Somatic embryogenesis in peach palm using the thin cell layer technique: induction, morpho-histological aspects and AFLP analysis of somaclonal variation

BACKGROUND AND AIMS: The thin cell layer (TCL) technique is based on the use of very small explants and has allowed enhanced in vitro morphogenesis in several plant species. The present study evaluated the TCL technique as a procedure for somatic embryo production and plantlet regeneration of peach palm. METHODS: TCL explants from different positions in the shoot apex and leaf sheath of peach palm were cultivated in MS culture medium supplemented with 0-600 microM Picloram in the presence of activated charcoal. The production of primary calli and embryogenic calli was evaluated in these different conditions. Histological and amplified fragment length polymorphism (AFLP) analyses were conducted to study in vitro morphogenetic responses and genetic stability, respectively, of the regenerated plantlets. KEY RESULTS: Abundant primary callus induction was observed from TCLs of the shoot meristem in culture media supplemented with 150-600 microM Picloram (83-97%, respectively). The production of embryogenic calli depends on Picloram concentration and explant position. The best response observed was 43% embryogenic callus production from shoot meristem TCL on 300 microM Picloram. In maturation conditions, 34+/-4 somatic embryos per embryogenic callus were obtained, and 45.0+/-3.4% of these fully developed somatic embryos were converted, resulting in plantlets ready for acclimatization, of which 80% survived. Histological studies revealed that the first cellular division events occurred in cells adjacent to vascular tissue, resulting in primary calli, whose growth was ensured by a meristematic zone. A multicellular origin of the resulting somatic embryos arising from the meristematic zone is suggested. During maturation, histological analyses revealed bipolarization of the somatic embryos, as well as the development of new somatic embryos. AFLP analyses revealed that 92% of the regenerated plantlets were true to type. The use of TCL explants considerably improves the number of calli and somatic embryos produced in comparison with previously described protocols for in vitro regeneration of peach palm. CONCLUSIONS: The present study suggests that the TCL somatic embryogenesis protocol developed is feasible, although it still requires further optimization for in vitro multiplication of peach palm, especially the use of similar explants obtained from adult palm trees.     

Stimulation of root development on buckwheat thin cell-layer explants by pectic fragments from pea stem cell walls

Buckwheat (Fagopyrum esculentum Moench.) thin cell-layers (TCLs) cultured individually in a liquid medium were used to test the root-inducing activity of pectic polysaccharides with a degree of polymerization (DP) of 20–25, isolated from pea (Pisum sativum L.) stem cell walls. These pectic fragments induced more rapid root formation on the explants in comparison with untreated controls. This pectic fragment treatment also promoted root growth as measured by both fresh and dry weights and about doubled the number of roots formed. This buckwheat TCL system is proposed as a new bioassay for oligosaccharins due to its sensitivity, reproducibility and ease of preparation.     

Versatile stem cells, young and old. A review

Both embryonic and somatic stem cells have been studied in recent years with particular regard to their differentiation potential. In vitro studies allow a considerable amplification of such cells in culture as well as the induction of commitment in different directions under proper stimulating factors. Moreover, a surprising versatility has been discovered,which makes possible a `reprogramming' of stem cells into a lineage pathway which may be completely different from the expected direction: for instance, a production of brain cells from blood progenitors has been obtained. It is thus possible to envisage methods of producing in culture sufficient amounts of stem cells, committed to a certain pathway, which can be transplanted in vivo to replace damaged tissues and organs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plant regeneration from pistil thin cell layers of Citrus

 Callus induction, somatic embryogenesis and plant regeneration were obtained in six different citrus species [Citrus deliciosa Ten. (cv 'Avana'), C.limon (L.) Burm. (cv 'Berna'), C.madurensis Lour. (cv 'CNR P9'), C.medica L. (cv 'Cedro di Trabia'), C.tardiva Hort. ex Tan. (cv 'CNR P6'), C.sinensis (L.) Osb. (cv 'Ugdulena 7')] from cultures of pistil transverse thin cell layer explants [(t)TCL]. Explants were cultured on three different media: the nutrients and vitamins of Murashige and Skoog medium alone (MS) or MS supplemented with either 500 mg l^–1 malt extract (MS I) or 500 mg l^–1 malt extract and 13.3 μM 6-benzylaminopurine (MS II). Sucrose (146 mM) was used as the carbon source. Somatic embryos were visible 2–5 months after culture initiation. The different genotypes showed a different embryogenic frequency from stigma, style and ovary (t)TCL explants. All of the cultivars regenerated somatic embryos. Percentages of style (t)TCL explants producing somatic embryos ranged from 0% (C.deliciosa, C.madurensis, C.sinensis and C.tardiva on the three different media) to 5.2% (C.limon on MS II). Embryo formation in stigma (t)TCL explants ranged from 0% (C.madurensis on MS and MS I, C.sinensis on MS, C.deliciosa and C.tardiva on the three different media) to 42.4% (C.limon on MS II). Embryo formation in ovary (t)TCL explants ranged from 0% (C.deliciosa on MS, C.limon, C.medica, and C.sinensis on the three different media) to 9.3% (C.tardiva on MS I). After about 12 weeks somatic embryos developed into plantlets at a high frequency. Received: 22 September 1998 / Revision received: 6 November 1998 / Accepted: 23 November 1998     

Histology,histochemistry and ultrastructure of pre-embryogenic cells determined for direct somatic embryogenesis in the palm tree Syagrus oleracea

Somatic embryogenesis in palm trees is, in general, a slow and highly complex process, with a predominance of the indirect route and, consequently, a lack of knowledge about the direct route. We present new knowledge related to the morphological, histochemical and ultrastructural aspects of the transition from somatic to embryogenic cells and direct formation of somatic embryos from mature zygotic embryos of Syagrus oleracea, a palm tree. The results support the general concept that 2,4-dichlorophenoxyacetic acid plays a critical role for the formation of somatic embryos of direct and multicellular origin. Seven days in medium with auxin were enough for the identification of embryogenic cells. These cells had a set of characteristics corresponding to totipotent stem cells. At 14 days on induction medium, nodular formations were observed in the distal region of inoculated embryos, which evolved into globular somatic embryos. At 120 days on induction medium, the quality of the somatic embryos was compromised. The dynamics of the mobilization of reserve compounds was also demonstrated, with emphasis on starch and protein as energy sources required for the embryogenic process. This study shows for the first time the anatomical and ultrastructural events involved in direct somatic embryogenesis in a palm tree and incites the scientific community to return to the discussion of classical concepts related to direct somatic embryogenesis, especially regarding the characteristics and location of determined pre-embryogenic cells.     

Establishment of male and female nuclear transfer embryonic stem cell lines from different mouse strains and tissues

Nuclear transfer can be used to generate embryonic stem cell lines from somatic cells, and these have great potential in regenerative medicine. However, it is still unclear whether any individual or cell type can be used to generate such lines. Here, we tested seven different male and female mouse genotypes and three cell types as sources of nuclei to determine the efficiency of establishing nuclear transfer embryonic stem cell lines. Lines were successfully established from all sources. Cumulus cell nuclei from F(1) mouse genotypes showed a significantly higher cumulative establishment rate from reconstructed oocytes than from other cells; however, there were no genotype differences in success rates from cloned blastocysts. Thus, the overall success depends on preimplantation development, and, once embryos have reached the blastocyst stage, the genotype differences disappear. All mouse genotypes that were tested demonstrated at least one cell line that subsequently contributed to germline transmission in chimeric mice, so these cell lines clearly possess the same potential as embryonic stem cells derived from fertilized embryos. Thus, nuclear transfer embryonic stem cells can be generated relatively easily from a variety of inbred mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly.     

Luminescent radio frequency radiation dosimetry

Thermoluminescent dosimetry has been the industry standard for ionizing radiation dosimetry because it is inexpensive, sensitive, and accurate. No such system exists for radio frequency radiation. This paper describes the state of the art of efforts toward developing such a system. Thermochemiluminescent (TCL) dosimetry, first reported in 1991, is a first step toward achieving this goal. However, it has had problems in the production of TCL materials and in conversion of the luminescent signal into specific absorption rate (SAR). The former problem has been solved by the development of a genetically engineered Escherichia coli bacterium (JM 109/plC20RNR_1.1), described herein, that produces the TCL material in a fermentation process. The latter problem stems from the difficulty in determining the structure of the currently best TCL material diazoluminomelanin. A theoretical approach for the solution of this problem has been achieved by combining equations for delayed fluorescence, temperature determination by TCL, and the free energy equation for equilibrium reactions. It has led to an explanation for the stable display of steady‐state energy disposition, illustrated by TCL, in phantoms without the expected disruption by thermal conduction or convection, at frequencies ranging from 2.06 GHz to 35 GHz. Bioelectromagnetics 20:46–51, 1999. Published 1999 Wiley‐Liss, Inc.     

HOX and TALE signatures specify human stromal stem cell populations from different sources

Human stromal stem cell populations reside in different tissues and anatomical sites, however a critical question related to their efficient use in regenerative medicine is whether they exhibit equivalent biological properties. Here, we compared cellular and molecular characteristics of stromal stem cells derived from the bone marrow, at different body sites (iliac crest, sternum, and vertebrae) and other tissues (dental pulp and colon). In particular, we investigated whether homeobox genes of the HOX and TALE subfamilies might provide suitable markers to identify distinct stromal cell populations, as HOX proteins control cell positional identity and, together with their co‐factors TALE, are involved in orchestrating differentiation of adult tissues. Our results show that stromal populations from different sources, although immunophenotypically similar, display distinct HOX and TALE signatures, as well as different growth and differentiation abilities. Stromal stem cells from different tissues are characterized by specific HOX profiles, differing in the number and type of active genes, as well as in their level of expression. Conversely, bone marrow‐derived cell populations can be essentially distinguished for the expression levels of specific HOX members, strongly suggesting that quantitative differences in HOX activity may be crucial. Taken together, our data indicate that the HOX and TALE profiles provide positional, embryological and hierarchical identity of human stromal stem cells. Furthermore, our data suggest that cell populations derived from different body sites may not represent equivalent cell sources for cell‐based therapeutical strategies for regeneration and repair of specific tissues. J. Cell. Physiol. 228: 879–889, 2013. © 2012 Wiley Periodicals, Inc.     

Surrogate production of genome-edited sperm from a different subfamily by spermatogonial stem cell transplantation

The surrogate reproduction technique, such as inter-specific spermatogonial stem cells (SSCs) transplantation (SSCT), provides a powerful tool for production of gametes derived from endangered species or those with desirable traits. However, generation of genome-edited gametes from a different species or production of gametes from a phylogenetically distant species such as from a different subfamily, by SSCT, has not succeeded. Here, using two small cyprinid fishes from different subfamilies, Chinese rare minnow (gobiocypris rarus, for brief: Gr) and zebrafish (danio rerio), we successfully obtained Gr-derived genome-edited sperm in zebrafish by an optimized SSCT procedure. The transplanted Gr SSCs supported the host gonadal development and underwent normal spermatogenesis, resulting in a reconstructed fertile testis containing Gr spermatids and zebrafish testicular somatic cells. Interestingly, the surrogate spermatozoa resembled those of host zebrafish but not donor Gr in morphology and swimming behavior. When pou5f3 and chd knockout Gr SSCs were transplanted, Gr-derived genome-edited sperm was successfully produced in zebrafish. This is the first report demonstrating surrogate production of gametes from a different subfamily by SSCT, and surrogate production of genome-edited gametes from another species as well. This method is feasible to be applied to future breeding of commercial fish and livestock.

     

Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei

Pluripotent human stem cells isolated from early embryos represent a potentially unlimited source of many different cell types for cell-based gene and tissue therapies [1-3]. Nevertheless, if the full potential of cell lines derived from donor embryos is to be realised, the problem of donor-recipient tissue matching needs to be overcome. One approach, which avoids the problem of transplant rejection, would be to establish stem cell lines from the patient's own cells through therapeutic cloning [3,4]. Recent studies have shown that it is possible to transfer the nucleus from an adult somatic cell to an unfertilised oocyte that is devoid of maternal chromosomes, and achieve embryonic development under the control of the transferred nucleus [5-7]. Stem cells isolated from such a cloned embryo would be genetically identical to the patient and pose no risk of immune rejection. Here, we report the isolation of pluripotent murine stem cells from reprogrammed adult somatic cell nuclei. Embryos were generated by direct injection of mechanically isolated cumulus cell nuclei into mature oocytes. Embryonic stem (ES) cells isolated from cumulus-cell-derived blastocysts displayed the characteristic morphology and marker expression of conventional ES cells and underwent extensive differentiation into all three embryonic germ layers (endoderm, mesoderm and ectoderm) in tumours and in chimaeric foetuses and pups. The ES cells were also shown to differentiate readily into neurons and muscle in culture. This study shows that pluripotent stem cells can be derived from nuclei of terminally differentiated adult somatic cells and offers a model system for the development of therapies that rely on autologous, human pluripotent stem cells.     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

Computer assisted image analysis to assess colonization of recipient seminiferous tubules by spermatogonial stem cells from transgenic donor mice

Transplantation of spermatogonial stem cells from fertile, transgenic donor mice to the testes of infertile recipients provides a unique system to study the biology of spermatogonial stem cells. To facilitate the investigation of treatment effects on colonization efficiency an analysis system was needed to quantify colonization of recipient mouse seminiferous tubules by donor stem cell‐derived spermatogenesis. In this study, a computer‐assisted morphometry system was developed and validated to analyze large numbers of samples. Donor spermatogenesis in recipient testes is identified by blue staining of donor‐derived spermatogenic cells expressing the E. coli lacZ structural gene. Images of seminiferous tubules from recipient testes collected three months after spermatogonial transplantation are captured, and stained seminiferous tubules containing donor‐derived spermatogenesis are selected for measurement based on their color by color thresholding. Colonization is measured as number, area, and length of stained tubules. Interactive, operator‐controlled color selection and sample preparation accounted for less than 10% variability for all collected parameters. Using this system, the relationship between number of transplanted cells and colonization efficiency was investigated. Transplantation of 10⁴ cells per testis only rarely resulted in colonization, whereas after transplantation of 10⁵ and 10⁶ cells per testis the extent of donor‐derived spermatogenesis was directly related to the number of transplanted donor cells. It appears that about 10% of transplanted spermatogonial stem cells result in colony formation in the recipient testis. The present study establishes a rapid, repeatable, semi‐interactive morphometry system to investigate treatment effects on colonization efficiency after spermatogonial transplantation in the mouse. Mol. Reprod. Dev. 53:142–148, 1999. © 1999 Wiley‐Liss, Inc.     

小鼠胚胎干细胞向生殖细胞分化的研究

小鼠胚胎干细胞(ESC)在体外可以分化为多种细胞类型,其中包括各阶段的生殖细胞,甚至精细胞和成熟卵母细胞。ESC向生殖细胞分化的效率受到包括生长因子、激素和体细胞等多种因素的影响,在体外形成的是雌性配子还是雄性配子与ESC是XX型还是XY型没有必然联系。简要综述了小鼠生殖细胞在体内外的分化发育、性别决定和增殖等,并总结和展望了ESC向生殖细胞分化研究面临的问题和应用前景。     

The importance of the explant on regeneration in thin cell layer technology

Summary The basic factor underlying the success of the tissue culture, large-scale micropropagation and genetic transformation of any plant species is regeneration. This has been achieved over the years through the use of various-sized explants ranging from protoplasts (small scale) to entire organs (large scale). Inherent problems underlie the use of either extreme, leading to both nonspecific morphogenic reactions in the latter, or to undesired necrosis in the former. This review investigates the importance of different aspects of a thin cell layer (TCL) explant, from its source to its size. TCLs, as a results of their size and origin, in combination with other controllable factors such as media and environmental conditions, have shown this system to be superior to the use of conventional explants. Numerous species that were previously unsuccessfully tissue-cultured have, with the use of TCL technology, resulted in their successful micropropagation and regeneration. These successes, based on the inherent qualities of the TCL explant—specific for a given species—are also examined.     

不同来源丹参药材酚酸类成分的分析研究

为比较不同来源的丹参(Salvia miltiorrhiza)药材的酚酸类成分,采用化学指纹图谱和定量分析的方法,对不同来源丹参药材中的酚酸类成分进行了系统分析。结果表明:产地、采收期、病害、根色、根的粗细以及药材部位等因素尽管对丹参酚酸类成分绝对含量的影响比较大,但对各成分相对含量的影响较小;不同来源丹参药材酚酸类成分指纹图谱相似性较高;8月份采收的药材,丹酚酸B含量较高;病害能够显著降低丹酚酸B的积累;与白根和褐色根相比,砖红色根中的丹酚酸B含量较高;根越粗,丹酚酸B含量也越高。这为丹参药材的品质评价和资源利用提供了依据。     

Induced pluripotent stem cells and Parkinson's disease: modelling and treatment

Many neurodegenerative disorders, such as Parkinson's disease (PD), are characterized by progressive neuronal loss in different regions of the central nervous system, contributing to brain dysfunction in the relevant patients. Stem cell therapy holds great promise for PD patients, including with foetal ventral mesencephalic cells, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Moreover, stem cells can be used to model neurodegenerative diseases in order to screen potential medication and explore their mechanisms of disease. However, related ethical issues, immunological rejection and lack of canonical grafting protocols limit common clinical use of stem cells. iPSCs, derived from reprogrammed somatic cells, provide new hope for cell replacement therapy. In this review, recent development in stem cell treatment for PD, using hiPSCs, as well as the potential value of hiPSCs in modelling for PD, have been summarized for application of iPSCs technology to clinical translation for PD treatment.     

Germline stem cell arrest inhibits the collapse of somatic proteostasis early in Caenorhabditis elegans adulthood

All cells rely on highly conserved protein folding and clearance pathways to detect and resolve protein damage and to maintain protein homeostasis (proteostasis). Because age is associated with an imbalance in proteostasis, there is a need to understand how protein folding is regulated in a multicellular organism that undergoes aging. We have observed that the ability of Caenorhabditis elegans to maintain proteostasis declines sharply following the onset of oocyte biomass production, suggesting that a restricted protein folding capacity may be linked to the onset of reproduction. To test this hypothesis, we monitored the effects of different sterile mutations on the maintenance of proteostasis in the soma of C. elegans. We found that germline stem cell (GSC) arrest rescued protein quality control, resulting in maintenance of robust proteostasis in different somatic tissues of adult animals. We further demonstrated that GSC‐dependent modulation of proteostasis requires several different signaling pathways, including hsf‐1 and daf‐16/kri‐1/tcer‐1, daf‐12, daf‐9, daf‐36, nhr‐80, and pha‐4 that differentially modulate somatic quality control functions, such that each signaling pathway affects different aspects of proteostasis and cannot functionally complement the other pathways. We propose that the effect of GSCs on the collapse of proteostasis at the transition to adulthood is due to a switch mechanism that links GSC status with maintenance of somatic proteostasis via regulation of the expression and function of different quality control machineries and cellular stress responses that progressively lead to a decline in the maintenance of proteostasis in adulthood, thereby linking reproduction to the maintenance of the soma.     

Plant regeneration from embryogenic cultures of Phragmites australis (Cav.) Trin. Ex Steud.

Embryogenic cultures of the common reed [Phragmites australis (Cav.) Trin. Ex. Steud.] were induced on Murashige and Skoog (1962)-based medium with 2% (w/v) sucrose, B5 vitamins and 4.5 μM 2,4-dichlorphenoxyacetic acid. Four independent culture lines, two initiated from stem nodes and two from roots, were established. These cultures underwent somatic embryogenesis. In one line of stem node origin, the somatic embryos germinated and developed into plants, following transfer of embryogenic cultures to Murashige and Skoog (1962)-based medium lacking growth regulators, with 108 ± 17 plants being recovered per 100 mg fresh weight of culture. In other lines, the somatic embryos developed roots, but not shoots. Shoot regeneration via somatic embryogenesis offers potential as anin vitro system for physiological studies, including assessments of the response of common reed to environmental pollutants. This revised version was published online in June 2006 with corrections to the Cover Date.