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Advances in cell fusion technology have propelled breeding into the realm of somatic hybridization, enabling the transfer of genetic material independent of sexual reproduction. This has facilitated genome recombination both within and between species. Despite its use in plant breeding for over fifty years, somatic hybridization has been limited by cumbersome procedures, such as protoplast isolation, hybridized-cell selection and cultivation, and regeneration, particularly in woody perennial species that are difficult to regenerate. This review summarizes the development of somatic hybridization, explores the challenges and solutions associated with cell fusion technology in woody perennials, and outlines the process of protoplast regeneration. Recent advancements in genome editing and plant cell regeneration present new opportunities for applying somatic hybridization in breeding. We offer a perspective on integrating these emerging technologies to enhance somatic hybridization in woody perennial plants. [ABSTRACT FROM AUTHOR]

In the world's flower trade, gladiolus (Gladiolus spp.) is ranked first among bulbous flowers and eighth among cut flowers, with more than 30,000 different cultivars being grown. Mass multiplication and commercialization are restricted by the traditional propagation methods. However, the large-scale proliferation and improvement of the gladiolus have been accomplished with the aid of plant tissue culture and other biotechnological techniques. The current review includes a thorough examination of the growth and development parameters required for successful in vitro gladiolus development as well as cormel formation. Moreover, focus is being given to various techniques and methods such as in vitro cytogenetic stability and modification of chromosome number, in vitro mutagenesis and selection of pest resistance, in vitro identification and selection to develop virus-free germplasm, cryopreservation, synthetic seed technology, identifying virus diseases by RT-PCR, somaclonal variation, and protoplast and somatic hybridization. Molecular markers and their applications for genetic diversity analysis, relationships between different genotypes, and clonal stability analysis in Gladiolus species have been conducted by several research groups worldwide and are also being discussed. The article also covers efforts to enhance the functionality of plant phenotypes through genetic transformation. Future prospects for further improvement of ornamental gladiolus are also explored. Overall, the current review provides insight into the applications of basic and advanced biotechnological tools for gladiolus improvement. [ABSTRACT FROM AUTHOR]

Advances in cell fusion technology have propelled breeding into the realm of somatic hybridization, enabling the transfer of genetic material independent of sexual reproduction. This has facilitated genome recombination both within and between species. Despite its use in plant breeding for over fifty years, somatic hybridization has been limited by cumbersome procedures, such as protoplast isolation, hybridized-cell selection and cultivation, and regeneration, particularly in woody perennial species that are difficult to regenerate. This review summarizes the development of somatic hybridization, explores the challenges and solutions associated with cell fusion technology in woody perennials, and outlines the process of protoplast regeneration. Recent advancements in genome editing and plant cell regeneration present new opportunities for applying somatic hybridization in breeding. We offer a perspective on integrating these emerging technologies to enhance somatic hybridization in woody perennial plants.

Somatic hybridization has emerged as a valuable tool for developing novel genetic combinations in citrus breeding programs, including the creation of salt-tolerant rootstocks. In this study, the performance of a tetraploid somatic hybrid, obtained by fusing protoplasts derived from salt-tolerant Cleopatra mandarin (Citrus reshni hort. ex Tanaka) and salt-sensitive Carrizo citrange (Citrus sinensis L. Osbeck × Poncirus trifoliata L. Raf), was assessed under in vitro salt stress. Hybrid plants were characterized by leaf morphology, and ploidy level by flow cytometry and molecular markers. In vitro shoots were generated from the micropropagation of mature stem pieces of the somatic hybrid and its parents, and these were challenged by exposure to NaCl (0, 50, or 100 mM) supplemented to the media for three weeks to induce salt stress. The leaves of the somatic hybrid display intermediate morphology compared to the parental Cleopatra mandarin and Carrizo citrange rootstocks. All molecular markers successfully amplified DNA from the three cultivars; however, only 11 of 14 unequivocally confirmed somatic hybridity. The physiological and biochemical parameters, including chlorophyll content, lipid peroxidation, total phenolic compounds, antioxidants activity and proline content, were measured in the leaves. The somatic hybrid exhibited superior salt stress tolerance compared to the parent varieties, as evidenced by the reduced cellular membrane damage indicated by the lower levels of malondialdehyde and electrolyte leakage, particularly under 100 mM NaCl treatment. The somatic tetraploid hybrid also displayed higher total phenolic content than either parent, while Cleopatra mandarin exhibited the highest proline levels under 50 mm NaCl. These results demonstrate the enhanced salinity stress tolerance of the somatic hybrid compared to its parent lines, highlighting its potential as a valuable candidate for developing salt-tolerant citrus rootstocks. [ABSTRACT FROM AUTHOR]

We applied a genomic in situ hybridization (GISH) to analyze the genomic constitution of and meiotic pairing in interspecific somatic hybrids, and in a wide subset of backcrossing derivatives (BC1–BC5), from three interspecific combinations involving the cultivated potato, Solanum tuberosum (AAAA genome), and three diploid (1 EBN) wild Mexican potato species (genome BB)—S. tarnii, S. pinnatisectum, and S. bulbocastanum. The theoretically expected genomic composition was detected in the somatic hybrids (AAAABB) and in the BC1 progeny (AAAAB), whereas in the subsequent BC2–BC4 generations, the partial loss of alien chromosomes was observed and almost all the BC5 genotypes showed a complete chromosome elimination of wild species. GISH revealed a homoeologous pairing between the chromosomes of the A- and the B-genomes in each of the hybrid progenies. Using GISH, we selected introgression lines with a single chromosome of the wild species in a potato genome background, as well as introgression lines with intergenomic recombinant chromosomes. Moreover, via molecular screening, BC hybrids with diagnostic markers for the R-genes conferring resistance to late blight disease and to the quarantine pest of the potato–Columbia root-knot nematode—were selected. The potential application of the results obtained for the planning of introgressive schemes directed to the breeding of advanced lines with multiple disease and pest resistance is discussed. [ABSTRACT FROM AUTHOR]

Citrus, belonging to the Rutaceae family, is a commercial fruit worldwide, and it is mainly recognized for its nutritional, anti-oxidant, and significant medicinal properties. Citruses are a group of multifaceted fruit crops with a rich traditional knowledge, deeply rooted in ethnic culture, and the fruits have been considered to be health-protecting and health-promoting food supplements since ancient times. The presence of secondary metabolites and their bioactivities has led to the development of new alternative drugs in recent years. Diverse secondary metabolites such as flavonoids, alkaloids, carotenoids, phenolic acids, and essential oils and their high bioactive properties have imparted great value to human health based on their anti-oxidative, anti-inflammatory, anti-cancer, cardiovascular protective, and neuroprotective effects. The indigenous Citrus species of India—mainly Northeast India—have distinctive and valuable genetic traits, such as resistance to biotic and abiotic stress, distinctive aroma, flavor, etc. Hence, these species are considered to be repertoires of valuable genes for molecular breeding aimed at quality improvement. There is a need for awareness and understanding among the citrus-producing countries of the exploitation of biodiversity and the conservation of Citrus for sustainable development and bioprospecting. The current review presents a holistic view of Citrus biodiversity from a global perspective, including phytochemical constituents and health benefits. Advanced biotechnological and genomic approaches for Citrus trait improvement have also been discussed to highlight their relevance in Citrus improvement. [ABSTRACT FROM AUTHOR]

Somatic hybridization has emerged as a valuable tool for developing novel genetic combinations in citrus breeding programs, including the creation of salt-tolerant rootstocks. In this study, the performance of a tetraploid somatic hybrid, obtained by fusing protoplasts derived from salt-tolerant Cleopatra mandarin (Citrus reshni hort. ex Tanaka) and salt-sensitive Carrizo citrange (Citrus sinensis L. Osbeck × Poncirus trifoliata L. Raf), was assessed under in vitro salt stress. Hybrid plants were characterized by leaf morphology, and ploidy level by flow cytometry and molecular markers. In vitro shoots were generated from the micropropagation of mature stem pieces of the somatic hybrid and its parents, and these were challenged by exposure to NaCl (0, 50, or 100 mM) supplemented to the media for three weeks to induce salt stress. The leaves of the somatic hybrid display intermediate morphology compared to the parental Cleopatra mandarin and Carrizo citrange rootstocks. All molecular markers successfully amplified DNA from the three cultivars; however, only 11 of 14 unequivocally confirmed somatic hybridity. The physiological and biochemical parameters, including chlorophyll content, lipid peroxidation, total phenolic compounds, antioxidants activity and proline content, were measured in the leaves. The somatic hybrid exhibited superior salt stress tolerance compared to the parent varieties, as evidenced by the reduced cellular membrane damage indicated by the lower levels of malondialdehyde and electrolyte leakage, particularly under 100 mM NaCl treatment. The somatic tetraploid hybrid also displayed higher total phenolic content than either parent, while Cleopatra mandarin exhibited the highest proline levels under 50 mm NaCl. These results demonstrate the enhanced salinity stress tolerance of the somatic hybrid compared to its parent lines, highlighting its potential as a valuable candidate for developing salt-tolerant citrus rootstocks.

Interspecific hybridization is a significant plant evolutionary process and, concomitantly, a frequently used method to broaden the genetic variability of species and genetically improve crops. However, in distant crosses, many prezygotic and postzygotic barriers are encountered that prevent free, uncontrolled gene flow between species. Therefore, various experimental methods exploiting tissue culture, such as in vitro fertilization, embryo rescue, and protoplast fusion, have been developed to raise hybrids that cannot be obtained naturally through conventional approaches. Professor Maciej Zenkteler of the Adam Mickiewicz University in Poznań pioneered the use of tissue culture in Poland to overcome barriers to sexual crossing. In the mid-1960s, he employed in vitro pollination in which pollen grains were directly applied onto the surface of exposed ovules as the primary method to bypass prezygotic barriers and in vitro culture of isolated ovules and developing embryos (embryo rescue) to overcome post-zygotic barriers. These approaches proved effective for species possessing numerous ovules and large placentas, such as representatives of the families Caryophyllaceae, Solanaceae, and Brassicaceae. Soon thereafter, these methods were extended to other Polish institutes and universities and applied for crop improvement, such as for the production of intergeneric hybrids between Salix and Populus species, resynthesis and broadening of the genetic variability of oilseed rape (Brassica napus L.), and the generation of new interspecific hybrids of Allium. In the late 1970s, Professor Zenkteler was the first to undertake protoplast fusion. At that time, it appeared that no incompatibility could exist at the fusion level and that somatic hybridization would supersede the possibilities offered by sexual hybridization; however, the reality turned out to be slightly harsh. Nonetheless, successful regeneration of different interspecific hybrids of potato, tomato, or gentians has been achieved by Polish research groups thus far.

We applied a genomic in situ hybridization (GISH) to analyze the genomic constitution of and meiotic pairing in interspecific somatic hybrids, and in a wide subset of backcrossing derivatives (BC1–BC5), from three interspecific combinations involving the cultivated potato, Solanum tuberosum (AAAA genome), and three diploid (1 EBN) wild Mexican potato species (genome BB)—S. tarnii, S. pinnatisectum, and S. bulbocastanum. The theoretically expected genomic composition was detected in the somatic hybrids (AAAABB) and in the BC1 progeny (AAAAB), whereas in the subsequent BC2–BC4 generations, the partial loss of alien chromosomes was observed and almost all the BC5 genotypes showed a complete chromosome elimination of wild species. GISH revealed a homoeologous pairing between the chromosomes of the A- and the B-genomes in each of the hybrid progenies. Using GISH, we selected introgression lines with a single chromosome of the wild species in a potato genome background, as well as introgression lines with intergenomic recombinant chromosomes. Moreover, via molecular screening, BC hybrids with diagnostic markers for the R-genes conferring resistance to late blight disease and to the quarantine pest of the potato–Columbia root-knot nematode—were selected. The potential application of the results obtained for the planning of introgressive schemes directed to the breeding of advanced lines with multiple disease and pest resistance is discussed.

Interspecific hybridization is a significant plant evolutionary process and, concomitantly, a frequently used method to broaden the genetic variability of species and genetically improve crops. However, in distant crosses, many prezygotic and postzygotic barriers are encountered that prevent free, uncontrolled gene flow between species. Therefore, various experimental methods exploiting tissue culture, such as in vitro fertilization, embryo rescue, and protoplast fusion, have been developed to raise hybrids that cannot be obtained naturally through conventional approaches. Professor Maciej Zenkteler of the Adam Mickiewicz University in Poznań pioneered the use of tissue culture in Poland to overcome barriers to sexual crossing. In the mid-1960s, he employed in vitro pollination in which pollen grains were directly applied onto the surface of exposed ovules as the primary method to bypass prezygotic barriers and in vitro culture of isolated ovules and developing embryos (embryo rescue) to overcome post-zygotic barriers. These approaches proved effective for species possessing numerous ovules and large placentas, such as representatives of the families Caryophyllaceae, Solanaceae, and Brassicaceae. Soon thereafter, these methods were extended to other Polish institutes and universities and applied for crop improvement, such as for the production of intergeneric hybrids between Salix and Populus species, resynthesis and broadening of the genetic variability of oilseed rape (Brassica napus L.), and the generation of new interspecific hybrids of Allium. In the late 1970s, Professor Zenkteler was the first to undertake protoplast fusion. At that time, it appeared that no incompatibility could exist at the fusion level and that somatic hybridization would supersede the possibilities offered by sexual hybridization; however, the reality turned out to be slightly harsh. Nonetheless, successful regeneration of different interspecific hybrids of potato, tomato, or gentians has been achieved by Polish research groups thus far. [ABSTRACT FROM AUTHOR]

This paper reviews the results of studies of interspecies hybridization, polyploidization, as well as phylogenetic relationships of Solanum species and members of closely related taxa by such molecular cytogenetic techniques as genomic (GISH) and fluorescent (FISH) DNA-DNA in situ hybridization. The latter was used to determine the genomic composition and origin of wild species of the Petota section, while the FISH technique was used for detecting intergenomic collinearity. The combination of these two types of research made possible a comparative analysis of karyotypes and genomes, thus allowing a better understanding of the meiotic interchromosomal interactions in hybrids. This review primarily focuses on the studies of wild allopolyploid potato species and artificially created intergeneric and interspecific hybrids of the genus Solanum and their offspring.

• Cabbage is an extensively grown vegetable crop because of its nutritional and medicinal value. • There is need to develop such crop enhancement initiatives that aim to provide improved crop varieties. • This comprehensive review deals with latest biotechnological techniques useful for cabbage improvement programs. Cabbage (Brassica oleracea var. capitata L.) is an extensively grown vegetable crop because of its nutritional and medicinal value. The crop is pervaded by various biotic and abiotic stresses and hence constant improvement of cabbage is of paramount importance. Crop improvement relies on gene and genome modulation, either directly or indirectly. Conventional breeding mainly constitutes the indirect approach; while as direct, approach includes RNAi, genome editing, overexpression etc. Conventional breeding methods are insufficient for cabbage improvement, because of being time-consuming and are limited by sexual incompatibility barriers. Therefore, biotechnological interventions become a major requirement. In order to address a range of biotic and abiotic stresses, it is essential to implement crop enhancement initiatives focused on the development of improved crop varieties capable of addressing the existing challenges. This comprehensive review deals with biotechnological techniques such as tissue culture, somatic hybridization, in vitro grafting, molecular assisted breeding, genetic engineering, RNAi, genome editing as well as some other approaches useful for cabbage improvement programmes. [ABSTRACT FROM AUTHOR]

Asymmetric somatic hybridization is an efficient strategy for crop breeding by introducing exogenous chromatin fragments, which leads to whole genomic shock and local chromosomal shock that induces genome-wide genetic variation including indel (insertion and deletion) and nucleotide substitution. Nucleotide substitution causes synonymous codon usage bias (SCUB), an indicator of genomic mutation and natural selection. However, how asymmetric somatic hybridization affects SCUB has not been addressed. Here, we explored this issue by comparing expressed sequence tags of a common wheat cultivar and its asymmetric somatic hybrid line. Asymmetric somatic hybridization affected SCUB and promoted the bias to A- and T-ending synonymous codon (SCs). SCUB frequencies in chromosomes introgressed with exogenous fragments were comparable to those in chromosomes without exogenous fragments, showing that exogenous fragments had no local chromosomal effect. Asymmetric somatic hybridization affected SCUB frequencies in indel-flanking sequences more strongly than in non-flanking sequences, and this stronger effect was present in both chromosomes with and without exogenous fragments. DNA methylation-driven SCUB shift was more pronounced than other SC pairs. SCUB shift was similar among seven groups of allelic chromosomes as well as three sub-genomes. Our work demonstrates that the SCUB shift induced by asymmetric somatic hybridization is attributed to the whole genomic shock, and DNA methylation is a putative force of SCUB shift during asymmetric somatic hybridization. Asymmetric somatic hybridization provides an available method for deepening the nature of SCUB shift and genetic variation induced by genomic shock.

Asymmetric somatic hybridization is an efficient strategy for crop breeding by introducing exogenous chromatin fragments, which leads to whole genomic shock and local chromosomal shock that induces genome-wide genetic variation including indel (insertion and deletion) and nucleotide substitution. Nucleotide substitution causes synonymous codon usage bias (SCUB), an indicator of genomic mutation and natural selection. However, how asymmetric somatic hybridization affects SCUB has not been addressed. Here, we explored this issue by comparing expressed sequence tags of a common wheat cultivar and its asymmetric somatic hybrid line. Asymmetric somatic hybridization affected SCUB and promoted the bias to A- and T-ending synonymous codon (SCs). SCUB frequencies in chromosomes introgressed with exogenous fragments were comparable to those in chromosomes without exogenous fragments, showing that exogenous fragments had no local chromosomal effect. Asymmetric somatic hybridization affected SCUB frequencies in indel-flanking sequences more strongly than in non-flanking sequences, and this stronger effect was present in both chromosomes with and without exogenous fragments. DNA methylation-driven SCUB shift was more pronounced than other SC pairs. SCUB shift was similar among seven groups of allelic chromosomes as well as three sub-genomes. Our work demonstrates that the SCUB shift induced by asymmetric somatic hybridization is attributed to the whole genomic shock, and DNA methylation is a putative force of SCUB shift during asymmetric somatic hybridization. Asymmetric somatic hybridization provides an available method for deepening the nature of SCUB shift and genetic variation induced by genomic shock. [ABSTRACT FROM AUTHOR]

In this study, we aimed to produce a citrus fruit with no seed and with edible peel. Therefore, the seedless Yoshida navel oranges (Citrus sinensis 'Yoshida navel') and Jangsil kumquat (Fortunella japonica) with edible peel were used as the starting materials. Since two varieties of different genera and different flowering periods would be difficult to crossbreed, protoplast fusion was conducted using the polyethylene glycol method. Yoshida navel orange and Jangsil kumquat were used as an embryogenic callus line and a mesophyll line, respectively. The regenerated plants were analyzed by flow cytometry to identify tetraploids, which were further evaluated by polymerase chain reaction, using simple sequence repeat markers specific for the nuclear and cytoplasmic organellar DNAs of the two parents, to confirm allotetraploids and cybrids. Sixteen allotetraploids were finally produced and characterized phenotypically for leaf morphology and fruit quality. All allotetraploids contained mitochondria originating from Yoshida navel orange; 12 contained chloroplasts derived from navel orange whereas four contained chloroplasts of kumquat origin. We investigated both plant and fruit characteristics of allotetraploids derived from the protoplast fusion of navel orange and kumquat. The sizes of the allotetraploid leaves were intermediate between those of both parents. However, in contrast to the spindle-shaped leaves of both parents, those of the allotetraploids were obovate. Petiole wings were absent in the allotetraploids, a known kumquat trait. Similar to navel oranges, flowering time was mid-May, and pollen was sterile, whereas fruit size, external shape, soluble solids content, and acidity were all intermediate between the two parents. Peel thickness and the number of segments were similar to those of kumquat, and flesh weight was similar to that of navel orange. Collectively, these results indicated that the intergeneric allotetraploids derived from navel orange and kumquat inherited favorable traits of both parents and could be produced and selected despite a lack of extensive parental variation. This is one of the first reports showing fruiting results of protoplast-fused citrus plants. The study reported characteristics of the fruits from both the original plants as well as from the protoplast-fused plant. Detailed characteristics of the allotetraploid fruit produced by protoplast fusion would be very helpful for future polyploid studies. [ABSTRACT FROM AUTHOR]

Plant biotechnologies have demonstrated to be a resource to cope with world population growth and environmental sustainability by increasing yield and partially reducing reliance upon agrochemicals. Nevertheless, despite several nutritionally enhanced crops have been obtained through genetic manipulation, their deployment in agriculture – to date – still remains marginal. In this article, we discuss the different GM approaches effectively employed to produce nutritionally improved food crops, as well as the future perspectives to overcome the barriers preventing the wide cultivation/commercialization of these products. [ABSTRACT FROM AUTHOR]

Protoplast fusion has great potential in citrus improvement. Although citrus mesophyll protoplasts usually cannot divide and regenerate, symmetric protoplast fusion of embryogenic callus protoplast + mesophyll protoplast sometimes results in the regeneration of mesophyll-parent-type cybrids. It suggested that mitochondrial DNA (mtDNA) from protoplasts of embryogenic callus parent plays an important role in stimulating division and regeneration of mesophyll protoplasts. Herein, somatic fusion was conducted via electrofusion between callus protoplasts isolated from Valencia orange [Citrus sinensis (L.) Osbeck] cell suspension cultures and transgenic GFP-tagged mesophyll protoplasts from the same genotype, i.e. transgenic Valencia orange plants containing the green fluorescent protein (GFP) gene, in an effort to elucidate whether mtDNA of callus line could stimulate the division and regeneration of mesophyll protoplasts from the same genotype. Two embryoids and one plantlet with GFP expression were successfully obtained and subsequent ploidy analysis by flow cytometry indicated that they were all diploids. The regenerated diploid embryoids and plantlet with GFP expression could be considered as ‘cybrids’ with mtDNA from the callus protoplasts of Valencia orange. The result indicated that citrus mesophyll-parent-type cybrid regeneration needed the stimulation of mtDNA from protoplasts of embryogenic callus parent regardless of their origin either from another genotype or the same genotype as the mesophyll parent.

Somatic hybridization is performed to obtain significant cytoplasmic male sterility (CMS) lines, whose CMS genes are derived either from the transfer of sterile genes from the mitochondrial genome of donor parent to the counterpart of receptor or production of new sterile genes caused by mitochondrial genome recombination of the biparent during protoplast fusion. In this study, a novel male sterile line, SaNa-1A, was obtained from the somatic hybridization between Brassica napus and Sinapis alba. The normal anther development of the maintainer line, SaNa-1B, and the abortive process of SaNa-1A were described through phenotypic observations and microtome sections. The floral organ of the sterile line SaNa-1A was sterile with a shortened filament and deflated anther. No detectable pollen grains were found on the surface of the sterile anthers. Semi-thin sections indicated that SaNa-1A aborted in the pollen mother cell (PMC) stage when vacuolization of the tapetum and PMCs began. The tapetum radically elongated and became highly vacuolated, occupying the entire locule together with the vacuolated microspores. Therefore, SaNa-1A is different from other CMS lines, such as ogu CMS, pol CMS and nap CMS as shown by the abortive process of the anther.