• Title/Summary/Keyword: hypocotyl explants

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Plant Regeneration via Adventitious Shoot Formation from Hypocotyl Explants of Groundcherry (Physalis angulata L.) (땅꽈리(Physalis angulata L.) 하배축 절편으로부터 신초 형성을 통한 식물체 재분화)

  • Koh, Seok Chan
    • Korean Journal of Plant Resources
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    • v.35 no.4
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    • pp.502-507
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    • 2022
  • In the present study, plant regeneration through adventitious shoot formation from hypocotyl segments of in vitro seedlings of groundcherry (Physalis angulata L.) was investigated to determine the optimum culture conditions for highly efficient regeneration of the species. Adventitious shoots in hypocotyl segments were efficiently induced on MS media with low concentrations of BAP, specifically, with 0.5-1.0 mg/L BAP singly or in combination with 0.1-0.5 mg/L NAA. The 1.0 mg/L BAP single treatment was most effective for forming multiple adventitious shoots. When the induced shoots were transferred to the root induction media, low concentrations of NAA, IBA, and IAA enhanced the development of adventitious roots from adventitious shoots, suggesting that low concentrations of auxins were optimal for producing regenerated plantlets. The number of roots per shoot was large (> 2.0), and the root length exceeded 8.0 cm. In particular, the development and the overall shape of the roots were ideal. Furthermore, the number and length of shoots exceeded 2 and 6.0 cm, respectively. When the regenerated plantlets were transferred to compost soil, the root and shoot systems had developed well to the point that all of the regenerated plantlets acclimated successfully, resulting in normal morphology and growth characteristics, similar to those of the mother plant. Therefore, plant regeneration via adventitious shoot formation is expected to be one of the main methods for producing groundcherry on a large scale for a stable supply of the raw materials.

Herbicide Resistant Cabbage (Brassica oleracea ssp. capitata) Plants by Agrobacterium-mediated Transformation

  • Lee, Yeon-Hee;Lee, Seung-Bum;Suh, Suk-Chul;Byun, Myung-Ok;Kim, Ho-Il
    • Journal of Plant Biotechnology
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    • v.2 no.1
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    • pp.35-41
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    • 2000
  • Transgenic cabbage (Brassica oleracea ssp. capitata) plants resistant to the commercial herbicide Bast $a^{R}$ were obtained by Agrobacterium tumefaciens - mediated transformation. Hypocotyl segments of in vitro grown plants were infected with Agrobacterium tumefaciens LBA 4404 harboring plasmid pMOG6-Bar which contains hpt and bar genes. Explants were cultured on callus induction medium (MS basal medium + 1 mg/L NAA + 2 mg/L BA + 2 mg/L AgN $O_3$+ 100 mg/L carbenicillin + 250 mg/L cefotaxime) supplemented with 15 mg/L hygromycin. Hygromycin resistant calluses were transferred to shoot regeneration medium (MS basal medium + 0.1 mg/L NAA + 2 mg/L BA + 3% sucrose + 2 mg/L AgN $O_3$+ 15 mg/L hygromycin + 250 mg/L cefotaxime + 100 mg/L carbenicillin). In order to induce roots, elongated shoots were placed on the MS medium without plant growth regulators and hygromycin. Southern blot analysis of several putative transgenic plants indicated that one to five intact copies of Apt and bar genes were incorporated into the genome. Expression of bar gene was confirmed by Northern blot analysis and by herbicide resistant phenotype. Seed progeny from self-pollinated transformants expressed the herbicide resistance and showed Mendelian segregation of the introduced gene.e.

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Development of Zygotic Embryos and Seedlings is Affected by Radiation Spectral Compositions from Light Emitting Diode (LED) System in Chestnut (Castanea crenata S. et Z.)

  • Park, So-Young;Kim, Man-Jo
    • Journal of Korean Society of Forest Science
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    • v.99 no.5
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    • pp.750-754
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    • 2010
  • Among the environmental conditions employed in micropropagation, light quality plays an important role in growth, specially morphogenesis and photosynthesis. The effect of radiation quality (350-740 nm) on the development and growth of zygotic embryos and in vitro plantlets of open-pollinated chestnut (Castanea crenata S. et Z.) were studied. Two types of explants were exposed for 4 weeks to cool white (W, as control), monochromatic red (R, peak emission 650 nm), monochromatic blue (B, peak emission 440 nm), red+blue (R+B, 1:1), or red+far-red (R+Fr, 1:1, far-red peak emission 720 nm) radiation from a light-emitting-diode (LED) system. While the zygotic embryos showed positive photoblastic behavior, their germination was inhibited by blue radiation. Hypocotyl elongation and root development were promoted by red radiation. The emergence of primary leaf and its expansion were faster under blue than under red radiation. In the plantlets, red and red+far-red radiation significantly increased the formation and growth of the root, whereas blue light reduced rooting. Therefore, radiation quality appears to influence some steps in the development of zygotic embryos and plantlets in the chestnut.

The apical bud as a novel explant for high-frequency in vitro plantlet regeneration of Perilla frutescens L. Britton

  • Hossain, H.M.M. Tariq;Kim, Yong-Ho;Lee, Young-Sang
    • Plant Biotechnology Reports
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    • v.4 no.3
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    • pp.229-235
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    • 2010
  • In this study, we established an in vitro regeneration system to maximize the recovery of leafy perilla (Perilla frutescens L. Britton) plantlets as part of developing a molecular biotechnology-based metabolic engineering program for this crop plant. Hypocotyl segments including the apical buds were used as explants for the direct production of shoots without an interim callus phase. The number of shoots produced from the apical buds peaked within 3-4 weeks, and the shoots were subsequently cultured on Murashige and Skoog (MS) media supplemented with 2 mg $1^{-1}$ benzylaminopurine (BA). Spontaneous rhizogenesis was observed after 7-10 days of culture on MS media without hormonal additives. The rooted shoots developed into normal plants in soil after hardening on distilled water for 3-4 days. The average plantlet regeneration frequency was higher for the apical buds (64.33%) than for the top (15.66%), middle (4%), and basal (1.33%) segments of the hypocotyls. This regeneration system demonstrates a capacity for high-frequency plantlet recovery and thus should be considered for use in the genetic manipulation of leafy perilla.

Comparison of Agrobacterium-mediated of Five Alfalfa (Medicago sativa L.) Cultivars Using the GUS Reporter Gene

  • Lee, Sang-Hoon;Kim, Ki-Yong;Park, Hyung Soo;Cha, Joon-Yung;Lee, Ki-Won
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.34 no.3
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    • pp.187-192
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    • 2014
  • Alfalfa (Medicago sativa L.) is one of the most important forage legumes in the world. It has been demanded to establish the efficient transformation system in commercial varieties of alfalfa for forage molecular breeding and production of varieties possessing new characteristics. To approach this, genetic transformation techniques have been developed and modified. This work was performed to establish conditions for effective transformation of commercial alfalfa cultivars, Xinjiang Daye, ABT405, Vernal, Wintergreen and Alfagraze. GUS gene was used as a transgene and cotyledon and hypocotyl as a source of explants. Transformation efficiencies differed from 0 to 7.9% among alfalfa cultivars. Highest transformation efficiencies were observed in the cultivar Xinjiang Daye. The integration and expression of the transgenes in the transformed alfalfa plants was confirmed by polymerase chain reaction (PCR) and histochemical GUS assay. These data demonstrate highly efficient Agrobacterium transformation of diverse alfalfa cultivars Xinjiang Daye, which enables routine production of transgenic alfalfa plants.

Optimization of Regeneration Condition Under Agrobacterium-mediated Transformation in in vitro Cultured Korean Soybean

  • Kantayos, Vipada;Bae, Chang-Hyu
    • Proceedings of the Plant Resources Society of Korea Conference
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    • 2018.04a
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    • pp.36-36
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    • 2018
  • Soybean is the important crop in Asian countries as protein source, oil production and animal feed. Improving soybean using genetic transformation is the principal tool in nowadays. Developing herbicide resistant transgenic soybean plants through Agrobacterium-mediated transformation has been worked in many previous studied. However, the transformation efficiency is still low. Many attempts try to find the optimum media condition for plant regeneration after infection. After transformation, the plant regeneration is very important condition to promote growth of transgenic plant. In this study, we optimized a regeneration condition for two Korean soybean cultivar, Dawonkong and Pungsannamulkong using cotyledon, cotyledonary nodes and hypocotyl as explant. The results showed that shoot regeneration of cotyledonary nodes on B5 medium containing 2 mg/L 6-benzylaminopurine showed the highest percentage of regeneration in Dawonkong (75.8%) while Pungsannamulkong presented high number of shoots 2.12 shoots per explant. For transformation condition, co-cultivation in 7 days showed a high number of GUS positive expression. Most of explants can survived under media including 5 mg/L of glufocinate which refers phosphinotricin for 2-week selection. Washing with 400 mg/L of cefotaxime in several times and selection in plant regeneration media with 400 mg/L of cefotaxime can prevent bacteria growth, effectively.

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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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Stable Transformation via Callus Formation and Rhizogenesis from the Cultures of Hypocotyl Explant of Chinese Cabbage (배추의 배축절편으로부터 캘러스와 뿌리 발생을 통한 안정적 형질전환)

  • Cho, Mi-Ae;Kim, Choon-Ae;Min, Sung-Ran;Ko, Suck-Min;Liu, Jang-Ryol;Choi, Pil-Son
    • Journal of Plant Biotechnology
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    • v.34 no.2
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    • pp.139-144
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    • 2007
  • Hypocotyl explants of Chinese cabbage (cvs. "Jeong Sang") produced transgenic calli on callus induction medium (MS salt, B5 vitamin, 5 mg/L acetosyringone, 1 mg/L 2,4-D, 3% sucrose, 400 mg/L cefotaxime, 100 mg/L paromomycin, pH 5.8) after cocultivation with strains of Agrobacterium tumefaciens (EHA101, LBA4404, GV3101) harboring the pPTN290 containing paromomycin-resistance gene as a selectable marker, and then they transferred to root induction medium (1/2MS salt, MS vitamins, 2% sucrose, 100 mg/L paromomycin, 100 mg/L cefotaxime, pH 5.8) and shoot induction medium (MS salt, B5 vitamin, 4 mg/L $AgNO_3$, 4 mg/L 6-benzyladenine, 3 mg/L alpha-naphthaleneacetic acid, 100 mg/L paromomycin, 100 mg/L cefotaxime, 3% sucrose, pH 5.8) in order. There was a significant difference in the frequency of transgenic calli depending on Agrobacterium strains. In particular, the highest frequency (6.1%) of transgenic calli was obtained from the hypocotyls cocultivated with EHA101 strains. Also, the frequency (%) of transgenic root and plants from each transgenic callus clone were obtained with 60.7% and 38.2% in EHA101, with 8.3% and 0% in LBA4404, with 20.5% and 85.7% in GV3101 strains, respectively. They were grown to maturity in a greenhouse and normally produced $T_2$ seeds. GUS histochemical assay for progeny ($T_2$) revealed that the transgenes was expressed in the plant genome, and progeny analysis from 7 independent transgenic events demonstrated that the transformants transmitted the transgene as a single or multiple functional locus.

Effects of different types and ages of explants and cytokinins on shoot regeneration in Brassica juncea L. (절편부위와 사이토키닌이 갓의 재분화에 미치는 영향)

  • Roh, Kyung Hee;Kwak, Bo-Kyung;Kim, Jong-Bum;Lee, Kyeong-Ryeol;Kim, Hyun Uk;Kim, Sun Hee;Kang, Han Chul
    • Journal of Plant Biotechnology
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    • v.40 no.2
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    • pp.72-78
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    • 2013
  • To establish an efficient protocol for plant regeneration of Brassica juncea L. Czern, the effects of explant types, explant ages and cytokinins on shoot regeneration were examined in this study. Shoot regeneration was markedly affected by the explant types used in the following order: cotyledon with petiole> hypocotyl> leaf with petiole> cotyledon> leaf. Five-day-old seedlings of cotyledon with petiole explants showed the maximum shoot regeneration frequency. Of the six cytokinins-6-${\gamma}$-${\gamma}$-Dimethylallylamino-purine (2-ip), 6-${\gamma}$-${\gamma}$-Dimethylallylamino-purine riboside (2-ip riboside), 6-Benzyl amino-purine (BAP), Thidiazuron (TDZ), Zeatin, Zeatin riboside-TDZ ($8{\mu}M$) was found to be the best cytokinin for shoot regeneration with the highest shoot induction frequency (80%) from cotyledon with petiole after 4 weeks. All the regenerated plantlets were developed well and they produced morphologically normal flowers.

Efficient Transformation Method of Soybean Using Meristematic Tissues of Germinating Seeds (발아종자의 분열조직을 이용한 효율적인 콩 형질전환 방법)

  • Kim, Yul-Ho;Park, Hyang-Mi;Choi, Man-Soo;Sohn, Soo-In;Shin, Dong-Bum;Lee, Jang-Yong
    • Korean Journal of Breeding Science
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    • v.40 no.3
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    • pp.278-285
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    • 2008
  • An efficient transformation method for soybean [Glycine max (L.) Merr.] using meristematic tissues of germinating seeds has been established. The embryonic axes were excised from germinating seeds of Korean soybean cultivar, Iksannamulkong and 0.5-2 cm long segment containing meristematic tissues were prepared by cutting hypocotyl region. The explants were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector with the bar gene as a selectable marker gene and a ${\beta}-glucuronidase$ (GUSINT) reporter gene, and then co-cultured for 7 days on co-cultivation medium (CCM). The meristematic tissues were cultured on shoot induction medium (SIMP6) supplemented with 0.4 mg/l $N_6-benzylaminopurine$ (BAP) and 0.1 mg/l indolebutyric acid (IBA) in the presence of 6 mg/l L-phosphinotricin (PPT) for 2 weeks and the surviving explants were transferred to shoot elongation medium (SEMP6). Transformation was confirmed by Southern blot analysis and the transformation efficiencies ranged from 1.48 to 2.07%. The new modified transformation method was successfully implemented for obtaining several transgenic lines with SMV-CP gene. It is expected that this method could efficiently be used for the transformation of recalcitrant soybean cultivars.