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This experiment was carried out to select resistant cultivar to CMV in cucumber using Elisa-test and protoplast isolation method. Twenty domestic cultivars or lines and 8 European cultivars were ested for resistance by Elisa test. Among the domestic cultivars, DADAKI group was found to be susceptible and CHEONGJANG group resistant. Among all the cultivars and lines tested, a European cultivar, DALIBOR and Janghyungheukjinju Korean line were found to be highly resistant. When comparing for the protoplast yield depending upon the positions of seedlings (cotyledon, young leaf and hypocotyl), the highest protoplast yield could be obtained from cotyledons in macerozyme 0.5% + cellulose 2.0%. Protoplast yields in susceptible cultivars were higher than those from resistant cultivars. Differences in cell wall thickness between susceptible and resistant cultivars were observed.
Three potato (Solanum tuberosum L.) cultivars `Superior', `Irish Cobbler', and `Jopung' were transformed by co-cultivation with tuber discs and disarmed Agrobacterium tumefaciens LBA4404 carrying modified vector pBI121, that contained PLRV coat protein (CP) gene and controlled by CaMV35S promoter. Putative transformants were selected and their genomic DNA and RNA transcripts were analyzed for the confirmation of genetic stability by RT-PCR, PCR, southern, and northern blot. The growth characteristics and viral resistance of progenies of transgenic potato plants were investigated. Twelve lines among the different seven-times manipulated transgenic lines were grown in greenhouse and isolates trial field. PLRV coat protein gene was stably inherited in `Superior', but not in `Jopung'. `Jopung' was less stable than `Irish Cobbler' and `Superior' at genetic stability of PLRV CP gene. And some of these transgenic lines were highly resisted in PLRV multiplication. The yield of transformants was reduced in `Irish Cobbler' but not in `Superior'. Possible explanations for these types of resistance are gene silencing and positional effects of transformed PLRV CP genes and that had cultivar specificity. We consider the appearance of escaped transformants in `Jopung' for emergence of chimeric explants from early selection stage.
Anti-bacterial peptide gene (shiva) was introduced into potato plants to improve the resistance to bacterial diseases. The 21 potato clones were selected in the medium containg 50 mg/L kanamycin and 13 transformants were confirmed by GUS activity assay using 4-methylumbellyferyl glucuronide (MUG) and PCR by NPTII specific primer sets. A 0.5-kb band was confirmed by PCR in the most transformants of T0, T1, and T2 generations. As a result of PCR with primer set chosen at shiva and GUS, expected 690-bp fragments were produced in the most transformants of T0, T1, and T2 generations. For southern blot analysis, potato genomic DNA digested with HindIII was separated on 0.7 % agarose gel, transferred to nylon membranes, and detected with nitroblue tetrazolium salt (NBT). As a result of southern analysis, different single bands were detected in the transformants with tPAL5 promoter and 1 to 3 bands were acquired in the transformants with CaMV35S promoter. To analysis protein level of transformants, NPTII ELISA kit were used. In several transformants, optical density (O.D.) values were 10- to 20-fold higher than non-transformants. Tubers were screened for resistance to Erwinia carotovora subsp. carotovora. The concentration of the inocula was 106 cells/mL. After inoculation, tuber slices were incubated aerobically for 48 h at 20 °C. The symptoms of soft rot in transgenic plants were considerably weakened in comparison with non-transformants.
Antimicrobial peptide gene (shiva) under the promoter of tomato phenylalanine ammonia-lyase (tPAL5) was transformed into potato (Solanum tuberosum L.) plants. Antimicrobial peptide gene was isolated originally from giant silk moth (Hyalophora cecropia) and modified its nucleotide seqnence to increase antimicrobial activity. Phenylalanine ammonia lyase 5 (PAL5) gene was known to express highly by wounding, irradiation, and infection by pathogens. It also expresses specifically on vessel tissues of young roots, stems, and leaves. The vector with shiva and CaMV35S promoter was also introduced into potatoes. The efficiency of regeneration was maximized at the medium containing Zeatin 2 mg/L, NAA 0.01 mg/L, GA3 0.1 mg/L. Putative transgenic potato plants were cultured on the media containing kanamycin 50 mg/L. From the tissue extracts of putative transgenic plants, GUS activity was assayed using 4-methylumbellyferyl glucuronide (MUG) as a substrate for GUS enzyme. In several transformant, GUS activity was 20- to 40-fold higher than non-transformants. Especially, one clone with CaMV35S promoter expressed ≈400-fold higher GUS activity than nontransformants. For histochemical in situ localization of GUS activity, chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide(X-gluc) was used for staining. GUS was highly expressed in the whole tissue of the transformants under CaMV35S promoter, but the other side GUS was expressed especially in the vascular tissues of stems and leaves of transformants with tPAL5 promoter. PCR was carried out at 94 °C for 20 s, 52 °C for 20 s, and 72 °C for 60 s with 45 cycles, using NPTII gene-specific primer set. PCR amplification by NPTII-specific primers confirmed 0.5-kb band in most transformants.
The principal bitter sesquiterpene lactones (BSLs; latucin, 8-deoxylactucin, and lactucopicrin) in six red and four green-pigmented leaf lettuce (Lactuca sativa L. var. crispa L.) cultivars were identified and quantified using high-performance liquid chromatography, proton nuclear magnetic resonance, and liquid chromatography–mass spectrometry and the contribution of each to the overall bitterness was determined. The concentration of each BSL and the total varied significantly among cultivars and there were significant differences resulting from leaf color (green versus red) and morphology (cut versus curled leaves) with red and curled leaf cultivars having higher BSL concentrations. The concentrations of lactucin, 8-deoxylactucin, and lactucopicrin ranged from 2.9 to 17.2, 2.8 to 17.1, and 8.8 to 36.1 μg·g−1 dry weight, respectively, with the total concentration ranging from 14.6 to 67.7 μg·g−1. Bitterness of the cultivars was assessed using a bitter activity value calculated using the concentration and bitterness threshold value for each BSL. Lactucopicrin was the primary contributor to bitterness as a result of its concentration and lower bitterness threshold; its relative proportion of the total bitterness activity value across all cultivars was over 72%. The concentration of individual BSLs differed with leaf location on the plant (i.e., basal, midstalk, and flower stalk). The concentrations in lactucin, 8-deoxylactucin, and lactucopicrin in flower stalk leaves were significantly higher (i.e., 2.9, 12.4, and 5.4 times, respectively) than in basal leaves, with the concentrations increasing acropetally. Genetic differences among cultivars and with leaf location on the plant contribute to the wide range in bitterness in lettuce.
This study was carried out to prove the new variety's originality by using Random Amplified Polymorphic DNA (RAPD) Analysis and to develope the specific markers for distinction new variety from others to database for improving the efficiency of germplasm conservation. The RAPD procedure was used to determine genetic diversity of 13 potato varieties including seven recommended varieties of Korea and six genotypes. Genomic DNAs from the 13 genotypes were amplified using PCR and URP 2F, 4R and 8R primers. URP primers which were 20-mers were received from NIAST (National Institute of Agricultural Science and Technology, Suwon, Korea) and they were shown very high reproducibility because of the high annealing temperature above 55 °C. So, they were known to be very desirable primers to examine the specificity between inter and intra species in various spectra. These 13 lines have many resemblances in plant characteristics each other because `Jopung', '92N09-6', `Daekwan 68', and `Daekwan 70' were originated from `Superior', `Atlantic', `Namsuh', and `Irish Cobbler' respectively. So, there are many difficulties to distinct new variety by the naked eye. The results of this study show that 2 sets of URP primers are very useful to distinct new variety and mutants from others.