fusion construction was to induce roots. When shoots were transferred to MS-0 or to MS-0 plus charcoal, no roots formed and the shoots degenerated. However, when shoots were transferred to MS-0 plus 50 # M indole-3-butyric acid, roots were induced on most, approx. 75%, of the shoots within 10 days. Over 60 plants were established in soil, and of these, 16 were characterized extensively using molecular analyses. These 16 individuals were each from a unique callus and represented a regenerant from a single protoplast or protoplast fusion product. Some
examples of the leaf shapes of these regenerants are shown in Fig. 1. None of the regenerants resembled either parent sufficiently to suggest they were either tomato protoplast regenerants orL. chilense protoplast regenerants. A sexually derived hybrid between these two species was not available, so we were unable to predict the expected phenotype of a hybrid individual. The leaves of all of the regenerants were coarse, thick and brittle, and smaller than the leaves on tomato and L. chilense. Many of the plants produced flowers and often seedless fruit.
Molecular analysis of nuclear genotype of the regenerants In order to determine if the regenerants we recovered were somatic hybrids we needed to test the genotype of the plants at the molecular level. We were unable to predict which regenerants might be hybrid based on the morphology of the plants. Five isozyme activities were tested for useful polymorphisms between the two fusion parents. We also scored 66 individual regeuerants with these five activities. No useful assays were determined primarily because the L. chilense parent was hybrid for these activities. L. chilense is a self-incompatible species (Rick 1979) and there was a great deal of polymorphism among L. chilense individuals. We had not expected this degree of heterozygosity in one of our fusion parents. Our next strategy was to use RFLPs to determine the genotype of the regenerants. Using cDNA and total genomic clones mapped on the tomato nuclear genome (Mutschler et al. 1987; Zamir and Tanksley 1988), species-specifc RFLPs were determined for 5 loci: CD15 and CD24 on chromosome 1, CD35 on chromosome 2, TG18 on chromosome 9, and TG30 on chromosome 11. Total DNA was isolated from 16 unique individual regenerants and the genotype determined at some or all of these five loci. The results of the analysis for TG18 are shown in Fig. 2. Tomato contains a 2.9 kb ECoR1 fragment which
631
Fig. 2. RFLP analysis at locus TG18. Total DNA was isolated, restricted with EcoR1 and probed with TG18. Lanes 117 contain DNA from individual regenerants, lane E contains DNA from L. esculentum, cv. UC82 and lane C contains DNA from L chilense LA1959. The species-specific fragments are indicated in kb.
hybridizes to the probe for this locus, while L. chilense contains a 12.5 kb EcoR1 fragment. All of the individuals tested contained the L. chilense fragment, and two also contained the tomato fragment (lanes 2, 3, and 8). Lanes 2 and 3 in Fig. 2 contain DNA from the same individual, 3D. Since this regenerant was variegated, DNA was isolated from dark and light green leaves separately. No difference was ever observed between the two samples from individual 3D. Regenerants 3D and 37A (lane 8) were scored as hybrid at this locus. The intensity of the tomato fragment in lane 8 is just detectable. For both hybrids, the intensity of the tomato-specific hybridization with this probe is not equal to the L. chilense-specific hybridization. The somatic hybrids may not have retained both of the two tomato chromosome 9 homologues.
the suspension cell line carried the 18.0 and 12.5 kb HindIII fragments. From these results, two individualswere scored as hybrid, lane 1 and lane 5, which contain DNA isolated from individuals 3D and 37A respectively. These two regenerants were the only ones to demonstrate any tomato RFLP signal at the other nuclear loci tested. All of the other regenerants scored as L. chilense at the tested loci. Table 1 lists the loci tested and the genotype determined for these two individuals. Two individuals in a population of 16 tested, suggests a reasonable recovery rate of fusion products, estimated at 12%. We never recovered any individuals which scored as tomato. Selection against one parent, using iodoacetate inactivation, was sufficient to allow for the recovery of somatic hybrids.
Table 1. Description of regenerated plants following culture of protoplast fusion between L. chilense and L. esculentum. Nuclear loci or organelles were scored as L. chilense (C), L. esculentum (E), or hybrid (H) using RFLP results, nd indicates not determined; the number in parentheses indicates the chromosomal location of the locus.
Plant 3D 6C 1ZA 35A 37A
nuclear DNA CD15 CD24 CD35TG18TG30 (1) (1) (2) (9) (11) H nd nd nd H
H nd nd nd H
H C C C H
H C C C H
H C C C H
organellar DNA cp mt E C C nd E
C C C C C
The genotype of the regenerants was scored at four other loci and the results of the analysis for TG30 are shown in Fig. 3. Tomato contains a 4.3 kb HindlII fragment which hybridizes to the probe for this locus. In this case some of the individual polymorphisms described at the isozyme level between L. chilense individuals are apparent in this Southern blot. The individual L. chilense plant used for the isolation of DNA is not the same plant used for the initiation of the suspension cell line. These two plants are hybrid and polymorphic at the TG30 locus. The lane marked C displays two HindIII fragments which hybridize to the probe for this locus, 15.0 and 12.5 kb, while the regenerants contain 18.0 and 12.5 kb fragments. We presume that the L. chilense used for the intiation of
Fig. 3. RFLP analysis at locus TG30. Total DNA was isolated, restricted with HindIII and probed with TG30. Lanes 1-5 contain DNA from individual regenerants, lane E contains DNA fromL. esculentum, cv. UC82 and lane C contains DNA from L. chilense LA1959. The species-specific fragments are indicated in kb.
The chromosome number of several of the regenerants was determined from root tip squashes. All of the individuals counted contained 48 chromosomes. Somatic hybrid 37A had 48 chromosomes; we were unable to determine the chromosome number in somatic hybrid 3D, this plant was not very vigorous and has since died. We suspect that the non-parental appearance of the L. chilense protoplast regenerants, i.e., 12A is due to the doubling
632
Fig. 4. RFLP analysis of cpDNA. Total DNA was isolated, restricted with EcoR1 and probed with cpDNA isolated from L. esculentum, cv. UC82. Lanes 1-17 contain DNA from individual regenerants, lane E contains DNA from L. esculentum, cv. UC82, lane C contains DNA from L. chilense LA1959, and lane cpE contains purified cpDNA isolated from UC82. The species-specific fragments are indicated in kb. Only the upper portion of the Southern (> 6 kb), which contains the RFLPs is shown.
of their chromosome number. Several of the L. chilense regenerants and both of the somatic hybrids, 3D and 37A developed flowers and produced seedless fruit. This was quite different from the behavior of the L. chilense parent, which never developed fruit. Organellar genomes
The chloroplast genotype of 16 regenerants was determined based on an EcoR1 species-specific polymorphism. Tomato chloroplast DNA (cpDNA) contains a 9.2 kb fragment missing in L. chilense, and L. chilense contains a 6.4 kb fragment missing in tomato (Fig. 4). All but two of the regenerants contained the cpDNA of L. chilense, regenerants 3D (lanes 2 and 3, Fig. 4) and 37A (lane 8, Fig. 4), both contained the cpDNA of tomato. The mitochondrial genotype of the regenerants was also characterized, however, only one genetic locus was examined, the ribosomal RNA genes 18S + 5S. These genes are linked and in tomato mitochondrial DNA (mtDNA) three copies reside on three HindIII fragments, 15, 10.5 and 8.8 kb, while L. chilense contains only two copies, 15.0 and 8.8 kb HindlII fragments (Fig. 5). The hybridization signal in the 7.9 kb HindlII fragment is from cross hybridization with sequences in cpDNA (data not shown). All of the regenerants tested contained the 18S + 5S ribosomal genes from L. chilense.
Fig. 5. RFLP analysis of mtDNA. Total DNA was isolated, restricted with Hindlll and probed with the tomato mitochondrial 18S + 5S ribosomal RNA gene. Lanes I-5 contain DNA from individual regenerants, lane E contains DNA from L. esculentum, cv. UC82 and lane C contains DNA from L. chilense LA1959. The sizes of the species-specific fragments are indicated in kb.
Conclusions
Tomato-L. chilense somatic hybrids could be recovered following protoplast fusion, however, the morphology of the regenerated somatic hybrids was not sufficiently different from tetraploid L. chilense protoplast regenerants to allow identification. Nuclear RFLPs were used to allow identification of the somatic hybrid plants. The somatic hybrid plants had inherited the tomato cpDNA and at least portions of L. chilense mtDNA. Fertility is required in the hybrid regenerants if the material is to be used for a crop improvement. While no seed was recovered following selfing of the somatic hybrids, a more agressive approach using either L. chilense, or tomato as parents in reciprocal crosses might yield seed. Acknowledgements. We thank Mary Lucero and Suzanne Rozario for technical assistance, and Steve Tanksley for the tomato nuclear RFLP markers. This work was supported by the New Mexico AES and USDA Competitive Grant 87-CRCR-t-2291.
References Austin S, Baer MA, Helgeson JP (1985) Plant Sci 39:75-82 Bonnema AB, Melzer JM, O'Connell MA (1991) Theor Appl Genet 81:339-348 Doyle .IF, Doyle JL (1989) Focus 12:13-14 Kemble ILl, Barsby T L Yarrow SA (1988) Mol Gen Genet 213:202-205 Kinsara A, Patnaik SN, Cocking EC, Power JB (1986) Plant Physiol 125:225-234 Kyozuka J, Kaneda T, Shimamoto K (1989) Biotech 7:1171-1174 Melzer JM, O'ConneU MA (1990) Theor Appl Genet 79:193-200 Melzer JM, O'ConneU MA (1991) Theor Appl Genet (in press) Morgan A, Maliga P (1987) Mol Gen Genet 209:240-246 Mutschler MA, Tanksley SD, Rick CM (1987) Tomato Genet Coop Pep 37:5-34 O'Connell MA, Hanson MR (1985) Theor Appl Genet 70:1-12 O'Connell MA, Hanson MR (1986) Theor Appl Genet 72:59-65 O'ConneU MA, Hanson MR (1987) Theor Appl Genet 75:83-89 Rick CM (1973) In: Srb AM (ed) Genes, Enzymes and Populations. Plenum Press, New York, pp 255-269 Rick CM (1979) In: Hawkes JG, Lester RN, Skelding AD (eds) The Biology and Taxonomy of the Solanaceae. Academic Press, London, pp 667-678 San LH, Vedel F, Sihacbakr D, Remy R (1990) Mol Gen Genet 221:17-26 Sjodin C, Glimelius K (1989) Theor Appl Genet 78:513-520 Wijbrandi J, Zabel P, Koornneef M (1990) Mol Gen Genet 222:270-277 Zamir D, Tanksley SD (1988) Mol Gen Genet 213:254-261
