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1. History and current status of embryogenic culture‐based tissue culture, transformation and gene editing of maize ( Zea mays L.)

   https://doi.org/10.1002/tpg2.20451  

   McFarland, Frank_L; Kaeppler, Heidi_F (April 2024, The Plant Genome)

   Abstract The production of embryogenic callus and somatic embryos is integral to the genetic improvement of crops via genetic transformation and gene editing. Regenerable embryogenic cultures also form the backbone of many micro‐propagation processes for crop species. In many species, including maize, the ability to produce embryogenic cultures is highly genotype dependent. While some modern transformation and genome editing methods reduce genotype dependence, these efforts ultimately fall short of producing truly genotype‐independent tissue culture methods. Recalcitrant genotypes are still identified in these genotype‐flexible processes, and their presence is magnified by the stark contrast with more amenable lines, which may respond more efficiently by orders of magnitude. This review aims to describe the history of research into somatic embryogenesis, embryogenic tissue cultures, and plant transformation, with particular attention paid to maize. Contemporary research into genotype‐flexible morphogenic gene‐based transformation and genome engineering is also covered in this review. The rapid evolution of plant biotechnology from nascent technologies in the latter half of the 20th century to well‐established, work‐horse production processes has, and will continue to, fundamentally changed agriculture and plant genetics research. 

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2. Eliminating tissue culture from plant gene editing in the near future: A wish or reality?

   https://doi.org/10.1016/j.cpb.2025.100433  

   Babaeianjelodar, Nadali; Trivedi, Jayati; Uhde-Stone, Claudia (March 2025, Current Plant Biology)

   Traditional plant breeding methods alone are insufficient to guarantee food security for a growing global population under a changing climate, necessitating more advanced approaches to develop productive and resilient crop varieties. The development of genome editing tools, particularly CRISPR/CAS, are significantly speeding up crop improvement by enabling targeted breeding in most crop species. However, for many crop species, the need for tissue culture remains a major bottle neck, slowing the progress of crop improvement. In this review, we are presenting and discussing approaches for delivering genome editing tools into a wide variety of crop plants, including perennials, and ideally without integration of transgenes. We suggest that efficient non-tissue culture delivery systems for high-performance genome editing are needed to fully reach the genome engineering potential in crop plants. 

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3. Biosystems Design to Accelerate C ₃ -to-CAM Progression

   https://doi.org/10.34133/2020/3686791  

   Yuan, Guoliang; Hassan, Md. Mahmudul; Liu, Degao; Lim, Sung Don; Yim, Won Cheol; Cushman, John C.; Markel, Kasey; Shih, Patrick M.; Lu, Haiwei; Weston, David J.; et al (October 2020, BioDesign Research)

   Global demand for food and bioenergy production has increased rapidly, while the area of arable land has been declining for decades due to damage caused by erosion, pollution, sea level rise, urban development, soil salinization, and water scarcity driven by global climate change. In order to overcome this conflict, there is an urgent need to adapt conventional agriculture to water-limited and hotter conditions with plant crop systems that display higher water-use efficiency (WUE). Crassulacean acid metabolism (CAM) species have substantially higher WUE than species performing C 3 or C 4 photosynthesis. CAM plants are derived from C 3 photosynthesis ancestors. However, it is extremely unlikely that the C 3 or C 4 crop plants would evolve rapidly into CAM photosynthesis without human intervention. Currently, there is growing interest in improving WUE through transferring CAM into C 3 crops. However, engineering a major metabolic plant pathway, like CAM, is challenging and requires a comprehensive deep understanding of the enzymatic reactions and regulatory networks in both C 3 and CAM photosynthesis, as well as overcoming physiometabolic limitations such as diurnal stomatal regulation. Recent advances in CAM evolutionary genomics research, genome editing, and synthetic biology have increased the likelihood of successful acceleration of C 3 -to-CAM progression. Here, we first summarize the systems biology-level understanding of the molecular processes in the CAM pathway. Then, we review the principles of CAM engineering in an evolutionary context. Lastly, we discuss the technical approaches to accelerate the C 3 -to-CAM transition in plants using synthetic biology toolboxes. 

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4. Highly efficient CRISPR systems for loss-of-function and gain-of-function research in pear calli

   https://doi.org/10.1093/hr/uhac148  

   Ming, Meiling; Long, Hongjun; Ye, Zhicheng; Pan, Changtian; Chen, Jiali; Tian, Rong; Sun, Congrui; Xue, Yongsong; Zhang, Yingxiao; Li, Jiaming; et al (June 2022, Horticulture Research)

   Abstract CRISPR/Cas systems have been widely used for genome engineering in many plant species, while their potentials have remained largely untapped in fruit crops, particularly in pear, due to the high levels of genomic heterozygosity and difficulties in tissue culture and stable transformation. To date, only few reports on application of CRISPR/Cas9 system in pear have been documented with a very low editing efficiency. Here, we report a highly efficient CRISPR toolbox for loss-of-function and gain-of-function research in pear. We compared four different CRISPR/Cas9 expression systems for loss-of-function analysis and identified a potent system that showed nearly 100% editing efficiency for multi-site mutagenesis. To expand targeting scope, we further tested different CRISPR/Cas12a and Cas12b systems in pear for the first time, albeit with low editing efficiency. In addition, we established a CRISPR activation (CRISPRa) system for multiplexed gene activation in pear calli for gain-of-function analysis. Furthermore, we successfully engineered the anthocyanin and lignin biosynthesis pathways using both CRISPR/Cas9 and CRISPRa systems in pear calli. Taken together, we build a highly efficient CRISPR toolbox for genome editing and gene regulation, paving the way for functional genomics studies as well as molecular breeding in pear. 

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5. Metabolomics should be deployed in the identification and characterization of gene‐edited crops

   https://doi.org/10.1111/tpj.14679  

   Fraser, Paul D.; Aharoni, Asaph; Hall, Robert D.; Huang, Sanwen; Giovannoni, James J.; Sonnewald, Uwe; Fernie, Alisdair R. (February 2020, The Plant Journal)

   Summary Gene‐editing techniques are currently revolutionizing biology, allowing far greater precision than previous mutagenic and transgenic approaches. They are becoming applicable to a wide range of plant species and biological processes. Gene editing can rapidly improve a range of crop traits, including disease resistance, abiotic stress tolerance, yield, nutritional quality and additional consumer traits. Unlike transgenic approaches, however, it is not facile to forensically detect gene‐editing events at the molecular level, as no foreign DNA exists in the elite line. These limitations in molecular detection approaches are likely to focus more attention on the products generated from the technology than on the process in itself. Rapid advances in sequencing and genome assembly increasingly facilitate genome sequencing as a means of characterizing new varieties generated by gene‐editing techniques. Nevertheless, subtle edits such as single base changes or small deletions may be difficult to distinguish from normal variation within a genotype. Given these emerging scenarios, downstream ‘omics’ technologies reflective of edited affects, such as metabolomics, need to be used in a more prominent manner to fully assess compositional changes in novel foodstuffs. To achieve this goal, metabolomics or ‘non‐targeted metabolite analysis’ needs to make significant advances to deliver greater representation across the metabolome. With the emergence of new edited crop varieties, we advocate: (i) concerted efforts in the advancement of ‘omics’ technologies, such as metabolomics, and (ii) an effort to redress the use of the technology in the regulatory assessment for metabolically engineered biotech crops. 

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