Enhanced Mechanical Properties of Polylactic Acid/Poly(Butylene Adipate-co-Terephthalate) Modified with Maleic Anhydride.

An increase in plastic waste pollution and the strengthening of global environmental policies have heightened the need for research on biodegradable plastics. In this regard, polylactic acid (PLA) and poly(butylene adipate-co-terephthalate) (PBAT) are notable examples, serving as alternatives to traditional plastics. In this study, the compatibility and mechanical properties of PLA/PBAT blends were improved by the chemical grafting of maleic anhydride (MAH). In addition, qualitative analyses were conducted, dynamic mechanical properties were investigated, and the structure and mechanical characteristics of the blends were analyzed. With an increase in the MAH concentration, the grafting yield of the blends increased, and significantly improved the compatibility of the PLA/PBAT blend, with an ~2 and 2.9 times increase in the tensile strength and elongation at break, respectively. These findings indicate that the modified PLA/PBAT blend demonstrates potential for applications that require sustainable plastic materials, thereby contributing to the development of environmentally friendly alternatives in the plastics industry.

Antibacterial effects of vinegar N6 and UV-C light-emitting diodes against Shiga toxin-producing and enterohemorrhagic Escherichia coli in fresh beef.

Some Escherichia coli serotypes cause diarrhea in infants and acute gastroenteritis. In this study, the incidence of Shiga toxin-producing (STEC) and enterohemorrhagic (EHEC) E. coli in 310 fresh raw beef samples and the presence of pathogenicity-associated virulence genes in the isolated strains were evaluated. The contamination rate reached 18.06% (STEC, 12.26%; EHEC, 5.81%). The highest rate of identified virulence genes was 8.38% for stx2 and 3.23% for stx2 and eae in STEC and EHEC, respectively. Vinegar N6 significantly lowered E. coli growth in beef samples, depending on its concentration (> 0.5%), treatment temperature (5 or 10 °C), and E. coli type (STEC, EHEC, or enteropathogenic), during 28 days of storage. However, no bactericidal effects were detected, unlike those observed for combined treatment with UV-C LED and vinegar N6. Treatment with vinegar N6 and UV-C LED together may significantly reduce E. coli growth in fresh beef, thereby improving food safety. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01260-x.

Hardening Properties of Cheeses by Latilactobacillus curvatus PD1 Isolated from Hardened Cheese-Ddukbokki Rice Cake.

Hardening of cheese is one of major issues that degrade the quality of Home Meal Replacement (HMR) foods containing cheese such as Cheese-ddukbokki rice cake (CD, stir-fried rice cakes with shredded cheese). The quality of cheese, such as pH, proteolytic, and flavor properties, depends on various lactic acid bacteria (LAB) used in cheese fermentation. The hardening of cheese is also caused by LAB. In this study, various LAB strains were isolated from CD samples that showed rapid hardening. The correlation of LAB with the hardening of cheese was investigated. Seven of the CD samples with different manufacturing dates were collected and tested for hardening properties of cheese. Among them, strong-hardening of cheese was confirmed for two samples and weak-hardening was confirmed for one sample. All LAB in two strong-hardening samples and 40% of LAB in one weak-hardening sample were identified as Latilactobacilluscurvatus. On the other hand, most LAB in normal cheese samples were identified as Leuconostoc mesenteroides and Lactobacillus casei. We prepared cheese samples in which L. curvatus (LC-CD) and L. mesenteroides (LM-CD) were most dominant, respectively. Each CD made of the prepared cheese was subjected to quality test for 50 days at 10 °C. Hardening of cheese with LC-CD dominant appeared at 30 days. However, hardening of cheese with LM-CD dominant did not appear until 50 days. The pH of the LC-CD was 5.18 ± 0.04 at 30 days, lower than that of LM-CD. The proteolytic activity of LC-CD sample was 2993.67 ± 246.17 units/g, higher than that of LM-CD sample (1421.67 ± 174.5 units/g). These results indicate that high acid production and high protease activity of L. curvatus might have caused hardening of cheese.

Isolation and Characterization of Kimchi Starters Leuconostoc mesenteroides PBio03 and Leuconostoc mesenteroides PBio104 for Manufacture of Commercial Kimchi.

This study was focused on developing and obtaining a kimchi starter for use in commercial kimchi production. Kimchi varieties made with selected starters are of high quality, have high levels of mannitol, and extended shelf life. The starters were screened for properties such as mannitol production, low gas/acid production, and acid resistance. Finally, kimchi fermentation testing was performed using selected LAB starters. Kimchi samples were prepared with lactic acid bacteria (LAB) starters, including Leuconostoc mesenteroides PBio03 and Leuconostoc mesenteroides PBio104. The LAB starters are isolated from kimchi and can grow under pH 3.0 and low temperature conditions of 5°C. Four kimchi samples were fermented and stored for 28 days at 5°C. The kimchi samples made with starters (PBio03 and PBio104) had better quality (production of mannitol and maintenance of heterofermentative LAB dominance) than the non-starter kimchi samples. In the starter kimchi, Leu. mesenteroides was the dominant LAB, comprising 80% and 70% of total LAB counts at 7 and 21 days, respectively. Mannitol content of the kimchi with Leu. mesenteroides PBio03 was 1,423 ± 19.1 mg/ 100 g at 28 days, which was higher than that of the non-starter kimchi sample (1,027 ± 12.2 mg/100 g). These results show the possibility of producing kimchi with improved qualities using Leu. mesenteroides PBio03 and PBio104 as starters.

Effectiveness of contrast-enhanced harmonic endoscopic ultrasound for the evaluation of solid pancreatic masses.

AIM: To evaluate the usefulness of contrast-enhanced harmonic endoscopic ultrasound (CH-EUS) in differentiating between pancreatic adenocarcinomas and other pancreatic disease. METHODS: This retrospective cohort study evaluated 90 patients who were seen between November 2010 and May 2013. All these patients had solid pancreatic masses that had a hypoechoic appearance on EUS. All patients underwent CH-EUS to evaluate this diagnostic method's usefulness. The mass lesions observed on CH-EUS were classified into three categories based on their echo intensity: hypoenhanced, isoenhanced, and hyperenhanced lesions. We adjusted the sensitivity and the specificity of each category for detecting malignancies. We also estimated the accuracy of CH-EUS by comparing it to a pathological diagnosis. RESULTS: Of the 90 patients, 62 had a pancreatic adenocarcinoma. Fifty-seven out of 62 pancreatic adenocarcinomas showed a hypoenhanced pattern on CH-EUS. The sensitivity was 92%, the specificity 68% and the accuracy approximately 82%. The area under the curve of the receiver operating characteristic analysis for CH-EUS was 0.799. There is a significant association between the hypoenhanced pattern on CH-EUS and pancreatic duct adenocarcinoma (χ(2) = 35.264, P < 0.001). In pathological examinations, the number of specimens for EUS-fine needle aspiration (EUS-FNA) was considered insufficient for diagnosis in three patients, and in two patients, the results were reported to be negative for malignancy. Pancreatic masses in all five patients revealed a hypoenhanced pattern with CH-EUS. Three patients were diagnosed with pancreatic adenocarcinoma based on the pathology results of a biopsy, and the remaining two patients were clinically diagnosed with malignancy. CONCLUSION: CH-EUS is useful for distinguishing between pancreatic adenocarcinoma and other pancreatic disease. When a pancreatic mass shows a hypoenhanced pattern on CH-EUS but involves either insufficient samples or negative results with EUS-FNA, clinicians might consider performing another pathologic diagnosis on the basis of an EUS-FNA sample or a biopsy.

Structure-function studies of a plant tyrosyl-DNA phosphodiesterase provide novel insights into DNA repair mechanisms of Arabidopsis thaliana.

TDP1 (tyrosyl-DNA phosphodiesterase 1), a member of the PLD (phospholipase D) superfamily, catalyses the hydrolysis of a phosphodiester bond between a tyrosine residue and the 3'-phosphate of DNA. We have previously identified and characterized the AtTDP gene in Arabidopsis thaliana, an orthologue of yeast and human TDP1 genes. Sequence alignment of TDP1 orthologues revealed that AtTDP has both a conserved C-terminal TDP domain and, uniquely, an N-terminal SMAD/FHA (forkhead-associated) domain. To help understand the function of this novel enzyme, we analysed the substrate saturation kinetics of full-length AtTDP compared with a truncated AtTDP mutant lacking the N-terminal FHA domain. The recombinant AtTDP protein hydrolysed a single-stranded DNA substrate with Km and kcat/Km values of 703±137 nM and (1.5±0.04)×10(9) M(-1)·min(-1) respectively. The AtTDP-(Δ1-122) protein (TDP domain) showed kinetic parameters that were equivalent to those of the full-length AtTDP protein. A basic amino acid sequence (RKKVKP) within the AtTDP-(Δ123-605) protein (FHA domain) was necessary for nuclear localization of AtTDP. Analysis of active-site mutations showed that a histidine and a lysine residue in each of the HKD motifs were critical for enzyme activity. Vanadates, inhibitors of phosphoryl transfer reactions, inhibited AtTDP enzymatic activity and retarded the growth of an Arabidopsis tdp mutant. Finally, we showed that expression of the AtTDP gene could complement a yeast tdp1Δrad1Δ mutant, rescuing the growth inhibitory effects of vanadate analogues and CPT (camptothecin). Taken together, the results of the present study demonstrate the structure-based function of AtTDP through which AtTDP can repair DNA strand breaks in plants.

Overexpression of EVE1, a novel ubiquitin family protein, arrests inflorescence stem development in Arabidopsis.

In Arabidopsis, inflorescence stem formation is a critical process in phase transition from the vegetative to the reproductive state. Although inflorescence stem development has been reported to depend on the expression of a variety of genes during floral induction and repression, little is known about the molecular mechanisms involved in the control of inflorescence stem formation. By activation T-DNA tagging mutagenesis of Arabidopsis, a dominant gain-of-function mutation, eve1-D (eternally vegetative phase1-Dominant), which has lost the ability to form an inflorescence stem, was isolated. The eve1-D mutation exhibited a dome-shaped primary shoot apical meristem (SAM) in the early vegetative stage, similar to that seen in the wild-type SAM. However, the SAM in the eve1-D mutation failed to transition into an inflorescence meristem (IM) and eventually reached senescence without ever leaving the vegetative phase. The eve1-D mutation also displayed pleiotropic phenotypes, including lobed and wavy rosette leaves, short petioles, and an increased number of rosette leaves. Genetic analysis indicated that the genomic location of the EVE1 gene in Arabidopsis thaliana corresponded to a bacterial artificial chromosome (BAC) F4C21 from chromosome IV at ∼17cM which encoded a novel ubiquitin family protein (At4g03350), consisting of a single exon. The EVE1 protein is composed of 263 amino acids, contains a 52 amino acid ubiquitin domain, and has no glycine residue related to ubiquitin activity at the C-terminus. The eve1-D mutation provides a way to study the regulatory mechanisms that control phase transition from the vegetative to the reproductive state.

Hemocyanin-derived phenoloxidase activity with broad temperature stability extending into the cold environment in hemocytes of the hair crab Erimacrus isenbeckii.

Phenoloxidase (PO) activity is a major component of the innate immune response in arthropods. In this study, we characterized PO activity from the hair crab Erimacrus isenbeckii, which inhabits very cold regions (2.4-3.4°C) of the Bering Sea. Hemocyte lysate supernatant (HLS) prepared from E. isenbeckii was inactive HLS until activated by nonspecific agents such as sodium dodecyl sulfate and trypsin, and elicitors such as lipopolysaccharide and lipoteichoic acid from the cell wall constituent of bacteria. The PO activity was maximal at 4°C, decreased slightly at temperatures up to 60°C, and fell rapidly at 80°C. Both L-DOPA and catechol were efficient substrates for the PO (EC 1.10.3.1), with K(m) values of 0.96 and 1.15mM, respectively, whereas tyrosine and hydroquinone were not. We isolated a protein fraction from HLS as a hexamer of 75kDa units with 216.7-fold higher PO activity than that of the HLS. The N-terminal amino acid analysis of an isolated protein revealed 80% sequence identity to hemocyanins from other crabs. These results suggest that cold-adapted hemocyanin-derived PO activity is important to the survival of these crabs. This is the first report of a crab PO activity with broad temperature stability extending into the cold environment.

Identification of tyrosyl-DNA phosphodiesterase as a novel DNA damage repair enzyme in Arabidopsis.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a key enzyme that hydrolyzes the phosphodiester bond between tyrosine of topoisomerase and 3'-phosphate of DNA and repairs topoisomerase-mediated DNA damage during chromosome metabolism. However, functional Tdp1 has only been described in yeast and human to date. In human, mutations of the Tdp1 gene are involved in the disease spinocerebellar ataxia with axonal neuropathy. In plants, we have identified the functional nuclear protein AtTDP, homolog to human Tdp1 from Arabidopsis (Arabidopsis thaliana). The recombinant AtTDP protein certainly hydrolyzes the 3'-phosphotyrosyl DNA substrates related to repairing in vivo topoisomerase I-DNA-induced damage. The loss-of-function AtTDP mutation displays developmental defects and dwarf phenotype in Arabidopsis. This phenotype is substantially caused by decreased cell numbers without any change of individual cell sizes. The tdp plants exhibit hypersensitivities to camptothecin, a potent topoisomerase I inhibitor, and show rigorous cell death in cotyledons and rosette leaves, suggesting the failure of DNA damage repair in tdp mutants. These results indicate that AtTDP plays a clear role in the repair of topoisomerase I-DNA complexes in Arabidopsis.

[A case of pulmonary thromboembolism in active ulcerative colitis].

Inflammatory bowel disease often involves extra-intestinal organs. Cerebral thrombosis, portal vein thrombosis and pulmonary thrombosis have been reported. Deep vein thrombosis and pulmonary thromboembolism are significant causes of mortality in patients with inflammatory bowel disease. A 48-year-old woman was diagnosed as inflammatory bowel disease on colonoscopy and histology. We used hydrocortisone and mesalazine for the treatment of disease. Nineteen days later, she complained of abrupt dyspnea. Pulmonary CT angiography revealed a thromboembolism in right pulmonary arteries. After the treatment of heparin therapy, follow-up pulmonary CT angiography showed significant improvement of previously thrombosed pulmonary arteries.

A case of MCTD overlapped by Takayasu's arteritis, presenting Raynaud's phenomenon as the initial manifestation of both diseases.

Raynaud's phenomenon is characteristic three-phase color change of digits that occurs when hands are exposed to cold and subsequently rewarmed. Raynaud's phenomenon has many possible causes, but evaluation tends to focus on a few notorious etiologies, such as, connective tissue diseases. Thus, having reached a diagnosis, detailed physical exam to rule out other possible causes is often not performed. The authors present a case of mixed connective tissue disease (MCTD) and Takayasu's arteritis overlap in a woman, who showed Raynaud's phenomenon as an initial manifestation. She was first diagnosed as having MCTD, but her treatment did not improve the persistent Raynaud's phenomenon. Several years later, follow-up chest CT showed underlying Takayasu's arteritis and a subsequent physical examination revealed that typical abnormalities consistent with Takayasu's arteritis were present. The authors advocate thorough history taking and complete physical examinations on a routine basis to help unearth other underlying causes.

An upstream region in the first intron of petunia actin-depolymerizing factor 1 affects tissue-specific expression in transgenic Arabidopsis (Arabidopsis thaliana).

The first intron of the petunia actin-depolymerizing factor 1 (PhADF1) gene was previously shown to induce strong and constitutive expression of that gene in vegetative tissues of transgenic Arabidopsis. To examine intron-mediated enhancement of PhADF1 gene expression in detail, the effects of splicing, deletion and promoter alteration on gene expression were analyzed in this study. Deletion of the 5' upstream region of the intron significantly reduced the level of enhancement, under the control of both the PhADF1 and the PhADF2 promoters. The ratio of pre-mRNA and mRNA does not correlate with the level of enhancement. To determine whether there is a promoter-intron interaction, the role of the intron was examined under the control of a heterogeneous promoter. The intron of PhADF1 induced GUS expression in vegetative tissues under the control of the reproductive tissue-specific Arabidopsis profilin 5 (PRF5) promoter. In transient assays, the presence of the intron increased GUS expression under control of the 35S minimal promoter. Our results suggest that the first intron of the PhADF1 gene alters tissue-specific expression by a post-transcriptional mechanism. In addition, we have also shown that intron-mediated enhancement is a conserved mechanism, which regulates the expression of the petunia and Arabidopsis ADF genes that are expressed in vegetative tissues.

Functional characterization of a gene encoding a dual domain for uridine kinase and uracil phosphoribosyltransferase in Arabidopsis thaliana.

Uridine kinase (UK) and uracil phosphoribosyltransferase (UPRT) are enzymes catalyzing the formation of uridine 5'-monophosphate (UMP) from uridine and adenine 5'-triphosphate (ATP) and from uracil and phosphoribosyl-alpha-l-pyrophosphate (PRPP), respectively, in the pyrimidine salvage pathway. Here, we report the characterization and functional analysis of a gene AtUK/UPRT1 from Arabidopsis thaliana. Sequencing of an expressed sequence tag clone of this gene revealed that it contains a full-length open reading frame of 1461 nucleotides and encodes a protein with a molecular mass of approximately 53 kDa. The sequence analysis revealed that the N-terminal region of AtUK/UPRT1 contains a UK domain and the C-terminal region consists of a UPRT domain. Expression of AtUK/UPRT1 in upp and upp-udk mutants of Escherichia coli supplied with 5-fluorouracil (5-FU) and 5-fluorouridine (5-FD) led to growth inhibition. Identical results were obtained with 5-FD and 5-FU treatments when the UK and UPRT domains were separated by the introduction of translation initiation and stop codons prior to complementation into the upp-udk and upp mutants. These results suggest that the AtUK/UPRT1 product can use uracil and uridine as substrates for the production of UMP. We also investigated the function of AtUK/UPRT1 in an Arabidopsis mutant. The wild-type Arabidopsis plants showed drastic growth retardation when they were treated with 5-FU and 5-FD while the growth of atuk/uprtl mutant plants was not significantly affected. These findings confirm that AtUK/UPRT1 has a dual role in coding for both uridine kinase and uracil phosphoribosyltransferase that form UMP through the pyrimidine salvage pathway in Arabidopsis.

Brassinosteroid signals control expression of the AXR3/IAA17 gene in the cross-talk point with auxin in root development.

Transgenic plants overexpressing AXR3/IAA17 were impaired in root growth. Specifically, they exhibited severe defects in lateral root and root hair development similar to the root phenotypes of epi-brassinolide (epiBL)-treated wild-type plants. Here, we investigated the involvement of AXR3/IAA17 gene expression in brassinosteroid (BR)-regulated root development. Exogenous epiBL application significantly induced expression of the AXR3/IAA17 gene as well as several Aux/IAA genes, such as AXR2/IAA7, SLR/IAA14, and IAA28. We analyzed the transcription levels of several Aux/IAA genes related to root development in the BR signaling mutant bri1 and the BR biosynthesis mutant det2. AXR3/IAA17 gene expression was significantly decreased in bri1 plants. In det2 plants, expression of AXR3/IAA17 slightly decreased. This in turn suggests that epiBL induced these Aux/IAA genes, and that these induced gene products might function as factors in root development. Furthermore, AXR3/IAA17 might be involved in the BR signaling pathway, suggesting an intersection node of BR-auxin signaling in root development.

Distinct roles of the first introns on the expression of Arabidopsis profilin gene family members.

Profilin is a small actin-binding protein that regulates cellular dynamics of the actin cytoskeleton. In Arabidopsis (Arabidopsis thaliana), five profilins were identified. The vegetative class profilins, PRF1, PRF2, and PRF3, are expressed in vegetative organs. The reproductive class profilins, PRF4 and PRF5, are mainly expressed in pollen. In this study, we examined the role of the first intron in the expression of the Arabidopsis profilin gene family using transgenic plants and a transient expression system. In transgenic plants, we examined PRF2 and PRF5, which represent vegetative and reproductive profilins. The expression of the PRF2 promoter fused with the beta-glucuronidase (GUS) gene was observed in the vascular bundles, but transgenic plants carrying the PRF2 promoter-GUS with its first intron showed constitutive expression throughout the vegetative tissues. However, the first intron of PRF5 had little effect on the reporter gene expression pattern. Transgenic plants containing PRF5 promoter-GUS fusion with or without its first intron showed reproductive tissue-specific expression. To further investigate the different roles of the first two introns on gene expression, the first introns were exchanged between PRF2 and PRF5. The first intron of PRF5 had no apparent effect on the expression pattern of the PRF2 promoter. But, unlike the intron of PRF5, the first intron of PRF2 greatly affected the reproductive tissue-specific expression of the PRF5 promoter, confirming a different role for these introns. The results of a transient expression assay indicated that the first intron of PRF1 and PRF2 enhances gene expression, whereas PRF4 and PRF5 do not. These results suggest that the first introns of profilin genes are functionally distinctive and the first introns are required for the strong and constitutive gene expression of PRF1 and PRF2 in vegetative tissues.

SUPPRESSOR OF FRIGIDA3 encodes a nuclear ACTIN-RELATED PROTEIN6 required for floral repression in Arabidopsis.

Flowering traits in winter annual Arabidopsis thaliana are conferred mainly by two genes, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). FLC acts as a flowering repressor and is regulated by multiple flowering pathways. We isolated an early-flowering mutant, suppressor of FRIGIDA3 (suf3), which also shows leaf serration, weak apical dominance, and infrequent conversion of the inflorescence shoot to a terminal flower. The suf3 mutation caused a decrease in the transcript level of FLC in both a FRI-containing line and autonomous pathway mutants. However, suf3 showed only a partial reduction of FLC transcript level, although it largely suppressed the late-flowering phenotype. In addition, the suf3 mutation caused acceleration of flowering in both 35S-FLC and a flc null mutant, indicating that SUF3 regulates additional factor(s) for the repression of flowering. SUF3 is highly expressed in the shoot apex, but the expression is not regulated by FRI, autonomous pathway genes, or vernalization. SUF3 encodes the nuclear ACTIN-RELATED PROTEIN6 (ARP6), the homolog of which in yeast is a component of an ATP-dependent chromatin-remodeling SWR1 complex. Our analyses showed that SUF3 regulates FLC expression independent of vernalization, FRI, and an autonomous pathway gene, all of which affect the histone modification of FLC chromatin. Subcellular localization using a green fluorescent protein fusion showed that Arabidopsis ARP6 is located at distinct regions of the nuclear periphery.

The SOC1 MADS-box gene integrates vernalization and gibberellin signals for flowering in Arabidopsis.

The floral transition in Arabidopsis is regulated by at least four flowering pathways: the long-day, autonomous, vernalization, and gibberellin (GA)-dependent pathways. Previously, we reported that the MADS-box transcription factor SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) integrates the long-day and vernalization/autonomous pathways. Here, we present evidences that SOC1 also integrates signaling from the GA-dependent pathway, a major flowering pathway under non-inductive short days. Under short days, the flowering time of GA-biosynthetic and -signaling mutants was well correlated with the level of SOC1 expression; overexpression of SOC1 rescued the non-flowering phenotype of ga1-3, and the soc1 null mutant showed reduced sensitivity to GA for flowering. In addition, we show that vernalization-induced repression of FLOWERING LOCUS C (FLC), an upstream negative regulator of SOC1, is not sufficient to activate SOC1; positive factors are also required. Under short days, the GA pathway provides a positive factor for SOC1 activation. In contrast to SOC1, the GA pathway does not regulate expression of other flowering integrators FLC and FT. Our results explain why the GA pathway has a strong effect on flowering under short days and how vernalization and GA interact at the molecular level.

Composition of seed storage proteins changed by glutathione treatment of soybeans.

The application of glutathione to immature soybean cotyledons reduced the accumulation of the beta subunit of beta-conglycinin, and increased the accumulation of most glycinins. Both reduced and oxidized forms of glutathione had these effects. The application of an inhibitor of glutathione synthesis, buthionine sulfoximine, increased accumulation of beta subunit. These results suggest that glutathione is important in affecting the composition of seed storage proteins.

Petunia actin-depolymerizing factor is mainly accumulated in vascular tissue and its gene expression is enhanced by the first intron.

Actin-depolymerizing factor (ADF) is one of the actin cytoskeleton-modulating proteins. We have characterized the accumulation pattern of petunia ADF proteins. PhADF proteins are accumulated in every petunia organ and their accumulation is differentially regulated by developmental signals. Their cellular localization is vascular tissue-preferential in vegetative organs, whereas somewhat different in reproductive organs. In reproductive organs, PhADFs are present in outer integument, endocarp of ovary wall, transmitting tissue of style, and epidermis and endothecium of young anther. From a petunia genomic library, we have isolated a genomic clone encoding PhADF1. Comparison to complementary DNA sequence revealed that the coding region of PhADF1 gene consists of three exons and two introns. Analysis of chimeric gene expression using beta-glucuronidase as a reporter gene in transgenic Arabidopsis revealed that PhADF1 was strongly expressed in every vegetative tissue except petal. In addition, expression of the gene was highly enhanced by its first intron. These results suggest that PhADF1 gene of petunia is mainly expressed in vascular tissues and its expression is regulated by intron-mediated enhancement mechanism.

Plant regeneration from callus cultures ofLithospermum erythrorhizon.

We previously studied the production of shikonin derivatives by cell lines ofLithospermum erythrorhizon. As a result, we have obtained a cell line LE 87, which exhibited high cell growth and high shikonin production. In the present study, the effects of auxins (2,4-D, IAA, picloram, and NAA) and cytokinins (BAP and kinetin) on organogenesis and somatic embryogenesis in this shikonin-producing cell line were investigated. The highest organogenic and embryogenic efficiency was obtained on MS medium supplemented with 10 µM NAA and 0.3 µM kinetin. Subcultured calli showed different morphogenic frequencies depending on the NAA and kinetin concentration. Morphologically normal plants have been regenerated via mostly organogenesis. Shoots subsequently produced roots on plant growth regulator-free MS medium and developed into plantlets. In most cases, a few thin roots were formed at the bases of the shoots after four weeks on the rooting medium. More than fifty green plantlets were transplanted to soil in pots and developed into phenotypically normal plants 8 weeks after being transferred to soil. The regenerated plants grew to maturity, flowered, and set seeds by only artificial pollination.