Background : Temperature is major factor for growth plant. Recently, because of global warming, abnormal temperature included drought, deluge, sudden temperature change and heavy snow damaged crops in the world. In Korea, crops have been sensitive to low temperature on early growth stage, e.g. fruit tree and ginseng, were damaged owing to sudden heavy snow and cold on Spring. Therefore, recently interest in cold resistance crops were increased in demand rapidly. This study was performed to establish transgenic Nicotiana benthamiana by transforming cold resistant gene related to cold tolerance S-adenosylmethionine synthetase (SAMS) isolated from Miscanthus sinensis. Methods and Results : Total RNA was extracted from leaves of M. sinensis using Trizol assay and isolated MsSAMS. Isolated MsSAMS was insert into SacⅠ- XbaⅠ sites of pMBP1 vector. The vector was transformed to Agrobacterium tumefaciens strain LBA4404 by DH5α. A. tumefaciens with binary plasmid were selected at YEP medium supplemented with kanamycin. Cut leaves of tobacco were co-cultured with selected A. tumefaciens. Co-cultured leaves was grown on regeneration medium for a month at dark condition, and transferred to at light condition. Regeneration shoot from callus were excised and transferred to root-induction medium. Approximately, 58% of leaves explant produced callus. Nearly, 30% of callus had shoot and approximately, 94% of shoots were rooted in root-induction medium. Conclusion : We established an efficient transformation system of N. benthamiana transformed by using MsSAMS gene related to cold tolerance isolated from M. sinensis. We may use the produced transgenic plants to prevent damages carried by cold.

Background : Miscanthus is a diploid hybrid and a temperate, perennial, cross-pollinating grass used as bioenergy plant, biomass production and high quality cellulose and ethanol production. This study was to determine an efficient transformation system for Miscanthus sinensis, and to optimize factors and conditions required for expression of MsCOMT–AS gene. Methods and Results : An efficient transformation of callus from M. sinensis was established using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pMBP1. In order to establish stable transformation system, we obtained high transformation rate from callus by various transformation factor explant type, strain, co-culture periods, acetosyringone concentration, and selective marker. Finally, in this study, seven putative transgenic plants were obtained. Through various tests including PCR analysis and southern blot were to detect antisense of COMT digested Xba I and Sac I restriction enzymes. The biomass of the control plant was superior than transgenic plants. Conclusion : This study was to develop transgenic Miscanthus sinensis by Agrobacterium tumerfeciens mediated transformation to produce high bioethanols and to reduce the lignin content of transgenic plants. Detailed characterization of the transgenic plants revealed interesting finding about COMT gene expression in the segregates

More than 300 Miscanthus accessions as a potential bioenergy crop were collected in Korea and their morphological traits were investigated at various growth stages. Among morphological traits, stem growth habit, the presence of awn in spikelet, and autumn new shoot are the most important key traits enabling to cluster Miscanthus accessions into M. sinensis and M. sacchariflorus groups. Miscanthus sinensis has bunch stem growth habit and awn in spikelet, and produces autumn new shoot, while M. sacchariflorus has scattering stem growth habit with no awn in spikelet and does not produce autumn new shoot. Interestingly, we found several Miscanthus accessions showing intermediate morphological traits. 7 M. sinensis accessions showed morphological traits similar to M. sacchariflorus and 17 M. sacchariflorus accessions showed morphological traits similar to M. sinensis. Flow cytometry and chromosome counting finally revealed 5 Miscanthus hybrids, suggesting that they are resulted from natural hybridization between M. sinensis and M. sacchariflorus. Therefore, these Miscanthus hybrids can be used to understand genetic recombination between these two Miscanthus species and our understanding may support future efforts for breeding new Miscanthus variety with high biomass productivity and environmental adaptability.

바이오에너지 작물로 주목받고 있는 억새(Miscanthus sinensis)의 성숙 종자로부터 캘러스를 유도하고 식물체를 재생시키는 방법을 개발하였다. 억새 종자로부터의 캘러스 유도에 적합한 조건을 알아보고자 기본배지와 2,4-D의 농도, proline의 농도, 배지고형제의 종류와 농도 처리를 각각 실시하였다. 기본배지로 MS배지와 N6배지를 두고 2,4-D를 농도별로 처리하였을 때, MS 배지에 2,4-D 4.0 mg/L를 첨가한 처리구에서 캘러스 형성율이 42.2%로 가장 높았다. Proline은 종자로부터 캘러스 형성을 증가시키는 효과가 있었고, 3.0 g/L 이하의 저농도에 비해 12.0 g/L를 첨가하는 것이 캘러스 형성에 좋았다. 배지 고형제는 agar에 비해 Phytagel이 효과적이었으며 Phytagel 2.25 g/L를 첨가한 처리구에서 캘러스 형성율이 86.1%로 가장 높았다. 억새의 종자로부터 유도된 캘러스는 4주 간격으로 계대배양하였고, 캘러스 유도 3개월 후 진한 노란색의 둥근 돔 형태를 갖는 캘러스를 분리하여 신초 재생에 이용하였다. 종자로부터 유도된 캘러스를 호르몬 무첨가 MS 배지 또는 MS 배지에 BA 1.0 mg/L를 첨가한 배지에서 배양하여 신초 재생을 유도할 수 있었다. 재생된 신초는 신장과 발근을 거쳐 소식물체로 발달하였으며, 소식물체는 성공적으로 활착되었고 정상적인 표현형을 나타냈다.

본 연구는 최근 한방자원, 사료자원, 바이오에너지 자원 등 다양하게 이용되는 국내 자생 억새(Miscanthus sinensis)의 대량생산 및 신품종 개발을 위한 조직배양체계 확립을 위해 수행되었다. 이를 위해 억새 완숙종자로부터의 캘러스 유도와 재분화를 위한 식물생장조절제의 적정농도를 규명하였다. 억새의 성숙종자유래 배발생 캘러스 유도를 위해 2,4-D, IBA, NAA를 1~10 mg·L-1의 농도로 단용 처리한 결과, 5 mg·L-1 2,4-D 처리에서 가장 높은 85.3%의 캘러스 유도율과 캘러스의 증식을 보였으며 조직배양 과정 중 갈변화율도 가장 낮았다. 또한, 캘러스의 재분화를 위해 옥신인 NAA와 Kinetin, 2-iP, 또는 BAP 등의 사이토키닌을 혼용 처리한 결과, 각각 19.0%~59.0%, 23.0%~67.3%, 14.7%~83.7%의 재분화율을 보여 NAA와 BAP의 혼용 처리구가 NAA와 Kinetin 또는 2-iP와 혼용 처리구보다 식물체 재분화에 효과적이었다. 특히 3 mg·L-1 NAA와 5 mg·L-1 BAP 혼용 처리된 배지에서의 재분화율이 83.7%로 가장 높게 나타났으며, 캘러스 당 재분화 식물체 개수도 5.5개로 동일농도의 2-iP 또는 Kinetin 혼용 처리 시 2.1 및 2.0개보다 많았다. 본 연구결과 억새 성숙 종자로부터의 배발생 캘러스 유도에는 5 mg·L-1 2,4-D가 그리고 캘러스의 재분화에는 3 mg·L-1 NAA와 5 mg·L-1 BAP 혼용 처리가 가장 효율적이었다. 본 연구를 통해 확립된 조직배양체계는 억새의 대량생산 및 신품종 개발에 유용할 것으로 판단된다.

Over the past few decades Miscanthus has been studied as a potential bioenergy crop in Europe but not much been studied in Korea although Korea has many native Miscanthus species and genetic resources. Because of their features, such as low demands of fertilizers and other agrochemicals, high water use efficiency and high productivity, Miscanthus can be cultivated in marginal lands. However, it is not known how Miscanthus can survive in saline soil and what extent Miscanthus can tolerate salt stress, one of main abiotic stresses in reclaimed lands or dry climates. Therefore, this study was conducted to investigate physiological response of Miscanthus species to salt stress and thus to establish a salt stress threshold. Miscanthus sinensis and M. sacchariflorus showed similar responses to salt stress. Chlorophyll contents, photosynthesis and plant growth were all significantly affected by salt stress. Both species were completely killed at > 100 mM NaCl. The GR50 values estimated by non-linear regression analysis with the logistic model were 116.2 mM and 63.1 mM for M. sinensis and M. sacchariflorus, respectively. These GR50 values can be used as their thresholds of salt stress. Further studies will be conducted to screen salt-tolerant Miscanthus genotypes.