Frequency Of Embryogenic Callus Induction Research Articles

Efficient protoplast isolation from ovule-derived embryogenic callus inCitrusvolkameriana

The present study reports on the isolation of viable protoplast from ovule-derived embryogenic calli of Volkameriana (Citrus volkameriana L.), which is a rootstock in high demand for lemon production.Ovules of C. volkameriana isolated at 3 different immature fruit stages, comprising4, 8, and 12 weeks after anthesis (WAA),were cultured on5 different media in order to produce embryogenic callus lines as a source material for protoplast isolation. EME medium (MT basal medium + 0.5 gL–1 malt extract), with the addition of phytohormones [kinetin (KIN), 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP)] at different concentrations, were tested for callogenesis. According to 2-way ANOVA, significant effects were determined as a result of the immature fruit stage and type of culture media (P ≤ 0.01) on the callogenesis and embryogenic callus induction frequency. First, callus formation was recorded after 4 WAA on medium comprising EME + 2,4-D (1.0 mg L–1) + BAP (0.5 mg L–1). Callus induction frequency was the highest (90.00%) in the same culture medium when the ovules wereculturedat8 WAA. In addition, culturing the ovules isolated from 12 WAA immature fruits of C. volkameriana resulted in the highest indirect somatic embryogenesis (24%). Embryogenic callus initiation was the highest (25.56%) using EME + KIN (1.0 mg L–1) and ovules cultured at 8 WAA (14%) resulted in the highest embryogenic callus formation. Effects of different enzyme concentrations on the efficiency of protoplast isolation were calculated using the hemocytometer cell counting method. The combination of 2% cellulase and 0.2% pectinase gave the highest numbers of protoplasts, at 12.33 × 105protoplast/mL. Embryogenic callus lines obtained by culturing ovules of C. volkameriana yielded high-quality protoplasts after isolation and could be useful as a protoplast source for further somatic hybridization studies.

Somatic embryogenesis and plant regeneration in Betula platyphalla

Betula platyphylla is a native tree species in northern China that has high economic and medicinal value. We developed an efficient protocol for the induction of somatic embryogenesis in B. platyphalla from immature zygotic embryos and assessed the effects of explant type, genotype, and plant growth regulators (PGRs) on embryogenic callus induction. Among the various explants evaluated, embryogenic callus was only produced from mature and immature zygotic embryos on medium with added 2,4-dichlorophenoxyacetic acid (2,4-D). Supplementation of 2,4-D-containing medium with cytokinins increased the frequency of embryogenic callus induction. On the 20 days after pollination, immature zygotic embryos that had been collected in mid-May yielded embryogenic tissue at the highest frequency (16.8%) when cultured on half-strength MS medium supplemented with 2.0 mg L−1 2,4-D and 0.2 mg L−1 6-benzylaminopurine (6-BA). The process of proliferation of embryogenic callus, somatic embryo formation, and subsequent plantlet conversion occurred under optimal culture conditions. When regenerated plants were transplanted to soil, 95% of them developed normally and grew vigorously. This somatic embryogenesis system required 3–4 months for the regeneration of B. platyphalla plantlets from immature zygotic embryos.

Callus Induction and Plant Regeneration from Immature Embryos of Sweet Sorghum (Sorghum bicolor Moench).

Background and ObjectiveRegeneration of plant through tissue culture technique is a critical process in transformation of plants. This study sought to establish the effect of genotype and auxin concentration on callus induction from different sweet sorghum genotypes (IESV92008DL, IESV92001DL, IESV92021DL, ICSV700 and ICSV93048).Materials and MethodsIn this study, MS medium supplemented with five levels of the hormone 2,4-D (0, 1, 2, 4 and 6 mg L-1) to assess the effect of the hormone 2,4-D on callus induction and regeneration was used.ResultsThe highest callus induction frequency was observed at 2 mg L-1 2,4-D for all the genotypes, IESV92008 gave the highest callus induction frequency among all the genotypes at 2 mg L-1 2,4-D. The lowest callus induction frequency was observed 0 and 6 mg L-1 for all the genotypes, the two genotypes ICSV700 and IESV92021 were observed to give the lowest callus induction frequency among all the genotypes used in this study. Embryogenic callus induction frequency was observed to be higher at 2 mg L-1 2,4-D and the genotype IESV92008 gave the highest callus induction frequency among all the genotypes used. Induction of shoots was achieved in MS medium supplemented with 3 mg L-1 6BA and 1 mg L-1 IAA, the highest regeneration efficiency was obtained from the three genotypes ICSV93048, IESV92008 and IESV92001, respectively.ConclusionThis study discovers the importance of the auxin 2,4-D on callus induction and regeneration of sweet sorghum and this will help the researcher to develop protocols for transformation of sweet sorghum.

Polyethylene glycol and chilling overcome Somatic embryogenesis obstacle in Pyrus communis

Abstract The purpose of this investigation is to achieve the procedure of somatic embryogenesis (SE) as an efficient propagation way in four pear cultivars: Natanzi, Ghosi, Williams and Dar Gazi, by focusing on two strategies: One is the application of competent explants, and the other one is stress employment. The results were variable because of cultivars behavior was highly genotype specific. However, the results identified that the highest frequency of embryogenic callus induction was obtained from the endosperm and immature cotyledonary segment explants of P. communis cultivars on Murashige and Skoog medium (MS) containing 8.05 μM 1-naphthaleneacetic acid (NAA), 13.32 μM N6-benzyladenine (BA) supplemented with 250 mg/L glutamine as a reduced nitrogen source and 30 g/L (w/v) sucrose in Ghosi (75.46%). The optimized media were different for each cultivar in the induction phase, namely, Natanzi and Ghosi in MS, Williams in Schenk and Hildebrandt (SH) and Dar Gazi in Nitsch and Nitsch (NN). Then, it was observed that when embryogenic calli were transferred to the media containing 1% (w/v) Polyethylene glycol (PEG), calli of some cultivars further differed into embryos while other cultivars produced in chilling at 4 °C. Also, maturation of embryos occurred at 4 °C for 4 months. Then passages using MS media caused germination of embryos and plantlets were produced. To sum up, the greatest frequency of embryogenic callus (75.46%) occurred in Ghosi, and the highest embryo maturation and germination (77.06% and 37.66%, respectively in mentioned cultivars) were observed in Natanzi.

Erratum to: In vitro clonal propagation and stable cryopreservation system for Platycladus orientalis via somatic embryogenesis

Platycladus orientalis is a widespread conifer, which is native in eastern Asia, and has recently attracted much attention due to its ornamental value for landscape and gardens. However, native P. orientalis populations have been in decline over the past century. Here, we established an in vitro propagation and cryopreservation system for P. orientalis via somatic embryogenesis (SE). Whole megagametophytes with four development stages (Early embryogeny: E1 and late embryogeny: L1, L2, and L3) of zygotic embryos from immature P. orientalis cones were used as initial explants and cultured on three different basal media such as initiation medium (IM), Litvay (LV), and Schenk and Hildebrandt (SH). Both the developmental stage of zygotic embryos and kind of basal medium had a significant effect on embryogenesis induction with IM (P < 0.001, respectively). The highest frequency of embryogenic callus induction was obtained in megagametophytes with zygotic embryos at L2 stage, which ranged as high as 30%. The maturation medium containing IM basal salts, vitamins and amino acids, 15 g l−1 abscisic acid (ABA), 50 g l−1 maltose, and 100 g l−1 polyethylene glycol 4000 (PEG) was found to be the suitable medium for production of somatic embryos. The frequency of somatic embryo formation from both non-cryopreserved and cryopreserved cell lines was also tested. There were no statistical differences on the production of somatic embryos between non-cryopreserved and cryopreserved cells (P = 0.523). Genetic fidelity of the plantlets regenerated from non-cryopreserved and cryopreserved embryogenic cell lines was assessed by both random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis. There was no genetic instability in the regenerated plantlets from cryopreserved embryogenic cell lines. Both the SE protocol and cryopreservation protocols described here have the potential to contribute the conservation and clonal propagation of P. orientalis germplasm.

Somatic embryogenesis and plant regeneration from zygotic embryo explants of onion

ABSTRACT: The study was undertaken to examine the effect of two synthetic auxins on somatic embryogenesis and plant regeneration from mature embryo explants of 16 onion cultivars. Cotyledons were removed from the embryos and remaining portions were cultured on MS medium supplemented with 1.0 mg/L 2,4-D and 5.0 mg/L picloram alone or in combination (1.0 mg/L 2,4-D + 2.5 mg/L picloram) to produce embryogenic callus. MS medium supplemented with 5.0 mg/L picloram was found to be the best one for both embryogenic callus induction (85%) and callus diameter (3.8 mm). Of the 16 cultivars studied, Yeoeuijuhwang exhibited the lowest frequency of embryogenic callus induction (50.5%), whereas all the other 15 cultivars showed more than 60% embryogenic callus induction. Scanning electron micrograph (SEM) analysis of embryogenic callus showed all stages of somatic embryos such as globular, scuetellar and coleoptilar. Plant regeneration was significantly affected by the composition of embryogenic callus induction medium. The greatest frequency of somatic embryo conversion was obtained from embryogenic callus developed in MS medium with 2,4-D (70.1%) followed by picloram (38.9%) and 2,4-D + picloram (34.5%). The germinated plantlets were further developed on the half-strength MS medium containing 3% sucrose and were acclimatized in the culture room with 98% survival rate.

Efficient plant regeneration of watermelon (Citrullus lanatus Thunb.) via somatic embryogenesis and assessment of genetic fidelity using ISSR markers

A simple and effective somatic embryogenic system was established for watermelon (Citrullus lanatus) cv. ‘Arka Manik’. Embryogenic callus was obtained from leaf explants of 20-d-old in vitro-grown seedlings cultured on embryogenic callus induction medium. The highest frequency of embryogenic callus induction (96.8%) occurred on Murashige and Skoog (MS) medium supplemented with 2.44 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.27 μM thidiazuron (TDZ). Transfer of embryogenic calluses with proembryogenic masses to embryo maturation medium led to the asynchronous development of somatic embryos (SEs), which progressed from the globular stage to the cotyledonary stage. The maximum number of SEs/explant (16.1 ± 0.24) was obtained on MS medium supplemented with 2.44 μM 2,4-D, 2.27 μM TDZ, and 30 g L−1 sucrose. Plantlet conversion from cotyledonary-stage SEs was tested on different strengths of MS medium (quarter-, half-, and full-strength) lacking plant growth regulators. The highest frequencies of germination (91.5%) and survivability (82.1%) of plantlets were achieved on full-strength MS medium. Transverse sections of embryogenic callus revealed SE development from callus cells near the epidermis. Secondary SEs occasionally formed from globular-shaped primary embryos. Genetic fidelity of mother plants and ex vitro plants was confirmed by inter-simple sequence repeat (ISSR) markers. The present study is the first report on the use of molecular markers in in vitro culture of watermelon. The developed protocol facilitates rapid production of true-to-type watermelon plants by somatic embryogenesis and thus could serve to generate effective target material for genetic transformation protocols.

Somatic embryogenesis and plant regeneration from zygotic embryo explants of onion

We investigated the effect of two synthetic auxins on somatic embryogenesis and plant regeneration from mature embryo explants of 16 onion cultivars. Cotyledons were removed from the embryos and remaining portions were cultured on MS medium supplemented with 1.0 mg L -1 2,4-D, 5.0 mg L -1 picloram or 1.0 mg L -1 2,4-D and 2.5 mg L -1 picloram. Among the media studied, MS medium supplemented with 5.0 mg L -1 picloram was found to be best for both embryogenic callus induction (85%) and callus growth (3.8 mm). Of the 16 cultivars studied, ‘Yeoeuijuhwang’ exhibited the lowest frequency of embryogenic callus induction (50.5%), whereas all the other 15 cultivars showed more than 60% embryogenic callus induction in all studied media. SEM analysis of embryogenic callus showed all stages of somatic embryos such as globular, scuetellar and coleoptilar. Plant regeneration was significantly affected by the embryogenic callus induction media. The greatest frequency of somatic embryo conversion was obtained from embryogenic callus develop in MS medium with 2,4-D (70.1%) followed by picloram (38.9%) and 2,4-D + picloram (34.5%). The germinated plantlets further developed on the half-strength MS medium containing 3% sucrose and were acclimatized in the culture room with 98% survival rate.

Micropropagation via somatic embryogenesis of rare and endangered species, Acanthopanax seoulenses Nakai

A somatic embryogenesis and plant regeneration study was conducted with a rare and endangered species, Acanthopanax seoulenses, and various factors affecting somatic embryo induction were evaluated. The frequency of embryogenic callus induction was slightly better on wounded seeds compared with non-wounded ones. The optimum medium to induce somatic embryos (SEs) from embryogenic cells was MS medium supplemented with 3% sucrose and 0.1–0.2 mg/l abscisic acid (ABA), or MS medium with 3% sucrose and 0.1 mg/l ABA+0.02% activated charcoal. Gibberellic acid resulted in a positive effect on SE germination, but there were no differences in the range of 0.1 to 1.0 mg/l. SE germination and plant conversion rate were different in the two gelling agents: agar-gelled medium was slightly better than gelite-gelled medium, and the highest plant conversion, 78±18.2%, was achieved in agar-gelled medium. Regenerated plantlets were successfully acclimatized in artificial soil mixture, and more than 98% of the plants survived in vermiculite or an equal volume mixture of vermiculite and peat moss. Results suggest that the rare and endangered species is able to propagate effectively via the somatic embryogenesis system.

New approaches to improve the efficiency of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.) from mature zygotic embryos

We developed an efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from mature zygotic embryos of oil palm. Embryogenic calli were induced from mature zygotic embryos of oil palm on modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid or picloram, alone or in combination with activated charcoal. The greatest frequency of embryogenic callus induction (97.5%) was obtained by culturing mature zygotic embryos on callus induction medium with 450 μM picloram and 2.5 g L−1 activated charcoal. Embryogenic calli proliferated on a medium with a reduced concentration of picloram. Embryogenic calli were then subcultured on a medium supplemented with 12.3 μM 2-isopentenyladenine and 0.54 μM naphthaleneacetic acid, with subcultures at 4-wk intervals. Somatic embryos were regenerated on a medium with Murashige and Skoog macro- and micronutrients at half-strength concentrations supplemented with 20 g L−1 sucrose, 2.5 g L−1 activated charcoal, and 2.5 g L−1 Phytagel. Detailed histological analysis revealed that somatic embryogenesis followed an indirect pathway. Primary calli were observed after 4–6 wk of culture and progressed to embryogenic calli at 12 wk. Embryogenic cells exhibited dense protoplasm, a high nucleoplasmic ratio, and small starch grains. Proembryos, which seemed to have a multicellular origin, formed after 16–20 wk of culture and successive cell divisions. Differentiated somatic embryos had a haustorium, a plumule, and the first and second foliar sheaths. In differentiated embryos, the radicular protrusion was not apparent because it generally does not appear until after the first true leaves emerge.

국내개발 이탈리안 라이그라스 품종 성숙종자의 식물체 재분화

In order to improve forage characteristics of Italian ryegrass by genetic transformation, an efficient callus induction from mature seed and optimal plant regeneration system were established using a domestic cultivar 'Kospeed'. Addition of 5 mg/L of 2,4-D showed highest frequency of embryogenic callus induction from mature seeds. N6 medium showed higher frequency of both callus induction and plant regeneration as compared with MS and SH medium. The highest plant regeneration frequency 67% was obtained when embryogenic calli were transferred to N6 medium containing 1 mg/L 2,4-D and 5 mg/L BA. Supplementation of regeneration medium with sucrose at 30 g/L level maximized regeneration frequency as compared to the other concentrations. These data would be very helpful for molecular breeding of domestic Italian ryegrass cultivar through genetic transformation.

High-frequency somatic embryogenesis from germinated zygotic embryos of Schisandra chinensis and evaluation of the effects of medium strength, sucrose, GA3, and BA on somatic embryo development

We developed a new protocol for highly efficient somatic embryogenesis and plantlet conversion of Schisandra chinensis. Friable embryogenic callus was induced from cotyledonary leaves and hypocotyls of germinated zygotic embryos on Murashige and Skoog (MS) agar medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Preculture of zygotic embryos on 2,4-D-containing medium increased embryogenic callus induction efficiency. The highest embryogenic callus induction frequency of 56.7% was obtained from shoot apical meristem-containing hypocotyl explants from 1-week-old germinated embryos on MS medium containing 4.0 mg l−1 2,4-D. Embryogenic callus proliferation, somatic embryo (SE) formation, and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose, gibberellic acid (GA3), and 6-benzyladenine (BA) on SE formation and plantlet conversion were evaluated. Low MS medium strength (1/4 to 1/2) was necessary for SE formation, and the optimal sucrose concentration was 2.0%. Supplementing medium with GA3 negatively impacted SE formation and subsequent development. BA significantly increased the number of SEs and the plantlet conversion capacity. One-third-strength MS medium with 1.0% sucrose and 0.5 mg l−1 BA produced the highest number of SEs (309 embryos from 9 mg embryogenic callus) and the highest frequency of plantlet conversion from germinated SEs (52.6%). When transplanted to soil, 90% of the regenerated plants developed into normal plants.

Influence of form of activated charcoal on embryogenic callus formation in coconut (Cocos nucifera)

Development of micropropagation protocols for Cocos nucifera has progressed slowly. Activated charcoal is included in the culture medium of each protocol, mainly to prevent tissue browning. Charcoal production procedures can affect the properties of different brands. In this study, eight types of activated charcoal were evaluated for their effects on free 2,4-dichlorophenoxyacetic acid level, pH, conductivity, and osmolarity of the culture medium and on the frequency of embryogenic callus induction. Moreover, the effect of particle size of the optimum charcoal type on embryogenic callus development was also studied. Charcoal type had a significant effect on (Y3) culture medium properties. Free 2,4-D was highest in Reactivos y Productos Quimicos Finos-containing medium and pH was lowest in MERCK-containing medium. Charcoal type also influenced embryogenic callus induction, with acid washed for plant cell and tissue culture-, DARCO- and United States Pharmacopeia-containing media promoting ~60% embryogenic callus, but with different optimal 2,4-D concentrations. Particle size profiles varied among all charcoal types, although small particle fraction (<38 μm) was abundant in all. Use of small particle fractions produced higher frequencies of embryogenic callus (70%) than either large particle or whole charcoal fractions.

들잔디 종자배양시 식물생장조절물질과 배지첨가물질이 배발생 캘러스 유도에 미치는 영향

In order to optimize tissue culture conditions for genetic transformation of zoysiagrass (Zoysia japonica Stued.), the effect of plant growth regulators and culture medium supplements on embryogenic callus induction from mature seeds of a cultivar `Zenith` were investigated. The optimal concentration and treatment period of NaOCl is 30% (v/v) for 60 minutes. Cultivation of mature seed on the callus Induction medium containing 3 mg/L 2,4-D and 3 mg/L dicamba showed 17.5% of embryogenic callus formation frequency. Supplementation of 1 g/L casein hydrolysate and 500 mg/L L-proline improved frequency of embryogenic callus induction. Audition of the medium with 5 mg/L and 20 mg/L cysteine enhanced frequencies of embryogenic callus induction. Efficient callus induction system established in this study will be useful for molecular breeding of Boysiagrass through genetic transformation.

High Frequency Somatic Embryogenic Callus Induction and Plant Regeneration from Various Indica Rice Genotypes

The paper evaluated the behavior of in vitro culture responses from a diverse set of Indica rice (Oryza sativa L.) genotypes. Significant differences were found in embryogenic callus induction frequency, callus growth and plant regeneration frequency when mature embryos of 11 cultivars, breeding lines and land races were compared. Genotype as well as plant growth regulator influenced the plant regeneration frequency. Callus induction frequency was not correlated with callus growth as well as plant regeneration frequency. The regenerated plants could grow to normal, fertile plants after they were successfully established in soil.

Agrobacterium-mediated transformation of seedling-derived maize callus

Efficient production of seedling-derived Type I callus was demonstrated for several corn genotypes including commercial inbred lines. Seeds were germinated on MS-based medium containing 10 mg l(-1) picloram and 3 mg l(-1) 6-benzylaminopurine, which induced the development of axillary buds in the area of coleoptilar node. Nodal sections of 7-10-day old seedlings were isolated, split longitudinally, and placed on callus induction medium supplemented with 2.2 mg l(-1) picloram and 0.5 mg l(-1) 2,4-dichlorophenoxyacetic acid. For lines L4 and L9 the frequency of embryogenic callus induction was 38-42% based on calli per split nodal section. Frequency of callus induction from split nodal sections of seeds germinated on media without growth regulators was 0-3%. Seedling-derived callus of five genotypes was used for Agrobacterium-mediated transformation. Two constructs containing the green fluorescence protein gene and genes for either neomycin phosphotransferase II or glyphosate selection were used in transformation experiments. Transformation frequency varied from 2 to 11% and about 60% of the T(0) plants had 1-2 copies of transgenes.

Plant Regeneration and Callus Type in Barley: Effects of Genotype and Culture Medium

To facilitate practical application of in vitro genetic manipulation to barley (Hordeum vulgare L.), methods for the in vitro culture of barley must be improved. Fifteen genotypes of barley, primarily cultivars or elite breeding lines adapted to North America, were examined for their ability to form regenerable callus from immature embryos on modified versions of MS, B5, and CC media. Regenerable calli were obtained from all genotypes. The frequency of embryogenic callus induction, the morphology and friability of embryogenic calli, the relative growth rate of embryogenic calli, and regenerability were influenced by genotype, culture medium, and genotype × culture medium interactions. Callus cultures that were embryogenic in appearance frequently were not regenerable, or regenerated only albino plantlets. The majority of green plants grown to maturity were fertile and normal in appearance. Genotype was the most important determinant of the in vitro response. Regenerability was transient for all genotypes, with few exceptions. The best in vitro performance, based on ability to form friable, regenerable callus, was observed for ‘Golden Promise’, ‘Hector’, ‘Piroline’, and ‘Klages’.