Use of secondary somatic embryos promotes genetic fidelity in cryopreservation of cocoa (Theobroma cacao L.)

The inability to conserve cocoa (Theobroma cacao L.) germplasm via sced storage and the vulnerability of field collections make the establishment of cryopreserved genebanks for the crop a priority. An effective encapsulation-dehydration based cryopreservation system has been developed for cocoa but because the somatic embryos used for freezing arise after a protracted period of callus culture there is concern about maintenance of genetic fidelity during the process. Microsatellite markers for seven of the 10 cocoa linkage groups were used to screen a population of 189 primary somatic embryo-derived emblings and the 43 secondary somatic embryos they gave rise to. Of the primary somatic embryos, 38.1% exhibited polymorphic microsatellite profiles while for secondary somatic embryos the frequency was 23.3%. The same microsatellite markers used to screen another population of 44 secondary somatic embryos cryopreserved through encapsulation-dehydration revealed no polymorphisms. Scanning electron microscopy showed the secondary somatic embryos were derived from cotyledonary epidermal cells rather than callus. The influence of embryo ontogeny on somaclonal variation is discussed.

Influence of freezable/non-freezable water and sucrose on the viability of Theobroma cacao somatic embryos following desiccation and freezing

Encapsulated cocoa (Theobroma cacao L.) somatic embryos subjected to 0.08-1.25 M sucrose treatments were analyzed for embryo soluble sugar content, non-freezable water content, moisture level after desiccation and viability after desiccation and freezing. Results indicated that the higher the sucrose concentration in the treatment medium, the greater was the extent of sucrose accumulation in the embryos. Sucrose treatment greatly assisted embryo post-desiccation recovery since only 40% of the control embryos survived desiccation, whereas a survival rate of 60-95% was recorded for embryos exposed to 0.5-1.25 M sucrose. The non-freezable water content of the embryos was estimated at between 0.26 and 0.61 g H2O g(-1)dw depending on the sucrose treatment, and no obvious relationship could be found between the endogenous sucrose level and the amount of non-freezable water in the embryos. Cocoa somatic embryos could withstand the loss of a fraction of their non-freezable water without losing viability following desiccation. Nevertheless, the complete removal of potentially freezable water was not sufficient for most embryos to survive freezing.

Headspace volatile markers for sensitivity of cocoa (Theobroma cacao L.) somatic embryos to cryopreservation

The mechanisms that reduce the viability of plant somatic embryos following cryopreservation are not known. The objective of the present study was to evaluate the sensitivity of cocoa (Theobroma cacao L.) somatic embryos at different stages of an encapsulation-dehydration protocol using stress-related volatile hydrocarbons as markers of injury and recovery. The plant stress hormone ethylene and volatile hydrocarbons derived from hydroxyl radicals (methane) and lipid peroxidation (ethane) were determined using gas chromatography headspace analysis. Ethylene and methane were the only volatiles detected, with both being produced after each step of the cryogenic protocol. Ethylene production was significantly reduced following exposure to liquid nitrogen, but then increased in parallel with embryo recovery. In contrast, the production of methane was cyclic during recovery, with the first cycle occurring earlier for embryos recovered from liquid nitrogen and desiccation than those recovered from earlier steps in the protocol. These results suggest that loss of somatic embryo viability during cryopreservation may be related to the oxidative status of the tissue, and its capacity to produce ethylene. This study has demonstrated that headspace volatile analysis provides a robust non-destructive analytical approach for assessing the survival and recovery of plant somatic embryos following cryopreservation.

The effectiveness of somatic embryogenesis in eliminating the cocoa swollen shoot virus from infected cocoa trees

Investigations were undertaken on the use of somatic embryogenesis to generate cocoa swollen shoot virus (CSSV) disease free clonal propagules, from infected trees. Polymerase chain reaction (PCR) capillary electrophoresis revealed the presence of CSSV in all the callus tissues induced from the CSSV-infected Amelonado cocoa trees (T1, T2 and T4). The virus was transmitted to primary somatic embryos induced from the infected callus tissues at the rate of 10 (19%), 18 (14%) and 16 (15%) for T1, T2 and T4, respectively. Virus free primary somatic embryos from the infected callus tissues converted into plantlets tested CSSV negative by PCR/capillary electrophoresis 2 years after weaning. Secondary somatic embryos induced from the CSSV-infected primary somatic embryos revealed the presence of viral fragments at the rate of 4 (4%) and 9 (9%) for T2 and T4, respectively. Real-time PCR revealed 23 of the 24 secondary somatic embryos contained no detectable virus. Based on these findings, it is proposed that progressive elimination of the CSSV in infected cocoa trees occurred from primary embryogenesis to secondary embryogenesis. (C) 2008 Elsevier B.V. All rights reserved.

Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos for long-term germplasm storage

Cryopreservation using encapsulation-dehydration was developed for the long-term conservation of cocoa (Theobroma cacao L.) germplasm. Survival of individually encapsulated somatic embryos after desiccation and cryopreservation was achieved through optimization of cryoprotectants (abscisic acid (ABA) and sugar), duration of osmotic and evaporative dehydration, and embryo development stage. Up to 63% of the genotype SPA4 early-cotyledonary somatic embryos survived cryopreservation following 7 days preculture with 1 M sucrose and 4 h silica exposure (16% moisture content in bead). This optimized protocol was successfully applied to three other genotypes, e.g. EET272, IMC14 and AMAZ12, with recovery frequencies of 25, 40 and 72%, respectively (but the latter two genotypes using 0.75 M sucrose). Recovered SPA4 somatic embryos converted to plants at a rate of 33% and the regenerated plants were phenotypically comparable to non-cryopreserved somatic embryo-derived plants.

Method for the production of cotton somatic embryos. United States Patent Application

The present invention provides Inter alia, a method for the production of cotton somatic embryos comprising (a) isolating a totipotent stomatal cell-containing epidermal explant from leaf material excised from a cotton plant; and (b) culturing said explant in a basal medium which comprises an embryogenic callus-inducing quantity of an auxin and a cytokinin under an embryogenic callus inducing intensity of light until embryogenic callus is formed; and (c) sub-culturing said embryogenic callus onto a somatic embryo differentiation media to produce said somatic embryos. Plants may be regenerated from the somatic embryos and in a particular embodiment of the invention said totipotent stomatal cell is transformed, prior to the inducement of embryogenic callus, with a polynucleotide that provides for a desired agronomic trait.

Detection of somaclonal variation during cocoa somatic embryogenesis characterised using cleaved amplified polymorphic sequence and the new freeware Artbio

The scarcity and stochastic nature of genetic mutations presents a significant challenge for scientists seeking to characterise de novo mutation frequency at specific loci. Such mutations can be particularly numerous during regeneration of plants from in vitro culture and can undermine the value of germplasm conservation efforts. We used cleaved amplified polymorphic sequence (CAPS) analysis to characterise new mutations amongst a clonal population of cocoa plants regenerated via a somatic embryogenesis protocol used previously for cocoa cryopreservation. Efficacy of the CAPS system for mutation detection was greatly improved after an ‘a priori’ in silico screen of reference target sequences for actual and potential restriction enzyme recognition sites using a new freely available software called Artbio. Artbio surveys known sequences for existing restriction enzyme recognition sites but also identifies all single nucleotide polymorphism (SNP) deviations from such motifs. Using this software, we performed an in silico screen of seven loci for restriction sites and their potential mutant SNP variants that were possible from 21 restriction enzymes. The four most informative locus-enzyme combinations were then used to survey the regenerant populations for de novo mutants. We characterised the pattern of point mutations and, using the outputs of Artbio, calculated the ratio of base substitution in 114 somatic embryo-derived cocoa regenerants originating from two explant genotypes. We found 49 polymorphisms, comprising 26.3% of the samples screened, with an inferred rate of 2.8 × 10−3 substitutions/screened base. This elevated rate is of a similar order of magnitude to previous reports of de novo microsatellite length mutations arising in the crop and suggests caution should be exercised when applying somatic embryogenesis for the conservation of plant germplasm.

Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos by vitrification

Losses of cultivated cocoa (Theobroma cacao L.) due to diseases and continued depletion of forests that harbour the wild progenitors of the crop make ex situ conservation of cocoa germplasm of paramount importance. In order to enhance security of in situ germplasm collections, 2-3 mm floral-derived secondary somatic embryos were cryopreserved by vitrification. This work demonstrates the most uncomplicated clonal cocoa cryopreservation. Optimal post-cryostorage survival (74.5%) was achieved by 5 d preculture of SSEs on 0.5 M sucrose medium followed by 60 min dehydration in cold PVS2. To minimise free radical related cryo-injury, cation sources were removed from the embryo development solution and/or the recovery medium, the former treatment resulting in a significant benefit. After optimisation with cocoa genotype AMAZ 15, the same protocol was effective across all five additional cocoa genotypes tested. For the multiplication of clones, embryos regenerated following cryopreservation were used as explant sources, and vitrification was found to maintain their embryogenic potential.

Highly efficient neural differentiation of human somatic stem cells, isolated by minimally invasive periodontal surgery

Neural stem cells (NSCs) are potential sources for cell therapy of neurodegenerative diseases and for drug screening. Despite their potential benefits, ethical and practical considerations limit the application of NSCs derived from human embryonic stem cells (ES) or adult brain tissue. Thus, alternative sources are required to satisfy the criteria of ready accessibility, rapid expansion in chemically defined media and reliable induction to a neuronal fate. We isolated somatic stem cells from the human periodontium that were collected during minimally invasive periodontal access flap surgery as part of guided tissue regeneration therapy. These cells could be propagated as neurospheres in serum-free medium, which underscores their cranial neural crest cell origin. Culture in the presence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) under serum-free conditions resulted in large numbers of nestin-positive/Sox-2-positive NSCs. These periodontium-derived (pd) NSCs are highly proliferative and migrate in response to chemokines that have been described as inducing NSC migration. We used immunocytochemical techniques and RT-PCR analysis to assess neural differentiation after treatment of the expanded cells with a novel induction medium. Adherence to substrate, growth factor deprivation, and retinoic acid treatment led to the acquisition of neuronal morphology and stable expression of markers of neuronal differentiation by more than 90% of the cells. Thus, our novel method might provide nearly limitless numbers of neuronal precursors from a readily accessible autologous adult human source, which could be used as a platform for further experimental studies and has potential therapeutic implications.

Relationship between the somatic cell count in raw milk and the casein fractions of UHT milk

The relationship between somatic cell count (SCC) in raw milk and casein fractions of 15 batches of the corresponding ultra-high-temperature (UHT) milk was examined. Raw milk was collected, pasteurised and submitted to UHT treatment. Samples of the UHT milk were taken on days 8, 30, 60, 90 and 120 of storage at room temperature and their casein fractions analysed by high performance liquid chromatography. SCC ranged from 197,000 to 800,000 cells/mL. No correlation (p>0.05) was found between SCC and K-casein concentrations in raw or UHT milks. The alpha(s2) and P-casein concentrations in raw milk were negatively correlated with SCC (p<0.05). In UHT milk, negative correlations were observed for a,1-casein (p<0.05) and beta-casein (p<0.05) on the 8th day, and for alpha S-2-casein (p<0.01) on the 60th day of storage. Results indicate that higher SSC in raw milk is associated with substantial degradation of beta-casein and alpha(s)-casein, which may lead to quality defects in UHT milk during storage. Aust. J. Dairy Technol. 63, 45-49

Loss of Methylation at H19 DMD Is Associated with Biallelic Expression and Reduced Development in Cattle Derived by Somatic Cell Nuclear Transfer

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

Effect of somatic cell counts on lipolysis, proteolysis and apparent viscosity of UHT milk during storage

In this work, lipolysis, proteolysis and viscosity of ultra-high temperature (UHT) milk containing different somatic cell counts (SCC) were investigated. UHT milks were analysed on days 8, 30, 60, 90 and 120 of storage. Lipolysis as measured by free fatty acids increase, casein degradation and viscosity of UHT milk were not affected by SCC but increased during storage. A negative relationship was observed between SCC and casein as a percentage of true protein on the 120th day of storage, hence indicating that high SCC increases the proteolysis of UHT milk by the end of its shelf life.

Viable Calves Produced by Somatic Cell Nuclear Transfer Using Meiotic-Blocked Oocytes

Somatic cell nuclear transfer (SCNT) has had an enormous impact on our understanding of biology and remains a unique tool for multiplying valuable laboratory and domestic animals. However, the complexity of the procedure and its poor efficiency are factors that limit a wider application of SCNT. In this context, oocyte meiotic arrest is an important option to make SCNT more flexible and increase the number of cloned embryos produced. Herein, we show that the use of butyrolactone I in association with brain-derived neurotrophic factor (BDNF) to arrest the meiotic division for 24 h prior to in vitro maturation provides bovine (Bos indicus) oocytes capable of supporting development of blastocysts and full-term cloned calves at least as efficiently as nonarrested oocytes. Furthermore, the procedure resulted in cloned blastocysts with an 1.5- and twofold increase of POU5F1 and IFNT2 expression, respectively, which are well-known markers of embryonic viability. Mitochondrial DNA (mtDNA) copy number was diminished by prematuration in immature oocytes (718,585 +/- 34,775 vs. 595,579 +/- 31,922, respectively, control and treated groups) but was unchanged in mature oocytes (522,179 +/- 45,617 vs. 498,771 +/- 33,231) and blastocysts (816,627 +/- 40,235 vs. 765,332 +/- 51,104). To our knowledge, this is the first report of cloned offspring born to prematured oocytes, indicating that meiotic arrest could have significant implications for laboratories working with SCNT and in vitro embryo production.

A novel regeneration system for a wild passion fruit species (Passiflora cincinnata Mast.) based on somatic embryogenesis from mature zygotic embryos

The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 mu M benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 mu M 2,4-D resulted in the largest number of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically, embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog`s medium with activated charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos, including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation protocol.