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The Coffea spp. plant is a significant crop in Latin America, Africa, and Asia, and recent advances in genomics and transcriptomics have opened possibilities for studying candidate genes and introducing new desirable traits through genetic engineering. While stable transformation of coffee plants has been reported using various techniques, it is a time-consuming and laborious process. To overcome this, transient transformation methods have been developed, which avoid the limitations of stable transformation. This chapter describes an ex vitro protocol for transient expression using A. tumefaciens-mediated infiltration of coffee leaves, which could be used to produce coffee plants expressing desirable traits against biotic and abiotic stresses, genes controlling biochemical and physiological traits, as well as for gene editing through CRISPR/Cas9.

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Cassava is a staple food used in many countries around the world, despite deficiencies in micronutrients such as provitamin A carotenoids. Unfortunately, improvement of the cassava nutritional content by use of conventional breeding is slow and difficult. Therefore, there is an urgent need to develop and standardize protocols using biotechnological tools to improve cassava. The Alliance of Biodiversity International and the International Center for Tropical Agriculture (CIAT) have worked on cassava genetic transformation over the last 30 years. Here, we describe, step by step, the procedures used for genetic transformation of cassava variety TMS60444, to improve carotenoids and other traits. This protocol includes stock setup, reagents, media preparation, materials, and equipment, for the genetic transformation of embryogenic tissues. The main expected output in publishing this protocol is to provide the basis for a reproducible and reliable method to genetically modify and/or gene edit Latin American and Asian cassava varieties.

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The recurring growth of bacterium in newly developed resistant cells and a minimal level of bacterial infection rate are the main limiting factors of Agrobacterium-mediated transformation experiments in Hevea brasiliensis. The current study aimed to optimize crucial factors of the transformation protocol in order to obtain an efficient transformation experimental model for Hevea using cotyledonary somatic embryos as explants. Transformation conditions such as antibiotic concentration, preculture duration, Agrobacterium concentration, sonication and cocultivation conditions were analyzed using the binary vector pCAMBIA2301. Transient transformation was confirmed by GUS histochemical staining. The best transformation efficiency was observed when the explants were not cultured on a preculture medium that contained acetosyringone at a level of 100 µM. The best results were obtained using a bacterial density of 0.45 at OD 600 nm, 50 s of sonication of explants in a bacterial liquid culture and a total incubation time of 18 min in the same bacterial suspension. Transmission electron microscopical analysis confirmed the impacts of sonication on bacterial infection efficiency. Cocultivation conditions of 22 °C and 84 h of darkness were optimal for the transfer of T-DNA. Agrobacterium was eliminated with 500 mg/L of timentin, and the selection of transformants was performed using 100 mg/L of kanamycin in the selection medium. The presence of transgene was confirmed in the resistant embryos by polymerase chain reaction (PCR). The improved method of genetic transformation established in the present study will be useful for the introduction of foreign genes of interest into the Hevea genome for the breeding of this economically important plant species in the future.

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In potato (Solanum tuberosum L.), protoplast techniques are limited to a few genotypes; thus, the use of regular regeneration procedures of multicellular explants causes us to face complexities associated to CRISPR/Cas9 gene editing efficiency and final identification of individuals. Geminivirus-based replicons contained in T-DNAs could provide an improvement to these procedures considering their cargo capability. We built a Bean yellow dwarf virus-derived replicon vector, pGEF-U, that expresses all the editing reagents under a multi-guide RNA condition, and the Green Fluorescent Protein (GFP) marker gene. Agrobacterium-mediated gene transfer experiments were carried out on 'Yagana-INIA', a relevant local variety with no previous regeneration protocol. Assays showed that pGEF-U had GFP transient expression for up to 10 days post-infiltration when leaf explants were used. A dedicated potato genome analysis tool allowed for the design of guide RNA pairs to induce double cuts of genes associated to enzymatic browning (StPPO1 and 2) and to cold-induced sweetening (StvacINV1 and StBAM1). Monitoring GFP at 7 days post-infiltration, explants led to vector validation as well as to selection for regeneration (34.3% of starting explants). Plant sets were evaluated for the targeted deletion, showing individuals edited for StPPO1 and StBAM1 genes (1 and 4 lines, respectively), although with a transgenic condition. While no targeted deletion was seen in StvacINV1 and StPPO2 plant sets, stable GFP-expressing calli were chosen for analysis; we observed different repair alternatives, ranging from the expected loss of large gene fragments to those showing punctual insertions/deletions at both cut sites or incomplete repairs along the target region. Results validate pGEF-U for gene editing coupled to regular regeneration protocols, and both targeted deletion and single site editings encourage further characterization of the set of plants already generated.

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The woody nature of grapevine (Vitis vinifera L.) has hindered the development of efficient gene editing strategies to improve this species. The lack of highly efficient gene transfer techniques, which, furthermore, are applied in multicellular explants such as somatic embryos, are additional technical handicaps to gene editing in the vine. The inclusion of geminivirus-based replicons in regular T-DNA vectors can enhance the expression of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) elements, thus enabling the use of these multicellular explants as starting materials. In this study, we used Bean yellow dwarf virus (BeYDV)-derived replicon vectors to express the key components of CRISPR/Cas9 system in vivo and evaluate their editing capability in individuals derived from Agrobacterium-mediated gene transfer experiments of 'Thompson Seedless' somatic embryos. Preliminary assays using a BeYDV-derived vector for green fluorescent protein reporter gene expression demonstrated marker visualization in embryos for up to 33 days post-infiltration. A universal BeYDV-based vector (pGMV-U) was assembled to produce all CRISPR/Cas9 components with up to four independent guide RNA (gRNA) expression cassettes. With a focus on fungal tolerance, we used gRNA pairs to address considerably large deletions of putative grape susceptibility genes, including AUXIN INDUCED IN ROOT CULTURE 12 (VviAIR12), SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER 4 (VviSWEET4), LESION INITIATION 2 (VviLIN2), and DIMERIZATION PARTNER-E2F-LIKE 1 (VviDEL1). The editing functionality of gRNA pairs in pGMV-U was evaluated by grapevine leaf agroinfiltration assays, thus enabling longer-term embryo transformations. These experiments allowed for the establishment of greenhouse individuals exhibiting a double-cut edited status for all targeted genes under different allele-editing conditions. After approximately 18 months, the edited grapevine plants were preliminary evaluated regarding its resistance to Erysiphe necator and Botrytis cinerea. Assays have shown that a transgene-free VviDEL1 double-cut edited line exhibits over 90% reduction in symptoms triggered by powdery mildew infection. These results point to the use of geminivirus-based replicons for gene editing in grapevine and other relevant fruit species.

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The first successful attempt to generate genetically modified plants expressing a transgene was preformed via T-DNA-based gene transfer employing Agrobacterium tumefaciens-mediated genetic transformation. Limitations over infectivity and in vitro tissue culture led to the development of other DNA delivery systems, such as the biolistic method. Herein, we developed a new one-step protocol for transgenic soybean recovery by combining the two different transformation methods. This protocol comprises the following steps: agrobacterial preparation, seed sterilization, soybean embryo excision, shoot-cell injury by tungsten-microparticle bombardment, A. tumefaciens-mediated transformation, embryo co-cultivation in vitro, and selection of transgenic plants. This protocol can be completed in approximately 30-40 weeks. The average efficiency of producing transgenic soybean germlines using this protocol was 9.84%, similar to other previously described protocols. However, we introduced a more cost-effective, more straightforward and shorter methodology for transgenic plant recovery, which allows co-cultivation and plant regeneration in a single step, decreasing the chances of contamination and making the manipulation easier. Finally, as a hallmark, our protocol does not generate plant chimeras, in contrast to traditional plant regeneration protocols applied in other Agrobacterium-mediated transformation methods. Therefore, this new approach of plant transformation is applicable for studies of gene function and the production of transgenic cultivars carrying different traits for precision-breeding programs.

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Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.

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Background: Banana (Musa spp.) is an important staple food, economic crop, and nutritional fruit worldwide. Conventional breeding has been seriously hampered by their long generation time, polyploidy, and sterility of most cultivated varieties. Establishment of an efficient regeneration and transformation system for banana is critical to its genetic improvement and functional genomics. Results: In this study, a vigorous and repeatable transformation system for banana using direct organogenesis was developed. The greatest number of shoots per explant for all five Musa varieties was obtained using Murashige and Skoog medium supplemented with 8.9 µM benzylaminopurine and 9.1 µM thidiazuron. One immature male flower could regenerate 380­456, 310­372, 200­240, 130­156, and 100­130 well-developed shoots in only 240­270 d for Gongjiao, Red banana, Rose banana, Baxi, and Xinglongnaijiao, respectively. Longitudinal sections of buds were transformed through particle bombardment combined with Agrobacterium-mediated transformation using a promoterless ß-glucuronidase (GUS) reporter gene; the highest transformation efficiency was 9.81% in regenerated Gongjiao plantlets in an optimized selection medium. Transgenic plants were confirmed by a histochemical assay of GUS, polymerase chain reaction, and Southern blot. Conclusions: Our robust transformation platform successfully generated hundreds of transgenic plants. Such a platform will facilitate molecular breeding and functional genomics of banana.

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Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone - AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 µM of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant-pathogen interaction.

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Soybean is one of humanity's major sources of plant protein. It is also very important for animal feed and as industrial raw material. Great advances have recently been achieved in its genetic transformation. This review provides a comprehensive discussion of important factors affecting Agrobacterium-mediated soybean transformation including target tissues, plant tissue health, wounding methods, regeneration systems, selectable markers and reporter genes.

A soja é uma das maiores fontes de proteína vegetal da humanidade. É também muito importante na alimentação animal e como matéria-prima industrial. Grandes avanços foram recentemente alcançados em sua transformação genética. O objetivo desta revisão é fornecer uma discussão acerca de importantes fatores que influenciam a transformação de soja mediada por Agrobaterium, incluindo tecidos alvo, condições do tecido vegetal, métodos de injúria, sistemas de regeneração, marcadores de seleção e genes repórter.

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Soybean is one of humanity's major sources of plant protein. It is also very important for animal feed and as industrial raw material. Great advances have recently been achieved in its genetic transformation. This review provides a comprehensive discussion of important factors affecting Agrobacterium-mediated soybean transformation including target tissues, plant tissue health, wounding methods, regeneration systems, selectable markers and reporter genes.

A soja é uma das maiores fontes de proteína vegetal da humanidade. É também muito importante na alimentação animal e como matéria-prima industrial. Grandes avanços foram recentemente alcançados em sua transformação genética. O objetivo desta revisão é fornecer uma discussão acerca de importantes fatores que influenciam a transformação de soja mediada por Agrobaterium, incluindo tecidos alvo, condições do tecido vegetal, métodos de injúria, sistemas de regeneração, marcadores de seleção e genes repórter.