Despite intensive optimization efforts, developing an efficient sequence-specific CRISPR/Cas-medi... more Despite intensive optimization efforts, developing an efficient sequence-specific CRISPR/Cas-mediated genome editing method remains a challenge, especially in polyploid cereal species such as wheat. Validating the efficacy of nuclease constructs prior to using them in planta is, thus, a major step of every editing experiment. Several construct evaluation strategies were proposed, with PEG-mediated plasmid transfection of seedling-derived protoplasts becoming the most popular. However, the usefulness of this approach is affected by associated construct copy number bias and chromatin relaxation, both influencing the outcome. Therefore, to achieve a reliable evaluation of CRISPR/Cas9 constructs, we proposed a system based on an Agrobacterium-mediated transformation of established wheat cell suspension cultures. This system was used for the evaluation of a CRISPR/Cas9 construct designed to target the ABA 8′-hydroxylase 1 gene. The efficiency of editing was verified by cost-effective mea...
1 Department of Animal Improvement, Polish Academy of Sciences Institute of Genetics and Animal B... more 1 Department of Animal Improvement, Polish Academy of Sciences Institute of Genetics and Animal Breeding, Jastrzebiec, 05-552 Wolka Kosowska, Poland 2 Department of Molecular Cytogenetics, Polish Academy of Sciences Institute of Genetics and Animal Breeding, Jatrzebiec, 05-552 Wolka Kosowska, Poland 3 Department of Plant Biotechnology and Cytogenetics, Plant Breeding and Acclimatization Institute, Radzikow, 05-870 Blonie, Poland 4 Chair of Sanitary Biology and Biotechnology, Bialystok Technical University, Wiejska 45a, 15-351 Bialystok. Poland
Czech Journal of Genetics and Plant Breeding, 2017
Isolated microspore culture is an excellent system for the production of doubled haploids in many... more Isolated microspore culture is an excellent system for the production of doubled haploids in many crops, including barley. In a more traditional barley anther culture method copper sulphate is known to enhance plant regeneration. Here we report that one hundred times higher concentration of copper sulphate in the isolated microspore culture of two spring barley genotypes compared to the standard content in the induction medium resulted in a 34% increase of total plant regeneration. Detailed analysis of plant regeneration showed that additional supplementation of copper sulphate increased not only the regeneration of green plants but also proportionately that of albino plants. Hence, the results from two studied genotypes do not support an assumption that the addition of copper reduces albinism in barley microspore culture.
Plant regeneration from in vitro cultures originating from different explants (anthers, young inf... more Plant regeneration from in vitro cultures originating from different explants (anthers, young inflorescences, immature embryos, leaf bases) has been achieved from all the major cereal crops. Regeneration is accomplished via either shoot-root morphogenesis or somatic embryogenesis. Special efforts have been made to establish embryogenic cultures, as somatic embryos are superior in producing single cell derived and true type regenerants. Embryogenic cultures have been induced from Zea mays, Oryza sativa, Hordeum vulgare, Triticum aestivum, Secale cereale and Triticale. The efficiency of culture initiation, long term maintenance of totipotent cultures, and plant regeneration is influenced by the genotype used, the media composition and physical culture conditions. Not all of the tissue culture derived plants exhibit a normal phenotype and those are classified as somaclonal variants. The regenerated plants and their progeny are analyzed genetically and at the molecular level. These studies include investigations as to whether gene expression in tissue culture derived plants is altered by the degree of methylation, or can be related to deletions, inversions or other rearrangements in known gene sequences.
... Such tech-niques have been described for human and animal chromosomes (Gosden and Hanratty 19... more ... Such tech-niques have been described for human and animal chromosomes (Gosden and Hanratty 1993; Terkelsen et al. ... 756 Page 9. Bedbrook JR, Jones J, O'Dell M, Thompson RD, Flavell RB (1980) A molecular description of telomeric heterochromatin in Secale species. ...
... 54, suppl. 1, 2012 Page 40. Prenatal development of the uterus in domestic cat: preliminary L... more ... 54, suppl. 1, 2012 Page 40. Prenatal development of the uterus in domestic cat: preliminary LM and SEM study Ewelina Prozorowska, Hanna Jackowiak Poznań University of Life Sciences, Department of Histology and Embryology. ...
At the Plant Breeding and Acclimatization Institute, National Research Institute (IHAR), research... more At the Plant Breeding and Acclimatization Institute, National Research Institute (IHAR), research on plant tissue cultures began in 1970s. Some in vitro methods, such as anther cultures or isolated microspore cultures, have been directly used to produce homozygous lines in plant breeding. These methods are also a prerequisite for genetic manipulation for both scientific and practical purposes. Here, we describe the achievements of the Department of Biotechnology and Cytogenetics over the last few decades in the area of somatic embryogenesis of cereals, genetic modifications, GMO detection and identification, and GMO legislation. This long-term research on plant regeneration systems has led to the development of the first transgenic triticale plants in the world and the first transgenic plants in Poland, which was followed by a multi-generation study of triticale transgene stability. The gene flow of transgenic triticale and transgenic maize investigated in field studies under Polish...
Plant tissue culture techniques are handy tools for obtaining unique plant materials that are dif... more Plant tissue culture techniques are handy tools for obtaining unique plant materials that are difficult to propagate or important for agriculture. Homozygous materials derived through in vitro cultures are invaluable and significantly accelerate the evaluation of new varieties, e.g., cereals. The induction of somatic embryogenesis/androgenesis and the regeneration and its efficiency can be influenced by the external conditions of tissue culture, such as the ingredients present in the induction or regeneration media. We have developed an approach based on biological system, molecular markers, Fourier Transform Infrared spectroscopy, and structural equation modeling technique to establish links between changes in sequence and DNA methylation at specific symmetric (CG, CHG) and asymmetric (CHH) sequences, glutathione, and green plant regeneration efficiency in the presence of variable supplementation of induction medium with copper ions. The methylation-sensitive Amplified Fragment Len...
Despite intensive optimization efforts, developing an efficient sequence-specific CRISPR/Cas-medi... more Despite intensive optimization efforts, developing an efficient sequence-specific CRISPR/Cas-mediated genome editing method remains a challenge, especially in polyploid cereal species such as wheat. Validating the efficacy of nuclease constructs prior to using them in planta is, thus, a major step of every editing experiment. Several construct evaluation strategies were proposed, with PEG-mediated plasmid transfection of seedling-derived protoplasts becoming the most popular. However, the usefulness of this approach is affected by associated construct copy number bias and chromatin relaxation, both influencing the outcome. Therefore, to achieve a reliable evaluation of CRISPR/Cas9 constructs, we proposed a system based on an Agrobacterium-mediated transformation of established wheat cell suspension cultures. This system was used for the evaluation of a CRISPR/Cas9 construct designed to target the ABA 8′-hydroxylase 1 gene. The efficiency of editing was verified by cost-effective mea...
1 Department of Animal Improvement, Polish Academy of Sciences Institute of Genetics and Animal B... more 1 Department of Animal Improvement, Polish Academy of Sciences Institute of Genetics and Animal Breeding, Jastrzebiec, 05-552 Wolka Kosowska, Poland 2 Department of Molecular Cytogenetics, Polish Academy of Sciences Institute of Genetics and Animal Breeding, Jatrzebiec, 05-552 Wolka Kosowska, Poland 3 Department of Plant Biotechnology and Cytogenetics, Plant Breeding and Acclimatization Institute, Radzikow, 05-870 Blonie, Poland 4 Chair of Sanitary Biology and Biotechnology, Bialystok Technical University, Wiejska 45a, 15-351 Bialystok. Poland
Czech Journal of Genetics and Plant Breeding, 2017
Isolated microspore culture is an excellent system for the production of doubled haploids in many... more Isolated microspore culture is an excellent system for the production of doubled haploids in many crops, including barley. In a more traditional barley anther culture method copper sulphate is known to enhance plant regeneration. Here we report that one hundred times higher concentration of copper sulphate in the isolated microspore culture of two spring barley genotypes compared to the standard content in the induction medium resulted in a 34% increase of total plant regeneration. Detailed analysis of plant regeneration showed that additional supplementation of copper sulphate increased not only the regeneration of green plants but also proportionately that of albino plants. Hence, the results from two studied genotypes do not support an assumption that the addition of copper reduces albinism in barley microspore culture.
Plant regeneration from in vitro cultures originating from different explants (anthers, young inf... more Plant regeneration from in vitro cultures originating from different explants (anthers, young inflorescences, immature embryos, leaf bases) has been achieved from all the major cereal crops. Regeneration is accomplished via either shoot-root morphogenesis or somatic embryogenesis. Special efforts have been made to establish embryogenic cultures, as somatic embryos are superior in producing single cell derived and true type regenerants. Embryogenic cultures have been induced from Zea mays, Oryza sativa, Hordeum vulgare, Triticum aestivum, Secale cereale and Triticale. The efficiency of culture initiation, long term maintenance of totipotent cultures, and plant regeneration is influenced by the genotype used, the media composition and physical culture conditions. Not all of the tissue culture derived plants exhibit a normal phenotype and those are classified as somaclonal variants. The regenerated plants and their progeny are analyzed genetically and at the molecular level. These studies include investigations as to whether gene expression in tissue culture derived plants is altered by the degree of methylation, or can be related to deletions, inversions or other rearrangements in known gene sequences.
... Such tech-niques have been described for human and animal chromosomes (Gosden and Hanratty 19... more ... Such tech-niques have been described for human and animal chromosomes (Gosden and Hanratty 1993; Terkelsen et al. ... 756 Page 9. Bedbrook JR, Jones J, O'Dell M, Thompson RD, Flavell RB (1980) A molecular description of telomeric heterochromatin in Secale species. ...
... 54, suppl. 1, 2012 Page 40. Prenatal development of the uterus in domestic cat: preliminary L... more ... 54, suppl. 1, 2012 Page 40. Prenatal development of the uterus in domestic cat: preliminary LM and SEM study Ewelina Prozorowska, Hanna Jackowiak Poznań University of Life Sciences, Department of Histology and Embryology. ...
At the Plant Breeding and Acclimatization Institute, National Research Institute (IHAR), research... more At the Plant Breeding and Acclimatization Institute, National Research Institute (IHAR), research on plant tissue cultures began in 1970s. Some in vitro methods, such as anther cultures or isolated microspore cultures, have been directly used to produce homozygous lines in plant breeding. These methods are also a prerequisite for genetic manipulation for both scientific and practical purposes. Here, we describe the achievements of the Department of Biotechnology and Cytogenetics over the last few decades in the area of somatic embryogenesis of cereals, genetic modifications, GMO detection and identification, and GMO legislation. This long-term research on plant regeneration systems has led to the development of the first transgenic triticale plants in the world and the first transgenic plants in Poland, which was followed by a multi-generation study of triticale transgene stability. The gene flow of transgenic triticale and transgenic maize investigated in field studies under Polish...
Plant tissue culture techniques are handy tools for obtaining unique plant materials that are dif... more Plant tissue culture techniques are handy tools for obtaining unique plant materials that are difficult to propagate or important for agriculture. Homozygous materials derived through in vitro cultures are invaluable and significantly accelerate the evaluation of new varieties, e.g., cereals. The induction of somatic embryogenesis/androgenesis and the regeneration and its efficiency can be influenced by the external conditions of tissue culture, such as the ingredients present in the induction or regeneration media. We have developed an approach based on biological system, molecular markers, Fourier Transform Infrared spectroscopy, and structural equation modeling technique to establish links between changes in sequence and DNA methylation at specific symmetric (CG, CHG) and asymmetric (CHH) sequences, glutathione, and green plant regeneration efficiency in the presence of variable supplementation of induction medium with copper ions. The methylation-sensitive Amplified Fragment Len...
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