Blastocysts derivation from somatic cell fusion with premature oocytes (prematuration somatic cell fusion)

This study was undertaken to investigate the development of immature oocytes after their fusion with male somatic cells expressing red fluorescence protein (RFP). RFP‐expressing cells were fused with immature oocytes, matured in vitro and then parthenogenetically activated. Somatic nuclei showed spindle formation, 1st polar body extrusion after in vitro maturation and protruded the 2nd polar body after parthenogenetic activation. RFP was expressed in the resultant embryos; two‐cell stage and blastocysts. Chromosomal analysis showed aneuploidy in 81.82% of the resulting blastocysts while 18.18% of the resulting blastocysts were diploid. Among eight RFP‐expressing blastocysts, Xist mRNAs was detected in six while Sry mRNA was detected in only one blastocyst. We propose “prematuration somatic cell fusion” as an approach to generate embryos using somatic cells instead of spermatozoa. The current approach, if improved, would assist production of embryos for couples where the male partner is sterile, however, genetic and chromosomal analysis of the resultant embryos are required before transfer to the mothers.     

Spaceflight reduces somatic embryogenesis in orchardgrass (Poaceae)

Somatic embryos initiate and develop from single mesophyll cells in in vitro cultured leaf segments of orchardgrass ( Dactylis glomerata L.). Segments were plated at time periods ranging from 21 to 0·9 d (21 h) prior to launch on an 11 d spaceflight (STS-64). Using a paired t -test, there was no significant difference in embryogenesis from preplating periods of 14 d and 21 d. However, embryogenesis was reduced by 70% in segments plated 21 h before launch and this treatment was significant at P = 0·0001. The initial cell divisions leading to embryo formation would be taking place during flight in this treatment. A higher ratio of anticlinal:periclinal first cell divisions observed in the flight compared to the control tissue suggests that microgravity affects axis determination and embryo polarity at a very early stage. A similar reduction in zygotic embryogenesis would reduce seed formation and have important implications for long-term space flight or colonization where seeds would be needed either for direct consumption or to grow another generation of plants.     

The sensitivity of transgenic spruce (Picea glauca (Moench) Voss) cotyledonary somatic embryos and somatic seedlings to kanamycin selection

Transformed white spruce cultures containing immature Stage I somatic embryos, were developed after particle bombardment of somatic embryos with pBI 426, carrying an expression cassette with a gus::nptII fusion gene. These Stage I cultures did not show tolerance to kanamycin concentrations above 3 to 5 mg l–1, although assays for GUS and NPTII showed that functional enzymes were present in the transgenic tissue. Embryonic liquid suspension cultures were initiated from this transformed tissue. After treatment on agar-solidified maturation medium with 48 m (±)-ABA high numbers of Stage III (cotyledonary) somatic embryos were produced. When subjected to an embryogenesis re-induction process with 2,4-D and BA, these Stage III embryos produced a new generation of Stage I embryogenic tissues which could tolerate 5--10 mg l–1 kanamycin. Stage III somatic embryos could alternatively be placed onto germination medium for the development of somatic seedlings. When germinated in the presence of 20 mg l–1 kanamycin, 77% of inoculants were resistant. The stability of integration of the gus::nptII fusion gene in the genome of white spruce Stage III somatic embryos and somatic seedlings was confirmed through Southern blot analysis     

Direct somatic embryogenesis and encapsulation of somatic embryos for in vitro conservation of Bacopa monnieri (L.) Wettst

Direct somatic embryogenesis and shoot organogenesis were achieved from leaf explants excised from microshoots of Bacopa monnieri cultured on Murashige and Skoog medium containing N6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D). The maximum frequency of explants differentiated somatic embryos and shoot buds on MS medium supplemented with 12.5 µM BA and 1 µM 2,4-D. The frequency of explants differentiating somatic embryos decreased with increasing concentration of 2,4-D. Light and scanning electron microscopy revealed direct differentiation of somatic embryos and shoot buds from explants, and various developmental stages of the somatic embryos were observed. Somatic embryos and apical shoot tips were encapsulated in sodium alginate gel to produce synthetic seeds. The storage of synthetic seeds produced by encapsulation was studied at 4 and 25?°C (room temperature) for a period of 140 days. Encapsulated somatic embryos were found to retain viability after 140 days of storage at both temperatures, whereas encapsulated apical shoot buds failed to germinate even after 40 days when stored at 4?°C. The viability of synthetic seeds was higher when stored at 25?°C. All amplified markers scored by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) were monomorphic for all the plants produced from synthetic seeds following different periods of storage, thus establishing the clonal fidelity of propagated plantlets.     

Scale-up of somatic embryogenesis in alfalfa (Medicago sativa L.) I subculture and indirect secondary somatic embryogenisis

Three methods of increasing the productivity of somatic embryogenesis in Medicago sativa L. were investigated. In the basic procedure, somatic embryos were initiated from young petioles and carried through several phases: callus formation, suspension culture, selection of the embryogenic fraction by sieving, development, maturation, desiccation and storage. The suspensions were normally separated into three fractions by sieving. Fraction I (<200 m) containing nonembryogenic cells or cell clusters was discarded. Fraction II (200–500 m) consisting of embryogenic cell clusters was collected for embryo development and maturation. Fraction III (over 500 M) containing the mixture of petiole residues with large pieces of calli and globular somatic embryos was usually discarded. Several methods to scale-up the suspension phase were unsuccessful. Direct subculture of the entire suspension by the addition of fresh liquid medium resulted in the loss of embryogenic capacity by the third subculture. Subculture of fraction II decreased embryogenic cell mass, and hence reduced total productivity. The recycling of fraction III back to fresh B₅g liquid medium resulted in high productivity in the first culture but further subculture of this fraction resulted in a rapid decline in the embryogenic capacity.As an alternative, somatic embryos from the first tissue culture cycle were also used as explants for the initiation of secondary embryogenic callus. The embryogenic capacity of these somatic embryo explants declined rapidly as they matured. More than 100 secondary somatic embryos could be induced from embryo explants removed from development medium at 10 days after sieving the suspension, but only 40 somatic embryos were produced from each mature somatic embryo explant, and 13 from desiccated embryos. The secondary somatic embryos were comparable to the primary embryos in quality according to germination tests. The implications of the results to the efficiency of somatic embryo production of Medicago are discussed.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - DAS days after sieving - PPF photosynthetic photon flux density - SE somatic embryo     

Induction of somatic embryogenesis in cashewnut (Anacardium occidentale L.)

Summary Somatic embryogenesis was induced in callus cultures derived from nucellar tissue of cashewnut (Anacardium occidentale L.). Callus was obtained from nucellar tissue after 3 wk of culture on semisolid Murashige and Skoog (MS) basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 5 μM)+gibberellic acid (GA₃, 15 μM)+N⁶-benzyladenine (BA, 5 μM). This callus gave rise to an embryogenic mass after 9 wk on maintenance medium containing 2,4-D (10 μM)+GA₃ (15 μM)+4% sucrose +0.5% activated charcoal +10% coconut water (CW) +0.05% casein hydrolysate (CH). The embryogenic mass, after transfer to medium supplemented with 2,4-D (5 μM)+GA₃ (30 μM)+4% sucrose +0.5% activated charcoal +10% CW +0.05% CH, gave rise to somatic embryos. The developmental stages of somatic embryos were observed using light and stereo microscopes. Histological study of somatic embryo development was also carried out. The present study would be useful for clonal propagation, and variety improvement in cashewnut, which is essential due to its increasing demand and export potential.     

Regeneration of Cladrastis lutea (Fabaceae) via somatic embryogenesis

Summary Immature embryos from 5 Cladrastis lutea (Michx.) K. Koch (yellowwood) trees were initially cultured on modified Schenk and Hildebrandt medium (SH) containing either 4.5, 9.0, 13.5 or 23 M 2,4-D. One-third of the explants were transferred to SH medium supplemented with 25.0 M NAA after 2 and 3 weeks respectively. The remaining explants were incubated on the initial 2,4-D containing media for 6 weeks. Groups of somatic embryos formed directly only at the proximal end of cotyledons; only a few formed as single embryos. The greatest numbers were formed from zygotic embryos explanted from 6–8 weeks post-anthesis and initially cultured on medium containing 9 or 13 M 2,4-D. However, all treatments supported somatic embryogenesis. In the second year, explants were initially cultured on SH medium containing either 9.0, 13.5, or 23 M 2,4-D and then transferred to SH medium containing 4.0 M ABA after 2 or 3 weeks. ABA did not affect the development of somatic embryos. Six of 21 somatic embryos germinated on half-strength SH medium without growth regulators. Three entire plantlets were formed, but only one was established in soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - ABA abscisic acid - CRAF III chromium trioxide-acetic acidformalin     

Characterization of somatic embryogenesis in sandalwood (Santalum album L.)

In the synchronous embryogenesis system of sandalwood developed in our laboratory, we observed that the early events of differentiation from freshly induced callus (stage 0) are accomplished in three distinct stages viz., preglobular masses (stage 1), globular embryos (stage 2), and bipolar embryos (stage 3). Transition from stage 0 to 1 was accomplished using 2,4-D and involves a stage specific appearance of two polypeptides of 15 and 30 kDa molecular weight. A 24 kDa polypeptide that was detected as a marked band in extracts of primary callus was not detected in stages 1, 2, and 3. Further, the tissue level of a 50 kDa glycoprotein decreased during transition from stage 2 to stage 3. However, the levels of glycoproteins in the medium were markedly higher in stage 0 cultures compared to those in stage 1. The activities of a protein kinase, glycosidase, and xylanase increased markedly with progressing embryogenesis. Our observations suggest that in addition to being controlled at the level of stage-specific gene expression, somatic embryogenesis in sandalwood is also regulated at the level of controls on cell wall flexibility and posttranslational changes in the pool of preexisting proteins.     

Interspecific somatic hybrids ofRudbeckia hirta andR. Laciniata (Compositae)

Interspecific somatic hybrid plants betweenRudbeckia hirta cv. Marmalade andR.laciniata cv. Irish Eyes were regenerated following the electro-fusion of mesophyll protoplasts ofR.hirta with callus protoplasts ofR.laciniata. A hybrid selection scheme was based on the fact that plant regeneration, from parental protoplasts ofR.hirta, was via shoot regeneration of callus, and only via rhizogenesis forR.laciniata. The other half of the selection strategy was based on the presence of anthocyanin-pigmented roots; a characteristic of theR.hirta parent only. Somatic hybrids were regenerated, via rhizogenesis, alongside normalR.laciniata but were distinguished by the presence of pigmented roots (a feature ofR.hirta). Hybrid plants had a floral morphology that was intermediate as compared to that of the two parents, with an expected somatic chromosome number of 2n=(2x+4x)=74. Pollen viability though was low. Esterase and peroxidase isozyme profiles confirmed the hybrid nature of the regenerated plants with pigmented roots, whilst chloroplast DNA restriction analysis showed that these hybrids had aR.laciniata chloroplast DNA. This demonstration of somatic hybridisation not only opens up the possibility of incorporating novel traits between such ornamentalCompositae species, but provides a selection strategy based on rhizogenesis as the route to plant regeneration coupled with heritable pigmentation production of roots as a confirmatory hybrid marker.ABBREVIATIONS BSA bovine serum albumin - EDTA ethylene diamine tetra acetic acid - FDA fluorescein diacetate - f.wt. fresh weight - IAA indole 3-acetic acid - MS Murashige and Skoog (1962) medium - TEMED N,N,N,N-Tetra methyl ethylene diamine - TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid)     

The production of fertile,triploid somatic hybrid plants (Nicotiana glutinosa (n) + N. tabacum (2n)) via gametic: somatic protoplast fusion

Summary The fusion of gametic protoplasts with somatic protoplasts giving rise to gametosomatic hybrid plants was investigated. Gametosomatic hybrid plants were regenerated following the fusion of nitrate reductase deficient (Nr^–) Nicotiana tabacum Nia-130 leaf mesophyll protoplasts with N. glutinosa tetrad protoplasts. The resulting plants were confirmed as hybrids, based on leaf and floral morphology, chromosome number, leaf esterase and leaf callus peroxidase zymograms and Fraction-1-protein analysis. The five gametosomatic hybrid plants had the expected pentaploid, but functionally triploid chromosome number of 3n=5x=60. The relevance of triploid gametosomatic hybrids in facilitating limited gene transfer, is discussed. The utilisation of tetrads as a generally available source of haploid protoplasts for fusion studies is proposed.     

Cryopreservation of bovine somatic cells using antifreeze polyamino-acid (carboxylated poly-l-lysine)

Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound, obtained by converting 65 mol% of amino groups to carboxyl groups after synthesizing ε-poly-l-lysine aqueous solution and succinic anhydride. CPLL has cryoprotective properties similar to those of anti-freeze protein. The addition of CPLL to freezing medium has been reported to improve the post-thawing survival rate of murine cells, human induced pluripotent stem (iPS) cells, embryonic stem (ES) cells and embryos. In this study, investigating CPLL for its effectiveness as a new cryoprotective material is aimed. In experiments with bovine somatic cells, CPLL was suggested to have an equal or superior cryoprotective effect to dimethyl sulfoxide (DMSO), the conventional material for cellular frozen storage, based on the results for post-thawing cell survival and proliferation rates. CPLL was demonstrated to have another advantage; thawed cells can be cultured without removing the cryopreservation medium when CPLL is used, but not when DMSO is used. These results suggest that CPLL could be used as cryoprotective material for bovine cells. It is also expected that CPLL can be applied to embryo and oocytes storage for cattle, and similar functions for cells and embryos of other animal species.     

Phosphinothricin stimulates somatic embryogenesis in grape (Vitis sp. L.)

The effect of phosphinothricin concentration on embryo production from an embryogenic callus of Chancellor (Vitis L. complex interspecific hybrid) was tested. Embryogenic callus was cultured on medium supplemented with nine phosphinothricin concentrations (0, 0.1, 0.5, 1, 1.5, 2, 3, 5, and 10 mg/l). The highest number of embryos per plate was observed at 0.5 mg/l phosphinothricin. The use of phosphinothricin to stimulate embryo production did not affect embryo germination and plantlet formation. Three germination techniques were compared. Embryo dehydration or growth on Transfergelsolidified medium gave higher germination rates than chilling treatments. Most germinated somatic embryos produced secondary embryos from the hypocotyl after a few weeks of culture. Regardless of the germination technique, the plantlet conversion rate was very low.Abbreviations AC activated charcoal - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - MS Murashige and Skoog (1962) basal medium - NN Nitsch and Nitsch (1969) medium - PPT phosphinothricin     

Targeting of somatic hypermutation

Somatic hypermutation (SHM) introduces mutations in the variable region of immunoglobulin genes at a rate of approximately 10(-3) mutations per base pair per cell division, which is 10(6)-fold higher than the spontaneous mutation rate in somatic cells. To ensure genomic integrity, SHM needs to be targeted specifically to immunoglobulin genes. The rare mistargeting of SHM can result in mutations and translocations in oncogenes, and is thought to contribute to the development of B-cell malignancies. Despite years of intensive investigation, the mechanism of SHM targeting is still unclear. We review and attempt to reconcile the numerous and sometimes conflicting studies on the targeting of SHM to immunoglobulin loci, and highlight areas that hold promise for further investigation.     

Plant regeneration via somatic embryogenesis in pea (Pisum sativum L.)

Whole plant regeneration via somatic embryogenesis was obtained in pea (Pisum sativum L.) using explants from immature embryos or shoot apex segments. The induction of somatic embryos required picloram or 2,4-D. Germination of fully-developed embryos was accomplished by subculture on medium with only cytokinin and then on medium supplemented with cytokinins in combination with a reduced auxin concentration. Plantlets obtained from both zygotic embryos and shoot apices were transferred to soil and were grown to maturity. Nine plants were examined cytologically, revealing three tetraploids (2n=4x=28) and six diploids (2n=2x=14).Abbreviations Picloram 4-amino-3,5,6-trichloropicolinic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzylaminopurine - IBA indole-3-butyric acid KAES Journal Article No. 87-3-4     

Enhancement of somatic embryogenesis in Norway spruce (Picea abies L.)

Summary Embryogenic callus developed in 55% of the mature embryo explants of Norway spruce (Picea abies L.) growing on a LP medium minus the amino acids and sugars (except sucrose). This is the highest reported yield of embryogenic callus from mature embryos of P. abies that has ever been reported. Callus induction from either the middle or the end of the hypocotyl of the embryos began after 2–3 weeks. Three types of calli were recovered: (a) globular, (b) light green-compact, (c) white mucilaginous. Only the white mucilaginous calli were embryogenic. The globular and light green-compact calli never become embryogenic, even after several subcultures. The development of somatic embryos was accomplished on half-strength macro-elements of NSIII medium containing 1 M -naphthaleneacetic acid, 1 M abscisic acid, and 3% sucrose. The addition of 10^–7 M buthionine sulfoximine to the medium increased the development of somatic embryos by three fold. These results suggest that there is a great potential for increasing the frequency and development of somatic embryos in P. abies. Careful selection of the genotype and modification of the culture medium is required.     

Factors controlling somatic embryogenesis

Histological and ultrastructural, molecular and elemental distribution changes were investigated during the induction of direct somatic embryogenesis using theCamellia japonica leaf culture system. In this culture system, direct somatic embryogenesis is induced in a controlled way in a specific leaf region (leaf blade) within a leaf. Embryogenic and non-embryogenic leaf regions have characteristic energy-dispersive X-ray spectra already before induction. According to these results electron probe X-ray microanalysis (EPMA) can be a tool for early diagnosis of embryogenic competence. Histological studies showed that severe fluctuations in the number of calcium oxalate crystals and in starch accumulation occur after induction but only in induced tissues. Changes in the cell wall composition of competent cells occur shortly after the induction treatment. The induction of morphogenesis is linked to the appearance of callose covering the surface cells of induced leaves and calluses. A 2^nd deposition of material (cutin) is necessary for normal somatic embryogenesis to occur. The involvement of lipid transfer proteins in the appearance of cutin in the embryogenic regions of the explant is suggested.     

Plant regeneration through somatic embryogenesis in peanut (Arachis hypogaea L.)

Somatic embryos were induced from immature cotyledons and immature embryonal axis ofArachis hypogaea L. on L-6 basal medium supplemented with NAA, picloram or 2,4-D at 5–50 mg 1^-1. Immature embryonal axis produced a higher number of somatic embryos in comparison with immature cotyledons. The highest number of responding cultures was produced on medium supplemented with NAA (50 mg 1^-1), while the highest average number of somatic embryos per culture was produced on medium with 2,4-D (10 or 20 mg 1^-1) and picloram (30 mg 1^-1) from cotyledons. The somatic embryos developed into plants on basal medium supplemented with activated charcoal and about 100 plants were successfully transferred to the field. Acknowledgement: The authors wish to thank Nuclear Agriculture Division, BARC for supplyingA. hypogaea seeds and Mr. R.M. Mudliar for photography.     

Regeneration of pineapple (Ananas comosus L.) plant through somatic embryogenesis

An efficient protocol for a complete plant regeneration by somatic embryogenesis was developed with Smooth Cayenne pineapple (Ananas comosus L.). Previous works showed that this species is responsive to somatic embryogenesis. In the present work the influence of components of culture medium in the induction, development and conversion of somatic embryos was investigate in order to establish a somatic embryogenesis protocol. Nodular callus (83.67%) was initiated from leaf explants of young plants on CIM₃ medium. The highest frequency (37.6%) of embryogenic callus induction was obtained from 4-week-old calluses on EIM₃ medium supplemented with 3.0 mg/l picloram. The highly organized callus induction and the development of somatic embryos were achieved after the transfer of callus clumps onto EIM₃ medium containing 1.0 mg/l BAP + 0.1 mg/l NAA. The frequency of somatic embryo formation was of 39.5?±?2.45 embryos per callus. Up to 97% of the somatic embryos were converted into complete plants within 4 week on MSB medium with 1.0 mg/l BAP + 0.05 mg/l GA₃ + 500 mg/l glutamine. The continuation of the elongation of the shoots occurred on this medium). Shoots obtained from all the above methods were rooted in MSB medium with activated charcoal. Complete plantlets were transferred onto specially made polyethylene bags containing soil mixture and transferred to the greenhouse. Survival rate of the plantlets under ex vitro conditions was 98% and maximum average number of plantlets (80?±?0.6). The well-developed plantlets were transferred to an open field where the plants produced normal fruits.