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Buckwheat (Fagopyrum esculentum Moench) is a healthy cereal with a short lifecycle, and an efficient transgenic method for gene function analysis of sweet buckwheat is lacking. In this study, we developed a simple Agrobacterium-mediated transformation protocol using a floral-dip. For this transformation experiment, the Agrobacterium strain GV3101 harboring binary vector pHZM69 with enhanced green fluorescent protein (eGFP) as well as a resistance gene to the herbicide Basta was used. The effect of different cell densities (OD600 = 0.4, 0.6, 0.8 and 1.0) applied for infection was measured, and maximum transformation efficiency (12.42%) was obtained with cell density of OD600 = 0.6, followed by OD600 = 0.4 (8.48%), OD600 = 0.8 (4.39%) and OD600 = 1.0 (3.62%). Here, sweet buckwheat was successfully transformed by Agrobacterium-mediated floral dip method, which could be a useful tool for the production of transgenic sweet buckwheat and promote genomic function analysis in sweet buckwheat. [ABSTRACT FROM AUTHOR]

Malaria remains a global health issue, especially in resource-limited regions. Artemisinin, a key antimalarial compound from Artemisia annua , is crucial for treatment, but low natural yields hinder large-scale production. In this study, we employed advanced transgenic technology to co-overexpress six key biosynthetic enzymes—Isopentenyl Diphosphate Isomerase (IDI), Farnesyl Pyrophosphate Synthase (FPS), Amorpha 4,11-diene Synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), cytochrome P450 oxidoreductase (AACPR) and artemisinic aldehyde D11 reductase (DBR2)—in A. annua to significantly enhance artemisinin production. Our innovative approach utilized a co-expression strategy to optimize the artemisinin biosynthetic pathway, leading to a remarkable up to 200 % increase in artemisinin content in T1 transgenic plants compared to non-transgenic controls. The stability and efficacy of this transformation were confirmed in subsequent generations (T2), achieving a potential 232 % increase in artemisinin levels. Additionally, we optimized transgene expression to maintain plant growth and development, and performed untargeted metabolite analysis using GC–MS, which revealed significant changes in metabolite composition among T2 lines, indicating effective diversion of farnesyl diphosphate into the artemisinin pathway. This metabolic engineering breakthrough offers a promising and scalable solution for enhancing artemisinin production, representing a major advancement in the field of plant biotechnology and a potential strategy for more cost-effective malaria treatment. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract The Agrobacterium-mediated floral dip protocol is the most extensively used transformation method for a model plant Arabidopsis thaliana. Several useful methods for Agrobacterium tumefaciens–mediated transformations of Arabidopsis are existing, but they are time consuming and with low transformation efficiency. Here, we developed a transgenic Arabidopsis lines TET12p::TET12-RFP in a short period of time and enhanced transformation efficiency by using a modified transformation method by applying drought stress after floral dip. In this protocol, Agrobacterium cells carrying TET12p::TET12-RFP recombinant vectors were resuspended in a solution of 5% sucrose, 0.05% (v/v) silwet L-77 to transform female gametes of developing Arabidopsis inflorescences. Treated Arabidopsis were then applied with different levels of drought stresses to stimulate plants for the utilization of maximum plant energy in seed maturation process. The applied stresses achieved the fast maturation of already treated inflorescences while stopped the growing of newly arising untreated inflorescence, thus decreased the chances of wrong collection of untransformed seeds. Consequently, the collected seeds were mostly transgenic with a transformation frequency of at least 10%, thus the screening for positive transformants selection was more advantageous on a selective medium as compared to a classical floral dip method. Within 2-3 months, two hundred of individual transgenic plants were produced from just 10 infiltrated plants. This study concludes that application of drought stresses in a specific stage of plant is a beneficial strategy for achieving the transgenic Arabidopsis in a short period of time with high transformation efficiency.

CRISPR/Cas9 genome editing has been used extensively in a wide variety of plant species. Creation of loss-of-function alleles, promoter variants and mutant collections are a few of the many uses of genome editing. In a typical workflow for sexually reproducing species, plants are generated that contain an integrated CRISPR/Cas9 transgene. After editing of the gene of interest, T-DNA null segregants can be identified in the next generation that contain only the desired edit. However, maintained presence of the CRISPR/Cas9 transgene and continued editing in the subsequent generations offer a range of applications for model plants and crops. In this review, we define transgenerational gene editing (TGE) as the continued editing of CRISPR/Cas9 after a genetic cross. We discuss the concept of TGE, summarize the current main applications, and highlight special cases to illustrate the importance of TGE for plant genome editing research and breeding.

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Abstract Background Rapid-cycling Brassica rapa (RCBr), also known as Wisconsin Fast Plants, are small robust plants with a short lifecycle that are widely used in biology teaching. RCBr have been used for decades but there are no published reports of RCBr genetic transformation. Agrobacterium-mediated vacuum infiltration has been used to transform pakchoi (Brassica rapa ssp. chinensis) and may be suitable for RCBr transformation. The floral dip transformation method, an improved version of vacuum infiltration, could make the procedure easier. Results Based on previous findings from Arabidopsis and pakchoi, plants of three different ages were inoculated with Agrobacterium. Kanamycin selection was suboptimal with RCBr; a GFP screen was used to identify candidate transformants. RCBr floral bud dissection showed that only buds with a diameter less than 1 mm carried unsealed carpels, a key point of successful floral dip transformation. Plants across a wide range of inflorescence maturities but containing these immature buds were successfully transformed, at an overall rate of 0.1% (one per 1000 T1 seeds). Transformation was successful using either vacuum infiltration or the floral dip method, as confirmed by PCR and Southern blot. Conclusion A genetic transformation system for RCBr was established in this study. This will promote development of new biology teaching tools as well as basic biology research on Brassica rapa.

Agrobacterium-mediated transformation methods have allowed the stable introduction of target genes into the nuclear genomes of recipient plants. Among them, the floral dip approach represents the simplest due to its straightforwardness and high transformation efficiency. In a standard floral dip protocol that most researchers follow, Agrobacterium cells are grown to stationary phase (OD 600≈2.0) in large cultures and resuspended in inoculation medium to OD 600≥0.8. Here, we tested the effects of low Agrobacterium inoculum on transformation rate. Our data revealed that the floral dip method still guarantees a relatively high transformation rate in the Arabidopsis thaliana Col-0 ecotype even with very low Agrobacterium inoculum (OD 600=0.002). Our finding thus simplifies the floral dipping protocol further, which allows transformation with small bacterial culture and enables high-throughput transformation of large numbers of constructs in parallel.

Agrobacterium-mediated transformation methods have allowed the stable introduction of target genes into the nuclear genomes of recipient plants. Among them, the floral dip approach represents the simplest due to its straightforwardness and high transformation efficiency. In a standard floral dip protocol that most researchers follow, Agrobacterium cells are grown to stationary phase (OD 600≈2.0) in large cultures and resuspended in inoculation medium to OD 600≥0.8. Here, we tested the effects of low Agrobacterium inoculum on transformation rate. Our data revealed that the floral dip method still guarantees relatively high transformation rate in Arabidopsis thaliana Col-0 ecotype even with very low Agrobacterium inoculum (OD 600=0.002). Our finding thus simplifies the floral dipping protocol further, which allows transformation with small bacterial culture and enables high-throughput transformation of large numbers of constructs in parallel.

In this review, the application of CRISPR/Cas9 plant genome editing using alternative transformation methods is discussed. Genome editing by the CRISPR/Cas9 system is usually implemented via the generation of transgenic plants carrying Cas9 and sgRNA genes in the genome. Transgenic plants are usually developed by in vitro regeneration from single transformed cells, which requires using different in vitro culture-based methods. Despite their common application, these methods have some disadvantages and limitations. Thus, some methods of plant transformation that do not depend on in vitro regeneration have been developed. These methods are known as "in planta" transformation. The main focus of this review is the so-called floral dip in planta transformation method, although other approaches are also described. The main features of in planta transformation in the context of CRISPR/Cas9 genome editing are discussed. Furthermore, multiple ways to increase the effectiveness of this approach and to broaden its use in different plant species are considered. [ABSTRACT FROM AUTHOR]

Gene transformation can be done in direct and indirect (Agrobacterium-mediated) ways. The most efficient method of gene transformation to date is Agrobacterium-mediated method. The main problem of Agrobacterium-method is that some plant species and mutant lines are recalcitrant to regeneration. Requirements for sterile conditions for plant regeneration are another problem of Agrobacterium-mediated transformation. Development of genotype-independent gene transformation method is of great interest in many plants. Some tissue culture-independent Agrobacterium-mediated gene transformation methods are reported in individual plants and crops. Generally, these methods are called in planta gene transformation. In planta transformation methods are free from somaclonal variation and easier, quicker, and simpler than tissue culture-based transformation methods. Vacuum infiltration, injection of Agrobacterium culture to plant tissues, pollen-tube pathway, floral dip and floral spray are the main methods of in planta transformation. Each of these methods has its own advantages and disadvantages. Simplicity and reliability are the primary reasons for the popularity of the in planta methods. These methods are much quicker than regular tissue culture-based Agrobacterium-mediated gene transformation and success can be achieved by non-experts. In the present review, we highlight all methods of in planta transformation comparing them with regular tissue culture-based Agrobacterium-mediated transformation methods and then recently successful transformations using these methods are presented.

Abiotic environmental stressors cause various types of damage to plants and cause significant loss in yield. Abiotic stress tolerance in plants refers to the ability to withstand environmental factors and maintain growth, development, and production. Since this tolerance is controlled by a gene or a set of genes, transgenic activating of these genes in plants often enhances tolerance under abiotic stress. Therefore, this methodology chapter describes a strategy and the corresponding protocols needed to induce a gene by an abiotic stressor, clone the corresponding cDNA into plasmids and Agrobacterium cells, and genetic transformation to the Arabidopsis plants using the floral dip method. The chapter also describes standard assays to evaluate the transgene's effect on the plant's tolerance. Finally, the techniques outlined in this chapter for cloning and generating transgenic plants tolerant to abiotic stress are a versatile approach that can be implemented across various plant species and genes., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Gene transformation can be done in direct and indirect (Agrobacterium-mediated) ways. The most efficient method of gene transformation to date is the Agrobacterium-mediated method. The main problem of this method is that some plant species and mutant lines are recalcitrant to regeneration. Requirements for sterile conditions for plant regeneration are another problem of Agrobacterium-mediated transformation. The development of a genotype-independent gene transformation method is of great interest in many plants. Some Agrobacteriummediated gene transformation methods independent of tissue culture are reported in individual plants and crops. Generally, these methods are called in planta gene transformation. In planta transformation methods are free from somaclonal variation and easier, quicker, and simpler than transformation methods based on tissue culture. Vacuum infiltration, injection of Agrobacterium culture into plant tissues, pollen-tube pathway, floral dip and floral spray are the main methods of in planta transformation. Each of these methods has its own advantages and disadvantages. Simplicity and reliability are the primary reasons for the popularity of the in planta methods. These methods are much faster than regular Agrobacterium-mediated gene transformation based on tissue culture and success can be achieved by non-experts. In the present review, we highlight all methods of in planta transformation comparing them with regular Agrobacterium-mediated transformation methods based on tissue culture. Finally, successful recent transformations using these methods are presented. [ABSTRACT FROM AUTHOR]

Yellow cosmos (Cosmos sulphureus Cav.) is a specific flowering plant and considered a suitable genetic engineering model. Agrobacterium -mediated plant transformation is commonly used for plant genetic engineering. Floral dip transformation is one of the plant genetic transformation methods, and it involves dipping flower buds into an Agrobacterium suspension. Studies on floral dip transformation of yellow cosmos have never been reported. Therefore, an efficient method in plant genetic engineering must be established. This study developed an effective and efficient floral dip transformation method for yellow cosmos. In this study, flower buds with sizes of 5–7 mm were used. Several parameters have been observed to optimize the floral dip method. These parameters included the optical density (OD 600) of Agrobacterium culture, concentration of surfactant, and duration of flower bud dipping into the Agrobacterium suspension. The results showed that the floral dip method was most efficient when the flower buds were dipped into Agrobacterium suspension with OD 600 = 0.8 and containing 5% sucrose and 0.1% Silwet L-77 for 30 s. This method enhanced the transformation efficiency at a rate of 12.78 ± 1.53%. The neomycin phosphotransferase II and green fluorescent protein genes with sizes of 550 and 736 bp, respectively, were confirmed by polymerase chain reaction. In addition, the transgenic plants were kanamycin resistant and fluorescent under ultraviolet light observation. This finding suggests that the proposed floral dip transformation provides new insights into efficient plant genetic engineering methods for yellow cosmos. [ABSTRACT FROM AUTHOR]

Tomato ( Solanum lycopersicum) is a model crop plant for the study of fruit ripening and disease resistance. Here we present a systemic study on in planta transformation of tomato with Agrobacterium tumefaciens strain LBA4404 harboring pCAMBIA1303 binary vector bearing HPTII as a plant selectable marker and mGFP/ GUS fusion as the reporter gene. We attempted the transformation of tomato at different developmental stages viz. during seed germination, seedling growth, and floral bud development. The imbibition of seeds with Agrobacterium suspension led to seed mortality. The vacuum infiltration of seedlings with Agrobacterium suspension led to sterility in surviving plants. Successful transformation could be achieved either by dipping of developing floral buds in the Agrobacterium suspension or by injecting Agrobacterium into the floral buds. Most floral buds subjected to dip as well as to injection either aborted or had arrested development. The pollination of surviving floral buds with pollen from wild-type plants yielded fruits bearing seeds. A transformation efficiency of 0.25-0.50% was obtained on floral dips/floral injections. Transgenic plants were selected by screening seedlings for hygromycin resistance. The presence of the transgene in genomic DNA was confirmed by Southern blot analysis and expression of the reporter gene up to the T generation. The amenability of tomato for in planta transformation simplifies the generation of transgenic tomato plants obviating intervening tissue culture. [ABSTRACT FROM AUTHOR]

Amaranth is a new and promising crop for the Russian climate, notable for its well-balanced amino acid composition. Yield increase using the methods of genetic engineering is a challenging task. We generated transgenic plants of amaranth with expression of the Arabidopsis thaliana ARGOS-LIKE gene under the control of the dahlia mosaic virus promoter. We achieved 1.4% transformation effectiveness. In comparison with wild-type amaranth, we observed a 21% increase in stem length, 79% increase in leaf length, and 190% increase in fresh weight of transgenic plants. It was shown that ARGOS-LIKE gene of A. thaliana along with the dahlia mosaic virus promoter can be used to increase primarily the green weight of shoot and leaf size of amaranth. [ABSTRACT FROM AUTHOR]

The CRISPR/Cas system has emerged as a versatile platform for sequence-specific genome engineering in plants. Beyond genome editing, CRISPR/Cas systems, based on nuclease-deficient Cas9 (dCas9), have been repurposed as an RNA-guided platform for transcriptional regulation. CRISPR activation (CRISPRa) represents a novel gain-of-function (GOF) strategy, conferring robust over-expression of the target gene within its native chromosomal context. The CRISPRa systems enable precise, scalable, and robust RNA-guided transcription activation, holding great potential for a variety of fundamental and translational research. In this chapter, we provide a step-by-step guide for efficient gene activation in Arabidopsis based on a highly robust CRISPRa system, CRISPR-Act3.0. We present detailed procedures on the sgRNA design, CRISPR-Act3.0 system construction, Agrobacterium-mediated transformation of Arabidopsis using the floral dip method, and identification of desired transgenic plants., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

PttKN1 (Populus tremula x tremuloides KNOTTED1) gene belongs to the KNOXI gene family. It plays an important role in plant development, typically in meristem initiation, maintenance and organogenesis, and potentially in plant coloration. To investigate the gene functions further, it was introduced into red leaf beet by the floral dip method mediated via Agrobacterium tumefaciens. The transformants demonstrated typical phenotypes as with other PttKN1 transformants. These alterations were very different from the morphology of the wild type. Among them, morphological modification of changed color throughout the entire plant from claret of wild type to yellowish green was the highlight in those transgenic PttKN1-beet plants. The result of spraying selection showed that the PttKN1-beet plants had kanamycin resistance. PCR assay of the 35S-Promoter, NPTII and PttKN1 gene, PCR-Southern analysis of the NPTII and PttKN1 gene showed that the foreign PttKN1 gene had successfully integrated into the genome of beet plant. Furthermore, the results of RT-PCR analysis showed that the gene was ectopic expressed in transgenic plants. These data suggested that there is a correlation between the ectopic expression of PttKN1 gene and morphological alterations of beet plants. Pigment content assay showed that betaxanthins concentrations shared little difference between wild type and transgenic lines, while betacyanins content in transgenic plants was sharply decreased, indicating that the altered plant coloration of the transgenic beet plants may be caused by the changed betacyanins content. The tyrosinase study suggested that the sharply decreased of betacyanins content in transgenic plants was caused via the decreased tyrosinase level. Therefore, the reason for the altered plant coloration may be due to partial inhibition of betacyanin biosynthesis that was induced via the pleiotropic roles of PttKN1 gene. [ABSTRACT FROM AUTHOR]

Being a model plant, Arabidopsis transformation is a frequent routine in genetics and molecular laboratories. Here, we explain a modified floral dip method routinely used in our laboratory for Arabidopsis transformation by applying drought stress after plant treatment with Agrobacterium. By using this method, hundreds of individual transgenic plants are produced from just 30 infiltrated plants by increasing transformation frequency from 1% to 10% within 2–3 months. • Drought stress and cutting of untransformed buds are used for a beneficial approach to enhance transformation efficiency. • Few unwanted floral buds are allowed to develop, thus minimize the chances of wrong collection of untransformed seeds. • The modified floral dip method reduces the time of transformation while increases the rate of Arabidopsis transformation. [ABSTRACT FROM AUTHOR]

The importance of using tissue-specific promoters in the genetic transformation of plants has been emphasized increasingly. Here, we report the isolation of a novel seed-specific promoter region from peanut and its validation in Arabidopsis and tobacco seeds. The reported promoter region referred to as groundnut seed promoter (GSP) confers seed-specific expression in heterologous systems, which include putative promoter regions of the peanut ( Arachis hypogaea L.) gene 8A4R19G1. This region was isolated, sequenced, and characterized using gel shift assays. Tobacco transgenics obtained using binary vectors carrying uidA reporter gene driven by GSP and/or cauliflower mosaic virus 35S promoters were confirmed through polymerase chain reaction (PCR), RT-PCR, and computational analysis of motifs which revealed the presence of TATA, CAAT boxes, and ATG signals. This seed-specific promoter region successfully targeted the reporter uidA gene to seed tissues in both Arabidopsis and tobacco model systems, where its expression was confirmed by histochemical analysis of the transgenic seeds. This promoter region is routinely being used in the genetic engineering studies in legumes aimed at targeting novel transgenes to the seeds, especially those involved in micronutrient enhancement, fungal resistance, and molecular pharming. [ABSTRACT FROM AUTHOR]

The purpose of this work was to evaluate two methods (floral dip and vacuum infiltration) of in planta transformation of Arabidopsis thaliana. The key issue of this work is the identification of the developmental stages of A. thaliana flower buds subjected to agroinfection, optimal for the successful transformation. Histological tests performed after agroinfection made it possible to establish the patterns of a GUSPlus reporter gene expression in the examined plants and thus precisely define the range of flower developmental stages most appropriate for efficient transformation. Two plasmids, CAMBIA 1305.1 and CAMBIA 2301, were used. Verification of the transgenic nature of plants was carried out by detection of CaMV ::gusA and CaMV ::GUSPlus transgenes and their expression in transgenic plants by appropriate molecular and histochemical methods. For the flower dip transformation, three concentrations of Silwet L-77 surfactant and two inoculation times were tested. The most efficient treatment appeared to be 2-min-long flower bud inoculation and 400 μl/l surfactant (pCAMBIA 1305.1 − 1.73%; pCAMBIA 2301 − 2.01%). In the case of vacuum infiltration method, the highest efficiency of the transformation occurred when the inoculation time was 4 min (pCAMBIA 2301 − 1.55%; pCAMBIA 1305.1 − 1.37%). [ABSTRACT FROM AUTHOR]

Plants are currently used as a cost-effective and safe heterologous expression system for the production of experimental vaccines. Here, we describe the expression of the HPVI6 L1 protein in Arabidopsis thaliana. The HPV16 major capsid gene L1 sequence was extracted by digestion from the plasmid pMOS-L1 HPV16 containing the template sequence and cloned into the binary vector pB1121 consecutively to obtain the plant expression plasmid pBI-L1. The T-DNA regions of the pBI-L1 binary vector were cloned under the control of the constitutive cauliflower mosaic virus 35S promoter and the neomycin phosphotransferase nptII gene; the vector allowed the selection of transformed plants using kanamycin. The Arabidopsis plants were transformed by floral dip method, using Agrobacterium tumefaciens GV3101, which harbored the plant expression plasmid. The generated transgenic Arabidopsis plants were selected on a kanamycin-added Murashige--Skoog medium, and the integration and the stability of the L1 gene in plant genome were confirmed by polymerase chain reaction (PCR) amplification in three successive generations. The evidence for mRNA expression was acquired by reverse transcriptase PCR analysis. To identify further integration of transgene into the genome of A. thaliana, Southern blot assay was carried out and showed that the HPV16 L1 gene was integrated stably into the genome of the transformed Arabidopsis plants. Furthermore, Western blot analysis showed that transformed Arabidopsis plants produce HPV16 L1 protein. Thus, HPV16 L1 protein expressed in transgenic Arabidopsis plants can be potentially used as an edible vaccine. [ABSTRACT FROM AUTHOR]

A number of in planta transformation protocols that avoid long culture under sterile conditions were developed for Arabidopsis thaliana. The most widely used methods are based on vacuum infiltration and floral dip. These methods were adapted for transformation of other species as well. Successful in planta transformations of alfalfa, radish, pakchoi and petunia were reported recently. In this short review we present several modified procedures originally developed for Arabidopsis thaliana and in some cases adapted to other species. We emphasize the crucial parameters involved in in planta transformation. We also describe here the studies attempting to shed light on the mechanisms and estimating the cellular target of transformation, which may help in transforming new plant species. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 genome editing has been used extensively in a wide variety of plant species. Creation of loss-of-function alleles, promoter variants and mutant collections are a few of the many uses of genome editing. In a typical workflow for sexually reproducing species, plants are generated that contain an integrated CRISPR/Cas9 transgene. After editing of the gene of interest, T-DNA null segregants can be identified in the next generation that contain only the desired edit. However, maintained presence of the CRISPR/Cas9 transgene and continued editing in the subsequent generations offer a range of applications for model plants and crops. In this review, we define transgenerational gene editing (TGE) as the continued editing of CRISPR/Cas9 after a genetic cross. We discuss the concept of TGE, summarize the current main applications, and highlight special cases to illustrate the importance of TGE for plant genome editing research and breeding., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Impens, Jacobs, Nelissen, Inzé and Pauwels.)

Arabidopsis thaliana (L.) Heynh. has emerged as the dominant plant species used in molecular genetics and functional genomics. Although it possesses several attributes of model organisms, the ease with which it can be transformed genetically is a paramount factor that has led to its widespread application as a tool for functional genomics. Progress made in the late 1990s has relieved users of the need to use a vacuum chamber for transformation, substituting its function with a surfactant known as Silwet L-77, to facilitate access to ovules by bacterial solution. Here, we evaluate the substitution of Silwet L-77 by XIAMETER OFX-0309. We demonstrate that transformation efficiency can be increased by an average of 3-fold (and up to 10-fold) solely by replacing Silwet L-77 with XIAMETER OFX-0309. Because downstream selection of transgenic plants, in either soil or Petri dishes, can be long and tedious, this substitution represents a significant reduction of time and materials required to retrieve transgenic lines. [ABSTRACT FROM AUTHOR]

Setaria viridis is an emerging model for C4 species, and it is an important model to validate some genes for further C4 crop transformation, such as sugarcane, maize, and wheat. Here, we describe two protocols for stable transformation of S. viridis mediated by Agrobacterium tumefaciens with three different reporter genes and two selectable markers. Routine transformation efficiency reaching 29% was achieved using embryogenic callus in S. viridis (accession A10.1). Alternatively, we developed a transformation method by floral dip with 0.6% efficiency. The developed protocols could be useful for genetic and genomics studies of important food-feed-fiber-fuel C4 crops.