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Yuri Yu. Gleba – One of the best experts on this subject based on the ideXlab platform.

  • Spontaneous extensive chromosome elimination in somatic hybrids between somatically congruent species Nicotiana tabacum L. and Atropa belladonna L.
    TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik, 1992
    Co-Authors: Elena Babiychuk, S Kushnir, Yuri Yu. Gleba

    Abstract:

    Mesophyll protoplasts of the kanamycin-resistant nightshade, Atropa belladonna, were fused with mesophyll protoplasts of the phosphinothricin resistant-tobacco, Nicotiana tabacum. A total of 447 colonies resistant to both inhibitors was selected. Most of them regenerated shoots with morphology similar to one of the earlier obtained and described symmetric somatic hybrids Nicotiana + Atropa. However, three colonies (0.2%) regenerated vigorously growing tobacco-like shoots; they readily rooted, and after transfer to soil, developed into normal, fertile plants. Unlike their tobacco parental line, BarD, the obtained plants are resistant to kanamycin [they root normally in the presence of kanamycin (200 mg/1)] and possess activity of neomycin phosphotransferase (NPT II) with the same electrophoretic mobility as the one of the nightshade line. According to Southern blot hybridization analysis carried out with the use of radioactively labeled cloned fragments of the Citrus lemon ribosomal DNA repeat, as well as with Nicotiana plumbaginifolia genus-specific, interspersed repeat Inp, the kanamycin-resistant plants under investigation have only species-specific hybridizing bands from tobacco. Cytological analysis of the chromosome sets shows that plants of all three lines possess 48 large chromosomes similar to Nicotiana tabacum ones (2n = 48), and one small extra chromosome (chromosome fragment) similar to Atropa belladonna ones (2n = 72). Available data allow the conclusion that highly asymmetric, normal fertile somatic hybrids with a whole diploid Nicotiana tabacum genome and only part (not more than 2.8%) of an Atropa belladonna genome have been obtained without any pretreatment of a donor genome, although both these species are somatically congruent.

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  • Nucleo-cytoplasmic incompatibility in cybrid plants possessing an Atropa genome and a Nicotiana plastome.
    Molecular Genetics and Genomics, 1991
    Co-Authors: Sergei Kushnir, Elena Babiychuk, M. A. Bannikova, V. P. Momot, I. K. Komarnitsky, N. N. Cherep, Yuri Yu. Gleba

    Abstract:

    Twenty-nine cybrids possessing an Atropa belladonna nuclear genome and a Nicotiana tabacum plastome were selected from two independent protoplast fusion experiments. In contrast to the previously described reciprocal, green and fertile cybrids with a Nicotiana nuclear genome and an Atropa plastome (Kushnir et al. 1987), the plants obtained were totally chlorophyll-deficient. An Atropa nuclear genome and a Nicotiana plastome from these chlorophyll-deficient cybrids were combined with an Atropa or a Scopolia plastome and a Nicotiana nuclear genome, respectively, in control fusion experiments. All of these nuclear genome/plastome combinations gave rise to normal, green plants. Therefore, we conclude that an N. tabacum plastome is incompatible with an A. belladonna nuclear genome.

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Zhihua Liao – One of the best experts on this subject based on the ideXlab platform.

  • Expression pattern of genes involved in tropane alkaloids biosynthesis and tropane alkaloids accumulation in Atropa belladonna
    China journal of Chinese materia medica, 2014
    Co-Authors: Wei Qiang, Ya-xiong Wang, Qiao-zhuo Zhang, Ke Xia, Zhihua Liao

    Abstract:

    Atropa belladonna is a medicinal plant and main commercial source of tropane alkaloids (TAs) including scopolamine and hyoscyamine, which are anticholine drugs widely used clinically. Based on the high throughput transcriptome sequencing results, the digital expression patterns of UniGenes representing 9 structural genes (ODC, ADC, AIH, CPA, SPDS, PMT, CYP80F1, H6H, TRII) involved in TAs biosynthesis were constructed, and simultaneously expression analysis of 4 released genes in NCBI (PMT, CYP80F1, H6H, TRII) for verification was performed using qPCR, as well as the TAs contents detection in 8 different tissues. Digital expression patterns results suggested that the 4 genes including ODC, ADC, AIH and CPA involved in the upstream pathway of TAs, and the 2 branch pathway genes including SPDS and TRII were found to be expressed in all the detected tissues with high expression level in secondary root. While the 3 TAs-pathway-specific genes including PMT, CYP80F1, H6H were only expressed in secondary roots and primary roots, mainly in secondary roots. The qPCR detection results of PMT, CYP80F1 and H6H were consistent with the digital expression patterns, but their expression levels in primary root were too low to be detected. The highest content of hyoscyamine was found in tender stems (3.364 mg x g(-1)), followed by tender leaves (1.526 mg x g(-1)), roots (1.598 mg x g(-1)), young fruits (1.271 mg x g(-1)) and fruit sepals (1.413 mg x g(-1)). The highest content of scopolamine was detected in fruit sepals (1.003 mg x g(-1)), then followed by tender stems (0.600 mg x g(-1)) and tender leaves (0.601 mg x g(-1)). Both old stems and old leaves had the lowest content of hyoscyamine and scopolamine. The gene expression profile and TAs accumulation indicated that TAs in Atropa belladonna were mainly biosynthesized in secondary root, and then transported and deposited in tender aerial parts. Screening Atropa belladonna secondary root transcriptome database will facilitate unveiling the unknown enzymatic reactions and the mechanisms of transcriptional control.

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  • Enhancing the scopolamine production in transgenic plants of Atropa belladonna by overexpressing pmt and h6h genes
    Physiologia Plantarum, 2011
    Co-Authors: Xirong Wang, Min Chen, Chunxian Yang, Xiaoqiang Liu, Lei Zhang, Xiaozhong Lan, Kexuan Tang, Zhihua Liao

    Abstract:

    Atropa belladonna is officially deemed as the commercial plant to produce scopolamine in China. In this study we report the simultaneous overexpression of two functional genes involved in biosynthesis of scopolamine, which encode the upstream key enzyme putrescine N-methyltransferase (PMT) and the downstream key enzyme hyoscyamine 6β-hydroxylase (H6H), respectively, in transgenic herbal plants Atropa belladonna. Analysis of gene expression profile indicated that both pmt and h6h were expressed at a higher level in transgenic lines, which would be favorable for biosynthesis of scopolamine. High-performance liquid chromatography result suggested that transgenic lines could produce higher accumulation of scopolamine at different levels compared with wild-type lines. Scopolamine content increased to 7.3-fold in transgenic line D9 compared with control lines. This study not only confirms that co-overexpression of pmt and h6h is an ideal method to improve the biosynthetic capacity of scopolamine but also successfully cultivates the transgenic line D9, which significantly enhanced the scopolamine accumulation. Our research can serve as an alternative choice to provide scopolamine resources for relative industry, which is more competitive than conventional market.

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  • Promoting scopolamine accumulation in transgenic plants of Atropa belladonna generated from hairy roots with over expression of pmt and h6h gene.
    Journal of Medicinal Plants Research, 2010
    Co-Authors: Xiaoqiang Liu, Zhihua Liao, Min Chen, Chunxian Yang, Kexuan Tang

    Abstract:

    Atropa belladonna is the most important commercial source for obtaining pharmaceutical tropane alkaloids such as scopolamine and hyoscyamine. In the present study, two rate-limiting enzyme genes including putrescine N-methyltransferase and hyoscyamine 6β-hydroxylase were introduced into A. belladonna, and integration of the pmt and h6h genes into the genomic DNA of transgenic plants were confirmed by genomic polymerase chain reaction (PCR) analysis. The scopolamine content of transgenic lines was increased to 1.3 – 2.5 folds than that in wild type, which was caused by over expression of the pmt andh6h genes in the transgenic plant lines of A. belladonna. The current study provides a more effective approach for commercially large-scale production of scopolamine by cultivating A. belladonna plants in large fields but not using the hairy root systems as bioreactors.

     

    Key words: Atropa belladonna, pmt, h6h, scopolamine, transformation, regeneration.

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Koichiro Shimomura – One of the best experts on this subject based on the ideXlab platform.

  • Genetic Transformation of Atropa belladonna
    Transgenic Medicinal Plants, 1999
    Co-Authors: Mondher El Jaziri, Kayo Yoshimatsu, Koichiro Shimomura

    Abstract:

    Leaves of Atropa belladonna L. (Solanaceae) are mainly used as a source for the tropane alkaloids, hyoscyamine and scopolamine, which are not easily produced by chemical synthesis. Thus because of its high morphogenetic potential, the in vitro culture of A. belladonna has been actively investigated as an alternative to field cultivation and as a possible source for tropane alkaloid production (for review see Bajaj and Simola 1991) Several studies have confirmed that the product levels in unorganized A. belladonna callus and suspension cultures are generally very low and that root morphology is required for synthesis of tropane alkaloid in vitro. However, molecular genetic technology is now available to achieve transformation and regeneration of transgenic A. belladonna plants in which morphological traits and/or particular metabolic step(s) are artificially modified. The hairy root system is demonstrated to be much more genetically stable than dedifferentiated normal (callus) or transformed (crown gall) plant cells. In this chapter some important recent studies related to the genetic transformation of A. belladonna are discussed.

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  • Traits of transgenic Atropa belladonna doubly transformed with different Agrobacterium rhizogenes strains
    Plant Cell Tissue and Organ Culture, 1994
    Co-Authors: Mondher El Jaziri, Kayo Yoshimatsu, Jacques Homes, Koichiro Shimomura

    Abstract:

    Hairy root cultures of Atropa belladonna L. were established by infection either with Agrobacterium rhizogenes ATCC 15834 or MAFF 03-01724, and transgenic plants were obtained from both hairy root cultures. Doubly transformed roots were induced by re-infection of the leaf segments of transgenic Atropa belladonna plants (A. rhizogenes 15834) with MAFF 03-01724. Shoots and viviparous leaves were regenerated from the doubly transformed roots. The genetic transformation was determined by the opine assay (agropine, mannopine and/or mikimopine) and polymerase chain reaction. Physiological changes and tropane alkaloid biosynthesis in the hairy roots (singly and doubly transformed) were investigated. The alkaloid content in the doubly transformed root strain was intermediate as compared to the root strains which were singly transformed. On the other hand endogenous IAA levels in doubly transformed roots were significantly decreased compared to both singly transformed roots.

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