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At least 19 recordsLinked to original sources
PubMed2026

Controlling GRF4-GIF1 expression for efficient, genotype-independent transformation across wheat cultivars.

Wheat is a staple crop critical for global food security, and its continuous genetic improvement is essential to meet the demands of a growing population. Efficient, genotype-independent transformation is a major bottleneck in wheat functional genomics and gene editing. The growth regulating factor (GRF)-GRF-interacting factor (GIF) fusion technology enhances regeneration efficiency and broadens the range of transformable cultivars, but constitutive expression can reduce fertility and spikelet number. Here, we present an optimised Agrobacterium-mediated wheat transformation protocol incorporating GRF4-GIF1, tested across multiple tetraploid and hexaploid cultivars. Transformation efficiency was improved through adjustments in selection pressure, zeatin concentration, and promoter choice, with GRF4-GIF1 consistently enabling successful transformation across genotypes. Tissue-specific promoters and heat-inducible excision strategies effectively minimised pleiotropic effects, such as reduced fertility, while maintaining high transformation rates. This refined system provides a robust and versatile platform for gene function studies and gene editing, advancing genotype-independent wheat transformation and supporting breeding efforts to improve crop productivity, resilience, and nutritional value.

Triticum↗
PubMed2025

An elegant co-transformation strategy for recalcitrant wheat using morphogenic regulators.

Common wheat (Triticum aestivum L.) is a vital global crop, but many elite cultivars remain recalcitrant to genetic transformation, hindering functional genomics and crop improvement. Here, we developed an efficient co-transformation strategy for recalcitrant wheat varieties (e.g., Aikang58 and Xinong979) using the morphogenic gene mTaGRF4-TaGIF1. This approach entails mixing Agrobacterium tumefaciens cultures carrying two separate vectors: a standard gene-of-interest (GOI) vector (containing a selectable marker) and a gene-of-co-transformation vector (GOC, expressing mTaGRF4-TaGIF1 without a selectable marker). Co-transformation enhanced regeneration efficiency to ~37.38% in AK58, a marked improvement over conventional methods, enabling consistent recovery of transgenic plants. Among regenerants, ~63.25% carried both GOI and GOC (GOI&GOC), while ~11.92% contained only the GOI. Only-GOI plants could also be obtained through progeny segregation from GOI&GOC lines. We successfully generated GUS- and RUBY-expressing transgenic lines, as well as CRISPR-Cas9-edited mutants targeting Q and Ph1 genes, confirming the method's efficacy for both gain-of-function and genome editing application. Furthermore, the strategy was successfully extended to another recalcitrant variety Xinong979, demonstrating its potential for broad applicability. Unlike existing methods dependent on complex excision systems or tissue-specific promoters, our co-transformation methodology significantly simplifies both vector design and procedural workflow while maintaining high efficiency. Collectively, these findings establish a technically advanced yet operationally simplified transformation platform that addresses the long-standing challenge of genetic transformation in recalcitrant wheat varieties, providing researchers with a powerful tool for functional genomics studies and accelerating precision breeding programs in elite wheat cultivars.

Triticum↗
PubMed2026

Impact of wheat GRF4-GIF1 morphogenic regulators on transformation and genome editing efficiency in elite barley cultivars.

INTRODUCTION: Efficient genetic transformation is essential for the delivery of the CRISPR/Cas9 genome editing system and thus represents an important technology for breeding-oriented research in barley (Hordeum vulgare L.). However, transformation and plant regeneration from tissue culture remain challenging in non-model barley genotypes. Previous studies demonstrated that expression of a chimeric fusion between two interacting transcription factors, GROWTH-REGULATING FACTOR 4 (GRF4) and GRF-INTERACTING FACTOR 1 (GIF1), enhances regeneration capacity in wheat and other species. METHODS: In this study, we evaluated the effect of the wheat-derived GRF4-GIF1 morphogenic regulators on biolistic transformation and genome editing efficiency in three commercial barley cultivars: Tselinniy 5, Aley, and G-23035. RESULTS: The JD633 construct carrying GRF4-GIF1 enabled recovery of stable transformants in all three genotypes, with efficiencies ranging from 2.5% to 5%, whereas the control construct lacking morphogenic regulators resulted in no transgenic events in any of the tested varieties. Among transformed T0 plantlets, genome editing efficiency reached 64.3%, with predominantly biallelic mutations that were stably inherited in the T1 generation. Molecular screening revealed the presence of plasmid-free edited plants in the T0 generation, likely arising from transient Cas9 expression, and provided evidence of tissue chimerism. DISCUSSION: These results demonstrate that the GRF-GIF system facilitates genome editing, providing a practical framework for accelerating precision breeding in barley.

CRISPR/Cas9↗
PubMed1975

Interaction of Lens culinaris lectin, concanavalin A, Ricinus communis agglutinin and wheat germ agglutinin with the cell surface of normal and transformed rat liver cells.

The observation of BOREK et al. (1973) on nonagglutinability of transformed rat liver cells by Lens culinaris lectin and our ultrastructural findings of a greater mobility of the Lens culinaris lectin receptors on transformed rat liver cells as compared to normal rat liver cells (ROTH 1975) initiated the present agglutination experiments on liver cells with lectins. For agglutination assay the microhemadsorption technique after FURMANSKI et al. (1973) was used with exception of several tests on EDTA-detached cells. The transformed rat liver cells exhibited, in contrast to the findings of BOREK et al. (1973), a positive microhemadsorption with Lens culinaris lectin as well as with Concanavalin A, Ricinus communis lectin and wheat germ agglutinin whereas the normal rat liver cells became positive only after a brief trypsin treatment. The significance of the difference in agglutinability of rat liver cells with Lens culinaris lectin and the other lectins used is discussed with regard to the cell-cell interaction mediated by lectins.

Agglutination Tests↗
PubMed1976

Monomer molecular weight of T antigen from simian virus 40-infected and transformed cells.

T-antigens from simian virus 40 (SV 40)-transformed and lytically infected cells have been isolated by immunoprecipitation and their molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. T-antigen from SV40-transformed mouse and hamster cells has an apparent molecular weight of 94,000 whereas that from several lines of SV40-infected monkey cells is 84,000. In a wheat germ cell-free system, mRNA from either transformed or productively infected cells is translated into a 94,000 species. Experiments with the protease inhibitors L-l-(tosylamide-2-phenyl)ethylchloromethyl ketone HCl and N-alpha-p-tosyl-L-lysylchloromethyl ketone HCl suggest that the 84,000 species of T-antigen found in infected cells is derived from the larger species by proteolytic cleavage. Further, the cleavage pathway probably involves a two-step reaction with an 89,000 intermediate. The biological significance of the two molecular weight forms of T-antigen is unknown, but the possibility that they have different physiological activities is discussed.

Antigens, Viral↗
PubMed1978

Similarities in the membrane fluidity of 3T3 and SV101-3T3 cells and its relation to concanavalin A- and wheat germ agglutinin-induced agglutination.

Intact, viable ultransformed 3T3 and transformed SV101-3T3 cells were labeled with fatty acid spin labels and with 2,2,6,6-tetramethylpiperidine-1-oxyl in order to measure the fluidity properties of membrane lipids. Both cell types were grown in regular calf serum and in a lipid-depleted serum supplemented with either oleate or elaidate. The temperature dependence of the spectra obtained revealed inflections that correlate with the temperature below which agglutination with concanavalin A is inhibited, and another inflection that correlates with the temperature below which agglutination with wheat germ agglutinin is inhibited, suggesting that (a) the lipid phase(s) in the vicinity of the receptor(s) for these two lectins differ, and (b) a fluid membrane in the vicinity of the lectin receptor(s) is necessary for agglutination with either concanavalin A or wheat germ agglutinin. Studies with a partially characterized plasma membrane fraction suggest that the plasma membrane fluidity parameters closely resemble those of the intact cell. 3T3 and SV101-3T3 cells show virtually identical fluidity profiles by all of the tests we have applied.

Agglutination↗
PubMed1979

Cholesterol levels and plasma membrane fluidity in 3T3 and SV101-3T3 cells.

Polyene antibiotics such as filipin selectively inhibit wheat germ agglutinin-induced agglutination of transformed and malignant cells compared to normal cells (Hatten ME, Burger MM: Biochemistry 18: 739, 1979). Since filipin binds specifically to cholesterol, we measured cholesterol levels in 3T3 cells and SV101-3T3 cells. SV101-3T3 cells contained 50-100% more cholesterol per cell than 3T3 cells. Both cell types were starved for cholesterol by growth in lipid-depleted medium plus 25-hydroxycholesterol. The cholesterol level of SV101-3T3 cells decreased by 30-50%, while the level in 3T3 cells remained constant. Filipin-stained SV101-3T3 cells revealed bright patches of filipin under fluorescence microscopy. These patches were absent in 3T3 cells and in SV101-3T3 and 3T3 cells starved for cholesterol. We selectively labeled plasma membranes of these cells with a spin label analog of phosphatidylcholine. The spin label indicated differences in plasma membrane fluidity that may be related to the different cholesterol levels in 3T3 and SV101-3T3 cells.

Animals↗
PubMed1977

Levels of translatable mRNAs for cell surface protein, collagen precursors, and two membrane proteins are altered in Rous sarcoma virus-transformed chick embryo fibroblasts.

Transformation of chick embryo fibroblasts by Rous sarcoma virus results in decreased amounts of a major cell surface protein and of collagen. To determine the mechanism accounting for the decreased production of these proteins, we have measured the relative amounts of functional mRNAs for these and other transformation-sensitive proteins. Total cellular RNAs extracted from normal cells and from cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus were translated in a cell-free system derived from wheat germ. Analysis of the in vitro translation products of RNAs from normal and transformed chick embryo fibroblasts shows a 5-fold reduction in the translatable mRNA for cell surface protein and a 10-fold reduction in translatable mRNA for two collagen precursors. In addition, increases in functional mRNA are observed for myosin and for two membrane polypeptides with molecular weights of 95,000 and 78,000; the latter two proteins increase on transformation, but the increases are in large part secondary to the depletion of glucose from the medium of transformed cells. Our data suggest that some of the major cellular changes induced by oncogenic viruses are due to changes in the activity of specific cellular genes.

Actins↗
PubMed1979

Effect of polyene antibiotics on the lectin-induced agglutination of transformed and untransformed cell lines.

Treatment of transformed Py3T3, SV101-3T3, and L1210 cells, as well as mitotic and Pronase-treated untransformed 3T3 cells, with the polyene antibiotics filipin, nystatin, and amphotericin B inhibited agglutination by wheat germ agglutinin. The effect of polyene antibiotic treatment was lectin and cell specific. Concanavalin A induced agglutination was not inhibited, wheat germ agglutination induced agglutination of untransformed 3T3 interphase cells was not influenced, and other aggregation phenomena, including those of erythrocytes with blood group specific antibodies or divalent cations, were unaffected by polyene treatments. This suggests that the formation of polyene-cholesterol complexes in transformed and erythrocyte cell membranes may specifically affect wheat germ agglutinin receptors and/or secondary events necessary for wheat germ agglutinin induced agglutination. Fluorescence studies of membrane filipin-cholesterol complexes showed that pretreating the cells with wheat germ agglutinin, but not concanavalin A, perturbed the fluorescence properties of filipin. Electron spin resonance studies with spin-labeled fatty acids revealed at best only a slight decrease in fatty acyl chain flexibility following filipin treatment. These studies indicate that there are not only quantitative differences between the agglutinability of transformed and untransformed cells with wheat germ agglutinin but that qualitative differences exist as well.

Agglutination↗
PubMed1975

Changes in cell surface structure by viral transformation studied by binding of lectins differing in sugar specificity.

Changes in cell surface structure by viral transformation were studied by examining changes in the binding of various lectins differing in carbohydrate specificities. Binding of lectins was assayed directly using cells grown in coverslips. The following 125I-lectins were used: Concanavalin-A (specific for glucose and mannose), wheat germ agglutinin (specific for N-acetylglucosamine), castor bean agglutinin (specific for galactose), Wistaria floribunda agglutinin (specific for N-acetylgalactosamine), and soybean agglutinin (specific for N-acetyl-galactosamine). Cells for a clone, SS7, transformed by bovine adenovirus type-3, were found to bind 5 to 6 times more Wistaria floribunda agglutinin than the normal counterpart cells (clone C31, from C3H mouse kidney). In contrast, the binding of soybean agglutinin, which has a sugar specificity similar to Wistaria floribunda agglutinin, to normal and transformed cells was similar. The binding of wheat germ agglutinin and castor bean agglutinin, respectively, to normal and transformed cells was also similar. However, normal cells bound twice as much concanavalin-A as transformed cells. Only half as much Wistaria floribunda agglutinin was bound to transformed cells when they had been dispersed with EDTA. These changes in the number of lectin binding sites on transformation are thought to reflect alteration of the cell surface structure. The amount of lectins bound per cell decreased with increase in cell density, especially in the case of binding of Wistaria floribunda agglutinin to normal cells.

Adenoviridae↗
PubMed1977

Changes in surface properties of normal and transformed cells caused by tunicamycin, an inhibitor of protein glycosylation.

Normal and virally transformed mouse (3T3) and human (WI-38) cells were treated with tunicamycin, an inhibitor of lipid-carrier-dependent glycosylation of proteins. Incubation of cells with tunicamycin (1 microgram/ml) caused detachment and death of simian virus 40- and polyoma-transformed cells within 24 hr; these effects were not seen with nontransformed cell lines. However, the proliferation of 3T3 cells was inhibited by tunicamycin and, after a few days, a distinct change from an epithelioid to an abnormally elongated shape was observed. Both inhibition of growth and the morphological changes were reversible. A marked decrease in concanavalin A agglutinability was observed in virally transformed cells treated with tunicamycin (0.5 microgram/ml), but agglutination by wheat germ agglutinin or soybean agglutinin was unaffected. Analysis of biosynthetically labeled proteins showed that a high-molecular-weight protein, presumed to be related to fibronectin, is markedly reduced in the medium of cells cultured in the presence of tunicamycin. These results suggest that tunicamycin interferes with the insertion or function of one or more cell-surface glycoproteins. Such cell-surface changes could affect a number of cellular properties, including attachment, cell shape, and agglutinability by some lectins.

Agglutination Tests↗
PubMed2026

The combination of morphogenic regulators BABY BOOM and GRF-GIF improves maize transformation efficiency and promotes leaf regeneration.

Transformation is an indispensable tool for plant genetics and functional genomics. Although stable transformation in maize is no longer a major obstacle, there remains a need for accessible and efficient methods for academic laboratories. Here, we present the GGB system, a rapid and efficient approach optimized for immature embryo transformation in B104 and other maize lines. This system combines two distinct morphogenetic regulators, the wheat GRF4-GIF1 chimera and the maize BABY BOOM (BBM) transcription factor (hence the name "GGB") with a modified QuickCorn protocol, enabling regeneration of transformed maize plantlets in c. 2 months with an efficiency 7-fold higher than when compared to either morphogenic factor used in isolation. Expression of both regulators did not significantly affect development, eliminating the need to excise them after regeneration. However, transmission of the transgenic GGB construct through pollen was significantly reduced, potentially aiding transgenic line containment. We show that the GGB system is adaptable for CRISPR-Cas9 editing and reporter line generation. Furthermore, stable GGB transformants exhibited high leaf regeneration capacity via somatic embryogenesis. RNA-seq time-course profiling of GGB leaf cultures identified additional factors that could promote regeneration and led to the discovery of asparagine and trehalose as additional media components that significantly enhanced leaf regeneration.

Zea mays↗
PubMed1978

Photochemical modifications of the tryptophan residues of wheat-germ agglutinin in the presence of trichloroethanol.

Trichloroethanol is an efficient quencher of indole fluorescence of model compounds and proteins [Eftink, M. R. and Ghiron, C. A. (1976) J. Phys. Chem. 80, 486--493]. At low quencher concentrations, the quenching follows the classical Stern-Volmer law. Bimolecular rate constants calculated from measured quenching constants and lifetimes are equal to 6 X 10(9) M-1s-1 and 1.2 X 10(9) M-1s-1 for N-acetyltrypotophanamide and wheat germ agglutinin, respectively. Upon ultraviolet irradiation in the presence of trichloroethanol, transformation of fluorescent tryptophan occurs, leading to a fluorescent photoproduct. This can be easily used as a method for the quantitative determination of fluorescent tryptophan residues in proteins. In good agreement with previous results, two fluorescent tryptophan residues per polypeptide chain are found in wheat germ agglutinin. Concomitantly with the photochemical reactions, the hemagglutinating protein activity and its affinity constant towards chitin oligomers are reduced. A probable location of tryptophan residues in the binding sites of wheat germ agglutinin is proposed.

Agglutinins↗
PubMed1977

Differences in agglutinability of adult and fetal human fibroblasts using phytohemagglutinin.

Whereas Concanavalin A (Con A) and Wheat Germ Agglutinin (WGA) detect differences in the agglutinability of transformed, established and secondary cultures, Phytohemagglutinin (PHA) detects differences between cultured adult and fetal human fibroblasts. Adult cells agglutinate with PHA to the same extent as transformed cells, whereas fetal cells show significant agglutination only after trypsinization. Differences in cell size, growth rate, surface architecture or binding of fluorescent PHA could not be demonstrated between adult and fetal cells. Although the basis for this apparent difference in agglutinability remains unknown, it is the first demonstration that fetal cells (even after prolonged in vitro culture) retain at least some surface properties not shared by adult or transformed cells.

Adult↗
PubMed2025

From convention to innovation: the role of genetic modification and genome editing in Australian wheat breeding.

Wheat is the most cultivated crop worldwide, and Australia consistently ranks among the top wheat-exporting countries. Although modern technology has expanded the speed and accuracy of conventional breeding, progress is constrained by limited genetic diversity and linkage drag, with new wheat varieties often taking 8-12 years to reach the market. Biotech methods involving the transformation of foreign DNA into genomes [genetic modification (GM)], or editing of native DNA [genome editing (GEd)], provide novel opportunities to efficiently improve traits alongside conventional breeding. In 2020, the world's first GM drought-tolerant bread wheat (HB4) hit the market in Argentina. The USA recently approved HB4 wheat for commercial cultivation, and human consumption of HB4 wheat has been approved by nine countries, including Australia. Currently, 25 countries, Australia included, have deregulated GEd crops in some form, and many other countries have indicated that they will follow suit. As of March 2025, no GM or GEd wheat is commercially grown in Australia. The rate at which private industry integrates GM and GEd into wheat breeding programmes will depend on several factors, including the regulatory consistency governing GM and GEd crops within Australia and among international trading partners, the return on investments relative to deregulation costs including licensing, the level of acceptance amongst growers and consumers, and technical considerations including wheat's amenability to tissue culture. This review contextualizes GM and GEd applications in wheat, often drawing on examples from crop species where biotechnology has been more widely employed, and considers the key stakeholders that will shape the future of GM and GEd wheat in Australia.

GMO↗
PubMed2026

Investigating the Role of MicroRNA396 (miR396) Gene in Regulating Wheat Yield and Grain Nitrogen Concentration.

Nitrogen (N) is essential for crop growth, yet excessive fertilization causes environmental issues, highlighting the need to sustain yield and grain N concentration under reduced N input. miR396s are known to regulate plant development and stress responses. Here, we examined whether and how miR396 affects wheat yield and N status under high and low N conditions. TaMIM396 (transforming with the target mimicry construct of miR396) overexpression significantly increased plant height, spike length, grain yield, and grain N concentration under both N treatments. Physiological data showed TaMIM396 enhanced dry matter (DM) and N accumulation at anthesis and maturity, as well as improved post-anthesis remobilization of DM and N to grains. RNA-seq analysis revealed that, under low N, TaMIM396 specifically upregulated key photosynthetic antenna genes, including Lhca3 and Lhcb1/2/3/5, which are critical for light harvesting, suggesting improved photosynthetic efficiency that promotes DM accumulation under N limitation. Collectively, our results demonstrate that TaMIM396 acts as a broad-spectrum N-efficiency gene, coordinating carbon and N remobilization while boosting photosynthetic capacity, thereby supporting stable yield and grain N concentration across N supply levels. Therefore, TaMIM396 is a promising candidate for breeding N-efficient wheat cultivars compatible with sustainable high-yield agriculture.

TaMIM396↗
PubMed1975

Receptor mobility and the binding of cells to lectin-coated fibers.

The ability of cells to bind to nylon fibers coated with lectin molecules interspaced with varying numbers of albumin molecules has been analyzed. The cells used were lymphoma cells, normal lymphocytes, myeloid leukemia cells, and normal and transformed fibroblasts, and the fibers were coated with different densities of concanavalin A or the lectins from soybean or wheat germ. Cells fixed with glutaraldehyde did not bind to lectin-coated fibers. The number of cells bound to fibers could be increased by increasing the density of lectin molecules on the fiber, the density of specific receptors on the cell, or the mobility of the receptors. It is suggested that binding of cells to fibers involves alignment and binding of specific cell surface receptors with lectin molecules immobilized on the fibers, and that this alignment requires short-range rapid lateral mobility (RLM) of the receptors. The titration of cell binding to fibers coated with different densities of lectin and albumin has been used to measure the relative RLM of unoccupied cell surface receptors for the lectin. The results indicate a relationship of RLM to lectin-induced cell-to-cell binding. The RLM or receptors for concanavalin A (Con A) was generally found to be higher than that of receptors for the lectins from wheat germ or soybean. Receptor RLM could be decreased by use of metabolic inhibitors or by lowering the temperature. Receptors for Con A had a lower RLM on normal fibroblasts than on SV40-transformed fibroblasts, and trypsinization of normal fibroblasts increased Con A receptor RLM. Normal lymphocytes, lymphoma cells, and lines of myeloid leukemia cells that can be induced to differentiate had a high receptor RLM, whereas lines of myeloid leukemia cells that could not be induced to differentiate had a low receptor RLM. These results suggest that the RLM of Con A receptors is related to the transformation of fibroblasts and the ability of myeloid leukemia cells to undergo differentiation

Animals↗