Efficient methods for gene transfer to maize were developed in the 1990s, first mediated by particle bombardment and then by Agrobacterium tumefaciens. Both methods can efficiently create high-quality events. Genetically modified varieties were commercialized in 1996 and are now planted in more than 90% of the US corn field. Tissue culture protocols for both methods have been well developed and widely employed. Thus, various factors, including handling before gene delivery, techniques to protect cells during gene delivery, and culture media, have been well optimized for various genotypes. Typical protocols for both methods are herein presented to show major outputs from the studies conducted since the early 1990s. As the bombardment protocols tended to be optimized specifically for limited genotypes, the one for B104, a new public inbred with favorable agronomic characteristics, is shown. The Agrobacterium protocol is suitable for various inbred lines, including B104. These protocols are also useful starting points in the optimization of tissue culture for gene editing. The rate-limiting step in both transformation and gene editing is in tissue culture and plant regeneration from modified cells in elite germplasm. Despite the prolonged efforts, large varietal differences in tissue culture responses remain a serious issue in maize. Recently, protocols using morphogenic regulator genes, such as Bbm and Wus2, have been developed that show a strong potential of efficiently transforming recalcitrant varieties.
The family Liliaceae (Cronquist system) contains various important ornamental plants. We have been examining for about 20 years the establishment of plant regeneration and genetic transformation systems in liliaceous ornamental plants for their biotechnological breeding and elucidation of the molecular mechanisms determining ornamental traits. In this review, studies on in vitro plant regeneration in 7 genera and on Agrobacterium-mediated production of transgenic plants in 4 genera are described. Plant regeneration was achieved via callus cultures in Agapanthus, Hemerocallis, Hosta, Lilium, Muscari and Tricyrtis. Auxins (2,4-dichrolophenoxyacetic acid, α-naphthaleneacetic acid and/or picloram) were effective for inducing regenerable calli. Tulipa species and cultivars were very recalcitrant to callus induction and plant regeneration. Agrobacterium-mediated transformation was examined in Agapanthus, Lilium, Muscari and Tricyrtis, and transgenic plants were obtained in all genera by using regenerable calli as a target material for Agrobacterium inoculation, inoculation and co-cultivation with Agrobacterium in the presence of acetosyringone, and selection of transgenic tissues and plantlets on hygromycin-containing media. Among 4 genera, Tricyrtis has several advantages for transformation studies: higher transformation efficiency, relatively small plant size, ease of cultivation, and taking only 1 year from in vitro regeneration to flowering. We are now investigating the molecular mechanisms for determining plant form, flower color and flower form by using Tricyrtis spp. as liliaceous model plants.
Cucumber (Cucumis sativus L.) and Cucurbita species (squashes, pumpkins, and gourds), belonging to the Cucurbitaceae family, are among the major vegetable crops in the world. Transgenic approaches could contribute to the accumulation of new knowledge of these species and to the development of elite cultivars. Despite this, research reports using transformants of these species are very limited so far. One of the reasons for this may be that although there are effective transformation methods, these methods are not well known among researchers. In the present review, we describe efficient protocols for the transformation of cucumber and squash plants and mention possible pitfalls in and advice for following these protocols. In addition, we discuss the current progress of genetic transformation research using cucumbers and squash, including genome editing.
Sugi (Cryptomeria japonica D. Don) is the most important afforestation coniferous tree in Japan. Coniferous trees normally have a long juvenile period and require a long cultivation time for breeding. Through a traditional breeding project that began in the 1950s, first generation plus trees with excellent traits were selected primarily from artificial forests and used as seedlings. Recently, the second generation plus trees obtained by crossing between plus trees have been selected. In light of this situation, the improvement of Sugi by a transgenic approach is effective in terms of shortening the breeding period compared with conventional crossing-dependent approaches. There are three key points to an efficient Agrobacterium-mediated transformation system: (1) establishment of explants with high regeneration ability, (2) optimal co-cultivation conditions for explants and Agrobacterium, and (3) efficient elimination of Agrobacterium. Here we describe a protocol for Agrobacterium-mediated transformation of Sugi that meets the above criteria using embryogenic tissues as explants isolated from immature seeds obtained by crossing.
Tall fescue (Festuca arundinacea Schreb.) is a major cool-season perennial grass grown for forage and turf. We have obtained transgenic tall fescue by Agrobacterium-mediated transformation to improve agronomically important traits. In our protocol, we use embryogenic calli derived from not only mature seeds but also shoot tips. Although tall fescue cultivars consist of various genotypes with different genetic variation, we can produce transgenic plants at any time with calli induced from shoot tips of in vitro-maintained responsive genotypes. When the hygromycin phosphotransferase gene is used as a selectable marker, transformants are selected by incubation with 100 mg l−1 hygromycin in both selection and regeneration media. Since tall fescue is an anemophilous species, the cultivation of transgenic plants poses the risk of transgenic pollen flow. Recently, it has been reported that genome-edited plants without the integration of foreign DNA fragments can be produced by an Agrobacterium-mediated transient gene expression system. We hope that our protocol will contribute to production of transgene-free genome-edited tall fescue.
Apple is one of precious fruit crop grown in temperate zone. In the post genomic era, the analysis of gene functions in horticultural crops such as apple is required for agricultural utilization. For analysis of such crops, the protocol establishment of tissue culture and transformation is essential. Although transformation efficiency in family Rosaceae is generally very low, some cultivars of Malus species have high transformation ability. Apple cultivars are usually clonally propagated by grafting on rootstocks, which can affect fruit quality and maturity and scion productivity. Apple rootstock cultivar Japan Morioka 2 (JM2) was produced at the Division of Apple Research, Institute of Fruit and Tea Science, NARO, in Japan. JM2, which was developed for dwarfing scions and improving disease resistance, is easily propagated by hardwood cutting. Furthermore, JM2 can be stably transformed at a high efficiency, which is better than other JM series rootstocks derived from the same parent. Leaflets of cultured shoots of JM2 have been transformed using Agrobacterium (Rhizobium) with a transducing gene. In this article, the JM2 transformation protocol is introduced in detail. Various genes and promoters have been confirmed to function as expected, with the resultant transformants exhibiting specific staining and fluorescent signals, and modified floral organ shapes, precious blooming and other characteristics. JM2 is thus a useful rootstock material for the enhancement of genetic research on apple and its relatives.
Transformation is a key step in modern breeding technology that involves genome editing. The requirement for in vitro tissue culture and regeneration hampers application of this technology to commercially important varieties of many crop species. To overcome this problem, we developed a simple and reproducible in planta transformation method in wheat (Tritticum aestivum L.). Our in planta particle bombardment (iPB) method utilizes the shoot apical meristem (SAM) as a target tissue. The SAM contains a subepidermal cell layer termed L2, from which germ cells later develop during floral organogenesis. The iPB method can also be used for genome editing through transient CRISPR/Cas9 expression or direct delivery of the CRISPR/Cas9 ribonucleoprotein. In this review, we describe the iPB technology and provide an overview of its current and future applications in plant transformation and genome editing.
Biolistic transformation systems are widely used to introduce foreign genes into common wheat (Triticum aestivum L.); however, these techniques often generate high transgene copy numbers and complex transgene integration patterns that hinder the stable expression of the transgenes. To improve the efficiency of stable transgene expression, we examined the effect of low-temperature pretreatment of wheat flower spikes and of high maltose concentration (HMC) in the medium during the subsequent callus culture. Tillers of the spring wheat cultivar Bobwhite were stored at 5°C without water for one week before the isolation of their immature scutellar tissues, and the resulting particle-bombarded explants were cultured on 15% maltose for a month. Together, these treatments significantly increased the number of recovered transgenic lines expressing the reporter gene. The low-temperature pretreatment eliminated the negative effects of HMC, and HMC improved the efficiency of stable transgene expression. Southern blot analysis revealed that transgenic lines recovered after HMC treatment integrated a lower copy number of transgenes than those cultured at normal (4%) maltose concentration. These findings suggest that the HMC-mediated reduction of the transgene copy number results from the suppression of plasmid DNA rearrangement before or during transgene integration into the wheat genome.
We established a method for embryogenic callus induction and highly efficient Agrobacterium-mediated genetic transformation of a table grape cultivar ‘Shine Muscat’ (Vitis labruscana). Embryogenic calli were induced using flower bud filaments from a dormant cane. Agrobacterium strain LBA4404 harboring the binary plasmid pBin19-sgfp, which contains the sgfp and nptII genes, was used to infect embryogenic calli. Infected calli were selected on 1/2 MS medium containing 5% maltose and 2% agar supplemented with 15 mg l−1 kanamycin. Efficiency of transformation of regenerated plants reached nearly 100% as determined by PCR and Southern blot analyses. The developed method will open a new avenue for genome editing of ‘Shine Muscat’ and contribute to the advancement of grape breeding.
The tea plant (Camellia sinensis) contains various metabolic substances, including catechins and caffeine, for which genetic transformation techniques are essential for investigating the associated metabolic pathways. In this study, we sought to optimize the conditions and culture process for particle bombardment-mediated transformation of tea plant somatic embryos. We describe somatic embryo pretreatment for effective transient transformation in biolistic bombardment and the posttreatment conditions of somatic embryos for accelerating differentiation after bombardment. For the purpose of transformation, we used the somatic embryos of C. sinensis var. assamica ‘Tingamira normal,’ which were cultured in Murashige and Skoog (MS) medium containing 2 mg l−1 indole-3-butyric acid (IBA) and 4 mg l−1 6-benzyladenine (BA) at 25°C ±2°C under a 16-h photoperiod. With respect to the optimization of particle bombardment conditions for tea somatic embryos, we examined the effects of different Au colloid particle diameters and bombardment pressures, and accordingly established bombardment with 1.0-µm-diameter DNA-coated Au colloid at 1,100 psi as optimal conditions for introducing DNA for the transient expression of GUS. Additionally, we found that transplantation of tea somatic embryos from IBA/BA medium to a hormone-free medium prior to bombardment and incubation in the dark post-bombardment increased the frequency of secondary embryo production. Furthermore, osmotic treatment by culturing the somatic embryos in medium supplemented with 0.4 M mannitol improved transient transformation efficiency. After transformation, the culture of somatic embryos on filter papers or Kimwipes soaked in MS medium facilitated rapid and effective development of the somatic embryos.
Genome editing using site-specific nucleases, such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat–CRISPR-associated protein 9 (CRISPR-Cas9), is a powerful technology for crop breeding. For plant genome editing, the genome-editing reagents are usually expressed in plant cells from stably integrated transgenes within the genome. This requires crossing processes to remove foreign nucleotides from the genome to generate null segregants. However, in highly heterozygous plants such as potato, the progeny lines have different agronomic traits from the parent cultivar and do not necessarily become elite lines. Agrobacteria can transfer exogenous genes on T-DNA into plant cells. This has been used both to transform plants stably and to express the genes transiently in plant cells. Here, we infected potato, with Agrobacterium tumefaciens harboring TALEN-expression vector targeting sterol side chain reductase 2 (SSR2) gene and regenerated shoots without selection. We obtained regenerated lines with disrupted-SSR2 gene and without transgene of the TALEN gene, revealing that their disruption should be caused by transient gene expression. The strategy using transient gene expression by Agrobacterium that we call Agrobacterial mutagenesis, developed here should accelerate the use of genome-editing technology to modify heterozygous plant genomes.
The CRISPR/Cas9 system is widely used for targeted mutagenesis in many organisms including plants. For application of this system, tissue culture methods need to be established. In this study, detailed methods for introduction of mutations in tomato and Nicotiana benthamiana plants using the CRISPR/Cas9 system are described. The methods include tissue culture protocols for tomato and N. benthamiana. We also demonstrate the methodology to generate Cas9-free genome edited tomato plants and use of one single guide RNA (sgRNA) to edit two orthologs in N. benthamiana. The examples of editing the PHYTOENE DESATURASE (PDS) genes in these plants are also provided. The Cas9-free tomato line was obtained when tomato plants were cultured on a non-selective medium after transformation with the CRISPR/Cas9 system. Two orthologs of PDS in N. benthamiana were mutated using a sgRNA, because these orthologs contain the same nucleotide sequences with PAM motif. These mutations were inherited to the next generation. The mutations in the PDS genes resulted in an albino phenotype in tomato and N. benthamiana plants. These results demonstrate that the non-selective method is one of the ways to obtain Cas9-free genome editing in tomato plants and that the two orthologs can be edited by one sgRNA in N. benthamiana.
Plastid transformants form biofactories that are able to produce extra proteins in plastids when they are in a homoplasmic state. To date, plastid transformation has been reported in about twenty plant species; however, the production of homoplasmic plastid transformants is not always successful or easy. Heteroplasmic plants that contain wild-type plastids produce fewer target proteins and do not always successfully transfer transgenes to progeny. In order to promote the generation of homoplasmic plants, we developed a novel system using barnase–barster to eliminate wild-type plastids from heteroplasmic cells systematically. In this system, a chemically inducible cytotoxic barnase under a plastid transit signal was introduced into nuclear DNA and barster, which inhibits barnase, was integrated into plastid DNA with the primary selection markers aminoglycoside 3′-adenylyltransferase (aadA) and green fluorescence protein (GFP) gene. As expected, the expression of the plastid barnase was lethal to cells as seen in leaf segments, but barster expression in plastids rescued them. We then investigated the regeneration frequency of homoplasmic shoots from heteroplasmic leaf segments with or without barnase expression. The regeneration frequency of homoplasmic-like shoots expressing barnase–barster system was higher than that of shoots not expressing this. We expect that the application of this novel strategy for transformation of plastids will be supportive to generate homoplasmic plastid transformants in other plant species.
We have developed a system using plastic culture bags with forced aeration system for both liquid medium and gaseous phase to produce microtubers of potato (Solanum tuberosum L.). The production of microtubers under sterile conditions is a good way to produce disease-free materials for crop production, and bioreactors have been used for this purpose. However, bioreactors are expensive and difficult to handle. The plastic culture bags are relatively inexpensive and are easy to store and sterilize because they can be flattened. Microtuber production involves two stages: plant proliferation in one medium, followed by microtuber production in a different medium. Both steps are carried out using the same culture bag. Using this system, we produced 100 to 300 microtubers per 8 l culture bag, depending on the potato cultivar. We varied the nutrient concentrations in the media and found that a lower sucrose concentration in the plant proliferation medium and lower nitrogen concentration in the microtuber production medium both increased the total numbers of microtubers per bag. Notably, a higher concentration of potassium phosphate increased the numbers of larger microtubers. This is beneficial because larger microtubers are much more tolerant to field conditions. We produced about 250,000 microtubers per year in a 66 m2 tissue culture room using the culture bag system. These microtubers have been planted directly in the field and utilized for seed potato production.
Cryptomeria japonica D. Don (common name is Sugi or Japanese cedar) is the most important forestation tree species in Japan, and 2nd generation plus trees with superior traits have been selected by breeding projects. Biotechnological approaches such as genetic transformation and genome editing are expected to accelerate to add useful traits (e.g., no-pollen traits) to superior trees in short time. To develop a platform for genetic transformation and genome editing of C. japonica superior trees, this study investigated the embryogenic potential of 2nd generation plus trees and obtained good cell lines with high embryogenic potential, which could be useful material for adding new and useful traits to superior trees by genetic transformation. However, the maintenance of embryogenic cell lines is laborious, and prolonged subculture leads to a loss of embryogenesis potential. Therefore, cell lines need to be cryopreserved for long without subculture. Therefore, in this study we made a simple cryopreservation protocol suitable for most C. japonica cell lines. We showed that cryopreserved cells using this protocol formed somatic embryos, which were then converted to plantlets. Transgenic cells produced from cryopreserved cells expressed transgene, gfp. These results indicated that our cryopreservation protocol can be used for prolonged storage of genetic transformation target materials in C. japonica.
Genome editing using CRISPR/Cas9 is useful for common wheat because common wheat has allohexaploid nature and it can induce mutations simultaneously in three homoeologous genes. Although Agrobacterium-mediated transformation has advantages in genome editing, it still has low efficiency and requires relatively long time in wheat. Therefore, the use of guide RNAs (gRNAs) with efficient mutagenesis in vivo is one of the critical factors for producing genome-edited mutant lines in a short time. In this study, we targeted three genes in common wheat and established a rapid method for detection of mutations induced by the biolistic transient expression system. Biolistic transient expression of the gRNAs and Cas9 was achieved in immature wheat embryos. Mutations were detected a week later using PCR-RFLP and verified by the sequencing of genomic clones. We confirmed several types of mutations that occurred at different rates depending on the target sequences. Furthermore, frequencies of mutations tended to be higher at the targets that were edited at higher rates in the plants transformed by Agrobacterium. These results show that this method of rapid detection of edited mutations could be used for variety of applications, such as screening of target sequences or modified vectors for efficient CRISPR/Cas9 genome editing in wheat.