Manifestation of agronomically valuable traits in the progeny of a sorghum mutant carrying the genetic construct for RNA silencing of the γ-kafirin gene.

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.

Influence of different types of sterile cytoplasms (A3, A4, 9E) on the combining ability of CMS lines of sorghum.

Investigation of the effect of the cytoplasm on the combining ability (CA) of lines with cytoplasmic male sterility (CMS) is of considerable interest in terms of understanding the genetic functions of the cytoplasm and for practical purposes to create hybrids with improved economically valuable traits. In order to investigate the effect of different types of sterile cytoplasm (A3, A4, 9E) on CA in sorghum, we studied the manifestation of a number of biological and agronomic traits in 54 F1 hybrid combinations obtained using iso-nuclear CMS lines with the nuclear genome of the line Zheltozernoye 10, differing only in the types of sterile cytoplasm (A3, A4 and 9E). Eighteen varieties and lines of grain sorghum developed at the Russian Research and Project-technological Institute of Sorghum and Maize were used as paternal parents. The CA was determined by the topcross method. F1 hybrids and their parents were grown in 2015-2017 in conditions of insufficient (2015-2016: HTC (hydro-thermal coefficient) = 0.32-0.66), or good water availability conditions (2017: HTC = 1.00). On average, for three years of testing, a positive effect of the 9E cytoplasm on the general combining ability (GCA) (0.63) and negative effects of the A3 and A4 cytoplasms (-0.32 and -0.31) for the inflorescence length were noted. In dry seasons, significant positive effects of the 9E cytoplasm on GCA for the length of the largest leaf, and positive effects of the A3 cytoplasm on GCA for the plant height, and negative effects of the A4 cytoplasm on GCA for these traits were observed. No differences were observed during the wet season. The type of CMS did not affect the GCA for the width of the largest leaf and grain yield. The dispersion of specific combining ability (SCA) in the dry seasons was significant for the following traits: leaf length, plant height, panicle length and width, and grain yield, the 9E cytoplasm had the highest SCA dispersion, whereas the A4 cytoplasm had the smallest one. The data obtained indicate that different types of sterile cytoplasm of sorghum make a different contribution to CA under conditions of drought stress.

[Inheritance of reversions to male fertility in male-sterile sorghum hybrids with 9E cytoplasm male sterility induced by environmental conditions].

Heritable phenotypic alterations occurring during plant ontogenesis under the influence of environmental factors are among the most intriguing genetic phenomena. It was found that male-sterile sorghum hybrids in the 9E cytoplasm from the F1 and F2 generations, which were obtained by crossing CMS lines with different fertile lines grown in field conditions, were transferred to greenhouse produce fertile tillers. Lines created by the self-pollination of revertant tillers exhibit complete male fertility upon cultivation under various environments (in the field, Tdry plot,(y) Tirrigated plot(y)). In a number of test-crosses of revertants to CMS lines in the 9E cytoplasm, restoration of male fertility in F1 hybrids was found, indicating that revertants possess functional fertility-restoring genes. A high positive correlation was found between the fertility level of the test-cross hybrids and the hydrothermal coefficient (the ratio of the sum of precipitation to the sum of temperatures) during the booting stage and pollen maturation (r = 0.75...0.91; P<0.01), suggesting that a high level of plant water availability is needed for the expression of fertility-restoring genes of revertants. These data show that the fertility-restoring genes for the 9E cytoplasm are dominant in conditions of high water availability and recessive in drought conditions; reversions to male fertility are due to up-regulation of fertility-restoring genes by a high level of water availability. Comparative MSAP-analysis of DNA of male-sterile and male-fertile test-cross hybrids using HpaII/MspI restrictases and primers to polygalacturonase gene ADPG2, which is required for cell separation during reproductive development, and gene MYB46, the transcription factor regulating secondary wall biosynthesis, revealed differences in the number and the length of amplified fragments. Changes in the methylation of these genes in conditions of drought stress are apparently the reason for male sterility of sorghum hybrids in the 9E cytoplasm. These data demonstrate that methylation of nuclear genes in sterility-inducing cytoplasm may be one of mechanisms causing the CMS phenomenon.

Heritable Effect of Plant Water Availability Conditions on Restoration of Male Fertility in the "9E" CMS-Inducing Cytoplasm of Sorghum.

Heritable changes of phenotype arising in plant ontogenesis by the influence of environmental factors belong to the most intriguing genetic phenomena. An unusual inheritance pattern was detected during examination of male fertility restoration in the CMS-inducing "9E" type cytoplasm of sorghum: Rf-genes were functional in self-pollinated progeny of F(1) hybrids yet were either not expressed or poorly expressed in backcrosses of these hybrids to CMS-lines with the same cytoplasm type. In experiments on parallel growing of the same F(1) hybrid combinations in the "dry plot" and in the "irrigated plot," it was found that high level of plant water availability during panicle and pollen developmental stages significantly increased male fertility of F(1) and test-cross hybrid populations, in which fertility-restoring genes were in heterozygote state, whereas in F(2) populations the influences of water availability conditions cause less pronounce effects. Similarly, male-sterile F(1) plants, being transferred from the "dry plot" to greenhouse, produced male-fertile panicles. In addition, male-sterile plants from F(2) families, which segregated-out as recessives, being transferred to greenhouse also produced male-fertile panicles. In the progenies of these revertants that were grown in field conditions and in the "dry plot," stable inheritance of male fertility for three cycles of self-pollination was observed, and a number of stable fertile lines in the "9E" cytoplasm were obtained. However, in test-crosses of these fertile lines to CMS-lines with the "9E" cytoplasm restoration of male fertility was not observed, except the progeny of one revertant that behaved as fertility-restorer line. These data suggest that the functional state of fertility-restoring genes for the "9E" sorghum cytoplasm is epigenetically regulated trait established by the influence of environmental factors and is transmitted to sexual generations.