Erianthus germplasm collection in Thailand: genetic structure and phylogenetic aspects of tetraploid and hexaploid accessions.

BACKGROUND: The genus Erianthus, which belongs to the "Saccharum complex", includes C4 warm-season grasses. Erianthus species are widely distributed throughout Southeast Asia, East Asia and South Asia. Erianthus arundinaceus (Retz.) Jeswiet is highly adaptable to the environment, has a high percentage of dry matter, and is highly productive. Recently, this species has attracted attention as a novel bioenergy crop and as a breeding material for sugarcane improvement. Such interest in E. arundinaceus has accelerated the collection and conservation of its genetic resources, mainly in Asian countries, and also evaluation of morphological, agricultural, and cytogenetic features in germplasm collections. In Thailand, genetic resources of E. arundinaceus have been collected over the past 20 years and their phenotypic traits have been evaluated. However, the genetic differences and relatedness of the germplasms are not fully understood. RESULTS: A set of 41 primer pairs for nuclear simple sequence repeats (SSRs) developed from E. arundinaceus were used to assess the genetic diversity of 121 Erianthus germplasms collected in Thailand; of these primer pairs, 28 detected a total of 316 alleles. A Bayesian clustering approach with these alleles classified the accessions into four main groups, generally corresponding to the previous classification based on phenotypic analysis. The results of principal coordinate analysis and phylogenetic analysis of the 121 accessions on the basis of the SSR markers showed the same trend as Bayesian clustering, whereas sequence variations of three non-coding regions of chloroplast DNA revealed eight haplotypes among the accessions. The analysis of genetic structure and phylogenetic relationships, however, found some accessions whose classification contradicted the results of previous phenotypic classification. CONCLUSIONS: The molecular approach used in this study characterized the genetic diversity and relatedness of Erianthus germplasms collected across Thailand. This knowledge would allow efficient maintenance and conservation of the genetic resources of this grass and would help to use Erianthus species as breeding materials for development of novel bioenergy crops and sugarcane improvement.

Assistant skill in gastric endoscopic submucosal dissection using a clutch cutter.

BACKGROUND: A clutch cutter is a scissor-type knife used in endoscopic submucosal dissection (ESD) for gastrointestinal tract tumors. The assistant during the ESD using a clutch cutter (ESD-C) needs to rotate the device and grasp the target tissue appropriately; therefore, the assistant's skill may affect the technical outcomes of ESD-C. AIM: To determine how assistant skill level affected the technical outcomes of gastric ESD-C using an ex vivo porcine training model. METHODS: In this pilot study, mock lesions of 15-30 mm in diameter were created in the middle or lower third of the porcine stomach. A total of 32 ESD-C procedures were performed by 16 trainees. Each trainee operator performed two ESD-C procedures; one ESD-C was assisted by an expert (ESD-C-E), and the other was assisted by a non-expert (ESD-C-NE). The total procedure time of the ESD was set as the primary outcome, and en bloc resection rate, complete procedure rate, perforation rate, and each procedure time/speed for mucosal incision or submucosal dissection were set as the secondary outcomes. In addition, we investigated factors associated with the difficulty of ESD including incompletion of ESD procedure, a long procedure time (≥ 20 min) or intraoperative perforation. RESULTS: The median total procedure time of the ESD-C-E was significantly shorter than that of the ESD-C-NE (12.9 min vs 21.9 min, P = 0.001). The en bloc resection rate was 100% in both groups. Complete resection rates of the ESD-C-E and ESD-C-NE groups were 100% and 93.8%, respectively. No intraoperative perforation was observed in both groups. In the multivariate analysis, assistant skill was significantly associated with the difficulty of ESD, with the highest odds ratio of 16.5. CONCLUSION: Assistance by an expert is an important factor when trainees perform ESD-C procedures.

Self-Completion Method of Endoscopic Submucosal Dissection Using Endosaber without Any Other Device or Assistance: An ex vivo Porcine Model Study.

BACKGROUND: Endoscopic submucosal dissection (ESD) is a standard treatment for tumors of the gastrointestinal tract. We developed a self-completion method of ESD using Endosaber to eliminate the need for an additional device or human assistance during the procedure. The aim of this study was to evaluate the technical feasibility and outcomes of this method in an ex vivo porcine training model. METHODS: This was a pilot study, and the procedures were performed by 4 experts. Mock lesions measuring 15 mm in diameter were prepared at the posterior wall in the middle or lower esophagus obtained from domestic pigs. Each operator performed ESD on the mock lesions in 3 models. The primary outcome was ESD completion rate using the self-completion method. The secondary outcomes were procedure time, en bloc resection rate, perforation rate, and number of injections during the procedure. RESULTS: All 12 ESDs were completed using the self-completion method. The median procedure time (interquartile range) was 483.5 (399-619.3) s (median incision time: 240.4 [168.3-332.5] s; median dissection time: 222 [182.8-257] s). En bloc resection rate was 100%. No perforation was noted during any of the procedures. The median number of injections was 10.5 (9-14.3). The procedure time decreased significantly with increase in experience (p = 0.020). CONCLUSIONS: The self-completion ESD method using one Endosaber without any assistance achieved a 100% en bloc resection rate without any perforation. The need for an additional device or assistance was successfully eliminated. This method may prove to be a simple and cost-effective ESD procedure for lesions in humans.

Efficacy of traction, using a clip-with-thread, for esophageal endoscopic submucosal dissection for esophageal lesions with fibrosis in an ex vivo pig training model.

BACKGROUND/AIMS: Endoscopic submucosal dissection (ESD) of recurrent esophageal carcinoma is technically difficult to perform due to submucosal fibrosis that develops after definitive chemoradiation therapy. Therefore, our aim was to evaluate the usefulness of clip-with-thread traction for ESD of esophageal lesions with submucosal fibrosis. MATERIALS AND METHODS: Four endoscopists excised 16 lesions by ESD in an ex vivo pig training model. Mock lesions (30 mm in diameter) were created, including a 10-mm area of submucosal fibrosis in the center of each lesion. Each endoscopist performed two ESDs with traction (ESD-T) and two without traction (ESD-N). The primary outcome was the time required for submucosal dissection. Secondary outcomes were the rate of en bloc (complete) resection and perforation during the procedure, and the total amount of solution injected. RESULTS: All esophageal ESDs were completed. The median dissection time was significantly shorter for the ESD-T group (median 12.5 min, interquartile range 10.2-14.5) when comparing to the ESD-N group (median 18.0 min, interquartile range 14.6-19.2) (P=0.040). The en bloc resection rate was 100% in both groups, with a rate of complete resection of 87.5% and a rate of perforation of 37.5% for both groups. The median amount of solution injected was not significantly different between the ESD-T (18.0 ml) and ESD-N (20.5 ml) groups (P=0.526). CONCLUSION: Clip-with-thread traction improved the performance of ESD for lesions with submucosal fibrosis. However, the method might not reduce the risk of perforation, which remains an important clinical issue to resolve.

Solid-type poorly differentiated adenocarcinoma of the stomach: Deficiency of mismatch repair and SWI/SNF complex.

ARID1A, one of the subunits in SWI/SNF chromatin remodeling complex, is frequently mutated in gastric cancers with microsatellite instability (MSI). The most frequent MSI in solid-type poorly differentiated adenocarcinoma (PDA) has been reported, but the SWI/SNF complex status in solid-type PDA is still largely unknown. We retrospectively analyzed 54 cases of solid-type PDA for the expressions of mismatch repair (MMR) proteins (MLH1, PMS2, MSH2, and MSH6), SWI/SNF complex subunits (ARID1A, INI1, BRG1, BRM, BAF155, and BAF170) and EBER, and mutations in KRAS and BRAF. We analyzed 40 cases of another histological type of gastric cancer as a control group. The solid-type PDAs showed coexisting glandular components (76%), MMR deficiency (39%), and complete/partial loss of ARID1A (31%/7%), INI1 (4%/4%), BRG1 (48%/30%), BRM (33%/33%), BAF155 (13%/41%), and BAF170 (6%/2%), EBER positivity (4%), KRAS mutation (2%), and BRAF mutation (2%). Compared to the control group, MMR deficiency and losses of ARID1A, BRG1, BRM, and BAF155 were significantly frequent in solid-type PDAs. Mismatch repair deficiency was associated with the losses of ARID1A, BRG1, and BAF155 in solid-type PDAs. In the MMR-deficient group, solid components showed significantly more frequent losses of ARID1A, BRG1, BRM, and BAF155 compared to glandular components (P = .0268, P = .0181, P = .0224, and P = .0071, respectively). In the MMR-proficient group, solid components showed significantly more frequent loss of BRG1 compared to glandular components (P = .012). In conclusion, solid-type PDAs showed frequent losses of MMR proteins and the SWI/SNF complex. We suggest that loss of the SWI/SNF complex could induce a morphological shift from differentiated-type adenocarcinoma to solid-type PDA.

Involvement of different receptor subtypes in prostaglandin E2-induced contraction and relaxation in the lower esophageal sphincter and esophageal body.

Prostaglandin E2 (PGE2) plays a role in the pathogenesis of gastro-esophageal reflux disease (GERD). There are 4 subtypes of PGE2, PGE2 receptor 1, 2, 3 and 4 (EP 1-4). In GERD patents, PGE2, EP2 and EP4 are upregulated. However, the effects of PGE2 on esophageal motility remain elusive. We examined how PGE2 regulates motility in the porcine circular smooth muscle of the lower esophageal sphincter (LES), and the circular and longitudinal smooth muscle of the esophagus body in organ bath. PGE2 induced tonic relaxation in the LES and circular smooth muscle, but transient contraction in longitudinal smooth muscle. The relaxation of the LES and circular smooth muscle was similar in pattern and mechanism, but was much larger in the LES. The relaxation was completely blocked by a voltage-gated K+ channel blocker or 40 mM K+ depolarization, indicating the involvement of K+ channel. Longitudinal smooth muscle contraction was completely blocked by an L-type Ca2+ channel blocker, showing the contribution of Ca2+ movement. The involvement of the EP receptor in motility was examined with selective receptor agonists and antagonists. Activation of EP2 and EP4 caused relaxation in the LES and circular smooth muscle. Compatible with PGE2, EP2 and EP4 agonists caused more significant relaxation in the LES than in circular smooth muscle. EP1 contributed to the longitudinal smooth muscle contraction. The different effects of PGE2 in the LES, circular and longitudinal smooth muscle contributes to esophageal motility, their impairment might increase the amount and frequency of esophageal reflux.

Gastric hepatoid adenocarcinomas are a genetically heterogenous group; most tumors show chromosomal instability, but MSI tumors do exist.

The Cancer Genome Atlas Research Network classified gastric adenocarcinoma into four molecular subtypes: (1) Epstein-Barr virus-positive (EBV), (2) microsatellite-instable (MSI), (3) chromosomal instable (CIN), and (4) genomically stable (GS). The molecular subtypes of gastric hepatoid adenocarcinomas are still largely unknown. We analyzed 52 hepatoid adenocarcinomas for the expression of surrogate markers of molecular subtypes (MLH1, p53, and EBER in situ hybridization) and some biomarkers (p21, p16, Rb, cyclin D1, cyclin E, ß-catenin, Bcl-2, IMP3, ARID1A and HER2), and mutations of TP53, CTNNB1, KRAS, and BRAF. We analyzed 36 solid-type poorly differentiated adenocarcinomas as a control group. Hepatoid adenocarcinomas were categorized as follows: EBV group (EBER-positive), no cases (0%); MSI group (MLH1 loss), three cases (6%); "CIN or GS" (CIN/GS) group (EBER-negative, MLH1 retained), 49 cases (94%). In the CIN/GS group, most of the tumors (59%) had either p53 overexpression or TP53 mutation and a coexisting tubular intestinal-type adenocarcinoma component (90%), suggesting that most hepatoid adenocarcinomas should be categorized as a true CIN group. Hepatoid adenocarcinomas showed relatively frequent expressions of HER2 (score 3+/2+: 21%/19%). Hepatoid adenocarcinomas showed shorter survival, more frequent overexpressions of p16 (67%) and IMP3 (98%) than the control group. None of hepatoid adenocarcinomas had KRAS or CTNNB1 mutations except for one case each, and no hepatoid adenocarcinomas had BRAF mutation. In conclusion, gastric hepatoid adenocarcinomas are a genetically heterogenous group. Most hepatoid adenocarcinomas are "CIN," but a small number of hepatoid adenocarcinomas with MSI do exist. Hepatoid adenocarcinomas are characterized by overexpressions of p16 and IMP3.

Mucosal incision-assisted biopsy versus endoscopic ultrasound-guided fine-needle aspiration with a rapid on-site evaluation for gastric subepithelial lesions: A randomized cross-over study.

OBJECTIVES: This study aimed to compare the diagnostic yield of mucosal incision-assisted biopsy (MIAB) and endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) with a rapid on-site evaluation (ROSE) for gastric subepithelial lesions (SEL) suspected of being gastrointestinal stromal tumors (GIST) with an intraluminal growth pattern. METHODS: This was a prospective randomized, cross-over multicenter study. The primary outcome was the diagnostic yield of EUS-FNA and MIAB. The secondary outcomes were the technical success rate, complication rate, procedure time and biopsy frequency. RESULTS: A total of 47 patients were randomized to the MIAB group (n = 23) and EUS-FNA group (n = 24). There was no significant difference in the diagnostic yield of MIAB and EUS-FNA (91.3% vs 70.8%, P = 0.0746). The complication rates of MIAB and EUS-FNA did not differ to a statistically significant extent. The mean procedure time in the MIAB group was significantly longer than that in the EUS-FNA group (34 vs 26 min, P = 0.0011). CONCLUSIONS: The diagnostic yield of MIAB was satisfactorily as high as EUS-FNA with ROSE for gastric SEL with an intraluminal growth pattern.

Propensity score-matching analysis to compare clinical outcomes of endoscopic submucosal dissection for early gastric cancer in the postoperative and non-operative stomachs.

BACKGROUND: Endoscopic submucosal dissection (ESD) of the postoperative stomach (ESD-P) for early gastric cancer (EGC) is considered a technically difficult procedure. However, it is difficult to compare the outcomes of ESD-P and ESD of the non-operative stomach (ESD-N) because their baseline characteristics are different. Therefore, we aimed to compare the technical outcomes of ESD-P with those of ESD-N using a propensity score-matching analysis to compensate for the differences. METHODS: The chart records of 1046 patients with EGC who were treated with ESD between January 2004 and July 2016 at Kitakyushu Municipal Medical Center in Japan were reviewed in this retrospective study. Multivariate analyses and propensity score-matching were performed for age, sex, lesion location, lesion size, tumor invasion, tumor size, ulcer (scar), and operator skill. The primary outcome was procedure time. Secondary outcomes were percentages of en bloc, complete, and curative resections, and percentages of adverse events, which were evaluated between the two groups. RESULTS: Forty-one patients were in the ESD-P group and 1005 patients were in the ESD-N group. Propensity score-matching created 41 matched pairs. According to the adjusted comparisons, ESD-P required a significantly longer procedure time (85 min vs 51 min, p < 0.001). Other treatment outcomes showed an en bloc resection rate of 100% for both groups (p = 1) and complete resection rates of 95.1 and 97.6% (p = 1), curative resection rates of 90.2 and 90.2% (p = 1), perforation during ESD rates of 2.4 and 0% (p = 1), and postprocedure bleeding rates of 2.4 and 2.4% (p = 1) for the ESD-P and ESD-N groups, respectively. For the ESD-P group, lesions on the suture line or anastomotic site were significantly associated with longer procedure times (p = 0.038). CONCLUSIONS: ESD-P was a more time-consuming procedure than ESD-N. However, ESD-P and ESD-N achieved high rates of curative resection with a low rate of adverse events for the treatment of EGC. ESD could be selected as the treatment for EGC even in the postoperative stomach provided that careful attention is given to lesions on the suture line or anastomotic site.

Non-familial juvenile polyposis of the stomach with gastric cancers: a case report.

BACKGROUND: Juvenile polyposis is an autosomal dominant inherited disease characterized by the development of numerous hamartomatous and nonneoplastic polyps of the gastrointestinal tract. Juvenile polyposis has also recently been reported as a predisposition for gastrointestinal cancer. CASE PRESENTATION: A 63-year-old man underwent esophagogastroduodenoscopy because of anemia and hypoalbuminemia during a follow-up for gastric polyposis, which showed multiple reddish polyps and two elevated lesions in the stomach. The elevated lesions were diagnosed as well-differentiated adenocarcinomas by biopsy. He had no specific physical findings or family history. Computed tomography showed gastric wall thickening without lymphadenopathy or distant metastasis. Colonoscopy showed an adenoma in the transverse colon. He underwent laparoscopy-assisted total gastrectomy with Roux-en-Y esophagojejunostomy. The resected specimen revealed numerous variously sized non-pedunculated polyps throughout the stomach, diagnosed histopathologically as hamartomatous polyps. The two elevated lesions were diagnosed as a well-differentiated adenocarcinoma restricted to the mucosa and a well-to-poorly differentiated adenocarcinoma invading the submucosa with prominent lymphatic permeation, respectively. Genetic analysis failed to identify any germline mutations in the genes usually associated with juvenile polyposis, including SMAD4 and BMPR1A. However, based on the few characteristic physical findings and histopathological features, the final diagnosis was juvenile polyposis restricted to the stomach. CONCLUSIONS: This patient represented a rare case of non-familial juvenile polyposis of the stomach with gastric cancers. Juvenile polyposis has malignant potential, and patients should therefore be carefully followed up. Surgical treatment, particularly total gastrectomy, is recommended as a standard treatment in patients with juvenile polyposis of the stomach with gastric cancer.

Clinicopathologic and Molecular Characteristics of Synchronous Colorectal Carcinoma With Mismatch Repair Deficiency.

Synchronous colorectal carcinoma (CRC) is a unique disease associated with a high prevalence (∼35%) of microsatellite instability and occasionally with Lynch syndrome. The clinicopathologic and molecular features of synchronous CRC are poorly understood, particularly in Japanese patients. We examined 118 Japanese patients (236 tumors) with synchronous CRC and 117 Japanese patients (117 tumors) with solitary CRC with immunohistochemical staining for TP53 and mismatch repair (MMR) protein (MLH1, MSH2, PMS2, and MSH6) and mutation analyses of KRAS and BRAF genes. The results revealed no significant differences in clinicopathologic, histologic, and molecular findings between the synchronous and solitary CRC groups. Among the 118 synchronous CRC patients, 15 (12.7%) showed loss of MMR protein(s) expression in at least 1 tumor, whereas 103 (87.3%) showed intact expression of all 4 MMR proteins in both tumors. Of note, all patients with MMR deficiency had excellent prognoses. The 15 patients were further subdivided into 2 groups: the Concordant group, with concordant MMR loss (n=9, 7.6%) and the Discordant group, with discordant MMR loss (n=6, 5.1%). The Concordant patients showed concurrent MLH1/PMS2 loss (n=3), concurrent MSH2/MSH6 loss (n=4) and isolated MSH6 loss (n=2) in both tumors, whereas the Discordant patients showed concurrent MLH1/PMS2 loss (n=2), isolated PMS2 loss (n=2) and isolated MSH6 loss (n=2) in a single tumor. On the basis of the MMR expression pattern and BRAF mutation, the Concordant and Discordant groups were suspected to include Lynch syndrome, Lynch-like syndrome and sporadic MLH1 promoter hypermethylated CRC. In addition, KRAS mutation was present in only 1 tumor in a single patient in each group. In conclusion, the frequency of MMR protein deficiency in synchronous CRC in the Japanese population may be lower compared with the reported data from Western populations. MMR protein loss and KRAS and BRAF mutations in synchronous CRCs were heterogenous even in an individual patient.

Clutch Cutter knife efficacy in endoscopic submucosal dissection for early gastric neoplasms.

AIM: To compare the outcomes of endoscopic submucosal dissection (ESD) for gastric neoplasms using Clutch Cutter (ESD-C) or other knives (ESD-O). METHODS: This was a single-center retrospective study. Gastric neoplasms treated by ESD between April 2016 and October 2017 at Kitakyushu Municipal Medical Center were reviewed. Multivariate analyses and propensity score matching were used to reduce biases. Covariates included factors that might affect outcomes of ESD, including age, sex, underlying disease, anti-thrombotic drugs use, tumor location, tumor position, tumor size, tumor depth, tumor morphology, tumor histology, ulcer (scar), and operator skill. The treatment outcomes were compared among two groups. The primary outcome was ESD procedure time. Secondary outcomes were en bloc, complete, and curative resection rates, and adverse events rates including perforation and delayed bleeding. RESULTS: A total of 155 patients were included in this study; 44 pairs were created by propensity score matching. Background characteristics were quite similar among two groups after matching. Procedure time was significantly shorter for ESD-C (median; 49 min) than for ESD-O (median; 88.5 min) (P < 0.01). However, there was no significant difference in treatment outcomes between ESD-C and ESD-O including en bloc resection rate (100% in both groups), complete resection rate (100% in both groups), curative resection rate (86.4% vs 88.6%, P = 0.730), delayed bleeding (2.3% vs 6.8%, P = 0.62) and perforation (0% in both groups). CONCLUSION: ESD-C achieved shorter procedure time without an increase in complication risk. Therefore, ESD-C could become an effective ESD option for gastric neoplasms.

Complete Chloroplast Genomes of Erianthus arundinaceus and Miscanthus sinensis: Comparative Genomics and Evolution of the Saccharum Complex.

The genera Erianthus and Miscanthus, both members of the Saccharum complex, are of interest as potential resources for sugarcane improvement and as bioenergy crops. Recent studies have mainly focused on the conservation and use of wild accessions of these genera as breeding materials. However, the sequence data are limited, which hampers the studies of phylogenetic relationships, population structure, and evolution of these grasses. Here, we determined the complete chloroplast genome sequences of Erianthus arundinaceus and Miscanthus sinensis by using 454 GS FLX pyrosequencing and Sanger sequencing. Alignment of the E. arundinaceus and M. sinensis chloroplast genome sequences with the known sequence of Saccharum officinarum demonstrated a high degree of conservation in gene content and order. Using the data sets of 76 chloroplast protein-coding genes, we performed phylogenetic analysis in 40 taxa including E. arundinaceus and M. sinensis. Our results show that S. officinarum is more closely related to M. sinensis than to E. arundinaceus. We estimated that E. arundinaceus diverged from the subtribe Sorghinae before the divergence of Sorghum bicolor and the common ancestor of S. officinarum and M. sinensis. This is the first report of the phylogenetic and evolutionary relationships inferred from maternally inherited variation in the Saccharum complex. Our study provides an important framework for understanding the phylogenetic relatedness of the economically important genera Erianthus, Miscanthus, and Saccharum.

[Comorbid Meckel's diverticulum and omphalomesenteric cyst evaluated by small bowel series under double-balloon enteroscopy: a case report].

A 59-year-old man was referred to our hospital for examination of intermittent abdominal pain. Computed tomography scan showed a cystic lesion adjoining the ileum, and small bowel series demonstrated a small bowel diverticulum. Double-balloon enteroscopy (DBE) revealed a diverticulum in the ileum and a soft and smooth elevated lesion with a small hole at the base of the diverticulum. Small bowel series under DBE demonstrated that the cystic lesion communicated with the diverticulum through the small hole. The diagnosis was Meckel's diverticulum and an omphalomesenteric cyst. This is the first reported case of a Meckel's diverticulum and omphalomesenteric cyst communicating through a small hole without a fibrous ligament. In addition, precise evaluation was possible by small bowel series and DBE.

Particle inflow gun-mediated transformation of multiple-shoot clumps in rhodes grass (Chloris gayana).

Rhodes grass (Chloris gayana) is one of the most important warm-season forage grasses. It is cultivated in tropical and subtropical parts of the world and is mostly used for grazing and hay production. We have established a particle-bombardment transformation protocol for rhodes grass using multiple-shoot clumps (MSCs) as the target tissue. A vector pAHC25 containing a herbicide-resistance gene (bar) together with the beta-glucuronidase (GUS) gene was used in transformation experiments. The most efficient recovery of bialaphos-resistant tissue was achieved when the bombarded MSCs were first cultured for 15 d on bialaphos-free medium before being subjected to selection pressure. The resistant tissues regenerated transgenic plants that displayed GUS gene expression. Under optimized conditions, 251 target pieces yielded 46 transgenic plants from 4 independent transgenic lines.

Analysis of expressed sequence tags in apomictic guineagrass (Panicum maximum).

Apomixis is an intriguing asexual mode of reproduction, because it produces maternal clones that permit vegetative reproduction through seeds. Guineagrass (Panicum maximum) has both facultative aposporous apomixis and obligate sexual modes of reproduction. Despite the importance of apomixis in guineagrass, expressed sequence tags (ESTs) for this condition have not been studied in this species. We constructed a guineagrass cDNA library from two aposporous strains, Ku5954 and GM64-3A, and utilized them as microarray probes. To find genes uniquely expressed in the immature pistils of apomicts, we performed a microarray analysis using target RNA from another apomict, OKI64. Of the 4608 probes in the microarray, only 394 showed clear gene expression in the immature pistils. Of the 394 expressed probes, 196 were successfully sequenced. Of these, 181 had significant homology with other species, including 10 ESTs with matches in a pistil cDNA library from another aposporous species, Cenchrus ciliaris. Of the remaining ESTs, three showed significant homology only with animal database sequences and the other 12 ESTs showed no homology with any previously registered sequence. In reverse-transcriptase PCR and real-time quantitative PCR, nine ESTs reliably detected ovary-specific gene expression. Of these, three revealed aposporous ovary-specific genes expressed in the early developmental stage, suggesting that these could be apomixis-related genes.

Transgenic superroots of Lotus corniculatus can be regenerated from superroot-derived leaves following Agrobacterium-mediated transformation.

Super-growing roots (superroots; SR), which have been established in the legume species Lotus corniculatus, are a fast-growing root culture that allows continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely growth regulator-free culture conditions. These features are unique for non-hairy root cultures, and they are now stably expressed since the culture was isolated more than 10 years ago (1997). Attempts to achieve direct and stable transformation of SR turned out to be unsuccessful. Making use of the supple regeneration plasticity of SR, we are reporting here an indirect transformation protocol. Leaf explants, derived from plants regenerated from SR, were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pBI121, which contains the neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes as selectable and visual markers, respectively. After co-cultivation, the explants were selected on solidified MS medium with 0.5 mg/L benzylamino purine (BAP), 100 mg/L kanamycin and 250 mg/L cefotaxime. Kanamycin-resistant calli were transferred to liquid rooting medium. The newly regenerated, kanamycin-resistant roots were harvested and SR cultures re-established, which exhibited all the characteristics of the original SR. Furthermore, kanamycin-resistant roots cultured onto solidified MS medium supplemented with 0.5 mg/L BAP produced plants at the same rate as control SR. Six months after gene transfer, PCR analysis and histochemical locating indicated that the NPTII gene was integrated into the genome and that the GUS gene was regularly expressed in leaves, roots and nodules, respectively. The protocol makes it now possible to produce transformed SR and nodules as well as transgenic plants from transformed SR.

Green, herbicide-resistant plants by particle inflow gun-mediated gene transfer to diploid bahiagrass (Paspalum notatum).

We have established an efficient particle-bombardment transformation protocol for the diploid non-apomictic genotype of the warm season forage crop Paspalum notatum (bahiagrass). A vector containing a herbicide resistance gene (bar) together with the GUS reporter gene was used in transformation experiments. The bar gene confers resistance to the herbicide bialaphos. An improved culture system, highly regenerative callus, dense in compact polyembryogenic clusters, was produced on medium with a high CuSO4 content at elevated temperature. Target tissue (360 calli) produced under these conditions yielded 52 rooted plants on herbicide-containing medium, and 22 of these plants were PCR-positive. DNA gel blot analysis revealed a copy number of 1-5 for the GUS gene in different independent transformants. There was no correlation between copy number and GUS activity. While conventional cultures yielded exclusively albino plants on herbicide-containing medium, improved culture conditions for the target tissue resulted in the recovery of 100% green transgenic plants. All green herbicide-resistant regenerants were morphological normal and fertile.