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NAA at a high concentration promotes efficient plant regeneration via direct somatic embryogenesis and SE-mediated transformation system in Ranunculus sceleratus.

ABSTRACT: The novel methods for efficient plant regeneration via direct somatic embryogenesis (SE) and SE-mediated transformation system under high concentration of NAA in Ranunculus sceleratus were established. On MS media containing a high concentration of NAA (10.0?mg/L) in the dark, all inoculated explants (root, stem and leaf) formed somatic embryos at high frequencies, respectively, 66.03, 126.47 and 213.63 embryoids per explant, and 100% of the embryoids developed into plantlets on 1/2 MS rooting media. Morphological and histological analyses revealed that SE in R. sceleratus followed a classical pattern. All inoculated explants can be used as receptors for genetic transformation in R. sceleratus, through direct SE-mediated method after Agrobacterium infection. RcLEC1-B, as a marker gene, changed the number and morphology of flower organs and the development of cuticle in R. sceleratus, which indicated that the efficient transgenic system of R. sceleratus was established. To our knowledge, this is the first observation that both direct SE and transgenic transformation system, via induction of a single plant growth regulator, have been successfully constructed in R. sceleratus.

SUBMITTER: Xu KD

PROVIDER: S-EPMC6892856 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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NAA at a high concentration promotes efficient plant regeneration via direct somatic embryogenesis and SE-mediated transformation system in Ranunculus sceleratus.

Xu Ke-Dong KD Wang Wei W Yu De-Shui DS Li Xiao-Li XL Chen Jia-Min JM Feng Bo-Jin BJ Zhao Ya-Wen YW Cheng Meng-Jia MJ Liu Xin-Xin XX Li Cheng-Wei CW

Scientific reports 20191204 1

The novel methods for efficient plant regeneration via direct somatic embryogenesis (SE) and SE-mediated transformation system under high concentration of NAA in Ranunculus sceleratus were established. On MS media containing a high concentration of NAA (10.0 mg/L) in the dark, all inoculated explants (root, stem and leaf) formed somatic embryos at high frequencies, respectively, 66.03, 126.47 and 213.63 embryoids per explant, and 100% of the embryoids developed into plantlets on 1/2 MS rooting m ...[more]

PMID: 31797961

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Project description:Camellia oleifera Abel. is an important woody oil species; however, the shortage of rapid and industrialized seedling culture is a large constraint on the development of the tea oil industry. Somatic embryogenesis (SE) is one of the main powerful biotechnological tools for plant mass regeneration, but the largely unknown SE in C. oleifera limits the scale production of clonal plants. In this study, we described a high-efficiency SE system via direct and indirect pathways in C. oleifera and investigated the effect of genotype, explant age and phytohormones on SE. In the direct pathway, somatic embryos were highly induced from immature seeds 220 days after full blossom, and the development of embryoids was achieved with a combination of 0.19 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg/L thidiazuron (TDZ). In the indirect pathway, embryogenic calli were induced from the same explants in medium containing 1.5 mg/L 2,4-D, while 0.75 mg/L 2,4-D treatment led to high proliferation rates for embryogenic calli. The addition of 0.19 mg/L 2,4-D alone stimulated the production of globular embryos while causing a 75% loss of the induction rate in the heart embryo stage. Upon transfer of the globular embryos to phytohormone-free medium, an optimal induction rate of 62.37% from globular embryos to cotyledonary embryos was obtained. These data suggest that the subsequent differentiation process after the globular embryo stage in ISE is more similar to an endogenous phytohormones-driven process. Mature embryos germinated to produce intact plantlets on half-strength MS basal medium with a regeneration rate of 63.67%. Histological analysis confirmed the vascular bundle isolation of embryoids from the mother tissue. We further studied the different varieties and found that there were no significant genotype differences for SE induction efficiency in C. oleifera. Thus, we established a high-efficiency induction system for direct and indirect somatic embryogenesis (ISE) in C. oleifera and regenerated intact plantlets via SE, not organogenesis. ISE has a more complicated induction and regulatory mechanism than direct somatic embryogenesis. The improved protocol of SE would benefit mass propagation and genetic manipulation in C. oleifera.

Project description:Somatic embryogenesis (SE), which occurs naturally in many plant species, serves as a model to elucidate cellular and molecular mechanisms of embryo patterning in plants. Decoding the regulatory landscape of SE is essential for its further application. Hence, the present study was aimed at employing Weighted Gene Correlation Network Analysis (WGCNA) to construct a gene coexpression network (GCN) for Arabidopsis SE and then identifying highly correlated gene modules to uncover the hub genes associated with SE that may serve as potential molecular targets. A total of 17,059 genes were filtered from a microarray dataset comprising four stages of SE, i.e., stage I (zygotic embryos), stage II (proliferating tissues at 7 days of induction), stage III (proliferating tissues at 14 days of induction), and stage IV (mature somatic embryos). This included 1,711 transcription factors and 445 EMBRYO DEFECTIVE genes. GCN analysis identified a total of 26 gene modules with the module size ranging from 35 to 3,418 genes using a dynamic cut tree algorithm. The module-trait analysis revealed that four, four, seven, and four modules were associated with stages I, II, III, and IV, respectively. Further, we identified a total of 260 hub genes based on the degree of intramodular connectivity. Validation of the hub genes using publicly available expression datasets demonstrated that at least 78 hub genes are potentially associated with embryogenesis; of these, many genes remain functionally uncharacterized thus far. In silico promoter analysis of these genes revealed the presence of cis-acting regulatory elements, "soybean embryo factor 4 (SEF4) binding site," and "E-box" of the napA storage-protein gene of Brassica napus; this suggests that these genes may play important roles in plant embryo development. The present study successfully applied WGCNA to construct a GCN for SE in Arabidopsis and identified hub genes involved in the development of somatic embryos. These hub genes could be used as molecular targets to further elucidate the molecular mechanisms underlying SE in plants.

Dataset Information

NAA at a high concentration promotes efficient plant regeneration via direct somatic embryogenesis and SE-mediated transformation system in Ranunculus sceleratus.

Publications

NAA at a high concentration promotes efficient plant regeneration via direct somatic embryogenesis and SE-mediated transformation system in Ranunculus sceleratus.

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