Novel electroporation-based genome editing of carnation plant tissues using RNPs targeting the anthocyanidin synthase gene.

MAIN CONCLUSION: A novel electroporation method for genome editing was performed using plant tissue samples by direct RNPs-introduction in carnation. Genome editing is becoming a very useful tool in plant breeding. In this study, a novel electroporation method was performed for genome editing using plant tissue samples. The objective was to create a flower color mutant using the pink-flowered carnation 'Kane Ainou 1-go'. For this purpose, a ribonucleoprotein consisting of guide RNA and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) was introduced into the stem tissue to induce mutations in the anthocyanidin synthase (ANS) gene, which is involved in anthocyanin biosynthesis. As the ANS of 'Kane Ainou 1-go' has not been previously isolated, we initially isolated the ANS gene from 'Kane Ainou 1-go' for characterization. Southern hybridization analysis confirmed that the ANS gene was present in the genome as a two-allele gene with a pair of homologous sequences (ANS-1 and 2); these sequences were used as the target for genome editing. Genome editing was performed by introducing #2_single-guide RNA into the stem tissue using the ribonucleoprotein. This molecule was used because it exhibited the highest efficiency in an analysis of cleavage activity against the target sequence in vitro. Cleaved amplified polymorphic sequence analysis of genomic DNA extracted from 85 regenerated individuals after genome editing was performed. The results indicated that mutations in the ANS gene may have been introduced into two lines. Cloning of the ANS gene in these two lines confirmed the introduction of a single nucleotide substitution mutation for ANS-1 in both lines, and a single amino acid substitution in one line. We discussed the possibility of color change by the amino acid substitution, and also the future applications of this technology.

Enhanced Resistance to Fungal and Bacterial Diseases Due to Overexpression of BSR1, a Rice RLCK, in Sugarcane, Tomato, and Torenia.

Sugarcane smut caused by Sporisorium scitamineum is one of the most devastating sugarcane diseases. Furthermore, Rhizoctonia solani causes severe diseases in various crops including rice, tomato, potato, sugar beet, tobacco, and torenia. However, effective disease-resistant genes against these pathogens have not been identified in target crops. Therefore, the transgenic approach can be used since conventional cross-breeding is not applicable. Herein, the overexpression of BROAD-SPECTRUM RESISTANCE 1 (BSR1), a rice receptor-like cytoplasmic kinase, was conducted in sugarcane, tomato and torenia. BSR1-overexpressing tomatoes exhibited resistance to the bacteria Pseudomonas syringae pv. tomato DC3000 and the fungus R. solani, whereas BSR1-overexpressing torenia showed resistance to R. solani in the growth room. Additionally, BSR1 overexpression conferred resistance to sugarcane smut in the greenhouse. These three BSR1-overexpressing crops exhibited normal growth and morphologies except in the case of exceedingly high levels of overexpression. These results indicate that BSR1 overexpression is a simple and effective tool for conferring broad-spectrum disease resistance to many crops.

Ectopic expression of AtNF-YA6-VP16 in petals results in a novel petal phenotype in Torenia fournieri.

MAIN CONCLUSION: A novel Torenia phenotype having separate petals was obtained by the combination of NF-YA6-VP16 with a floral organ-specific promoter. Genetic engineering techniques helped in obtaining novel flower colors and shapes, in particular, by introducing functionally modified transcription factors (TFs) to ornamental flower species. Herein, we used functionally modified Arabidopsis TFs fused with the repression domain SRDX and the activation domain VP16 to screen for novel floral traits in Torenia fournieri Lind (torenia). We avoided undesired phenotypes unrelated to flowers by expressing these TFs through a floral organ-specific promoter belonging to the class-B genes, GLOBOSA (TfGLO). Fourteen constructs were produced to express functionally modified Arabidopsis TFs in which each of SRDX and VP16 was fused into 7 TFs that were used for the collective transformation of Torenia plants. Among the obtained transgenic plants, phenotypes with novel floral traits reflected in separate petals within normally gamopetalous flower lines. Sequencing analysis revealed that the transgenic plants contained nuclear factor-YA6 (NF-YA6) fused with the VP16. In the margin between the lips of the petals and tube in the TfGLOp:NF-YA6-VP16 plants, staminoid organs have been developed to separate petals. In the petals of the TfGLOp:NF-YA6-VP16 plants, the expression of a Torenia class C gene, PLENA (TfPLE), was found to be ectopically increased. Moreover, expression of TfPLE-VP16 under the control of the TfGLO promoter brought a similar staminoid phenotype observed in the TfGLOp:NF-YA6-VP16 plants. These results suggest that the introduction of the TfGLOp:NF-YA6-VP16 induced TfPLE expression, resulting in the formation of staminoid petals and separation of them.

Overexpression of Rice BSR2 Confers Disease Resistance and Induces Enlarged Flowers in Torenia fournieri Lind.

Plant pathogens evade basal defense systems and attack different organs and tissues of plants. Genetic engineering of plants with genes that confer resistance against pathogens is very effective in pathogen control. Conventional breeding for disease resistance in ornamental crops is difficult and lagging relative to that in non-ornamental crops due to an inadequate number of disease-resistant genes. Therefore, genetic engineering of these plants with defense-conferring genes is a practical approach. We used rice BSR2 encoding CYP78A15 for developing transgenic Torenia fournieri Lind. lines. The overexpression of BSR2 conferred resistance against two devastating fungal pathogens, Rhizoctonia solani and Botrytis cinerea. In addition, BSR2 overexpression resulted in enlarged flowers with enlarged floral organs. Histological observation of the petal cells suggested that the enlargement in the floral organs could be due to the elongation and expansion of the cells. Therefore, the overexpression of BSR2 confers broad-spectrum disease resistance and induces the production of enlarged flowers simultaneously. Therefore, this could be an effective strategy for developing ornamental crops that are disease-resistant and economically more valuable.

Production of petaloid phenotype in the reproductive organs of compound flowerheads by the co-suppression of class-C genes in hexaploid Chrysanthemum morifolium.

MAIN CONCLUSION: Functional suppression of two types of class-C genes caused transformation of pistils and stamens into petaloid organs that exhibit novel phenotypes, which gives a distinct gorgeous impression in the florets of chrysanthemum. The multiple-petal trait is a breeding objective for many horticultural plants. The loss of function of class-C genes causes the multiple-petal trait in several plant species. However, mechanisms involved in the generation of the multiple-petal trait are unknown in Chrysanthemum morifolium (chrysanthemum). Here, we isolated 14 class-C AGAMOUS (AG) genes, which were classified into two types of class-C genes, in chrysanthemum. Seven of these were categorized into CAG type 1 genes (CAG1s) and seven into CAG type 2 genes (CAG2s). Functions of class-C genes were co-suppressed by chimeric repressors and simultaneously knocked-down by RNAi to produce the multiple-petal phenotype in chrysanthemum. The expression of chimeric repressors of CAG1s and CAG2s caused morphological alteration of the pistils and stamens into petaloid organs in the ray and disk florets. Interestingly, the reproductive organs of the disk florets were transformed into petaloid organs similar to the petals of the disk florets, and those of the ray florets were transformed into petaloid organs such as the petals of the ray florets. Simultaneous knockdown of CAG1s and CAG2s expression by RNAi also exhibited a petaloid phenotype as observed in transgenic plants obtained by chimeric repressors. These results showed that CAG1s and CAG2s play important roles in the development of pistils and stamens, and the simultaneous repression of CAG1s and CAG2s resulted in a multiple-petal phenotype in chrysanthemum.

Production of multi-petaled Torenia fournieri flowers by functional disruption of two class-C MADS-box genes.

MAIN CONCLUSION: Simultaneous knockdown or knockout of Torenia fournieri PLENA (TfPLE) and FALINELLI (TfFAR) genes with RNAi or genome-editing technologies generated a multi-petal phenotype in torenia. The MADS-box gene AGAMOUS (AG) is well known to play important roles in the development of stamens and carpels in Arabidopsis. Mutations in AG cause the morphological transformation of stamens and carpels into petaloid organs. In contrast, torenia (Torenia fournieri Lind.) has two types of class-C MADS-box genes, PLENA (PLE) and FALINELLI (FAR); however, their functions were previously undetermined. To examine the function of TfPLE and TfFAR in torenia, we used RNAi to knockdown expression of these two genes. TfPLE and TfFAR double-knockdown transgenic torenia plants had morphologically altered stamens and carpels that developed into petaloid organs. TfPLE knockdown transgenic plants also exhibited morphological transformations that included shortened styles, enlarged ovaries, and absent stigmata. Furthermore, simultaneous disruption of TfPLE and TfFAR genes by CRISPR/Cas9-mediated genome editing also resulted in the conversion of stamens and carpels into petaloid organs as was observed in the double-knockdown transgenic plants mediated by RNAi. In addition, the carpels of one TfPLE knockout mutant had the same morphological abnormalities as TfPLE knockdown transgenic plants. TfFAR knockdown genome-edited mutants had no morphological changes in their floral organs. These results clearly show that TfPLE and TfFAR cooperatively play important roles in the development of stamens and carpels. Simultaneous disruption of TfPLE and TfFAR functions caused a multi-petal phenotype, which is expected to be a highly valuable commercial floral trait in horticultural flowers.

Distribution of cell layers in floral organs of chrysanthemum analyzed with periclinal chimeras carrying a transgene encoding fluorescent protein.

KEY MESSAGE: A fluorescent protein visualized distributions of cell layers in floral organs of chrysanthemum using transgenic periclinal chimeras carrying a gene encoding a fluorescent compound. Plant meristems have three cell layers: the outermost layer (L1), the second layer (L2), and the inner layer (L3). The layers are maintained during development but there is limited knowledge of the details of cell layer patterns within floral organs. In this study, we visualized the distributions of cell layers in floral organs of chrysanthemum using periclinal chimeras carrying a gene encoding a fluorescent compound in the L1 or the L2/L3 layers. The L1 layer contributed most of the epidermal cells of organs including the receptacle, petal, anther, filament, style, stigma, and ovule. The transmitting tissue in the pistil and most of the internal area of the ovule were also derived from the L1. In crossing experiments, no progeny of the L1-chimeric plants showed fluorescence, indicating that the germ cells of chrysanthemum are not derived from the L1 layer. Since anthocyanin pigment is present only in the L1-derived epidermal cells of petals, L1-specific gene integration could be used to alter flower color in commercial cultivars, with a reduced risk of transgene flow from the transgenic chrysanthemums to wild relatives.

Parsley ubiquitin promoter displays higher activity than the CaMV 35S promoter and the chrysanthemum actin 2 promoter for productive, constitutive, and durable expression of a transgene in Chrysanthemum morifolium.

The chrysanthemum (Chrysanthemum morifolium) is one of the most popular ornamental plants in the world. Genetic transformation is a promising tool for improving traits, editing genomes, and studying plant physiology. Promoters are vital components for efficient transformation, determining the level, location, and timing of transgene expression. The cauliflower mosaic virus (CaMV) 35S promoter is most frequently used in dicotyledonous plants but is less efficient in chrysanthemums than in tobacco or torenia plants. Previously, we used the parsley ubiquitin (PcUbi) promoter in chrysanthemums for the first time and analyzed its activity in transgenic calli. To expand the variety of constitutive promoters in chrysanthemums, we cloned the upstream region of the actin 2 (CmACT2) gene and compared its promoter activity with the 35S and PcUbi promoters in several organs, as well as its durability for long-term cultivation. The CmACT2 promoter has higher activity than the 35S promoter in calli but is less durable. The PcUbi promoter has the highest activity not only in calli but also in leaves, ray florets, and disk florets, and retains its activity after long-term cultivation. In conclusion, we have provided useful information and an additional type of promoter available for transgene expression in chrysanthemums.

Utilization of transcription factors for controlling floral morphogenesis in horticultural plants.

Transcription factors play important roles not only in the development of floral organs but also in the formation of floral characteristics in various plant species. Therefore, transcription factors are reasonable targets for modifying these floral traits and generating new flower cultivars. However, it has been difficult to control the functions of transcription factors because most plant genes, including those encoding transcription factors, exhibit redundancy. In particular, it has been difficult to understand the functions of these redundant genes by genetic analysis. Thus, a breakthrough silencing method called chimeric repressor gene silencing technology (CRES-T) was developed specifically for plant transcription factors. This method transforms transcriptional activators into dominant repressors, and the artificial chimeric repressors suppress the function of transcription factors regardless of their redundancy. Among these chimeric repressors, some were found to be inappropriate for expression throughout the plant body because they resulted in deformities. For these chimeric repressors, utilization of floral organ-specific promoters overcomes this problem by avoiding expression throughout the plant body. In contrast, attachment of viral activation domain VP16 to transcriptional repressors effectively alters into transcriptional activators. This review presents the importance of transcription factors for characterizing floral traits, describes techniques for controlling the functions of transcription factors.

Correction to: Generation of expressed sequence tags for discovery of genes responsible for floral traits of Chrysanthemum morifolium by next-generation sequencing technology.

After publication of the original article [1] the authors noted that the following errors had occurred.

Generation of expressed sequence tags for discovery of genes responsible for floral traits of Chrysanthemum morifolium by next-generation sequencing technology.

BACKGROUND: Chrysanthemum morifolium is one of the most economically valuable ornamental plants worldwide. Chrysanthemum is an allohexaploid plant with a large genome that is commercially propagated by vegetative reproduction. New cultivars with different floral traits, such as color, morphology, and scent, have been generated mainly by classical cross-breeding and mutation breeding. However, only limited genetic resources and their genome information are available for the generation of new floral traits. RESULTS: To obtain useful information about molecular bases for floral traits of chrysanthemums, we read expressed sequence tags (ESTs) of chrysanthemums by high-throughput sequencing using the 454 pyrosequencing technology. We constructed normalized cDNA libraries, consisting of full-length, 3'-UTR, and 5'-UTR cDNAs derived from various tissues of chrysanthemums. These libraries produced a total number of 3,772,677 high-quality reads, which were assembled into 213,204 contigs. By comparing the data obtained with those of full genome-sequenced species, we confirmed that our chrysanthemum contig set contained the majority of all expressed genes, which was sufficient for further molecular analysis in chrysanthemums. CONCLUSION: We confirmed that our chrysanthemum EST set (contigs) contained a number of contigs that encoded transcription factors and enzymes involved in pigment and aroma compound metabolism that was comparable to that of other species. This information can serve as an informative resource for identifying genes involved in various biological processes in chrysanthemums. Moreover, the findings of our study will contribute to a better understanding of the floral characteristics of chrysanthemums including the myriad cultivars at the molecular level.

Generation of Gene-Edited Chrysanthemum morifolium Using Multicopy Transgenes as Targets and Markers.

The most widely used gene editing technology-the CRISPR/Cas9 system-employs a bacterial monomeric DNA endonuclease known as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) and single-guide RNA (sgRNA) that directs Cas9 to a complementary target DNA. However, introducing mutations into higher polyploid plant species, especially for species without genome information, has been difficult. Chrysanthemum morifolium (chrysanthemum) is one of the most important ornamental plants, but it is a hexaploid with a large genome; moreover, it lacks whole-genome information. These characteristics hinder genome editing in chrysanthemum. In the present study, we attempted to perform gene editing using the CRISPR/Cas9 system to introduce mutations into chrysanthemum. We constructed transgenic chrysanthemum plants expressing the yellowish-green fluorescent protein gene from Chiridius poppei (CpYGFP) and targeted CpYGFP for gene editing. We compared the activity of a Cauliflower mosaic virus (CaMV) 35S promoter and parsley ubiquitin promoter in chrysanthemum calli and chose the parsley ubiquitin promoter to drive Cas9. We selected two sgRNAs to target different positions in the CpYGFP gene and obtained transgenic calli containing mutated CpYGFP genes (CRISPR-CpYGFP-chrysanthemum). A DNA sequencing analysis and fluorescence observations indicated that cells containing the mutated CpYGFP gene grew independently of cells containing the original CpYGFP gene in one callus. We finally obtained the CRISPR-CpYGFP-chrysanthemum shoot containing a mutation in the CpYGFP sequence. This is the first report of gene editing using the CRISPR/Cas9 system in chrysanthemum and sheds light on chrysanthemum genome editing.

Transcriptome analysis in petals and leaves of chrysanthemums with different chlorophyll levels.

BACKGROUND: Chlorophylls (Chls) are magnesium-containing tetrapyrrole macromolecules responsible for the green color in plants. The Chl metabolic pathway has been intensively studied and nearly all the enzymes involved in the pathway have been identified and characterized. Synthesis and activity of these enzymes are tightly regulated in tissue- and developmental stage-specific manners. Leaves contain substantial amounts of Chls because Chls are indispensable for photosynthesis. In contrast, petals generally contain only trace amounts of Chls, which if present would mask the bright petal color. Limited information is available about the mechanisms that control such tissue-specific accumulation of Chls. RESULTS: To identify the regulatory steps that control Chl accumulation, we compared gene expression in petals and leaves of chrysanthemum cultivars with different Chl levels. Microarray and quantitative real-time PCR analyses showed that the expression levels of Chl biosynthesis genes encoding glutamyl-tRNA reductase, Mg-protoporphyrin IX chelatase, Mg-protoporphyrin IX monomethylester cyclase, and protochlorophyllide oxidoreductase were well associated with Chl content: their expression levels were lower in white petals than in green petals, and were highest in leaves. Among Chl catabolic genes, expression of STAY-GREEN, encoding Mg-dechelatase, which is a key enzyme controlling Chl degradation, was considerably higher in white and green petals than in leaves. We searched for transcription factor genes whose expression was well related to Chl level in petals and leaves and found three such genes encoding MYB113, CONSTANS-like 16, and DREB and EAR motif protein. CONCLUSIONS: From our transcriptome analysis, we assume that a low rate of Chl biosynthesis and a high rate of Chl degradation lead to the absence of Chls in white chrysanthemum petals. We identified several candidate transcription factors that might affect Chl accumulation in chrysanthemum petals. Functional analysis of these transcription factors will provide a basis for future molecular studies of tissue-specific Chl accumulation.

Generation of Novel Floral Traits Using a Combination of Floral Organ-Specific Promoters and a Chimeric Repressor in Torenia fournieri Lind.

In this study, we attempted to develop a new biotechnological method for the efficient modification of floral traits. Because transcription factors play an important role in determining floral traits, chimeric repressors, which are generated by attaching a short transcriptional repressor domain to transcription factors, have been widely used as effective tools for modifying floral traits in many plant species. However, the overexpression of these chimeric repressors by the Cauliflower mosaic virus 35S promoter sometimes causes undesirable morphological alterations to other organs. We attempted simultaneously to generate new floral traits and avoid such quality loss by examining five additional floral organ-specific promoters, one Arabidopsis thaliana promoter and four Torenia fournieri promoters, for the expression of the chimeric repressor of Arabidopsis TCP3 (AtTCP3), whose overexpression drastically alters floral traits but also generates dwarf phenotypes and deformed leaves. We found that the four torenia promoters exhibited particularly strong activity in the petals but not in the leaves, and that the combination of these floral organ-specific promoters with the chimeric repressor of AtTCP3 caused changes in the color, color patterns and cell shapes of petals, whilst avoiding other unfavorable phenotypes. Interestingly, each promoter that we used in this study generated characteristic and distinguishable floral traits. Thus, the use of different floral organ-specific promoters with different properties enables us to generate diverse floral traits using a single chimeric repressor without changing the phenotypes of other organs.

Co-modification of class B genes TfDEF and TfGLO in Torenia fournieri Lind. alters both flower morphology and inflorescence architecture.

The class B genes DEFICIENS (DEF)/APETALA3 (AP3) and GLOBOSA (GLO)/PISTILLATA (PI), encoding MADS-box transcription factors, and their functions in petal and stamen development have been intensely studied in Arabidopsis and Antirrhinum. However, the functions of class B genes in other plants, including ornamental species exhibiting floral morphology different from these model plants, have not received nearly as much attention. Here, we examine the cooperative functions of TfDEF and TfGLO on floral organ development in the ornamental plant torenia (Torenia fournieri Lind.). Torenia plants co-overexpressing TfDEF and TfGLO showed a morphological alteration of sepals to petaloid organs. Phenotypically, these petaloid sepals were nearly identical to petals but had no stamens or yellow patches like those of wild-type petals. Furthermore, the inflorescence architecture in the co-overexpressing torenias showed a characteristic change in which, unlike the wild-types, their flowers developed without peduncles. Evaluation of the petaloid sepals showed that these attained a petal-like nature in terms of floral organ phenotype, cell shape, pigment composition, and the expression patterns of anthocyanin biosynthesis-related genes. In contrast, torenias in which TfDEF and TfGLO were co-suppressed exhibited sepaloid petals in the second whorl. The sepaloid petals also attained a sepal-like nature, in the same way as the petaloid sepals. The results clearly demonstrate that TfDEF and TfGLO play important cooperative roles in petal development in torenia. Furthermore, the unique transgenic phenotypes produced create a valuable new way through which characteristics of petal development and inflorescence architecture can be investigated in torenia.

Faster, safer, and better DNA purification by ultracentrifugation using GelRed stain and development of mismatch oligo DNA for genome walking.

Purification of plant DNA involves lengthy ultracentrifugation using ethidium bromide. Here, ultracentrifugation method is improved by staining with GelRed. The resulting method is faster, safer and of higher sensitivity. Purified DNA quality was confirmed by treatment with restriction enzymes and isolation of gene promoters. New type of long adaptor with mismatch sequence was also developed for promoter isolation.

Mutation in Torenia fournieri Lind. UFO homolog confers loss of TfLFY interaction and results in a petal to sepal transformation.

We identified a Torenia fournieri Lind. mutant (no. 252) that exhibited a sepaloid phenotype in which the second whorls were changed to sepal-like organs. This mutant had no stamens, and the floral organs consisted of sepals and carpels. Although the expression of a torenia class B MADS-box gene, GLOBOSA (TfGLO), was abolished in the 252 mutant, no mutation of TfGLO was found. Among torenia homologs such as APETALA1 (AP1), LEAFY (LFY), and UNUSUAL FLORAL ORGANS (UFO), which regulate expression of class B genes in Arabidopsis, only accumulation of the TfUFO transcript was diminished in the 252 mutant. Furthermore, a missense mutation was found in the coding region of the mutant TfUFO. Intact TfUFO complemented the mutant phenotype whereas mutated TfUFO did not; in addition, the transgenic phenotype of TfUFO-knockdown torenias coincided with the mutant phenotype. Yeast two-hybrid analysis revealed that the mutated TfUFO lost its ability to interact with TfLFY protein. In situ hybridization analysis indicated that the transcripts of TfUFO and TfLFY were partially accumulated in the same region. These results clearly demonstrate that the defect in TfUFO caused the sepaloid phenotype in the 252 mutant due to the loss of interaction with TfLFY.

Overcoming Difficulties in Molecular Biological Analysis through a Combination of Genetic Engineering, Genome Editing, and Genome Analysis in Hexaploid Chrysanthemum morifolium.

Chrysanthemum is one of the most commercially important ornamental plants globally, of which many new varieties are produced annually. Among these new varieties, many are the result of crossbreeding, while some are the result of mutation breeding. Recent advances in gene and genome sequencing technology have raised expectations about the use of biotechnology and genome breeding to efficiently breed new varieties. However, some features of chrysanthemum complicate molecular biological analysis. For example, chrysanthemum is a hexaploid hyperploid plant with a large genome, while its genome is heterogeneous because of the difficulty of obtaining pure lines due to self-incompatibility. Despite these difficulties, an increased number of reports on transcriptome analysis in chrysanthemum have been published as a result of recent technological advances in gene sequencing, which should deepen our understanding of the properties of these plants. In this review, we discuss recent studies using gene engineering, genome editing, and genome analysis, including transcriptome analysis, to analyze chrysanthemum, as well as the current status of and future prospects for chrysanthemum.

Detection of Transcription Factors Related to Axillary Bud Development after Exposure to Cold Conditions in Hexaploid Chrysanthemum morifolium Using Arabidopsis Information.

Chrysanthemum is one of the most commercially used ornamental flowering plants in the world. As chrysanthemum is self-incompatible, the propagation of identical varieties is carried out through cuttings rather than through seed. Axillary bud development can be controlled by changing the temperature; for instance, axillary bud development in some varieties is suppressed at high temperatures. In this study, we focused on the simultaneous axillary bud growth from multiple lines of chrysanthemum upon changing conditions from low to normal temperature. Transcriptome analysis was conducted on the Chrysanthemum morifolium cultivar 'Jinba' to identify the important genes for axillary bud development seen when moved from low-temperature treatment to normal cultivation temperature. We performed RNA-Seq analysis on plants after cold conditions in two-day time-course experiments. Under these settings, we constructed a transcriptome of 415,923 C. morifolium and extracted 7357 differentially expressed genes. Our understanding of Arabidopsis axillary meristem development and growth showed that at least 101 genes in our dataset were homologous to transcription factors involved in the biological process. In addition, six genes exhibited statistically significant variations in expression throughout conditions. We hypothesized that these genes were involved in the formation of axillary buds in C. morifolium after cold conditions.

Excision of DNA fragments with the piggyBac system in Chrysanthemum morifolium.

Chrysanthemum morifolium is one of the most popular ornamental plants in the world. However, as C. morifolium is a segmental hexaploid, self-incompatible, and has a sizable heterologous genome, it is difficult to modify its trait systematically. Genome editing technology is one of the attractive methods for modifying traits systematically. For the commercial use of genetically modified C. morifolium, rigorous stabilization of its quality is essential. This trait stability can be achieved by avoiding further genome modification after suitable trait modification by genome editing. Since C. morifolium is a vegetatively propagated plant, an approach for removing genome editing tools is required. In this study, we attempted to use the piggyBac transposon system to remove specific DNA sequences from the C. morifolium genome. Using the luminescence as a visible marker, we demonstrated that inoculation of Agrobacterium harboring hyperactive piggyBac transposase removes inserted 2.6 kb DNA, which harbors piggyBac recognition sequences, from the modified Eluc sequence.