Search

The phenolics concentrations of rapeseed are important because they are antinutritional com-pound or directly positive related to antioxidant actives. In this study, single nucleotide poly-morphisms (SNPs) identified from genotyping-by-sequencing (GBS), and genome-wide associa-tion study of phenolic contents were investigated in 95 rapeseed mutant lines derived from gamma rays and their original cultivar. A total of 3,196,318 SNPs were detected, identifying a set of 70,208 union SNPs used to perform association studies. Six phenolics and glucosinolate (pro-goitrin) were identified, sinapine was the major phenolic compound (91.2–99.2%) in all geno-types. The association study was conducted for six phenolic compounds, progoitrin, total sinap-ine content (TSC), total phenolic content (TPC) and seed yield. The association study identified 241 SNPs that were significantly associated with six phenolic compounds, progoitrin, TSC and TPC. Based on the detected SNP markers, BnaA06g31740D, a gene associated progoitrin, BnaC-nng39930D, a gene associated trans-sinapine 1, BnaA07g31720D, a gene associated trans-sinapine 2, BnaC03g31950D and BnaA06g09180D, a gene associated TSC, and BnaAnng27700D, a gene associ-ated TPC, were selected as candidate genes. The SNP makers associated major phenolic com-pounds identified and could be used as targets for breeding programs aimed at improving anti-oxidant contents and reducing antinutritional components.

Rapeseed (Brassica napus L.) is one of the most oil crop, and its commercial value is contingent upon its agronomic characteristics and oil quality. In this study, 73,226 single nucleotide polymorphisms (SNPs) across 95 rapeseed mutant lines derived from gamma rays and their original cultivar (‘Tamra’) obtained from genotyping-by-sequencing (GBS) was investigated gene ontology (GO) analysis and genome-wide association study (GWAS). GWAS was conducted on agronomic (plant height, ear length, thousand seed weight and seed yield) and oil (fatty acid and crude fat) traits. GO analysis showed that many genes displaying SNPs were involved in cellular processes, intracellular anatomical structures and organic cyclic compound binding. A total of 149 significant SNPs were associated with the agronomic traits (76 SNPs) and oil traits (73 SNPs). Bna.A05p02350D (SFGH) and Bna.C02p22490D (MDN1) were selected as novel candid genes for thousand seed weight. In addition, Bna.C03p14350D (EXO70) and Bna.A09p05630D (PI4Kα1) were selected as novel candidate genes for the erucic acid and crude fat content, respectively. These findings could facilitate the identification of optimal genotypes for breeding new cultivars, and association studies represent an economically efficient tool for mutant screening and the selection of elite rapeseed-breeding lines.

Soybean seeds are consumed worldwide owing to their nutritional value and health benefits. In this study we investigated the metabolic properties of 208 soybean mutant diversity pool (MDP) lines by measuring the isoflavone and fatty acid contents of the seed. The total isoflavone content (TIC) ranged from 0.88 mg/g to 7.12 mg/g and averaged 3.08 mg/g. The proportion of oleic acid among total fatty acids (TFA) ranged from 0.38% to 24.66% and averaged 11.02%. Based on the TIC and TFA among the 208 MDP lines, we selected six lines with altered isoflavone content and six lines with altered oleic acid content compared with those of the corresponding wild-types for measuring gene expression. Each of twelve genes from the isoflavone and fatty acid biosynthesis pathways were analyzed at three different seed developmental stages. Isoflavone biosynthetic genes, including CHI1A, IFS1, and IFS2, showed differences in stages and expression patterns among individuals and wild-types, whereas MaT7 showed consistently higher expression levels in three mutants with increased isoflavone content at stage 1. Expression patterns of the 12 fatty acid biosynthetic genes were classifiable into two groups that reflected the developmental stages of the seeds. The results will be useful for functional analysis of the regulatory genes involved in the isoflavone and fatty acid biosynthetic pathways in soybean.

Mutation breeding is useful for improving agronomic characteristics of various crops. In this study, we conducted a genetic diversity and association analysis of soybean mutants to assess elite mutant lines. On the basis of phenotypic traits, we chose 208 soybean mutants as a mutant diversity pool (MDP). We then investigated the genetic diversity and inter-relationships of these MDP lines using target region amplification polymorphism (TRAP) markers. Among the different TRAP primer combinations, polymorphism levels and polymorphism information content (PIC) values averaged 59.71% and 0.15, respectively. Dendrogram and population structure analyses divided the MDP lines into four major groups. According to an analysis of molecular variance (AMOVA), the percentage of inter-population variation among mutants was 11.320 (20.6%), whereas mutant intra-population variation ranged from 0.231 (0.4%) to 14.324 (26.1%). Overall, intra-population genetic similarity was higher than that of inter-populations. In an analysis of the association between TRAP markers and agronomic traits using three different statistical approaches based on the single factor analysis (SFA), the Q general linear model (GLM), and the mixed linear model (Q+K MLM), we detected six significant marker&ndash, trait associations involving five phenotypic traits. Our results suggest that the MDP has great potential for soybean genetic resources and that TRAP markers are useful for the selection of soybean mutants for soybean mutation breeding.

A fusion gene coding for trehalose-6-phosphate synthase/phosphatase (TPSP) derived from Escherichia coli was introduced into tomato plants (Solanum lycopersicum cv. Joyful) to generate overexpressing lines and enhance the level of trehalose in the seeds. Trehalose has been implicated in tolerance to abiotic stress in plants, so the response to heat stress of seeds from the transgenic lines and seeds from wild-type plants were compared. Wild-type heat-treated seeds had a germination rate of 17%, while four transgenic lines had germination rates over 50% following heat stress. In one heat-treated transgenic line, 100% of the seeds germinated, and therefore had a germination rate six times that of seeds from wild-type plants. Moreover, quantitative PCR revealed that the expression of diverse genes that respond to heat stress was enhanced in TPSP transgenic seeds compared to wild-type seeds 150 min after the onset of heat stress. The enhanced germination rate and expression of these genes in the transgenic seeds were essentially mimicked in wild-type seeds treated with 1 mM exogenous trehalose. Therefore, accumulated trehalose and associated metabolites may act as signaling molecules that enhance the expression of heat stress-responsive genes and confer heat-stress tolerance to seeds.

Kenaf is an economically important crop that contains various functional compounds. The objective of this study was to evaluate the nutritional properties of leaves, stem bark, flowers, and seeds collected from three mutant kenaf cultivars (Jangdae, Baekma, and Jeokbong) and two original cultivars (Jinju and C14). For all analyzed tissues, there were no significant differences in the proximate compositions (moisture, crude protein, crude fiber, crude fat, ash, and nitrogen-free extract) among the tested genotypes, except for the leaf crude protein content, which was highest in Jinju plants. Of the tested minerals, potassium and calcium were the most abundant in all tissues. Additionally, we detected significant differences in the mineral contents (e.g., calcium, phosphorus, potassium, magnesium, zinc, and iron) of various tissues in all cultivars. Proline and phenylalanine were the major amino acids detected in leaves, and the highest total amino acid (TAA) and essential amino acid (EAA) contents were observed in Jinju plants. The TAA and EAA contents were lower in stem bark than in the other tissues, except in Baekma plants, in which the lowest levels were observed in flowers. Moreover, the TAA and EAA contents were approximately 20 times lower in white flowers (Baekma) than in ivory flowers. Furthermore, the highest TAA and EAA concentrations were observed in Jangdae seeds. These results may be useful for identifying the optimal cultivar and tissues for use in food products.

Rice blast (Magnaporthe oryza B.) is one of the most important diseases in rice that causing great yield losses every year around the world. It is important to screen valuable genetic resources for improving blast resistance. This study was conducted to identify the blast resistance in 279 Korean rice landraces using blast nursery tests and isolate inoculum screening. The results showed that 11 landrace accessions found to be resistant to rice blast in blast nursery and inoculation screening tests and the degree of lesions in most accessions showed that they were susceptible to reactions. In order to find the distribution of blast resistant genes, a molecular survey was conducted to identify the presence of major blast resistance (R) gene in 279 Korean landraces. The results revealed that their frequency distribution was Pik-m (36.2%), Piz (25.4%), Pit (13.6%), and Pik (10%). Besides, the frequency distribution of Piz-t, Pii, Pik-m/Pik-p, Pi-39(t), Pib, Pi-d(t)2, Pita/Pita-2 and Pi-ta genes were identified as less than 10%. The results did not consist with the reactions against blast diseases between genotypes and phenotypic part of the nursery tests and isolate inoculation. For concluding these results, we used genome-wide SSR markers that have closely been located with resistance genes. The PCoA analysis showed that the landrace accessions formed largely two distinct groups according to their degree of blast resistance. By comparing genetic diversities using polymorphic information contents (PIC) value among the resistant, total and susceptible landraces, we found that PIC values decreased in four SSR markers and increased in six markers in the resistant accessions, which showed contrary to total and susceptible groups. These regions might be linked to resistance alleles. In this study, we evaluated the degree of blast resistance and the information about the distribution of rice blast resistant genes in Korean rice landraces. This study might be the basis for association analysis of blast resistance in rice.

This study was carried out to address an efficient in vitro regeneration system from seed-derived callus of Phragmites communis, and to evaluate genetic variations of the regenerants using ISSR markers. Shoot regeneration via calli was greatly influenced by N6 medium compared with MS medium, and plant regeneration frequency was 90% in N6 supplemented with BA 0.25 mg/L and BA 0.5 mg/L. According to ISSR analysis of the thirty regenerants, out of 94 loci detected overall, 16 were identified to be polymorphic with a rate (PR) of 17.0%. The mean gene diversity (h) of different in vitro condition was 0.03 and ranged from 0.008 for N6 with BA 5 mg/L, to 0.040 for MS with IAA 0.1 mg/L+kinetin 2 mg/L. The results indicate that the regenerants have a low genetic variation, and ISSR analysis is effective to detect genetic variation of regenerants.

The study was carried out to evaluate growth and genetic diversity among in vitro cultured protocorms of Cymbidium spp. that irradiated with 10 Gy~300 Gy of electron beam. Shoot induction and shoot length were decreased by an increase of intensity of electron beam, and shoot induction was significantly decreased over 200 Gy of electron beam irradiation in 10 weeks culture. As a result of ISSR (inter-simple sequence repeats) analysis, out of 80 bands detected overall, 10 bands were identified to be polymorphic with a rate of 12.5% at control group, and 90 bands detected overall, 38 bands were identified to be polymorphic with a rate of 42.2% at electron beam treatment. According to the RAPD (random amplified polymorphic DNA) analysis, out of 92 bands detected overall, 12 bands were identified to be polymorphic with a rate of 13.0% at control group, and 121 bands detected overall, 53 bands were identified to be polymorphic with a rate of 43.8% at electron beam treatment. Genetic similarity matrix (GSM) of electron beam treatment was lower than that of control. The result indicates that the beam treatment accelerated genetic diversity by promoting polymorphism of DNA. According to the cluster analysis by using both ISSR and RAPD markers, individuals of C. spp. were divided into control and electron beam treatment group. Our results indicate that the electron beam irradiated in vitro cultured C. spp. might be resulted in promoting higher polymorphism but decreasing GSM.

The objective of this study was to analyze the genetic diversity using SSR marker and investigate the fatty acid composition of perilla (P. frutescens var. frutescens) germplasm. Genetic diversity among 95 accessions, which consisted of 29 weedy types and 66 landrace accessions, was evaluated based on 12 SSR markers carrying 91 alleles. The mean values of observed (HO) and expected heterozygosities (HE) were 0.574 and 0.640, respectively, indicating a considerable amount of polymorphism within this collection. A genetic distance-based phylogeny grouped into two distinct groups, which were the landrace, moderate and weedy type, genetic distance (GD) value was 0.609. The physicochemical traits about crude oil contents and fatty acid compositions were analyzed using GC. Among tested germplasm, the total average oil contents (%) showed a range from 28.57 to 49.67 %. Five fatty acids and their contents in the crude oils are as follows: α-linolenic acid (41.12%-51.81%), linoleic acid (15.38%-16.43%), oleic acid (18.93%-27.28%), stearic acid (2.56%-4.01%), and palmitic acid (7.38%-10.77%). The average oil content of wild types was lower than landrace, and the oil content of middle genotype accessions was higher than other germplasm, but no significant variation between landrace and wild types was shown. Nevertheless, IT117174, landrace of Korea, was highest in crude oil content (47.11%) and linolenic acid composition (64.58%) among the used germplasm. These traits of the selected accessions will be helped for new functional plant breeding in perilla crop.

Dandelion has been widely utilized for medicinal and edible purposes. This research was conducted to evaluate the effect of supplemental LED (light-emitting diode) light on germination, growth characteristics and anthocyanin content of dandelion (Taraxacum officinale) seedling using LED blue (460 nm), red (660 nm, R), blue + red (B:R=6:4) and fluorescent lamp light treatment. By LED illumination to T. officinale seed germination speed was delayed, and germination rate was the highest in the fluorescent light. The growth characteristics (plant height, number of leaves, root length and fresh weight) were greatly influenced by supplemental LED light compared with control treatment, and the growth promotion was the most effective in the red LED illumination. After 60 days of red and mixed LED light treatments, anthocyanin content of dandelion plants was significantly changed. The anthocyanin content was increased by 12~19 mg/100 g under the red LED and the mixed light conditions compared with the control and the blue LED. Results indicate that illumination with red and mix LEDs, compared with other light treatments, is beneficial for promotion of growth and anthocyanin content in dandelion.

Genus Taraxacum has been widely used as a folkloric medicine for treatment of diverse diseases. The genetic diversity and relationship among 32 accessions belonging to five Taraxacum species (T. mongolicum T. coreanum, T. coreanum var. flavescens, T. officinale and T. laevigatum) which collected from field, mountain, island and seaside of Korea were evaluated using ISSR markers. A total of 142 ISSR loci detected in the overall species were all polymorphic loci (100%) and interspecies polymorphisms obtained from Korean native and naturalized species were 98.2% and 94.5%, respectively. The genetic similarity matrix (GSM) among 32 accessions ranged from 0.025 to 0.860 with an average of 0.303. According to the clustering analysis, the Korean native species and naturalized species were divided two major clusters. In addition, the different species were divided into independent groups except for the T. coreanum and T. coreanum var. flavescens, and all the 32 accessions could be classified into 7 categories. The study findings indicate that Taraxacum accessions have a high genetic diversity and the dandelion accessions as breeding materials can be effectively utilized for the improvement of Taraxacum breeding.

This study was conducted to evaluate the effect of light spectrum using red (660 nm), blue (460 nm), red and blue mixed (Red : Blue

A laboratory experiment was conducted to determine the content of phenolics and flavonoids, antioxidant activity and cytotoxicity from methanol extracts of different plant parts of T. officinale F. H. Wigg. Total phenolics (mg chlorogenic acid equivalents (FAE) kg -1 DW) was highest in flower extracts (72.0 mg kg -1 ), followed by leaf, root, and stalk extracts of T. officinale (p < 0.05). The result of total flavonoid level (mg naringin equivalents kg -1 DW) had same tendency to differential total phenolics contents among plant parts, but showed lower ranges of amount. The antioxidant activity of the methanol extracts from all the plant parts dose-dependently increased. DPPH (1,1-diphenyl-2-picryl hydrazyl radical) free radical scavenging activity was highest in flower extracts (IC50 value = 624.3 mg kg -1 ), and followed by leaf, root, and stalk extracts of T. officinale (p < 0.05). By means of MTT (3-(4,5-dimethylthiazol -2-yl)-2,5-diphenyltetrazolium bromide) assay, cell viability of Calu-6 for human pulmonary carcinoma and SNU-601 for human gastric carcinoma showed the lowest IC50 value in the flower extracts (IC50 value = 85.7 and 311.4 mg kg -1 , respectively), indicating the highest cytotoxicity. The results suggested that total phenolics content and total flavonoids level in different plant parts of T. officinale were highly correlated with antioxidative (r 2 =0.7280 to 0.9971) or with cytotoxic activities (r 2 =0.5795 to 0.9515).

This study was carried out to evaluate genetic variation of early-heading rice (Oryza sativa L. cv. Dongjin 1) lines derived from gamma-ray ( 60 Co, 300 Gy) irradiation. The average heading date of the 5 early-heading lines in M7 and M8 generation was faster than that of untreated control as 11 (line γ-2), 10 (line γ-5), 6 (γ-1 line), 5 (γ-3) and 4 days (line γ-4), respectively. According to ISSR analysis, polymorphic rate of the early-heading lines (from 5.9% to 23.4%) was higher than that of control (4.3%). The result indicates that the gamma-ray promote variation at DNA level. When genetic variations of rps16-trnK region were evaluated by nucleotide analysis, nucleotide length of the rps16-trnK region was 664 bp in all the early-heading lines and control. Out of 5 sites of nucleotide transposition detected in the region, however, 2 sites were appeared only in the early-heading lines.

Naehanyuchae and Youngsanyuchae were originated from the cross between “Erra”(♀) and “Tower”(♂). The cultivars were made at Bioenergy Crop Research Center, National Institute of crop science, Rural Development Administration, Korea in order to develop the high cold tolerant and yielding rapeseed variety with non-erucic acid and non-glucosinolate as good seed quality indicator. The superiorities of these varieties have been shown through the regional performance trials and demonstration farmers fields. Naehanyuchae and Youngsanyuchae had good agronomic characteristics related to yield potential such as many valuable branches and pods, and long pod length. The seeds of these new varieties composed of the 80% of oleic acid and linoleic acid without erucic acid, and glucosinolate was not extracted in the oil cake. The average grain yields increased by the from 16% to 22% according to the regional performance trials compared to check variety. Key words - Rapeseed, Combining ability, Quality and Yielding, Variety

This experiment was carried out to confirm characteristics of macro granule (GG) for herbicidal efficacy by using remote-controlled aerial application (RCAA) to control annual and perennial weeds in rice paddy field, and phytotoxicity to rice and non target plants of formulation types. Herbicidal efficacy of GG applied by using RCAA was 93.6 ∼96.6% in flooded direct sowing cultivation. There was no difference in phytotoxicity between the hand and the power applicator methods in terms of height, number of tiller and yield component. Herbicidal efficacy of GG applied by using RCAA was 92.7 ∼97.3% in machine transplanting rice field. There was no difference in phytotoxicity between the hand and RCAA methods in terms of height, number of tiller and yield component. When suspension concentrate (SC) and GG were applied directly on Chinese cabbage, lettuce, cucumber, pepper, soybean and wild sesame, several symptoms of injury such as, the inhibition of growth, yellowish, leaf fall and withering was observed.

The genetic diversity and the genetic relationship among 30 genetic resources of T. officinale and T. coreanum collected from 20 regions in Korea were evaluated by using ISSR markers. Out of 127 loci detected overall, 122 were identified to be polymorphic with a rate of 96.0% at the 30 individuals. The intraspecific polymorphism between T. officinale and T. coreanum was 92.6% and 88.2%, respectively. The genetic similarity matrix (GSM) revealed a wide range of variablility among the 30 accessions, spanning from 0.179 to 922. According to the clustering analysis, different species T. officinale and T. coreanum, were divided into independent groups and all of the accessions could be classified into 7 categories. Especially, all of the mountain collected accessions belonged to independent groups. The study findings indicate that T. officinale and T. coreanum accessions have a high genetic diversity and accordingly carry a germ-plasm qualifying as good genetic resources for breeding.

RAPD (random amplified polymorphic DNA) analysis was examined to detect variation of in vitro cultured 30 rhizomes of Cymbidium goeringii Lindley and Cymbidium kanran Makino, with long-term (8 years) subculture, respectively. Out of 151 DNA bands detected, the 40 were polymorphic with a polymorphic rate 26.4% in the C. goeringii. Out of 155 DNA bands detected, the 56 were polymorphic with a polymorphic rate 36.1% in the C. kanran. Genetic similarity matrix (GSM) shows from 0.825 to 1.00 with an average of 0.944 in the rhizomes of C. goeringii and 0.812 to 1.00 with an average of 0.913 in the C. kanran. According to the clustering analysis, C. goeringii was divided into 1 group and 2 independent individuals and its structure of clustering was simple than that of C. kanran. The higher polymorphism and the decreased GSM were showed in the long-term in vitro cultured C. goeringii and C. kanran supplemented with growth regulators. The results provide as fundamental data to develop a new materials for plant breeding and resources plant.

Ion-beam mutagenesis is a highly effective way to rapidly create new cultivars. To optimize conditions for heavy ion mutagenesis, we irradiated tobacco (Nicotiana tabacum L.) tissues at various developmental stages with heavy ion beams of various doses and examined the effects of irradiation by monitoring plant growth and mutation induction. The effects differed among irradiated tissues. Sensitivity to heavy ion-beam irradiation increased in the following order: dry seeds, imbibed seeds, and culture tissues. We isolated three white flower mutants. One, BWF1, was found to be a novel mutant, in which the synthesis of proanthocyanidin was up-regulated. The others may have a mutation in some regulatory genes involved in the flavonoid biosynthetic pathway. These results suggest that the developmental state of plant tissues is critical for efficient plant mutagenesis, and that the broad spectrum of mutations may be induced by heavy ion-beam irradiation at molecular level.

We have developed a homemade particle inflow gun (PIG) system which has simple operation method, low price and high gene introduction efficiency into rice callus. Rice callus were inflowed with gold particles containing DNA of a plasmid, pIG121Hm, harboring intron GUS (-glucuronidase) gene, NPTII gene and HPT gene. For optimal GUS transient expression, the effects of parameters on DNA delivery efficiency of the PIG system was investigated by scoring transient GUS expression. The highest number of blue spots was observed at 16 mM of spemidine and 1.5 M of calcium chloride, respectively. And the amount of gold particles required for the best GUD expression was 2 mg. Optimum GUS transient expression was observed at target distance of 12 cm and helium pressure of 3.5 bar (50 psi). Gene introduction efficiency of the PIG system was observed almost similar to that of the Biolistic Gun (Bio-Rad Company). Since PIG system is simple to operate and one doesn`t need disposable accessaries, the PIG system can be easily applied to various replication experiments.

This study has been focused on improving transformation efficiency of rice using Agrobacterium tumefaciens. We have demonstrated the effect of this system when the GPAT gene related to the cold-resistance was transferred by Agrobacterium tumefaciens in rice. Transformation conditions were modified using intron -glucuronidase (GUS) expression as a reporter gene in the rice. In this study, mature seed-derived calli of rice (Oruza sativa L. cv. Dongjin) were pre-cultured for 3 days and then infected with Agrobacterium. When this infected calli were cultured in the dark for 10 days on co-cu]lure medium containing 50 mg/L of CaCl, 30 mg/L of acetosyringone, 2 mg/L of 2,4-D, 120 mg/L of betaine, high GUS expression was observed. In the present transformation system, the efficiency of transformation of GPAT gene was about 54%. Stable integration of GPAT gene into chromosomal DNA was proven by southern blot analysis of genomic DNA isolated from T progenies. The progenies (T1 generation) derived from primary transformant of 5 lines were segregated with a 3 (resistant) : 1 (sensitive ratio) in medium containing hygromycin. This high frequency transformation system can be used as a useful tool in transformation of another monocotyledon.n.

A solution-based vitrification protocol is a process of sequentially changing-solutions from which both influx of cryoprotectants (loading) and efflux of water (dehydration) were accomplished before cryo-exposure. Hence, we need to properly control the concentration /composition of the cryoprotectant solutions.The study was, using a systematic approach, to develop a protocol for Rubia akane hairy roots, a very sensitive material to cytotoxicity of vitrification solutions.Due to the poor response of 10-year in vitro maintained R. akane hairy roots to already established cryopreservation protocols, the following sets of experiments were designed: 1) combinational effect of preculture, osmoprotection and cryoprotection with PVS2-based (A3-70%) and PVS3-based (B5-80%) vitrification solutions; 2) different cooling/warming rates and warming temperature; 3) varying unloading solutions (25%, 35%and 45% sucrose) and durations (7 min and 30 min) with or without changing the unloading solutions.Preculture and osmoprotection treatments were necessary to acquire cytotoxicity tolerance in both vitrification solutions tested and osmoprotection treatment was more critical, especially in B5-80%. A sequential osmoprotection treatment (C10-50%) following conventional osmoprotection (C4-35%) was needed to increase the post-cryopreservation regrowth. Aluminum foil strips were superior to cryovials, but the warming temperature tested (20 degree C and 40 degree C) did not affect post-cryopreservation recovery. In the unloading procedure, a longer duration (30 min) with a higher sucrose solution (S-45%) was harmful, possibly due to osmotic stress.R. akane hairy roots are very sensitive to cytotoxicity (both osmotic stress and chemical toxicity) and thus a proper process (preculture, osmoprotection, cryoprotection and unloading) is necessary for higher post-cryopreservation recovery.

An albino mutant designated ali was isolated from an M 2 population derived from a tobacco ( Nicotiana tabacum L. ‘BY-4’) plant which had been irradiated with a 14 N-ion beam at an early stage of embryonic development. Leaf mesophyll cells of ali were devoid of developed chloroplasts, and no stacked thylakoid membranes could be detected in the plastids. The reduction of pigment and the arrest of chloroplast development in ali were associated with distinct alterations in gene expression. Expression of plastid-encoded genes for the photosynthesis system ( rbcL , psbA ) was greatly reduced in ali , but at the transcript level plastid-encoded genes for 23S rDNA and 16S rDNA expression were only slightly reduced. Transcription of the nuclear-encoded Lhc and rbcS genes in the mutant was similar to that in the wild-type. There was no accumulation of Rubisco-L, Rubisco-S, and PSII-D1 polypeptides in the ali mutant. The number of chloroplasts per cell was similar in both ali and wild-type plants. These results suggest that ali is a novel albino mutant in which chloroplast gene expression is affected.

In the present work, the male-associated DNA sequence was analyzed in a dioecious plant, Schisandra nigra. The randomly amplified polymorphic DNA (RAPD) method was used to detect potential male-specific markers. Genomic DNA of Schisandra nigra was isolated from male and female plants and subjected to polymerase chain reaction (PCR) with decamer nucleotide primers of arbitrary sequence. Out of 120 primers tested, only one primer, OPA-17, produced a nucleotide segment of approximately 800 bp that appeared to be male specific. This male-specific segment was cloned and used as a probe for hybridization with genomic DNA. When the male DNA and female DNA were allowed to hybridize, this probe was found to be male specific. Analysis of the nucleotide sequence of the male-specific RAPD marker did not show any significant homology with previously reported sequences. The nucleotide sequence was used to design a sequence characterized amplified region (SCAR) marker of 486 bp in length and male-specific. This SCAR could be useful for a precise, early and rapid identification of male plants during breeding programs of Schisandra nigra.

Restriction Landmark Genomic Scanning (RLGS) profiles, which are based on the concept of using restriction enzyme sites as landmarks and are generated by two-dimensional gel electrophoresis, were applied to the analysis of plant genomes. This study identified landmark enzymes in RLGS profiles of rice, tobacco, and arabidopsis, because the success of RLGS analysis depends on finding useful landmarks and these are the most frequently studied higher plants. As the results demonstrate, in each plant RLGS analysis various landmark enzymes were identified as useful landmark enzymes. However, the number of spots in a single profile was smaller than in mouse RLGS profiles and differed remarkably in the various plants. In addition, we demonstrate the optimal electrophoresis conditions and a convenient spot-cloning method.

A plant isolated from a population of tobacco (Nicotiana tabacum L. cv. BY-4) lines, which were generated with a heavy-ion beam, exhibited green and white variegation on its leaves, stems, and calyces. Fluorescence microscopic analysis of white and green sections of the leaves of the mutant showed an absence of developed chloroplasts in the epidermis and palisade layer. There were undeveloped chloroplasts in one or two layers of spongy parenchyma cells in the green sections of the mutant. In the F1 generation, the segregation ratio of green to white was 6:1163 and reciprocal crosses showed that the variegation was not maternally inherited. These results suggest that the mutant is a periclinal chimera with a White-White-Green histogenic composition. The mRNA levels of the photosynthetic genes rbcL and psbA in the green areas were normal, while they were significantly reduced in the white areas.

Herbicide-resistant zoysiagrass (Zoysia japonica Steud.) has been developed by Agrobacterium-mediated transformation. A callus-type transformation system was established by optimizing several factors that affect the rate of transformation, including co-cultivation period and concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), CaCl2 and acetosyringone. Maximal GUS expression was observed when a Type 3 callus was co-cultivated on 2,4-D-free co-cultivation medium for 9 d. In addition, removal of calcium and addition of 50-100 mg/L acetosyringone during co-cultivation enhanced GUS expression. When this optimized protocol was applied to the transformation of the bialaphos resistance gene (bar), four plants per 700 mg of infected calluses survived on the selective medium. DNA gel-blot analysis showed that two copies of the transgene had been integrated. After application of 2 g/L bialaphos for a week the transgenic plants survived herbicide spraying, while untransformed zoysiagrasses and invading weeds died. The herbicide-tolerant zoysiagrass will permit more efficient weed control in this widely cultivated turf grass.