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Intracellular protein delivery in plant tissues is becoming an important tool for addressing both basic and applied research questions by plant biologists, especially in the era of genome editing. The ability to deliver proteins or protein/RNA complexes into cells allows for producing gene-edited plants that are free of transgene integration in the genome. Here we describe a protocol for the delivery of a protein/gold particle mixture in plant cells through biolistics. The key for the delivery is the drying of the protein/gold suspension directly onto the gene-gun cartridge or macrocarrier. The intracellular protein delivery into plant cells is achieved through the bombardment using the Bio-Rad PDS-1000/He particle delivery device. Samotolisib chemical structure We termed this methodology "proteolistics."Biotechnological methods for targeted gene transfers into plants are key for successful breeding in the twenty-first century and thus essential for the survival of humanity. Two decades ago, genetic transformation of crop plants was not routine, and it was all but impossible with important cereals such as barley and wheat. The recent focus on crop plant genomics-yet based on the Arabidopsis toolbox-boosted the research for more efficient plant transformation protocols, thereby considerably widened the number of genetically tractable crops. Moreover, modern genome editing methods such as the CRISPR/Cas technique are game changers in plant breeding, though heavily dependent on technical optimization of plant transformation. Basically, there are two successful ways of introducing DNA into plant cells one is making use of a living DNA vector, namely, microbes such as the soil bacterium Agrobacterium tumefaciens that infects plants and naturally transfers and subsequently integrates DNA into the plant genome. The other method uses a direct physical transfer of DNA by means of microinjection, microprojectile bombardment, or polymers such as polyethylene glycol. Both ways subsequently require sophisticated strategies for selecting and multiplying the transformed cells under tissue culture conditions to develop into a fully functional plant with the new desirable characteristics. Here we discuss practical and theoretical aspects of cereal crop plant transformation by Agrobacterium-mediated transformation and microparticle bombardment. Using immature embryos as explants, the efficiency of cereal transformation is compelling, reaching today up to 80% transformation efficiency.There are specific advantages of using microspores as explants (1) A small number of explant donors are required to obtain the desired number of pollen embryoids for genetic transformation and (2) microspores constitute a synchronous mass of haploid cells, which are transformable by various means and convertible to doubled haploids therefore allow production of homozygous genotypes in a single generation. Additionally, it has been demonstrated in wheat and other crops that microspores can be easily induced to produce embryoids and biolistic approach to produce a large number of transformants. In view of these listed advantages, we optimized the use of microspore-derived calli for biolistic transformation of wheat. The procedure takes about 6 months to obtain the viable transformants in the spring wheat background. In the present communication, we demonstrated the use of this method to produce the reduced immunogenicity wheat genotypes.We describe a protocol for the establishment and preparation of creeping bentgrass (Agrostis stolonifera L.) cultivar "Penn A-4" embryonic calli, biolistic transformation, selection, and regeneration of transgenic plants. The embryonic callus is initiated from mature seeds, maintained by visual selection under the dissecting microscope and subjected to bombardment with plasmid DNA containing a bialaphos-resistance (bar) gene. PCR, Southern, and Northern blot analyses are used to confirm the transgene integration and expression.The following protocol describes the genetic transformation of wheat using the BioRad PDS/1000-He particle delivery system. Immature embryos are isolated 12-16 days post-anthesis, the embryonic axis is removed, and the immature scutella are precultured for 1-2 days prior to particle bombardment. Gold particles are coated with plasmid DNA containing the gene(s) of interest plus a selectable marker gene, in this instance bar (bialaphos resistance), and are fired into the cells to deliver the DNA. Subsequent tissue culture and regeneration steps allow recovery of plantlets, assisted by the inclusion of PPT (phosphinothricin tripeptide), the active ingredient of glufosinate-ammonium containing herbicides, to help select transformants. This updated method introduces selection earlier in the regeneration process which provides a shortened protocol while maintaining high transformation efficiencies.Biolistic transformation is one of two popular methods for introducing genes into sugarcane. However, unlike Agrobacterium-mediated transformation, the efficiency of gene transfer into sugarcane cells, using the biolistic method is very high. In addition to this, the biolistic transformation method is independent of the explant genotype or tissue. It also has the advantage that a minimum DNA sequence of linearized plasmid can be used, thus eliminating the introduction of undesirable plasmid derived genes, delivering low-copy transgenic events. In this chapter, we describe the method for efficient delivery of genes into sugarcane cells using a biolistic approach.Biolistic DNA delivery has been considered a universal tool for genetic manipulation to transfer exotic genes to cells or tissues due to its simplicity, versatility, and high efficiency. It has been a preferred method for investigating plant gene function in most monocot crops. The first transgenic sorghum plants were successfully regenerated through biolistic DNA delivery in 1993, with a relatively low transformation efficiency of 0.3%. Since then, tremendous progress has been made in recent years where the highest transformation efficiency was reported at 46.6%. Overall, the successful biolistic DNA delivery system is credited to three fundamental cornerstones robust tissue culture system, effective gene expression in sorghum, and optimal parameters of DNA delivery. In this chapter, the history, application, and current development of biolistic DNA delivery in sorghum are reviewed, and the prospect of sorghum genetic engineering is discussed.