• Title/Summary/Keyword: microspore culture

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Selection of 5-Methyltryptophan and S-(2-Aminoethyl)-L-Cysteine Resistant Microspore-Derived Rice Cell Lines Irradiated with Gamma Rays

  • Kim, Dong-Sub;Lee, In-Sok;Jang, Cheol-Seong;Hyun, Do-Yoon;Lee, Sang-Jae;Seo, Yong-Weon;Lee, Young-Il
    • 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.

Effects of Several Factors on Embryogenesis in Anther Culture of Broccoli(Brassica oleracea L. var. italica Plenck.) (녹색꽃양배추(Broccoli)의 葯培養에 있어서 배발생에 미치는 몇가지 要因分析)

  • 이광식;박재복;안춘희;윤여중
    • Korean Journal of Plant Tissue Culture
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    • v.25 no.4
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    • pp.251-256
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    • 1998
  • The effect of several factors on efficiency of anther culture of broccoli was studied. The medium supplemented with 14% sucrose, 125mg/L of silver nitrate and 0.1mg/L of each of NAA and 2,4-D was best in embryogenesis in all varieties tested. The effect of auxin was affected by silver nitrate, that is, it lowered embryogenesis when the concentration of NAA and 2,4-D was low, but it enhanced when their concentrations were high. Benzyl aminopurine increased embryogenesis in some varieties, but decreased in others. Although microspore embryogenesis was observed in anthers of which heigest was 1 to 0.6 - 1.0 of petal heigest in the bud, which corresponds to the early uninucleate to early binucleate stage of the microspores, it was much better when the ratio of heigest was 1 : 0.8. More embryos ware inducted from culture in March than in February in general. Because there was a big yearly variation, it was assumed that microspore embryogenesis was influenced greatly by the culture condition and the physiological condition of donor plants.

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Callus and Embryo Formation from Microspore Culture of Peony(Paeonia lactiflora Pall.) (작약(芍藥)의 화분소포자(花粉小胞子)로부터 캘러스와 배(胚) 형성(形成))

  • Sohn, Jae Keun;Kim, Kyung Min;Kwon, Yong Sham
    • Current Research on Agriculture and Life Sciences
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    • v.12
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    • pp.51-55
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    • 1994
  • Pollen microspores isolated from peony anthers were cultured by agarose embedding method in the MS medium with 2,4-D(1mg/l) or phenylacetic acid(1, 10, 100mg/l), and without plant hormone. It was observed that pollen microspores cultured on hormone-free medium were directly developed into embryos. Callus formation was enhanced from microspores which were cultured on medium supplemented with 1mg/l PAA. Embryos were also formed from the calli transferred into the hormone-free medium.

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Efficiency of microspore embryogenesis in Brassica rapa using different genotypes and culture conditions (배추 유전자원의 소포자 유래 배 발생 효율에 미치는 배양 조건 구명)

  • Seo, Mi-Suk;Sohn, Seong-Han;Park, Beom-Seok;Ko, Ho-Cheol;Jin, Mina
    • 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.

Production of doubled haploid population derived from the microspore culture of rapeseed (Brassica napus L.) F1 generation and analysis of fatty acid composition (유채 잡종 1세대의 소포자 배양에 의한 배가반수체 집단 선발 및 지방산 조성 분석)

  • Lee, Ji Eun;Park, Ju Hyun;Kim, Kwang Soo;An, Da Hee;Cha, Young Lok
    • 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.

Stress as a Trigger of Pollen Embryogenesis

  • Zarsky, Viktor;Soukupova, Hana
    • Korean Journal of Plant Tissue Culture
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    • v.27 no.5
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    • pp.411-413
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    • 2000
  • The ability of microspores or young pollen grains (male gametophytes) to undergo developmetal switch to embryogenic (sporophytic) pathway exemplifies the concept of totipotency as applied to haploid posmeiotic cells. As a first step pollen is devoid of positional information provided in situ by the intact anther - by isolation and cultivation in vitro in artificial media. This is inevitably accompanied by some degree of stress response in microspore/pollen. It has been shown in both monocots and dicots that intentional stress treatment (mostly starvation or heat shock) greatly stimulates embryo induction rate. Using transgenic sHSP antisense Nicotiana tabacum we show that expression of small heat shock proteins is an integral part of successful embryo and later haploid plant production from pollen grains. Our recently published data show that sHSP chaperone function is optimal in the absence of ATP.

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STUDIES ON THE TISSUE CULTURE OF PANAX GINSENG

  • Harn C
    • Proceedings of the Ginseng society Conference
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    • 1974.09a
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    • pp.9-22
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    • 1974
  • Unlike the tissue culture in animals and human being, in higher plants various parts of the plant are cultured for varied purposes, and they are named variously depending on which parts are used as explants or what purposes they are cultured for. Followings are some of the names of culture used frequently: organ culture, tissue culture, callus culture, single cell culture, meristem culture, mericlone culture, ovary culture, ovule culture, embryo culture, endosperm culture, anther culture, pollen culture, protoplast culture, etc.. As the names of the culture indicate, in some kinds of culture the explants used for culture are actually not tissues, but organs, single cells, or protoplasts. It seems, however, convenient to call all of the above-mentioned cultures grossly as tissue culture. Several kinds of tissue culture were attempted using Panax ginseng as material and some of the results were summarized below. 1. Callus culture After dormancy of the sed was broken, whole embryo or parts (hypocotyl, cotyledon and epicotyl) of partly grown embryo were cultured in the media supplemented with growth regulators. Rapid swelling occurred in a few weeks, but most of the swelling was observed only in the basal part of epicotyl, changes in the other parts of embryo appearing in much later stages. The swelling or increase in size, however, was resulted not from the divisions of cells, but from the mere expansion of cell. Real calli were formed about two months after inoculation of explants. Callus tissues developed from cortex, pith, and vascular bundle in the cases of hypo- and epicotyl, from mesophyl tissue in the case of cotyledon. Shoots developed more easily from cotyledons regardless of whether they are detached from or attached to the embryo proper. 2. Culture in the Knudson C medium When cotyledons, detached from or attached to the embryo proper, were cultured in the growth regulator-free Knudson C medium comprision only several kinds of mineral compounds and sucrose, shoot primordium or callus developed profusely and finally plantlets were produced directly from shoot primordium or indirectly through callus. In this medium epidermal cells as well as mesophyl cells of the cotyledon became meristematic and divided, changing into multinucleate cells or multicellular bodies, developing eventually into either shoot primordia or calli. 3. Anther culture Anthers were cultured in the media supplemented with various growth regulators applied singly or in combinations. Callus was formed mostly in the connective tissue of anther. Cells of anther wall layers changed in appearance, but no division occurred. Microspores of all stages in development were not changed, ruling out the possibility that microspore-originated callus might be formed. 4. Isolation of protoplast Protoplasts were isolated from young root, leaf, and epicotyl, using 0.7M D-mannitols as osmoticum and using macerozyme and cellulase respectively for maceration and digestion of the cell wall. Production in large number of naked intact protoplast was rather difficult as compared with other plant species. Fusion of protoplasts occurred infrequently mainly due to the fewer number of naked protoplasts in the solution.

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Studies on the Anther Culture of Prunus mume S. et Z. and the Other Three Species (Prunus mume S. et Z. 외(外) 삼종(三種)의 약배양(葯培養)에 관(關)한 연구(硏究))

  • Kim, Jai Saing
    • Journal of Korean Society of Forest Science
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    • v.31 no.1
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    • pp.1-7
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    • 1976
  • When haploid plant would be appeared by the anther culture, the large quantity of young plant multiplied maternal inheritance and the same pure line rapidly in the short length of time, which will be effected to cut down much expences efforts and time for the production of seeds or seedlings. Therefore, the development of the technique for this would be much profited in the country industry. In the late of a few years studies were early attempted in this field, but up this time there were a few success of plants only and none of perennial plant. In this status of the country condition required earnestly for the development of the green industry, this researcher attempted to culture the anther of late uninucleate microspore or early binucleate microspore of the Prunus mume and other three psecies, economic trees estimated specially economic, on the place of Modified Murashige and Skoog's medium supliment with Kinetine, 2.4-D, and N.A.A for inducing haploid plants. The obtained results were as follows: 1. 2,000 anthers were cultured and there were shown that 2N callus in Prunus mume had 82 as 4.1%, 2N callus in Prunus tomentosa 15 as 0.8%, 2 N callus in Prunus salisina 75 as 4% 2. N callus had shown 40 as 2% from Prunus armeniaca var. ansu only, and the other trees showed all 2N callus. 3. Callus had appeared in every tree but 2N callus appeared was all filaments and there showed from only connective tissue N callus appeared was all from anther locule inside. 4. Then Prunus armeniaca var. ansu only was not callus of somatic anther tissue origin, but as there was callus origined from microspore which was changed in to swollen microspores or polynucleate microspores, it was certained to need haploid plant.

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Flower and Microspore Development in 'Campbell Early' (Vitis labruscana) and 'Tamnara' (V. spp.) Grapes ('캠벨얼리'와 '탐나라' 포도의 꽃과 소포자 발달)

  • Yim, Bomi;Mun, Jeong-Hwan;Jeong, Young-Min;Hur, Youn Young;Yu, Hee-Ju
    • 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.

High Frequency Production of Doubled-haploid Plants by Colchicine Application in Anther Cultures of Rice (벼 약배양에서 콜히친이 식물체 배수성에 미치는 영양)

  • 권용삼;이형규;박규환;손재근
    • 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.

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