Yoshihito Takahata

Highly divergent sequences of the pollen self-incompatibility (S) gene in class-I S haplotypes of Brassica campestris (syn. rapa) L.

Self‐incompatibility (SI) enables flowering plants to discriminate between self‐ and non‐self‐pollen. In Brassica, SI is controlled by the highly polymorphic S locus. The recently identified male determinant, termed SP11 or SCR, is thought to be the ligand of S receptor kinase, the female determinant. To examine functional and evolutionary properties of SP11, we cloned 14 alleles from class‐I S haplotypes of Brassica campestris and carried out sequence analyses. The sequences of mature SP11 proteins are highly divergent, except for the presence of conserved cysteines. The phylogenetic trees suggest possible co‐evolution of the genes encoding the male and female determinants.

High frequency embryogenesis and plant regeneration in isolated microspore culture of Brassica oleracea L.

Abstract Conditions for reliable induction of embryogenesis from isolated microspores of Brassica oleracea var. italica were identified. An initial culture period at elevated temperature prior to maintenance at 25°C was required for induction of embryogenesis with incubation at 32.5°C for 1 day being the most effective. Neither double layer nor diluted culture methods were effective, rather they decreased embryo yield in comparison with liquid medium. Variation between genotypes and between bud sizes were significant in relation to embryo yield. Microspores from buds 4–5 mm in size from the B31-18 strain of broccoli were most responsive. With the best conditions, 400–700 embryo/bud could be routinely obtained. Approximately 60% of the embryos germinated normally when subcultured onto a filter paper placed on top of agar medium. This frequency was 1.5-fold higher than those subcultured onto agar medium. More than 80% of the regenerated plants were haploid.

Draft Sequences of the Radish (Raphanus sativus L.) Genome

Radish (Raphanus sativus L., n = 9) is one of the major vegetables in Asia. Since the genomes of Brassica and related species including radish underwent genome rearrangement, it is quite difficult to perform functional analysis based on the reported genomic sequence of Brassica rapa. Therefore, we performed genome sequencing of radish. Short reads of genomic sequences of 191.1 Gb were obtained by next-generation sequencing (NGS) for a radish inbred line, and 76,592 scaffolds of ≥300 bp were constructed along with the bacterial artificial chromosome-end sequences. Finally, the whole draft genomic sequence of 402 Mb spanning 75.9% of the estimated genomic size and containing 61,572 predicted genes was obtained. Subsequently, 221 single nucleotide polymorphism markers and 768 PCR-RFLP markers were used together with the 746 markers produced in our previous study for the construction of a linkage map. The map was combined further with another radish linkage map constructed mainly with expressed sequence tag-simple sequence repeat markers into a high-density integrated map of 1,166 cM with 2,553 DNA markers. A total of 1,345 scaffolds were assigned to the linkage map, spanning 116.0 Mb. Bulked PCR products amplified by 2,880 primer pairs were sequenced by NGS, and SNPs in eight inbred lines were identified.

Medium and genotype factors influencing shoot regeneration from cotyledonary explants of Chinese cabbage (Brassica campestris L. ssp. pekinensis)

Abstract Medium conditions for reliable shoot regeneration from cotyledonary explants of Chinese cabbage were examined. Maximum shoot regeneration was obtained in the presence of 5 mg/l BA and 0.5 mg/l NAA. Shoot induction was further improved by the addition of AgNO3 as well as higher concentrations (1.2–1.6%) of agar in the regeneration medium. When 123 genotypes were tested, a large variation in regeneration frequency was observed, ranging from 95% to 0%. Shoot regeneration frequency was not related to origin and days to maturity of the genotypes. Ethylene production from cultured explants seemed to play an important role in shoot regeneration. Explants of highly responsive genotypes or if cultured on the medium solidified with a higher concentration of agar generally showed low levels of ethylene production. However, AgNO3, which also enhanced shoot induction, resulted in an increase in ethylene production. The possible interaction between ethylene and shoot regeneration is discussed.

Identification and characterization of genes expressed in early embryogenesis from microspores of Brassica napus

To understand the mechanism in induction of embryogenesis from microspores of Brassica napus, we isolated exhaustively the genes expressed differentially during the early stage of microspore culture. A subtracted cDNA library composed of up-regulated genes during androgenic initiation was produced by suppression subtractive hybridization followed by differential screening by dot blot hybridization, and a total of 136 non-redundant expressed sequence tags were identified. Analysis of the potential functions of the genes showed that 64% of these genes were homologous to known genes, and the remaining ones have not been previously reported to participate in embryogenesis. Many embryo-specific genes were contained in the isolated genes, for example, genes cording lipid transfer protein, napin, cruciferin, oleosin, and phytosulfokine. Real-time RT-PCR analysis for 15 selected genes, which are understood to not be related with embryogenesis, demonstrated that all genes were expressed highly in the early stage of microspore embryogenesis. A few genes also showed higher expression in microspores cultured in non-embryogenic condition or in later stages of embryos. A principal component analysis based on expression profiles of the 15 genes demonstrated that these genes were classified into 2 groups, one characterized by their high expression in initiation of embryogenesis, and the other characterized by their expression in the early to middle stage of embryogenesis. The expressions of these genes were confirmed in zygotic embryos. The identification and characterization of the genes isolated in the present study provide novel information on microspore embryogenesis in Brassica.

Arabidopsis EMBRYOMAKER encoding an AP2 domain transcription factor plays a key role in developmental change from vegetative to embryonic phase.

Although several types of plant cells retain the competence to enter into embryonic development without fertilization, the molecular mechanism(s) underlying ectopic embryogenesis is largely unknown. To gain insight into this mechanism, in a previous study we identified 136 ESTs specifically expressed in microspore embryogenesis of Brassica napus. Here, we describe the characterization of the ArabidopsisEMBRYOMAKER (EMK) gene, which is homologous to one of the identified Brassica ESTs (BnGemb-58) and encodes an AP2 domain transcription factor. The AtEMK was expressed in developing and mature embryos, but its rapid disappearance occurred during germination. After germination, the expression of AtEMK was found in the root apical meristem and the distal parts of cotyledons. Although a mutant lacking AtEMK exhibited no distinctive defects in the embryo, ectopic expression of AtEMK induced embryo-like structures from cotyledons. The embryo-like structures contained high concentration of lipids, expressed several embryo-specific genes, and could convert into independent plants, indicating that the structures are somatic embryos. In vitro culture, AtEMK enhanced the efficiency of somatic embryogenesis. Furthermore, ectopic expression of AtEMK caused the formation of trichomes on cotyledons, dedifferentiated several tissues into calli, and retarded root development, demonstrating that AtEMK is harmful for the normal development of plants after germination. From these results, we conclude that the AtEMK is a key player to maintain embryonic identity, and the rapid disappearance of AtEMK expression during germination is essential for the developmental transition between the embryonic and vegetative phases in plants.

The advantages of cDNA microarray as an effective tool for identification of reproductive organ-specific genes in a model legume, Lotus japonicus.

To understand the molecular mechanisms intrinsic to reproductive organ development a cDNA microarray, fabricated from flower bud cDNA clones, was used to isolate genes, which are specifically expressed during the development of the anther and pistil in Lotus japonicus. Cluster analysis of the microarray data revealed 21 and 111 independent cDNA groups, which were specifically expressed in immature and mature anthers, respectively. RT‐PCR was performed to provide a direct assessment of the accuracy and reproducibility of our approach. Confirmation of our results suggests that cDNA microarray technology is an effective tool for identification of novel reproductive organ‐specific genes.

Effect of genotype on shoot regeneration from cotyledonary explants of rapeseed (Brassica napus L.)

SummaryAbility of shoot regeneration from cotyledonary explants of rapeseed (B. napus) was surveyed for 100 cultivars. Effects of explant age and plant growth regulators were determined before screening the genotypes. The optimal condition required 4-day-old cotyledons as explant and 4.0 mg/l benzylaminopurine as plant growth regulator. Gas-permeable tape as sealing material was more effective for shoot regeneration than Parafilm. When testing cultivars, shoot regeneration response was strongly influenced by genotype with a range of variation from 97% (percentage of explants regenerating shoots) in ‘Arabella’ and ‘Norin 26’ to 0% in ‘Norin 18’ and ‘Norin 30’. The response was not dependent on origin and cropping type (spring vs. winter type). The ability of shoot regeneration was not related to that of microspore embryogenesis. The regenerants rooted on medium containing 2.0 mg/l indole-3-butyric acid and after planting in soil flowered and set seeds. Histological studies showed that shoot meristems developed in callus which had grown from the vascular bundle tissue within 8 days.

Efficient plant regeneration from leaves of rapeseed (Brassica napus L.): the influence of AgNO3 and genotype

Factors influencing reliable shoot regeneration from leaf explants of rapeseed (Brassica napus L.) were examined. Addition of AgNO3 to callus induction medium was significantly effective for shoot regeneration in all three genotypes initially tested. When 48 genotypes subsequently were surveyed, a large variation of shoot regenerability was observed, ranging from 100 to 0% in frequency of bud formation and from 7.5 to 0 in the number of buds per explant. A significant correlation (r=0.84) was observed between the frequency of bud formation and the number of buds per explant. The shoot regenerability from leaf explants was not related to that from cotyledonary explants (r=0.28). Histological observations showed that an organized structure developed from calluses produced at vascular bundle tissues after 7 days of culture on callus induction medium, and they developed shoot apical meristems one week after transfer onto shoot induction medium. Regenerated plantlets were obtained 2 months after the initiation of culture and they normally flowered and set seeds. No alterations of morphology or DNA contents were observed in regenerated plants and their S1 progenies.

Agrobacterium-mediated transformation of cotyledonary explants of Chinese cabbage (Brassica campestris L. ssp. pekinensis)

Abstract A procedure for producing transgenic Chinese cabbage plants by inoculating cotyledonary explants with Agrobacterium tumefaciens strain EHA101 carrying a binary vector pIG121Hm, which contains kanamycin-resistance and hygromycin-resistance genes and the GUS reporter gene, is described. Infection was most effective (highest infection frequency) when explants were infected with Agrobacterium for 15 min and co-cultivated for 3 days in co-cultivation medium at pH 5.2 supplemented with 10 mg/l acetosyringone. Transgenic plants of all three cultivars used were obtained with frequencies of 1.6–2.7% when the explants were regenerated in shoot regeneration medium solidified with 1.6% agar. A histochemical GUS assay and PCR and Southern blot analyses confirmed that transformation had occurred. Genetic analysis of T1 progeny showed that the transgenes were inherited in a Mendelian fashion.