Background The aim of this study was to evaluate and characterize the mutations induced by two TA... more Background The aim of this study was to evaluate and characterize the mutations induced by two TALE-based approaches, double-strand break (DSB) induction by the FokI nuclease (mitoTALEN) and targeted base editing by the DddA cytidine deaminase (mitoTALECD), to edit, for the first time, the mitochondrial genome of potato, a vegetatively propagated crop. The two methods were used to knock out the same mitochondrial target sequence (orf125). Targeted chondriome deletions of different sizes (236-1066 bp) were induced by mitoTALEN due to DSB repair through ectopic homologous recombination of short direct repeats (11-12 bp) present in the target region. Furthermore, in one case, the induced DSB and subsequent repair resulted in the amplification of an already present substoichiometric molecule showing a 4288 bp deletion spanning the target sequence. With the mitoTALECD approach, both nonsense and missense mutations could be induced by base substitution. The deletions and single nucleotide mutations were either homoplasmic or heteroplasmic. The former were stably inherited in vegetative offspring. Conclusions Both editing approaches allowed us to obtain plants with precisely modified mitochondrial genomes at high frequency. The use of the same plant genotype and mtDNA region allowed us to compare the two methods for efficiency, accuracy, type of modifications induced and stability after vegetative propagation.
Somatic fusion of mesophyll protoplasts was used to produce hybrids between the frost-tolerant sp... more Somatic fusion of mesophyll protoplasts was used to produce hybrids between the frost-tolerant species Solanum commersonii (2n = 2x = 24) and dihaploid S. tuberosum (2n = 2x = 24). This is a sexually incompatible combination due to the difference in EBN (Endosperm Balance Number, Johnston et al. 1980). Species with different EBNs as a rule are sexually incompatible. Fifty-seven hybrids were analysed for variation in chromosome number, morphological traits, fertility and frost tolerance. About 70% of the hybrids were tetraploid, and 30% hexaploid. Chloroplast counts in stomatal guard cells revealed a low frequency of cytochimeras. The frequency of aneuploids was relatively higher at the hexaploid level (hypohexaploids) than at the tetraploid level (hypotetraploids). The somatic hybrids were much more vigorous than the parents, and showed an intermediate phenotype for several morphological traits and moderate to profuse flowering. Hexaploid hybrid clones were less vigorous and had a lower degree of flowering than the tetraploid hybrid clones. All of the hybrids were female fertile but male sterile except for one, which was fully fertile and self-compatible. Many seeds were produced on the latter clone by selfing and on the male-sterile clones by crossing. The somatic hybrid plants showed an introgression of genes for frost tolerance and an adaptability to cold from S. commersonii. Therefore, the use of these somatic hybrids in breeding for and in genetic research on frost tolerance and cold-hardening is suggested.
Vegetables include high-value crops with health-promoting effects and reduced environmental impac... more Vegetables include high-value crops with health-promoting effects and reduced environmental impact. The availability of genomic and biotechnological tools in certain species, coupled with the recent development of new breeding techniques based on precise editing of DNA, provides unique opportunities to finally take advantage of the past decades of detailed genetic analyses, thus making improvement of traits related to quality and stress tolerance achievable in a reasonable time frame. Recent reports of such approaches in vegetables illustrate the feasibility of obtaining multiple homozygous mutations in a single generation, heritable by the progeny, using stable or transient transformation approaches, which may not rely on the integration of unwanted foreign DNA. Application of these approaches to currently non-sequenced/tissue culture recalcitrant crops will contribute to meet the challenges posed by the increase in population and climate change.
Current plant science and biotechnology in agriculture, 1999
Somatic hybridization is a useful technique for gene transfer between Solanum species. By contras... more Somatic hybridization is a useful technique for gene transfer between Solanum species. By contrast with other methodologies, it is also a powerful method to create new nuclear-cytoplasmic organelle assortments and induce variability in organellar DNA (Earle 1995). In potato, male sterility and several important traits are controlled by cytoplasmic genes or their interaction with nuclear ones. However, the genetic control of these traits is not well known. In some interspecific sexual hybrids of Solanum spp., nuclear factors from the wild species interact with cytoplasmic factors of the cultivated one to cause male sterility (CMS) (Kaul 1988). Genetic analysis has revealed that one or few nuclear genes are involved in this interaction; in contrast the cytoplasmic factors involved have not yet been characterized. As a matter of fact, while the chloroplast genome of some Solanum species has been extensively studied, only recently has the organization of the mitochondrial genome of Solanum tuberosum been examined, and very little is known about other Solanum species.
Novel and potentially useful genetic variation in cytoplasmic genomes can be induced by interspec... more Novel and potentially useful genetic variation in cytoplasmic genomes can be induced by interspecific somatic hybridization in plants. To evaluate such variability and correlate it with nuclear-cytoplasmic interactions leading to male sterility in Solanum spp., we examined progeny of male-sterile and male-fertile somatic hybrids between Solanum tuberosum (tbr), the common potato, and S. commersonii (cmm), a wild species showing sexual incongruity with tbr, for fertility and organelle DNA composition. Uniform male-fertile and male-sterile progenies were obtained by selfing the male-fertile hybrid and crossing the male-sterile ones, indicating maternal inheritance of the fertility phenotype. The two fusion partners were only slightly differentiated in the plastidial genome. MtDNA polymorphism between the species was greater, although its extent varied with the genomic region investigated. All somatic hybrids had non-parental organelle genomes, with reassorted organelles and/or rearranged mitochondria (i.e., cmm-specific bands for some regions and tbr-specific bands for others). Mitochondria reassorted independently from chloroplasts. Most hybrids showed the cmm cpDNA hybridization pattern, indicating non-random transmission of chloroplasts. Most male-sterile hybrids showed preferential inheritance of tbr mtDNA fragments. The male-fertile somatic hybrid clone had predominantly cmm mtDNA fragments. This result suggests that a tbr-derived region involved in nuclear-cytoplasmic incompatibility and male sterility has been lost by rearrangement; however, no clear correlation between a specific mitochondrial region and male sterility has been found so far.
The possibility to produce plants edited in multiple genes by means of DNA-free approaches opens ... more The possibility to produce plants edited in multiple genes by means of DNA-free approaches opens new perspectives for breeding purposes and acceptance of resultant genotypes. In this work, we have explored the polyethylene glycol (PEG)mediated delivery of ribonucleoproteins (RNPs) in tomato protoplasts using a multiplexing approach (i.e. two genes targeted simultaneously using two sgRNAs per gene) for the first time. We have analysed the editing outcome in fully developed green calli and demonstrated that tomato protoplasts are a valid cell target for RNP-mediated multiplexed genome editing with high efficiency. Key message RNP could be applied with high efficiency in a multiplexing genome editing approach in tomato protoplasts. Keywords Tomato • Ribonucleoprotein • Genome editing • Protoplast Abbreviations RNP Ribonucleoprotein HFRA High resolution fragment analysis PEG Polyethylene glycol CRISPR Clustered regularly interspaced short palindromic repeats Cas9 CRISPR associated protein 9 Tomato (Solanum lycopersicum L.) is the most cultivated vegetable worldwide after potato and is grown for both fresh and processing markets. It is considered a model plant for research because of its simple genetic nature (2n = 2x = 24), medium-size genome, short generation time, routine transformation technology in place and a high availability of plant genetic resources. With the advent of genome editing techniques and CRISPR/Cas9 in particular, many papers have been published on tomato and other vegetables (Cardi et al. 2017a; Wang et al. 2019), the vast majority relying on Agrobacterium tumefaciens (currently renamed to Rhizobium radiobacter) mediated transformation of multicellular explants for the delivery of editing reagents. In tomato, transformation and shoot regeneration is routinely achieved using cotyledonary explants, while alternative direct approaches, such as protoplast or biolistics-based delivery methods, are either not available or have limited applicability (Cardi et al. 2017b; Van Eck 2017). The delivery in plant cells of in vitro pre-assembled ribonucloprotein (RNP) complexes, constituted of protein (i.e. Cas9) bound to ribonucleic acid (i.e. the single guide RNA, sgRNA), has been demonstrated in several species (
Somatic hybridization between Solanum commersonii and S. tuberosum resulted in the production of ... more Somatic hybridization between Solanum commersonii and S. tuberosum resulted in the production of male-sterile hybrid plants, except for one fully male-fertile hybrid. The male-sterile hybrids exhibited a“pollen-less” phenotype, with rare pollen grains which were abnormal in shape and exine sculpture. Microsporogenesis and tapetal development were investigated both in male-sterile and male-fertile somatic hybrids to assess the cytological events that were
The possibility of scaling ploidy levels up and down offers several advantages for genetic studie... more The possibility of scaling ploidy levels up and down offers several advantages for genetic studies and breeding work in potato. Shoot regeneration and chromosome doubling in plants regenerated from in vitro cultures were investigated in 4 diploid (2n= 2x= 24) and 2 triploid (2n= 3x= 36) clones. Internode and leaf explants were taken from plants propagated either in vitro as shoot cultures, or in vivo in a greenhouse. Two regeneration procedures were compared. Regeneration frequencies were significantly higher using a two-step regeneration protocol and from leaf explants, while doubling was more efficient starting from in vivo grown plants. No doubling was observed in triploid clones. Considering altogether the percentage of regeneration and doubling, and the number of shoots regenerated per explant, the most efficient conditions were considered leaf explants taken from in vivo grown plants, and cultured according to a two-step regeneration protocol.
Capsicum is one of the major vegetable crops grown worldwide. Current subdivision in clades and s... more Capsicum is one of the major vegetable crops grown worldwide. Current subdivision in clades and species is based on morphological traits and coarse sets of genetic markers. Broad variability of fruits has been driven by breeding programs and has been mainly studied by linkage analysis. We discovered 746k variable sites by sequencing 1.8% of the genome in a collection of 373 accessions belonging to 11 Capsicum species from 51 countries. We describe genomic variation at population-level, confirm major subdivision in clades and species, and show that the known major subdivision of C. annuum separates large and bulky fruits from small ones. In C. annuum, we identify four novel loci associated with phenotypes determining the fruit shape, including a non-synonymous mutation in the gene Longifolia 1-like (CA03g16080). Our collection covers all the economically important species of Capsicum widely used in breeding programs and represent the widest and largest study so far in terms of the number of species and number of genetic variants analyzed. We identified a large set of markers that can be used for population genetic studies and genetic association analyses. our results provide a comprehensive and precise perspective on genomic variability in Capsicum at population-level and suggest that future fine genetic association studies will yield useful results for breeding.
Summary Solanum commersonii Dun. is a diploid (2n=2x=24, 1EBN) wild species of potential value fo... more Summary Solanum commersonii Dun. is a diploid (2n=2x=24, 1EBN) wild species of potential value for potato breeding. It is a reproductively isolated species and cannot be crossed with Tuberosum haploids (2n=2x=24, 2EBN) or other diploid 2EBNSolanum species. In order to overcome the EBN barriers, triploid hybrids were produced between Phureja-Tuberosum haploid hybrids, which form 2n pollen grains by parallel spindles,
Solanum commersonii (Cmm) is a tuber-bearing wild relative of the common potato (S. tuberosum). I... more Solanum commersonii (Cmm) is a tuber-bearing wild relative of the common potato (S. tuberosum). It is widespread in Paraguay and along the coasts of Argentina, Uruguay, and southern Brazil, from sea level up to about 400m (Correll 1962; Hawkes 1990). Typically, it is a small, bushy plant, with flowers (deep mauve to purple on the outer surface, paler within) arranged in compact inflorescences. Generally, it is diploid (2n = 2x = 24) with an endosperm balance number (EBN) equal to 1, but autotriploid forms (2n = 3x = 36) are also known (Hanneman and Bamberg 1986).
Current plant science and biotechnology in agriculture, 1999
In earlier days of plant tissue culture, protoplast fusion provided the only method of introducin... more In earlier days of plant tissue culture, protoplast fusion provided the only method of introducing or combining genes somewhat beyond the limits of sexual reproduction. Protoplast fusion is now rather overshadowed by plant transformation, a very powerful method that permits transfer into plants of genes from any taxonomic group. In view of the success of gene transfer, is protoplast fusion still of interest and value?
Solanum commersonii (+) S. tuberosum hybrids with different endosperm balance numbers (EBN) and p... more Solanum commersonii (+) S. tuberosum hybrids with different endosperm balance numbers (EBN) and ploidy were used in various crossing schemes with 4EBN S. tuberosum cultivars and a 3EBN somatic hybrid to test their behavior in intra-and inter-EBN crosses and to derive a BC 1 population for potato breeding. The somatic hybrids included 12 tetraploids (2n = 48, 3EBN), 18 hypotetraploids (2n = 43-47, 3EBN), 2 hexaploids (2n = 72, 4 or 5EBN), and 5 hypohexaploids (2n = 63-70, 4 or 5EBN). The results confirmed that in the potato, EBN is more important than actual ploidy in determining the success or failure of crosses. Indeed, crosses between some hypohexaploid hybrids and S. tuberosum cultivars gave the highest number of viable seeds per berry, and seed viability was similar to that of intra-EBN crosses between varieties. Inter-EBN crosses demonstrated that exceptions to the 2:1 maternal-paternal EBN ratio in the hybrid endosperm can sometimes be tolerated. However, an excess in maternal EBN dosage was better tolerated than an excess in paternal EBN.
Background The aim of this study was to evaluate and characterize the mutations induced by two TA... more Background The aim of this study was to evaluate and characterize the mutations induced by two TALE-based approaches, double-strand break (DSB) induction by the FokI nuclease (mitoTALEN) and targeted base editing by the DddA cytidine deaminase (mitoTALECD), to edit, for the first time, the mitochondrial genome of potato, a vegetatively propagated crop. The two methods were used to knock out the same mitochondrial target sequence (orf125). Targeted chondriome deletions of different sizes (236-1066 bp) were induced by mitoTALEN due to DSB repair through ectopic homologous recombination of short direct repeats (11-12 bp) present in the target region. Furthermore, in one case, the induced DSB and subsequent repair resulted in the amplification of an already present substoichiometric molecule showing a 4288 bp deletion spanning the target sequence. With the mitoTALECD approach, both nonsense and missense mutations could be induced by base substitution. The deletions and single nucleotide mutations were either homoplasmic or heteroplasmic. The former were stably inherited in vegetative offspring. Conclusions Both editing approaches allowed us to obtain plants with precisely modified mitochondrial genomes at high frequency. The use of the same plant genotype and mtDNA region allowed us to compare the two methods for efficiency, accuracy, type of modifications induced and stability after vegetative propagation.
Somatic fusion of mesophyll protoplasts was used to produce hybrids between the frost-tolerant sp... more Somatic fusion of mesophyll protoplasts was used to produce hybrids between the frost-tolerant species Solanum commersonii (2n = 2x = 24) and dihaploid S. tuberosum (2n = 2x = 24). This is a sexually incompatible combination due to the difference in EBN (Endosperm Balance Number, Johnston et al. 1980). Species with different EBNs as a rule are sexually incompatible. Fifty-seven hybrids were analysed for variation in chromosome number, morphological traits, fertility and frost tolerance. About 70% of the hybrids were tetraploid, and 30% hexaploid. Chloroplast counts in stomatal guard cells revealed a low frequency of cytochimeras. The frequency of aneuploids was relatively higher at the hexaploid level (hypohexaploids) than at the tetraploid level (hypotetraploids). The somatic hybrids were much more vigorous than the parents, and showed an intermediate phenotype for several morphological traits and moderate to profuse flowering. Hexaploid hybrid clones were less vigorous and had a lower degree of flowering than the tetraploid hybrid clones. All of the hybrids were female fertile but male sterile except for one, which was fully fertile and self-compatible. Many seeds were produced on the latter clone by selfing and on the male-sterile clones by crossing. The somatic hybrid plants showed an introgression of genes for frost tolerance and an adaptability to cold from S. commersonii. Therefore, the use of these somatic hybrids in breeding for and in genetic research on frost tolerance and cold-hardening is suggested.
Vegetables include high-value crops with health-promoting effects and reduced environmental impac... more Vegetables include high-value crops with health-promoting effects and reduced environmental impact. The availability of genomic and biotechnological tools in certain species, coupled with the recent development of new breeding techniques based on precise editing of DNA, provides unique opportunities to finally take advantage of the past decades of detailed genetic analyses, thus making improvement of traits related to quality and stress tolerance achievable in a reasonable time frame. Recent reports of such approaches in vegetables illustrate the feasibility of obtaining multiple homozygous mutations in a single generation, heritable by the progeny, using stable or transient transformation approaches, which may not rely on the integration of unwanted foreign DNA. Application of these approaches to currently non-sequenced/tissue culture recalcitrant crops will contribute to meet the challenges posed by the increase in population and climate change.
Current plant science and biotechnology in agriculture, 1999
Somatic hybridization is a useful technique for gene transfer between Solanum species. By contras... more Somatic hybridization is a useful technique for gene transfer between Solanum species. By contrast with other methodologies, it is also a powerful method to create new nuclear-cytoplasmic organelle assortments and induce variability in organellar DNA (Earle 1995). In potato, male sterility and several important traits are controlled by cytoplasmic genes or their interaction with nuclear ones. However, the genetic control of these traits is not well known. In some interspecific sexual hybrids of Solanum spp., nuclear factors from the wild species interact with cytoplasmic factors of the cultivated one to cause male sterility (CMS) (Kaul 1988). Genetic analysis has revealed that one or few nuclear genes are involved in this interaction; in contrast the cytoplasmic factors involved have not yet been characterized. As a matter of fact, while the chloroplast genome of some Solanum species has been extensively studied, only recently has the organization of the mitochondrial genome of Solanum tuberosum been examined, and very little is known about other Solanum species.
Novel and potentially useful genetic variation in cytoplasmic genomes can be induced by interspec... more Novel and potentially useful genetic variation in cytoplasmic genomes can be induced by interspecific somatic hybridization in plants. To evaluate such variability and correlate it with nuclear-cytoplasmic interactions leading to male sterility in Solanum spp., we examined progeny of male-sterile and male-fertile somatic hybrids between Solanum tuberosum (tbr), the common potato, and S. commersonii (cmm), a wild species showing sexual incongruity with tbr, for fertility and organelle DNA composition. Uniform male-fertile and male-sterile progenies were obtained by selfing the male-fertile hybrid and crossing the male-sterile ones, indicating maternal inheritance of the fertility phenotype. The two fusion partners were only slightly differentiated in the plastidial genome. MtDNA polymorphism between the species was greater, although its extent varied with the genomic region investigated. All somatic hybrids had non-parental organelle genomes, with reassorted organelles and/or rearranged mitochondria (i.e., cmm-specific bands for some regions and tbr-specific bands for others). Mitochondria reassorted independently from chloroplasts. Most hybrids showed the cmm cpDNA hybridization pattern, indicating non-random transmission of chloroplasts. Most male-sterile hybrids showed preferential inheritance of tbr mtDNA fragments. The male-fertile somatic hybrid clone had predominantly cmm mtDNA fragments. This result suggests that a tbr-derived region involved in nuclear-cytoplasmic incompatibility and male sterility has been lost by rearrangement; however, no clear correlation between a specific mitochondrial region and male sterility has been found so far.
The possibility to produce plants edited in multiple genes by means of DNA-free approaches opens ... more The possibility to produce plants edited in multiple genes by means of DNA-free approaches opens new perspectives for breeding purposes and acceptance of resultant genotypes. In this work, we have explored the polyethylene glycol (PEG)mediated delivery of ribonucleoproteins (RNPs) in tomato protoplasts using a multiplexing approach (i.e. two genes targeted simultaneously using two sgRNAs per gene) for the first time. We have analysed the editing outcome in fully developed green calli and demonstrated that tomato protoplasts are a valid cell target for RNP-mediated multiplexed genome editing with high efficiency. Key message RNP could be applied with high efficiency in a multiplexing genome editing approach in tomato protoplasts. Keywords Tomato • Ribonucleoprotein • Genome editing • Protoplast Abbreviations RNP Ribonucleoprotein HFRA High resolution fragment analysis PEG Polyethylene glycol CRISPR Clustered regularly interspaced short palindromic repeats Cas9 CRISPR associated protein 9 Tomato (Solanum lycopersicum L.) is the most cultivated vegetable worldwide after potato and is grown for both fresh and processing markets. It is considered a model plant for research because of its simple genetic nature (2n = 2x = 24), medium-size genome, short generation time, routine transformation technology in place and a high availability of plant genetic resources. With the advent of genome editing techniques and CRISPR/Cas9 in particular, many papers have been published on tomato and other vegetables (Cardi et al. 2017a; Wang et al. 2019), the vast majority relying on Agrobacterium tumefaciens (currently renamed to Rhizobium radiobacter) mediated transformation of multicellular explants for the delivery of editing reagents. In tomato, transformation and shoot regeneration is routinely achieved using cotyledonary explants, while alternative direct approaches, such as protoplast or biolistics-based delivery methods, are either not available or have limited applicability (Cardi et al. 2017b; Van Eck 2017). The delivery in plant cells of in vitro pre-assembled ribonucloprotein (RNP) complexes, constituted of protein (i.e. Cas9) bound to ribonucleic acid (i.e. the single guide RNA, sgRNA), has been demonstrated in several species (
Somatic hybridization between Solanum commersonii and S. tuberosum resulted in the production of ... more Somatic hybridization between Solanum commersonii and S. tuberosum resulted in the production of male-sterile hybrid plants, except for one fully male-fertile hybrid. The male-sterile hybrids exhibited a“pollen-less” phenotype, with rare pollen grains which were abnormal in shape and exine sculpture. Microsporogenesis and tapetal development were investigated both in male-sterile and male-fertile somatic hybrids to assess the cytological events that were
The possibility of scaling ploidy levels up and down offers several advantages for genetic studie... more The possibility of scaling ploidy levels up and down offers several advantages for genetic studies and breeding work in potato. Shoot regeneration and chromosome doubling in plants regenerated from in vitro cultures were investigated in 4 diploid (2n= 2x= 24) and 2 triploid (2n= 3x= 36) clones. Internode and leaf explants were taken from plants propagated either in vitro as shoot cultures, or in vivo in a greenhouse. Two regeneration procedures were compared. Regeneration frequencies were significantly higher using a two-step regeneration protocol and from leaf explants, while doubling was more efficient starting from in vivo grown plants. No doubling was observed in triploid clones. Considering altogether the percentage of regeneration and doubling, and the number of shoots regenerated per explant, the most efficient conditions were considered leaf explants taken from in vivo grown plants, and cultured according to a two-step regeneration protocol.
Capsicum is one of the major vegetable crops grown worldwide. Current subdivision in clades and s... more Capsicum is one of the major vegetable crops grown worldwide. Current subdivision in clades and species is based on morphological traits and coarse sets of genetic markers. Broad variability of fruits has been driven by breeding programs and has been mainly studied by linkage analysis. We discovered 746k variable sites by sequencing 1.8% of the genome in a collection of 373 accessions belonging to 11 Capsicum species from 51 countries. We describe genomic variation at population-level, confirm major subdivision in clades and species, and show that the known major subdivision of C. annuum separates large and bulky fruits from small ones. In C. annuum, we identify four novel loci associated with phenotypes determining the fruit shape, including a non-synonymous mutation in the gene Longifolia 1-like (CA03g16080). Our collection covers all the economically important species of Capsicum widely used in breeding programs and represent the widest and largest study so far in terms of the number of species and number of genetic variants analyzed. We identified a large set of markers that can be used for population genetic studies and genetic association analyses. our results provide a comprehensive and precise perspective on genomic variability in Capsicum at population-level and suggest that future fine genetic association studies will yield useful results for breeding.
Summary Solanum commersonii Dun. is a diploid (2n=2x=24, 1EBN) wild species of potential value fo... more Summary Solanum commersonii Dun. is a diploid (2n=2x=24, 1EBN) wild species of potential value for potato breeding. It is a reproductively isolated species and cannot be crossed with Tuberosum haploids (2n=2x=24, 2EBN) or other diploid 2EBNSolanum species. In order to overcome the EBN barriers, triploid hybrids were produced between Phureja-Tuberosum haploid hybrids, which form 2n pollen grains by parallel spindles,
Solanum commersonii (Cmm) is a tuber-bearing wild relative of the common potato (S. tuberosum). I... more Solanum commersonii (Cmm) is a tuber-bearing wild relative of the common potato (S. tuberosum). It is widespread in Paraguay and along the coasts of Argentina, Uruguay, and southern Brazil, from sea level up to about 400m (Correll 1962; Hawkes 1990). Typically, it is a small, bushy plant, with flowers (deep mauve to purple on the outer surface, paler within) arranged in compact inflorescences. Generally, it is diploid (2n = 2x = 24) with an endosperm balance number (EBN) equal to 1, but autotriploid forms (2n = 3x = 36) are also known (Hanneman and Bamberg 1986).
Current plant science and biotechnology in agriculture, 1999
In earlier days of plant tissue culture, protoplast fusion provided the only method of introducin... more In earlier days of plant tissue culture, protoplast fusion provided the only method of introducing or combining genes somewhat beyond the limits of sexual reproduction. Protoplast fusion is now rather overshadowed by plant transformation, a very powerful method that permits transfer into plants of genes from any taxonomic group. In view of the success of gene transfer, is protoplast fusion still of interest and value?
Solanum commersonii (+) S. tuberosum hybrids with different endosperm balance numbers (EBN) and p... more Solanum commersonii (+) S. tuberosum hybrids with different endosperm balance numbers (EBN) and ploidy were used in various crossing schemes with 4EBN S. tuberosum cultivars and a 3EBN somatic hybrid to test their behavior in intra-and inter-EBN crosses and to derive a BC 1 population for potato breeding. The somatic hybrids included 12 tetraploids (2n = 48, 3EBN), 18 hypotetraploids (2n = 43-47, 3EBN), 2 hexaploids (2n = 72, 4 or 5EBN), and 5 hypohexaploids (2n = 63-70, 4 or 5EBN). The results confirmed that in the potato, EBN is more important than actual ploidy in determining the success or failure of crosses. Indeed, crosses between some hypohexaploid hybrids and S. tuberosum cultivars gave the highest number of viable seeds per berry, and seed viability was similar to that of intra-EBN crosses between varieties. Inter-EBN crosses demonstrated that exceptions to the 2:1 maternal-paternal EBN ratio in the hybrid endosperm can sometimes be tolerated. However, an excess in maternal EBN dosage was better tolerated than an excess in paternal EBN.
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