Lev A. Elkonin

Site-directed mutagenesis using genetic constructs carrying the CRISPR/Cas system is an effective technology that is actively used to solve a variety of problems in plant genetics and breeding. One of these problems is to improve the nutritional value of grain sorghum, a high-yielding heat- and drought-tolerant cereal crop that is becoming increasingly important in the conditions of climate aridization. The main reason for the relatively low nutritional value of sorghum grain is the resistance of its storage proteins, kafirins, to proteolytic digestion. We have previously obtained mutants with improved kafirin in vitro digestibility using the CRISPR/Cas technology in grain sorghum variety Avance. The nucleotide sequence of one of the genes (k1C5) of the gene family encoding the signal polypeptide of 22 kDa α-kafirin was used as a target. The aim of this study was to investigate the manifestation of the main agronomically-important traits in the progeny of these mutants and inheritance of high in vitro protein digestibility, and also sequencing nucleotide sequences encoding the 22 kDa α-kafirin signal polypeptide in a number of plants from the T ₀ generation and their T ₁ progeny. It was revealed that four of the six studied T ₀ plants, as well as their progeny, had the same mutation: a T→C substitution in the 23rd position of the nucleotide sequence of the k1C5 gene encoding the signal polypeptide, which led to a substitution of the coding triplet CTC→CCC (Leu→Pro). This mutation is located off-target, 3’ from the PAM sequence. It is suggested that this mutation may have arisen as a result of Cas9 nuclease errors caused by the presence of multiple PAM sequences located close to each other. It was found that the progeny of two of the three studied mutants (T ₂ and T ₃ families), grown in the experimental field conditions, differed from the original variety by a reduced plant height (by 12.4–15.5 %). The peduncle length, 1,000-grain mass, and grain mass per panicle did not differ from the original variety, with the exception of the progeny of the 2C-1.2.5b mutant, which had a reduced grain yield per panicle. Unlike the original variety, plants from the T ₂ and T ₃ generations had kernels with a modified type of endosperm (completely floury, or floury with inclusions of vitreous endosperm, or with a thin vitreous layer). The level of grain protein digestibility in the progeny of mutants 2C-2.1.1 #13 and 2C-1.2.5a #14 varied from 77 to 84 %, significantly exceeding the original variety (63.4 ± 2.3 %, p < 0.05). The level of protein digestibility from kernels with modified endosperm was higher than that of kernels with normal vitreous endosperm (84–93 %, p < 0.05). The reasons for the variation in endosperm texture in the progeny of the mutants and its relationship with the high digestibility of kafirins are discussed.

Sorghum is a heat- and drought-tolerant cereal crop used for feed and food purposes in more than 100 countries. Sorghum becomes highly important in conditions of climate warming. The use of genome editing technologies is of paramount importance for this crop, which has a number of constraints in key agronomic traits that are difficult for improvement using conventional breeding. Application of genome editing for sorghum is limited due to problems of genetic transformation; however, recent advances in this field have increased the efficiency of the genome editing procedure in sorghum. This review summarizes recent achievements in the field of sorghum genetic transformation and results of site-directed mutagenesis in this crop, such as obtaining mutants with increased kafirin digestibility, apomixis, resistance to Striga hermontica, modification of plant architecture and “stay-green” trait, fragrant leaves and seeds, and others. The review also discusses the biosafety of edited plants and outlines prospects for further work on the genetic improvement of sorghum using genome editing technologies, and its application in practical breeding.

Improving the nutritional value of grain sorghum, a drought- and heat-tolerant grain crop, is an important task in the context of global warming. One of the reasons for the low nutritional value of sorghum grain is the resistance of its storage proteins (kafirins) to proteolytic digestion, which is due, among other things, to the structural organization of protein bodies, in which γ-kafirin, the most resistant to proteases, is located on the periphery, encapsulating more easily digested α-kafirins. The introduction of genetic constructs capable of inducing RNA silencing of the γ-kafirin (gKAF1) gene opens up prospects for solving this problem. Using Agrobacterium-mediated genetic transformation of immature embryos of the grain sorghum cv. Avans we have obtained a mutant with improved digestibility of endosperm proteins (up to 92 %) carrying a genetic construct for RNA silencing of the gKAF1 gene. The goal of this work was to study the stability of inheritance of the introduced genetic construct in T₂–T₄ generations, to identify the number of its copies, as well as to trace the manifestation of agronomically valuable traits in the offspring of the mutant. The mutant lines were grown in experimental plots in three randomized blocks. The studied lines were characterized by improved digestibility of kafirins, a modified type of endosperm, completely or partially devoid of the vitreous layer, an increased percentage of lysine (by 75 %), reduced plant height, peduncle length, 1000-grains weight, and grain yield from the panicle. In T₂, a line with monogenic control of GA resistance was selected. qPCR analysis showed that in different T₃ and T₄ plants, the genetic construct was present in 2–4 copies. In T₃, a line with a high digestibility of endosperm proteins (81 %) and a minimal decrease in agronomically valuable traits (by 5–7 %) was selected.

The use of genome editing technologies opens wide opportunities for the targeted mutagenesis in important agricultural crops. In the context of global warming, sorghum, an important drought- and heat-tolerant crop is of particular importance. However, compared to other cereals, sorghum grain has a lower nutritional value, due to the resistance of its storage proteins (kafirins) to proteolytic digestion. A decrease in the synthesis of kafirins as a result of mutations or the expression of the RNAi genetic constructs modifies the ultrastructure of protein bodies and improves their digestibility by proteases. To obtain mutants with improved protein digestibility, we created four binary vectors for site-directed mutagenesis of the k1C5 and gKAF1 genes encoding α- and γ-kafirin, respectively. Each of these vectors contained the cas9 endonuclease gene and a guide RNA targeted the nucleotide sequences encoding the kafirin signal polypeptides. By means of agrobacterial transformation, the created vectors were introduced into the genome of the grain sorghum cv. Avans. 14 transgenic plants were regenerated. Sequencing of 5 regenerants obtained using a vector for the k1C5 mutagenesis revealed 3 plants with mutations. The offspring of these mutants had a higher digestibility of grain proteins in vitro (86–92%) compared to the initial cv. Avans (63–67%). Notably, the T1 plants lacked the cas9 gene and the bar marker gene, which indicates the production of mutants with the edited k1C5 gene sequence, which lack the genetic construct that induced this mutation. Two mutants with mutations in the gKAF1 sequence were obtained. Thus, using the CRISPR/Cas technology, we have obtained mutants with improved digestibility of kafirins, which can be used in practical sorghum breeding.

One of the main components of apomictic plant reproduction is the formation of unreduced embryo sacs (ESs). Heteroploid crosses, in which maternal diploid plants are pollinated by pollen of tetraploid paternal parent, can be used as an effective tool for identifying the ability to form unreduced ESs. In maize, in crosses 2n(♀) × 4n(♂), as a rule, shrunken kernels with a triploid embryo are formed, in which the development of the endosperm is impaired due to the deviation of the balance of the maternal (m) to paternal (p) genomes from the ratio of 2m : 1p. In our experiments, in several diploid maize lines, after their pollination with tetraploid pollen, the formation of large plump kernels was observed, from which diploid maternal-type plants developed, the maternal-type phenotype, as well as the hybrid endosperm phenotype, were confirmed by the expression of genetic markers. It has been suggested that the plump kernels in 2n × 4n crosses arise on the basis of unreduced embryo sacs (ESs) and pseudogamous apomixis, since the fusion of diploid sperms with diploid polar nuclei provides a 2 : 1 ratio of maternal to paternal genomes in the endosperm. In order to clarify the genetic nature of diploid plants phenotypically similar to maternal lines that developed from plump kernels in 2n × 4n crosses, we carried out their genotyping for all 10 chromosomes of the maize genome using polymorphic codominant SSR- and Indel-markers that differentiate the paternal line from maternal lines. As maternal lines, we used HPL AT, which has the ability to haploid parthenogenesis, KM, YuV-11, as well as the F2 B47/HPL AT hybrid plants; and as a paternal line – Chernaya Tetra(4n). It was found that in all the plants studied, when markers of five chromosomes (1, 2, 3, 4, and 9) were used, amplification of only maternal alleles was observed. However, in each of the studied plant, when using markers of other chromosomes, cases of amplification of alleles characteristic of the paternal line were noted. A hypothesis is put forward on the formation of diploid plants in 2n × 4n crosses in maize as a result of fertilization of the unreduced ESs and the subsequent elimination of chromosomes, predominantly of the pollen parent.

One of the main components of apomictic plant reproduction is the formation of unreduced embryo sacs (ESs). Heteroploid crosses, in which maternal diploid plants are pollinated by pollen of tetraploids, can be used as an effective tool for identifying the ability to form such ESs. In maize, in 2n(♀) × 4n(♂) crosses, as a rule, shrunken kernels with a triploid embryo are formed, in which the development of the endosperm is impaired due to the deviation of the balance of the maternal (m) to paternal (p) genomes from the ratio of 2m : 1p. In our experiments, the formation of large plump kernels, from which diploid maternal-type plants developed, was observed in several diploid maize lines under their pollination with tetraploid pollen, while the maternal-type phenotype of plants, as well as the hybrid endosperm phenotype was confirmed by expression of genetic markers. It has been suggested that the plump kernels in 2n × 4n crosses arise based on unreduced ESs and pseudogamous apomixis, since the fusion of two diploid polar nuclei with a diploid sperm provides a ratio of maternal to paternal genomes in the endosperm of 2m : 1p. In order to clarify the genetic nature of diploid plants that are phenotypically similar to maternal lines that developed from plump kernels in 2n × 4n crosses, we carried out their genotyping for all ten chromosomes of the maize genome using polymorphic codominant SSR and Indel markers that differentiate the paternal pollinator line from maternal lines. As maternal forms, we used the HPL AT line, which has the ability for haploid parthenogenesis, plants from the F₂ generation of the hybrid between the lines B47 capable of forming unreduced ESs and HPL AT. In addition, the Korichnevyi Marker (KM) line carrying the marker gene B of brown stem coloring, as well as the YuV11 line was included in the crosses. As the paternal form, the Chernaya Tetra (ChT) tetraploid corn carrying the marker gene A1 of the black coloring of kernels was used. It was found that in all studied plants amplification of only maternal alleles was observed when using markers of five chromosomes: chromosome 1 (iDp525), chromosome 2 (iDP4004), chromosome 3 (JY_3:457), chromosome 4 (UflDP4_31.55), and chromosome 9 (INDEL_139329242). However, in each of the studied plants, cases of amplification of alleles characteristic of the paternal line were noted when using markers of other chromosomes. A hypothesis on the formation of diploid plants in 2n × 4n crosses in maize as a result of fertilization of unreduced ESs and the subsequent elimination of chromosomes predominantly of the paternal line is proposed.

Main conclusion AS-3 line of Sorghum bicolor possesses functional components of apomixis—apospory, parthenogenesis and autonomous endospermogenesis. The data obtained indicate efficiency of selection for apomixis components in diploid species of cultivated crops. Abstract Apomixis (seed formation without fertilization) is one of most attractive phenomena in plant biology. In this paper, we provide the results of long-term selection for apomixis components in the progeny of grain sorghum (Sorghum bicolor (L.) Moench) hybrid plants with male sterility mutation. Selection was carried out for a high frequency of aposporous embryo sacs (ESs), autonomous pro-embryos, and the presence of maternal-type plants in test crosses with the line Volzhskoe-4v (V4v) homozygous for the Rs1 genes determining the red color of the leaves and stem of the hybrids. As a result of using this approach, the line, AS-3, was created, in which the frequency of ovaries with parthenogenetic embryos reached 42–45%. The autonomous development of embryos and endosperm was observed in the panicles of each of the 10 cytologically studied plants of this line. The frequency of parthenogenesis positively correlated with the high average daily air temperature during the first five out of 10 days preceding the onset of flowering (r = 0.75; P > 0.01). Genotyping of the plants from the progeny of hand-emasculated panicles of AS-3 pollinated with V4v performed using co-dominant SSR markers revealed that the F₁ hybrids carrying the Rs1 gene (chromosome 6) possessed both paternal and maternal alleles of Sb1-10 (chromosome 4) and Xtxp320 (chromosome 10) markers, while in the maternal-type plants (rs1rs1), only the maternal alleles of these markers were present. In the endosperm of the kernels from which the maternal-type seedlings were obtained, only the maternal alleles were present, while in the endosperm of the kernels that produced hybrid seedlings, both the paternal and maternal alleles were observed. The data obtained indicate the presence of functional components of apomixis (apospory, parthenogenesis, autonomous endospermogenesis) in the grain sorghum line AS-3, and the efficiency of selection for apomixis in functionally diploid species of cultivated crops.

Modification of the composition of grain storage proteins is an intensively developing area of plant biotechnology, which is of particular importance for sorghum – high-yielding drought tolerant crop. Compared to other cereals, the majority of sorghum cultivars and hybrids are characterized by reduced nutritional value that is caused by a low content of essential amino acids in the seed storage proteins (kafirins), and resistance of kafirins to protease digestion. RNA interference (RNAi) by suppressing synthesis of individual kafirin subclasses may be an effective approach to solve this problem. In this chapter, we review published reports on RNAi silencing of the kafirin-encoding genes. In addition, we present new experimental data on phenotypic effects of RNAi-silencing of γ-KAFIRIN-1 gene in sorghum cv. Avans. To obtain RNAi mutants with γ-KAFIRIN-1 gene silencing we used Agrobacterium-mediated genetic transformation. Transgenic kernels had modified endosperm type with reduced vitreous layer and significantly improved in vitro protein digestibility (93% vs. 57%, according to the densitometry of SDS-PAGE patterns). SDS-PAGE of transgenic kernels showed lowered level of kafirins and appearance of globulin proteins, which were not observed in the original cultivar. For the first time, the cases of instability of inserted genetic construct were identified: elimination of ubi1-intron that is a constituent part of the genetic construct for RNAi silencing, or nos-promotor governing expression of the marker gene (bar) (in the RNAi mutants of cv. Zheltozernoe 10). The research findings presented in this chapter provide strong evidence that RNA interference can be used for improvement of the nutritional properties of sorghum grain.