In both cases the proximal region relative to the vascular bundles is composed of parenchymatic cells while the middle region is characterized by having a meristematic activity, forming new cells in both proximal and distal directions. A similar pattern in the cambium- like zone of nodular callus derived from perivascular cells, has been described by other authors (Ozias-Akins & Vasil 1982, Ho & Vasil 1983, Schwendiman et al. 1988, Buffard-Morel et al. 1992, Guerra & Handro 1998). The distal region is formed by potentially detachable cells, either by being converted in somatic embryos or by being exfoliated in root caps. In both cases the cells of this region exhibited starch grains which were proposed to be somaticembryogenesis markers (Buffard-Morel et al. 1992) while in normal roots they are known to act as statholiths (Mauseth 1995). The presence of starch grains in root primordia has also been reported in several other somaticembryogenesis systems (Thomas et al. 1972, Street & Withers 1974, Ozias-Akins & Vasil 1982, Lu & Vasil 1985, Schwendiman et al. 1988).
Plant micropropagation systems on liquid medium associated to membrane rafts has been successfully used in several works (Watad et al., 1995; Desamero et al., 1993; Adelberg et al., 1992). However, as far as we know this is the first report describing the use of such system for somaticembryogenesis induction.
The total absence of protein in somatic embryos at a more advanced stage of development agrees with past research on palms (Goh et al. 2001, Sané et al. 2006) and emphasizes the necessity for optimization of the maturation conditions, for example, by manipulation of the components of the medium culture. The latter enables the accumulation of sufficient reserves for satisfactory germination of somatic embryos obtained. According to Joy IV et al. (1991), several factors may influence the pattern of reserve deposition during somaticembryogenesis, such as the absence of maternal influence, water content, growth regulators, the concentration of salts, carbon source, and stability of the osmotic environment.
Despite the economic importance of the THB cultivar, few studies in the literature have investigated its propagation. Commercially, papaya trees are propagated from seeds; however, seeds can be used for other purposes. For example, zygotic embryos (Ascencio-Cabral et al. 2008, Farzana et al. 2008, Malabadi et al. 2011, Anandan et al. 2012) or segments of seedlings (Yu et al. 2003, Saha et al. 2004, Farzana et al. 2008, Homhuan et al. 2008) can be used in tissue culture to produce explants and somatic embryos via somaticembryogenesis. This technique produces large numbers of somatic embryos that can be transformed into plantlets, identified as hermaphrodite ex vitro and subsequently rescued in vitro and used in the field.
et al., 1997; Somleva et al., 2000; Hecht et al., 2001). Since that time SERK gene expression has been associated with somaticembryogenesis (SE) and organogenesis in numerous species ( NOLAN et al., 2003; ZAKIZADEH et al., 2010). In Arabidopsis five SERK genes have been identified ( HECHT et al., 2001) (AtSERKs 1-5) and the gene functioning in SE is AtSERK1 (locus At1g71830). As understanding of the roles of the different members of the SERK gene family has increased, it has become apparent that these genes function in diverse signaling pathways with roles from development to defense. The Arabidopsis SERK gene family is subdivided into two subfamilies, generated from an ancestral gene duplication event. The first subfamily consists of AtSERKs 1 and 2 (SERK1/2) and the second subfamily, AtSERKs 3, 4 and 5 (SERK3/ 4/5) ( HECHT et al., 2001; HE et al., 2007; ALBRECHT et al., 2008).
Most genes expressed differentially during somaticembryogenesis belong to the late embryo-abundant (lea) genes. Proposed functions for the products of this family of genes arethe protection of cellular structures in mature embryos during seed desiccation and prevention of precocious germination of the zygotic embryos during seed development (reviewed by Wilde et al., 1995 and Dong & Dunstan, 2000). Several genes expressed in carrot somatic embryos code for secreted extracellular proteins. One gene product (EP1), with homology to Brassica S-locus glycoproteins, is present in nonembryogenic callus but not in somatic embryos themselves. Another gene that produces a lipid transfer protein (EP2) has been particularly useful as a marker for epidermal cell differentiation during embryogenesis. The precise role of these extracellular proteins remains to be established, but they may be involved in the regulation of cell expansion and the maintenance of biophysical features required for morphogenesis. Perhaps the most unexpected finding involves a secreted glycoprotein (EP3) that rescues a temperature- sensitive mutant of carrot (ts11) that fails to complete the transition from a globular to heart stage of somaticembryogenesis (Meinke, 1995; Sugiyama, 2000). Another group of genes that are developmentally regulated in carrot suspension cultures is constituted by the Dc3, Dc8, J4e and ECP31 genes. They are expressed at different momentsduring embryo development and localized in different cell groups within the PEMs and embryos (Wilde et al., 1995). Dudits et al. (1995) indicate that the gene expression is expected to be different during the processes of embryogenic commitment in primary explants or fully differentiated somatic cells from those that act in suspension cultures with proembryogenic structures, such as in the case of carrot.
Abstract – The aim of this work was to identify Brazilian soybean (Glycine max) genotypes with potential to respond to in vitro culture stimuli for primary somatic embryo induction, secondary embryo proliferation and plant regeneration. Differences among eight tested cultivars were observed at each stage. Two cultivars, IAS-5 and BRSMG 68 Vencedora, were selected for the evaluation of the capacity for embryo differentiation and plant regeneration. These cultivars had high embryo induction frequencies, repetitive embryogenic proliferation, and low precocious embryo germination in the initial experiment. The effect of abscisic acid (ABA) and charcoal addition on plant regeneration was investigated. The addition of ABA to proliferation medium and of ABA and activated charcoal to maturation medium increased embryo differentiation rates, which resulted in a higher number of regenerated plants. The BRSMG 68 Vencedora cultivar was found to have a high potential for embryo induction, embryo proliferation and plant regeneration. The potential of this cultivar for somaticembryogenesis was similar to that observed for cultivar IAS-5, which is currently used for soybean transformation in Brazil. BRSMG 68 Vencedora may be a good alternative genotype for soybean genetic engineering via somaticembryogenesis protocols.
Among the carbohydrates, galactose and lactose induced the highest number of somatic embryos for ‘Ponkan’ at 75 mM of lactose (Table 1). Similar responses were observed for the varieties ‘Valencia’ sweet orange, and ‘Kinnow’ mandarin, with the exception of the ‘Cravo’ mandarin, which had the lowest number of embryos. The best concentrations of lactose for ‘Valencia’, ‘Kinnow’ and ‘Cravo’ were 37 mM, 75 mM and 37 mM, respectively (Table 1). These results are similar to that documented by Kochba et al. (1982), who studied somaticembryogenesis of callus obtained from several varieties of Citrus and found that galactose or lactose was very effective at inducing the embryo formation. Also, according to them, galactose induces the production of ethylene and that high level of ethylene inhibits the biosynthesis of auxins, which favors the formation of somatic embryos in embryogenic calli. This fact might explain the responses observed in the citrus varieties mentioned above.
somaticembryogenesis. This illustrates that various culture stages may be affected differently by the formulation of the basal media. This was also demonstrated by Capuana et al. (2007) in common ash (Fraxinus excelsior L.) where the best proliferation of embryogenic tissue was obtained when the material was subcultured on MS medium; whereas, WPM medium appeared to be more conducive to faster embryo maturation. In a study on the induction potential of somatic embryos in Eucalyptus globulus involving MS, B5, WPM and DKW (Driver Kuniyuki Walnut medium; Driver and Kuniyuki, 1984), Pinto et al. (2008a) demonstrated that MS and B5 media were the best. In a subsequent study, Pinto et al. (2008b) found that MS medium and B5 were equally effective in the formation of secondary globular somatic embryos; however, MS was more efficient for the germination stage as compared to B5 medium. In the same way, Fisichella et al. (2000) reported that MS medium appeared to be the most favorable for somatic embryo formation in quince (Cydonia oblonga Mill.) as compared to W, WPM, DKW, QL (Quoirin and Lepoivre, 1977), SH, B5 and NN. However, root formation was higher on WPM and SH media.
Abstract – The aim of this work was to quantify the protein, starch and total sugars levels during histodifferentiation and development of somatic embryos of Acca sellowiana Berg. For histological observations, the samples were dehydrated in a battery of ethanol, embedded in historesin and stained with toluidine blue (morphology), coomassie blue (protein bodies) and periodic acid–Schiff (starch). Proteins were extracted using a buffer solution, precipitated using ethanol and quantified using the Bradford reagent. Total sugars were extracted using a methanol-chloroform-water (12:5:3) solution and quantified by a reaction with anthrone at 0.2%. Starch was extracted using a 30% perchloric acid solution and quantified by a reaction with anthrone at 0.2%. During the somaticembryogenesis’ in vitro morphogenesis and differentiation processes, the total protein levels decreased and the soluble sugars levels increased during the first 30 days in culture and remained stable until the 120 th day.
Abstract: The objective of this study was to evaluate the genotype x environment interaction and to estimate the genetic components of variance and mean us- ing mixed models in early selection of 238 clones of Pinus taeda propagated by somaticembryogenesis. The experiment consisted of a complete blocks design, with 12 replications, with one plant per plot, in four environments, at 1, 3, and 4 years of age. Estimates of heritability and of genetic gains in the evaluated environments showed good prospects for selection of superior genotypes. The effect of genotype x environment interaction was pronounced for all traits in- vestigated. With the simultaneous selection for stability and adaptability, 10% genetic gain was obtained in relation to the mean of the commercial controls. This estimated gain indicates that the somaticembryogenesis technique has been effective in propagation of clones with good productive potential. Key words: Forestry improvement, clonal silviculture, genetic selection, early selection.
induction and somaticembryogenesis. Since higher concentration of 2,4-D had negative effect on somatic embryo development, low concentration of 2,4-D in combination with high concentration of picloram, were used for callus induction and somaticembryogenesis. As a result, the highest percentage of plant regeneration was obtained from embryogenic calli induced on MS medium supplemented with 1 mg/L 2,4-D, 2 mg/L picloram and 200 mg/L casein hydrolysate (Table 2 and 3). This results is consistent with the reports of Przetakiewicz et al. . They reported that dicamba alone or in combination with picloram and 2,4-D were the best medium for embryogenic callus formation in the wheat genotypes. Kachhwaha et al.  compared the picloram and 2,4-D effects on somaticembryogenesis and plant regeneration of barley and showed that picloram gave much better results. Tao et al.  reported that substitution of 2,4-D with dicamba enhances the growth and regeneration capacity of wheat. Kordestani and Karami  reported that the maximum embryogenesis in strawberry was obtained by using 2 mg/L picloram.
culture; the same results were observed when the explants were cultured on medium containing picloran (CHENGALRAYAN et al., 2005). In a study of the sugarcane cultivar CoJ88t, an increase in the concentration of 2,4-D from 18.1 to 36.2 μM reduced callus induction from 86.4 to 76.0% (SINGH et al., 2008). In another study, dicamba (3,6-dichloro-2 hydroxybenzoic acid) was found to be more effective at inducing callus formation in the Nco310 sugarcane cultivar compared to 2,4-D, naphthaleneacetic acid (NAA) and 4-fluorophenoxyacetic acid (4-FPA) (BRISIBE et al., 1994). However, most studies showed that 2,4-D or 4-chlorophenoxyacetic acid (pCPA) were superior to other regulators in terms of the induction of somaticembryogenesis in sugarcane (LAKSHMANAN, 2006). Shoot regeneration from somaticembryogenesis of the sugarcane cultivar Q165 A was found to occur in MS medium
In maize culture, the immature zygotic embryo is the most widely used explant for the establishment of embryogenic callus cultures and genetic transformation (FRAME et al., 2002, 2006, 2011; ISHIDA et al., 1996, 2007; LEE et al., 2007; VEGA et al., 2008) because the immature embryo presents cells that are competent for somaticembryogenesis, unlike other tissues that are more differentiated. However, the production of immature embryos is a demanding process that requires time and painstaking activities for the growth of plants and pollination. Thus, the use of more readily available alternative explants, such as immature tassels, can accelerate the obtainment of in vitro plants.
The success of plant regeneration via somaticembryogenesis from protoplasts of C. harnandiana is strongly depend on BA and DAP. Cytokinin was necessary to recover organs and plants from protoplasts to plantlets [1, 10, 14, 15, 30, 33]. This efficient protoplast-to-plant system for this species could facilitate the transfer of nucellar and cytoplasmic genes of a source of time tolerance from this species into cultivated Citrus through protoplast fusion. While Citrus relatives have been difficult or impossible to hybridize with Citrus by conventional methods [7, 11].
References 40 and 41 are incorrectly switched. The correct reference 40 is: Lebrecka R (2008) Host-pathogen interaction between Phytophthora infestans and Solanum nigrum, S. villosum and S. scabrum. European J of Plant Pathol 120(3): 233–240. doi: 10.5580/e27. The correct ref- erence 41 is: Rohani ER, Ismanizan I, Noor NM (2012) Somaticembryogenesis of mangosteen. Plant Cell Tiss Org Cult 110(2): 251–259. doi: 10.1007/s11240-012-0147-4.
A. sellowiana somaticembryogenesis was firstly reported by Cruz et al., (1990). High frequency of somatic embryos was further obtained (Canhoto & Cruz, 1994, 1996a). In Brazil the first results on somaticembryogenesis in this species were reported by Guerra et al. (1997) which showed that this morphogenetic route was dependent on the culture medium composition and the genotype employed. Dal Vesco & Guerra (2001) showed that different nitrogen sources in the culture medium affected the number of somatic embryos developed from zygotic embryos. Guerra et al. (2001) demonstrated that somaticembryogenesis in this species was affected by 2,4-D pulses, and histological studies revealed abnormalities on the development of somatic embryos. Stefanello et al. (2005) reported embryogenic competence from floral tissues, and Cangahula-Inocente et al. (2007) included the technology of synthetic seeds in the somaticembryogenesis protocol.
A somaticembryogenesis receptor kinase like (SERKL) cDNA, designated PhSERKL , was isolated from date palm (Phoenix Dactylifera L) using RACE PCR. PhSERKL protein shared all the characteristic domains of the SERK family, including five leucine-rich repeats, one proline-rich region motif, a transmembrane domain, and kinase domains. Phylogenetic analyses using PHYLIP and Notung 2.7 programs suggest that the SERK proteins of some plant species resulted from relatively ancient duplication events. We predict an ancestor protein of monocots and dicots SERK using FASTML program. Somatic embryogenic cultures of date palm were established following transfer of callus cultures to medium containing 2, 4-dichlorophenoxyacetic acid. The role of PhSERKL gene during establishment of somaticembryogenesis in culture was investigated using quantitative real-time PCR. PhSERKL gene was highly expressed during embryogenic competence acquisition and globular embryo formation in culture. Overall, levels of expression of PhSERKL gene were lower in nonembryogenic tissues and organs than in embryogenic callus.
After the establishment of somaticembryogenesis and plant regeneration protocols, some embryogenic calluses from cell suspension of banana cv. Maçã were bombarded with plasmid constructs, containing the gene for kanamycin resistance and the gus-A gene for β-glucoronidase. Ten Petri dishes were used containing 0.5 mL of packed cell suspension for each plasmid construction. The following constructs were used: pBI426 (70S promoter, uid-A/neo), pFF19 (70S promoter, uid-A) and pCAMBIA1303 (35S promoter, uid-A, Hygromycin). The DNA of each plasmid construction was isolated from the Escherichia coli DH5α strain by alkaline lysis, and purified by polyethylene glycol precipitation (Concert kit; Invitrogen). Tungsten particles were prepared using 5 µL of DNA (1 ng DNA µL -1 ), 20 µL of spermidine and
The study was undertaken to examine the effect of two synthetic auxins on somaticembryogenesis 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.