Doo-Hwan Kim

Stable transformation of protocorm-like bodies in Phalaenopsis orchid mediated by Agrobacterium tumefaciens

Genetically transformed plantlets of Phalaenopsis were regenerated after co-cultivation of protocorm-like bodies (PLBs) with Agrobacterium tumefaciens strain LBA4404 containing the vector pTOK233 that harbors genes for β-glucuronidase and hygromycin resistance. Four lines of Phalaenopsis orchid (T0, T5, T10 and Hikaru) were tested on three multiplication media for the production of PLBs. Two types of explants, intact and transversely bisected PLBs, were checked for efficiency of multiplication, and it was found that transversely bisected PLBs were much better than intact ones. The effect of hygromycin concentrations on rooting of PLBs was also established. Initial recovery of the PLBs following co-cultivation was allowed without selection for 6 weeks. The first selection was carried out on regeneration medium containing 3 mg/l hygromycin for 2 months. New PLBs, as well as shoots and roots, were formed from original PLBs when they were regenerated under hygromycin selection. Leaves, roots, original and some newly formed PLBs were taken from hygromycin-resistant rooted plantlets and then subjected to GUS assay. Newly formed PLBs from GUS-positive plantlets were then regenerated on the above-mentioned regeneration medium but containing a lower concentration of hygromycin (1.5 mg/l). When the PLBs regenerated into plantlets, original PLBs were subjected to GUS assay, and again newly formed PLBs were regenerated and selected as described above. The procedures were repeated monthly for four cycles and it was found that a long selection period with the low hygromycin concentration yielded stable transformants. Histochemical GUS assay indicated successful transformation, which was further confirmed by PCR analysis and Southern hybridization of transformants.

Transgenic Plants of Colonial Bentgrass from Embryogenic Callus via Agrobacterium-mediated Transformation

Colonial bentgrass (Agrostis tenuis Sibth. Fl. Oxen.) is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable and repeatable approach in transforming the grass using Agrobacterium (strain LBA4404), in which β-glucuronidase (gus) gene was used as a reporter and hygromycin phosphotransferase (hpt) gene as a selectable marker. This vector was effective in transforming 7-week-old calluses derived from mature seeds cultured on MS medium supplemented with 2,4-D. A two-step solid medium selection with increasing hygromycin concentration (from 50 to 70 mg l−1) was used to obtain resistant calluses. Hundreds of transgenic plants have been produced from several independent transformed calluses. The presence of functional β-glucuronidase (GUS) was detected in hygromycin-resistant calluses, young leaves and roots of transgenic plants. The transgenic plants collected from greenhouse showed strong resistance to 50 mg l−1 hygromycin solution. Four putative transgenic plants and one control plant were randomly chosen and analyzed by Southern blot analysis. Bands corresponding to the hpt gene were clearly shown in transgenic plants.

Bactericidal activity of green tea extracts: the importance of catechin containing nano particles.

When we drink green tea infusion, we believe we are drinking the extract of the green tea leaves. While practically each tea bag infused in 300 mL water contains about 50 mg of suspended green tea leaf particles. What is the role of these particles in the green tea effect is the objective of this study. These particles (three different size ranges) were isolated via varying speed centrifugation and their respective inputs evaluated. Live oral bacterial samples from human volunteers have been screened against green tea extracts and macro, micro and nano sized green tea particles. The results showed that the presence/absence of the macro and mico sized tea particles in the green tea extract did not contribute much. However, the nano sized particles were characterized to be nature’s nano stores of the bioactive catechins. Eradication of these nano tea particles resulted in decrease in the bactericidal property of the green tea extracts. This is a curtain raiser investigation, busting the nano as well as green tea leaf particle contribution in green tea extracts.

Onion extract structural changes during in vitro digestion and its potential antioxidant effect on brain lipids obtained from low- and high-fat-fed mice.

Abstract This study investigated the effects of onion (Allium cepa, L.) extract on the antioxidant activity of lipids in low-and high-fat-fed mouse brain lipids and its structural change during in vitro human digestion. The onion extracts were passed through an in vitro human digestion model that simulated the composition of the mouth, stomach, and small intestine juice. The brain lipids were collected from low- and high-fat-fed mouse brain and then incubated with the in vitro-digested onion extracts to determine the lipid oxidation. The results confirmed that the main phenolics of onion extract were kaempferol, myricetin, quercetin, and quercitrin. The quercetin content increased with digestion of the onion extract. Antioxidant activity was strongly influenced by in vitro human digestion of both onion extract and quercetin standard. After digestion by the small intestine, the antioxidant activity values were dramatically increased, whereas the antioxidant activity was less influenced by digestion in the stomach for both onion extract and quercetin standard. The inhibitory effect of lipid oxidation of onion extract in mouse brain lipids increased after digestion in the stomach. The inhibitory effect of lipid oxidation of onion extract was higher in the high-fat-fed mouse brain lipids than that in the low-fat-fed mouse brain lipids. The major study finding is that the antioxidative effect of onion extract may be higher in high-fat-fed mouse brain lipids than that in low-fat-fed mouse brain lipids. Thus, dietary onion may have important applications as a natural antioxidant agent in a high-fat diet.

Agrobacterium tumefaciens-mediated hairy root production from seedlings of Chinese cabbage

Cruciferous hairy roots are often used for improving drought adaptability, peroxidase production, andin vitro subculturing ofPlasmodiophora brassicae. For metabolic engineering,Agrobacterium tumefaciens-mediated systems have previously been developed for hairy root production in other plant species. Here, we used therolABC gene binary construct inA. tumefaciens strain GV3101 to establish cultures of Chinese cabbage hairy roots. On both solid and liquid media, therolABC hairy root lines exhibited a wild-type hairy root syndrome in terms of their growth and morphology. This demonstrates that those three genes are sufficient to induce high-quality hairy roots in Chinese cabbage. Such a system could be useful for the stable production of secondary metabolites in that species.