Immature Wheat Embryos Research Articles

Improved biolistic transformation and genome editing in wheat by using trehalose for high osmotic treatment.

The tissue culture process is usually involved in gene transfer and genome editing in plants. Like other species, there is enormous variation among wheat genotypes in tissue culture response. In the rapidly advancing system of CRISPR/Cas9 for genome editing, particle bombardment has received increasing attention as a delivery method for a large amount of nucleic acids and RNA-protein complexes. However, the efficiency of transformation by particle bombardment has been low in wheat, and only a limited number of varieties have been transformed. In this study, replacement of maltose with trehalose as an osmolyte for high osmotic treatment for the protection of tissues from physical impacts improved callus formation in immature wheat embryos and efficiency of transformation and genome editing in varieties that are relatively poor in tissue culture response. The range of varieties amenable to biolistic transformation and genome editing may be expanded by this modification.

Functional Analysis of Seed Dormancy Genes by Biolistic Transient Gene Expression in Immature Embryos of Wheat.

Seed dormancy genes typically suppress germination and cell division. Therefore, overexpressing these genes can negatively affect tissue culture, interfering with the generation of transgenic plants and thus hampering the analysis of gene function. Transient expression in target cells is a useful approach for studying the function of seed dormancy genes. Here, we describe a protocol for transiently expressing genes related to seed dormancy in the scutellum of immature wheat (Triticum aestivum) embryos to analyze their effects on germination.

Comprehensive Transcriptomic and Metabolic Profiling of Agrobacterium-tumefaciens-Infected Immature Wheat Embryos.

The transformation efficiency (TE) was improved by a series of special chemical and physical methods using immature embryos from the cultivar Fielder, with the PureWheat technique. To analyze the reaction of immature embryos infected, which seemed to provide the necessary by Agrobacterium tumefaciens in PureWheat, a combination of scanning electron microscopy (SEM), complete transcriptome analysis, and metabolome analysis was conducted to understand the progress. The results of the SEM analysis revealed that Agrobacterium tumefaciens were deposited under the damaged cortex of immature embryos as a result of pretreatment and contacted the receptor cells to improve the TE. Transcriptome analysis indicated that the differentially expressed genes were mainly enriched in phenylpropanoid biosynthesis, starch and sucrose metabolism, plant-pathogen interaction, plant hormone signal transduction, and the MAPK (Mitogen-activated protein kinase) signaling pathway. By analyzing the correlation between differentially expressed genes and metabolites, the expression of many genes and the accumulation of metabolites were changed in glucose metabolism and the TCA cycle (Citrate cycle), as well as the amino acid metabolism; this suggests that the infection of wheat embryos with Agrobacterium is an energy-demanding process. The shikimate pathway may act as a hub between glucose metabolism and phenylpropanoid metabolism during Agrobacterium infection. The downregulation of the F5H gene and upregulation of the CCR gene led to the accumulation of lignin precursors through phenylpropanoid metabolism. In addition, several metabolic pathways and oxidases were found to be involved in the infection treatment, including melatonin biosynthesis, benzoxazinoid biosynthesis, betaine biosynthesis, superoxide dismutase, and peroxidase, suggesting that wheat embryos may be under the stress of Agrobacterium and, thus, undergo an oxidative stress response. These findings explore the physiological and molecular changes of immature embryos during the co-culture stage of the PureWheat technique and provide insights for Agrobacterium-mediated transgenic wheat experiments.

МОРФОГИСТОЛОГИЧЕСКИЕ ОСОБЕННОСТИ РИЗОГЕНЕЗА IN VITRO В ЗАРОДЫШЕВЫХ КАЛЛУСАХ ПШЕНИЦЫ

In modern biotechnological studies of economically valuable plants, numerous developments aimed at obtaining calluses are presented, in which various pathways and types of morphogenesis are induced in vitro. At the same time, such the type of morphogenesis as rhizogenesis in vitro, consisting in the formation and development of roots, but without further regeneration of plants, has not been studied sufficiently. Nevertheless, it is important to identify the tissue mechanisms of formation and development in calluses the roots as rhizogenesis structures. Using the example of calluses obtained from immature wheat embryos, it was revealed that the formation of morphogenetic foci is the basis of rhizogenesis in vitro. Anatomical characteristics of the emerging roots have been revealed, which largely coincide with similar characteristics of the cereal roots under in planta conditions. It is proposed to use morphogenic calluses in vitro as model systems for studying the rhizogenesis of intact plants.

Optimization of highly efficient exogenous-DNA-free Cas9-ribonucleoprotein mediated gene editing in disease susceptibility loci in wheat (Triticum aestivum L.).

The advancement of precision engineering for crop trait improvement is important in the face of rapid population growth, climate change, and disease. To this end, targeted double-stranded break technology using RNA-guided Cas9 has been adopted widely for genome editing in plants. Agrobacterium or particle bombardment-based delivery of plasmids encoding Cas9 and guide RNA (gRNA) is common, but requires optimization of expression and often results in random integration of plasmid DNA into the plant genome. Recent advances have described gene editing by the delivery of Cas9 and gRNA as pre-assembled ribonucleoproteins (RNPs) into various plant tissues, but with moderate efficiency in resulting regenerated plants. In this report we describe significant improvements to Cas9-RNP mediated gene editing in wheat. We demonstrate that Cas9-RNP assays in protoplasts are a fast and effective tool for rational selection of optimal gRNAs for gene editing in regenerable immature embryos (IEs), and that high temperature treatment enhances gene editing rates in both tissue types. We also show that Cas9-mediated editing persists for at least 14 days in gold particle bombarded wheat IEs. The regenerated edited wheat plants in this work are recovered at high rates in the absence of exogenous DNA and selection. With this method, we produce knockouts of a set of three homoeologous genes and two pathogenic effector susceptibility genes, engineering insensitivity to corresponding necrotrophic effectors produced by Parastagonospora nodorum. The establishment of highly efficient, exogenous DNA-free gene editing technology holds promise for accelerated trait diversity production in an expansive array of crops.

ФОРМИРОВАНИЕ МОРФОГЕНЕТИЧЕСКИХ ОЧАГОВ КАК ОСНОВА ГЕММОРИЗОГЕНЕЗА IN VITRO В ЗАРОДЫШЕВЫХ КАЛЛУСАХ ПШЕНИЦЫ

Biotechnological elaborations for breeding purposes aimed at creating highly productive wheat varieties require a significant number of morphogenic calli, in which the manifestation of various pathways of morphogenesis in vitro are demonstrated. Of great practical interest is the gemmorhizogenesis in vitro, associated with multiple regeneration of full–fledged plants. It is methodically important to identify the dependence of the induction of this morphogenesis pathway in wheat calli in vitro on the duration of their cultivation as well cell/tissue mechanisms of gemmorhizogenic structures formation. On the example of morphogenic calli obtained from immature wheat embryos the process of gemmorhizogenic structures forming is significantly stretched over time, and the formation of morphogenetic zones lays at the basis of in vitro gemmorhizogenesis.

Lipopolysaccharide and flagellin of Azospirillum brasilense Sp7 influence callus morphogenesis and plant regeneration in wheat.

In vitro somatic callus culturing is used widely in plant biotechnology, but its effectiveness depends largely on the donor plant genotype. Bacteria or components of their cells are rarely used to activate morphogenesis. In this work, inoculation of explants from immature wheat (Triticum aestivum L.) embryos with a suspension of living cells of the bacterium Azospirillum brasilense Sp7 resulted in callus death after 7days of growth, in contrast to explant treatment with a suspension of heat-killed whole cells of Sp7. The experiments used two wheat lines, LRht-B1a and LRht-B1c, which differ in morphogenic activity. Growing calluses with the lipopolysaccharide of A. brasilense Sp7 increased the yield of regenerated plants 2- to 3.5-fold in both lines. This increase was through the activation of regenerant formation from morphogenic calluses. We have demonstrated for the first time the effects of bacterial flagellin on plant tissue culture. The polar-flagellum flagellin of A. brasilense Sp7 leveled the genotypic differences in the morphogenic ability of callus tissue. Specifically, it increased the yield of morphogenic calluses in the weakly morphogenic line LRht-B1a to the yield value in the highly morphogenic line LRht-B1c but lowered the yield of regenerants in the highly morphogenic line LRht-B1c to the yield value in the weakly morphogenic line LRht-B1a. Thus, bacterial lipopolysaccharides and flagellins can be used to regulate the formation of morphogenic calluses and regenerants in plant tissue culturing in vitro.

ПРОДОЛЖИТЕЛЬНОСТЬ КУЛЬТИВИРОВАНИЯ IN VITRO ЗАРОДЫШЕВЫХ КАЛЛУСОВ ПШЕНИЦЫ ВЛИЯЕТ НА ПРОЯВЛЕНИЕ ИХ МОРФОГЕНЕТИЧЕСКОГО И РЕГЕНЕРАЦИОННОГО ПОТЕНЦИАЛА

Biotechnological developments for breeding purposes aimed at creating highly productive wheat varieties require a significant number of morphogenic callus, in which the manifestation of various pathways of morphogenesis in vitro associated with the regeneration of full-fledged plants is induced. It is methodically important to identify the dependence of the induction of a specific pathway of morphogenesis in wheat callus in vitro on the duration of their cultivation. On the example of morphogenic calli obtained from immature wheat embryos at the stage of early organogenesis on an induction medium containing 2.0 mg/l of synthetic auxin 2,4-D, depending on the duration of cultivation, the following sequence of induction of morphogenesis pathways in vitro was established: gemmogenesis, rhizogenesis, gemmorhizogenesis, somatic embryogenesis (or embryoidogenesis). These pathways of morphogenesis were identified morphologically by the appearance of the corresponding morphogenic structures: buds, roots, gemmorhizogenic structures, somatic embryos. It is shown that the process of laying and forming morphogenic structures is significantly stretched over time, and morphogenic structures may be present in the same callus at different stages of development.

Obtaining Wheat (Triticum aestivum L.) Lines with Yeast Genes for Trehalose Biosynthesis

Yeast (Saccharomyces cerevisiae) genes for trehalose biosynthesis (TPS1 and TPS2) were transferred into genomes of several common wheat cultivars using two methods of Agrobacterium-mediated transformation (in vitro and in planta) to enhance drought tolerance. For this purpose, vectors pBract214-TPS1 and pBract214-TPS2 were constructed using the Gateway-cloning technique. Both the vectors contained TPS1 and TPS2 genes under control of the constitutive maize ubiquitin promoter (PUbi) and selectable marker hygromycin-phosphotransferase (hpt) gene. Three- to five-day-old calluses obtained from immature wheat embryos were used as explants for the transformation in vitro. Selection of transgenic plants was carried out on nutrient medium supplemented with 30 mg/L hygromycin (as a selectable agent). Seeds of wheat (transgenic generation T1) were obtained by the in planta method of transformation. Integration and the presence of yeast genes in wheat genomic DNA isolated from transgenic plants were confirmed by PCR analysis using primers specific to TPS1 and TPS2 genes.

A rapid method for detection of mutations induced by CRISPR/Cas9-based genome editing in common wheat.

Genome editing using CRISPR/Cas9 is useful for common wheat because common wheat has allohexaploid nature and it can induce mutations simultaneously in three homoeologous genes. Although Agrobacterium-mediated transformation has advantages in genome editing, it still has low efficiency and requires relatively long time in wheat. Therefore, the use of guide RNAs (gRNAs) with efficient mutagenesis in vivo is one of the critical factors for producing genome-edited mutant lines in a short time. In this study, we targeted three genes in common wheat and established a rapid method for detection of mutations induced by the biolistic transient expression system. Biolistic transient expression of the gRNAs and Cas9 was achieved in immature wheat embryos. Mutations were detected a week later using PCR-RFLP and verified by the sequencing of genomic clones. We confirmed several types of mutations that occurred at different rates depending on the target sequences. Furthermore, frequencies of mutations tended to be higher at the targets that were edited at higher rates in the plants transformed by Agrobacterium. These results show that this method of rapid detection of edited mutations could be used for variety of applications, such as screening of target sequences or modified vectors for efficient CRISPR/Cas9 genome editing in wheat.

CADMIUMIONSINCELLSELECTION FOR OBTAINING WHEAT CELL FORMS TOLERANT TO WATER STRESS

Introduction. Water deficit significantly decrease the plant development and crop production. Genetic effects that increased the genotype tolerance abilities are the aims of various investigations. Cell selection is the appropriate biotechnology for obtaining plant forms that challenged abiotic stresses. It is known that Cd 2+ cations significantly destroy various plant compartments and tissues. There was detected that Cd 2+ injures the water status of the organism. Purpose. The aim of the investigation was the promotion of cell selection with Cd 2+ cations for obtaining wheat cell lines tolerant to water stress. Methods. Selective systems with lethal doses of cadmium ions (Cd 2+ ) for obtaining wheat cell forms tolerant to water stress are proposed and elaborated. The minimum Cd 2+ concentration that eliminates wild type cell population was established as lethal doses. The water stress was conducted by the addition of manitol. Manitol is usually used for simulation water deficit in vitro. Callus and suspension cultures were initiated from immature embryos of winter wheat, ( Triticum aestivum L .), cv. Favoritka. Cell suspension (wild type) was placed on agar cultural B5 medium with the addition of lethal doses of cadmium ions (“plating procedure”). Such doses were deduced during preliminary tests. Only Cd-resistant cell survive under lethal ion stress pressure. In Cd 2+ resistant cell lines relative fresh weight and free proline levels were estimated. Result. Resistant cells formed primary minicolonies. Such colonies are considered to be wheat resistant cell lines (Cd-RCL). Cd-RCL grew at Cd 2+ ions presence during 3 passages. Then callus was cut and transferred to fresh media: basal medium (normal conditions) and selective media (stress conditions). There were established two variants of selective systems: medium with the addition of Cd 2+ cations, (stress I); cultural medium with the addition of manitol (stress II). Cd-RCL maintained their viability under any stress pressure. Genetic basis of Cd-RCL combined stress resistance was confirmed via media rotations. The changes were: normal conditions → stresses I, II; stresses I, II → normal conditions; stress I → stress II or other way roads. The type of cultural medium and the number of passages were always free. As proliferation marker calli relative fresh mass growth (RFW, Δm) was used. It was always positive. This parameter measured under normal conditions exceeded (40-45%) biomass RFW estimated under manitol pressure. But normal data were lower (more than three times) than data measured during calli cultivation at Cd 2+ presence. It is assumed that such events are the exhibitions of combined resistance. The levels of free proline (pro) were estimated in Cd-RCL. Under normal conditions wheat cell cultures accumulated pro in various amounts. In wild type callus the proline level was the highest. In Cd-RCL cultivated at manitol presence pro contents increased. We suppose that elevated pro levels in Cd-RCL under water stress were due to activity of system of its synthesis. The wild type cell cultures eliminated at the end of any passage. Proline levels were lower than level of determination. Conclusion . Cell lines of winter wheat with combined stress resistance were obtained via cell selection with Cd 2+ cations. Cd 2+ -resistant cell lines tolerated both lethal ion and water stresses. Under water stress pressure callus RFW of Cd 2+ -resistant cell lines was lower and under Cd 2+ affect was higher than normal parameters. The growth under water was cooperated with free proline accumulation. Cell selection with heavy metal ions is the perspective approach for obtaining cell variants with higher tolerance to osmotic stresses.

Efficient In Vitro Somatic Embryogenesis and Plant Regeneration from Mature and Immature Embryos of Wheat ( Triticum aestivum L.)

ABSTRACT An efficient regeneration system is a pre-requisite for the application of genetic transformation and functional genomics study of important plants. In this study, the effect of different factors (plant growth regulators, casein hydrolysate, aspartic acid and ascorbic acid) on in vitro embryogenesis and regeneration of Arta, Bahar and Zagros cultivars from mature and immature explants were investigated. Immature and mature embryos were dissected from disinfected seeds 20-25 days after pollination and imbedded mature seeds, respectively, and cultured on MS (Murashige and Skoog) medium supplemented with different compounds. The results showed that immature embryos expose high capacity of embryogenesis and regeneration in comparison with mature embryos. There were significant differences between cultivars in terms of the percentage of callus induction and regeneration. Plant growth regulators had significant effect on percentage of callus induction in mature explants and percentage of regeneration from both explants. In immature explants, the highest percentage of regeneration (65%) was achieved with the Arta cultivar calli derived from MS medium supplemented with 1mg/L 2,4-D, 2 mg/L Picloram and 200 mg/L casein hydrolysate, and subcultured on MS medium. Also, the highest percentage of regeneration (52.38%) from mature embryo explants was achieved in the Arta cultivar with callus induction on MS medium supplemented with 1 mg/L 2,4-D, 2 mg/L Picloram and 200 mg/L casein hydrolysate and regeneration on MS medium containing 0.05 mg/L NAA.

De novo assembly and comparative analysis of the transcriptome of embryogenic callus formation in bread wheat (Triticum aestivum L.)

BackgroundDuring asexual reproduction the embryogenic callus can differentiate into a new plantlet, offering great potential for fostering in vitro culture efficiency in plants. The immature embryos (IMEs) of wheat (Triticum aestivum L.) are more easily able to generate embryogenic callus than mature embryos (MEs). To understand the molecular process of embryogenic callus formation in wheat, de novo transcriptome sequencing was used to generate transcriptome sequences from calli derived from IMEs and MEs after 3d, 6d, or 15d of culture (DC).ResultsIn total, 155 million high quality paired-end reads were obtained from the 6 cDNA libraries. Our de novo assembly generated 142,221 unigenes, of which 59,976 (42.17%) were annotated with a significant Blastx against nr, Pfam, Swissprot, KOG, KEGG, GO and COG/KOG databases. Comparative transcriptome analysis indicated that a total of 5194 differentially expressed genes (DEGs) were identified in the comparisons of IME vs. ME at the three stages, including 3181, 2085 and 1468 DEGs at 3, 6 and 15 DC, respectively. Of them, 283 overlapped in all the three comparisons. Furthermore, 4731 DEGs were identified in the comparisons between stages in IMEs and MEs. Functional analysis revealed that 271transcription factor (TF) genes (10 overlapped in all 3 comparisons of IME vs. ME) and 346 somatic embryogenesis related genes (SSEGs; 35 overlapped in all 3 comparisons of IME vs. ME) were differentially expressed in at least one comparison of IME vs. ME. In addition, of the 283 overlapped DEGs in the 3 comparisons of IME vs. ME, excluding the SSEGs and TFs, 39 possessed a higher rate of involvement in biological processes relating to response to stimuli, in multi-organism processes, reproductive processes and reproduction. Furthermore, 7 were simultaneously differentially expressed in the 2 comparisons between the stages in IMEs, but not MEs, suggesting that they may be related to embryogenic callus formation. The expression levels of genes, which were validated by qRT-PCR, showed a high correlation with the RNA-seq value.ConclusionsThis study provides new insights into the role of the transcriptome in embryogenic callus formation in wheat, and will serve as a valuable resource for further studies addressing embryogenic callus formation in plants.

Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes

Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized.

Effects of the wheat UDP-glucosyltransferase gene TaUGT-B2 on Agrobacterium-mediated plant transformation

UDP-glucosyltransferase enzymes (UGTs) are known to function in plant disease defense responses. In our previous RNA-seq analysis, TaUGT expression was found to be strongly up-regulated in Agrobacterium-infected wheat immature embryos, as compared to untreated controls. In this study, we isolated the TaUGT-B2, located on wheat 2BS chromosome, and characterized its function in tobacco, Arabidopsis, and wheat. Transient overexpression of TaUGT-B2 reduced the number of Agrobacterium cells attached to the surface of wheat cells, and hindered Agrobacterium-mediated GUS transient expression in wheat cells. Transiently silenced TaUGT-B2 expression resulted in an increased rate of shoot regeneration from Agrobacterium-infected wheat tissues. Therefore, we concluded that the overexpression of TaUGT-B2 impeded T-DNA transformation by reducing number of Agrobacterium cells attached to wheat tissues and lowering the regeneration efficiency of transgenic plantlets. In order to further investigate the function of TaUGT-B2, it was introduced into tobacco and Arabidopsis, and transgenic plants were then re-transformed with another vector carrying the GUS gene. The results of these experiments indicated that the expression of TaUGT-B2 in tobacco and Arabidopsis led to a reduction in the efficiency of Agrobacterium-mediated transformation. GUS transient expression was also impeded following overexpression of TaUGT-B2 in tobacco and Arabidopsis. Therefore, we conclude that TaUGT-B2 plays a negative role in Agrobacterium-mediated transformation of plants, especially in wheat.

Identification and Comparative Analysis of microRNA in Wheat (Triticum aestivum L.) Callus Derived from Mature and Immature Embryos during In vitro Culture.

Feasible and efficient tissue culture plays an important role in plant genetic engineering. Wheat (Triticum aestivum L.) immature embryos (IMEs) are preferred for tissue culture to mature embryos (MEs) because IMEs easily generate embryogenic callus, producing large number of plants. The molecular mechanisms of regulation and the biological pathways involved in embryogenic callus formation in wheat remain unclear. Here, microRNAs (miRNAs) potentially involved in embryogenic callus formation and somatic embryogenesis were identified through deep sequencing of small RNAs (sRNAs) and analyzed with bioinformatics tools. Six sRNA libraries derived from calli of IMEs and MEs after 3, 6, or 15 d of culture (DC) were constructed and sequenced. A total of 85 known miRNAs were identified, of which 30, 33, and 18 were differentially expressed (P < 0.05) between the IME and ME libraries at 3, 6, and 15 DC, respectively. Additionally, 171 novel and 41 candidate miRNAs were also identified, of the novel miRNA, 69, 67, and 37 were differentially expressed (P < 0.05) between the two types of libraries at 3, 6, and 15 DC, respectively. The expression patterns of eight known and eight novel miRNAs were validated using quantitative real-time polymerase chain reaction. Gene ontology annotation of differentially expressed miRNA targets provided information regarding the underlying molecular functions, biological processes, and cellular components involved in embryogenic callus development. Functional miRNAs, such as miR156, miR164, miR1432, miR398, and miR397, differentially expressed in IMEs and MEs might be related to embryogenic callus formation and somatic embryogenesis. This study suggests that miRNA plays an important role in embryogenic callus formation and somatic embryogenesis in wheat, and our data provide a useful resource for further research.

Changes in distribution of zeatin and indole-3-acetic acid in cells during callus induction and organogenesis in vitro in immature embryo culture of wheat

Plant organogenesis remains one of the most essential questions of plant developmental biology. Callus tissue in vitro is a valuable tool for the studies on hormonal aspects of plant organogenesis, especially its early events, and immunohistochemical analysis is one of the few approaches offering information on the localization and role of hormones during organ development. The localization of endogenous zeatin and indole-3-acetic acid was investigated during simultaneous bud and root formation in calluses derived from immature embryos of wheat (Triticum aestivum L.). Calluses were induced on Murashige and Skoog (MS) medium supplemented with 2.0 mg L−1 2,4-dichlorophenoxyacetic acid. To stimulate simultaneous bud and root formation, calluses were transferred onto MS medium supplemented with 0.2 mg L−1 kinetin and 0.2 mg L−1 indoleacetic acid. Strong immunostaining for both hormones was detected in proliferating callus tissue, in developing meristematic centers and meristematic zones (whose cells were shown to be involved in organ formation), and at the sites of shoot and root apex initiation. During further development, shoot apexes with leaf primordia were heavily immunostained for zeatin, while immunostaining for indole-3-acetic acid was more intense at the sites of leaf primordia initiation and incipient primordia themselves. In the developing roots, immunostaining for both hormones reached a maximum in the root apex and gradually declined with increasing distance from the apex. Cells of developing procambial strands were also strongly stained for both zeatin and indole-3-acetic acid. These data suggest considerable similarity between patterns of hormone distribution in organs in vitro and in vivo. Thus, callus culture is a convenient and useful model for the study of fundamental biological questions such as how hormones regulate development.

Allelic state and effects of VRN genes on soft wheat in in vivo and in vitro systems

The article investigates the effects of the main manifestations of the genetic system controlling the type and rate of wheat development – VRN (vernalization) at the level of an integrated system of plant organism in vivo and callus certain population of cells in vitro. The paper studies the system of allelic VRN genes, which determine the need or insensitivity Triticum aestivum L. to vernalization and predetermination process of callusogenesis by the present system in vitro. In the experiments the modern model system of nearly isogenic lines (NILs) of spring type was used. The NILs were created in genotypes of the winter varieties Myronivska 808 and Olvia. Molecular genetic analysis of allelic loci of VRN genes was carried out by PCR analysis on grains and secondary callus culture using five pairs of specific primers (Grain Gene Mass Wheat). In the course of the experiments, it was found that the genetic system controlling the wheat rate determined the frequency of callusogenesis. However, the studied genetic system did not affect the morphological characteristics of primary and secondary callus tissue. In both hexaploid wheat cultivars the maximum frequency of callusogenesis appeared to be characterized in the Vrn 2 isogenic line and the original variety, slowly developing and intensely accumulating vegetative mass in vivo. The minimal frequency of callusogenesis was determined in the VRN 1 and VRN 3 isogenic lines, characterized by the rapid development of vegetation. The callus was derived from immature wheat embryos by morphological features analysis. Calluses with low water content, mainly, amorphous, compact, transparent, white or with yellowish tint were identified. Using PCR analysis, in grains in vivo and in the callus culture in vitro the almost identical allelic status of VRN genes was revealed. In grains and callus of Vrn 1 isogenic lines of both wheat varieties the presence of a dominant gene VRN A1 and recessive VRN B1 and VRN D1 was detected, whereas in VRN 2 – a dominant gene VRN B1 and recessive VRN A1 and VRN D1. However, the dominant allele VRN D1 in the studied NILs was detected. Therefore, in varieties of grains and callus cultures all genes are represented only by recessive alleles VRN A1, VRN B1 and VRN D1. Differences were found in the callus culture of VRN 3 isogenic line of Myronivska 808 on the allelic state of theVRN B1 gene. The obtained results could be associated with genomic reconstructions during callusogenesis induction. Our studies indicate the unidirectional system of VRN genes functioning, which appears to be the main control system of type and rate of soft wheat development in the system in vivo and in vitro. This allows us to assume the role of the VRN genetic system in the determination of callusogenesis, and also the adequacy of the functioning of the system in vitro processes, determining the system of integration of plants. Thus, our study confirms that the callus tissues cells of higher plants are able to preserve the cells properties of the whole organism along with the acquisition of new specific properties. Moreover, the culture in vitro is an adequate system for the study of the plant organism properties as a system.

Isolation and characterization of rubisco small subunit gene promoter from common wheat (Triticum aestivum L.)

Choice of an appropriate promoter is critical to express target genes in intended tissues and developmental stages. However, promoters capable of directing gene expression in specific tissues and stages are not well characterized in monocot species. To identify such a promoter in wheat, this study isolated a partial sequence of the wheat small subunit of RuBisCO (TarbcS) promoter. In silico analysis revealed the presence of elements that are characteristic to rbcS promoters of other, mainly dicot, species. Transient expression of the TarbcS:GUS in immature wheat embryos and tobacco leaves but not in the wheat roots indicate the functionality of the TarbcS promoter fragment in directing the expression of target genes in green plant tissues.

Effects of inter-culture, arabinogalactan proteins, and hydrogen peroxide on the plant regeneration of wheat immature embryos

The regeneration rate of wheat immature embryo varies among genotypes, howbeit many elite agriculture wheat varieties have low regeneration rates. Optimization of tissue culture conditions and attempts of adding signal molecules are effective ways to increase plant regeneration rate. Inter-culture is one of ways that have not been investigated in plant tissue culture. Moreover, the use of arabinogalactan proteins (AGPs) and hydrogen peroxide (H2O2) have been reported to increase regeneration rate in a few plant species other than wheat. The current research pioneeringly uses inter-culture of immature embryos of different wheat genotypes, and also investigates impacts of AGP and H2O2 on the induction of embryogenic calli and plant regeneration. As a result, high-frequency regeneration wheat cultivars Kenong 199 (KN199) and Xinchun 9 (XC9), together with low-frequency regeneration wheat line Chinese Spring (CS), presented striking increase in the induction of embryogenic calli and plant regeneration rate of CS through inter-culture strategy, up to 52.19 and 67.98%, respectively. Adding 50 to 200 mg L−1 AGP or 0.005 to 0.01 ‰ H2O2 to the callus induction medium, enhanced growth of embryogenic calli and plant regeneration rate in quite a few wheat genotypes. At 50 mg L−1 AGP application level in callus induction medium plant regeneration rates of 8.49, 409.06 and 283.16% were achieved for Jimai 22 (JM22), Jingdong 18 (JD18) and Yangmai 18 (YM18), respectively; whereas at 100 mg L−1 AGP level, CS (105.44%), Chuannong 16 (CN16) (80.60%) and Ningchun 4 (NC4) (62.87%) acted the best. Moreover CS (79.05%), JM22 (7.55%), CN16 (101.87%), YM18 (365.56%), Yangmai 20 (YM20) (10.48%), and CB301 (187.40%) were more responsive to 0.005 ‰ of H2O2, and NC4 (35.37%) obtained the highest shoot regeneration rates at 0.01 ‰ of H2O2. Overall, these two methods, inter-culture and AGP (or H2O2) application, can be further applied to wheat transgenic research.