• Plant Biotechnology Reports
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• v.5 no.2
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• pp.187-195
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• 2011

The gesneriaceous perennial plant Titanotrichum oldhamii has beautiful foliage and attractive bright yellow flowers. However, breeding of T. oldhamii by conventional sexual hybridization may be difficult because sexual reproduction of this species is very rare. In the present study, plant regeneration systems via both direct and indirect formation of adventitious shoots from leaf explants were established as the first step toward breeding T. oldhamii by using biotechnological techniques. Adventitious shoots were formed efficiently on medium containing $0.1mg\;l^{-1}$ benzyladenine. Histological observation showed that shoot formation on this medium occurred directly from leaf epidermal cells without callus formation. On the other hand, leaf explants formed calluses on medium containing $0.1mg\;l^{-1}$ 2,4-dichlorophenoxyacetic acid. The calluses could be maintained by monthly subculturing to fresh medium of the same composition. When the calluses were transferred to plant growth regulator-free medium, they formed adventitious shoots. Directly and indirectly formed shoots rooted well on medium containing $0.1mg\;l^{-1}$ indole-3-butyric acid. Plantlets thus obtained were successfully acclimatized and grew vigorously in the greenhouse. Flow cytometry analysis indicated that no variation in the ploidy level was observed in plants regenerated via direct shoot formation, whereas chromosome doubling occurred in several plants regenerated via indirect shoot formation. Regenerated plants with the same ploidy level as the mother plants showed almost the same phenotype as the mother plants, whereas chromosome-doubled plants showed apparent morphological alterations: they had small and crispate flowers, and round and deep green leaves.

• Journal of Plant Biotechnology
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• v.29 no.4
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• pp.259-264
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• 2002

Induction of embryogenic callus from Allium wakegi Araki explants was promoted on medium containing 2,4-D, and production of abnormal embryos from the embryogenic callus increased as 2,4-D concentration was raised. Shoot tip was found to be the best explant source for embryogenic callus formation followed in the order by bulb scale and leaf section. Medium containing 0.09 M sucrose was effective for embryogenic callus production. The regenerants from embryogenic callus on medium containing 2,4-D and BA at different concentrations was consisted of diploids, tetraploids and a few mixaploids of 2n＋4n, and their chromosomal aberration rate ranged from 8.0 to 33.3%. Frequency of chromosomal aberrants was the highest (18.7%) in the regenerants obtained from bulb scale-derived embryogenic callus among others. Plant regeneration rate was high (33.5%) from the shoot tip-derived embryogenic callus and the frequency of chromosomal aberrants was very low (7.0%). The plantlets regenerated on medium containing 0.26 M sucrose resulted in high chromosomal aberrants. The regenerants on medium containing sucrose at 0.09∼0.20 M produced chromosomal aberrants at around 15.2∼16.6%.

• Horticultural Science & Technology
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• v.32 no.3
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• pp.366-374
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• 2014

The study was conducted to investigate the possibility that epigenetic DNA methylation causes tree phenotypic variation in autotetraploids through evaluating the phenotypic variation and DNA methylation in autotetraploids occurred spontaneously from diploid trifoliate orange. Chromosome analysis confirmed that fourteen trifoliate orange trees of selected by flow cytometry were tetraploids (2n = 4X = 36) without any aneuploids. Chromomycin A3 staining determined that these trees were all autotetraploid with doubled chromosome set. Tree phenotypes, such as tree height and width, branching number, length, and angle, internode length, and leaf characteristics, varied in the autotetraploids. Chlorophyll indices were diverse in the autotetraploids, but photosynthetic rates were not significantly different. In addition, a wide range of variation was observed in stomatal density and guard cell length. Analysis of global cytosine DNA methylation showed that there was a variation of the methylation level in autotetraploids. More than half of 14 autotetraploids had at least 2 times higher methylation level than diploid trifoliate orange. The results indicate that tree phenotypic variation in autotetraploids might be related to global DNA methylation for reducing gene redundancy.

• Development and Reproduction
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• v.22 no.3
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• pp.225-234
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• 2018

A new embryonic cell line (OFEC-17FEN) derived from olive flounder Paralichthys olivaceus was developed. OFEC-17FEN cells were subcultured for <30 passages over ~200 days. OFEC-17FEN cells had a doubling time of 114.34 h and modal diploid chromosome number was 48. The pluripotency genes POU5f1 and NANOG were expressed in OFEC-17FEN cells. However, the lack of several pluripotency-related genes expression indicates that OFEC-17FEN cells are not stem cells. OFEC-17FEN cells transfected with plasmid pEGFP-c1 exhibited a strong green fluorescent signal at 48 h after transfection. Accordingly, OFEC-17FEN cells may be useful for both basic research and biotechnological application.

• Current Research on Agriculture and Life Sciences
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• v.31 no.2
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• pp.75-82
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• 2013

Haploids are plants with a gametophytic number of chromosomes in their sporophytes. Androgenesis occurs from asymmetric division of pollen grains into generative cells and vegetative cells, followed by re-entry of the vegetative cell during S-phase, which causes microspores progress into G2/M transition in culture. One of the most interesting features of haploids is the possibility to produce doubled haploid (DH) individuals. Doubled haploidy is extremely useful to plant breeders because it enables shortened breeding periods and efficiency in selection of useful recessive agronomic traits. Doubled-haploid technology is not only applicable to breeding, but also to transformation programs of desired genes. In addition to practical breeding programs, DH lines provide useful materials of fundamental genetics including exploitation of QTLs and genes conferred with various agronomic traits by establishing DH populations. This paper provides historical overviews on androgenesis and describes several mechanisms associated with pollen embryogenesis, including mode of actions in pollen embryogenesis, mechanisms of chromosome doubling and factors affecting androgenesis. We also discuss recent progress in application of haploids to breeding, genes associated with in vitro response and drawbacks to anther culture for application of doubled haploids in crop breeding.

• Fisheries and Aquatic Sciences
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• v.16 no.3
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• pp.177-185
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• 2013

The development of species-specific fish cell lines has become a valuable tool for biological research. In recent years, marine medaka Oryzias dancena has been recognized as a good experimental model fish but there are no reports of establishment of cell lines from this fish. In this study, two cell lines from O. dancena blastula embryos were established from 41 total trials (4.9%). The two cell lines displayed typical in vitro morphology and have been cultured for >121 passages, which corresponds to 293 days. The doubling times of the cell lines were 29.84 and 28.59 h, respectively, and both possessed the potential to expand in a clonal manner, albeit with significant differences between the two cell lines. The absence of any of the four main medium supplements; i.e., fish serum, fetal bovine serum, basic fibroblast growth factor, and medaka embryo extract, significantly inhibited growth. The proportion of cells possessing normal chromosome number was 45% and 46.7% of the cell lines, respectively. Taken together, two cell lines that proliferate continuously were established from marine medaka and these cell lines may provide a basic tool for characterizing the unique features of this fish species.

• Korean Journal of Breeding Science
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• v.42 no.1
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• pp.100-108
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• 2010

This study was carried out to confirm the chromosome constitution and homoeologous recombination of progenies derived from various cross combination using tetraploid OA interspecific hybrid originated from mitotic chromosome doubling. Based on the chromosome analysis of progenies crossed reciprocally, there were only triploid progenies when crossed with diploid Asiatics as male or female parent. While only tetraploid progenies were produced when crossed tetraploid Asiatics or tetraploid OA hybrid with tetraploid OA hybrid, respectively. However, two types of progenies, that is, diploid and triploid plants, were produced from cross combinations between diploid Oriental hybrid and tetraploid OA hybrid. From the GISH analysis of OA hybrid, it was confirmed that diploid $F_1$ OA hybrid was consisted of 24 chromosomes (12 Oriental and 12 Asiatics) showing authentic OA hybrid. On the other hand, it was notified that triploid plants (3x=36) were consisted of 24 Asiatics lily chromosomes and 12 Oriental lily chromosomes by analysis of backcross progenies derived from either $A{\times}OA$ or $OA{\times}A$ crosses. In cross between tetraploid OA and OA, all the progenies were tetraploid with equal number of chromosomes without any homoeologous recombination, i.e. each 24 chromosomes of Oriental and Asiatics. In 2x-4x ($O{\times}OA$) cross combination, some progenies had 2x=24 chromosomes originated from only Oriental hybrid, and other progenies had 3x=36 chromosomes derived from 24 chromosomes of Oriental hybrid and 12 chromosomes of Asiatic hybrid. Only tetraploid Asiatics chromosomes without any Oriental one were produced in all the progenies from 4x-4x ($AA{\times}OA$) cross combination.

• Korean Journal of Plant Tissue Culture
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• v.28 no.1
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• pp.37-40
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• 2001

To optimize the in uitro chromosome doubling procedure in anther cultures of rice, anthers were cultured on callus induction medium with 0.001 to 0.1 mg/L colchicine for 30 days. The addition of colchicine slightly reduced callus formation and plant regeneration in comparison to the colchicine-free medium (control medium). This reduction was greater with higher concentration colchicine. Microspore-derived rice plants in control medium were found to be mainly haploid (50 to 58.8%) and doubled-haploid (31 to 40%) in anther culture of 3 Japonica and 1 Tonsil type cultivars. The application of 0.001 mg/L colchicine was increased to 54.3∼60.0% in the frequency of fertile doubled-haploid plants. These results indicate that the addition of colchicine to the callus induction medium is an efficient means to obtain doubled-haploid plants in anther cultures of rice.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.273-273
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• 2017

Polyploidy has opened a new horizon for selection to sculpt a variety of new gene functions, traits, and lineages. The aim of this study was to investigate the efficacy of the colchicine concentration, temporal changes, and suitable material for inducing effective tetraploid plants of Codonopsis lanceolata. A total of 180 individuals from 16 treatment groups were germinated, and exposed to different concentrations of Colchicine. The plant height of the diploid (18.1 cm) was slightly shorter than that of the tetraploid (13.4 cm). The fresh weight of the main root in the diploid (0.5 g) was 4-fold higher than the tetraploid (2.2 g). The colchicine-treated plant regeneration rate in C. lanceolata was decreased at the elevated concentration of colchicine. A total of 126 individual plants were regenerated in the entire treatment group and tetraploid (2n=4x=32) plants were obtained. In particular, 5 individuals of the tetraploid plant were induced in the 0.05% colchicine for 6h, which is a higher rate (29.4%) than other regenerated plants. As in the seed treatment result, the plant height of the diploid was significantly higher (10.4 cm) than tetraploid. The root length of the tetraploid (10.1 cm) was longer than the diploid, and the root was also thicker. Taken together, the results obtained from the present study may be helpful for the efficient recovery of such polyploid plants through the in vitro application of colchicine, and may improve the productivity and breeding of C. lanceolata.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.4
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• pp.492-499
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• 2009

A Silkie Bantam embryo fibroblast line (named SBF59 line) was successfully established by using direct explant culture and cryopreservation techniques. Cell morphology, viability, dynamic growth and contamination were tested and the karyotype and levels of isoenzymes of lactic dehydrogenase and malic dehydrogenase were analyzed. Four kinds of fluorescent protein extrogenes, including $pEGFP-N_3$, $pECFP-N_1$, $pEYFP-N_1$ and $pDsRed1-N_1$ were transfected into the cells. The results showed that the cells were healthy and possessed a fibrous structure without a change in morphology. The average viability of the cells was 96% before freezing and 90.5% after thawing. The growth curve appeared as typical "S" shape and the cell growth passed through a detention phase, a logarithmic phase and a platform phase; the estimated population doubling time (PDT) was 38.5 h; assays for the presence of bacteria, fungi, viruses and mycoplasmas were negative; the cell line showed no cross contamination when assessed by isoenzyme analysis; the chromosome number was 2n = 78 on more than 88% of occasions; four kinds of fluorescent protein extro-genes appeared to be expressed effectively with a high transfection efficiency between 18.3% and 42.3%. The cell line met the required quality control standard. It not only preserves the genetic resources of the important Silkie Bantam at the cellular level but also provides valuable materials for genomic, post-genomic, somatic cell cloning research and other applications.