We have achieved routine transformation of maize (Zea mays) using an Agrobacterium tumefaciens standard binary (non-super binary) vector system. Immature zygotic embryos of the hybrid line Hi II were infected with A. tumefaciens strain EHA101 harboring a standard binary vector and cocultivated in the presence of 400 mg L-1 L-cysteine. Inclusion of L-cysteine in cocultivation medium lead to an improvement in transient beta-glucuronidase expression observed in targeted cells and a significant increase in stable transformation efficiency, but was associated with a decrease in embryo response after cocultivation. The average stable transformation efficiency (no. of bialaphos-resistant events recovered per 100 embryos infected) of the present protocol was 5.5%. Southern-blot and progeny analyses confirmed the integration, expression, and inheritance of the bar and gus transgenes in R0, R1, and R2 generations of transgenic events. To our knowledge, this represents the first report in which fertile, stable transgenic maize has been routinely produced using an A. tumefaciens standard binary vector system.

Maize may be transformed very efficiently using Agrobacterium tumefaciens-mediated methods. The most critical factor in the transformation protocol is the co-cultivation of healthy immature embryos of the correct developmental stage with A. tumefaciens; the embryos should be collected only from vigorous plants grown in well-conditioned glasshouses. With the protocol described here, approximately 50% of immature embryos from the inbred line A188 and 15% from inbred lines A634, H99 and W117 will produce transformants. About half of the transformed plants are expected to carry one or two copies of the transgenes, which are inherited by the progeny in a mendelian fashion. More than 90% of transformants are expected to be normal in morphology. The protocol takes about 3 months from the start of co-cultivation to the planting of transformants into pots.

A novel, efficient maize genetic transformation system was developed using Agrobacterium-mediated transformation of embryo explants from mature seeds. Seeds from field grown plants were sterilized and crushed to isolate embryo explants consisting of the coleoptile, leaf primordia, and shoot apical meristem which were then purified from the ground seed bulk preparation. The infection of relevant tissues of seed embryo explants (SEEs) by Agrobacterium was improved by the centrifugation of the explants. Transgenic plants were obtained by multiple bud induction on high cytokinin media, followed by plant regeneration on hormone-free medium. Three different selectable markers (cp4 epsps, aadA, and nptII) were successfully used for producing transgenic plants. Stable integration of transgenes in the maize genome was demonstrated by molecular analyses and germline transmission of the inserted transgenes to the next generation was confirmed by pollen segregation and progeny analysis. Phenotypic evidence for chimeric transgenic tissue was frequently observed in initial experiments but was significantly reduced by including a second bud induction step with optimized cytokinin concentration. Additional improvements, including culturing explants at an elevated temperature during bud induction led to the development of a revolutionary system for efficient transgenic plant production and genome editing. To our knowledge, this is the first report of successful transgenic plant regeneration through Agrobacterium-mediated transformation of maize mature SEEs. This system starts with mature seed that can be produced in large volumes and the SEEs explants are storable. It has significant advantages in terms of scalability and flexibility over methods that rely on immature explants.

Transformation in grass species has traditionally relied on immature embryos and has therefore been limited to a few major Poaceae crops. Other transformation explants, including leaf tissue, have been explored but with low success rates, which is one of the major factors hindering the broad application of genome editing for crop improvement. Recently, leaf transformation using morphogenic genes Wuschel2 (Wus2) and Babyboom (Bbm) has been successfully used for Cas9-mediated mutagenesis, but complex genome editing applications, requiring large numbers of regenerated plants to be screened, remain elusive. Here we demonstrate that enhanced Wus2/Bbm expression substantially improves leaf transformation in maize and sorghum, allowing the recovery of plants with Cas9-mediated gene dropouts and targeted gene insertion. Moreover, using a maize-optimized Wus2/Bbm construct, embryogenic callus and regenerated plantlets were successfully produced in eight species spanning four grass subfamilies, suggesting that this may lead to a universal family-wide method for transformation and genome editing across the Poaceae.© 2023. The Author(s).

Efficient methods for in vitro propagation, regeneration, and transformation of plants are of pivotal importance to both basic and applied research. While being the world's major food crops, cereals are among the most difficult-to-handle plants in tissue culture which severely limits genetic engineering approaches. In maize, immature zygotic embryos provide the predominantly used material for establishing regeneration-competent cell or callus cultures for genetic transformation experiments. The procedures involved are demanding, laborious and time consuming and depend on greenhouse facilities. We have developed a novel tissue culture and plant regeneration system that uses maize leaf tissue and thus is independent of zygotic embryos and greenhouse facilities. We report here: (i) a protocol for the efficient induction of regeneration-competent callus from maize leaves in the dark, (ii) a protocol for inducing highly regenerable callus in the light, and (iii) the use of leaf-derived callus for the generation of stably transformed maize plants.

Efficient production of seedling-derived Type I callus was demonstrated for several corn genotypes including commercial inbred lines. Seeds were germinated on MS-based medium containing 10 mg l(-1) picloram and 3 mg l(-1) 6-benzylaminopurine, which induced the development of axillary buds in the area of coleoptilar node. Nodal sections of 7-10-day old seedlings were isolated, split longitudinally, and placed on callus induction medium supplemented with 2.2 mg l(-1) picloram and 0.5 mg l(-1) 2,4-dichlorophenoxyacetic acid. For lines L4 and L9 the frequency of embryogenic callus induction was 38-42% based on calli per split nodal section. Frequency of callus induction from split nodal sections of seeds germinated on media without growth regulators was 0-3%. Seedling-derived callus of five genotypes was used for Agrobacterium-mediated transformation. Two constructs containing the green fluorescence protein gene and genes for either neomycin phosphotransferase II or glyphosate selection were used in transformation experiments. Transformation frequency varied from 2 to 11% and about 60% of the T(0) plants had 1-2 copies of transgenes.

The lack of efficient delivery methods is a major barrier to clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-mediated genome editing in many plant species. Combinations of morphogenic regulator (MR) genes and ternary vector systems are promising solutions to this problem. In this study, we first demonstrated that MR vectors greatly enhance maize () transformation. We then tested a CRISPR/Cas9 MR vector in maize and found that the MR and CRISPR/Cas9 modules have no negative influence on each other. Finally, we developed a novel ternary vector system to integrate the MR and CRISPR/Cas modules. Our ternary vector system is composed of new pGreen-like binary vectors, here named pGreen3, and a pVS1-based virulence helper plasmid, which also functions as a replication helper for the pGreen3 vectors in The pGreen3 vectors were derived from the plasmid pRK2 and display advantages over pGreen2 vectors regarding both compatibility and stability. We demonstrated that the union of our ternary vector system with MR gene modules has additive effects in enhancing maize transformation and that this enhancement is especially evident in the transformation of recalcitrant maize inbred lines. Collectively, our ternary vector system-based tools provide a user-friendly solution to the low efficiency of CRISPR/Cas delivery in maize and represent a basic platform for developing efficient delivery tools to use in other plant species recalcitrant to transformation.© 2019 American Society of Plant Biologists. All Rights Reserved.

For maize genome-editing and bioengineering, genetic transformation of inbred genotypes is most desired due to the uniformity of genetic background in their progenies. However, most maize inbred lines are recalcitrant to tissue culture and transformation. A public, transformable maize inbred B104 has been widely used for genome editing in recent years. This is primarily due to its high degree of genetic similarity shared with B73, an inbred of the reference genome and parent of many breeding populations. Conventional B104 maize transformation protocol requires 16–22 weeks to produce rooted transgenic plants with an average of 4% transformation frequency (number of T0 plants per 100 infected embryos). In this Method paper, we describe an advanced B104 transformation protocol that requires only 7–10 weeks to generate transgenic plants with an average of 6.4% transformation frequency. Over 66% of transgenic plants carried CRISPR/Cas9-induced indel mutations on the target gene, demonstrating that this protocol can be used for genome editing applications. Following the detailed and stepwise procedure described here, this quick and simplified method using the Agrobacterium ternary vector system consisting of a T-DNA binary vector and a compatible helper plasmid can be readily transferable to interested researchers.

Agrobacterium-mediated transformation is an essential tool for functional genomics studies and crop improvements. Recently developed ternary vector systems, which consist of a T-DNA vector and a compatible virulence (vir) gene helper plasmid (ternary helper), demonstrated that including an additional vir gene helper plasmid into disarmed Agrobacterium strains significantly improves T-DNA delivery efficiency, enhancing plant transformation. Here, we report the development of a new ternary helper and thymidine auxotrophic Agrobacterium strains to boost Agrobacterium-mediated plant transformation efficiency. Auxotrophic Agrobacterium strains are useful in reducing Agrobacterium overgrowth after the co-cultivation period because they can be easily removed from the explants due to their dependence on essential nutrient supplementation. We generated thymidine auxotrophic strains from public Agrobacterium strains EHA101, EHA105, EHA105D, and LBA4404. These strains exhibited thymidine-dependent growth in the bacterial medium, and transient GUS expression assay using Arabidopsis seedlings showed that they retain similar T-DNA transfer capability as their original strains. Auxotrophic strains EHA105Thy- and LBA4404T1 were tested for maize B104 immature embryo transformation using our rapid transformation method, and both strains demonstrated comparable transformation frequencies to the control strain LBA4404Thy-. In addition, our new ternary helper pKL2299A, which carries the virA gene from pTiBo542 in addition to other vir gene operons (virG, virB, virC, virD, virE, and virJ), demonstrated consistently improved maize B104 immature embryo transformation frequencies compared to the original version of pKL2299 (33.3% vs 25.6%, respectively). Therefore, our improved Agrobacterium system, including auxotrophic disarmed Agrobacterium strains and a new ternary helper plasmid, can be useful for enhancing plant transformation and genome editing applications.

Auxotrophic strains of Agrobacterium tumefaciens can contribute to the development of more efficient transformation systems, especially for crops historically considered recalcitrant. Homologous recombination was used to derive methionine auxotrophs of two common A. tumefaciens strains, LBA4404 and EHA105. The EHA105 strains were more efficient for switchgrass transformation, while both the EHA105 and LBA4404 strains worked equally well for the rice control. Event quality, as measured by transgene copy number, was not affected by auxotrophy, but was higher for the LBA4404 strains than the EHA105 strains. Ultimately, the use of auxotrophs reduced bacterial overgrowth during co-cultivation and decreased the need for antibiotics.© 2022. The Author(s).

Rye is second only to wheat among grains most widely used in the making of bread and is also a very important gene resource for breeding and improvement of wheat and other cereal crops owing to tolerance to abiotic stress factors such as low temperatures, drought and poor soil conditions. However, application of biotechnologies has been limited in rye breeding since it is one of the most recalcitrant species in tissue culture. A simple and fast regeneration system from leaf-base segment explant of rye was developed in this study. Basal media, carbohydrate source, combination of plant growth regulators and the leaf segment locations were evaluated for callus and shoot formation. The highest callus formation (10.39%) and shoot formation (4.53%) were achieved from first basal segments 3-4 days old seedlings. MS (Murashige and Skoog, in Physiol Plant 15:473-497, 1962) medium supplemented with 30 g/L sucrose and 2 mg/L 2,4-D (2-4 dichlorophenoxyacetic acid) + 1 mg/L TDZ (Thidiazuran) was the best medium for shoot formation (18.75%) in first leaf base segment culture. Regenerated plants were phenotypically normal and set seed after they were successfully transferred to soil. The results indicate that this regeneration method can be used for genetic transformation in rye.

A reliable and reproducible protocol has been developed for high frequency plant regeneration from 4-5 mm long leaf base segments of 4 days old in vitro germinated seedlings of indica rice (Oryza sativa) cultivar Rasi. The effect of age of seedlings, position of leaf base segments and optimum concentration of 2,4-D on callus induction frequency was investigated with a future aim to use leaf bases for biolistic and Agrobacterium-mediated transformation experiments. Friable, nodular and white to pale yellow embryogenic callus cultures (206 mg fresh weight /explant) were obtained from the first basal segments of rice seedlings on Linsmaier and Skoog (LS) medium supplemented with 2,4-D (11.3 microM) and 3.0 microM thiamine-HCL. Plant regeneration was achieved after the transfer of 54 days old embryogenic callus cultures to Murashige and Skoog (MS) medium supplemented with BAP (2.2 microM) and NAA (0.27 microM). In vitro regenerated plants with multiple shoots and roots transferred to sterile soil in growth chamber and maintained in greenhouse exhibited normal growth and were phenotypically similar to plants germinated from seeds.

The key agricultural species of Urochloa P.Beauv. (signal grass), important as tropical forage grasses, are characterised by asexual seed formation (apomixis), and this presents a challenge for breeding programs. Biotechnological approaches could be an option to develop improved cultivars. We evaluated the regenerative potential from three commercial genotypes, U. brizantha cv. Marandu, U. decumbens cv. Basilisk and U. ruziziensis cv. Ruziziensis, by using leaf-base segments as explants. We tested two auxins (2,4-D and picloram) and one cytokinin (TDZ) at four concentrations (1, 2, 3 and 4 mg L–1). Seeds were scarified, peeled and disinfected before inoculation on half-strength MS media in the dark for 14 days. Leaf-base explants were sectioned in thin slices and inoculated into the media. We analysed the number of primary calluses, number of calluses with shoots clusters and the average of regenerated plants. The lowest concentration of auxins tested (1 mg L–1) yielded the highest number of regenerated plants for Marandú and Basilisk, whereas the optimum for Ruziziensis was 2 mg L–1. Medium with higher concentrations of TDZ (4 mg L–1) was required to produce high frequency of plants for all genotypes. Explants cultured on media with TDZ produced very few calluses. These results indicate that the auxins and cytokinin tested can induce plant regeneration from Urochloa leaf-base segments, and may be used to produce transgenic plants in genetic transformation studies.