<Emphasis Type="Italic">LEAFY COTYLEDON1</Emphasis>, <Emphasis Type="Italic">FUSCA3</Emphasis> expression and auxin treatment in relation to somatic embryogenesis induction in Arabidopsis

The expression pattern of the LEC1 and FUS3 genes during somatic embryogenesis in Arabidopsis explants (immature zygotic embryos) induced in vitro was analysed, using Real-time quantitative PCR (qRT-PCR). The analysis revealed differential expression of LEC1 but not FUS3 within a 30 day time course of somatic embryo development, and a significant auxin-dependent upregulation of LEC1 was found over the time course. In contrast to embryogenic culture, the level of LEC1 and FUS3 expression was noticeably lower in non-embryogenic callus of Col-0 and hormonal mutants (cbp20 and axr4-1) with low SE-efficiency. In addition, the expression profile of LEC1 and FUS3 was followed in the embryogenic culture derived from 35S::LEC2-GR explants. A significant increase of LEC1 but not FUS3 activity was observed under LEC2 overexpression induced in auxin-treated explants. The work provides further experimental evidence on LEC gene involvement in the embryogenic response in Arabidopsis somatic cells, and also implicates LEC1 function in more advanced stages of SE culture in relation to somatic embryo differentiation and development.     

Leafy cotyledon genes are essential for induction of somatic embryogenesis of <Emphasis Type="Italic">Arabidopsis</Emphasis>

The capacity for somatic embryogenesis was studied in lec1, lec2 and fus3 mutants of Arabidopsis thaliana (L.) Heynh. It was found that contrary to the response of wild-type cultures, which produced somatic embryos via an efficient, direct process (65–94% of responding explants), lec mutants were strongly impaired in their embryogenic response. Cultures of the mutants formed somatic embryos at a low frequency, ranging from 0.0 to 3.9%. Moreover, somatic embryos were formed from callus tissue through an indirect route in the lec mutants. Total repression of embryogenic potential was observed in double (lec1 lec2, lec1 fus3, lec2 fus3) and triple (fus3 lec1 lec2) mutants. Additionally, mutants were found to exhibit efficient shoot regenerability via organogenesis from root explants. These results provide evidence that, besides their key role in controlling many different aspects of Arabidopsis zygotic embryogenesis, LEC/FUS genes are also essential for in vitro somatic embryogenesis induction. Furthermore, temporal and spatial patterns of auxin distribution during somatic embryogenesis induction were analyzed using transgenic Arabidopsis plants expressing GUS driven by the DR5 promoter. Analysis of data indicated auxin accumulation was rapid in all tissues of the explants of both wild type and the lec2-1 mutant, cultured on somatic embryogenesis induction medium containing 2,4-D. This observation suggests that loss of embryogenic potential in the lec2 mutant in vitro is not related to the distribution of exogenously applied auxin and LEC genes likely function downstream in auxin-induced somatic embryogenesis.     

Histological analysis of direct somatic embryogenesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.) Heynh

In Arabidopsis the in vitro culture of immature zygotic embryos (IZEs) at a late stage of development, on the solid medium containing synthetic auxin, leads to formation of somatic embryos via direct somatic embryogenesis (DSE). The presented results provide evidence that in IZE cells competent for DSE are located in the protodermis and subprotodermis of the adaxial side of cotyledons and somatic embryos displayed a single- or multicellular origin. Transgenic Arabidopsis lines expressing the GUS reporter gene, driven by the DR5 and LEC2 promoters, were used to analyse the distribution of auxin to mark embryogenic cells in cultured explants and develop somatic embryos. The analysis showed that at the start of the culture auxin was accumulated in all explant tissues, but from the fourth day onwards its location shifted to the protodermis and subprotodermis of the explant cotyledons. In globular somatic embryos auxin was detected in all cells, with a higher concentration in the protodermis, and in the heart stage its activity was mainly displayed in the shoot, root pole and cotyledon primordia. The embryogenic nature of dividing protodermal and subprotodermal cells accumulating auxin was confirmed by high expression of promoter activity of LEC2 in these cells. Analysis of symplasmic tracer (CFDA) distribution indicated symplasmic isolation between tissues engaged in DSE and other parts of an explant. Symplasmic isolation of somatic embryos from the explant was also detected.     

<Emphasis Type="Italic">Arabidopsis tanmei/emb2757</Emphasis> embryo mutant is defective for in vitro plant morphogenesis

A mutation in the Arabidopsis TANMEI/EMB2757 (TAN) gene with an embryo defective phenotype was analysed for its effect on the morphogenic potential of somatic tissue cultured under in vitro conditions. The capacity for in vitro morphogenesis was evaluated using cultures of immature zygotic embryos, and seedling explants of the tan mutant and the parental Col-0 genotype. The explants were cultured on media supplemented with different plant growth regulators, and the capacity for two alternative pathways of morphogenesis, somatic embryogenesis (SE) and shoot organogenesis, was evaluated. Reporter genes (GUS, GFP) were used to monitor auxin and LEC2 and FUS3 gene activity in the tan explants. Moreover, the expression pattern of the TAN gene was analyzed during SE and in callus tissue of Col-0. It was indicated that the tan mutation resulted in a total lost of embryogenic and organogenic capacity of cultured tissues, suggesting the involvement of the TAN gene in basic cellular processes related to cell growth and differentiation. However, differential expression of the TAN gene during SE, and its increased activity at advanced stages of embryogenesis, implicate a specific role for the gene in the development of somatic embryos.     

Effect of polyamines on in vitro somatic embryogenesis in <Emphasis Type="Italic">Momordica</Emphasis><Emphasis Type="Italic">charantia</Emphasis> L.

The effects of exogenous polyamines (PAs) on enhancement of somatic embryogenic calli was investigated in Momordica charantia L. in vitro. Induction of somatic embryogenesis (SE) in leaf explants of M. charantia after 21 days of culture in Murashige and Skoog (MS) medium was determined using scanning electron microscopy. During induction of SE there were high titers of Putrescine (Put) as compared to Spermidine (Spd) and Spermine (Spm), a prerequisite for cell division. Addition of PAs to the embryogenic media resulted in an increase in fresh weights and number of somatic embryos of 21-day old embryogenic calli. Put at a concentration of 1 mM showed maximum increase in fresh weights of embryogenic calli (5 fold) and number of somatic embryos produced per 0.2 g of callus (2.5 fold). Moreover addition of PAs to the embryogenic media resulted in lowering of endogenous free PA level of 21-day old embryogenic calli. Thus, when the media was supplemented with exogenous PAs a positive correlation was found to exist between Somatic Embryogenesis enhancement and decrease in endogenous free PA levels.     

Somatic embryogenesis in tulip (<Emphasis Type="Italic">Tulipa gesneriana</Emphasis> L.) flower stem cultures

In the present study, the procedures for induction of somatic embryogenesis (SE) in an in vitro culture of the tulip have been developed. SE was initiated on flower stem explants isolated from “Apeldoorn” bulbs during their low-temperature treatment. Bulbs had not been chilled or had been chilled for 12 or 24 weeks at 5°C. The explants were cultured with exogenous auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (Picloram), α-naphthaleneacetic acid (NAA) at 1–100 μM and cytokinins: benzyladenine (BA) and zeatin (ZEA) at 0.5–50 μM. Increase in auxin concentrations caused an intensive enlargement of the explant parenchyma, which changed into homogenous colorless callus. On the same media, vein bundles developed into yellowish, nodular callus. Picloram was more efficient in inducing the formation of embryogenic nodular callus than 2,4-D, whereas the latter stimulated formation of colorless callus. The base of the lower part of the flower stem isolated from bulbs chilled for 12 weeks proved to be the best explant for callus formation. The highest number of somatic embryos was produced on medium with 25 μM Picloram and 0.5 μM BA. Development of adventitious roots was noticed in the presence of 2,4-D. Globular embryos developed into torpedo stage embryos under the influence of BA (5 μM) and NAA (0.5 μM). Morphological and anatomical data describing development of callus and somatic embryos are presented.     

<Emphasis Type="Italic">CDPK</Emphasis> gene expression in somatic embryos of <Emphasis Type="Italic">Panax ginseng</Emphasis> expressing <Emphasis Type="Italic">rolC</Emphasis>

A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium (MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) after 4 weeks of culture in darkness. A higher response (66%) of embryogenic callus was induced on 0.45 μM 2,4-d. Higher numbers of globular- (31), heart- (17), torpedo- (12), and cotyledon-stage (8) embryos per explant were obtained by culturing embryogenic callus on MS with 3% sucrose, 0.24% Phytagel™, and devoid of growth regulators after 8 weeks culture in darkness. Continuous sub-culturing of embryogenic callus on medium containing 2,4-d yielded only compact callus. Desiccation of embryos for 3 days in darkness at 25 ± 2°C followed by cold storage at 4°C in darkness for 8 weeks favored embryo germination and development of plantlets. Cotyledon-stage embryos subjected to desiccation and chilling treatment cultured on MS with 3% sucrose, 0.24 Phytagel™, 0.44 μM 6-benzylaminopurine (BA), and 0.29 μM gibberellic acid germinated at a higher frequency (61%) than with 0.44 μM BA alone and control cultures. Germinated plantlets developed a shoot and root, were acclimatized successfully, and maintained in a growth room for plantlet development.     

Induction of somatic embryos from cultures of <Emphasis Type="Italic">Agave vera-cruz</Emphasis> Mill.

Somatic embryogenesis from cultures of shoot apices, cotyledon and young leaves of in vitro shoots of Agave vera-cruz Mill. was studied. Embryogenic callus was obtained when explants were cultured on Murashige and Skoog’s (MS) medium (1962) supplemented with L₂ vitamins, 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-d) or 5.37 μM ∝-naphthalene acetic acid (NAA). Somatic embryos differentiated from this embryogenic callus upon subculture to maturation/conversion medium containing cytokinin either alone or with auxin and l-glutamine. The best combination of growth regulators for development of somatic embryos was found to be 5.37 μM naphthalene acetic acid plus 0.91 μM zeatin and 40 g/l sucrose. The conversion frequency of somatic embryos to plantlets varied from 46–50%. Rooted plantlets were transferred directly to pots containing a soil, sand, and manure mixture without any hardening phase with 96–98% survival of the plantlets. Based on the histological observations, the potential origin of the somatic embryo is discussed.     

Genetic transformation of <Emphasis Type="Italic">Coffea canephora</Emphasis> by particle bombardment

Stable transformation of Coffea canephora P. was obtained by particle bombardment of embryogenic tissue. Leaf explants were cultured on medium supplemented with 5 µM isopentenyl-adenosine to induce direct embryogenesis. Explants with somatic embryos were transferred to half strength MS medium with 9 µM 2,4 dichlorophenoxyacetic acid. After 2 weeks, the explants with somatic embryos and embryogenic tissue were bombarded with tungsten particles (M-25) carrying the plasmid pCambia3301 (containing the bar and uidA genes) using a high pressure helium microprojectile device. The bombarded explants were submitted to selection on medium containing 5 µM ammonium glufosinate herbicide as selective agent. After 6 months, putative transgenic embryos were transferred to a growth regulator-free medium for germination. The regenerated plantlets were β-glucuronidase (GUS) positive whereas no GUS activity was observed in non-transgenic controls. Incorporation of the bar gene into the genome was confirmed by PCR and Southern blot analysis of the regenerated transformed plants. Greenhouse grown transgenic coffee plants were found to withstand the recommended level of the herbicide Finale™ for weed control.This research was supported by the Consorcio Brasileiro de Pesquisa e Desenvolvimento do Cafe (CBP&D-Cafe).     

Evaluation of key factors influencing <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of somatic embryos of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.)

Key factors influencing the efficiency of transformation of embryogenic cultures, induced from immature zygotic embryos, of avocado cv. ‘Duke 7’ were evaluated. Initially, the sensitivity of somatic embryos to the antibiotics kanamycin, used for selection, carbenicillin, cefotaxime and timentin, all used for elimination of Agrobacterium cells, were evaluated. Isolated globular somatic embryos were more sensitive to kanamycin than embryogenic masses, and 25 mg l⁻¹ kanamycin completely restricted callus proliferation. Cefotaxime at 500 mg l⁻¹ partially inhibited proliferation of embryogenic cultures, while both carbenicillin and timentin did not affect callus growth. For genetic transformation, somatic embryos were infected with A. tumefaciens containing the pBINUbiGUSint plasmid. After 2 days, the embryos were transferred to selection medium supplemented with 50 mg l⁻¹ kanamycin and 250 mg l⁻¹ timentin for 2 months. Then, kanamycin level was increased to 100 mg l⁻¹ for two additional months. The A. tumefaciens strain AGL1 yielded higher transformation rates, 6%, than EHA105 or LBA4404, 1.2%. The percentage of kanamycin resistant calli obtained was significantly influenced by the embryogenic line used as source of explants. Genetic transformation was confirmed by PCR and Southern blot analysis. A significant improvement in the germination rate was obtained when transgenic embryos were cultured in liquid MS medium with 4.44 μM BA and 2.89 μM GA₃ for 3 days in a roller drum and later transferred to the same medium gelled with 7 g l⁻¹ agar. Plants from five independent transgenic lines were acclimated and grown in the greenhouse, being phenotipically similar to control plants.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration through somatic embryogenesis and organogenesis using cotyledons of <Emphasis Type="Italic">Cassia angustifolia</Emphasis> Vahl

In vitro regeneration through somatic embryogenesis as well as organogenesis using cotyledon of a woody medicinal legume, Cassia angustifolia is reported. The cotyledons dissected from semi-mature seeds, if inoculated on Murashige and Skoog’s medium (MS) supplemented with auxin alone or in combination with cytokinin, produced direct and indirect somatic embryos. A maximum of 14.36 ± 2.26 somatic embryos per 20 mg of explants including callus were produced in 70% cultures on MS medium with 2.5 μM benzyladenine (BA) + 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Although the percentage of embryogenic cultures was higher (83.33%) at 10 μM 2,4-D + 1 μM BA, the average number of somatic embryos was much less (7.6 ± 0.85) at this level, whereas at 2.5 μM BA and 5 μM 2,4-D, there was a simultaneous formation of both somatic embryos and shoots. The somatic embryos, although started germinating on the same medium, developed into full plantlets only if transferred to MS basal with 2% sucrose. Cytokinins alone did not induce somatic embryogenesis, but formed multiple shoots. Five micromolar BA proved optimum for recurrently inducing shoots in the competent callus with a maximum average of 12.04 ± 2.10 shoots and shoot length of 2.26 ± 0.03 cm. Nearly 91.6% shoots (2–2.5 cm in size) organized an average of 5.12 ± 0.58 roots on half strength MS + 10 μM indole-3-butyric acid. All the plantlets have been transferred successfully to soil. Types of auxin and its interaction with cytokinin significantly influenced somatic embryogenesis.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-Mediated genetic transformation in tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> [L.] O. Kuntze)

We have developed a system to produce transgenic plants in tea (Camelia sinensis [L.] O. Kuntze) viaAgrobacterium tumefaciens-mediated transformation of embryogenic calli. Cotyledon-derived embryogenic callus cultures were cocultivated with anA. tumefaciens strain (AGL 1) harboring a binary vector carrying the hygromycin phosphotransferase (hpt II), glucuronidase (uid A), and green fluorescent protein (GFP) genes in the tDNA region. Following cocultivation, embryogenic calli were cultured in medium containing 500 mg/L carbenicillin for 1 wk and cultured on an antibiotic selection medium containing 75 mg/L hygromycin for 8–10 wk. Hygromycin-resistant somatic embryos were selected. The highest production efficiency of hygromycin-resistant calli occurred with cocultivation for 6–7 d in the presence of 400 μM acetosyringone (AS). Hygromycin-resistant somatic embryos developed into complete plantlets in regeneration medium containing half-strength Murashige and Skoog (MS) salts with 1 mg/L benzyl amino purine (BAP) and 9 mg/L giberellic acid (GA₃). Transformants were subjected to GFP expression analysis, β-glucuronidase (GUS) histochemical assay, PCR analysis, and Southern hybridization to confirm gene integration.     

Biosynthesis of Dibenzyl Trisulfide (DTS) from somatic embryos and rhizogenous/embryogenic callus derived from Guinea hen weed (<Emphasis Type="Italic">Petiveria alliacea</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) leaf explants

A procedure for producing somatic embryos enriched with dibenzyl trisulfide (DTS) using a hormone-dependent culture system is reported for Petiveria alliacea L. (Guinea hen weed). Leaf explants were cultured on a Murashige and Skoog medium supplemented with a range of naphthaleneacetic acid (NAA) concentrations and a fixed concentration of benzyladenine (BAP) at 11.0 μM and sucrose or glucose at 30 g l⁻¹. Leaf explants cultured on all media types started to form callus at the cut surfaces of the discs 10–14 d after initiation. The type of sugar used influenced average fresh weight, the propensity to form roots, as well as the embryogenic response. The highest mean fresh weight (337.7 ± 26.18 mg) and mean root number (23.7 ± 1.69) was produced on media enriched with sucrose and supplemented with 53.7 μM NAA and 11.0 μM BAP. An ethanol extract of rhizogenic/embryogenic callus or somatic embryos was subjected to high-performance liquid chromatography analysis, which revealed the presence of DTS in both extracts. UV spectral analysis and the use of standard quantitation procedures showed that the quantity of DTS in the somatic embryo extract, at 0.16% (w/v), was approximately 30-fold higher than in rhizogenic/embryogenic callus (0.0055% w/v) of similar fresh weight. These results indicate that it is possible to biosynthesize approximately 6 mg of natural DTS from 3,808 mg of fresh somatic embryos within 10 wk from less than three leaf explants.     

Plant regeneration <Emphasis Type="Italic">via</Emphasis> somatic embryogenesis in <Emphasis Type="Italic">Pinus kesiya</Emphasis> (Rolye ex. Gord.) influenced by triacontanol

Embryogenic cultures were initiated and established for the first time in 3 different genotypes of Pinus kesiya using mature zygotic embryos and triacontanol. Mature zygotic embryos produced white-mucilaginous embryogenic callus when cultured on half strength MSG (Becwar et al. 1990) basal medium supplemented with 90 mM maltose, 2.0 g l⁻¹ Gellan gum, 9.0 M 2, 4-D and 10 g l⁻¹triacontanol. On subculture of such embryogenic callus on the maintenance medium (II) containing 2.0 M 2,4-D and 2.0 g l⁻¹ triacontanol induced cleavage polyembryogenesis with proembryos. The percentage of somatic embryogenesis was not similar in all the three genotypes. The highest percentage of somatic embryogenesis (88.5 %) was recorded in PK04 genotype. Somatic embryos were successfully germinated on half strength MSG basal medium without growth regulators. Somatic seedlings showed fast growth and a survival rate of 95%. This work for the first time reveals that triacontanol can be used as an effective growth regulator for inducing somatic embryogenesis in conifers.     

Isolation and characterization of four somatic embryogenesis receptor-like kinase (<Emphasis Type="Italic">RhSERK</Emphasis>) genes from miniature potted rose (<Emphasis Type="Italic">Rosa hybrida</Emphasis> cv. Linda)

The isolation and expression analysis of four partial gene sequences from rose (Rosa hybrida cv. Linda) belonging to the receptor-like kinase gene superfamily are reported. These genes have been designated RhSERK1 to RhSERK4 (Accession No. EF631967 to EF631970) as they exhibit high sequence identities with genes from the somatic embryogenesis receptor-like kinase (SERK) family in other plant species. The RhSERK genes are differentially expressed in non-embryogenic callus, embryogenic callus, mature somatic embryos and a range of tissues from intact plants, indicating a broad role in plant growth and development. However, the expressions of RhSERK3 and RhSERK4 were approximately fivefold higher in embryogenic callus than in non-embryogenic callus, and they are even higher when compared to tissues from intact plants. In addition, RhSERK4 expression was approximately eightfold higher in somatic embryos than in embryogenic callus. These results suggest that the expression pattern of RhSERK3 and RhSERK4 may be used as a marker of somatic embryogenesis.     

Plant regeneration via somatic embryogenesis from protoplasts of six explants in Coker 201 (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>)

Fertile regenerated plants were obtained from protoplasts via somatic embryogenesis in Coker 201 (Gossypium hirsutum L.). Protoplasts were isolated from six different explantsleaves, hypocotyls, young roots, embryogenic callus, immature somatic embryos and suspension cultures and cultured in liquid thin layer KM8P medium. Callus-forming percentage of 20–50% was obtained in protoplast cultures from embryogenic callus, immature embryos and suspension cultures, and visible callus formed within 2 months. Callus-forming percentage of 5–20% in protoplast cultures from young roots, hypocotyls and leaves, and visible callus formed in 3 months. NAA 5.371 μM/kinetin 0.929 μM was effective to stimulate protoplast division and callus formation from six explants. Percentage of callus formation in the medium with 2,4-D 0.452 μM/kinetin 0.465 μM was over 40% from suspension cultures and immature embryos, 25% from embryogenic callus and 10% from hypocotyls. Callus from protoplasts developed into plantlets via somatic embryogenesis. Over 100 plantlets were obtained from protoplasts derived from 6 explants. Ten plants have been transferred to the soil, where they all have set seeds.