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.

The identification of a new gene for leaf pubescence introgressed into bread wheat from Triticum timopheevii Zhuk. and its manifestation in a different genotypic background

Leaf pubescence is widespread among higher plants. In bread wheat, a relationship was found between this trait and the efficiency of photosynthetic processes and productivity. In this work, we established the chromosomal localization of the gene for leaf pubescence introgressed from Triticum timopheevii into a bread wheat line 821 and studied its expression in the genetic background of two wheat cultivars differing in genetic control and phenotypic expression of pubescence. To obtain quantitative characteristics of pubescence in cultivars and hybrid populations, the LHDetect2 program was used, which makes it possible to estimate the length and number of trichomes on a leaf fold. A genetic analysis showed the dominant inheritance of the gene. Monosomic analysis F2 was used to establish chromosome localization and investigate the expression of the gene in cultivars Saratovskaya S29 (S29) and Diamant 2 (Dm2). As a result, the gene Hltt, introgressed from T. timopheevii, was identified and localized in the distal region of the long arm of 5A chromosome for the first time. In both F2 populations, the gene reduced the density of trichomes and formed long trichomes, uncharacteristic for the two recipient cultivars S29 and Dm2. A larger number of long trichomes was formed in the genetic background of S29, which carry the bread wheat gene Hl1 and Hl3 for leaf pubescence, than in Dm2. Development of substitution and isogenic lines with the fragment of introgression carrying the gene Hltt will allow determining function and assessing the adaptive significance of the gene more precisely.

Genetic mapping of male sterility and pollen fertility QTLs in triticale with sterilizing Triticum timopheevii cytoplasm

Cytoplasmic male sterility (CMS) phenomenon is widely exploited in commercial hybrid seed production in economically important crop species, including rye, wheat, maize, rice, sorghum, cotton, sugar beets, and many vegetables. Although some commercial successes, little is known about QTLs responsible for the trait in case of triticale with sterilizing Triticum timopheevii (Tt) cytoplasm. Recombinant inbred line (RIL) F6 mapping population encompassing 182 individuals derived from the cross of individual plants representing the HT352 line and cv Borwo was employed for genetic map construction using SNP markers and identification of QTLs conferring pollen sterility in triticale with CMS Tt. The phenotypes of the F1 lines resulting from crossing of the HT352 (Tt) with HT352 (maintainer) × Borwo were determined by assessing the number of the F2 seeds per spike. A genetic map with 21 linkage groups encompasses 29,737 markers and spanned over the distance of 2549 cM. Composite (CIM) and multiple (MIM) interval mappings delivered comparable results. Single QTLs mapped to the 1A, 1B, 2A, 2R, 3B, 3R, 4B, and 5B chromosomes, whereas the 5R and 6B chromosomes shared 3 and 2 QTLs, respectively. The QTLs with the highest LOD score mapped to the 5R, 3R, 1B, and 4B chromosomes; however, the QRft-5R.3 has the highest explained variance of the trait.

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.