Embryogenic Suspension Cultures Research Articles

Somatic hybrids between Gossypium hirsutum L. (4×) and G. davidsonii Kellog (2×) produced by protoplast fusion

Somatic hybrids were obtained from electrofused protoplasts derived from embryogenic suspension cultures of tetraploid cotton (G. hirsutum L. cv. Coker 201) and embryogenic callus of diploid wild cotton G. davidsonii. The regenerants were initially identified as hybrids by RAPD (random amplified polymorphic DNA) analysis. Subsequently, observation on chromosome counting, morphology and SSR (simple sequence repeat) confirmed the hybrid status. Cytological investigation of the metaphase root-tip cells of the regenerated plants revealed there were 74 to 84 chromosomes in the plants, close to the expected 78 chromosomes. SSR analysis revealed the regenerated plants contained specific genomic fragments from both fusion partners, further confirmed their hybridity. The morphology of the plants was intermediate between the two fusion partners. The regenerants were difficult to develop into mature plants because their roots browned and they wilted from the stem apex before forming 3 to 5 true leaves. The hybrid plants were transferred to soil by grafting in vitro onto rootstocks.

Efficient plant regeneration from protoplasts isolated from embryogenic suspension cultures of Cyclamen persicum Mill.

A protocol for plant regeneration from protoplasts has been developed, and then successfully applied to different genotypes of Cyclamen persicum Mill. Protoplasts were isolated from embryogenic suspension cultures by enzymatic digestion in 2% cellulase R10 and 0.5% macerozyme R10. Yields obtained varied between 1 and 5 × 105 protoplasts per gram fresh mass depending on the genotype. Protoplasts were immobilized in alginate films, which promoted proper cell wall regeneration. The highest cell division frequencies were found in modified Kao and Michayluk (1975, Planta 126:105–110) medium containing the same types and concentrations of plant growth regulators that were applied for suspension culture (2.0 mg l−1 2,4-dichlorophenoxyacetic acid and 0.8 mg l−1 6-(γ,γ-dimethylallylamino)purine). Cell division was recorded for all 11 tested genotypes in frequencies of up to 12% and 18% after 7 and 14 days, respectively. However, cell division frequency varied strongly between different genotypes. After 4–6 weeks calluses were released from the alginate films and further cultured on hormone-containing medium for continued growth or transferred to hormone-free medium for regeneration of somatic embryos. Plant regeneration via somatic embryogenesis succeeded in 9 out of the 11 genotypes under investigation. Up to now protoplast-derived plants from four genotypes have been successfully transferred to soil.

Development of an embryogenic suspension culture of bitter melon (Momordica charantia L.)

Abstract We have optimized a system for the somatic embryogenesis via embryogenic suspension cultures in bitter melon (Momordica charantia L.). Friable calli could be induced in 30-day-old leaves on semi-solid MS [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497.] medium supplemented with 1.0 mg/l 2,4-D. Large number of globular embryos (24.6%) were noticed when the calli was subcultured in liquid medium containing 1.5 mg/l 2,4-D. The complete removal of 2,4-D in the later stages of culture, stimulated their further development to heart and torpedo stages. Microscopic examination revealed the ontogeny of somatic cell development via the formation of cell clusters, which then enlarged to pro-embryos, and gave rise to heart and torpedo stages within a period of 2 weeks. Somatic embryos successfully germinated on agarified MS medium with no additional growth regulators. An effect of media, other components and stimulating factors such as carbohydrates, amino acids has also been evaluated for somatic embryogenesis. The full strength MS medium containing 50 mg/l PVP and 40 mg/l glutamine was effective to achieve a high frequency of somatic embryo induction, maturation and further development. An average of 6.2% young plants was achieved from friable callus and was phenotypically normal. To our knowledge, there is no published report on somatic embryogenesis of bitter melon (M. charantia L.) via embryogenic callus or cell suspension cultures. These results are likely to facilitate genetic transformation of bitter melon.

Polyamine effects on the endogenous polyamine contents, nitric oxide release, growth and differentiation of embryogenic suspension cultures of Araucaria angustifolia (Bert.) O. Ktze.

In the present work we determined the effects of exogenous polyamines (PAs), supplemented to the culture medium, on the endogenous PA contents, nitric oxide (NO) release, culture growth and organization of proembryogenic masses (PEMs), in embryogenic suspension cultures of Araucaria angustifolia. We observed that PAs (Putrescine (Put), spermidine (Spd) and spermine (Spm)) had a different effect on growth, morphological evolution and NO biosynthesis in embryogenic cultures. Spd (1.0 mM) and Spm (1.0 mM) supplemented into the culture medium reduced cellular growth and allowed a morphogenetic evolution of aggregates from PEM II to PEM III stages. Put increased NO release from the embryogenic culture, and Spd and Spm had an inhibitory effect on NO release when compared with the control treatment. In the control and Put (1.0 mM) treatments, it was observed that embryonic cells of A. angustifolia accumulated more NO than the suspensor cells, suggesting that NO biosynthesis might be related to the maintenance of the polarity (embryonic–suspensor cells) present at the PEM II stage.

Bioproduction of Diosgenin in Callus Cultures of Balanites aegyptiaca: Effect of Growth Regulators, Explants and Somatic Embryogenesis

This study describes the effects of plant growth regulators, explants, and somatic embryogenesis on in vitro production of the steroidal sapogenin, diosgenin, in callus cultures of the Balanites aegyptiaca (L.) Del.(desert date). Root, shoot, hypocotyl, and epicotyl callus culture of B. aegyptiaca, were raised on MS basal media supplemented with various combinations of either 2,4-D and NAA alone, or with BAP. The diosgenin content (on a dry weight basis) was found to be highest when calli were cultured in MS basal medium supplemented with 1.0 mg l−1 2,4-D alone and/or in combination with 0.5 mg l−1 BAP. However, the callus growth was highest in media supplemented with 2.5 or 3.0 mg l−1 2,4-D. MS basal media supplemented with 2,4-D 2.5 mg l−1 alone and in combination with 0.5 mg l−1 BAP induced pre-embryogenic callus formation on root cultures. When these pre-embryogenic callus cultures were used to establish cell suspension cultures, two growth densities were obtained in embryogenic suspension cultures, inducing clusters of somatic embryos at various stages of development. The maximum number of somatic embryos were obtained at the fifth week on the medium supplemented with 1.0 mg l−1 2,4-D. However, the diosgenin content in these somatic cells was found to be lower compared to the explant calluses. This study revealed that production of diosgenin in callus cultures of B. aegyptiaca is possible, but the amount is significantly affected by the growth regulators, type of explants, and somatic embryogenesis.

Secretion of a chitinase-like protein in embryogenic suspension cultures of Dactylis glomerata L.

A chitinase-like 32 kDa acidic protein with a potential chitinase activity has been identified in the medium of embryogenic suspension cultures of Dactylis glomerata L. using an antiserum raised against endochitinase EP3 from Daucus carota L. The presence of this protein discriminates between Dactylis glomerata L. embryogenic and nonembryogenic suspension cultures and thus could be possibly used as a marker for embryogenic potential.

Transgenic Phalaenopsis plants with resistance to Erwinia carotovora produced by introducing wasabi defensin gene using Agrobacterium method

Transgenic plants over-expressing wasabi defensin gene were successfully produced in Phalaenopsis orchid by Agrobacterium-mediated transformation method. Embryogenic cell suspension culture of Phalaenopsis Wataboushi '#6.13' was infected with A. tumefaciens strain EHA101 carrying a plasmid containing wasabi defensin gene and selectable marker nptII, hpt genes. Plantlets were regenerated through somatic embryogenesis from the calli selected on hygromycin- containing medium. Transformation of plantlets with wasabi defensin gene was confirmed by PCR analysis. Southern blot analysis confirmed successful integration of 1-4 copies of the gene. Production of the 5 kDa wasabi defensin protein with varying levels was confirmed in the leaf extracts of different transgenic clones using Western blot analysis. Most of the transgenic plants showed strong resistance to Erwinia carotovora, which causes soft rot disease in the control plant. These results suggest the usefulness of this gene for conferring the resistance to various diseases of Phalaenopsis and possibly other orchids.

Establishment and plant regeneration of somatic embryogenic cell suspension cultures of the Zingiber officinale Rosc.

Somatic embryogenic cell suspension cultures of four ginger cultivars were established. Somatic embryogenic calli were induced from ginger shoot tips on MS agar medium supplemented with 1.0 mg l −1 2,4-D and 0.2 mg l −1 Kn, which contained only half concentration of NH 4NO 3. Rapid-growing and well-dispersed suspension cultures were established by subculturing this kind of callus in the same liquid MSN medium. The suspension cultures (about 1–2 mm in diameter) were placed on the MSN agar medium for callus proliferation. Thereafter embryogenic callus (1.5 cm 2) was transferred to solid media (MS + 0.2 mg l −1 2,4-D + 5.0 mg l −1 BA + 3% sucrose + 0.7% agar). Somatic embryos produced shoots and roots, and shoots developed into complete plantlets on solid MS medium supplemented with 3.0 mg l −1 BA and 0.1 mg l −1 NAA. The relationship between the DW of suspension cultures and pH changes in medium is also discussed. The suspension cultures still kept their vitalities after subculture for 8 months.

Monoclonal antibody against a cell wall marker protein for embryogenic potential of Dactylis glomerata L. suspension cultures

We identified and isolated a monoclonal antibody (MAb 3G2) raised against extracellular proteins from microcluster cells of orchard grass (Dactylis glomerata L.) embryogenic suspension culture. MAb 3G2 recognized with high specificity an antigen ionically bound within the primary cell wall and in the culture medium of microcluster cells. Two-dimensional polyacrylamide gel analysis and blotting of proteins on PVDF membrane showed that MAb 3G2 detected a single polypeptide of apparent molecular mass of 48 kDa and an isoelectric point (pI) of 5.2, designated EP48. A transient expression during somatic embryogenesis was observed for EP48. Indirect immunofluorescence showed that this protein highly accumulated in the cell walls of some single cells, microclusters and partly in proembryogenic masses (PEMs), but not in globular embryos of the embryogenic cell line and microclusters from the non-embryogenic cell line. Signal intensity varied between individual cells of the same population and in successive stages of somatic embryo development. Screening of several D. glomerata L. embryogenic and non-embryogenic cell lines with MAb 3G2 indicated the presence of ECP48 in only embryogenic suspension cultures at early stages of embryo development long before morphological changes have taken place and thus it could serve as an early marker for embryogenic potential in D. glomerata L. suspension cultures.

Proliferation and germination of somatic embryos from embryogenic suspension cultures in Ph?nix dactylifera

The present study demonstrates a procedure for the rapid development of a high number of somatic embryos from embryogenic suspension culture. This method might be efficient for mass propagation of Phœnix dactylifera L. Embryogenic callus placed in liquid medium with 10−5 M ABA yielded an average 72 embryos per 100 ml of culture medium within 2 months, while those placed on solid medium yielded an average of 33, 20 and 16 embryos per 100 ml of culture medium respectively for 10−7, 10−6 and 10−5 M ABA after 4 months. The combination of 2,4-DIchlorophenoxyacetic acid (2,4-D) (4.5 × 10−7 M), glutamine (6.7 × 10−4 M), and ABA (10−5 M) (L8 liquid medium) showed a beneficial effect on somatic embryos production compared to 2,4- D and glutamine alone, while this combination significantly ( p D and 10−5 M ABA (L6 liquid medium) accumulated more sugars (292 and 265 mg g−1 DW respectively for Jihel and Bousthami noir) than those matured on any other liquid medium. Histological studies revealed that somatic embryos (developed in L6 and L8 liquid media) accumulated less reserve compounds (proteins and sugars) than zygotic embryos. The addition of activated charcoal (0.25 and 0.5 g l−1) and phytagel® (2.5 g l−1) to the germination medium may be useful for enhancing the germination of Phœnix dactyliferasomatic embryos.

Tolerance of embryogenic suspension cultures from two Phragmites communis (reed) ecotypes to salt: Changes of respiration pathway

In the present work, an intensive tolerance to high salt stress of embryogenic suspension cultures from two reed (Phragmites communis Trin.) ecotypes, dune reed and swamp reed, was found and studied. The dune reed was more resistant than the swamp reed to salt. Aiming at better understanding the characteristic tolerance to salt stress in reed plants, changes of respiration and respiratory pathways in both reed suspension cultures exposed to 50-800 mM NaCl stress were measured and analyzed. The results demonstrated that the respiration rates of both reed cultures increase significantly during the 50-400 mM NaCl treatment. Inhibition tests with cyanide and salicylhydroxamic acid showed that the increasing respiration rate resulted from high operation of the cytochrome pathway in the dune reed, and cooperation of the cytochrome pathway and an alternative pathway in the swamp reed. The respiration rates of both reed cultures decreased dramatically following 800 mM NaCl treatment. The results were due to the l...

Protoplast transformation and regeneration of transgenic Valencia sweet orange plants containing a juice quality-related pectin methylesterase gene

Valencia orange [Citrus sinensis (L.) Osbeck] is the leading commercial citrus species in the world for processed juice products; however, the presence of thermostable pectin methylesterase (TSPME) reduces its juice quality. A long-term strategy of this work is to eliminate or greatly reduce TSPME activity in Valencia orange. Previous work resulted in the isolation of a putative TSPME gene, CsPME4, associated with a thermostable protein fraction of Valencia orange juice. To begin research designed to overexpress CsPME4 to verify the thermostability of the protein product and/or to downregulate the gene, a sense gene cassette containing a gene-specific sequence from a putative TSPME cDNA and the enhanced green fluorescent protein (GFP) as a selectable marker was constructed (M2.1). In the work reported here, M2.1 plasmid DNA was transformed (polyethylene glycol-mediated) into protoplasts isolated from an embryogenic suspension culture of Valencia somaclone line B6-68, in an effort to obtain transgenic Valencia lines. A vigorous transformed line was identified via GFP expression, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. One transgenic proembryo expressing GFP was recovered and multiple shoots were regenerated. The recovery of multiple transgenic plants was expedited by in vitro grafting. Polymerase chain reaction analysis revealed the presence of the PME gene in transgenic plants, and subsequent Southern blot analysis confirmed the presence of the eGFP gene. These transgenic plants show normal growth and minor morphological variation. The thermostability of PME in these plants will be assessed after flowering and fruit set. This is the first successful transfer of a target fruit-quality gene by protoplast transformation with recovery of transgenic plants in citrus. This method of transformation has the advantage over Agrobacterium-mediated transformation in that it requires no antibiotic-resistance genes.

Enhancement of American chestnut somatic seedling production

Somatic embryogenesis holds promise for mass propagation of American chestnut trees bred or genetically engineered for resistance to chestnut blight. However, low germination frequency of chestnut somatic embryos has limited somatic seedling production for this forest tree. We tested the effects of culture regime (semi-solid versus liquid), cold treatment, AC and somatic embryo morphology (i.e., cotyledon number) on germination and conversion of the somatic embryos. Cold treatment for 12 weeks was critical for conversion of chestnut somatic embryos to somatic seedlings, raising conversion frequencies for one line to 47%, compared to 7% with no cold treatment. AC improved germination and conversion frequency for one line to 77% and 59%, respectively, and kept roots from darkening. For two lines that produced embryos with one, two or three-plus cotyledons, cotyledon number did not affect germination or conversion frequency. We also established embryogenic American chestnut suspension cultures and adapted a fractionation/plating system that allowed us to produce populations of relatively synchronous somatic embryos for multiple lines. Embryos derived from suspension cultures of two lines tested had higher conversion frequencies (46% and 48%) than those from cultures maintained on semi-solid medium (7% and 30%). The improvements in manipulation of American chestnut embryogenic cultures described in this study have allowed over a 100-fold increase in somatic seedling production efficiency over what we reported previously and thus constitute a substantial advance toward the application of somatic embryogenesis for mass clonal propagation of the tree.

Establishment of embryogenic cell suspension cultures of garlic (Allium sativum L.), plant regeneration and biochemical analyses

Embryogenic cell suspension cultures of garlic (Allium sativum L.) were initiated in liquid medium from friable embryogenic tissue. The optimal parameters for culture maintenance were: (1) an initial cell density of 1-4% (v/v); (2) medium renewal every 14 days and subculturing every 28 days; (3) a low 2,4-dichlorophenoxyacetic acid concentration (0.1-0.3 mg/l). Cultures regenerated during a 14-month period. The cell suspension cultures differentiated embryos following transfer to a semi-solid embryo induction medium, with histological studies confirming and characterising the embryogenic nature of the process. Forty percent of these embryos converted into plantlets, which produced micro bulbs in vitro. The composition of the sulphur compounds of the micro bulbs obtained from cell suspension embryo-derived plantlets differed slightly from those produced by in vitro shoot proliferation-derived plantlets, but after two cycles of multiplication in the field these differences had disappeared.

Stable transformation of plant cells by particle bombardment/biolistics.

Particle bombardment, or biolistics, is a commonly used method for genetic transformation of plants and other organisms. Millions of DNA-coated metal particles are shot at target cells or tissues using a biolistic device or gene gun. The DNA elutes off the particles that lodge inside the cells, and a portion may be stably incorporated in the host chromosomes. A protocol for the generation of transgenic grapevines via biolistic transformation of embryogenic cell suspension cultures is detailed in this chapter. In a typical experiment, transient gene expression averaged nearly 8000 "hits" per bombarded plate. Five months after bombardment, there were nearly five putative transgenic embryos per bombarded plate. About half of the embryos were regenerated into confirmed transgenic plants. The basic bombardment procedures described are applicable to a wide range of plant genotypes, especially those for which embryogenic cell cultures are available. All users of particle bombardment technology will find numerous useful tips to maximize the success of transformation.

Establishment of embryogenic cell suspensions and plant regeneration of the dessert banana ‘Williams’ (Musa AAA group)

Summary‘Williams’ is one of the most important dessert banana cultivars in the World. Improvement through genetic engineering depends on the development of embryogenic cell suspension (ECS) cultures. Yellow, compact meristematic globules and yellow-whiteish, friable embryogenic calli were induced from scalps (i.e., the top layer of highly proliferating meristem cultures). About 10% of induced explants gave rise to embryogenic calli. Embryogenic calli were used subsequently to establish ECS cultures. After 3 months of selection and sub-culturing, homogeneous ECS cultures with a high regeneration capacity were obtained. More than 80% of the cell culture consisted of embryogenic cell aggregates. Regenerable somatic embryos emerged about 3 weeks after the ECS was plated onto regeneration medium. Their weight increased 4.8- to 13-fold after 8 weeks of culture. The number of somatic embryos regenerated per ml settled cell volume (SCV) of embryogenic cells (i.e., somatic embryo recovery frequency) ranged between 0.92 – 2.17 105, depending on incubation and regeneration conditions. The conversion frequency from somatic embryo to plant varied between 42.5 – 55.5%, and was independent of regeneration conditions.

Generation of Specific Antibodies Against Stress-Related Proteins from Nacl Adapted Embryogenic Suspension Cultures of Dactylis Glomerata L.

ABSTRACTWe describe a method for the generation of antibodies against plant stress-related proteins in crude biochemical samples, such as proteins in suspension medium, by two-dimensional electrophoresis/phage panning (2D-PP). 2D-PP involves two-dimensional resolution of proteins, blotting of the proteins onto a nitrocellulose membrane, and screening of a phage antibody library. Phage antibodies were selected through 4 rounds of panning from the human synthetic single-chain Fv (scFv) library (Griffin.1). These antibodies are functional in Western blotting and are potential candidates as markers for the loss of regeneration capacity.

Evolution of endogenous hormone concentration in embryogenic cultures of carrot during early expression of somatic embryogenesis.

Embryogenic callus and suspension cultures of carrot (Daucus carota L., cv. Nantaise), growing on/in medium including 1 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), were transferred to medium with or without this plant growth regulator, to impair or induce, respectively, further development of somatic embryos. The endogenous hormone levels of the cultures were determined over 7 days by means of radio-immunoassay, to characterize their evolution in the initial stages of embryo development. In general, levels of indoleacetic acid (IAA) and abscisic acid (ABA) showed only short-lived differences among treatments during this time in both types of tissue analyzed (i.e., a peak of IAA in callus cultures in the absence of 2,4-D, 48 h after medium change, and higher ABA contents 144 h after subculture of suspension cultures in the presence of 2,4-D). Gibberellins (1, 3 and 20) were detected only in suspension cultures devoid of 2,4-D, starting 24 h after subculture. Concerning the evaluated cytokinins-zeatin/zeatin riboside and N6(Delta2-isopentenyl) adenine/N6(Delta2-isopentenyl) adenosine-the most remarkable observation is that high levels of the former generally coincided with low concentrations of the latter, indicating a shift from precursor to the active form, and vice versa.

Regeneration of sweet potato transgenic plants withoryzacystatin-I(OCI) gene

AbstractRegeneration of sweet potato (Ipomoea batatascv. Lizixiang) transgenic plants withoryzacystatin-I(OCI) gene was achieved using anAgrobacterium tumefaciens-mediated method.A. tumefaciensstrain LBA4404, harbouring a binary vector pBinh withneomycin phosphotransferase IIandOCIgenes, was used. After 3 days of subculture, Lizixiang embryogenic suspension cultures were co-cultivated with LBA4404 (OD600nm=0.5) for 4 days. Next, the infected suspension cultures were first cultured for 5 days in MS medium with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 300 mg/l carbencillin (Carb) but without kanamycin (Kan) and then transferred to MS medium supplemented with 2 mg/l 2,4-D, 50 mg/l Kan and 300 mg/l Carb for the selection culture. Four weeks after selection, 200 Kan-resistant cell aggregates (∼1 mm in size) from the embryogenic suspension cultures were transferred to MS solid medium supplemented with 2 mg/l 2,4-D, 50 mg/l Kan and 300 mg/l Carb, and eight embryogenic calluses were obtained. After transferring to MS medium supplemented with 1 mg/l ABA, 50 mg/l Kan and 100 mg/l Carb, these embryogenic calluses formed 13 plantlets via somatic embryogenesis. PCR and PCR–Southern blot analysis indicated that seven of the 13 plantlets were transgenic.

Establishment and Growth of Embryogenic Suspension Cultures of Ocotea catharinensis Mez. (Lauraceae)

The focus of this study was to test the effects of 2,4-D, sucrose, culture media and initial inocula on the development of embryogenic suspension cultures of Ocotea catharinensis Mez. (Lauraceae). Suspension cultures were established in half-strength MS medium supplemented with 2% (w/v) sucrose either in the absence or in the presence of 2.2 µM 2,4-D, when higher cell viability was achieved. Under this culture condition the maximum fresh weight increase occurred in the fourth week. The cultures were yellow and consisted of a mixture of highly cytoplasmic single cells and small cell aggregates (<0.25 mm). The best proportion of inoculum per volume of medium for suspension culture development was 5% (w/w). Suspension cultures consisting of somatic embryos at the globular and cotyledonary stages (structures ranging from 1 to 3 mm) were successfully established on half-strength MS supplemented with 2% (w/w) sucrose through repetitive embryogenesis from the desiccated mature somatic embryos used as initial inoculum. The failure to initiate liquid cultures from non-desiccated mature somatic embryos was overcome by pre-treatment with air desiccation and reduction of the water content to 6.1 g H2O g−1 dry weight.