• Journal of Plant Biotechnology
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• v.44 no.1
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• pp.35-41
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• 2017

One of the most important species, Brassica rapa encompasses a variety of commercial vegetables, such as the Chinese cabbage, pak choi and oilseed crops. The LP08 of yellow sarson (Brassica rapa ssp, tricolaris) have a distinct morphology, with yellow seed color and a unique tetralocular ovary. LP21 of pak choi (Brassica rapa ssp, chinensis) have a dark brown seed color and bilocular ovary. In this study, we generated double haploid plants by crossing the LP08 (maternal variety) and LP21 (paternal variety), using microspore culture. A total of 66 accessions with various morphological characteristics were used for content analysis of flavonoids. The three flavonoids, quercetin, naringenin and kaempferol, showed differing contents in the two crossing parents. The Chinese cabbage type 'Chiifu' was used as the control. The highest accumulation of total flavonoids was observed in LP08. The lowest mean total flavonoids were found in 'Chiifu'. Among the 66 DH accessions, the quercetin contents of 18 accessions showed higher content than LP08. Kaempferol content was also high, and was found to be 79.7% of the total flavonoid content. Naringenin content was low at 2.8%, and was not detected in 22 accessions. Interestingly, the quercetin content positively correlated with the kaempferol content. These results can be used to identify genetic locus and genes related to useful traits. Phenotypic analysis of 66 DH accessions can further be used for natural selection of good breeding materials in B. rapa.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.313-313
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• 2017

Cold stress is one of the most influenced factors to rice yield. In order to identify genes related to cold stress in fertility stage, genome-wide association study (GWAS) was conducted. Cultivated 129 rice germplasm were moved in the growth chamber under the condition of $12^{\circ}C/RH70%$(12h day/12h night when the rice plant was grown in 10 DBH(days before heading). Also, rice plant as control was moved in the green house under condition of $28^{\circ}C/RH70%$(12h day/12h night). After 4 days the plants were moved in a greenhouse. The fertility of rice plant were monitored after the grain were fully grown. The most tolerant rice germplasm to cold stress were Cheongdo-Hwayang-12 and IR38 as 63.1 and 61.8 of fertility and the most recessive rice germplasm were Danyang38 and 8 rice germplasm as 0. As a result of GWAS with re-sequencing data and fertility after cold treatment germplasm using genome association and prediction integrated tool (GAPIT), 99 single-nucleotide polymorphisms (SNPs) were observed by applying a significance threshold of -logP>4.5 determined by QQ plot. With SNPs region, 14 candidate genes responded to cold stress in fertility stage were identified.

• Journal of Plant Biotechnology
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• v.5 no.1
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• pp.33-41
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• 2003

Microspore-derived cell lines resistant to 5-methyltryptophan (5MT, a tryptophan analog) or S-(2-aminoethyl)-L-cysteine (AEC, a Iysine analog) were selected in rice by in vitro mutagenesis. For selection of 5MT or AEC resistant cell lines, suspension-cultured cells were irradiated with gamma rays. Thirteen 5MT resistant cell lines were selected and they were able to grow stably at 2 times higher 5MT concentration. A feedback insensitive form of anthranilate synthesis, the pathway specific control enzyme for tryptophan synthesis, was detected from the 5MT resistant lines. Contents of the free amino acids in five resistant lines (MR12-1 to MR12-5) showed a 7.4 to 46.6 times greater level than that in the control culture. Tryptophan, phenylalanine, and tyrosine levels in the shikimate pathway were 28.1 and 22.5 times higher in MR12-3 and MR12 4, respectively, than that measured in the control cells. Four AEC resistant cell lines were isolated from cultures grown on medium containing 1 mM AEC, They were able to grow stably with 2 mM AEC, while sensitive calli were inhibited by 0.5 mM AEC. Aspartate kinase activities of the resistant lines were insensitive to the natural inhibitor, Iysine, and accumulated 2.2 to 12.9-fold higher levels of free Iysine than that of the control cells. Especially, the levels of aspartate, asparagine, and methionine in the aspartate pathway showed higher accumulation in the AEC resistant lines than that in the control cells.

• Korean Journal of Plant Tissue Culture
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• v.26 no.3
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• pp.149-156
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• 1999

In order to investigate the effect of low temperature pretreatment on pollen dimorphism and embryo formation in anther culture of Platycodon grandiflorum, the anthers with microspore at the uninucleate stage were cultured on Murashige and Skoog medium supplemented with 0.5mg/L NAA and 1.0 mg/L BA. The low temperature pretreatment have clear effect on the frequencies of S pollen grains, symmetrical binucleate microspores (B type of S pollen), multinucleate and multicelled pollen grains. Especially, after low temperature pretreatment at 8$^{\circ}C$ for 5 days increased the frequency of S pollen grain (20.6%) in vivo. In addition, the highest frequency of callus induction (54.9%) and embryo formation (9.9%) were obtained from the anther pretreatment at 8$^{\circ}C$ or 5 days. Three distinct pathways could be recognized in the androgenesis, one involving mainly the vegetative cell, the second starting with the vegetative and the generative cell, respectively, and the third accompaning with two equal vegetative type cells in the pollen grains.

• Korean Journal of Plant Resources
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• v.18 no.2
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• pp.302-308
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• 2005

This study was carried out to obtain the basic informations on the characteristics of gametophytes formation and embryo developement in Dicentra spectabilis. Microspore mother cells developed from archesporial cells, start meiosis when flower bud length reaches around 1 mm, formed tetrahedral type tetrad. The 4 microspores were separated. They were developed to male gametophytes, respectively. Megaspore mother cells were observed when flower bud length was $4{\sim}5\;mm$. The developemental type of megaspore was polygonum and embryo sac was amphitropous. Three large and distinctive antipodals did not degenerated and remained after embryo sac was developed. When the male and female gametophytes was fully developed, the length of stamen and style was very similar or stamen was shorter about 0.5 mm than that of style. This result indicates that self-fertilization can be occurred in this species. After fertilization, developing zygotic embryos showed various stages of development from globular to cotyledonary embryos, and zygotic embryo in seed scattering time seemed to have an early cotyledonary stage.

• The Journal of Natural Sciences
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• v.6 no.1
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• pp.71-78
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• 1993

Five $F_1$ hybrids of radish(Raphanus sativus L.) were used in the study for induction of microspore derived embryos. Anthers from the mid-uninucleate to early bicellular stage were inoculated on the modified B5 medium and modified Nitch-Nitch medium supplemented with several growth regulators. The efficiency of anther culture was dspendent on the genotype of donor plants and we obtained various culture efficiency from different genotypes. Induction of embryos from microspore was best result on Nitsch-Nitsch media supplemented with 0.1mg/l NAA and 0.05mg/l BAP. Heat treatments of anthers at $35^{\circ}C$-2days and combined with pretreatment of $4^{\circ}C$ for 2, 8, 12 and 16days . Among the treatments, $35^{\circ}C$-2 days treatment combined with $4^{\circ}C$-2days pretreatment treatment were the most effective in developing embryos from microspores.

• Journal of Practical Agriculture & Fisheries Research
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• v.25 no.4
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• pp.90-102
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• 2024

Haploid/double haploid plants developed from isolated microspores can significantly accelerate plant breeding. Haploid plants can naturally double their chromosomes to create a pure homozygous line of diploid plants. We present a method for producing embryos from isolated microspores of hot peppers (Capsicum annuumL.). We analyzed the polyploidization levels of the regenerated plants. The donor plants produced the optimal stage of microspores following short-term growth under low-intensity light, which resulted in high rates of embryogenesis and cotyledonary embryogenesis. To find an efficient culture method, liquid, doubled-layer, and 2-step cultures were tested. Liquid culture yielded the highest number of embryos, whereas the highest efficiency for cotyledonary embryogenesis was afforded by the doubled-layer culture. When normal cotyledonary embryos were transplanted onto a regeneration medium, they developed into complete plants. From these, 208 plants were tested via flow cytometric analysis, and 35.6% and 72.7% of the chromosomes from the Milyang-jare and LV2319 genotypes, respectively, were found to be spontaneous double haploids. These results are the same as those obtained on analyzing horticultural characteristics, including the size of leaves and the size and shape of fruits. The present study provides information on the practical application of isolated microspore culture of hot peppers, factors that affect embryogenesis, and methods for polyploidy testing.

• Horticultural Science & Technology
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• v.33 no.3
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• pp.420-428
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• 2015

The majority of cultivated varieties of grape have perfect flowers that are clustered in an individual inflorescence. Grape flower has a single pistil, five stamens, a protective flower cap (calyptra), and a calyx. After fertilization, an individual flower develops into a single berry. Although there are a number of reported studies focusing on berry formation, berry enlargement, and sugar accumulation in grape, the morphological studies of flower, including gametophyte morphogenesis and structural change in floral organs, have not yet been studied in detail. In this study, we investigated the flower structure and development characteristics of grape using microscopy and defined the floral development stages 9 to 13 based on microspore or male gametophyte development stage from tetrad to mature pollen. We used seeded diploid table grapes 'Campbell Early' (Vitis labruscana) and 'Tamnara' (V. spp.) as plant materials. At floral development stage 9, pollen mother cells develop to tetrads. During floral development stages 10 to 11, unicellular microspore develop to mid bicellular pollen. At the end of floral stage 12, male gametophyte develops to mature tricelluar pollen. In floral stage 13, the flower cap falls off and flower bud opens. During floral development stages 9 to 12, there were no major changes in calyx length, whereas the length of the flower cap continuously increased. The flower cap-to-calyx length ratio was 2.0, 3.0, 4.5, and 6.5 at floral stages 9, 10, 11, and 12, respectively. The flower cap-to-calyx length ratio was consistent in the two grape cultivars, suggesting that the ratio is a morphological character representing floral development stage. This study provides a reference for determining floral development stage of the two grape cultivars. It will be useful for the determination of optimum time for microspore culture needed to generate doubled haploid lines and appropriate gibberellic acid treatment needed to induce parthenocarpic fruit development in 'Tamnara' grape.

• Journal of Plant Biotechnology
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• v.41 no.3
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• pp.116-122
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• 2014

Total of fifty accessions of Brassica rapa with various morphological characteristics were used for production of double haploid plants though microspore culture in Brassica rapa. Among them, only 30 accessions induced embryos from microspores. The highest efficiency of embryo induction of 1.194 per bud was obtained from IT135449 of turnip type, while 3 accessions of sarson (winter oil) type did not generate embryo. The effect of heat shock periods for embryogenesis was also investigated with 4 accessions (IT135449; Turnip type, IT199710; Chinese cabbage type, IT212886; Pak choi type, IT218043; Summer oil type). The high productions of embryos were observed in IT135449, IT199710 and IT212886 when microspores were pre-cultured to $32^{\circ}C$ for 2 days. In IT218043, high embryogenesis was observed at the 3 days of heat shock treatment. The optimal condition of shoot regeneration for IT199710 was observed in MS medium supplemented with NAA $0.5mg{\cdot}L^{-1}$ and BAP $1mg{\cdot}L^{-1}$. In contrast, the IT135449 and IT212886 were observed high regeneration frequency in MS medium without plant growth regulators. All the plantlets regenerated from microspore-derived embryos have been successfully transplanted to soil, and bud self-pollinated seeds were produced from doubled haploid plants. This indicated that double-haploid genotype was likely generated naturally during embryogenesis process.

• Journal of Plant Biotechnology
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• v.49 no.1
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• pp.74-81
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• 2022

Brassica napus, an oil crop that produces rapeseed oil, is an allotetraploid (AACC, 2n = 38) produced by natural hybridization between B. rapa and B. oleracea. In this study, microspore was cultured using the F₁ developed from a cross between 'EMS26' line with high oleic acid content and 'J8634-B-30' lines. The flower bud size showing the nuclear development at the late uninucleate and binucleate stage with high embryogenesis rate was 2.6 ~ 3.5 mm. Microspores were cultured using only this size and after then most microspore embryo developed into secondary embryos and then regeneration plants obtained from the developed multilobe. The analysis of the ploidy of the plants revealed that 66.7% and 27.8% of the total lines were tetraploids and octoploids, respectively. The sizes of stomatal cells in tetraploids, octoploids, and diploids were 25.5, 35.6, and 19.9 ㎛, respectively, indicating that ploidy level was positively correlated with cell size. Furthermore, 62 tetraploid doubled haploid (DH) lines were selected. The average oleic acid (C18:1) and linolenic acid (C18:3) concentrations of DH were 72.3% and 6.2%, respectively. Oleic acid and linolenic acid concentrations exceeded the two parental values in 5 and 14 DH lines, respectively, suggesting that these two fatty acids had transgressive segregation. Therefore, the DH population can be utilized for the biosynthesis of unsaturated fatty acids in rapeseed and related genes. It can also be used as a breeding material for varieties with high oleic acid concentrations.