CRISPR/Cas9-mediated мultiplexed multi-allelic mutagenesis of genes located on A, B and R subgenomes of hexaploid triticale.

KEY MESSAGE: The first-time generation of hexaploid triticale plants harbouring variable panels of novel mutations in gene families involved in starch biosynthesis has been achieved by the subgenome-independent multiplexed CRISPR/Cas9-mediated editing.

Endogenously Produced Jasmonates Affect Leaf Growth and Improve Osmotic Stress Tolerance in Emmer Wheat.

In light of recent climate change, with its rising temperatures and precipitation changes, we are facing the need to increase the valuable crop's tolerance against unfavorable environmental conditions. Emmer wheat is a cereal crop with high nutritional value. We investigated the possibility of improving the stress tolerance of emmer wheat by activating the synthesis of the stress hormone jasmonate by overexpressing two genes of the jasmonate biosynthetic pathway from Arabidopsis thaliana, ALLENE OXIDE SYNTHASE (AtAOS) and OXOPHYTODIENOATE REDUCTASE 3 (AtOPR3). Analyses of jasmonates in intact and mechanically wounded leaves of non-transgenic and transgenic plants showed that the overexpression of each of the two genes resulted in increased wounding-induced levels of jasmonic acid and jasmonate-isoleucine. Against all expectations, the overexpression of AtAOS, encoding a chloroplast-localized enzyme, does not lead to an increased level of the chloroplast-formed 12-oxo-phytodienoic acid (OPDA), suggesting an effective conversion of OPDA to downstream products in wounded emmer wheat leaves. Transgenic plants overexpressing AtAOS or AtOPR3 with increased jasmonate levels show a similar phenotype, manifested by shortening of the first and second leaves and elongation of the fourth leaf, as well as increased tolerance to osmotic stress induced by the presence of the polyethylene glycol (PEG) 6000.

12-Oxophytodienoate Reductase Overexpression Compromises Tolerance to Botrytis cinerea in Hexaploid and Tetraploid Wheat.

12-Oxophytodienoate reductase is the enzyme involved in the biosynthesis of phytohormone jasmonates, which are considered to be the major regulators of plant tolerance to biotic challenges, especially necrotrophic pathogens. However, we observe compromised tolerance to the necrotrophic fungal pathogen Botrytis cinerea in transgenic hexaploid bread wheat and tetraploid emmer wheat plants overexpressing 12-OXOPHYTODIENOATE REDUCTASE-3 gene from Arabidopsis thaliana, while in Arabidopsis plants themselves, endogenously produced and exogenously applied jasmonates exert a strong protective effect against B. cinerea. Exogenous application of methyl jasmonate on hexaploid and tetraploid wheat leaves suppresses tolerance to B. cinerea and induces the formation of chlorotic damages. Exogenous treatment with methyl jasmonate in concentrations of 100 µM and higher causes leaf yellowing even in the absence of the pathogen, in agreement with findings on the role of jasmonates in the regulation of leaf senescence. Thereby, the present study demonstrates the negative role of the jasmonate system in hexaploid and tetraploid wheat tolerance to B. cinerea and reveals previously unknown jasmonate-mediated responses.

CRISPR/Cas9-induced modification of the conservative promoter region of VRN-A1 alters the heading time of hexaploid bread wheat.

In cereals, the vernalization-related gene network plays an important role in regulating the transition from the vegetative to the reproductive phase to ensure optimal reproduction in a temperate climate. In hexaploid bread wheat (Triticum aestivum L.), the spring growth habit is associated with the presence of at least one dominant locus of VERNALIZATION 1 gene (VRN-1), which usually differs from recessive alleles due to mutations in the regulatory sequences of the promoter or/and the first intron. VRN-1 gene is a key regulator of floral initiation; various combinations of dominant and recessive alleles, especially VRN-A1 homeologs, determine the differences in the timing of wheat heading/flowering. In the present study, we attempt to expand the types of VRN-A1 alleles using CRISPR/Cas9 targeted modification of the promoter sequence. Several mono- and biallelic changes were achieved within the 125-117 bp upstream sequence of the start codon of the recessive vrn-A1 gene in plants of semi-winter cv. 'Chinese Spring'. New mutations stably inherited in subsequent progenies and transgene-free homozygous plants carrying novel VRN-A1 variants were generated. Minor changes in the promoter sequence, such as 1-4 nucleotide insertions/deletions, had no effect on the heading time of plants, whereas the CRISPR/Cas9-mediated 8 bp deletion between -125 and -117 bp of the vrn-A1 promoter shortened the time of head emergence by up to 2-3 days. Such a growth habit was consistently observed in homozygous mutant plants under nonvernalized cultivation using different long day regimes (16, 18, or 22 h), whereas the cold treatment (from two weeks and more) completely leveled the effect of the 8 bp deletion. Importantly, comparison with wild-type plants showed that the implemented alteration has no negative effects on main yield characteristics. Our results demonstrate the potential to manipulate the heading time of wheat through targeted editing of the VRN-A1 gene promoter sequence on an otherwise unchanged genetic background.

The Effect of Daminozide, Dark/Light Schedule and Copper Sulphate in Tissue Culture of Triticum timopheevii.

Triticum timopheevii Zhuk. is a tetraploid wheat that is utilized worldwide as a valuable breeding source for wheat improvement. Gene-based biotechnologies can contribute to this field; however, T. timopheevii exhibits recalcitrance and albinism in tissue cultures, making this species of little use for manipulation through genetic engineering and genome editing. This study tested various approaches to increasing in vitro somatic embryogenesis and plant regeneration, while reducing the portion of albinos in cultures derived from immature embryos (IEs) of T. timopheevii. They included (i) adjusting the balance between 2,4-D and daminozide in callus induction medium; (ii) cultivation using various darkness/illumination schedules; and (iii) inclusion of additional concentrations of copper ions in the tissue culture medium. We achieved a 2.5-fold increase in somatic embryogenesis (up to 80%) when 50 mg L-1 daminozide was included in the callus induction medium together with 3 mg L-1 2,4-D. It was found that the dark cultivation for 20-30 days was superior in terms of achieving maximum culture efficiency; moreover, switching to light in under 2 weeks from culture initiation significantly increased the number of albino plants, suppressed somatic embryogenesis, and decreased the regeneration of green plants. Media containing higher levels of copper ions did not have a positive effect on the regeneration of green plants; contrarily, the elevated concentrations caused albinism in plantlets. The results and relevant conclusions of the present study might be valuable for establishing an improved protocol for the regeneration of green plants in tissue cultures of T. timopheevii.

Effect of Grafting on Viral Resistance of Non-transgenic Plum Scion Combined With Transgenic PPV-Resistant Rootstock.

In stone fruit trees, resistance to Plum pox virus (PPV) can be achieved through the specific degradation of viral RNA by the mechanism of RNA interference (RNAi). Transgenic virus-resistant plants, however, raise serious biosafety concerns due to the insertion and expression of hairpin constructs that usually contain various selective foreign genes. Since a mature stone tree represents a combination of scion and rootstock, grafting commercial varieties onto transgenic virus-tolerant rootstocks is a possible approach to mitigate biosafety problems. The present study was aimed at answering the following question: To what extent are molecular RNAi silencing signals transmitted across graft junctions in transgrafted plum trees and how much does it affect PPV resistance in genetically modified (GM)/non-transgenic (NT) counterparts? Two combinations, NT:GM and GM:NT (scion:rootstock), were studied, with an emphasis on the first transgrafting scenario. Viral inoculation was carried out on either the scion or the rootstock. The interspecific rootstock "Elita" [(Prunus pumila L. × P. salicina Lindl.) × (P. cerasifera Ehrh.)] was combined with cv. "Startovaya" (Prunus domestica L.) as a scion. Transgenic plum lines of both cultivars were transformed with a PPV-coat protein (CP)-derived intron-separate hairpin-RNA construct and displayed substantial viral resistance. High-throughput sequence data of small RNA (sRNA) pools indicated that the accumulation of construct-specific small interfering RNA (siRNA) in transgenic plum rootstock reached over 2%. The elevated siRNA level enabled the resistance to PPV and blocked the movement of the virus through the GM tissues into the NT partner when the transgenic tissues were inoculated. At the same time, the mobile siRNA signal was not moved from the GM rootstock to the target NT tissue to a level sufficient to trigger silencing of PPV transcripts and provide reliable viral resistance. The lack of mobility of transgene-derived siRNA molecules was accompanied by the transfer of various endogenous rootstock-specific sRNAs into the NT scion, indicating the exceptional transitivity failure of the studied RNAi signal. The results presented here indicate that transgrafting in woody fruit trees remains an unpredictable practice and needs further in-depth examination to deliver molecular silencing signals.

Regulation of Sixth Seminal Root Formation by Jasmonate in Triticum aestivum L.

A well-developed root system is an important characteristic of crop plants, which largely determines their productivity, especially under conditions of water and nutrients deficiency. Being Poaceous, wheat has more than one seminal root. The number of grown seminal roots varies in different wheat accessions and is regulated by environmental factors. Currently, the molecular mechanisms determining the number of germinated seminal roots remain poorly understood. The analysis of the root system development in germinating seeds of genetically modified hexaploid wheat plants with altered activity of jasmonate biosynthesis pathway and seeds exogenously treated with methyl jasmonate revealed the role of jasmonates in the regulation of sixth seminal root development. This regulatory effect strongly depends on the jasmonate concentration and the duration of the exposure to this hormone. The maximum stimulatory effect of exogenously applied methyl jasmonate on the formation of the sixth seminal root was achieved at 200 µM concentration after 48 h of treatment. Further increase in concentration and exposure time does not increase the stimulating effect. While 95% of non-transgenic plants under non-stress conditions possess five or fewer seminal roots, the number of plants with developed sixth seminal root reaches up to 100% when selected transgenic lines are treated with methyl jasmonate.

Evaluation of Plant-Derived Promoters for Constitutive and Tissue-Specific Gene Expression in Potato.

Various plant-derived promoters can be used to regulate ectopic gene expression in potato. In the present study, four promoters derived from the potato genome have been characterized by the expression of identical cassettes carrying the fusion with the reporter ß-glucuronidase (gusA) gene. The strengths of StUbi, StGBSS, StPat, and StLhca3 promoters were compared with the conventional constitutive CaMV 35S promoter in various organs (leaves, stems, roots, and tubers) of greenhouse-grown plants. The final amount of gene product was determined at the post-transcriptional level using histochemical analysis, fluorometric measurements, and Western blot analysis. The promoter strength comparison demonstrated that the StUbi promoter generally provided a higher level of constitutive ß-glucuronidase accumulation than the viral CaMV 35S promoter. Although the StLhca3 promoter was predominantly expressed in a green tissue-specific manner (leaves and stems) while StGBSS and StPat mainly provided tuber-specific activity, a "promoter leakage" was also found. However, the degree of unspecific activity depended on the particular transgenic line and tissue. According to fluorometric data, the functional activity of promoters in leaves could be arranged as follows: StLhca3 > StUbi > CaMV 35S > StPat > StGBSS (from highest to lowest). In tubers, the higher expression was detected in transgenic plants expressing StPat-gusA fusion construct, and the strength order was as follows: StPat > StGBSS > StUbi > CaMV 35S > StLhca3. The observed differences between expression patterns are discussed considering the benefits and limitations for the usage of each promoter to regulate the expression of genes in a particular potato tissue.

A competence of embryo-derived tissues of tetraploid cultivated wheat species Triticum dicoccum and Triticum timopheevii for efficient and stable transgenesis mediated by particle inflow gun.

BACKGROUND: The ability to engineer cereal crops by gene transfer technology is a powerful and informative tool for discovering and studying functions of genes controlling environmental adaptability and nutritional value. Tetraploid wheat species such as emmer wheat and Timopheevi wheat are the oldest cereal crops cultivated in various world areas long before the Christian era. Nowadays, these hulled wheat species are gaining new interest as donors for gene pools responsible for the improved grain yield and quality, tolerance for abiotic and biotic stress, resistance to pests and disease. The establishing of efficient gene transfer techniques for emmer and Timopheevi wheat may help in creation of modern polyploid wheat varieties. RESULTS: In the present study, we describe a robust protocol for the production of fertile transgenic plants of cultivated emmer wheat (Russian cv. 'Runo') using a biolistic delivery of a plasmid encoding the gene of green fluorescent protein (GFP) and an herbicide resistance gene (BAR). Both the origin of target tissues (mature or immature embryos) and the type of morphogenic calli (white or translucent) influenced the efficiency of stable transgenic plant production in emmer wheat. The bombardment of nodular white compact calluses is a major factor allowed to achieve the highest transformation efficiency of emmer wheat (on average, 12.9%) confirmed by fluorescence, PCR, and Southern blot. In the absence of donor plants for isolation of immature embryos, mature embryo-derived calluses could be used as alternative tissues for recovering transgenic emmer plants with a frequency of 2.1%. The biolistic procedure based on the bombardment of immature embryo-derived calluses was also successful for the generation of transgenic Triticum timopheevii wheat plants (transformation efficiency of 0.5%). Most of the primary events transmitted the transgene expression to the sexual progeny. CONCLUSION: The procedures described here can be further used to study the functional biology and contribute to the agronomic improvement of wheat. We also recommend involving in such research the Russian emmer wheat cv. 'Runo', which demonstrates a high capacity for biolistic-mediated transformation, exceeding the previously reported values for different genotypes of polyploid wheat.

Agrobacterium-Mediated Transformation of Russian Commercial Plum cv. "Startovaya" (Prunus domestica L.) With Virus-Derived Hairpin RNA Construct Confers Durable Resistance to PPV Infection in Mature Plants.

In modern horticulture Plum pox virus (PPV) imposes serious threats to commercial plantations of a wide range of fruit species belonging to genera Prunus. Given the lack of natural genetic resources, which display reliable resistance to PPV infection, there has been considerable interest in using genetic engineering methods for targeted genome modification of stone fruit trees to control Sharka disease caused by PPV. Among the many virus defense mechanisms, RNA interference is shown to be the most promising transgenic disease-control strategy in plant biotechnology. The present study describes the production of transgenic PPV resistant European plum "Startovaya" (P. domestica L.) through the Agrobacterium-mediated transformation of in vitro leaf explants. Due to organogenesis from leaves, the established protocol allows the genetic engineering of the plum genome without losing clonal fidelity of original cultivar. Seven independent transgenic plum lines containing the self-complementary fragments of PPV-CP gene sequence separated by a PDK intron were generated using hpt as a selective gene and uidA as a reporter gene. The transformation was verified through the histochemical staining for ß-glucuronidase activity, PCR amplification of appropriate vector products from isolated genomic DNA and Southern blot analysis of hairpin PPV-CP gene fragments. To clarify the virus resistance, plum buds infected by PPV-M strain were grafted onto 1-year-old transgenic plants, which further were grown into mature trees in the greenhouse. As evaluated by RT-PCR, DAS-ELISA, Western blot, ImmunoStrip test, and visual observations, GM plum trees remained uninfected over 9 years. Infected branches that developed from grafted buds displayed obvious symptoms of Sharka disease over the years and maintained the high level of virus accumulation, whereby host transgenic trees had been constantly challenged with the pathogen. Since the virus was unable to spread to transgenic tissues, the stable expression of PPV-derived gene construct encoding intron-spliced hairpin RNAs provided a highly effective protection of plum trees against permanent viral infection. At the same time, this observation indicates the lack of the systemic spread of resistance from GM tissues to an infected plum graft even after years of joint growth.

Overexpression of Arabidopsis OPR3 in Hexaploid Wheat (Triticum aestivum L.) Alters Plant Development and Freezing Tolerance.

Jasmonates are plant hormones that are involved in the regulation of different aspects of plant life, wherein their functions and molecular mechanisms of action in wheat are still poorly studied. With the aim of gaining more insights into the role of jasmonic acid (JA) in wheat growth, development, and responses to environmental stresses, we have generated transgenic bread wheat plants overexpressing Arabidopsis 12-OXOPHYTODIENOATE REDUCTASE 3 (AtOPR3), one of the key genes of the JA biosynthesis pathway. Analysis of transgenic plants showed that AtOPR3 overexpression affects wheat development, including germination, growth, flowering time, senescence, and alters tolerance to environmental stresses. Transgenic wheat plants with high AtOPR3 expression levels have increased basal levels of JA, and up-regulated expression of ALLENE OXIDE SYNTHASE, a jasmonate biosynthesis pathway gene that is known to be regulated by a positive feedback loop that maintains and boosts JA levels. Transgenic wheat plants with high AtOPR3 expression levels are characterized by delayed germination, slower growth, late flowering and senescence, and improved tolerance to short-term freezing. The work demonstrates that genetic modification of the jasmonate pathway is a suitable tool for the modulation of developmental traits and stress responses in wheat.

Genetic transformation of einkorn (Triticum monococcum L. ssp. monococcum L.), a diploid cultivated wheat species.

BACKGROUND: Domesticated einkorn (Triticum monococcum L.) is one of the oldest cultivated cereal crops in the world. Its small genome size (~ 5.7 GB), low ploidy (2n = 2x = 14, AmAm) and high genetic polymorphism make this species very attractive for use as a diploid model for understanding the genomics and proteomics of Triticeae. Einkorn, however, is still a recalcitrant monocotyledonous species for the application of modern biotechnologies, including transgenesis. This paper reports the factors that may influence transgene delivery, integration, expression and inheritance in einkorn. RESULTS: In this study, we report the successful genetic transformation of einkorn using biolistic-mediated DNA delivery. Immature embryo-derived tissues of spring einkorn were bombarded with a plasmid containing the reporter gene GFP (green fluorescent protein) driven by the rice actin promoter (act1) and the selectable bar gene (bialaphos resistance gene) driven by the maize ubiquitin promoter (ubi1). Adjustments to various parameters such as gas pressure, microcarrier size and developmental stage of target tissue were essential for successful transient and stable transformation. Bombarded einkorn tissues are recalcitrant to regenerating plants, but certain modifications of the culture medium have been shown to increase the production of transgenic events. In various experiments, independent transgenic plants were produced at frequencies ranging from 0.0 to 0.6%. Molecular analysis, marker gene expression and herbicide treatment demonstrated that gfp/bar genes were stably integrated into the einkorn genome and successfully inherited over several generations. The transgenes, as dominant loci, segregated in both Mendelian and non-Mendelian fashion due to multiple insertions. Fertile homozygous T1-T2 populations of transgenic einkorn that are resistant to herbicides were selected. CONCLUSION: To the best of our knowledge, this is the first report of the production of genetically modified einkorn plants. We believe that the results of our research could be a starting point for the application of the current biotechnological-based technologies, such as transgenesis and genome editing, to accelerate comparative functional genomics in einkorn and other cereals.

Protocol for efficient regulation of in vitro morphogenesis in einkorn (Triticum monococcum L.), a recalcitrant diploid wheat species.

Einkorn (Triticum monococcum L.) is A-genome diploid wheat that has a potential to become a useful model for understanding the biology and genomics in Triticeae. Unfortunately, the application of modern technologies such as genetic engineering, RNAi-based gene silencing and genome editing is not available for einkorn as there is no efficient in vitro tissue culture and plant regeneration system. In the present study an efficient and simple protocol for plant regeneration via direct or indirect somatic embryogenesis and organogenesis has been developed. Various auxins used as sole inductors in einkorn displayed low effect for morphogenesis (0-8%) and plant regeneration (1-2 shoots per explant). The addition of Daminozide, the inhibitor of biosynthesis of gibberellins, together with auxin significantly improved the formation of morphogenic structures, especially when Dicamba (51.4%) and Picloram (56.6%) were used for combination; furthermore, the simultaneous addition of cytokinin into induction medium significantly promoted in vitro performance. Among the tested cytokinins, the urea-type substances, such as TDZ and CPPU were more effective than the adenine type ones, BA and Zeatin, for the regulation of morphogenesis; especially, TDZ was more effective than CPPU for shoot formation (11.73 vs. 7.04 per regenerating callus). The highest morphogenic response of 90.2% with the production of more than 10 shoots per initial explant was observed when 3.0 mg/L Dicamba, 50.0 mg/L Daminozide and 0.25 mg/L TDZ were combined together. Along with the identification of appropriate induction medium, the optimal developmental stage for einkorn was found as partially transparent immature embryo in size of around 1.0 mm. Although in the present study the critical balance between plant growth regulators was established for einkorn only, we assume that further the proposed strategy could be successfully applied to other recalcitrant cereal species and genotypes.