Sorghum (<i>Sorghum</i><span> <i>bicolor</i></span> (L.) Moench) is one of the world’s leading cereal crops in agricultural production, which has a special importance in the arid r...Sorghum (<i>Sorghum</i><span> <i>bicolor</i></span> (L.) Moench) is one of the world’s leading cereal crops in agricultural production, which has a special importance in the arid regions. However, unlike other cereals, sorghum grain has a lower nutritional value, which is caused, inter alia, by the resistance of its seed storage proteins (kafirins) to protease digestion. One of the effective approaches to improve the nutritional value of sorghum grain is to obtain mutants with partially or completely suppressed synthesis or altered amino acid composition of kafirins. The employment of genome editing may allow to solve this problem by introducing mutations into the nucleotide sequences of the <i>α</i>- and <i>γ</i>-kafirin genes. In this study, genomic target motifs (23 bp sequences) were selected for the introduction of mutations into the <i>α-</i> and <i>γ-KAFIRIN</i> genes of sorg<span>hum. The design of the gRNAs was conducted using the online tools</span> CRISPROR and CHOPCHOP. <a name="_Hlk55317737"></a>Two most suitable targets were chosen for <i>α-KAFIRIN</i> (<i>k</i><span>1<i>C</i>5</span>) and two for <i>γ-KAFIRIN</i> (<i>gKAF</i><span>1</span>) genes. The insertion of respective sequences in the generic vector pSH121 was performed at the <i>BsaI</i> (<i>Eco</i><span>31<i>I</i></span>) sites. Validation of the cloning procedure was performed by DNA sequencing. Subcloning of the resulting constructs was performed using the <i>SfiI</i> restriction sites into the compatible binary vector B479p7oUZm-LH. The correct assembly of binary vectors was confirmed by restriction analysis using the <i>MluI</i> and <i>SfiI</i> cleavage sites. The four vectors created (1C</span><span style="font-family:""> </span><span style="font-family:"">-</span><span style="font-family:""> </span><span style="font-family:"">4C) were transferred by electroporation into the <i>Agrobacterium</i><span> <i>tumefaciens</i></span> strain AGL0. Currently, this vector series is used for stable transformation of sorghum using immature embryo explants.展开更多
Investigation of male sterility mutations is an effective approach for identification of genes involved in anther and pollen development. The comparison of “cytological phenotypes” of newly induced mutants with phen...Investigation of male sterility mutations is an effective approach for identification of genes involved in anther and pollen development. The comparison of “cytological phenotypes” of newly induced mutants with phenotypes determined by already known genes favors elucidation of genetic control of diverse microsporo- and gametogenesis stages. In this paper, we describe pollen development in the grain sorghum line Zh10-asc1 with mutation of male sterility. This line was obtained from callus culture treated by sodium ascorbate. A wide spectrum of abnormalities in microsporogenesis have been found, such as cytomixis, chromosomal laggards, chromosome disjunction, adhesion of chromosomes, disturbed cytokinesis, and others. In tapetum, the cells with one nucleus, with unequal nuclei, and with micronuclei have been observed. During pollen grain (PG) maturation abnormalities in starch accumulation and delay of development often took place. In mature anthers, a variety of pollen grain types have been revealed: fertile, of irregular shape, incompletely filled with starch, PGs delayed at the uni-nucleate or bi-nucleate gametophyte stages, with partially or fully degenerated contents, and with abnormal coloration. Variation in spectrum and the frequency of disturbances between the flowers of one and the same plant have been revealed. The reasons for significant genetic and epigenetic instability are discussed.展开更多
文摘Sorghum (<i>Sorghum</i><span> <i>bicolor</i></span> (L.) Moench) is one of the world’s leading cereal crops in agricultural production, which has a special importance in the arid regions. However, unlike other cereals, sorghum grain has a lower nutritional value, which is caused, inter alia, by the resistance of its seed storage proteins (kafirins) to protease digestion. One of the effective approaches to improve the nutritional value of sorghum grain is to obtain mutants with partially or completely suppressed synthesis or altered amino acid composition of kafirins. The employment of genome editing may allow to solve this problem by introducing mutations into the nucleotide sequences of the <i>α</i>- and <i>γ</i>-kafirin genes. In this study, genomic target motifs (23 bp sequences) were selected for the introduction of mutations into the <i>α-</i> and <i>γ-KAFIRIN</i> genes of sorg<span>hum. The design of the gRNAs was conducted using the online tools</span> CRISPROR and CHOPCHOP. <a name="_Hlk55317737"></a>Two most suitable targets were chosen for <i>α-KAFIRIN</i> (<i>k</i><span>1<i>C</i>5</span>) and two for <i>γ-KAFIRIN</i> (<i>gKAF</i><span>1</span>) genes. The insertion of respective sequences in the generic vector pSH121 was performed at the <i>BsaI</i> (<i>Eco</i><span>31<i>I</i></span>) sites. Validation of the cloning procedure was performed by DNA sequencing. Subcloning of the resulting constructs was performed using the <i>SfiI</i> restriction sites into the compatible binary vector B479p7oUZm-LH. The correct assembly of binary vectors was confirmed by restriction analysis using the <i>MluI</i> and <i>SfiI</i> cleavage sites. The four vectors created (1C</span><span style="font-family:""> </span><span style="font-family:"">-</span><span style="font-family:""> </span><span style="font-family:"">4C) were transferred by electroporation into the <i>Agrobacterium</i><span> <i>tumefaciens</i></span> strain AGL0. Currently, this vector series is used for stable transformation of sorghum using immature embryo explants.
文摘Investigation of male sterility mutations is an effective approach for identification of genes involved in anther and pollen development. The comparison of “cytological phenotypes” of newly induced mutants with phenotypes determined by already known genes favors elucidation of genetic control of diverse microsporo- and gametogenesis stages. In this paper, we describe pollen development in the grain sorghum line Zh10-asc1 with mutation of male sterility. This line was obtained from callus culture treated by sodium ascorbate. A wide spectrum of abnormalities in microsporogenesis have been found, such as cytomixis, chromosomal laggards, chromosome disjunction, adhesion of chromosomes, disturbed cytokinesis, and others. In tapetum, the cells with one nucleus, with unequal nuclei, and with micronuclei have been observed. During pollen grain (PG) maturation abnormalities in starch accumulation and delay of development often took place. In mature anthers, a variety of pollen grain types have been revealed: fertile, of irregular shape, incompletely filled with starch, PGs delayed at the uni-nucleate or bi-nucleate gametophyte stages, with partially or fully degenerated contents, and with abnormal coloration. Variation in spectrum and the frequency of disturbances between the flowers of one and the same plant have been revealed. The reasons for significant genetic and epigenetic instability are discussed.
