To develop transgenic forage crops with enhanced tolerance to abiotic stress, we introduced an alfalfa Hsp23 gene expression vector construct through Agrobacterium-mediated transformation. Integration and expression of the transgene were confirmed by PCR, northern blot, and western blot analyses. Under normal growth conditions, there was no significant difference in the growth of the transgenic plants and the non-transgenic controls. However, when exposed to various stresses such as salt or arsenic, transgenic plants showed a significantly lower accumulation of hydrogen peroxide and thiobarbituric acid reactive substances than control plants. The reduced accumulation of thiobarbituric acid reactive substances indicates that the transgenic plants possessed a more efficient reactive oxygen species-scavenging system. We speculate that the high levels of MsHsp23 proteins in the transgenic plants protect leaves from oxidative damage through chaperon and antioxidant activities. These results suggest that MsHsp23 confers abiotic stress tolerance in transgenic forage crops and may be useful in developing stress tolerance in other crops.

Abiotic stress is the major limiting factor of forage crops growth and yields. The objective of this work was to study the stress tolerance and regeneration capability of transgenic forage crops carrying a MsHSP23 gene, encoding a alfalfa mitochondrial sHSP protein. The expression of the MsHSP23 gene was confirmed in bacteria, recombinant mHSP23 conferred tolerance to salinity and arsenic stress. Furthermore, mHSP23 was cloned in a plant expressing vector and transformed into forage crops such as alfalfa, tall fescue and bent grass. The transgenic plants exhibited enhanced tolerance to salinity and arsenic stress conditions. In comparison to wild type plants, transgenic plants were exhibited significantly lower electrolyte leakage. Moreover, the transgenic plants had superior germination rates when placed on medium containing arsenic. Taken together, these overexpression results imply that mHSP23 plays an important role in salinity and arsenic stress tolerance in transgenic forage crops. This approach could be useful to develop stress-tolerant plants including forage crops.

The molecular responses to various abiotic stresses were investigated by the approaches with transcriptomic analysis based on an ACP system. Here we identified differentially expressed genes under abiotic stresses in alfalfa seedlings and they were mostly unknown genes and a few common stress-related genes. Among them, mitochondrial small HSP23 was responded by the diverse stress treatment such as heat, salt, As stresses and thus it could be a strong candidate that may confer the abiotic stress tolerance to plants. When expressed in bacteria, recombinant MsHSP23 conferred tolerance to salinity and arsenic stress. Furthermore, MsHSP23 was cloned in a plant expressing vector and transformed into tobacco, a eukaryotic model organism. The transgenic plants exhibited enhanced tolerance to salinity and arsenic stress under ex vitro conditions. In comparison to wild type plants, the transgenic plants exhibited significantly lower electrolyte leakage. Moreover, the transgenic plants had superior germination rates when placed on medium containing arsenic. Taken together, these overexpression results imply that MsHSP23 plays an important role in salinity and arsenic stress tolerance in transgenic tobacco. The results of the present study show that overexpression of alfalfa mitochondrial MsHSP23 in both eukaryotic and prokaryotic model systems confers enhanced tolerance to salt and arsenic stress. This indicates that MsHSP23 could be used potentially for the development of stress tolerant transgenic crops, such as forages.

Tall fescue (Festuca arundinacea Schreb.) is an important cool season forage plant that is not well suited to extreme heat, salts, or heavy metals. To develop transgenic tall fescue plants with enhanced tolerance to abiotic stress, we introduced a MsHsp23 gene expression vector construct through Agrobacterium-mediated transformation. Integration and expression of the transgene were confirmed by PCR, northern blot, and western blot analyses. Under normal growth conditions, there was no significant difference in the growth of the transgenic plants and the non-transgenic controls. However, when exposed to various stresses such as salt or arsenic, transgenic plants showed a significantly lower accumulation of hydrogen peroxide and thiobarbituric acid reactive substances than control plants. We speculate that the high levels of MsHsp23 proteins in the transgenic plants protect leaves from oxidative damage through chaperon and antioxidant activities. These results suggest that MsHsp23 confers abiotic stress tolerance in transgenic tall fescue and may be useful in developing stress tolerance in other crops. Compared with traditional plant breeding, genetic engineering provides a relatively fast and precise means of achieving improved stress tolerance of forage crops. Development of forage crops that are more tolerant to various abiotic stresses could lead to the use of more new lands for cultivation.

Italian ryegrass (Lolium multiflorum Lam.) is one of important forage crop grass widely cultivated in Korea. Progress in breeding using conventional selection procedure is very slow, since Italian ryegrass is highly self-infertile. Biotechnological approaches, therefore, may contribute to the development of improved cultivars for forage crops. In an effort to optimize tissue culture responses of Italian ryegrass (Lolium multiflorum Lam.) for future genetic manipulations to improve forage characteristics, the effects of culture medium supplements on tissue culture responses were investigated with mature seeds of Korean Italian ryegrass (Lolium multiflorum Lam.) seven cultivars as explant tissues.

Microcystis aeruginosa is common form of cyanobacteria (blue-green algae) capable of producing toxic heptapeptide (microcystin) that cause illness or death. The comparison of molecular genetic method with the morphological characteristics of cyanobacteria was conducted. We have designed PCR primers (JJM98F, JJM1141R) for cyanobacterial 16S rRNA and phycocyanin intergenic spacer (PC-IGS) gene domain. To confirm the production of microcystins, PCR primers for the N-methyltransferase (NMT) domain of microcystin synthetase gene mcyA were designed using 21 cyanobacteria strains Most of isolated strains from the Nakdong River was classified as Microcystis aeruginosa and the similarities were 99% with M. aeruginosa AF 139292. 38.1% of isolated strains contained microcystin synthesis gene. NMT (N-methyltransferase) were not detected in isolated strain in several strains, which means non-toxic. However, the NMTs of the strains were detected during the cultivation.