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.

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.

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.

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.

Global scale transcriptome analysis of Arabidopsis embryogenesis in vitro

Background Somatic embryogenesis (SE) in plants is a process by which embryos are generated directly from somatic cells, rather than from the fused products of male and female gametes. Despite the detailed expression analysis of several somatic-to-embryonic marker genes, a comprehensive understanding of SE at a molecular level is still lacking. The present study was designed to generate high resolution transcriptome datasets for early SE providing the way for future research to understand the underlying molecular mechanisms that regulate this process. We sequenced Arabidopsis thaliana somatic embryos collected from three distinct developmental time-points (5, 10 and 15 d after in vitro culture) using the Illumina HiSeq 2000 platform. Results This study yielded a total of 426,001,826 sequence reads mapped to 26,520 genes in the A. thaliana reference genome. Analysis of embryonic cultures after 5 and 10 d showed differential expression of 1,195 genes; these included 778 genes that were more highly expressed after 5 d as compared to 10 d. Moreover, 1,718 genes were differentially expressed in embryonic cultures between 10 and 15 d. Our data also showed at least eight different expression patterns during early SE; the majority of genes are transcriptionally more active in embryos after 5 d. Comparison of transcriptomes derived from somatic embryos and leaf tissues revealed that at least 4,951 genes are transcriptionally more active in embryos than in the leaf; increased expression of genes involved in DNA cytosine methylation and histone deacetylation were noted in embryogenic tissues. In silico expression analysis based on microarray data found that approximately 5% of these genes are transcriptionally more active in somatic embryos than in actively dividing callus and non-dividing leaf tissues. Moreover, this identified 49 genes expressed at a higher level in somatic embryos than in other tissues. This included several genes with unknown function, as well as others related to oxidative and osmotic stress, and auxin signalling. Conclusions The transcriptome information provided here will form the foundation for future research on genetic and epigenetic control of plant embryogenesis at a molecular level. In follow-up studies, these data could be used to construct a regulatory network for SE; the genes more highly expressed in somatic embryos than in vegetative tissues can be considered as potential candidates to validate these networks.

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.

Overexpression of coconut AINTEGUMENTA-like gene, CnANT, promotes in vitro regeneration in transgenic Arabidopsis

Knowledge of the molecular biological changes underlying the process of embryogenesis is important for the improvement of somatic embryogenesis of coconut. Among the transcription factors that control the transition from vegetative to embryogenic growth, members of APETALA2/Ethylene-responsive element binding protein domain family play an important role in promoting embryo development. Significant insights into the role of AP2 genes have been obtained by the ectopic expression of AP2 sub family genes in transgenic Arabidopsis. A homolog of the AINTEGUMENTA-like gene that encodes the two AP2 domains and the linker region was identified in the coconut genome. Phylogenetic analysis showed that this gene, CnANT, encodes a protein that branched with BABY BOOM/PLETHORA clade in the AINTEGUMENTA-like major clade and was similar to the oil palm EgAP2-1 protein. According to real time RT-PCR results, higher expression of CnANT was observed in more mature zygotic embryos. Also, high CnANT expression was recorded in embryogenic callus compared to other stages of somatic embryogenesis. We examined the effect of ectopic CnANT expression on the development and regenerative capacity of transgenic Arabidopsis. Overexpression of CnANT in Arabidopsis induced hormone free regeneration of explants. Furthermore, ectopic expression of CnANT enhanced regeneration in vitro and suggested a role for this gene in cell proliferation during in vitro culture.

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.

Improved method for ex ovo-cultivation of developing chicken embryos for human stem cell xenografts

The characterization of human stem cells for the usability in regenerative medicine is particularly based on investigations regarding their differentiation potential in vivo. In this regard, the chicken embryo model represents an ideal model organism. However, the access to the chicken embryo is only achievable by windowing the eggshell resulting in limited visibility and accessibility in subsequent experiments. On the contrary, ex ovo-culture systems avoid such negative side effects. Here, we present an improved ex ovo-cultivation method enabling the embryos to survive 13 days in vitro. Optimized cultivation of chicken embryos resulted in a normal development regarding their size and weight. Our ex ovo-approach closely resembles the development of chicken embryos in ovo, as demonstrated by properly developed nervous system, bones, and cartilage at expected time points. Finally, we investigated the usability of our method for trans-species transplantation of adult stem cells by injecting human neural crest-derived stem cells into late Hamburger and Hamilton stages (HH26-HH28/E5-E6) of ex ovo-incubated embryos. We demonstrated the integration of human cells allowing experimentally easy investigation of the differentiation potential in the proper developmental context. Taken together, this ex ovo-method supports the prolonged cultivation of properly developing chicken embryos enabling integration studies of xenografted mammalian stem cells at late developmental stages.

Three dimensional analysis of cardiovascular development in mouse embryos using X-ray microcomputed tomography

Micro-computed tomography (μCT) has been successfully used to study the cardiovascular system of mouse embryos in situ. With the use of barium as a suitable contrast agent, blood vessels have been imaged and analysed quantitatively such as blood volume and vessel sizes on embryos of ages 14.5 to 16.5 days old. The advantage of using this imaging modality is that it has provided three dimensional information whilst leaving samples intact for further study.