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Phenotyping root systems provide essential information for plant breeding, particularly aiming for better abiotic stress resistance. D-Cycloserine Rhizobox systems provide a field-near growth environment for in situ imaging of root systems in soil. A protocol for RGB and hyperspectral imaging of rhizobox-grown plants is presented that enables gathering of root structural (morphology, architecture) as well as functional (water content, decomposition) information. The protocol exemplifies the setup of a root phenotyping platform combining low-cost RGB with advanced short-wave infrared hyperspectral imaging. For both types of imaging approach, the essential steps of an image analysis pipeline are provided to retrieve biological information on breeding-relevant traits from the imaging datasets.For centuries, combining useful traits into a single tomato plant has been done by selective crossbreeding that resulted in hundreds of extant modern cultivars. However, crossbreeding is a labor-intensive process that requires between 5 and 7 years to develop a new variety. More recently, genome editing with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been established as an efficient method to accelerate the breeding process by introducing targeted modifications to plant genomes via generation of targeted double-strand breaks (DSBs). CRISPR/Cas9 has been used to generate a variety of specific changes ranging from gene knockouts to gene replacements, and can also be easily multiplexed to modify several targets simultaneously. Given that (1) generating knockout mutations only requires a DSB that is frequently repaired by the error-prone nonhomologous end joining (NHEJ) pathway resulting in gene function inactivation, and (2) the genetic basis of many useful agronomic traits consists of loss of gene function, multiple traits can be created in a plant in one generation by simultaneously introducing DSBs into multiple genes of interest. On the other hand, more precise modifications, such as allele replacement, can be achieved by gene targeting-a less efficient process in which an external template is used to repair the DSB by homologous recombination (HR). These technical breakthroughs allow the design and customization of plant traits to achieve the ideal plant type (“ideotype”). Here, we describe protocols to assemble CRISPR/Cas9 constructs for both single and multiplex gene knockouts as well as gene targeting and to generate and identify genome-edited tomato plants via Agrobacterium-mediated transformation in tissue culture.Transcription activator-like effector (TALE) is a DNA-binding domain that can be paired with a nuclease to create DNA double-strand breaks, or with an effector protein to alter gene transcription. The ability to precisely alter plant genomes and transcriptomes has provided many insights into gene function and has recently been utilized for crop improvement. Easy design and construction of TALE make the tool more accessible to a variety of researchers. Here, we describe two TALE-based systems transcription activator-like effector nucleases (TALEN), for creating targeted mutations in a gene of interest, and multiplex TALE activation (mTALE-Act), for activating one or a few genes of interest at the transcription level. Assembly of these tools is based on Golden Gate cloning and Gateway recombination, which are cost-effective and streamlined cloning methods.The use of antimitotic agents such as colchicine has been common to obtain polyploid organisms. However, this approach entails certain problems, from its toxicity to the operators for being carcinogenic compounds to the instability of the individuals obtained, and the consequent reversion to its original ploidy because the individuals obtained in most cases are chimeric. In vitro culture allows taking advantage of the full potential offered by the cellular totipotence of plant organisms. Based on this, we present a new in vitro culture protocol to obtain polyploid organisms using zeatin riboside (ZR) and eggplant as a model organism. Flow cytometry is used to identify tetraploid regenerants. The regeneration of whole plants from the appropriate tissues using ZR allowed developing polyploid individuals in eggplant, a crop that tends to be recalcitrant to in vitro organogenesis. Thanks to the use of the polysomatic pattern of the explants, we have been able to develop a methodology that allows to obtain stable non-chimeric polyploid individuals from organogenic processes.Homozygous lines occur for plant breeding programs and for studies about gene expression and genetic mapping and they can be derived from anther culture. In this chapter, the method to obtain androgenic plants from an ornamental cut flower, Anemone coronaria belonging to the Ranunculaceae family, is described. In this species, androgenic plants were obtained culturing anthers with responsive microspores in Petri dishes containing a double layer of substrate with specific composition. Moreover, thermic treatment has been applied to induce the switch from pollen development program to embryo development program. The method allows to produce both double-haploid plants from diploid mothers (2n) and di-haploid plants from tetraploid mothers (4n).The cultivated potato is tetraploid with four probably equivalent loci for each gene. A potato variety is furthermore commonly genetically heterogeneous and selected based on a beneficial genetic context which is maintained by clonal propagation. When introducing genetic changes by genome editing it is then desirable to achieve edits in all four loci for a certain gene target. This is in order to avoid crosses to achieve homozygosity for edited gene loci and at the same time reduce risk of inbreeding depression. In such a context transient transfection of protoplasts for the introduction of mutations, avoiding stable insertion of foreign DNA, would be very attractive. The protocol of this chapter has been shown to be applicable for the introduction of mutations by DNA vectors containing expression cassettes of TALEN, Cas9, and Cas9 deaminase fusions together with sgRNA expression cassettes on either single or separate vectors. Furthermore, the protoplast-based system has been shown to work very efficiently for mutations introduced by in vitro-produced and transfected RNP (ribonucleoprotein) complexes.