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The ectopic overexpression of developmental regulator (DR) genes has been reported to improve the transformation in recalcitrant plant species because of the promotion of cellular differentiation during cell culture processes. In other words, the external plant growth regulator (PGR) application during the tissue and cell culture process is still required in cases utilizing DR genes for plant regeneration. Here, the effect of Arabidopsis BABY BOOM (BBM) and WUSCHEL (WUS) on the differentiation of tobacco transgenic cells was examined. We found that the SRDX fusion to WUS, when co-expressed with the BBM-VP16 fusion gene, significantly influenced the induction of autonomous differentiation under PGR-free culture conditions, with similar effects in some other plant species. Furthermore, to understand the endogenous background underlying cell differentiation toward regeneration, phytohormone and RNA-seq analyses were performed using tobacco leaf explants in which transgenic cells were autonomously differentiating. The levels of active auxins, cytokinins, abscisic acid, and inactive gibberellins increased as cell differentiation proceeded toward organogenesis. Gene Ontology terms related to phytohormones and organogenesis were identified as differentially expressed genes, in addition to those related to polysaccharide and nitrate metabolism. The qRT-PCR four selected genes as DEGs supported the RNA-seq data. This differentiation induction system and the reported phytohormone and transcript profiles provide a foundation for the development of PGR-free tissue cultures of various plant species, facilitating future biotechnological breeding. [ABSTRACT FROM AUTHOR]

Modern plant biotechnology often faces the problem of obtaining a stable and powerful vector for gene overexpression. It is known that introns carry different regulatory elements whose effects on transgene expression have been poorly studied. To study the effect of an intron on transgene expression, the stilbene synthase 11 (VaSTS11) gene of grapevine Vitis amurensis Rupr. was selected and overexpressed in grapevine callus cell cultures and several plant generations of Arabidopsis thaliana as two forms, intronless VaSTS11c and intron-containing VaSTS11d. The STS genes play an important role in the biosynthesis of stilbenes, valuable plant secondary metabolites. VaSTS11d contained two exons and one intron, while VaSTS11c contained only two exons, which corresponded to the mature transcript. It has been shown that the intron-containing VaSTS11d was better expressed in several generations of transgenic A. thaliana than VaSTS11c and also exhibited a lower level of cytosine methylation. As a result, the content of stilbenes in the VaSTS11d-transgenic plants was much higher than in the VaSTS11c-transgenic plants. Similarly, the best efficiency in increasing the content of stilbenes was also observed in grapevine cell cultures overexpressing the intron-containing VaSTS11d transcript. Thus, the results indicate that an intron sequence with regulatory elements can have a strong positive effect on both transgene expression level and its biological functions in plants and plant cell cultures. [ABSTRACT FROM AUTHOR]

This review is devoted to the prospects for the use of fundamentally important approaches and methods for the correction and therapy of genodermatoses, a group of inherited skin diseases. The greatest number of methods was applicable for the group of inherited epidermolysis bullosa. Gene replacement using viral and non-viral methods of delivery to cells has been replaced by genome editing using programmable nucleases used both in vitro and in vivo. The focus is on more widely used methods applied in vitro to various cell types. The description of the methods used is classified based on the use of DNA break repair pathways: the canonical non-homologous end-reconnection pathway—cNHEJ, and directed homologous recombination—HDR. The choice of editing strategy depends on the type of mutation causing the disease, the type of mutation inheritance, and the nucleotide environment of the mutation. Animal disease models obtained by genome editing are considered. The experience of developing methods for editing the genome and their application for the treatment of genodermatoses, previously recognized as incurable, is summarized. [ABSTRACT FROM AUTHOR]

Stilbenes are plant defense compounds known to rapidly accumulate in grapevine and some other plant species in response to microbial infection and several abiotic stresses. Stilbenes have attracted considerable attention due to valuable biological effects with multi-spectrum therapeutic application. However, there is a lack of information on natural signaling pathways and transcription factors regulating stilbene biosynthesis. It has been previously shown that MYB R2R3 transcription factor genes VaMyb40 and VaMyb60 were up-regulated in cell cultures of wild-growing grapevine Vitis amurensis Rupr. in response to UV irradiation. In this study, the effects of VaMyb40 or VaMyb60 overexpression in cell cultures of V. amurensis on their capability to produce stilbenes were investigated. Overexpression of the VaMyb60 gene led to a considerable increase in the content of stilbenes in three independently transformed transgenic lines in 5.9–13.9 times, while overexpression of the VaMyb40 gene also increased the content of stilbenes, although to a lesser extent (in 3.4–4.0 times) in comparison with stilbene levels in the empty vector-transformed calli. Stilbene content and stilbene production in the VaMyb60-transgenic calli reached 18.8 mg/g of dry weight (DW) and 150.8 mg/L, respectively. Using HPLC analysis, we detected eight individual stilbenes: t-resveratrol diglucoside, t-piceid, t-resveratrol, ε-viniferin, δ-viniferin, cis-resveratrol, cis-piceid, t-piceatannol. T-resveratrol prevailed over other stilbenoid compounds (53.1–89.5% of all stilbenes) in the VaMyb-overexpressing cell cultures. Moreover, the VaMyb40- and VaMyb60-transformed calli were capable of producing anthocyanins up to 0.035 mg/g DW, while the control calli did not produce anthocyanins. These findings show that the VaMyb40 and VaMyb60 genes positively regulate the stilbene biosynthesis as strong positive transcription regulators and can be used in biotechnological applications for stilbene production or high-quality viticulture and winemaking. [ABSTRACT FROM AUTHOR]

Modern plant biotechnology often faces the problem of obtaining a stable and powerful vector for gene overexpression. It is known that introns carry different regulatory elements whose effects on transgene expression have been poorly studied. To study the effect of an intron on transgene expression, the stilbene synthase 11 (VaSTS11) gene of grapevine Vitis amurensis Rupr. was selected and overexpressed in grapevine callus cell cultures and several plant generations of Arabidopsis thaliana as two forms, intronless VaSTS11c and intron-containing VaSTS11d. The STS genes play an important role in the biosynthesis of stilbenes, valuable plant secondary metabolites. VaSTS11d contained two exons and one intron, while VaSTS11c contained only two exons, which corresponded to the mature transcript. It has been shown that the intron-containing VaSTS11d was better expressed in several generations of transgenic A. thaliana than VaSTS11c and also exhibited a lower level of cytosine methylation. As a result, the content of stilbenes in the VaSTS11d-transgenic plants was much higher than in the VaSTS11c-transgenic plants. Similarly, the best efficiency in increasing the content of stilbenes was also observed in grapevine cell cultures overexpressing the intron-containing VaSTS11d transcript. Thus, the results indicate that an intron sequence with regulatory elements can have a strong positive effect on both transgene expression level and its biological functions in plants and plant cell cultures.

Coronaviridae is a family of single-stranded RNA (ssRNA) viruses that can cause diseases with high mortality rates. SARS-CoV-1 and MERS-CoV appeared in 2002‒2003 and 2012, respectively. A novel coronavirus, SARS-CoV-2, emerged in 2019 in Wuhan (China) and has caused more than 5 million deaths in worldwide. The entry of SARS-CoV-1 into the cell is due to the interaction of the viral spike (S) protein and the cell protein, angiotensin-converting enzyme 2 (ACE2). After infection, virus assembly occurs in Golgi apparatus-derived vesicles during exocytosis. One of the possible participants in this process is LAMP1 protein. We established transgenic Vero cell lines with increased expression of human LAMP1 gene and evaluated SARS-CoV-1 and SARS-CoV-2 production. An increase in the production of both viruses in LAMP1-expressing cells when compared with Vero cells was observed, especially in the presence of trypsin during infection. From these results it can be assumed that LAMP1 promotes SARS-CoV-1 and SARS-CoV-2 production due to enhanced exocytosis. [ABSTRACT FROM AUTHOR]

Stilbenes are plant defense compounds known to rapidly accumulate in grapevine and some other plant species in response to microbial infection and several abiotic stresses. Stilbenes have attracted considerable attention due to valuable biological effects with multi-spectrum therapeutic application. However, there is a lack of information on natural signaling pathways and transcription factors regulating stilbene biosynthesis. It has been previously shown that MYB R2R3 transcription factor genes VaMyb40 and VaMyb60 were up-regulated in cell cultures of wild-growing grapevine Vitis amurensis Rupr. in response to UV irradiation. In this study, the effects of VaMyb40 or VaMyb60 overexpression in cell cultures of V. amurensis on their capability to produce stilbenes were investigated. Overexpression of the VaMyb60 gene led to a considerable increase in the content of stilbenes in three independently transformed transgenic lines in 5.9–13.9 times, while overexpression of the VaMyb40 gene also increased the content of stilbenes, although to a lesser extent (in 3.4–4.0 times) in comparison with stilbene levels in the empty vector-transformed calli. Stilbene content and stilbene production in the VaMyb60-transgenic calli reached 18.8 mg/g of dry weight (DW) and 150.8 mg/L, respectively. Using HPLC analysis, we detected eight individual stilbenes: t-resveratrol diglucoside, t-piceid, t-resveratrol, ε-viniferin, δ-viniferin, cis-resveratrol, cis-piceid, t-piceatannol. T-resveratrol prevailed over other stilbenoid compounds (53.1–89.5% of all stilbenes) in the VaMyb-overexpressing cell cultures. Moreover, the VaMyb40- and VaMyb60-transformed calli were capable of producing anthocyanins up to 0.035 mg/g DW, while the control calli did not produce anthocyanins. These findings show that the VaMyb40 and VaMyb60 genes positively regulate the stilbene biosynthesis as strong positive transcription regulators and can be used in biotechnological applications for stilbene production or high-quality viticulture and winemaking.

The ectopic overexpression of developmental regulator (DR) genes has been reported to improve the transformation in recalcitrant plant species because of the promotion of cellular differentiation during cell culture processes. In other words, the external plant growth regulator (PGR) application during the tissue and cell culture process is still required in cases utilizing DR genes for plant regeneration. Here, the effect of Arabidopsis BABY BOOM ( BBM ) and WUSCHEL ( WUS ) on the differentiation of tobacco transgenic cells was examined. We found that the SRDX fusion to WUS , when co-expressed with the BBM - VP16 fusion gene, significantly influenced the induction of autonomous differentiation under PGR-free culture conditions, with similar effects in some other plant species. Furthermore, to understand the endogenous background underlying cell differentiation toward regeneration, phytohormone and RNA-seq analyses were performed using tobacco leaf explants in which transgenic cells were autonomously differentiating. The levels of active auxins, cytokinins, abscisic acid, and inactive gibberellins increased as cell differentiation proceeded toward organogenesis. Gene Ontology terms related to phytohormones and organogenesis were identified as differentially expressed genes, in addition to those related to polysaccharide and nitrate metabolism. The qRT-PCR four selected genes as DEGs supported the RNA-seq data. This differentiation induction system and the reported phytohormone and transcript profiles provide a foundation for the development of PGR-free tissue cultures of various plant species, facilitating future biotechnological breeding., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sato, Minamikawa, Pratama, Koyama, Kojima, Takebayashi, Sakakibara and Igawa.)

Stilbenes are plant phenolics known to rapidly accumulate in grapevine and other plants in response to injury or pathogen attack and to exhibit a great variety of healing beneficial effects. It has previously been shown that several calmodulin-like protein (CML) genes were highly up-regulated in cell cultures of wild-growing grapevine Vitis amurensis Rupr. in response to stilbene-modulating conditions, such as stress hormones, UV-C, and stilbene precursors. Both CML functions and stilbene biosynthesis regulation are still poorly understood. In this study, we investigated the effect of overexpression of five VaCML genes on stilbene and biomass accumulation in the transformed cell cultures of V. amurensis. We obtained 16 transgenic cell lines transformed with the VaCML52, VaCML65, VaCML86, VaCML93, and VaCML95 genes (3–4 independent lines per gene) under the control of the double CaMV 35S promoter. HPLC-MS analysis showed that overexpression of the VaCML65 led to a considerable and consistent increase in the content of stilbenes of 3.8–23.7 times in all transformed lines in comparison with the control calli, while biomass accumulation was not affected. Transformation of the V. amurensis cells with other analyzed VaCML genes did not lead to a consistent and considerable effect on stilbene biosynthesis in the cell lines. The results indicate that the VaCML65 gene is implicated in the signaling pathway regulating stilbene biosynthesis as a strong positive regulator and can be useful in viticulture and winemaking for obtaining grape cultivars with a high content of stilbenes and stress resistance.

The homeodomain transcription factor Shox2 controls the development and function of the native cardiac pacemaker, the sinoatrial node (SAN). Moreover, SHOX2 mutations have been associated with cardiac arrhythmias in humans. For detailed examination of Shox2-dependent developmental mechanisms in SAN cells, we established a murine embryonic stem cell (ESC)-based model using Shox2 as a molecular tool. Shox2+/+ and Shox2−/− ESC clones were isolated and differentiated according to five different protocols in order to evaluate the most efficient enrichment of SAN-like cells. Expression analysis of cell subtype-specific marker genes revealed most efficient enrichment after CD166-based cell sorting. Comparative cardiac expression profiles of Shox2+/+ and Shox2−/− ESCs were examined by nCounter technology. Among other genes, we identified Nppb as a novel putative Shox2 target during differentiation in ESCs. Differential expression of Nppb could be confirmed in heart tissue of Shox2−/− embryos. Taken together, we established an ESC-based cardiac differentiation model and successfully purified Shox2+/+ and Shox2−/− SAN-like cells. This now provides an excellent basis for the investigation of molecular mechanisms under physiological and pathophysiological conditions for evaluating novel therapeutic approaches.

Transgene-mediated programming is a preeminent strategy to direct cellular identity. To facilitate cell fate switching, lineage regulating genes must be efficiently and uniformly induced. However, gene expression is often heterogeneous in transgenic systems. Consistent with this notion, a non-uniform reporter gene expression was detected in our doxycycline (DOX)-regulated, murine embryonic stem (ES) cell clones. Interestingly, a significant fraction of cells within each clone failed to produce any reporter signals upon DOX treatment. We found that the majority of these non-responsive cells neither carry reporter transgene nor geneticin/G418 resistance. This observation suggested that our ES cell clones contained non-recombined cells that survived the G418 selection which was carried out during the establishment of these clones. We successfully eliminated most of these corrupted cells with repeated chemical (G418) selection, however, even after prolonged G418 treatments, a few cells remained non-responsive due to epigenetic silencing. We found that cell sorting has been the most efficient approach to select those cells which can uniformly and stably induce the integrated transgene in this ES cell based platform. Together, our data revealed that post-cloning chemical re-selection or cell sorting strongly facilitate the production of ES cell lines with a uniform transgene induction capacity.

The homeodomain transcription factor Shox2 controls the development and function of the native cardiac pacemaker, the sinoatrial node (SAN). Moreover, SHOX2 mutations have been associated with cardiac arrhythmias in humans. For detailed examination of Shox2-dependent developmental mechanisms in SAN cells, we established a murine embryonic stem cell (ESC)-based model using Shox2 as a molecular tool. Shox2 +/+ and Shox2 −/− ESC clones were isolated and differentiated according to five different protocols in order to evaluate the most efficient enrichment of SAN-like cells. Expression analysis of cell subtype-specific marker genes revealed most efficient enrichment after CD166-based cell sorting. Comparative cardiac expression profiles of Shox2 +/+ and Shox2 −/− ESCs were examined by nCounter technology. Among other genes, we identified Nppb as a novel putative Shox2 target during differentiation in ESCs. Differential expression of Nppb could be confirmed in heart tissue of Shox2 −/− embryos. Taken together, we established an ESC-based cardiac differentiation model and successfully purified Shox2 +/+ and Shox2 −/− SAN-like cells. This now provides an excellent basis for the investigation of molecular mechanisms under physiological and pathophysiological conditions for evaluating novel therapeutic approaches. [ABSTRACT FROM AUTHOR]

Background Iron deficiency related gene, Femu2 , encodes protein homologous to a C 2 H 2 -type zinc finger protein, which participates in the regulation of FOX1 gene induced by iron (Fe) deficiency in Chlamydomonas reinhardtii . In this study, we investigate the gene function of Femu2 in response to salt stress in C. reinhardtii . Methods Femu2 -overexpressing and Femu2 -silencing transgenic cells were analyzed under salt stress. Several physiological indices were measured, and global changes in gene expression were investigated via RNA-seq. Results Compared with that of the non-treated control, the transcript levels of Femu2 were dramatically induced by iron deficiency and can also be significantly induced after algal cell exposure to Tris–acetate–phosphate (TAP) medium with 100 and 150 mM NaCl. The promoter also responded to NaCl induction. Femu2 -overexpressing transgenic algal cells exhibited significantly enhanced tolerance to salt stress. Conversely, Femu2 -silencing cells showed higher sensitivity to salt stress than the control. Physiological analyses revealed that the overexpression of Femu2 increased the contents of proline and soluble sugars in transgenic cells under high salinity and that silencing Femu2 resulted in increased malondialdehyde level and decreased superoxide dismutase activity. RNA-seq results showed that a total of 248 genes have opposite expression profiles and that 5508 and 2120 genes were distinctly up-regulated or down-regulated in Femu2 -overexpressing and Femu2 -silencing transgenic cells under salt stress, respectively. Conclusion Femu2 may play an important positive role in protecting C. reinhardtii against salt stress. General Significance The results of this study indicated that Femu2 may be useful in improving plant salt tolerance. [ABSTRACT FROM AUTHOR]

Transgene-mediated programming is a preeminent strategy to direct cellular identity. To facilitate cell fate switching, lineage regulating genes must be efficiently and uniformly induced. However, gene expression is often heterogeneous in transgenic systems. Consistent with this notion, a non-uniform reporter gene expression was detected in our doxycycline (DOX)-regulated, murine embryonic stem (ES) cell clones. Interestingly, a significant fraction of cells within each clone failed to produce any reporter signals upon DOX treatment. We found that the majority of these non-responsive cells neither carry reporter transgene nor geneticin/G418 resistance. This observation suggested that our ES cell clones contained non-recombined cells that survived the G418 selection which was carried out during the establishment of these clones. We successfully eliminated most of these corrupted cells with repeated chemical (G418) selection, however, even after prolonged G418 treatments, a few cells remained non-responsive due to epigenetic silencing. We found that cell sorting has been the most efficient approach to select those cells which can uniformly and stably induce the integrated transgene in this ES cell based platform. Together, our data revealed that post-cloning chemical re-selection or cell sorting strongly facilitate the production of ES cell lines with a uniform transgene induction capacity. [ABSTRACT FROM AUTHOR]

Class I histone deacetylases (HDACs) are important regulators of cellular functions in health and disease. HDAC1, HDAC2, HDAC3, and HDAC8 are promising targets for the treatment of cancer, neurological, and immunological disorders. These enzymes have both catalytic and non-catalytic functions in the regulation of gene expression. We here describe the generation of a genetic toolbox by the CRISPR/Cas9 methodology in nearly haploid human tumor cells. This novel model system allows to discriminate between catalytic and structural functions of class I HDAC enzymes and to mimic the treatment with specific HDAC inhibitors., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Tobacco (Nicotiana tabacum) cv. 'Bright Yellow 2' (BY2) cell suspension is a useful system to study the structure and function of plant cell. However, low efficiency of Agrobacterium-mediated transformation, and transgene silencing during subculture limit its application. Here we present optimization of the traditional protocols of Agrobacterium-mediated transformation and genomic DNA extraction. The transforming efficiency and recovery ratio of genomic DNA extraction were substantially increased by these improvements. Southern assay demonstrated that copy number of transgene could be determined unambiguously. Meanwhile by monitoring the GFP fluorescence we found that the GFP expression can keep stable in suspension culture cells for at least 20 days in liquid medium. Finally, applicability of constitutive promoters of Arabidopsis thaliana UBIQUITIN10 (AtUBQIO) and ARABIDOPSIS SKP1 HOMOLOGUEI (AtASKl) also can drive stable GFP expression in vivo of BY2 cells like CaMV 35S promoter in this plant system. [ABSTRACT FROM AUTHOR]

Under the leadership of Corresponding Member of the Russian Academy of Sciences L.I. Korochkin, the Laboratory of Neurogenetics and Developmental Genetics (Institute of Gene Biology, Russian Academy of Sciences) for many years has been conducting studies of nervous system development, neural cell differentiation, and application of gene and cell technology to cure neurodegenerative diseases. The results of the study initiated by L.I. Korochkin and continued by his scientific successors support the direction of allocation of transgenic neurotrofic factors and heat-shock proteins as neuroprotectors for cell therapy. Potential for usage of promoter of HSP70 heat-shock gene of Drosophila to create transgenic constructs for therapy has been shown. Further improvement of technology of nonvirus transfer for therapeutic genes, as well as production of multicomponent genetic constructs coding several therapeutic factors with synergy effect, would stimulate creation of efficient cell medicals to cure neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

This review summarizes progress on the genetic transformation of millets and discusses the future prospects for the development of improved varieties. Only a limited number of studies have been carried out on genetic improvement of millets despite their nutritional importance in supplying minerals, calories and protein. Most genetic transformation studies of millets have been restricted to pearl millet and bahiagrass and most studies have been limited to the assessment of reporter and marker gene expression. Biolistic-mediated gene delivery has been frequently used for the transformation of millets but Agrobacterium-mediated transformation is still lagging. Improved transformation of millets, allied to relevant gene targets which may offer, for example, improved nutritional quality, resistance to abiotic and biotic stresses, and resistance to fungal infection will play important roles in millet improvement. [ABSTRACT FROM AUTHOR]

Stilbenes are plant phenolics known to rapidly accumulate in grapevine and other plants in response to injury or pathogen attack and to exhibit a great variety of healing beneficial effects. It has previously been shown that several calmodulin-like protein (CML) genes were highly up-regulated in cell cultures of wild-growing grapevine Vitis amurensis Rupr. in response to stilbene-modulating conditions, such as stress hormones, UV-C, and stilbene precursors. Both CML functions and stilbene biosynthesis regulation are still poorly understood. In this study, we investigated the effect of overexpression of five VaCML genes on stilbene and biomass accumulation in the transformed cell cultures of V. amurensis. We obtained 16 transgenic cell lines transformed with the VaCML52, VaCML65, VaCML86, VaCML93, and VaCML95 genes (3–4 independent lines per gene) under the control of the double CaMV 35S promoter. HPLC-MS analysis showed that overexpression of the VaCML65 led to a considerable and consistent increase in the content of stilbenes of 3.8–23.7 times in all transformed lines in comparison with the control calli, while biomass accumulation was not affected. Transformation of the V. amurensis cells with other analyzed VaCML genes did not lead to a consistent and considerable effect on stilbene biosynthesis in the cell lines. The results indicate that the VaCML65 gene is implicated in the signaling pathway regulating stilbene biosynthesis as a strong positive regulator and can be useful in viticulture and winemaking for obtaining grape cultivars with a high content of stilbenes and stress resistance. [ABSTRACT FROM AUTHOR]

Cochlear implants (CIs) can restore hearing in deaf patients by electrical stimulation of the auditory nerve. To optimize the electrical stimulation, the number of independent channels must be increased by reduction of connective tissue growth on the electrode surface and selective neuronal cell contact. The femtosecond laser microstructuring of the electrode surfaces was performed to investigate the effect of fibroblast growth on the implant material. A cell culture model system was established to evaluate cell–material interactions on these microstructured CI‐electrode materials. Fibroblasts were used as a cell culture model for connective tissue formation, and differentiating neuronal‐like cells were employed to represent neuronal cells. For nondestructive microscopic examination of living cells on the structured surfaces, the cells were genetically modified to express green fluorescent protein. To investigate the special interaction between the electrode material and the tissue we used electrode material which is originally used for manufacturing CI for human applications, namely platinum (contact material) and silicone carrier material (LSR 30, HCRP 50). Microstructures of various dimensions (groove width 1–10 μm) were generated by using femtosecond laser ablation. The highest fibroblast growth rate was observed on platinum, but cell growth rates on the silicone carrier material were lower. Microstructuring reduced fibroblast cell growth on platinum significantly. On the microstructured silicone, a trend to lower cell growth rates was observed. In addition, microgrooves on platinum surfaces can direct neurite outgrowth parallel to the grooves. The implications of the results are discussed with respect to the design of a microstructured CI surface. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 2008 [ABSTRACT FROM AUTHOR]

Transgenic cell lines of Papaver somniferum have been obtained via Agrobacterium tumefaciens. Papaver somniferum is known to be genetically unstable in in vitro culture conditions. Cryopreservation at ultra-low temperature is an appropriate strategy for long-term preservation of germplasm. We have studied the effects of osmotic stress due to cryoprotectants during pretreatment and of storage at –196 °C on the stability and the expression of the foreign sam1 gene from Arabidopsis thaliana. We established that the integrity, transcription of the transgene and enzymatic activity of its product were not affected by cryopreservation in liquid nitrogen [ABSTRACT FROM AUTHOR]

Expression of α-amylase genes during seedling development plays a key role in production of sugar from the starch stored in the cereal seed. Rice α-amylase Amy3D promoter/GUS constructs in transgenic rice cell lines were studied to identify cis elements in the promoter of this metabolite-regulated gene. Three sequences having the greatest effects on Amy3D gene expression included the amylase element (TATCCAT), the CGACG element, and a G box-related element (CTACGTGGCCA). These promoter cis elements are needed for high-level expression of Amy3D under conditions of sugar starvation. The involvement of G box cis-elements in environmental stress responses suggest a link between the nutrient stress and the environmental stress responses of the plant. [ABSTRACT FROM AUTHOR]