Chrysanthemum Genome Research Articles

CGD: a multi-omics database for Chrysanthemum genomic and biological research.

Asteraceae is the largest family of dicotyledons and includes Chrysanthemum and Helianthus, two important genera of ornamental plants. The genus Chrysanthemum consists of more than 30 species and contains many economically important ornamental, medicinal, and industrial plants. To more effectively promote Chrysanthemum research, we constructed the CGD, a Chrysanthemum genome database containing a large amount of data and useful tools. The CGD hosts well-assembled reference genome data for six Chrysanthemum species. These genomic data were fully annotated by comparison with various protein and domain data. Transcriptome data for nine different tissues, five flower developmental stages, and five treatments were subsequently added to the CGD. A fully functional 'RNA data' module was designed to provide complete and visual expression profile data. In addition, the CGD also provides many of the latest bioinformatics analysis tools, such as the efficient sgRNA search tool for Chrysanthemum. In conclusion, the CGD provides the latest, richest, and most complete multi-omics resources and powerful tools for Chrysanthemum. Collectively, the CGD will become the central gateway for Chrysanthemum genomics and genetic breeding research and will aid in the study of polyploid evolution.

Developing a UV-visible reporter-assisted CRISPR/Cas9 gene editing system to alter flowering time in Chrysanthemum indicum.

Developing a UV-visible reporter-assisted CRISPR/Cas9 gene editing system to alter flowering time in Chrysanthemum indicum.

Identification of CmbHLH Transcription Factor Family and Excavation of CmbHLHs Resistant to Necrotrophic Fungus Alternaria in Chrysanthemum.

Chrysanthemum morifolium Ramat. 'Huaihuang' is a traditional Chinese medicinal plant. However, a black spot disease caused by Alternaria sp., a typical necrotrophic fungus, has a serious damaging influence on the field growth, yield, and quality of the plant. 'Huaiju 2#' being bred from 'Huaihuang', shows resistance to Alternaria sp. bHLH transcription factor has been widely studied because of their functions in growth development, signal transduction, and abiotic stress. However, the function of bHLH in biotic stress has rarely been studied. To characterize the resistance genes, the CmbHLH family was surveyed in 'Huaiju 2#'. On the basis of the transcriptome database of 'Huaiju 2#' after Alternaria sp. inoculation, with the aid of the Chrysanthemum genome database, 71 CmbHLH genes were identified and divided into 17 subfamilies. Most (64.8%) of the CmbHLH proteins were rich in negatively charged amino acids. CmbHLH proteins are generally hydrophilic proteins with a high aliphatic amino acid content. Among the 71 CmbHLH proteins, five CmbHLHs were significantly upregulated by Alternaria sp. infection, and the expression of CmbHLH18 was the most significant. Furthermore, heterologous overexpression of CmbHLH18 could improve the resistance of Arabidopsis thaliana to necrotrophic fungus Alternaria brassicicola by enhancing callose deposition, preventing spores from entering leaves, reducing ROS accumulation, increasing the activities of antioxidant enzymes and defense enzymes, and promoting their gene expression levels. These results indicate that the five CmbHLHs, especially CmbHLH18, may be considered candidate genes for resistance to necrotrophic fungus. These findings not only increase our understanding of the role CmbHLHs play in biotic stress but also provide a basis by using CmbHLHs to breed a new variety of Chrysanthemum with high resistance to necrotrophic fungus.

The nature and genomic landscape of repetitive DNA classes in Chrysanthemum nankingense shows recent genomic changes.

Tandemly repeated DNA and transposable elements represent most of the DNA in higher plant genomes. High-throughput sequencing allows a survey of the DNA in a genome, but whole-genome assembly can miss a substantial fraction of highly repeated sequence motifs. Chrysanthemum nankingense (2n = 2x = 18; genome size = 3.07 Gb; Asteraceae), a diploid reference for the many auto- and allopolyploids in the genus, was considered as an ancestral species and serves as an ornamental plant and high-value food. We aimed to characterize the major repetitive DNA motifs, understand their structure and identify key features that are shaped by genome and sequence evolution. Graph-based clustering with RepeatExplorer was used to identify and classify repetitive motifs in 2.14 millions of 250-bp paired-end Illumina reads from total genomic DNA of C. nankingense. Independently, the frequency of all canonical motifs k-bases long was counted in the raw read data and abundant k-mers (16, 21, 32, 64 and 128) were extracted and assembled to generate longer contigs for repetitive motif identification. For comparison, long terminal repeat retrotransposons were checked in the published C. nankingense reference genome. Fluorescent in situ hybridization was performed to show the chromosomal distribution of the main types of repetitive motifs. Apart from rDNA (0.86 % of the total genome), a few microsatellites (0.16 %), and telomeric sequences, no highly abundant tandem repeats were identified. There were many transposable elements: 40 % of the genome had sequences with recognizable domains related to transposable elements. Long terminal repeat retrotransposons showed widespread distribution over chromosomes, although different sequence families had characteristic features such as abundance at or exclusion from centromeric or subtelomeric regions. Another group of very abundant repetitive motifs, including those most identified as low-complexity sequences (9.07 %) in the genome, showed no similarity to known sequence motifs or tandemly repeated elements. The Chrysanthemum genome has an unusual structure with a very low proportion of tandemly repeated sequences (~1.02 %) in the genome, and a high proportion of low-complexity sequences, most likely degenerated remains of transposable elements. Identifying the presence, nature and genomic organization of major genome fractions enables inference of the evolutionary history of sequences, including degeneration and loss, critical to understanding biodiversity and diversification processes in the genomes of diploid and polyploid Chrysanthemum, Asteraceae and plants more widely.

De novo whole-genome assembly of Chrysanthemum makinoi, a key wild chrysanthemum.

Chrysanthemum is among the top 10 cut, potted, and perennial garden flowers in the world. Despite this, to date, only the genomes of two wild diploid chrysanthemums have been sequenced and assembled. Here, we present the most complete and contiguous chrysanthemum de novo assembly published so far, as well as a corresponding ab initio annotation. The cultivated hexaploid varieties are thought to originate from a hybrid of wild chrysanthemums, among which the diploid Chrysanthemum makinoi has been mentioned. Using a combination of Oxford Nanopore long reads, Pacific Biosciences long reads, Illumina short reads, Dovetail sequences, and a genetic map, we assembled 3.1 Gb of its sequence into nine pseudochromosomes, with an N50 of 330 Mb and a BUSCO complete score of 92.1%. Our ab initio annotation pipeline predicted 95,074 genes and marked 80.0% of the genome as repetitive. This genome assembly of C. makinoi provides an important step forward in understanding the chrysanthemum genome, evolution, and history.

The Isolation and Identification of Anthocyanin-Related GSTs in Chrysanthemum

Anthocyanin is the crucial pigment for the coloration of red chrysanthemum flowers, which synthesizes in the cytosol and is transported to the vacuole for stable storage. In general, glutathione S-transferases (GSTs) play a vital role in this transport. To date, there is no functional GST reported in chrysanthemums. Here, a total of 94 CmGSTs were isolated from the chrysanthemum genome, with phylogenetic analysis suggesting that 16 members of them were clustered into the Phi subgroup which was related to anthocyanin transport. Among them, the expression of CmGST1 was positively correlated with anthocyanin accumulation. Protein sequence alignment revealed that CmGST1 included anthocyanin-related GST-specific amino acid residues. Further transient overexpression experiments in tobacco leaves showed that CmGST1 could promote anthocyanin accumulation. In addition, a dual-luciferase assay demonstrated that CmGST1 could be regulated by CmMYB6, CmbHLH2 and CmMYB#7, which was reported to be related to anthocyanin biosynthesis. Taken together, we suggested that CmGST1 played a key role in anthocyanin transport and accumulation in chrysanthemums.

Agrobacterium-Mediated Transformation of Chrysanthemum with Artemisinin Biosynthesis Pathway Genes.

Artemisinin-based drugs are the most effective medicine for the malaria treatment. To date, the main method of artemisinin production is its extraction from wormwood plants Artemisia annua L. Due to the limitation of this source, considerable efforts are now directed to the development of methods for artemisinin production using heterologous expression systems. Artemisinin is a sesquiterpene lactone, synthesized through the cyclization of farnesyl diphosphate involved in other sesquiterpene biosynthetic systems. Chrysanthemum species as well as A. annua, belong to Asteraceae family, and had been characterized by containing highly content of sesquiterpenes and their precursors. This makes chrysanthemum a promising target for the production of artemisinin in heterologous host plants. Chrysanthemum (C. morifolium Ramat.) was transformed by Agrobacterium tumefaciens carrying with the binary vectors p1240 and p1250, bearing artemisinin biosynthesis genes coding: amorpha-4,11-diene synthase, artemisinic aldehyde Δ11(13) reductase, amorpha-4,11-diene monooxygenase (p1240 was targeted to the mitochondria and p1250 was targeted to the cytosol), cytochrome P450 reductase from A. annua, as well as yeast truncated 3-hydroxy-3-methylglutarylcoenzyme A reductase. This study obtained 8 kanamycin-resistant lines after transformation with the p1240 and 2 lines from p1250. All target genes were detected in 2 and 1 transgenic lines of the 2 vectors. The transformation frequency of all target genes were 0.33% and 0.17% for p1240 and p1250, relative to the total transformed explant numbers. RT-PCR analysis revealed the transcription of all transferred genes in two lines obtained after transformation with the p1240 vector, confirming the possibility of transferring genetic modules encoding entire biochemical pathways into the chrysanthemum genome. This holds promise for the development of a chrysanthemum-based expression system to produce non-protein substances, such as artemisinin.

Tools for Chrysanthemum genetic research and breeding: Is genotyping-by-sequencing (GBS) the best approach?

Chrysanthemum, which belongs to the Asteraceae family, is a floriculture crop with ornamental, industrial, and medicinal value. Chrysanthemum breeding programs have generated genetic linkage maps and identified molecular markers for important traits. Chrysanthemum species have a complex genetic background, and several studies on Chrysanthemum species have been published in the last 3 decades. The Chrysanthemum genome was published by Amway Botanical RD however, the molecular breeding of Asteraceae is not well studied yet. Next-generation sequencing (NGS) technologies, genotyping-by-sequencing (GBS), and association mapping have facilitated the identification of novel genetic markers. Chrysanthemum species have a very large genome and include diploid and polyploid species (such as allohexaploids) with complex hereditary features such as self-incompatibility systems and inbreeding depression. The development of NGS technologies allowed the advance of high-throughput DNA sequencing for detecting and single nucleotide polymorphisms (SNP) in various plant species. Through NGS, a large number of SNPs detected via whole-genome sequencing can be used as markers in breeding and genetic studies. While SNP markers allow rapid analyses, contribute to the low cost of genome-wide scans, and are sequenced by numerous efficient platforms in diverse genetic backgrounds, GBS can be advantageous for the study of gene function, genetic variation, and heredity, specifically in modern plant breeding. In addition, GBS can reduce genome complexity, allowing SNP discovery and genotyping of sequences without a reference genome. This review focuses on the tools used for genomic research and breeding in Chrysanthemum, and discusses the processes in SNP development and study of the evolutionary processes in Chrysanthemum species.

CmFTL2 is involved in the photoperiod- and sucrose-mediated control of flowering time in chrysanthemum

The chrysanthemum genome harbors three FT-like genes: CmFTL1 and CmFTL3 are thought to act as regulators of floral induction under long-day (LD) and short-day (SD) conditions, respectively, whereas the function of CmFTL2 is currently unclear. The objective of the present research was to explore the function of CmFTL2 in the determination of flowering time of the photo-insensitive chrysanthemum cultivar ‘Floral Yuuka’, both in response to variation in the photoperiod and to the exogenous provision of sucrose. Spraying leaves of ‘Floral Yuuka’ plants with 50 mM sucrose accelerated flowering and increased the level of CmFTL2 transcription in the leaf more strongly than either CmFTL1 or FTL3 under both long and SD conditions. Transcription profiling indicated that all three CmFTL genes were upregulated during floral induction. The relationship of the CmFTL2 sequence with that of other members of the PEBP family suggested that its product contributes to the florigen rather than to the anti-florigen complex. The heterologous expression of CmFTL2 in the Arabidopsis thaliana ft-10 mutant rescued the mutant phenotype, showing that CmFTL2 could compensate for the absence of FT. These results suggest that CmFTL2 acts as a regulator of floral transition and responds to both the photoperiod and sucrose.

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.

Cloning of chrysanthemum high-affinity nitrate transporter family (CmNRT2) and characterization of CmNRT2.1.

The family of NITRATE TRANSPORTER 2 (NRT2) proteins belongs to the high affinity transport system (HATS) proteins which acts at low nitrate concentrations. The relevant gene content of the chrysanthemum genome was explored here by isolating the full length sequences of six distinct CmNRT2 genes. One of these (CmNRT2.1) was investigated at the functional level. Its transcription level was inducible by low concentrations of both nitrate and ammonium. A yeast two hybrid assay showed that CmNRT2.1 interacts with CmNAR2, while a BiFC assay demonstrated that the interaction occurs at the plasma membrane. Arabidopsis thaliana plants heterologously expressing CmNRT2.1 displayed an enhanced rate of labeled nitrogen uptake, suggesting that CmNRT2.1 represents a high affinity root nitrate transporter.

Assessing the Genetic Diversity of Chrysanthemum Cultivars with Microsatellites

The morphological characteristics of chrysanthemum (Chrysanthemum ×morifolium) are rich in variation. However, as a result of the aneuploid polyploidy of the chrysanthemum genome and the lack of proper tools, the genomic information of this crop is limited. Development of microsatellite markers has been an increasing trend in crop genetic studies because of the applicability of these markers in breeding programs. In this study, we reported the development of a simple sequence repeat in chrysanthemums using a magnetic beads enrichment method. An enriched genomic library with AC and GT microsatellite motifs was constructed, and 53 positive clones were detected by a colony polymerase chain reaction (PCR) technique. Of these clones, 35 showed high-quality sequences, and 35 primer pairs were designed accordingly. Twenty-six (74.29%) of the 35 primer pairs revealed polymorphisms on a set of 40 chrysanthemum cultivars. There were 172 alleles amplified over 26 loci with an average of 6.615 alleles per locus. The mean values of gene diversity corrected for the sample size and the inbreeding coefficient were 0.609 and 0.119 over 26 loci, respectively, which indicated that the majority of the microsatellite loci is highly informative. Cluster analysis based on 26 polymorphic loci demonstrated that the selected cultivars were clustered according to geographical origin. This study shows the isolation efficiency of the magnetic beads technique; the abundance of microsatellites in chrysanthemum; and the potential application for the cultivar classification, the studies on genetic diversity, and molecular breeding of chrysanthemums, which is beneficial to promoting the conservation and sustainable use of this crop.

감마선 처리에 의한 스프레이형 국화 화색변이체로부터 Flavonoid 3'-Hydroxylase(F3'H) 유전자의 분리 및 특성 구명

스프레이 국화품종 'Argus'와 감마선 조사에 의해 화색변이가 일어난 돌연변이체의 꽃잎으로부터 안토시아닌 생합성 경로에서 중요한 역할을 담당하는 신규 <TEX>$DgF3'H$</TEX>의 전장 cDNA와 genomic DNA를 분리하였다. 전장 cDNA는 1,527bp(509 아미노산)의 ORF를 포함하고 있으며, 원품종 'Argus'와 화색변이체 사이의 염기서열 상동성은 97% 이상을 나타내었다. Genomic DNA의 크기는 야생형 'Argus'에서 3,831bp이었고, 3가지 화색 변이체에서는 3,828부터 3,838bp의 크기를 나타내었다. <TEX>$DgF3'H$</TEX> 유전자는 세 개의 exon사이에 두개의 intron을 갖고 있는 구조이고, 3'과 5' UTR 부분을 제외한 intron의 크기는 야생형 'Argus'에서 2,157bp이지만 3가지 화색 변이체에서는 2,155부터 2,159bp의 크기를 갖고 있었다. 이것은 감마선 조사에 의해 intron 부분의 유전자가 결실 또는 삽입된 것으로 추정된다. Southern 분석 결과 국화의 genome 내에서는 복수의 F3'H 유전자를 갖는 것이 확인되었다. <TEX>$DgF3'H$</TEX> 유전자의 발현 정도를 분석한 결과, 연분홍의 'Argus'와 두 개의 보라색 변이체(AM1 and AM3)에서 높게 발현되었으나 흰색 변이체(AM2)에서는 매우 약하게 발현되었으며, 염기서열 변이에 의한 F3'H 유전자의 구조적 차이가 화색의 변이에 관련된 것으로 추정되었다. 국화 'Argus' 및 화색 변이체를 이용하여 본 연구에서 분리한 신규 F3'H 유전자의 구조 및 유전자 발현 등을 포함하는 유전정보들은 화색 변이의 유전적 기작을 밝히는데 중요한 자료로 이용될 것으로 기대되나 향후 다른 유전적 발현요소들이 국화의 F3'H 유전자의 발현에 관여하는지에 관한 추가적인 연구가 필요하다고 하겠다. The objectives of this study were to isolate and the sequence of novel <TEX>$F3'H$</TEX> gene related to an anthocyanin pathway, and to confirm the expression patterns of the gene involved in the flower color variations of chrysanthemum mutants. In this study, we isolated the full-length cDNAs and the genomic DNAs of an <TEX>$F3'H$</TEX> gene from a wild type (WT) chrysanthemum (cv. Argus) and its three color mutants. The sequence analysis revealed a putative open reading frame of 1,527 bp that encodes a polypeptide of 509 amino acids. Sequence homology ranged from 97% to 99% between 'Argus' and its three color mutants. The sequence analysis from the genomic DNA revealed that the chrysanthemum <TEX>$DgF3'H$</TEX> gene consisted of three exons and two introns spanning a 3,830 bp length. The sizes of the gene for three mutants ranged from a shorter size of 3,828 bp to a longer size of 3,838 bp when compared to the size of WT. The total size of the two introns was 2,157 bp for WT, but those for three color mutants ranged from 2,154 bp to 2,159 bp. A result of an RT-PCR analysis indicated that the color variations of the mutants AM1 and AM2 can be partly explained by the structural modification derived from the sequencial changes in the gene caused by gamma ray. A Southern blot analysis revealed that the <TEX>$DgF3'H$</TEX> gene existing as multiple copies in the chrysanthemum genome. A systemic study will be further needed to provide a genetic mechanism responsible for the color mutation and to uncover any involvement of genetic elements for the expression of the <TEX>$DgF3'H$</TEX> gene for the color variation in chrysanthemum.

Isolation of a gibberellin 20‐oxidase cDNA from and characterization of its expression in chrysanthemum

With 5 figures and 1 table AbstractA PCR strategy was used to isolate a full‐length GA20‐oxidase cDNA from chrysanthemum. The gene putatively encodes a 377 residue polypeptide, which shares 87% homology with lettuce GA20ox. A Southern blot analysis indicated that the hexaploid chrysanthemum genome probably contains several GA20ox copies. The DgGA20ox gene is expressed in the leaf, stem, flower and root, but most strongly in the young leaf. The application of the GA biosynthesis inhibitor paclobutrazol to the fourth internode increased the expression of DgGA20ox, while exogenously applied GA3 decreased it. This supports the commonly observed phenomenon that the expression level of GA20ox genes is regulated by the local level of physiologically active GA. The relevance of the DgGA20ox in relation to chrysanthemum breeding was discussed as well.

Transgenic chrysanthemum plants expressing a harpinXoo gene demonstrate induced resistance to alternaria leaf spot and accelerated development

In order to improve resistance to pathogens in chrysanthemum, we generated transgenic chrysanthemum plants heterogeneously expressing hpaG Xoo gene from Xanthomonas oryzae pv. oryzae via leaf-disc mediated Agrobacterium tumefaciens (EHA105) transformation. Polymerase chain reaction (PCR), Southern blot, and Reverse transcription PCR (RT-PCR) analysis showed that the hpaG Xoo gene was successfully integrated into the chrysanthemum genome and expressed in transgenic lines. An inoculation assay indicated that the presence of the transgene was associated with an improved level of resistance to alternaria leaf spot. The transgenic introduction of pathogen elicitor-encoding genes has a considerable potential for improving the disease resistance of ornamental plants. Furthermore, transgenic chrysanthemums flowered significantly earlier than the wild-type plants did, suggesting that the hpaG Xoo gene probably accelerated chrysanthemum development.

Cloning, characterization and expression of a Lateral suppressor- like gene from chrysanthemum ( Dendranthema grandiflorum Kitamura)

Conventionally, the lateral shoots of chrysanthemum are removed manually, which is time consuming and uneconomical. The development of branchless chrysanthemum will economize its commercial cultivation. To investigate the regulatory mechanism of branchlessness, we undertook cloning of Dendranthema grandiflorum Kitamura Lateral suppressor-like ( DgLsL) gene for development of lateral shoot in chrysanthemum. A full-length cDNA of DgLsL gene was isolated by screening cDNA library and performing Rapid Amplification of cDNA Ends (RACE) PCR. Phylogenetic analysis showed that the DgLsL gene is closely related to Lateral suppressor that encodes transcriptional regulator proteins belonging to GRAS family of known transcription factor. Southern blot analysis revealed that DgLsL gene in chrysanthemum genome has one copy. DgLsL expression was apparently up-regulated by ethephon treatment. The expression patterns revealed that DgLsL transcripts were detected in all organs, but showed their highest level in stems.

Agrobacterium rhizogenes is a useful transporter for introducing T-DNA of the binary plasmid into the chrysanthemum, Dendranthema grandiflorum Kitamura, genome

Agrobacterium rhizogenes was used for efficient transformation of chrysanthemum. Two types of Agrobacterium, A. rhizogenes (A-13) and A. tumefaciens (LBA4404), which harbor pIG121-Hm, were employed for infection. In the A. rhizogenes-infected explants, hairy roots were not observed on any tested medium or culture condition. When explants were cultured on shoot induction medium, calli were formed at the cutting edge within 4–6 weeks of culture, and shoot primordia were observed on the callus surface after 2 weeks of callus formation. Consequently, with gus introduction, a significantly higher transformation rate was observed for A. rhizogenes (6.0%) compared with A. tumefaciens (3.3%). However, only 0.6% of the frequency of rol insertion was exhibited in A. rhizogenes mediation. These results indicate that A. rhizogenes effectively introduces T-DNA of the binary plasmid into the chrysanthemum genome by introducing Ri T-DNA at a low frequency. It also indicates that the system is a useful alternative for the transformation of chrysanthemum.

Biolistic transformation of chrysanthemum with the nucleocapsid gene of tomato spotted wilt virus

In vitro regeneration and biolistic transformation procedures were developed for several commercial chrysanthemum Dendranthema grandiflora Tzvelev, syn. Chrysanthemum morifolium Ramat. cultivars using leaf and stem explants. Studies on the effect of several growth regulators and kanamycin on chrysanthemum regeneration were conducted, and a step-wise procedure to optimize kanamycin selection and recovery of transgenic plants was developed. A population of putative transformed chrysanthemum plants cvs. Blush, Dark Bronze Charm, Iridon, and Tara, was obtained after bombardment with tungsten microprojectiles coated with the binary plasmid pBIN19 containing the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) and the marker gene neomycin phosphotransferase (NPT II). PCR analysis of 82 putative transgenic plants selected on kanamycin indicated that the majority of the lines (89%) were transformed and contained both genes (71%). However, some transgenic lines contained only one of the genes: either the NPT II (15%) or the TSWV (N) gene (14%). Southern blot analysis on selected transgenic lines confirmed the integration of the TSWV (N) gene into the chrysanthemum genome. These results demonstrate the development of an efficient procedure to transfer genetic material into the chrysanthemum genome and selectively regenerate transgenic chrysanthemum plants at frequencies higher than previously reported.