Genetic control of somatic embryogenesis induction in <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> Labill.

A reproducible protocol for somatic embryogenesis (SE) induction in Eucalyptus globulus from mature zygotic embryos is available since 2002. However, for the use of SE in tree breeding programs, the frequency of SE initiation needs to be improved and controlled, and this was investigated in 13 open-pollinated (OP) families over three consecutive years. A diallel mating design with five parent trees was used to study genetic control of SE induction. Results showed that SE induction varies across E. globulus families and over the years of seed production tested. Somatic embryogenesis was initiated on explants from 84% of the OP families tested in 2002 and 100% of the families tested in 2003 and 2004. The year 2003 gave best results for percentage of induction and total number of somatic embryos produced. Results concerning genetic control showed that SE induction is under the control of additive genetic effects, as 22.0% of variation in SE initiation was due to general combining ability (GCA) effect, whereas 6.4% was due to maternal effects. Neither specific combining ability (SCA) nor reciprocal effects were significant.     

NH4 + and NO3 − requirement for wheat somatic embryogenesis

The influence of three nitrogen salts: NH₄NO₃, KNO₃ and NH₄Cl on wheat in vitro cultures was investigated. Both NO ₃ ⁻ and NH ₄ ⁺ ions were indispensable for proliferation of embryogenic calli and development of wheat somatic embryos. It is possible to obtain wheat somatic embryos when the medium is enriched with NH₄NO₃ only as a source of inorganic nitrogen. The results of the statistical analysis showed that the level of NH₄NO₃ and KNO₃ in the medium had a great influence on the efficiency of somatic embryogenesis. We observed tendency that calli on media containing 50 mM NH₄NO₃ and 0 to 20 mM KNO₃ turned out to be more embryogenic than on control MS medium. High concentrations of KNO₃- 100 mM inhibited somatic embryogenesis, while 100 mM NH₄NO₃ did not. The level of total N did not have significant influence on wheat somatic embryogenesis. Ratio NO ₃ ⁻ :NH ₄ ⁺ also turned out to be not substantial. We observed that mutual connection of concentration levels between NH₄NO₃ and KNO₃ and between NH₄Cl and KNO₃ was more important. The efficiency of somatic embriogenesis obtained in the experiment with NH₄Cl and KNO₃ was significantly lower than in experiment with NH₄NO₃ and KNO₃.     

The effect of auxins, time exposure to auxin and genotypes on somatic embryogenesis from mature embryos of wheat

The effects of different factors on the embryogenesis and plant regeneration from mature embryos of Russian spring and winter genotypes were studied. Embryogenic callus induction was achieved on MS medium supplemented with different concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) or Dicamba (3,6-dichloro-o-anisic acid). Although all auxins were able to induce callus from explants with high frequency (98–100%), Dicamba was more effective for the induction of embryogenic callus (21.8–38.3%). Maximum embryogenic callus formation and high number of regenerated plants were observed at 12 mg l⁻¹ of Dicamba. The time exposure to Dicamba (7, 14, 21 and 28 days) had a significant effect on efficiency of somatic embryogenesis. When contact of explants with callus induction medium was increased from 7 to 21 days the rate of somatic embryogenesis and number of regenerated plants per embryogenic callus gradually increased from 13.0 to 38.4% and 3.6 to 8.0%, respectively. Supplement of additional auxins (indoleacetic acid (IAA), indolebutyric acid (IBA), and naphthaleneacetic acid (NAA)) to callus induction medium with Dicamba had a positive effect on the rate of embryogenic callus formation, while the average number of regenerated shoots was not affected. The best rate of somatic embryogenesis was observed at the addition of 0.5 mg l⁻¹ IAA with Dicamba (61.0%). The optimum combination of Dicamba and IAA increased the efficiency of somatic embryogenesis and plant regeneration from seven spring and winter wheat genotypes, thought overall morphogenic capacity was still genotype dependent.     

A novel method to induce direct somatic embryogenesis,secondary embryogenesis and regeneration of fertile green cereal plants

A direct somatic embryogenesis and secondary embryogenesis protocol was developed for seven cereal species, thus providing a new vista for in vitro plant genetic transformation or propagation. This paper describes a novel process that has been successfully developed for efficient regeneration of a wide range of cereal species and genotypes. This tissue culture and regeneration system does not require formation of callus tissues and takes approximately 2 months to complete, shorter than any of the currently available systems requiring 3-4 months. Rapid induction of direct somatic embryogenesis in barley (Hordeum vulgare), common wheat (Triticum aestivum), durum wheat (T. durum) and derived amphiploids, wild wheat (T. monococcum and T. urartu), rye (Secale cereale) and oats (Avena sativa) was induced from excised immature scutellum on DSEM medium. Newly developed globular embryos were cultured on SEM medium for a second cycle of embryogenesis followed by germination (GEM medium) and regeneration of embryos into normally growing green and fertile plants. In vitro techniques to induce direct somatic embryogenesis, secondary embryogenesis and plant regeneration from these cereals require a specific sequence of defined media and controlled environments. The sequence and the timing of the media used, as well as their hormonal composition and balance are critical aspects of this process. The organic and mineral compositions of these media are not new but are important for supporting and sustaining rapid growth of the tissues.     

Micropropagation ofPanax notoginseng by somatic embryogenesis and RAPD analysis of regenerated plantlets

Somatic embryogenesis was induced in callus tissues derived from young flower buds ofPanax notoginseng via callus within 18 weeks of culture. The mature somatic embryos were germinated on half-strength Murashige and Skoog's (MS) medium supplemented with gibberellic acid A₃(GA) and 6-benzyladenine (BA). The most suitable medium for optimal root formation proved to be MS medium supplemented with 1-naphthaleneacetic acid (NAA). Total DNA was extracted from the leaves of the regenerated plantlets ofP. notoginseng. Analysis of random-amplified polymorphic DNA (RAPD) using 21 arbitrary oligonucleotide 10-mers, showed the genetic homogeneity ofP. notoginseng. The amplification products were monomorphic for all of the plantlets ofP. notoginseng regenerated by embryogenesis, suggesting that somatic embryogenesis can be used for clonal micropropagation of this plant.     

Lectin levels in tissues of cultured immature wheat embryos

Levels of wheat germ agglutinin have been determined by radioimmunoassay in tissues of immature wheat embryos cultured under different conditions in order to determine the suitability of the lectin as a marker for somatic embryogenesis. Embryos cultured on media favouring continued embryo development accumulated lectin in a similar manner to zygotic embryos in planta unless precocious germination occurred. Embryos cultured on media containing 2,4-D produced callus, and some of this developed somatic embryos. Both embryogenic and non-embryogenic callus contained WGA, that in non-embryogenic callus possibly arising from developmentally arrested root primordia.Abbreviations ABA abscisic acid - dpa days post anthesis - PBS phosphate buffered saline, (10 mM KH₂PO₄ K₂HPO₄, 145 mM NaCl, pH 7.4) - RIA radioimmunoassay - WGA wheat germ agglutinin - 2,4-D 2,4-dichlorophenoxyacetic acid     

Role of calcium–calmodulin in auxin-induced somatic embryogenesis in leaf base cultures of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> var. HD 2329)

Wheat leaf bases cultured for 1 day on 2,4-d (10 μM) display the induction of somatic embryogenesis. The induction of somatic embryogenesis by 2,4-d appears to be calcium-mediated as treatment of leaf bases with the calcium chelator, EGTA, prior to 2,4-d treatment, inhibited the induction of somatic embryogenesis. This sensitivity of auxin to reduced calcium levels can be reversed by calcium ions alone and not any other divalent cation like magnesium or zinc. Additionally, the expression of the three calcium-regulated genes, Triticum aestivum calmodulin binding protein kinase, calcium-dependent protein kinase, and putative calcium binding protein was analyzed in wheat leaf bases which suggest a specific role for Ca²⁺ in somatic embryogenesis. Application of the calcium ionophore, A23187, either alone or along with 2,4-d, induced somatic embryogenesis. This specificity for calcium was verified both by treatment with the calcium antagonist TMB8, and the elimination of calcium from the medium, resulting in reduction of somatic embryogenesis by 80%. Treatment with calcium channel blockers like verapamil and nifedipine, calcium antagonist, lanthanum, and calmodulin inhibitors chlorpromazine and fluphenazine, prior to the 2,4-d treatment, inhibited induction of somatic embryogenesis. The present study thus provides evidence for the involvement of calcium–calmodulin in the stimulus–response coupling of auxin-induced somatic embryogenesis in wheat leaf base system.     

Identification of QTL underlying somatic embryogenesis capacity of immature embryos in soybean (Glycine max (L.) Merr.)

High embryogenesis capacity of soybean (Glycine max (L.) Merr.) in vitro possessed potential for effective genetic engineering and tissue culture. The objects of this study were to identify quantitative trait loci (QTL) underlying embryogenesis traits and to identify genotypes with higher somatic embryogenesis capacity. A mapping population, consisting of 126 F_5:6 recombinant inbred lines, was advanced by single-seed-descent from cross between Peking (higher primary and secondary embryogenesis) and Keburi (lower primary and secondary embryogenesis). This population was evaluated for primary embryogenesis capacity from immature embryo cultures by measuring the frequency of somatic embryogenesis (FSE), the somatic embryo number per explant (EPE) and the efficiency of somatic embryogenesis (ESE). A total of 89 simple sequence repeat markers were used to construct a genetic linkage map. Six QTL were associated with somatic embryogenesis. Two QTL for FSE were found, QFSE-1 (Satt307) and QFSE-2 (Satt286), and both were located on linkage group C2 that explained 45.21 and 25.97% of the phenotypic variation, respectively. Four QTL for EPE (QEPE-1 on MLG H, QEPE-2 on MLG G and QEPE-3 on MLG G) were found, which explained 7.11, 7.56 and 6.12% of phenotypic variation, respectively. One QTL for ESE, QESE-1 (Satt427), was found on linkage group G that explained 6.99% of the phenotypic variation. QEPE-2 and QESE-1 were located in the similar region of MLG G. These QTL provide potential for marker assistant selection of genotypes with higher embryogenesis.     

Chromosomal location of genes controlling short-term and long-term somatic embryogenesis in wheat revealed by immature embryo culture of aneuploid lines

The expression of essential genes during somatic embryogenesis can be analysed by inducing aneuploid cells to undergo embryogenesis during immature embryo culture and then determining whether defects occur. Triticum aestivum disomic and aneuploid stocks, including 36 ditelosomics and 7 nullitetrasomic Chinese Spring wheats, were compared for their ability to undergo somatic embryogenesis after 2 months of in vitro immature embryo culture. Their regeneration capacity was observed after 4 and 14 months of in vitro culture to determine which chromosome arms influence the process. The large range of variation found among the tested aneuploids suggested that genetic control of the somatic tissue culture ability is polygenic. Our results indicate that genes affecting somatic embryo-genesis and regeneration are located in all of the homoeologous chromosome groups. The lack of chromosome arms 1AL (DT 1AS) and 3DL (DT 3DS) practically suppresses somatic embryogenesis, demonstrating that major genes on wheat chromosome arms 1AL and 3DL control regeneration capacity. Results suggest that plants were mainly produced from somatic embryo development. Although the control of somatic embryogenesis and regeneration is polygenic, the genes located on the long arms of homoeologous group 3 chromosomes have a major effect. We also have evidence of chromosome arms that determine the time required for regeneration.     

Identification of a RAPD marker associated with somatic embryogenesis in alfalfa

The current study was conducted to identify random amplified polymorphic DNA (RAPD) markers linked to genes controlling somatic embryogenesis in alfalfa. Segregation analyses of the somatic embryogenesis trait and the RAPD markers in an F₁ population of 83 plants, derived from a cross between embryogenic A70-34 and non-embryogenic Arrow36 alfalfa plants, identified a polymorphic band that is associated with somatic embryogenesis. Based on the assumptions that somatic embryogenesis in alfalfa is controlled by two dominant genes with complementary effects and that the genotypes of A70-34 and Arrow36 are AAaaBbbb and aaaabbbb, respectively, the segregation data for the marker and the somatic embryogenesis trait in the F₁s indicate that the marker is linked to the A locus. The maximum recombination fraction estimated for the linkage between the marker and the gene is 36.3%.     

Studies on genetic transformation of Theobroma cacao L.: evaluation of different polyamines and antibiotics on somatic embryogenesis and the efficiency of uidA gene transfer by Agrobacterium tumefaciens

In order to develop a more efficient genetic transformation system for cacao somatic embryos, the effects of polyamines and β-lactam antibiotics on somatic embryogenesis, hygromycin as selective agent, and different factors affecting uidA gene transfer have been evaluated. The polyamines putrescine, spermidine, and spermine significantly improved secondary somatic embryogenesis in cacao. Spermine at 1,000 μM provided the best responses, increasing 6.7× the percentage of embryogenic callus and 2.5× the average number of embryos per embryogenic callus. The β-lactam antibiotics timentin and meropenem, used for Agrobacterium tumefaciens counter-selection, had a non-detrimental effect on secondary somatic embryogenesis, depending on their concentration, whereas the commonly used β-lactam cefotaxime inhibited it, irrespective of the tested concentration. Hygromycin showed a strong inhibitory effect on secondary somatic embryogenesis of cacao, impairing completely the embryo production at 20 mg l⁻¹. Following the criterion of GUS activity, the best conditions for T-DNA transfer into cotyledon explants from primary somatic embryos of cacao were a sonication of the explants for 100 s, a 20-min incubation period in Agrobacterium solution, an Agrobacterium concentration of 1.0 (OD₆₀₀), and cocultivation of the explants on tobacco feeder layers. These findings will have important implications for studies on functional genomics of cacao.     

Effect of MgSO4 and K2SO4 on somatic embryo differentiation in Theobroma cacao L.

Somatic embryogenesis in cacao is difficult and this species is considered as recalcitrant. Therefore, reformulation of culture media might be a breakthrough to improve its somatic embryogenesis. In cacao, acquisition of somatic embryogenesis competence involves three main stages: induction of primary callus, induction of secondary callus and embryo development. Screening for MgSO₄ and K₂SO₄ concentrations for somatic embryo differentiation was conducted on three genotypes (Sca6, IMC67 and C151-61) at the three stages. The effect of these two salts in culture media appears to be most efficient at the embryo development stage. At this stage, high MgSO₄ (24 mM) and K₂SO₄ (71.568 mM) in the culture media induced direct somatic embryos on staminodes and petals of the Sca6 and IMC67 genotypes. Media supplemented with 6.0 mM and 12.0 mM MgSO₄ enabled high responsive of explants and produced high proportion of embryos. The positive effect of MgSO₄ and K₂SO₄ on the acquisition of embryogenesis competence was further tested on seven cacao genotypes reputed as non embryogenic: SNK12, ICS40, POR, IMC67, PA121, SNK64 and SNK10. All these genotypes were able to produce somatic embryos depending on the MgSO₄ concentration. Thus, our results showed that the recalcitrance of cacao to somatic embryo differentiation can be overcome by screening for the suitable MgSO₄ or K₂SO₄ concentration. Studies of the influence of different K⁺/Mg²⁺ ratios (at normal sulphate concentration) on somatic embryo differentiation revealed that sulphate supply was the main factor promoting responsive explants and the proportion of embryos. Cysteine synthase isoforms showed patterns related to morphogenetic structures sustaining that sulphur supply and its assimilation improve somatic embryogenesis in cacao.     

Inheritance of somatic embryogenesis and plantlet regeneration from primary (type 1) callus in maize

Summary Genetic factors controlling the differential expression of somatic embryogenesis and plant regeneration of maize from tissue culture were studied in two crosses. Inbred, hybrid, F2 and backcross generations developed from crossing maize inbred A188 with two commercially important inbred maize lines (B73 and Mo17) demonstrated genetic and environmental effects on somatic embryogenesis and plant regeneration when immature zygotic embryos were cultured on MS medium. Additive gene effects were more important in both crosses than dominant gene effects for precent somatic embryogenesis and percent or number of plants regenerated per embryo when generation means were analyzed. In backcross generations of each cross, cytoplasmic, maternal and/or paternal effects were significant for frequency of somatic embryos three weeks after culture as well as frequency, or number of plants regenerated per embryo, nine weeks after culture. Analysis of genetic variances suggests at least one gene (or block of genes) controls the expression of the frequency of somatic embryogenesis in these crosses. Differences in somatic embryogenesis and plant regeneration between B73 and Mo17 are discussed. This is Journal Paper No. 11,435 of the Purdue University Agricultural Experiment Station.     

Influence of in vitro environment on somatic embryogenesis of Coffea arabica L. cv. Caturra rojo: the effects of carbon dioxide on embryogenic cell suspensions

The influence of environment in the culture vessel is a factor that has very little study in the process of somatic embryogenesis. The present research was carried out with the objective to determine the effects of carbon dioxide on somatic embryogenesis of Coffea arabica cv. Caturra rojo. Embryogenic cell suspensions were cultured under different carbon dioxide concentrations (2.5%, 5.0%, and 10.0%) in the gases mixture and two control treatments, one with passive exchange and the other with forced ventilation. The results demonstrated that there were a larger number of somatic embryos formed with a concentration of 2.5% CO_2. The differentiation of these somatic embryos of coffee in embryogenic cell suspensions (130 × 10³ SE l⁻¹) was also stimulated. The effects of CO₂ on somatic embryogenesis were demonstrated when the control with passive exchange was compared with forced ventilation control, because in the former, where there was an accumulation of CO₂, the production of somatic embryos was greater. CO₂ could stimulate the formation and differentiation of somatic embryos directly, which led to a modification of the pH patterns of the culture medium or indirectly when producing changes in the pH that favored the somatic embryogenesis process.     

Inheritance of Fusarium head blight resistance in the soft red winter wheat Ernie

Fusarium head blight (FHB), caused by Fusarium graminearum Schwabe [telomorph:Gibberella zeae Schw. (Petch)], is an increasingly important disease of wheat (Triticum aestivum L.). Host-plant resistance is considered to be the most economical means of control, but a lack of unique sources of resistance has hindered efforts to breed resistant varieties. The soft red winter wheat, Ernie, has moderately high FHB resistance and is widely used in U.S. breeding programs; however, the genetics of resistance have not been studied. The objectives of this study were to estimate the genetic effects, gene numbers, and heritability for traits related to FHB resistance in Ernie through generation means analyses and variance analyses of 243 F₃-derived F₈ and F₉ recombinant inbred lines (RILs). Replicated experiments were grown in the greenhouse, inoculated with F. graminearum, and evaluated for disease spread and the FHB index (FHBI). The latter was calculated as the percentage of diseased spikelets in inoculated spikes and is often referred to as type-II resistance. Gene action for both disease spread and FHBI was primarily additive with partial dominance for low disease. Broad-sense heritabilities for spread and FHBI were 78.2% and 78.3%, respectively, while the narrow-sense heritabilities were 51.3% and 55.4%, respectively. Line-mean heritabilities from analyses of variance of RILs were 0.70 and 0.87 for spread and FHBI, respectively. A minimum of four genes conditioned both disease spread and FHBI. These results suggest that breeders should be able to enhance FHB resistance by combining the resistance in Ernie with other complementary additive sources of resistance.     

Efficient somatic embryogenesis in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) breeding lines

Efficient regeneration via somatic embryogenesis (SE) would be a valuable system for the micropropagation and genetic transformation of sugar beet. This study evaluated the effects of basic culture media (MS and PGo), plant growth regulators, sugars and the starting plant material on somatic embryogenesis in nine sugar beet breeding lines. Somatic embryos were induced from seedlings of several genotypes via an intervening callus phase on PGo medium containing N⁶-benzylaminopurine (BAP). Calli were mainly induced from cotyledons. Maltose was more effective for the induction of somatic embryogenesis than was sucrose. There were significant differences between genotypes. HB 526 and SDM 3, which produced embryogenic calli at frequencies of 25–50%, performed better than SDM 2, 8, 9 and 11. The embryogenic calli and embryos produced by this method were multiplied by repeated subculture. Histological analysis of embryogenic callus cultures indicated that somatic embryos were derived from single- or a small number of cells. 2,4-dichlorophenoxyacetic acid (2,4-D) was ineffective for the induction of somatic embryogenesis from seedlings but induced direct somatic embryogenesis from immature zygotic embryos (IEs). Somatic embryos were mainly initiated from hypocotyls derived from the cultured IEs in line HB 526. Rapid and efficient regeneration of plants via somatic embryogenesis may provide a system for studying the molecular mechanism of SE and a route for the genetic transformation of sugar beet.     

The embryogenic response of immature embryo cultures of durum wheat (Triticum durum Desf.): histology and improvement by AgNO3

Immature zygotic embryos of durum wheat cv Ardente were cultured vitro on 2,4-D to induce somatic embryogenesis. Five days after culture initiation, somatic proembryos were directly initiated from the scutellum of immature embryos. After 28 days, somatic embryos were fully developed with a scutellum-like structure. Histological observations between the first and the eighty day in culture showed a clear unicelllar origin for a few of these somatic embryos, whilst most of them originated from a meristematic multilayer. Furthermore, estimation of the mitotic index of outer epithelium, subepithelium and inner epithelium of the scutellum during the first week of culture, showed a strict epidermal origin of these early developed structures. The addition of 1 mg·L^–1 of AgNO₃ enhanced the induction of direct somatic embryogenesis (a more than 22 fold increase), affecting both the percentage of embryogenic explants and the number of somatic embryos per explant, suggesting the possible involvement of ethylene.     

The use of somatic embryogenesis for plant propagation in cassava

In cassava, somatic embryogenesis starts with the culture of leaf explants on solid Murashige and Skoog-based medium supplemented with auxins. Mature somatic embryos are formed within 6 wk. The cotyledons of the primary somatic embryos are used as explants for a new cycle of somatic embryogenesis. The cotyledons undergo secondary somatic embryogenesis on both liquid and solid Murashige and Skoog-based medium supplemented with auxins. Depending on the auxin, new somatic embryos are formed after 14–30 d after which they can be used for a new cycle of somatic embryogenesis. In liquid medium, more than 20 secondary somatic embryos are formed per initial cultured embryo. In both primary and secondary somatic embryogenesis, the somatic embryos originate directly from the explants. Transfer of clumps of somatic embryos to a Greshoff and Doy-based medium supplemented with auxins results in indirect somatic embryogenesis. The direct form of somatic embryogenesis has a high potential for use in plant propagation, whereas the indirect has a high potential for use in genetic modification of cassava. Mature somatic embryos germinate into plants after desiccation and culture on a Murashige and Skoog-based medium supplemented with benzylaminopurine (BA). Depending on the used BA concentration, plants can either be transferred either directly to the greenhouse or after using standard multiplication protocols.