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Effect of drought stress on male fertility restoration in A3 CMS-inducing cytoplasm of sorghum

The Crop Journal 2017年第4期5卷 282-289页

作者： Valentin V.Kozhemyakin lev a.elkonin Jeffery A.Dahlberg Department of Biotechnology Agricultural Research Institute of South-East Region Tulaikov Street 7 Saratov 410010 Russia Kearney Agricultural Research & Extension (KARE) 9240 South Riverbend Avenue Parlier CA 93648 USA

Use of cytoplasmic male sterility (CMS) in hybrid breeding requires effective male fertility-restoring lines. In sorghum, very few restoring lines that can restore fertility in A_3 CMS have been reported. To identify the reasons for this deficiency, F_1 and F_2 hybrids of an A_3 CMS line crossed with the line IS1112C, a donor of fertility-restoring (Rf) genes for A_3 cytoplasm, and testcrosses of fertile plants to A_3CMS lines were grown under contrasting water availability regimes in dryland and irrigated field plots. In the irrigated plots the frequency of fertile plants in testcrosses was twice that in dryland plots (P < 0.05). Fertile plants from the F_2 family grown in the irrigated plots showed significantly higher restoration ability than fertile plants from the same family grown in dryland plots. F_3 plants from the F_2 family grown in irrigated plots yielded on average a sixfold higherfrequency of fertile plants in testcrosses than F_3 plants derived from dryland plots (P < 0.01).Fertility of testcross hybrids correlated negatively with air vapor pressure deficit (VPD) at flowering (r = - 0.96; P < 0.01) suggesting that VPD is a trigger for downregulation of Rf genes for A_3 cytoplasm.

关键词： Sorghum bicolor (L.) Moench Cytoplasmic male sterility A3 cytoplasm Fertility-restoring genes Epigenetics Vapor pressure deficit

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Development of sorghum mutants with improved in vitro protein digestibility by CRISPR/Cas9 editing of kafirin genes

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作物学报:英文版 2023年第5期11卷 1411-1418页

作者： lev a.elkonin Grigoriy A.Gerashchenkov Natalie V.Borisenko Odyssey A.Kenzhegulov Saule Kh.Sarsenova Natalya A.Rozhnova Valery M.Panin Federal Centre of Agriculture Research of South-East Region Saratov 410010Russia Institute of Biochemistry and Genetics-Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences Ufa 450054Russia

Sorghum(Sorghum bicolor(L.) Moench) is a major world crop that is a reliable source of fodder and food grain in arid regions. However, unlike other cereals, sorghum grain has low nutritional value, owing mainly to the resistance of its storage proteins(kafirins) to protease digestion. Changing the composition of kafirins or their primary structure may address this problem. To induce mutations in kafirin-encoding genes that were expected to disturb their accumulation in endosperm cells, we used a genome-editing approach. By Agrobacterium-mediated genetic transformation of immature embryos of cv. Avans, we obtained 14 transgenic plants with genetic constructs for site-directed mutagenesis of the k1C5 and g KAF1 genes encoding 22 k Da a-and 28 kDa γ-kafirins, respectively. Sequencing of 5 regenerants obtained by using k1C5-addressing vector revealed two plants with mutations. T_1 progeny of these mutants had higher in vitro digestibility of endosperm proteins(86%–92%), in comparison with the donor Avans(63%–67%). The kernels of these plants had a thick vitreous endosperm. A mutant with increased in vitro protein digestibility and vitreous endosperm, carrying a mutation in the target sequence, was also obtained by use of the gKAF1-addressing vector. Thus, using genome editing technology, we have obtained mutants with improved kafirin digestibility that can be used in sorghum breeding.

关键词： Sorghum CRISPR/Cas Kafirins In vitro protein digestibility Vitreous endosperm

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Development of sorghum mutants with improved in vitro protein digestibility by CRISPR/Cas9 editing of kafirin genes

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The Crop Journal 2023年第5期

作者： lev a.elkonin Grigoriy A.Gerashchenkov Natalie V.Borisenko Odyssey A.Kenzhegulov Saule Kh.Sarsenova Natalya A.Rozhnova Valery M.Panin

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Pseudogamous Apomixis in Maize and Sorghum in Diploid-Tetraploid Crosses

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Phyton-International Journal of Experimental Botany 2019年第4期88卷 389-401页

作者： Marina Tsvetova lev elkonin Yulia Italianskaya Agricultural Research Institute of South-East Region Department of BiotechnologySaratov410010Russia

Apomictic seed development is a complex process including formation of unreduced embryo sac,parthenogenetic embryo development from the egg cell,and endosperm formation either autonomously,or due to fertilization of polar nuclei by the sperm(under pseudogamous form of apomixis).In the latter case,an obstacle to the normal endosperm development is disturbance of maternal(m)-to-paternal(p)genomic ratio 2m:1p that occurs in the cases of pollination of unreduced embryo sac with haploid *** of tetraploid pollinators can overcome this problem because in such crosses maternal-to-paternal genomic ratio is 4m:2p that provides formation of kernels with plump *** tetraploid lines as pollen parents we observed formation of plump kernels on the ears and panicles of diploid maize and sorghum *** kernels had hybrid endosperm and diploid maternal-type embryo or hybrid embryo with different ploidy level(2n,3n,4n).The frequencies of plump kernels on the ear ranged from 0.2-0.3%to 5.7-6.2%counting from the number of ***-type plants were found in two maize lines,their frequency varying from 10.7 to 37.5%of the progeny *** CMS-lines of sorghum pollinated with tetraploid sorghum accessions,the frequency of plump kernels ranged from 0.6 to 14.0%counting from the number of ovaries;the frequency of maternal-type plants varied from 33.0 up to 96.1%.The hybrid nature of endosperm of the kernels that gave rise to maternal-type plants has been proved by marker gene expression and by SDS-electrophoresis of endosperm *** data testify to variable modes of seed formation under diploid×tetraploid crosses in maize and sorghum both by amphi-and by ***,usage of tetraploid pollinators might be a promising approach for isolation of apomixis in maize and sorghum accessions.

关键词： Interploidy crosses genomic balance unreduced embryo sac pseudogamous apomixis Zea mays L Sorghum bicolor(L.)Moench

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Cytological Investigation of Pollen Development in Sorghum Line with Male Sterility Induced by Sodium Ascorbate in Tissue Culture

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American Journal of Plant Sciences 2013年第7期4卷 11-18页

作者： Marina I. Tsvetova lev A. elkonin Department of Biotechnology Agricultural Research Institute for South-East Region Saratov Russia.

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.

关键词： Male Sterility Microsporogenesis Sodium Ascorbate In Vitro Mutagenesis Genetic Instability Sorghum bicolor (L.) Moench

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Binary Vector Construction for Site-Directed Mutagenesis of <i>Kafirin</i>Genes in Sorghum

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American Journal of Plant Sciences 2021年第8期12卷 1276-1287页

作者： Grigoriy A. Gerashchenkov lev A. elkonin Kirill G. Gerashchenkov Natalia A. Rozhnova Stefan Hiekel Jochen Kumlehn Alexey V. Chemeris Institute of Biochemistry and Genetics Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences Ufa Russia Federal Centre of Agriculture Research of South-East Region Saratov Russia Kazan Federal University Institute of Fundamental Medicine and Biology Kazan Russia Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben Germany

Sorghum (Sorghum bicolor (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 α- and γ-kafirin genes. In this study, genomic target motifs (23 bp sequences) were selected for the introduction of mutations into the α- and γ-KAFIRIN genes of sorghum. The design of the gRNAs was conducted using the online tools CRISPROR and CHOPCHOP. Two most suitable targets were chosen for α-KAFIRIN (k1C5) and two for γ-KAFIRIN (gKAF1) genes. The insertion of respective sequences in the generic vector pSH121 was performed at the BsaI (Eco31I) sites. Validation of the cloning procedure was performed by DNA sequencing. Subcloning of the resulting constructs was performed using the SfiI restriction sites into the compatible binary vector B479p7oUZm-LH. The correct assembly of binary vectors was confirmed by restriction analysis using the MluI and SfiI cleavage sites. The four vectors created (1C - 4C) were transferred by electroporation into the Agrobacterium tumefaciens strain AGL0. Currently, this vector series is used for stable

关键词： Sorghum bicolor (L.) Moench CRISPR/Cas Genome Editing α-Kafirin γ-Kafirin Genetic Engineering Grain Quality

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Binary Vector Construction for Site-Directed Mutagenesis of Kafirin Genes in Sorghum

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American Journal of Plant Sciences（美国植物学期刊） 2021年第8期

作者： Grigoriy A. Gerashchenkov lev A. elkonin Kirill G. Gerashchenkov Natalia A. Rozhnova Stefan Hiekel Jochen Kumlehn Alexey V. Chemeris

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