Histological analysis of hard white scutellum-like structures of <i>Triticum monococcum</i> L. induced on the medium supplemented with 3 mg/L DIC and 50 mg/L DAM.

<p>(A) Section from developing scutellum-like structures bordered by well-developed protoderm (the outer unicellular layer) at an early stage of the ‘storage-like’ differentiation (asterisks), 15 days of culture, <i>bar</i> 100 μm. (B) General view of the entire longitudinal section from the hard scutellum-like structures at the end of callus induction phase, 25 days of culture, <i>bar</i> 100 μm. (C) Detailed view from the part of longitudinal section from the scutellum-like structures, the presence of cells with various shapes and differentiation is occurred, <i>bar</i> 50 μm. (D) The presence of vacuolated big cell types with the larger intracellular spaces, starch granules (closed arrow) and stock substances on the vacuoles surfaces (open arrow), <i>bar</i> 20 μm. (E) The presence of small granules concentrated in cytoplasm along the cell walls, <i>bar</i> 20 μm. <i>p</i>–protoderm, <i>m</i>.<i>c</i>.–meristematic centers.</p

The effect of PGRs combinations added to induction medium on morphogenesis and plant regeneration from immature embryos of einkorn (<i>Tritcum monococcum</i> L.).

<p>The effect of PGRs combinations added to induction medium on morphogenesis and plant regeneration from immature embryos of einkorn (<i>Tritcum monococcum</i> L.).</p

Histological study of morphogenesis in immature embryo-derived culture of <i>Triticum monococcum</i> L. on the medium supplemented with 3 mg/L DIC, 0.25 mg/L TDZ and 50 mg/L DAM.

<p>(A) Initial divisions of peripheral cells with dense cytoplasm and big nuclei in the contrast to cells deeper in the scutellar tissue that remained vacuolated, 7 days of culture, <i>bar</i> 20 μm. (B) Nodular structures progressed from active cell divisions of the inner part of cultured scutellum, 15 days of culture, <i>bar</i> 200 μm. (C) Details of nodular structure showing both pericline and anticline divisions of isodiametric cytoplasm-rich cells, <i>bar</i> 100 μm. (D) Longitudinal section of leafy-like structures presenting a connection with the original tissue of scutellum, 19 days of culture, <i>bar</i> 200 μm. (E) The axis of shoot primordia vascularly connected with the base of leaf-like structure, 40 days of culture, <i>bar</i> 200 μm. (F) Direct development of embryo-like structure without vascular connection with the original tissue, 17 days of culture, <i>bar</i> 100 μm.</p

Morphological aspects of morphogenesis and plant regeneration in <i>Triticum monococcum</i> L.

<p><b>(A-C).</b> Effect of PGRs on the regenerable callus induction from immature embryos on the medium supplemented with 3 mg/L DIC (A), 3 mg/L DIC and 25 mg/L DAM (B), 3 mg/L DIC, 50 mg/L DAM and 0.25 mg/L TDZ (C), 6 weeks of culture. (D-H, J-K) Details of morphogenesis in immature embryo cultures induced on the medium supplemented with 3 mg/L DIC, 50 mg/L DAM and 0.25 mg/L TDZ; (D) swelling at the inner part of cultured immature scutellum, 7 days of culture; (E) appearance of nodular structures on the scutellum, 14 days of culture; (F) formation of leaf-like structures (asterisks), 20 days of culture, and (G) their development by the end of the callus induction phase; (H) differentiation of multiple plantlets on regeneration medium under 16 h of photoperiod, 40 days of culture; (J) shoot apices formation at the base of leaf-like structure on regeneration medium, 50 days of culture; (K) pro-morphogenic granular masses (black arrows) appeared on the same explant concomitantly with the development of plantlets and leaf-like structures (asterisks), 35 days of culture. (I) Callus induced on the medium supplemented with 3 mg/L DIC and 50 mg/L DAM displaying the formation of non translucent scutellum-like (white arrows) and translucent off-white nodular (black arrows) morphotypes, 30 days of culture. (L) In vitro plant regeneration from morphogenic callus, 25 days of culture on medium lack of growth regulators under 16-hour photoperiod. (M) Mature regenerated plants growing under greenhouse conditions.</p

The influence of various cytokinin concentrations on morphogenesis and plant regeneration from immature embryos of einkorn (<i>Tritcum monococcum</i> L.).

<p>The influence of various cytokinin concentrations on morphogenesis and plant regeneration from immature embryos of einkorn (<i>Tritcum monococcum</i> L.).</p

The influence of cytokinin type on plant regeneration from morphogenic calluses of einkorn (<i>Tritcum monococcum</i> L.).

<p>The influence of cytokinin type on plant regeneration from morphogenic calluses of einkorn (<i>Tritcum monococcum</i> L.).</p

The influence of DAM concentrations on morphogenesis and plant regeneration from immature embryos of einkorn (<i>Tritcum monococcum</i> L.)

<p>The influence of DAM concentrations on morphogenesis and plant regeneration from immature embryos of einkorn (<i>Tritcum monococcum</i> L.)</p

Histology of <i>Triticum monococuum</i> L. somatic embryos at different stages of development.

<p>(A) Transversal section of meristematic centers delineated from the surrounding non-organized, dispersed and vacuolated cells, <i>bar</i> 100 μm. (B) Thickened cell walls proembryo arising from friable callus tissue, <i>bar</i> 50 μm. (C) Section of globular somatic embryo with suspensor displaying transition to bilateral symmetrical orientation (open arrows), <i>bar</i> 100 μm. (D) Longitudinal section of somatic embryo at a later developmental stage showing clear bilateral symmetrical orientation, <i>bar</i> 100 μm. <i>p</i>–protoderm, <i>sm</i>–shoot meristem, <i>cr</i>–coleorhizae, <i>co–</i>coleoptile, <i>sc</i>.–scutellum, <i>s</i>–suspencer, <i>ve</i>–ventral; <i>do</i>–dorsal; <i>di</i>–distal; <i>pr</i>–proximal;</p

DataSheet_1_CRISPR/Cas9-induced modification of the conservative promoter region of VRN-A1 alters the heading time of hexaploid bread wheat.pdf

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.</p