Secondary Embryogenesis Research Articles

Identification of differentially expressed sequences in pre-embryogenic tissue of oilseed rape by suppression subtractive hybridisation (SSH)

A subtracted cDNA library comprising of 576 clones were constructed to isolate differentially expressed sequences in the pre-embryogenic tissue (PEC) of winter oilseed repe. After differential screening, only 16 clones were identified as potential positives. Eventually, only three clones: BNPE DG3, BNPE AE4 and BNPE EG1 were confirmed by Northern analysis as upregulated in␣PEC of the oilseed rape embryogenic culture. This is the first study to report Ae4, Dg3 and Eg1 sequences as preferentially expressed in the oilseed␣rape PEC and associated with the induction of somatic embryogenesis in B.napus secondary embryogenesis. Ae4 encodes a putative proline-rich protein, Dg3 encodes a lipid transfer-like protein and Eg1 encodes a napin, member of the BNMNAP subfamily.

Direct somatic embryogenesis and plant regeneration from leaf explants of Phalaenopsis amabilis

Leaf explants of Phalaenopsis amabilis var. formosa formed clusters of somatic embryos directly from epidermal cells without an intervening callus within 20 – 30 d when cultured on 1/2-strength modified Murashige and Skoog medium supplemented with 0.1, 1 and 3 mg dm−3 TDZ. Repetitive production of embryos involved secondary embryogenesis could be obtained by culturing segments of embryogenic masses on TDZ-containing media. Plantlet conversion from embryos was successfully achieved on regulator-free growth medium.

Optimizing Agrobacterium-mediated transformation of grapevine

Summary A translational fusion between the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (NPTII) genes was used to optimize parameters influencing Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless. The corresponding bifunctional protein produced from this EGFP/NPTII fusion gene allowed for a single promoter to drive expression of both green fluorescence and kanamycin resistance, thus conserving promoter resources and eliminating potential promoter‐promoter interactions. The fusion gene, driven by either a double cauliflower mosaic virus 35S (CaMV 35S) promoter or a double cassava vein mosaic virus (CsVMV) promoter, was immobilized into Agrobacterium strain EHA 105. Somatic embryos capable of direct secondary embryogenesis were used as target tissues to recover transgenic plants. Simultaneous visualization of GFP fluorescence and kanamycin selection of transgenic cells, tissues, somatic embryos, and plants were achieved. GFP expression and recovery of embryogenic culture lines were used as indicators to optimize transformation parameters. Preculturing of somatic embryos for 7d on fresh medium prior to transformation minimized Agrobacterium-induced tissue browning/necrosis. Alternatively, browning/necrosis was reduced by adding 1gl 21 of the antioxidant dithiothreitol (DTT) to post co-cultivation wash media. While combining preculture with antioxidant treatments did not result in a synergistic improvement in response, either treatment resulted in recovery of more stable embryogenic lines than did the control. A 48h co-cultivation period combined with 75mgl 21 kanamycin in selection medium was optimal. DNA analysis confirmed stable integration of transgenes into the grape genome; 63% had single gene insertions, 27% had two inserts, and 7 and 3% had three and four inserts, respectively. Utilizing optimized procedures, over 1400 stable independent transgenic embryogenic culture lines were obtained, of which 795 developed into whole plants. Transgenic grapevines have exhibited normal vegetative morphology and stable transgene expression for over 5yr.

Identification of a potential structural marker for embryogenic competency in the Brassica napus spp. oleifera embryogenic tissue

The Brassica napus secondary embryogenesis system requires no exogenous growth regulator to stimulate embryo development. It is stable embryogenically over a long period of culture and has a distinct pre-embryogenic stage. This system was used to investigate the morphological and cellular changes occurring in the embryogenic tissue compared to non-embryogenic tissue using various microscopy techniques. A unique ultrastructural feature designated the extracellular matrix (ECM) was observed on the surface of pre-embryogenic embryoids but not on the non-embryogenic individuals. The ECM layer was found to be dominant in the pre-embryogenic stage and reduced to fragments during embryo growth and development in mature embryogenic tissue. This is a novel aspect of the phenotype previously unreported in the Brassica system. This structure might be linked to acquisition of embryogenic competence.

Somatic embryogenesis from immature zygotic embryos of Rosa bourboniana desp

A protocol for the inducton of somatic embryogenesis from immature zygotic embryos of Rosa bourboniana, a scented rose species, was established. Somatic embryos were induced after 8wk of inoculation of zygotic embryos on MS medium supplemented with different concentrations of 2,4-dichlorophenoxy acetic acid (5–15 μM). In addition to 2,4-dichlorophenoxy acetic acid concentrations, somatic embryogenesis was also influenced by the month of collection of the explant and the stage of maturity of the hip. Maximum embryogenic response (16.6%) was observed using 2,4-dichlorophenoxy acetic acid (15 μM), from green hips in the month of September. The use of l-proline (800 mg l−1) was found to be optimum for secondary embryogenesis. On periodic subculturing, the cultures formed somatic embryos sustainably over a period of 18 mo. For somatic embryo germination, 6-benzylaminopurine (5 μM) was found to be most suitable. Rooted plants were transferred successfully to soil and appear morphologically normal under greenhouse conditions. Transfer of plants for hardening was most suitable during the active growth period between June and September.

Somatic embryogenesis from stem nodal sections of grapevine

Indirect somatic embryogenesis was obtained for 11 clones of 6 Vitis vinifera cultivars: Cabernet-Sauvignon, Chardonnay, Gewurztraminer, Grenache, Merlot and Sauvignon, and for the rootstock Fercal [( Vitis berlandieri x Vitis colombard ) x ( Vitis vinifera x Vitis berlandieri )], starting from vegetative explants of in vitro plantlets. Embryogenic callus was recovered from nodal explants of every tested clone, while leaf explants led to embryogenesis only for the rootstock Fercal. We thus showed that axillary bud microcuttings are valuable explants for inducing somatic embryogenesis in V. vinifera and Fercal. Embryogenic cell lines have been maintained through secondary embryogenesis, and some embryos were converted into whole plantlets. A complete protocol for somatic embryogenesis and plant regeneration was therefore designed, using this very simple method.

Regeneration of androgenic embryos in apple (Malus x domestica Brokh.) via anther and microspore culture

Based on optimized protocols for anther and microspore culture in apple (Malus x domestica Borkh.), the regeneration phase and the efficiency of the processes in general were compared by using the same androgenic material of two experimental years. Microspore culture resulted in an increase in embryo induction depending on the genotype (Hofer 2004), however anther culture was superior to microspore culture in the total number of regenerated plants. The regeneration process in anther and microspore culture is similar. Two developmental pathways were observed: 1) secondary embryogenesis followed by adventitious shoot formation and 2) direct adventitious shoot formation from primary embryos. Induction and regeneration processes are delayed in microspore culture as compared with anther culture. The reasons for the reduced regeneration efficiency in microspore culture are discussed.

High efficiency plant production of North American ginseng via somatic embryogenesis from cotyledon explants

An efficient in vitro protocol for plant production of North American ginseng has been established. The pretreatment of cotyledon explants with 1.0 M sucrose at 4 degrees C resulted in an improvement of embryo quality and, combined with a higher sucrose content (7%) in induction medium, improved the embryogenesis frequency from 40% to 75% and the number of embryos per explant from 10 to 21. The frequency of secondary embryogenesis from somatic embryo-derived tissues cultured on MS medium with 1.0 mg l(-1) 2, 4-D and 1.0 mg l(-1) NAA is up to 90%. Somatic embryos can further develop to maturity on SH medium supplemented with 1% activated charcoal and half of them can germinate. About 85% of the germinated embryos will convert into plants with well-developed taproot systems on 1/2 SH medium with 0.5% activated charcoal. The growth chamber and field establishment rates were 95.6 and 93.7%, respectively. The plants transplanted to growth chambers and field plots appear normal.

High-frequency plant regeneration through cyclic secondary somatic embryogenesis in black pepper (Piper nigrum L.)

A high-frequency plantlet regeneration protocol was developed for black pepper (Piper nigrum L.) through cyclic secondary somatic embryogenesis. Secondary embryos formed from the radicular end of the primary somatic embryos which were originally derived from micropylar tissues of germinating seeds on growth regulator-free SH medium in the absence of light. The process of secondary embryogenesis continued in a cyclic manner from the root pole of newly formed embryos resulting in clumps of somatic embryos. Strength of the medium and sucrose concentration influenced the process of secondary embryogenesis and fresh weight of somatic embryo clumps. Full-strength SH medium supplemented with 1.5% sucrose produced significantly higher fresh weight and numbers of secondary somatic embryos while 3.0 and 4.5% sucrose in the medium favored further development of proliferated embryos into plantlets. Ontogeny of secondary embryos was established by histological analysis. Secondary embryogenic potential was influenced by the developmental stage of the explanted somatic embryo and stages up to "torpedo" were more suitable. A single-flask system was standardized for proliferation, maturation, germination and conversion of secondary somatic embryos in suspension cultures. The system of cyclic secondary somatic embryogenesis in black pepper described here represents a permanent source of embryogenic material that can be used for genetic manipulations of this crop species.

Efficient plant regeneration through direct somatic embryogenesis from leaf explants of Phalaenopsis ‘Little Steve’

Leaf segments of the orchid sp. Phalaenopsis ‘Little Steve’ were used as explants testing the effects of 2,4-dichlorophenoxyacetic acid (2,4-D; 0.45, 2.26, 4.52 μM), 6-furfurylaminopurine (kinetin; 2.32, 4.65, 13.95 μM), N6-benzyladenine (BA; 2.22, 4.44, 13.32 μM), and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ; 2.27, 4.54, 13.62μM) on the induction of direct somatic embryogenesis. After 20–30 d of culture in darkness, clusters of somatic embryos formed from leaf surfaces and wounded regions of explants on half-strength Murashige and Skoog medium supplemented with BA and TDZ. However, kinetin had no response on direct embryo induction. In addition, 2,4-D highly retarded the frequency of embryogenesis that was induced by TDZ. Generally, adaxial surfaces near wounded regions had the highest embryogenic competency compared to other regions of explants. Histological sections revealed that somatic embryos mostly arose from epidermal cell layers of the explants. Secondary embryogenesis occurred at basal parts of embryos, and originated from outer cell layers. Following transfer of regenerated embryos onto growth regulator-free medium for 3.5–4 mo., plantlets with three to four leaves and several roots were obtained. This protocol provides a simple way to regenerate plants through direct somatic embryogenesis, and is suitable for further studies on embryo development and genetic transformation of Phalaenopsis.

Possible involvement of abscisic acid in the induction of secondary somatic embryogenesis on seed-coat-derived carrot somatic embryos

When seed coats (pericarps) were picked from 14-day-old carrot (Daucus carota) seedlings and cultured on agar plates, embryogenic cell clusters were produced very rapidly at a high frequency on the open side edge. Embryo induction progressed without auxin treatment; indeed treatment caused the formation of non-embryogenic callus. The embryogenic tissues (primary embryos) developed normally until the torpedo stage; however, after this a number of secondary somatic embryos were produced in the hypocotyl and root regions. "Tertiary" embryos were formed on some of the secondary embryos, but many developed into normal plantlets. The primary embryos contained significantly higher levels of abscisic acid (ABA) than the hypocotyl-derived normal and seed-coat-derived secondary embryos. Fluridone inhibited the induction of secondary embryogenesis, while exogenously supplied ABA induced not only "tertiary" embryogenesis on the seed-coat-derived secondary embryos, but also secondary embryos on the hypocotyl-derived normal somatic embryos. These results indicate that ABA is one of the important endogenous factors for the induction of secondary embryogenesis on carrot somatic embryos. Higher levels of indole-3-acetic acid (IAA) in primary embryos also suggest the presence of some concerted effect of ABA and IAA on the induction of secondary embryogenesis in primary embryos.

Somatic embryogenesis from ovaries, developing ovules and immature zygotic embryos, and improved embryo development of Castanea sativa

Somatic embryogenesis of European chestnut (Castanea sativa Mill.) was obtained using juvenile tissue cultured on P24 medium with 5 μM 2,4-dichlorophenoxyacetic acid plus 0.5 μM 6-benzylaminopurine (BA) for three weeks and then cultured on 0.89 μM BA. Induction frequency with ovaries ranged from 2.0 to 19.1 % and was observed in tissue collected 2 to 8 weeks postanthesis, ovules used as a starting tissue gained 0.8 to 7.8 %, 3 to 9 weeks postanthesis. Zygotic embryos collected 5 to 10 weeks postanthesis formed 10.5 to 57.1 % somatic embryos, respectively. The culture lines were maintained via secondary embryogenesis on P24 medium with 0.89 μM BA. Development and maturation were stimulated on P24 medium with increased agar concentration (1.1 %). Five plantlets were transferred to substrate and acclimatized successfully in greenhouse.

Effect of exogenous ABA on embryo maturation and quantification of endogenous levels of ABA and IAA in Quercus suber somatic embryos

Knowledge of the relationship between indole-3-acetic acid (IAA) and abscisic acid (ABA) is relevant to control the development and the maturation of cork oak (Quercus suber L.) somatic embryos. The addition of 1 μM ABA to the culture medium significantly promoted somatic embryo maturation and increased both fresh and dry matter without affecting the relative water content. This effect was parallel to the pattern of variation observed in the endogenous ABA level, which increased from the immature to the mature stage. Endogenous ABA content during the occurrence of secondary embryogenesis was similar to that of the immature stage, showing that embryos with lower ABA levels produced secondary embryos. In contrast, IAA showed the highest concentration during early embryo development and decreased afterwards. Only in somatic embryos subjected to 1-week desiccation followed by stratification at 4 °C for 2 weeks, was a moderate increment of endogenous IAA content observed. IAA and ABA showed opposite levels during the development and maturation of cork oak somatic embryos and characterised specific stages of the embryonic development.

An Efficient Transformation Method to Regenerate a High Number of Transgenic Plants Using a New Embryogenic Line of Medicago truncatula cv. Jemalong

A simple and efficient regeneration–transformation method was established to obtain transgenic plants of the model legume Medicago truncatula cv. Jemalong. This method takes advantage of a new highly embryogenic line (M9-10a) isolated in our laboratory. Leaflets of in vitro grown M9-10a plants were co-cultured with Agrobacterium tumefaciens EHA105. Plasmid constructs containing the oat arginine decarboxylase gene, Adc and the GUS reporter gene (p35SAdc–Gus) or ELIP-like drought stress protein 22 (DSP22) encoding gene from Craterostigma plantagineum (p35SDsp22) were used. Both constructs include the nptII gene as selection marker. Embryogenic calli (100–97%) were obtained on embryo induction medium containing 100 mg l −1 kanamycin and 500 mg l−1 carbenicillin. Using a two-fold increase in kanamycin concentration, instead of 50 mg l−1 usually used, we reduced the number of emerging false kanamycin-resistant (KanR) embryos, which is an important improvement to the method, making it less laborious and very efficient. Isolation of late torpedo/cotyledonary-stage embryos to lower carbenicillin/agar media reduced secondary embryogenesis and prevents hyperhydricity, improving embryo conversion. Primary transformants (T0) were regenerated within 3–4 months and those that were able to root in a 50 mg l−1 kanamycin medium were transferred to the greenhouse to produce seeds. Southern blot hybridisation analysis confirmed the integration of either the Adc or Dsp22 transgenes in the genome of the T0 transformants. Detection of β-glucuronidase (GUS) activity in Adc–Gus T0 plants demonstrated the expression of the inserted transgene. In average, 1–2 independent transgenic lines are obtained per KanR embryogenic callus, independently of the plasmid construct used for transformation. Inheritance of the transgenes is shown to be stable in the T1 generation.

EMBRYOGENESIS AND PLANT REGENERATION IN VITIS VINIFERA L BY ANTHER CULTURE

Different culture media were studied to obtain embryos from anther culture of two clones of the cultivar “Viosinho” from Vitis vinifera L., in order to regenerate plants from them. Six different culture media were used: five of them solidified with agar, ACM (anther culture medium) supplemented with 2,4 – D (1.0 and 1.5mg.L-1) and BAP (0.25 mg.L-1); ACM with NOA (1.0 and 1.5 mg.L-1) and BAP (0.25 mg.L-1); ACM with 2,4 – D (0.25 mg.L-1) and BAP (1.0 mg.L-1); one solidified with phytagel, and ACM with 2,4 – D (1.0 mg.L-1) and BAP (0.25 mg.L-1). In all the media tested, anthers with calli and embryos were produced and secondary embryogenesis obtained. After 10 weeks of in vitro culture, a non-significant effect (P>0.05) of clone and a significant effect (P<0.001) of culture medium composition were observed for all three parameters evaluated – namely the number of anthers with calli, the number of embryogenic calli/anther and the number of embryos/embryogenic calli. The highest number of anthers with calli was obtained in the medium containing 1.0 mg.L-1 NOA and 0.25 mg.L-1 BAP and the highest percentage of the embryogenic calli (35.2%) was observed in the medium containing 2,4 - D at 1.0 mg.L-1 and BAP at 0.25 mg.L-1. The ACM medium supplemented with 1.5 mg.L-1 2,4 - D and 0.25 mg.L-1 BAP proved to be the best for embryo production with 3.2 embryos formed per embryogenic calli.

Factors affecting secondary somatic embryogenesis and embryo maturation in Morus alba L

A procedure for optimum secondary embryogenesis and maturation of Morus alba somatic embryos is described. The primary somatic embryos were inoculated on MS medium with different plant growth regulators and complex additives in various combinations and concentrations. Best results were obtained on the medium supplemented with 0.05 mg 2,4-D + 0.1 mg l −1 BAP and 6% sucrose, where percentage of secondary embryogenesis was 98.93 with 17% well-matured cotyledonary embryos. On this medium, the embryogenic competence was maintained for more than a year by repeated subculture.

Factors affecting somatic embryogenesis in Prunus incisa cv. February Pink

Factors affecting somatic embryogenesis from root explants of Prunus incisa Thunb. cv. February Pink were investigated. Using a medium containing Murashige and Skoog salts and vitamins supplemented with 10 microM 2,4-dichlorophenoxyacetic (2,4-D), we evaluated the effects of light, growth regulators, amino acids, carbohydrate source, and root induction medium. Explants cultured under light or dark conditions both resulted in the formation of embryos. Embryogenesis was inhibited by the addition of 6-benzyladenine, thidiazuron, or gibberellic acid to the medium. Amino acids were not effective in promoting embryogenesis, with high levels of amino acids actually inhibiting it. Sucrose and glucose effectively induced embryogenesis, while sorbitol and mannitol completely inhibited it. Sucrose and glucose also promoted secondary embryogenesis. Embryos that formed in medium containing 4% or 5% sucrose were abnormally shaped and did not fully develop, while those that formed in medium with sucrose concentrations of 2% or 3% were much more vigorous. Root explants that were induced on medium containing 1.0 micro M indole-3-butyric acid (IBA) produced more somatic embryos than explants induced on medium without IBA. Approximately 50% of the roots induced on medium containing 1.0 microM IBA produced somatic embryos on medium containing 10 microM 2,4-D and 3% sucrose.

Somatic Embryogenesis in Mature Quercus Robur Trees

Somatic embryo induction and plant regeneration have been obtained in tissues from mature Quercus robur L. trees. Epicormic shoots were forced to flush in branch segments collected from the crown of trees growing in selected stands on different collection dates. Expanding leaves from five genotypes, cultured following a multistage treatment procedure, produced somatic embryos at frequencies ranging from 0.3 to 3.6% of leaf explants, depending on genotype and collection date. After being induced, somatic embryos started a recurrent process by secondary embryogenesis which amplified the 15 embryogenic lines established. Plant recovery was achieved in 60% and 17% of matured embryos from two genotypes.

In vitro morphogenesis in zygotic embryo cultures of neem (Azadirachta indica A. Juss.).

Immature zygotic embryo cultures of neem yielded highly regenerative cultures, with the response varying with the embryo stage at culture. Early dicotyledonous stage embryos were the most responsive followed by torpedo stage embryos. The embryo cultures differentiated three types of regenerants: somatic embryos (SEs), shoot buds and neomorphs. SEs exhibited morphological abnormalities such as pluricotyledony, fusion of cotyledons and absence of cotyledons. Although these SEs showed secondary embryogenesis, the occurrence of normal dicotyledonous embryos was extremely rare. On MS basal medium 3% of SEs developed a long tap root but a plumular shoot did not appear. However, it was possible to regenerate plantlets from immature zygotic embryo cultures of neem via neomorph formation and adventitious shoot bud formation. The transplantation survival of these plants was more than 80%.

In-vitro regeneration of olive tree by somatic embryogenesis

We induced somatic embryogenesis from the cotyledon segments ofOlea europaea (L) cvs. ‘Chetoui’, ‘Chemleli’, and ‘Arbequina’. Calli were established from all three cultvars on OMc media supplemented with IBA and 2i-R The greatest success was obtained with media that contained zero or low concentrations of growth regulators. High levels of hormones (i.e.,>0.5 mgL-1 IBA and 2i-P) inhibited embryogenesis. Embryos at different maturation stages were observed with continuously proliferating secondary embryogenesis. Abnormally shaped embryos and teratoma were also noted. Four weeks was the optimal incubation period for inducing embryogenesis on the auxin-containing medium. In addition, 30 to 40 gL-1 sucrose was more effective than glucose in stimulating the growth and maturation of somatic embryos. Embryogeic efficiency was also higher when multivariate combinations of nitrogen sources (inorganic and organic nitrogen forms) were used. The plantlets that were derived from our germinating somatic embryos were similar to those obtained from axillary buds.