• Journal of Life Science
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• v.11 no.4
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• pp.340-345
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• 2001

Effects of growth regulators(2,4-D, IAA, NAA, BA and kinetin) and chilling treatment on callus induction. embryogenesis and regeneration through anther cultures of B. falcatum L. were examined. Frequency of callus induction and embryogenesis was effectively increased by the treatment of chilling at 5$^{\circ}C$ for 10 days before anther inoculation. Optimal level of growth regulator for callus induction and embryogenesis from anther was 2,4-D 1.0 mg/L in Murashige and Skoog(MS) basal medium supplemented with 30 g/L sucrose, 8 g/L agar. Frequency of embryogenesis from anther derived callus was increased up to 48% or 45% by addition of IAA 0.1+ kinetin 1.0 mg/L of IAA 0.1+ BA 1.0 mg/L in MS medium, respectively, Optimal medium for obtaining green callus was MS basal supplemented with BA 1.0mg/L. Addition of auxins(IAA or NAA) inhibited the formation of green callus from anther derived callus.

• Journal of Crop Science and Biotechnology
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• v.11 no.1
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• pp.23-30
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• 2008

The relationship between three phenological parameters and somatic embryogenesis was investigated during a two-year period. Staminodes and petals from six hybrids and two clones as controls were sowed on three distinct primary callus growth media. Flowering level, fructification level, and leaf thrusts rhythm as phenological parameters were measured simultaneously during the weekly harvest of flower buds. Mean and coefficient of variation of the measured parameters highlighted stable phenological phases. The relationship between phenological parameters and somatic embryogenesis was investigated first by comparing the variation of somatic embryogenesis and that of the phenological parameters, and second by using Pearson's linear correlation. Except for the fructification level in both control clones the first year, the other parameters recorded stable phenological phases, regardless of the genotype and year. Favorable and unfavorable phases for the somatic embryogenesis were identified. In hybrids, favorable phases included February, August, September, and October. In both control clones, time interval propitious to embryogenesis stretched from February to December. The significance of the coefficient of correlation seemed to establish a relationship between somatic embryogenesis and phenology. However, a causal link could not be established. Leaf thrusts rhythm was revealed to be the phenological parameter most linked to somatic embryogenesis. Attempts to optimize embryogenesis during unfavorable phases, showed that a correction of 2.4 D/TDZ concentration is not the solution.

• Plant Biotechnology Reports
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• v.2 no.3
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• pp.179-189
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• 2008

We established an efficient plant regeneration system for Catharanthus roseus L. (G.) Don through somatic embryogenesis. Embryogenic callus was induced from hypocotyl of seed germinated in vitro. Somatic embryogenesis in Catharanthus has been categorized into three distinct stages: (1) initiation and proliferation of embryo; (2) maturation, and; (3) germination or plantlet conversion. Beside plant growth regulators, various stages of embryogenesis were screened for their response to a wide variety of factors (pH, gelrite, light, sugar alcohols, polyethyleneglycol and amino acids), which affect embryogenesis. All of the tested factors had a small to marked influence on embryogeny and eventual conversion to plantlets. The plantlets were acclimatized successfully in a greenhouse. To our knowledge, this is the first report describing a detailed study of various cultural factors which regulate embryogenesis in C. roseus. The results discussed in this paper may be used in mass propagation to produce medicinal raw material, and the embryo precursor cells could be used in genetic modification programmes that aim to improve the alkaloid yield as well.

• Journal of Plant Biotechnology
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• v.2 no.3
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• pp.123-128
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• 2000

To enhance in vitro plantlet regeneration efficiency of soybean through embryogenesis, the culture conditions such as material part and size of immature seed, 2,4-D, pH and solidifying agents for somatic embryogenesis were investigated. Somatic embryogenesis was induced from the immature embryo, immature cotyledon and embryonic axis explants of the immature seed on MS medium supplemented with 2.0 mg/L 2,4-D. The highest rate (up to 22.9%) of somatic embryogenesis was obtained from the immature cotyledon, following embryonic axis and the immature embryo. The rate varied with the developmental stages of seed. The maximum rate (25.4%) of embryogenesis was obtained from 3-4 mm length of the seed (after 25 days of flowering). The optimum concentration of 2,4-D for embryogenesis was 10 mg/L. The optimum pH was at 5.8 and solidifying agent for medium was better with 0.4% gelrite than with agar. For rapid multiplication of shoot tips from the germinating somatic embryos, they were cultured on MS medium containing 2 mg/L indole-3-butyyic acid (IBA) and 1 mg/L 6-benzyladenine (BA). After then somatic embryos with one and three cotyledons were transferred to the growth regulator free medium. The medium exhibited the higher rate (ca. 50%) of development than the multiplication medium.

• Journal of Plant Biotechnology
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• v.6 no.1
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• pp.55-61
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• 2004

Callus induction and somatic embryogenesis are fundamental to cotton tissue culture biotechnology. An efficient protocol for callus induction, somatic embryogenesis and their maturation have been developed to regenerate plantlets from cotton (Gossypium hirsutum L.) variety coker 312. Embryogenic callus was initiated from hypo-cotyl region that was used as an explant at seedling stage when it was about 7-8 days old. Callus induction was achieved through culturing hypocotyls (5-7mm) on $MS_{1a} medium supplemented with 2,4-D (0.1 mg/L) and KT (0.5 mg/L) for six weeks. A friable, colorless, bulky and well proliferating callus becomes greenish with the addition of NAA (2.0 mg/L), ZT (0.1 mg/L) and removal of 2,4-D (M $S_{1b}$) cultured for two weeks then again transferred to $MS_{1a}. 2,4-dichlorophenoxyacetic acid (2,4-D) promoted the proliferation of embryogenic callus, but had a negative effect on the differentiation and germination of somatic embryos. ZT (0.1mg/L) and activated charcoal (2g/L), both hormones play an important role in differentiation and germination of somatic embryos in hypocotyls derived embryogenic callus but in case of cotton, such a capability have been observed on MS medium with 1.92 g/L $KNO_3$, but it is considered to attain somewhat more improvement. High embryogenesis frequency was achieved through nutrient deficient stress treatment. The frequency of globular embryogenesis (two-three folds) was achieved when well proliferating callus was (from $MS_{1a}$ media) cultured on MS (1/5 strength) medium for four weeks. Here the development of anthocyanins is the best indicator for somatic embryogenesis. However, when embryoid callus was cultured on MS (full strength) medium, the globular embryos were developed into normal plantlets immediately. In this procedure 27.49% cotyledenary embryos were developed. Of that 70% cotyledenary embryos were developed not only into normal plantlets but rooted simultaneously, when cultured on MS (with 0.05 mgg/L giberrelic acid) medium. So complete plants could be regenerated through somatic embryogenesis from hypocotyl explants within 6 months.s.

• Development and Reproduction
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• v.21 no.2
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• pp.205-213
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• 2017

The aim of this study was to determine the effect of additional alpha-linolenic acid (ALA) supplementation during in vitro maturation (IVM) and culture (IVC) on nucleic maturation and embryo development of pigs. Cumulus-oocyte complexes (COCs) were incubated in IVM medium containing different concentration of ALA (25, 50 and $100{\mu}M$) for 44 h. After in vitro maturation, nuclear maturation of oocytes were evaluated by aceto-orcein stain. Mature oocytes with $50{\mu}M$ ALA were fertilized and cultured in IVC medium with ALA (25, 50 and $100{\mu}M$) during early-embryogenesis (48 hours after fertilization). Then, embryos were cultured with $25{\mu}M$ ALA during early embryogenesis and/or late embryogenesis (120 hours after early-embryogenesis). In results, oocyte maturation were significantly increased by $50{\mu}M$ ALA treatment groups compared with control groups (p<0.05). Treatment of $25{\mu}M$ ALA during early-embryogenesis enhanced cleavage rate of embryo compared with other groups (p<0.05), whereas formation and total cell number of blastocyst had no significant difference. Similarly, cleavage rate of embryos were increased by $25{\mu}M$ ALA supplement during early- or late-embryogenesis than ALA treatment both stage of embryogenesis (p<0.05), but did not influence to blastocyst formation. Interestingly, total cell number of blastocyst were enhanced in ALA treatment group during early-embryogenesis. These findings indicated that ALA supplement enhance the nuclear maturation of oocyte and embryo development, however, excessive ALA could negatively influence. Therefore, we suggest that ALA is used for improvement of in vitro production of mammalian embryo and further study regarding with functional mechanism of ALA is needed.

• Journal of Crop Science and Biotechnology
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• v.11 no.2
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• pp.119-126
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• 2008

Since somatic embryogenesis combined with ethylmethane sulfonate(EMS) treatments is the most efficient technique for mutagenesis, the embryogenic capacity of four soybean cultivars was evaluated at different EMS concentrations, treatment times, and preculture durations. Two to 4 mm long immature cotyledons were placed in induction medium after EMS treatment, and the numbers of somatic embryos formed per explant were counted four weeks after culture initiation. We observed genotypic differences in the efficiency of somatic embryogenesis from immature embryos among four cultivars treated with different concentrations of EMS for six hours. Cultivars, Sinpaldalkong 2 and Jack, displayed highly efficient somatic embryogenesis regardless of EMS concentration, whereas very low efficiency or no survival was observed in Jinju 1 and Iksannamulkong cultivars. Preculture duration did not influence the efficiency of somatic embryogenesis. Because Sinpaldalkong 2 exhibited the best somatic embryogenesis, much higher concentrations of EMS were used to test somatic embryo formation under different periods of time in this cultivar. Three and six hour treatments with both 1 and 2 mM EMS yielded higher embryo formation than longer periods of time. Increasing the time with embryos in 2 mM EMS caused a reduction in somatic embryogenesis in Sinpaldalkong 2, but many chlorophyll-deficient soybean variants were identified in the $M_1R_0$ and $M_2R_1$ generations. In addition to Jack, Sinpaldalkong 2 is a good genotype for plant regeneration from EMS-treated immature embryo cultures.

• Korean Journal of Plant Tissue Culture
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• v.27 no.4
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• pp.245-258
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• 2000

Somatic embryogenesis has become a major tool in the study of plant embryology, as it is possible in culture to manipulate cells of many plant species to produce somatic embryos in a process that is remarkably similar to zygotic embryogenesis. Traditionally, the process has been studied by an examination of the ex vitro factors which influence embryo formation. Later structural, physiological and biochemical approaches have been applied. Host recently, molecular tools are being used. Together, these various approaches are giving valuable information on the process. This article gives an overview of somatic embryogenesis by reviewing information on the morphogenesis, physiology, biochemistry and molecular biology of the process. Topics covered include a brief description of the factors involved in the production of embryogenic cells. Carrot cell suspension is most commonly used, and the development of a high frequency and synchronous system is outlined. At the physiological and biochemical lev-els various topics, including the reactivation of the cell cycle, changes in endogenous growth regulators, amino acid, polyamine, DNA, RNA and protein metabolism, and embryogenic factors in conditioned medium are all discussed. Lastly, recent information on genes and molecular markers of the embryogenic process are outlined. Somatic embryogenesis, the best example of totipotency in plant cells, is not only an important tool in studies in basic biology, but is potentially of equal significance in the micropropagation of economically important plants.

• Journal of Plant Biology
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• v.33 no.4
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• pp.259-269
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• 1990

Changes of peroxidase activity, polyamine content and ethylene production during somatic embryogenesis in suspension-cultured carrot (Daucus carota L.) cells were investigated. As compared with nonembyrogenic cells and their medium, embryogenic cells and their medium were characterized by higher levels of peroxidase at all times of culture period. Peroxidase in embryogenic cells showed higher oxidation activity of IAA than in nonembryogenic cells at the torpedo stage, but the IAA oxidation activity of peroxidase released into embryogenic medium was lower than that of peroxidase released into nonembryogenic medium. Peroxidase patterns of embryogenic and nonembryogenic cells showed three cathodic bands, and one anodic band, while peroxidase patterns released into embryogenic and nonembryogenic media did not show any anodic bands and the isoelectric points of cathodic peroxidase were pH 7.7, 7.5 and 6.6. Compared with nonembryogenic cells, polyamine content in embryogenic cells was increased by 15% at the torpedo stage, but polyamine ratio was constant, and ethylene production was extremely low at all times of culture period. Therefore, it is suggested that the peroxidase in embryogenic cells is correlated with embryogenesis by regulating hormone ratios through IAA oxidation, while the peroxidase isozyme patterns may be used as a biochemical marker of embryogenesis. The increase of polyamine content and the decrease of ethylene production suggest an interaction between polyamine and ethlyene during embryogenesis.

• Journal of Plant Biotechnology
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• v.4 no.2
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• pp.71-76
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• 2002

In order to achieve repetitive somatic embryogenesis in cacao (Theobroma cacao L.), callus derived from floral tissues were continuously cultured in a medium containing 2,4-D. In 5% of the explants, repetitive somatic embryogenesis was observed after 8 weeks and maintained in a globular stage for several weeks. This is the first report showing repetitive somatic embryogenesis in cacao. The calli were bombarded with a plasmid containing $\beta$-glucuronidase (gus) as reporter gene. Two week old calli showed the high average number of cells expressing the us gene. The effect of osmotic agents (mannitol, sorbitol and sucrose) on gene expression was evaluated. Pre-treatment during 16 h with 0.25 M mannitol revealed an improvement in gene expression. The potential utilization of the repetitive embryogenesis, combined with osmotic treatment, is discussed as an alternative to achieve stable transgenic cacao plants.