BACKGROUND: The Prime Editing (PE) system is a precise and versatile genome editing tool with great potential in plant breeding and plant synthetic biology. However, low PE efficiency severely restricts its application, especially in dicots. PE can introduce small tags to trace target protein or cis-element to regulate gene transcription which is an expertise superior to other gene editing tools. Owing to low efficiency, PE adaption in stably transformed Arabidopsis is lacking.
OBJECTIVE: This study aimed to investigate the issue of low PE efficiency in dicots and develop systematic solutions to improve it. Currently, PE in dicots is undetectable and inconsistent, and this study seeks to address it. Split PE into several parts showed better performance in some target sites in mammal cells. We plan to discover the optimal split PE combination in dicot.
METHODS: We conducted large-scale transformation experiments in dicot model plants Arabidopsis thaliana (At) and Nicotiana benthamiana (Nb) by Agrobacterium-mediated transformation with deep amplicon sequencing (0.2-0.5 million clean total reads).
RESULTS: The editing efficiency decreased upon using a fused reverse transcriptase (RT) or an extended pegRNA separately and further decreased dramatically when these were used together. With the help of the pol II strategy to express PE gRNA (pegRNA), we named the most effective split PE combination as a multi-modular assembled prime editing system (mPE). mPE exhibited improved precise editing efficiency on most gene sites with various editing types, ranging from 1.3-fold to 1288.5-fold and achieved PE on some sites that could not be edited by original PE2. Especially, mPE showed superiority for multi-base insertion with an average improvement of 197.9-fold.
CONCLUSIONS: The original PE architecture strongly inhibited the cleavage activity of Cas9. Split PE improved PE efficiency extensively and was in favor of introducing small insertions in dicot plants, indicating that different PE variants might have their own expertise.

Given the challenges imposed by climate change and societal challenges, the European Union established ambitious goals as part of its Farm to Fork (F2F) strategy. Focussed on accelerating the transition to systems of sustainable food production, processing and consumption, a key element of F2F is to reduce the use of fertilisers by at least 20% and plant protection products by up to 50% by 2030. In recent years, a substantial body of research has highlighted the potential impact of microbial-based applications to support crop production practices through both biotic/abiotic stresses via maintaining or even improving yields and reducing reliance on intensive chemical inputs. Here, we have characterised the ability of a new soil-borne free-living bacterium strain Ensifer adhaerens OV14 (EaOV14) to significantly enhance crop vigour index by up to 50% for monocot (wheat, Triticum aestivum L., p < 0.0001) and by up to 40% for dicot (oilseed rape, Brassica napus L., p < 0.0001) species under in-vitro conditions (n = 360 seedlings/treatment). The beneficial effect was further studied under controlled glasshouse growing conditions (n = 60 plants/treatment) where EaOV14 induced significantly increased seed yield of spring oilseed rape compared to the controls (p < 0.0001). Moreover, using bespoke rhizoboxes, enhanced root architecture (density, roots orientation, roots thickness etc.) was observed for spring oilseed rape and winter wheat, with the median number of roots 55% and 33% higher for oilseed rape and wheat respectively, following EaOV14 seed treatment compared to the control. In addition, EaOV14 treatment increased root tip formation and root volume, suggesting the formation of a more robust root system architecture post-seed treatment. However, like other microbial formulations, the trade-offs associated with field translation, such as loss or limited functionality due to inoculum formulation or environmental distress, need further investigation. Moreover, the delivery method requires further optimisation to identify the optimal inoculum formulation that will maximise the expected beneficial impact on yield under field growing conditions.

The mechanical structure of plant tissues has recently attracted a lot of attention. The present study aims to evaluate the importance of collenchymatous and sclerenchymatous tissues in supporting plant species in their harsh environments like road and street plant habitats. Dicots and monocots are classified into different models according to the types of supporting mechanisms. Mass cell percentage and soil analysis are used in this investigation. The tissues are distributed with different percentage masses and arrangements to overcome various severe conditions. Statistical analyses enhance the role of these tissues and clarify their significant values. The gear support mechanism is claimed to be the perfect mechanical method used.

Soybean is an important model crop for photoperiodic response studies in plants and contributes significantly to the study of plant development and physiology in the past century. Because soybean plant is much bigger in size and longer in life cycle than Arabidopsis, it needs much more space for growth and time for investigation, which significantly hamper the efficiency of research. In the current study, we tested the photoperiodic response of a distinctive artificially-made cotyledon-only plant (COP) using a photoperiod-sensitive soybean variety Zigongdongdou (ZGDD) and other varieties with diverse sensitivity to photoperiod. ZGDD COPs flowered 39.4 ± 2.5 d after emergence under short-day conditions but maintained vegetative growth under long-day and night break conditions, which is similar to the case in the intact ZGDD plants. The COPs of early-maturing and medium-maturing soybean varieties also grew and flowered normally under natural day-length conditions. At the molecular level, the key genes in the photoperiodic pathway such as E1, GmFT1a, GmFT2a, and GmFT5a in the COPs also showed the same photoperiod sensitivity as in the intact plants. In addition, a simpler material of COP with only one cotyledon and root was generated and found to be sensitive to photoperiod as well. Notably, the COPs are only one-fifth the height of intact plants and one-third the maximum diameter of the intact plants grown in chambers 30 d after emergence. Based on COPs, we established a novel experimental system characterized by an entire photoperiodic response and longer longevity of cotyledons in addition to small plant size, ensuring the consistency, reliability, and stability of plant materials. COPs have the potential to be a novel model material for studies of the developmental biology of soybean and other dicots.

Simulation-based training (SBT) programs are commonly employed by organizations to train individuals and teams for effective workplace cognitive and psychomotor skills in a broad range of applications. Distributed cognition has become a popular cognitive framework for the design and evaluation of these SBT environments, with structured methodologies such as Distributed Cognition for Teamwork (DiCoT) used for analysis. However, the analysis and evaluations generated by such distributed cognition frameworks require extensive domain-knowledge and manual coding and interpretation, and the analysis is primarily qualitative. In this work, we propose and develop the application of multimodal learning analysis techniques to SBT scenarios. Using these analysis methods, we can use the rich multimodal data collected in SBT environments to generate more automated interpretations of trainee performance that supplement and extend traditional DiCoT analysis. To demonstrate the use of these methods, we present a case study of nurses training in a mixed-reality manikin-based (MRMB) training environment. We show how the combined analysis of the video, speech, and eye-tracking data collected as the nurses train in the MRMB environment supports and enhances traditional qualitative DiCoT analysis. By applying such quantitative data-driven analysis methods, we can better analyze trainee activities online in SBT and MRMB environments. With continued development, these analysis methods could be used to provide targeted feedback to learners, a detailed review of training performance to the instructors, and data-driven evidence for improving the environment to simulation designers.

Plants require zinc (Zn) as an essential cofactor for diverse molecular, cellular and physiological functions. Zn is crucial for crop yield, but is one of the most limiting micronutrients in soils. Grasses like rice, wheat, maize and barley are crucial sources of food and nutrients for humans. Zn deficiency in these species therefore not only reduces annual yield but also directly results in Zn malnutrition of more than two billion people in the world. There has been good progress in understanding Zn homeostasis and Zn deficiency mechanisms in plants. However, our current knowledge of monocots, including grasses, remains insufficient. In this review, we provide a summary of our knowledge of molecular Zn homeostasis mechanisms in monocots, with a focus on important cereal crops. We additionally highlight divergences in Zn homeostasis of monocots and the dicot model Arabidopsis thaliana, as well as important gaps in our knowledge that need to be addressed in future research on Zn homeostasis in cereal monocots.

As an efficient tool for functional genomics, VIGS (virus-induced gene silencing) has been widely used in reverse and forward genetics to identify genes involved in various biology processes in many plant species. Up to now, at least 50 VIGS vectors based on RNA viruses, DNA viruses or satellites have been developed for either dicots or monocots or both. Silencing specific genes using VIGS vector involves five major steps including, first, choosing an appropriate VIGS vector for the plant; second, selecting a fragment of targeted host gene; third, cloning the fragment into viral VIGS vector; forth, inoculating and infecting the appropriate plant; and fifth, quantifying silencing effects including recording silencing phenotypes and determining silencing efficiency of the target gene. In this chapter, we introduce these steps for VIGS assay in dicots and monocots, by taking a cucumber mosaic virus-based VIGS vector for Nicotiana benthamiana and maize plants as an example. Moreover, we list available VIGS vectors for monocots.

Plants are exposed to a wide range of temperatures during their life cycle and need to continuously adapt. These adaptations need to deal with temperature changes on a daily and seasonal level and with temperatures affected by climate change. Increasing global temperatures negatively impact crop performance, and several physiological, biochemical, morphological and developmental responses to increased temperature have been described that allow plants to mitigate this. In this review, we assess various growth, development, and yield-related responses of crops to extreme and moderate high temperature, focusing on knowledge gained from both monocot (e.g. wheat, barley, maize, rice) and dicot crops (e.g. soybean and tomato) and incorporating information from model plants (e.g. Arabidopsis and Brachypodium). This revealed common and different responses between dicot and monocot crops, and defined different temperature thresholds depending on the species, growth stage and organ.

Successful regeneration of genetically modified plants from cell culture is highly dependent on the species, genotype, and tissue-type being targeted for transformation. Studies in some plant species have shown that when expression is altered, some genes regulating developmental processes are capable of triggering plant regeneration in a variety of plant cells and tissue-types previously identified as being recalcitrant to regeneration. In the present research, we report that developmental genes encoding GROWTH-REGULATING FACTORS positively enhance regeneration and transformation in both monocot and dicot species. In sugar beet (Beta vulgaris ssp. vulgaris), ectopic expression of Arabidopsis GRF5 (AtGRF5) in callus cells accelerates shoot formation and dramatically increases transformation efficiency. More importantly, overexpression of AtGRF5 enables the production of stable transformants in recalcitrant sugar beet varieties. The introduction of AtGRF5 and GRF5 orthologs into canola (Brassica napus L.), soybean (Glycine max L.), and sunflower (Helianthus annuus L.) results in significant increases in genetic transformation of the explant tissue. A positive effect on proliferation of transgenic callus cells in canola was observed upon overexpression of GRF5 genes and AtGRF6 and AtGRF9. In soybean and sunflower, the overexpression of GRF5 genes seems to increase the proliferation of transformed cells, promoting transgenic shoot formation. In addition, the transformation of two putative AtGRF5 orthologs in maize (Zea mays L.) significantly boosts transformation efficiency and resulted in fully fertile transgenic plants. Overall, the results suggest that overexpression of GRF genes render cells and tissues more competent to regeneration across a wide variety of crop species and regeneration processes. This sets GRFs apart from other developmental regulators and, therefore, they can potentially be applied to improve transformation of monocot and dicot plant species.

Tricin (3\',5\'-dimethoxylated flavone) is a predominant flavonoid amongst monocots but occurs only in isolated and unrelated dicot lineages. Although tricin biosynthesis has been intensively studied in monocots, it has remained largely elusive in tricin-accumulating dicots. We investigated a subgroup of cytochrome P450 (CYP) 75B subfamily flavonoid B-ring hydroxylases (FBHs) from two tricin-accumulating legumes, Medicago truncatula and alfalfa (Medicago sativa), by phylogenetic, molecular, biochemical and mutant analyses. Five Medicago cytochrome P450 CYP75B FBHs are phylogenetically distant from other legume CYP75B members. Among them, MtFBH-4, MsFBH-4 and MsFBH-10 were expressed in tricin-accumulating vegetative tissues. In vitro and in planta analyses demonstrated that these proteins catalyze 3\'- and 5\'-hydroxylations critical to tricin biosynthesis. A key amino acid polymorphism, T492G, at their substrate recognition site 6 domain is required for the novel 5\'-hydroxylation activities. Medicago truncatula mtfbh-4 mutants were tricin-deficient, indicating that MtFBH-4 is indispensable for tricin biosynthesis. Our results revealed that these Medicago legumes had acquired the tricin pathway through molecular evolution of CYP75B FBHs subsequent to speciation from other nontricin-accumulating legumes. Moreover, their evolution is independent of that of grass-specific CYP75B apigenin 3\'-hydroxylases/chrysoeriol 5\'-hydroxylases dedicated to tricin production and Asteraceae CYP75B flavonoid 3\',5\'-hydroxylases catalyzing the production of delphinidin-based pigments.