Masato Tsuro

Development of genomic and EST-SSR markers in radish (Raphanus sativus L.).

Radish (Raphanus sativus L.) belongs to Brassicaceae family and is a close relative of Brassica. This species shows a wide morphological diversity, and is an important vegetable especially in Asia. However, molecular research of radish is behind compared to that of Brassica. For example, reports on SSR (simple sequence repeat) markers are limited. Here, we designed 417 radish SSR markers from SSR-enriched genomic libraries and the cDNA data. Of the 256 SSR markers succeeded in PCR, 130 showed clear polymorphisms between two radish lines; a rat-tail radish and a Japanese cultivar, ‘Harufuku’. As a test case for evaluation of the present SSRs, we conducted two studies. First, we selected 16 SSRs to calculate polymorphism information contents (PICs) using 16 radish cultivars and four other Brassicaceae species. These markers detected 3–15 alleles (average = 9.6). PIC values ranged from 0.54 to 0.92 (average = 0.78). Second, part of the present SSRs were tested for mapping using our previously-examined mapping population. The map spanned 672.7 cM with nine linkage groups (LGs). The 21 radish SSR markers were distributed throughout the LGs. The SSR markers developed here would be informative and useful for genetic analysis in radish and its related species.

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

AbstractAgrobacterium 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.

The siRNAs targeting the left or right terminal region of chrysanthemum stunt viroid (CSVd) sequence suppress the development of disease symptoms caused by CSVd infection of chrysanthemum, but do not suppress viroid propagation

ABSTRACT Transgenic chrysanthemum plants harbouring the genes producing small interfering RNAs (siRNAs) whose sequence was designed based on the chrysanthemum stunt viroid (CSVd) genome were established. The siRNAs are expected to target and decompose CSVd RNA through a silencing mechanism. The CSVd-infected plants were grafted as scions onto the stocks of either non-transgenic or transgenic plants, and axial shoots that elongated from the stocks were rooted and grown to investigate if the plants are resistant to CSVd under greenhouse conditions. The non-transgenic plants infected with CSVd showed severe disease symptoms, i.e. stunted growth and spotty chlorotic lesions on leaves, but no such symptoms were observed in the transgenic plants. The real-time quantitative reverse transcription PCR for monitoring the amounts of CSVd indicated that CSVd equally accumulated in both non-transgenic and transgenic plants. The present results suggest that the resistant feature of the transgenic plants to CSVd was due to interference with the pathway of symptom development, and not the inhibition of CSVd replication.

In vitro induction of autotetraploid of Roman chamomile (Chamaemelum nobile L.) by colchicine treatment and essential oil productivity of its capitulum

Chamomile is well-known medicinal plant, comprise of ca. 100 species or varieties, cultivated in Europe, Asia, Australia, and North and South America (Hikawa 1998; Franke and Schilcher 2005). Two of these species, the annual herbaceous German chamomile (Matricaria recutita L., syn. Chamomilla recutita L.) and the perennial Roman chamomile (Chamaemelum nobile L., syn. Anthemis nobilis L.) are the most economically important in the production of essential oils that are used for pharmaceuticals and cosmetics (Franke and Schilcher 2005). The composition of essential oils is quite different between these two species. The major components of essential oil of German chamomile are sesquiterpenoids such as chamazulene, bisabolol, and its oxide, while those of Roman chamomile are hemiterpenoids such as angelic acid and its esters, and monoterpenoids like α-pinene (Omidbaigi et al. 2004; Franke and Schilcher 2005). It is of interest to breed to increase essential oil productivity in both species because of their low oil yield by steam distillation ca. 0.2~1.5% (v/w) (Hikawa 1998). Chromosome doubling is a powerful breeding method for increasing the productivity of secondary metabolites. To date, several plants have been successfully bred to increase production of secondary metabolites by chromosome doubling. For example, the productivity of tropane alkaloids has been increased to 122.5% in tetraploid plants compared to diploids in Hyoscyamus niger (Lavania and Srivastava 1991). In Artemisia annua, tetraploid hairy roots produced up to six times more sesquiterpene artemisinin than the diploid original (Jesus-Gonzalez and Weathers 2003). Moreover, enhanced productivities of essential oil as a secondary metabolite in tetraploid herbal plants have been reported, e.g., caraway (Dijkstra and Speckmann 1980) and Japanese peppermint (Janaki-Ammal and Sobti 1962). In German chamomile, tetraploid plants have also been bred since 1960s because tetraploid plants were well known for their large-sizedmorphology and increased productivity of essential oils (Franke and Schilcher 2005). However, the productivity of essential oil by tetraploid plants and the establishment of efficient formation of tetraploid plants in Roman chamomile remain unreported. In this paper, we tried to establish an efficient chromosome doubling system in Roman chamomile by using an in vitro culture system. We also characterized the morphological phenotypes of mature regenerated tetraploid plantlets. In addition, the content and composition of essential oils in those plants are discussed.