Lee Byung-Hyun

Effects of Plant Growth Regulators and Culture Medium Supplements on Embryogenic Callus Induction from Seeds of Zoysiagrass

In order to optimize tissue culture conditions for genetic transformation of zoysiagrass (Zoysia japonica Stued.), the effect of plant growth regulators and culture medium supplements on embryogenic callus induction from mature seeds of a cultivar `Zenith` were investigated. The optimal concentration and treatment period of NaOCl is 30% (v/v) for 60 minutes. Cultivation of mature seed on the callus Induction medium containing 3 mg/L 2,4-D and 3 mg/L dicamba showed 17.5% of embryogenic callus formation frequency. Supplementation of 1 g/L casein hydrolysate and 500 mg/L L-proline improved frequency of embryogenic callus induction. Audition of the medium with 5 mg/L and 20 mg/L cysteine enhanced frequencies of embryogenic callus induction. Efficient callus induction system established in this study will be useful for molecular breeding of Boysiagrass through genetic transformation.

Effect of Plant Growth Regulators and Antioxidants on Callus Induction and Plant Regeneration from Seed Culture of Orchardgrass

In order to optimize tissue culture conditions for genetic transformation of orchardgrass, the effects of culture medium supplements on tissue culture responses were investigated with mature seeds of 3 cultivars, `Frode`. `Roughrider` and `Frontier` as explant tissues. Callus induction medium containing 3mg/L 2,4-D or 3mg/L dicamba each with 0.1mg/L BA was optimal fer embryogenic callus formation from mature seed and had a strong effect on successive plant regeneration. The regeneration frequency from embryogenic callus among cultivars were descending order of Roughrider > Frode > Frontier. Supplementation of the regeneration media with 10mg/L and 40mg/L cysteine enhanced frequency of plant regeneration. Efficient regeneration system established in this study will be useful fur molecular breeding of orchardgrass through genetic transformation.

Effect of plant growth regulators on callus induction and plant regeneration from mature seed culture of Miscanthus sinensis.

In order to optimize tissue culture conditions for genetic transformation of Miscanthus sinensis , we investigated the effects of different plant growth regulators on callus induction and plant regeneration using mature seeds as explant. Dehusked mature seeds were cultured on MS medium supplemented with 3 to 10 mg/L 2,4-D, dicamba or NAA, 30 g/L sucrose and 750 mg/L MgCl 2 6H 2O. A number of combinations of auxin and cytokinin (BA, kinetin) were also used. MS medium containing 3 mg/L 2,4-D was found optimal for embryogenic callus induction (75.7%) from mature seed. The highest number of plants were regenerated (44.6%) upon transferring the embryogenic callus to MS medium supplemented with 1 mg/L 2,4-D plus 2 mg/L BA. This high efficient plant regeneration system could be useful to use for molecular breeding of new cultivars by genetic transformation. (Key words : Callus, Plant growth regulators, Miscanthus )

Proteome analysis of alfalfa roots in response to water deficit stress

Abstract To evaluate the response of alfalfa to water deficit (WD) stress, WD-induced candidates were investigated through a proteomic approach. Alfalfa seedlings were exposed to WD stress for 12 and 15 days respectively, followed by 3 days re-watering. Water deficit increased H2O2 content, lipid peroxidation, DPPH (1,1-diphenyl-2-picrylhydrazyl)-radical scavenging activity, and the free proline level in alfalfa roots. Root proteins were extracted and separated by two-dimentional polyacrylamide gel electrophoresis (2-DE). A total of 49 WD-responsive proteins were identified in alfalfa roots; 25 proteins were reproducibly found to be up-regulated and 24 were down-regulated. Two proteins, namely cytosolic ascorbate peroxidase (APx2) and putative F-box protein were newly detected on 2-DE maps of WD-treated plants. We identified several proteins including agamous-like 65, albumin b-32, inward rectifying potassium channel, and auxin-independent growth promoter. The identified proteins are involved in a variety of cellular functions including calcium signaling, abacisic acid (ABA) biosynthesis, reactive oxygen species (ROS) regulation, transcription/ translation, antioxidant/detoxification/stress defense, energy metabolism, signal transduction, and storage. These results indicate the potential candidates were responsible for adaptive response in alfalfa roots.

Effect of Callus Type and Antioxidants on Plant Regeneration and Transformation of Tall Fescue

An efficient transformation system for the production of transgenic plants has been developed for tall fescue (Festuca arundinacea Schreb.) via Agrobacterium-mediated transformation of seed-derived callus. From the point of morphogenetic capacity, three types of callus were selected. High frequency of plant regeneration was obtained by selection of type II callus, and the plant regeneration frequency was 52.6% when embryogenic callus were cultured on the regeneration medium. Supplementation of the media with 10 mg/L and 40 mg/L cysteine enhanced frequencies of plant regeneration up to 65.3%. The highest transformation efficiency was also obtained when type II callus were inoculated with Agrobacterium. Southern blot analysis of PCR products of transgenic plants demonstrated that transgenes were successfully integrated into the genome of tall fescue. Efficient regeneration system and transformation established in this study will be useful for molecular breeding of tall fescue through genetic transformation.

Effect of Plant Growth Regulators and Medium Supplements on Plant Regeneration of Kentucky Bluegrass

To optimize tissue culture responses for genetic transformation of Kentucky bluegrass, the effects of culture medium supplements on tissue culture responses were investigated with mature seeds of a cultivar `Newport` as explant tissues. The optimal concentration of 2,4-D (2.4-dichloro phenoxy acetic acid) for the induction of embryogenic callus from mature seed was 3 mg/L. Plant regeneration frequency was 54% when embryogenic callus was cultured on the regeneration medium supplemented with 1 mg/L 2,4-D and 3 mg/L of BA (6-benzyladenine). Addition of 1 g/L of casein hydrolysate and 500 mg/L of L-proline improved frequencies of embryogenic callus induction and plant regeneration up to 60.8% and 58.3%, respectively. Regenerated plants were grown normally when shoots transplanted to the soil. A rapid and efficient plant regeneration system established in this study. We suggest that the results may be useful for molecular breeding of Kentucky bluegrass through genetic transformation.

Production of Transgenic Birdsfoot Trefoil (Lotus corniculatus L.) Plants by Introduction of E35S Promoter + AtNDPK2 Gene

To develop transgenic birdsfoot trefoil (Lotus corniculatus L.) plants tolerant to environmental stress, Arabidopsis NDPK gene (AtNDPK) was introduced into birdsfoot trefoil plants using Agrobacterium-mediated transformation and expressed powerfully under the control of the E35S promoter. The expression vector, pEN-K was used for introduction of AtNDPK gene into birdsfoot trefoil plaits. The transformed calli were selected on kanamycin containing medium and then regenerated. The transformed birdsfoot trefoil plants were cultivated for 4 months on BOi2Y medium. Genomic DNA PCR and Southern blot analysis confirmed the incorporation of AtNDPK into the birdsfoot trefoil genome.

Development of High Quality Forage Grass by Down-regulating Lignin Biosynthetic Gene

To develop a new variety of orchardgrass with improved digestibility, caffeic acid O-methyltransferase (Dgcomt), which is a methylation enzyme involved in the early stages of lignin biosynthesis, was isolated and characterized. Dgcomt was expressed not only in leaves but also in stems and roots. The expression levels of transcripts were high in stems and roots which are the most lignified tissues, and only moderate levels of transcripts were expressed in leaves. To develop transgenic orchardgrass plants by down-regulating the Dgcomt gene, an RNAi suppression vector with partial Dgcomt DNA fragment was constructed and transferred into the genome of orchardgrass via Agrobacterium-mediated gene transfer method. PCR and Southern blot analyses with genomic DNAs from putative transgenic plants revealed that the T-DNA region containing RNAi construct was successfully integrated into the genome of orchardgrass. Northern blot analysis revealed that the majority of the down-regulated transgenic lines showed significant reduction in Dgcomt gene expression. These RNAi transgenic orchardgrass will be useful for molecular breeding of new variety with improved digestibility by down-regulating lignin biosynthetic enzyme.