Gentian Plants Research Articles

Content of Gentiopicroside in Natural Populations of Gentiana cruciata L., Growing in the Territory of the Republic of Tatarstan

This report contains the results of quantitative analysis of primary iridoid gentiopicroside, phenolic compounds, and flavonoids in the shoots, rhizomes, and roots of cross gentian (Gentiana cruciata L.) plants collected in natural phytocenoses of Alekseevskii, Zelenodol’skii, and Apastovskii raions in the Republic of Tatarstan. The content of phenolic compounds and flavonoids in herbs was determined spectrophotometrically. It was shown that the greatest content of phenolic compounds and flavonoids in above-ground parts was 19.4 and 10.80 mg/g in Zelenodol’skii and Alekseevskii raions and that in the rhizomes and roots was 8.53 and 1.96 mg/g in Apastovskii raion. By the method of high-performance liquid chromatography, it was shown that the greatest quantity of gentiopicroside (more than 9%) was accumulated in the underground part of plants, whereas the above-ground part contained approximately 6% of this compound. Thus, cross gentian growing in plant communities located in these raions differed in the content of biologically active substances, which probably depended on the joint effect of ecological and cenotic characteristics of habitats. The results of phytochemical analysis suggest that Apastovskii raion in the Republic of Tatarstan may be a promising locality for picking and preparing medicinal herbs with a high content of main iridoid gentiopicroside.

Efficient double-flowered gentian plant production using the CRISPR/Cas9 system.

Japanese cultivated gentians are highly valued ornamental flowers in Japan, but the flower shape is mostly limited to the single-flower type, unlike other flowers such as roses and carnations. To overcome this limitation, we used the CRISPR/Cas9 genome editing system to increase double-flowered genetic resources in gentians. Our approach targeted an AGAMOUS (AG) floral homeotic gene (AG1), which is responsible for the natural mutation that causes double flowers in gentians. We designed two targets in exon 1 of AG1 for genome editing and found that 9 of 12 herbicide-resistant shoots had biallelic mutations in the target regions of AG1. These nine lines all produced double flowers, with stamens converted into petaloid organs, similar to the natural mutant. We also analyzed the off-target effects of AG2, which is homologous to AG1, and found that such effects occurred in gentian genome editing but with low frequency. Furthermore, we successfully produced transgene-free genome-edited plants (null segregants) by crossing with wild-type pollen. F1 seedlings were subjected to PCR analysis to determine whether foreign DNA sequences, two partial regions of the CaMV35S promoter and Cas9 gene, were present in the genome. As a result, foreign genes were segregated at a 1 : 1 ratio, indicating successful null segregant production. Using PCR analysis, we confirmed that four representative null segregants did not contain transfer DNA. In summary, our study demonstrates that the CRISPR/Cas9 system can efficiently produce double-flowered gentians, and null segregants can also be obtained. These genome-edited plants are valuable genetic resources for future gentian breeding programs.

Biological control of gentian spot blight by the combination of endophytic fungi fermentation broth and traditional Chinese medicine extractions

Gentian spot blight is a disease that decreases yields. Soil treatment can improve both plant growth and resistance to this disease. We investigated the effectiveness of endophytic fungi fermentation broth combined with traditional Chinese medicine (TCM) extractions in the biological control of gentian spot blight. We tested the anti-pathogen and growth-promoting properties of the endophytic fungi 3714, 272 fermentation broth, and 12 different TCM extractions in various solvents alone or in combination. The experiment was conducted in the field by treating gentian with various mixtures and examining their effect in controlling gentian spot blight. The combination of TCM water extractions and endophytic fungi fermentation broth, Anemarrhena asphodeloides Bge (ZM), ZM-272, Cnidium monnieri (L.). Cuss. (SCZ)-3714 and ZM-SCZ-3714 significantly controlled gentian spot blight at a control rate of more than 70%. Analyses of physiological and biochemical indexes, defense enzymes, soil enzymes, and soil properties suggest a mechanism for disease resistance and growth promotion. Specifically, different combination generally increases the number of microorganisms and soil enzyme activities in the rhizosphere of gentian, resulting in improvements in the accumulation of nutrients in soil, as well as the living environment of plants. The accumulation of nutrients in soil was improved by activating defense enzymes. The levels of secondary metabolites such as gentiopicrin increased, thus improving the disease resistance of gentian. Disease resistance was accomplished by directly suppressing the pathogen causing gentian spot blight on the surface of gentian plants and in the soil. These results provide a theoretical foundation for the biological control of gentian spot blight.

Production of yellow-flowered gentian plants by genetic engineering of betaxanthin pigments.

Genetic engineering of flower color provides biotechnological products such as blue carnations or roses by accumulating delphinidin-based anthocyanins not naturally existing in these plant species. Betalains are another class of pigments that in plants are only synthesized in the order Caryophyllales. Although they have been engineered in several plant species, especially red-violet betacyanins, the yellow betaxanthins have yet to be engineered in ornamental plants. We attempted to produce yellow-flowered gentians by genetic engineering of betaxanthin pigments. First, white-flowered gentian lines were produced by knocking out the dihydroflavonol 4-reductase (DFR) gene using CRISPR/Cas9-mediated genome editing. Beta vulgaris BvCYP76AD6 and Mirabilis jalapa MjDOD, driven by gentian petal-specific promoters, flavonoid 3',5'-hydroxylase (F3'5'H) and anthocyanin 5,3'-aromatic acyltransferase (AT), respectively, were transformed into the above DFR-knockout white-flowered line; the resultant gentian plants had vivid yellow flowers. Expression analysis and pigment analysis revealed petal-specific expression and accumulation of seven known betaxanthins in their petals to c. 0.06-0.08 μmol g FW-1 . Genetic engineering of vivid yellow-flowered plants can be achieved by combining genome editing and a suitable expression of betaxanthin-biosynthetic genes in ornamental plants.

De Novo Transcriptome Analysis Reveals Flowering-Related Genes That Potentially Contribute to Flowering-Time Control in the Japanese Cultivated Gentian Gentiana triflora.

Japanese cultivated gentians are perennial plants that flower in early summer to late autumn in Japan, depending on the cultivar. Several flowering-related genes, including GtFT1 and GtTFL1, are known to be involved in regulating flowering time, but many such genes remain unidentified. In this study, we obtained transcriptome profiling data using the Gentiana triflora cultivar ‘Maciry’, which typically flowers in late July. We conducted deep RNA sequencing analysis using gentian plants grown under natural field conditions for three months before flowering. To investigate diurnal changes, the plants were sampled at 4 h intervals over 24 h. Using these transcriptome data, we determined the expression profiles of leaves based on homology searches against the Flowering-Interactive Database of Arabidopsis. In particular, we focused on transcription factor genes, belonging to the BBX and MADS-box families, and analyzed their developmental and diurnal variation. The expression levels of representative BBX genes were also analyzed under long- and short-day conditions using in-vitro-grown seedlings, and the expression patterns of some BBX genes differed. Clustering analysis revealed that the transcription factor genes were coexpressed with GtFT1. Overall, these expression profiles will facilitate further analysis of the molecular mechanisms underlying the control of flowering time in gentians.

Molecular characterization of an anthocyanin-related glutathione S-transferase gene in Japanese gentian with the CRISPR/Cas9 system

BackgroundThe blue pigmentation of Japanese gentian flowers is due to a polyacylated anthocyanin, gentiodelphin, and all associated biosynthesis genes and several regulatory genes have been cloned and characterized. However, the final step involving the accumulation of anthocyanins in petal vacuoles remains unclear. We cloned and analyzed the glutathione S-transferases (GSTs) in Japanese gentian that are known to be involved in anthocyanin transport in other plant species.ResultsWe cloned GST1, which is expressed in gentian flower petals. Additionally, this gene belongs to the Phi-type GST clade related to anthocyanin biosynthesis. We used the CRISPR/Cas9-mediated genome editing system to generate loss-of-function GST1 alleles. The edited alleles were confirmed by Sanger and next-generation sequencing analyses. The GST1 genome-edited lines exhibited two types of mutant flower phenotypes, severe (almost white) and mild (pale blue). The phenotypes were associated with decreased anthocyanin accumulation in flower petals. In the GST1 genome-edited lines, sugar-induced stress conditions inhibited the accumulation of anthocyanins in stems and leaves, suggestvhing that GST1 is necessary for stress-related anthocyanin accumulation in organs other than flowers. These observations clearly demonstrate that GST1 is the gene responsible for anthocyanin transport in Japanese gentian, and is necessary for the accumulation of gentiodelphin in flowers.ConclusionsIn this study, an anthocyanin-related GST gene in Japanese gentian was functionally characterized. Unlike other biosynthesis genes, the functions of GST genes are difficult to examine in in vitro studies. Thus, the genome-editing strategy described herein may be useful for in vivo investigations of the roles of transport-related genes in gentian plants.

Effects of knocking out three anthocyanin modification genes on the blue pigmentation of gentian flowers

Genome editing by the CRISPR/Cas9 system has recently been used to produce gene knockout lines in many plant species. We applied this system to analyze Japanese gentian plants that produce blue flowers because of the accumulation of a polyacylated anthocyanin, gentiodelphin. Mutant lines in which anthocyanin modification genes were knocked out were examined to assess the contribution of each gene to the blue pigmentation of flowers. The targeted genes encoded anthocyanin 5-O-glycosyltransferase (Gt5GT), anthocyanin 3′-O-glycosyltransferase (Gt3′GT), and anthocyanin 5/3′-aromatic acyltransferase (Gt5/3′AT). The Gt5GT knockout lines accumulated delphinidin 3G, whereas the Gt3′GT knockout lines accumulated delphinidin 3G-5CafG as the major flower pigment. Knocking out Gt5/3′AT resulted in the accumulation of delphinidin 3G-5G-3′G and delphinidin 3G-5G as the primary and secondary pigments, respectively. These results indicated the existence of two pathways mediating the modification of delphinidin 3G-5G in flowers, with one involving a glycosylation by 3′GT and the other involving an acylation by 5/3′AT. The Gt5GT, Gt3′GT, and Gt5/3′AT transformants produced pale red violet, dull pink, and pale mauve flowers, respectively, unlike the vivid blue flowers of wild-type plants. Thus, the glycosylation and subsequent acylation of the 3′-hydroxy group of the B-ring in delphinidin aglycone is essential for the development of blue gentian flowers.

Stem and root rot of gentian caused by Fusarium avenaceum

Stem and root rot was observed in gentian plants (Gentiana triflora) in Aomori Prefecture, Japan, in 2016. A fungal isolate from diseased root was identified as Fusariumavenaceum based on morphological characteristics and DNA sequence data. Inoculation of gentian plants with this isolate produced a similar stem and root rot. F.avenaceum is thus proposed as a new pathogen causing stem and root rot of gentian.

The Host Stomatal Density Determines Resistance to Septoria gentianae in Japanese Gentian.

Plant stomata represent the main battlefield for host plants and the pathogens that enter plant tissues via stomata. Septoria spp., a group of ascomycete fungi, use host plant stomata for invasion and cause serious damage to agricultural plants. There is no evidence, however, showing the involvement of stomata in defense systems against Septoria infection. In this study, we isolated Septoria gentianae 20-35 (Sg20-35) from Gentiana triflora showing gentian leaf blight disease symptoms in the field. Establishment of an infection system using gentian plants cultured in vitro enabled us to observe the Sg20-35 infection process and estimate its virulence in several gentian cultivars or lines. Sg20-35 also entered gentian tissues via stomata and showed increased virulence in G. triflora compared with G. scabra and their interspecific hybrid. Notably, the susceptibility of gentian cultivars to Sg20-35 was associated with their stomatal density on the adaxial but not abaxial leaf surface. Treatment of EPIDERMAL PATTERNING FACTOR-LIKE 9 (EPFL9/STOMAGEN) peptides, a small secreted peptide controlling stomatal density in Arabidopsis thaliana, increased stomatal density on the adaxial side of gentian leaves as well. Consequently, treated plants showed enhanced susceptibility to Sg20-35. These results indicate that stomatal density on the adaxial leaf surface is one of the major factors determining the susceptibility of gentian cultivars to S. gentianae and suggest that stomatal density control may represent an effective strategy to confer Septoria resistance.

Gentian (Gentiana triflora) prevents transmission of apple latent spherical virus (ALSV) vector to progeny seeds.

Gentian plants ( Gentiana triflora ) severely restrict apple latent spherical virus (ALSV) invasion to the gametes (pollens and ovules) and block seed transmission to progeny plants. Early flowering of horticultural plants can be induced by infection of ALSV vector expressing Flowering Locus T (FT) gene. In the present study, flowering of gentian plants was induced by infection with an ALSV vector expressing a gentian FT gene and the patterns of seed transmission of ALSV in gentian were compared with those in apple and Nicotiana benthamiana. Infection of gentian progeny plants with ALSV was examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), reverse transcription-loop-mediated isothermal amplification (RT-LAMP), and enzyme-linked immunosorbent assay (ELISA). ALSV was not transmitted to the progeny gentian plants, whereas small proportions of apple and N. benthamiana progeny plants are infected with ALSV. The in situ hybridization analyses indicated that ALSVs are not present in gentian pollen and ovules, but detected in most of gametes in apple and N. benthamiana. Collectively, these results suggest that seed transmission of ALSV is blocked in gentian plants through the unknown barriers present in their gametes. On the other hand, apple and N. benthamiana seem to minimize ALSV seed transmission by inhibiting viral propagation in embryos.

Biological effects of ion beam irradiation on perennial gentian and apple.

The development of new varieties of perennial plants generally requires lengthy and laborious procedures. In this study, we used ion beam irradiation mutagenesis in an attempt to accelerate the breeding process for perennial plants. We evaluated the biological effects of five ion beam sources (carbon, neon, argon, silicon, and iron) and neutron irradiation on Japanese gentian and apple. These treatments were applied at the National Institute of Radiological Sciences (NIRS) using the Heavy Ion Medical Accelerator in Chiba (HIMAC) and the Neutron-exposure Accelerator System for Biological Effect Experiments (NASBEE). Biological effects were observed in in vitro gentian plants after irradiation with ion beams at <10 Gy, whereas apple trees were less sensitive to ion beam irradiation. The growth of gentians in vitro was repressed by 3 Gy neutron irradiation, while that of grafted apple trees was not affected by 4 Gy neutron irradiation. During in vitro proliferation, seven pink-flowered lines were obtained from originally blue-flowered gentian after C and Ne ion beam irradiation treatments. Genomic and reverse transcription-PCR analyses of these lines suggested that the mutations occurred in the genomic region containing F3'5'H (encoding flavonoid 3',5'-hydroxylase). These results provide useful information for the mutagenesis and breeding of gentian, apple, and other perennial plants.

Symptom development, in planta distribution, and transmission of Impatiens necrotic spot virus in gentian: evidence for survival in roots and winter buds

Impatiens necrotic spot virus (INSV) causes serious damage to gentian (Gentiana spp.). Symptom development, in planta distribution, and transmission of INSV were studied after mechanical inoculation of gentian plants and propagation of shoot cuttings from infected stock plants. When young gentian plants at the 2nd-leaf stage were inoculated with INSV, plants developed systemic symptoms that were restricted to a few upper leaves. However, older plants at the 6th-leaf stage did not develop systemic symptoms. After plant inoculation, INSV was detected using DAS-ELISA in symptomatic upper leaves, and rootlets and winter buds, but not in asymptomatic leaves. When asymptomatic shoot cuttings from infected stock plants were vegetatively propagated in a thrips-free glasshouse, 44.4% of those obtained from the apical shoot and 20.6% of those obtained from the middle section of the plant developed systemic symptoms. These results indicated that when gentian plants were infected with INSV, the virus was preferentially transported from the infected leaves to the root and winter buds. However, even asymptomatic shoot cuttings may develop systemic symptoms when obtained from infected stock plants. Therefore, vegetative propagation from infected stock plants can be a source of INSV infection.

Investigating the Role of Endophytic Fungi in Gentiana scabra bge. by Cross-Growth Period Inoculation.

Gentiana scabra Bge. (gentian) is a Chinese medicinal plant. Endophytic fungi from the roots of gentian were isolated and cross-growth period inoculation was performed to study the roles of three Trichoderma spp. strains (F1, F2, and F9) in their original host plant. In treatments inoculated with F1, F2, and F9, gentiopicroside content increased 33.6, 23.7 and 13% than that in the control. Strains F1, F2, and F9 could also improve polysaccharide content by more than 6.6, 18.7 and 30% compared to the control. The incidence of spot blight in gentian inoculated with F1, F2, and F9 decreased by 31.2, 26.7 and 8.5%. Inconsistent changes in the activity of the three enzymes (superoxide dismutase, catalase and peroxidase) were observed when the plants were attacked by pathogens or inoculated with fungi. High enzymatic activity did not reflect mild disease. Cross-growth period inoculation, which takes into account the original living environment (gentian plant as "substrate" and different microorganisms as symbionts) of endophytic fungi, provides a new idea for studying effects of endophytes on their original hosts. This is the first research about the role of endophytic fungi in Gentiana scabra bge. in vivo.

リンドウこぶ症発生圃場におけるリンドウこぶ症関連ウイルス（GKaV）感染株の分布

リンドウこぶ症発生圃場におけるリンドウこぶ症関連ウイルス（GKaV）感染株の分布

A 64-bp sequence containing the GAAGA motif is essential for CaMV-35S promoter methylation in gentian

This study investigated sequence specificity and perenniality of DNA methylation in the cauliflower mosaic virus (CaMV) 35S promoter of transgenic gentian (Gentiana triflora×G. scabra) plants. Unlike conventional transgene silencing models, 35S promoter hypermethylation in gentian is species-specific and occurs irrespective of the T-DNA copy number and genomic location. Modified 35S promoters were introduced into gentian, and single-copy transgenic lines were selected for methylation analysis. Modified 35S promoter lacking a core (−90) region [35S(Δcore)] in gentian conferred hypermethylation and high levels of de novo methylation of the CpHpH/CpCpG sites in the 35S enhancer regions (−298 to −241 and −148 to −85). Therefore, promoter transcription may not be an absolute requirement for the methylation machinery. In vitro, de novo methylation persisted for more than eight years. In another modified 35S promoter, two “GAAGA” motifs (−268 to −264 and −135 to −131) were replaced by “GTTCA” in the two highly de novo methylated regions. It did not support hypermethylation and showed transgene expression. A 64-bp fragment of the 35S enhancer region (−148 to −85) was introduced into gentian and the resultant transgenic lines analyzed. The 64-bp region exhibited hypermethylation at the CpG/CpWpG sites, but the CpHpH/CpCpG methylation frequency was lower than those of the unmodified 35S- and 35S(Δcore) promoters. Nevertheless, a distinct CpHpH/CpCpG methylation peak was found in the 64-bp region of all single-copy transgenic lines. These results suggest that the 64-bp region may contain an element required for 35S methylation but insufficient for high de novo methylation compared with those in the unmodified 35S and 35S(Δcore) promoters.

Development of molecular markers for breeding of double flowers in Japanese gentian

Double flowers are valuable floral traits in most floricultural plants. We recently revealed that a double-flowered mutant of Gentiana scabra was caused by an insertion of a retrotransposable element (Tgs1) into GsAG1, one of the C-class MADS-box genes in gentian. In this study, we developed a PCR-based molecular DNA marker to distinguish double- and single-flower phenotypes at the young seedling stage in Japanese gentian plants. To test the validity of the markers, 17 F2 populations were produced by selfing F1 plants crossed between the double-flower mutant and seven breeding lines. Multiplex PCR demonstrated that the Tgs1 insertion in GsAG1 cosegregated with the double-flower phenotype in two F2 populations, indicating that the PCR-based DNA marker was useful to discriminate between double- and single-flower phenotypes in advance of flowering in Japanese gentian. Given that Japanese gentians lack variation in flower shape and require a long breeding period, the DNA marker developed here will be helpful for efficient breeding of double-flowered cultivars in the future.

Horizontal pollen transmission of Gentian ovary ring-spot virus is initiated during penetration of the stigma and style by infected pollen tubes

Gentian ovary ring-spot virus (GORV) infected gentian plants by pollination with GORV-infected gentian pollen grains, but the virus was not horizontally transmitted to gentian plants by transfer of pollen from GORV-infected Nicotiana benthamiana plants. However, N. benthamiana plants were infected with the virus by pollination with infected gentian pollen as well as by pollination with infected N. benthamiana pollen. When infected gentian pollen grains were placed on N. benthamiana stigmas, germinating pollen tubes penetrated into the stigmas and the styles (stigma-style). Virus infection occurred during penetration of the stigma-style, and the virus subsequently spread systemically to the mother plant. On the other hand, most infected N. benthamiana pollen grains failed to germinate on gentian stigmas, and virus infections were not detected in the stigma-style.

Development of a Broad bean wilt virus 2-based expression vector for gentian

Japanese gentian is one of the most important ornamental flowers in Japan. Its most attractive trait is the vivid blue flower, whose color results from polyacylated anthocyanins. Other important traits in gentian breeding include flower shape, flowering time, and overwintering characteristics. To show the molecular mechanism by which these traits are regulated, transgenic gentian plants are often generated by Agrobacterium-mediated transformation. However, this method is not suitable for all gentian cultivars because of the difficulties in regeneration and/or transformation. Also, it takes a long time to obtain transgenic plants and analyze their phenotypes. Therefore, it is important to develop a more efficient method to analyze gene function in gentian. In the present study, we developed a novel viral expression vector for efficient gene functional analysis in gentian using Broad bean wilt virus 2 (BBWV-2) infectious cDNA. First, we expressed two reporter genes, GFP and GUS, in gentian using the BBWV-2 vector. Fluorescence from GFP was observed in various organs including the leaf, flower, root, and overwintering bud. β-Glucuronidase (GUS) activity was detected in the inoculated leaf, but not in upper uninoculated leaves because the GUS insert was unstable in the BBWV-2 vector. Next, we inoculated the BBWV-2 vector carrying GtMYB3, a transcription factor regulating anthocyanin biosynthesis, fused with SRDX, which encodes the EAR transcriptional repression domain. Expression of GtMYB3-SRDX via the BBWV-2 vector partly changed the gentian petal color from blue to white. The expression levels of genes encoding anthocyanin biosynthetic enzymes (flavanone 3-hydroxylase, dihydroflavonol-4-reductase, anthocyanidin synthase, flavonoid 3′,5′-hydroxylase, and anthocyanin 5,3′-aromatic acyltransferase) were markedly lower in the white parts of petals than in the blue parts. These results clearly indicated that GtMYB3-SRDX expressed via the BBWV-2 vector was functional in gentian flowers. This BBWV-2-based gene expression system enabled us to analyze a phenotypic change in gentian flower color within a few months.

Functional characterization of duplicated B-class MADS-box genes in Japanese gentian.

The heterodimer formation between B-class MADS-box proteins of GsAP3a and GsPI2 proteins plays a core role for petal formation in Japanese gentian plants. We previously isolated six B-class MADS-box genes (GsAP3a, GsAP3b, GsTM6, GsPI1, GsPI2, and GsPI3) from Japanese gentian (Gentiana scabra). To study the roles of these MADS-box genes in determining floral organ identities, we investigated protein-protein interactions among them and produced transgenic Arabidopsis and gentian plants overexpressing GsPI2 alone or in combination with GsAP3a or GsTM6. Yeast two-hybrid and bimolecular fluorescence complementation analyses revealed that among the GsPI proteins, GsPI2 interacted with both GsAP3a and GsTM6, and that these heterodimers were localized to the nuclei. The heterologous expression of GsPI2 partially converted sepals into petaloid organs in transgenic Arabidopsis, and this petaloid conversion phenomenon was accelerated by combined expression with GsAP3a but not with GsTM6. In contrast, there were no differences in morphology between vector-control plants and transgenic Arabidopsis plants expressing GsAP3a or GsTM6 alone. Transgenic gentian ectopically expressing GsPI2 produced an elongated tubular structure that consisted of an elongated petaloid organ in the first whorl and stunted inner floral organs. These results imply that the heterodimer formation between GsPI2 and GsAP3a plays a core role in determining petal and stamen identities in Japanese gentian, but other B-function genes might be important for the complete development of petal organs.

CaMV-35S promoter sequence-specific DNA methylation in lettuce.

We found 35S promoter sequence-specific DNA methylation in lettuce. Additionally, transgenic lettuce plants having a modified 35S promoter lost methylation, suggesting the modified sequence is subjected to the methylation machinery. We previously reported that cauliflower mosaic virus 35S promoter-specific DNA methylation in transgenic gentian (Gentiana triflora × G. scabra) plants occurs irrespective of the copy number and the genomic location of T-DNA, and causes strong gene silencing. To confirm whether 35S-specific methylation can occur in other plant species, transgenic lettuce (Lactuca sativa L.) plants with a single copy of the 35S promoter-driven sGFP gene were produced and analyzed. Among 10 lines of transgenic plants, 3, 4, and 3 lines showed strong, weak, and no expression of sGFP mRNA, respectively. Bisulfite genomic sequencing of the 35S promoter region showed hypermethylation at CpG and CpWpG (where W is A or T) sites in 9 of 10 lines. Gentian-type de novo methylation pattern, consisting of methylated cytosines at CpHpH (where H is A, C, or T) sites, was also observed in the transgenic lettuce lines, suggesting that lettuce and gentian share similar methylation machinery. Four of five transgenic lettuce lines having a single copy of a modified 35S promoter, which was modified in the proposed core target of de novo methylation in gentian, exhibited 35S hypomethylation, indicating that the modified sequence may be the target of the 35S-specific methylation machinery.