The phylogenetic relationships between Korean endemic, Hanabusaya asiatica, and its allied groups, including four genera and nine species, were investigated at the DNA level using randomly amplified polymorphic DNA (RAPD) method. Ten primers out of 80 primers (10-mer) screened gave rise to very high polymorphism (99%) in all of the tested plants, producing 153 randomly amplified DNA fragments. H. asiatica was differentiated from its allied groups at the 0.62 of similarity index of RAPDs. This results were in accordance with previous classification based on palynological studies. It was confirmed that H. asiatica could be placed into Korean endemic and suggested that RAPD technique be used as an additional method of phylogenetic relationship for plant systematics.
Randomly and specifically amplified polymorphic DNA banding patterns based on polymerase chain reaction (PCR) analysis were used to assess the intraspecific genetic variations and relationships within Epimedium koreanum populations. A collection of 21 individuals were classified as different accessions by morphological characters such as leaflet number, shape of leaf base, cauline length, plant height, and leaf area. PCR amplification using 12 primers out of 62 [60 random (10-mer) primers, one 15-mer primer (M13 core sequence), and (GGAT)4] resulted in 89 amplified DNA fragments with polymorphisms (80.9%) in all of the tested plants. Similarity indices between accessions were computed from PCR data, and genetic relationships among intraspecific variations were closely related at the levels ranging from 0.66 to 0.93. These DNA data were not matched well with those of morphological characters because they were divided into two major groups at the similarity coefficient value of 0.74. Primers (VII, VIII) gave rise to monomorphic bands in all of examined plants, but specific primers (M13 core and (GGAT)4 sequences) were found to be very valuable molecular markers to evaluate the interspecific variations in Epimedium koreanum.
Five edible mountain vegetables (Saussurea sp., Aster tataricus, A. scaber, Synurus deltoides, Ligularia fischeri) were investigated on the basis of amplified DNA polymorphisms resulted from PCR–polymerase chain reaction analysis. The sampled plants consisted of 38 individuals in five taxa. Only 10 primers out of 62 [60 random (10-mer) primers, one 15-mer-M13 core sequence, and (GGAT)4 sequence] tested gave rise to polymorphisms in all of the tested plants, producing 176 DNA fragments amplified randomly and specifically. Intraspecific polymorphisms found in each taxa showed intra-variety constancy (31.1% to 40.9%) in the banding patterns of individual plants—Saussurea sp., 31.1%, 15 bands; Aster tataricus, 40.9%, 18 bands; A. scaber, 38.5%, 15 bands; Synurus deltoides, 34.7%, 17 bands; Ligularia fischeri, 38.9%; 22 bands, respectively. All five species were well-differentiated from each other at the 0.93 level of similarity index value. Genetic relationships among intraspecific and interspecific variations were closely related at the levels ranging from 0.62 to 0.99. Based on these results, our PCR analyses support the previous data derived from external morphology of the five edible mountain vegetables, but very low levels of intraspecific variations were detected in all of these taxa.
Lim Hak-Tae and Choi Yu Mi
This study was conducted to find out the effect of sprouting inhibitors under different storage temperatures and reconditioning conditions on the processing quality of potato tubers produced in the alpine area of Korea. A higher sprouting ratio was observed in potatoes stored at 15°C than those at 5°C. In particular, 1% CIPC, was effective in the inhibition of sprouting, keeping the sprouted shoots in less than 2 mm, while rosette-shaped shoots, 12–17 mm, were observed in the CMH (100%) treatment. Atlantic was, in general, lower in reducing sugar contents compared to Superior. Reducing sugar contents in potatoes stored at 15°C were not increased, while potatoes stored at 5°C showed a 1% increase in reducing sugar contents for 180 days after storage. As far as chip color “L” value was concerned, no difference was detected among potato cultivars and sprout inhibitor treatments. Potato chip color was found to be the best from potatoes stored at 15°C for 180 days of storage. However, potatoes stored at 5°C gave rise to poor quality of potato chips with browning and bitter taste. Reconditioning had different effects on potato cultivars in that Atlantic potatoes produced more sprouts when they were reconditioned compared to the control of 15°C potato storage. In terms of the effect of reconditioning on reducing sugar contents, Atlantic sugar contents was reduced reconditioning went on. Sugar contents of Superior, however, was increased after undergoing the decrease for some time. Changes in potato chip color as influenced by reconditioning were in accordance with changes in reducing sugar. Atlantic was much better in chip color than Superior, showing a chip color “L” value of more than 50 in all treatments.
Hak Tae Lim and E.J. Park
The regeneration medium supplemented with 2.0 mg/L BAP and 0.1 mg/L IAA allowed high efficient shoot regeneration from leaf discs and petioles of Cichorium intybus L. var. sativus. Multiple shoots ranged from 10 to 14 per explant were observed only 10 to 15 days after the initial culture. Reduced nitrogen and sucrose levels influenced on shoot regeneration frequency and growth rates. Especially, in C. Intybus L. var. sativus cv. Cesare explants cultured in the medium containing 50 mg/L MS macroelement and 1.5% sucrose displayed high regeneration frequency of 100%.
Hak Tae Lim and Jong Kuk Na
A genetic transformation in Solanum spp. was performed using Agrobacterium tumefaciens: C58:pGV2260:Athb-7. Athb-7 gene, known to be related to water stress and ABA level, one of Arabidopsis thaliana homeobox genes was inserted into pBin-Hyg-Tx. Explants were placed on callus induction medium for 14 days, and then transferred on shoot induction medium. Shoot primordium appeared on callus surface after 2 weeks of culture. About 6 weeks later, 100 putatively transgenic plants were obtained, and DNA was extracted from each plant for PCR analysis. Twenty out of 100 putatively transgenic plantlets turned to be positive, having a band of 800 bp in M.W. corresponding to the hygromycin gene. Both PCR and genomic Southern hybridizations using HPTII and Athb-7 genes as probes showed that these genes were inserted into plant genome.
Hak-Tae Lim, Haeng-Soon Lee, and Tage Eriksson
Plant regeneration of ginseng has been known to be difficult, and there are a few reports on plant regeneration of ginseng via somatic embryogenesis. In vitro flowering has, however, been one of the major drawbacks in these regeneration systems in which BA and GA3 were included in germination and shoot multiplication media. Multiplication of adventitious shoots from a single somatic embryo, abnormal morphology, and vitrified shoots were also observed. All these facts have made successful acclimatization of ginseng plantlets difficult. The purposes of this study were 1) to establish the plant regeneration system via organogenesis, 2) to improve normal plant regeneration via somatic embryogenesis, 3) to improve the efficiency of plant regeneration from protoplast culture, 4) to understand the acclimatization process, 5) to develop effective genetic transformation protocol. Data in relation with all these studies are presented in detail.
Hak-Tae Lim, Haeng-Soon Lee, and Tage Eriksson
Plant regeneration ability of ginseng (Panax ginseng) via organogenesis was studied morphologically and anatomically. Compact callus was introduced from four different types of explants—leaf, petiole, flower stalk, and root—of in vitro-grown plantlets. Petioles were found to be the best material for callus induction. Calli induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (1.0 mg·L–1) and kinetin (0.1 mg·L–1) were conditioned for 2 weeks on the same medium. These calli differentiated into adventitious shoots when cultured on half-strength MS basal medium plus kinetin at 1.0 mg·L–1 and STS at 2.5 mg·L–1. An addition of GA3 (1.0 mg·L–1) and BA (1.0 mg·L–1) to MS basal medium, however, induced high-frequency in vitro flowering (86.1%) and multiple shoot budding, which affected the normal, complete development of plantlets. Plantlets with well-developed root systems were obtained 6 weeks after regenerated shoots had been transplanted to half-strength MS20 medium containing IBA at 0.25 mg·L–1. Nuclear DNA content was measured to check the stability of their ploidy level. Based on DNA flow cytometric analysis, all of the regenerants were typically diploids as were the mothers plants, indicating that nuclear DNA content remained stable during cell differentiation.
Hak-Tae Lim, Eun-Ae Lee, and Won-Bae Kim
This study was conducted to investigate the possibility of obtaining plantlets via somatic embryogenesis and organogenesis as means of in vitro mass propagation in Allium victorialis var. platyphyllum Makino, one of the most popular wild vegetable plants in Korea. Shoots formed directly when bulb explants of A. victorialis were cultured on MS medium containing 0.2 mg·L–1 NAA and 2.0 mg·L–1 zeatin under 16 hours (light)/8 hours (dark) illumination. The use of leaf and shoot tip explants was not successful, largely due to explant senescence in the present of plant growth regulators. Embryogenic calli were obtained from the bulb explants of A. victorialis on MS medium supplemented with 0.2 mg·L–1 NAA, 0.2 mg·L–1 BAP, and 1.0 mg·L–1 picloram after 4–5 weeks of culture in the dark at 27°C. Upon transfer to shoot-induced MS medium containing 0.2 mg·L–1 NAA and 2.0 mg·L–1 zeatin, embryogenic calli gave rise to numerous somatic embryos, which subsequently developed into multiple shoots after 3 months of culture under 16 hours(light)/8 hours (dark) illumination. For root induction, regenerated shoots were transferred to MS medium added with 1.0 mg·L–1 NAA. Regenerants with well-developed roots were potted in an artificial soil mixture of vermiculite (1) and perlite (1).
Hak Tae Lim, Y.S. You, and E.J. Park
In order to regenerate explants of Brassica campestris ssp. pekinensis, known to be one of the most difficult crops to regenerate via organogenesis, three different explants, cotyledon, hypocotyl, and leaf, were cultured on MS basal medium supplemented with several plant growth regulators. In the medium containing NAA at 0.5 mg/L and BAP at 3.0 mg/L, the shoot regeneration, when hypocotyl was used as explant, was found to be quite effective. In the case of cotyledon, the most suitable combination of plant growth regulators was NAA at 1.0 mg/L and BAP at 3.0 mg/L. Treatment of AgNO3 (1.0 mg/L) for shoot regeneration gave positive results in general. Zeatin at 2.0 mg/L was very effective in shoot induction of leaf explant, especially when combined with BAP at 2.0 mg/L, NAA at 1.0 mg/L, and AgNO3 at 0.5 mg/L. A system to produce transgenic plants in Brassica spp. has also been developed using hypocotyl and cotyledonary-petiole segments and shoot-tips. An explants from 4-day-old seedlings were inoculated with an Agrobacterium tumefaciens strain containing a disarmed tumor-inducing plasmid pTiT37-SE carrying a chimaeric bacterial gene encoding hygromycin and kanamycin resistance, along with other genes of interests. The explants were co-cultured for 2 to 6 days before transfer to hygromycin and kanamycin selection media. Shoots regenerated directly from the explants in 1 to 4 weeks and were excised, transferred to shoot elongation medium, rooted in root induction medium, and planted in soil. Genetic transformation was confirmed by kanamycin or hygromycin resistance, GUS activities, and Southern blotting.