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Induction and flow cytometry identification of tetraploids from seed-derived explants through colchicine treatments in Catharanthus roseus (L.) G. Don.

ABSTRACT: The tetraploid plants of Catharanthus roseus (L.) G. Don was obtained by colchicine induction from seeds explants, and the ploidy of the plants was identified by flow cytometry. The optimal treatment is 0.2% colchicine solution treated for 24 hours, and the induction rate reaches up to 30%. Comparing with morphological characteristics and growth habits between tetraploids and the control, we found that tetraploids of C. roseus had larger stoma and more branches and leaves. HPLC analysis showed tetraploidization could increase the contents of terpenoid indole alkaloids in C. roseus. Thus, tetraploidization could be used to produce higher alkaloids lines for commercial use. QRT-PCR results showed that the expression of enzymes involved in terpenoid indole alkaloids biosynthesis pathway had increased in the tetraploid plants. To our knowledge, this was the first paper to explore the secondary metabolism in autotetraploid C. roseus induced by colchicine.

SUBMITTER: Xing SH

PROVIDER: S-EPMC3110335 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Induction and flow cytometry identification of tetraploids from seed-derived explants through colchicine treatments in Catharanthus roseus (L.) G. Don.

Xing Shi-Hai SH Guo Xin-Bo XB Wang Quan Q Pan Qi-Fang QF Tian Yue-Sheng YS Liu Pin P Zhao Jing-Ya JY Wang Guo-Feng GF Sun Xiao-Fen XF Tang Ke-Xuan KX

Journal of biomedicine & biotechnology 20110529

The tetraploid plants of Catharanthus roseus (L.) G. Don was obtained by colchicine induction from seeds explants, and the ploidy of the plants was identified by flow cytometry. The optimal treatment is 0.2% colchicine solution treated for 24 hours, and the induction rate reaches up to 30%. Comparing with morphological characteristics and growth habits between tetraploids and the control, we found that tetraploids of C. roseus had larger stoma and more branches and leaves. HPLC analysis showed t ...[more]

PMID: 21660143

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Project description:Available information associated with Calotropis procera posted its phytotoxic effect as bio-herbicide scarce works studied its stimulatory/nutritive effect. A pot experiment was performed to assess the validity of using Calotropis procera (C. procera) leaves extract as a bio-stimulant for the growth and quality of a medicinal plant Catharanthus roseus (C. roseus) evaluated by some physio-biochemical indices. Different types of C. procera leaves extracts (CLEs) (methanolic, cold water and autoclaved water extracts) were delivered by two different modes of application. The results revealed that application of CLEs as irrigation or foliar spraying caused a stimulation effect on C. roseus plant. Root and shoot length, dry and fresh weight were significantly improved due to CLEs applications. C. roseus bioactive molecules such as anthocyanins, phenolics, flavonoids, alkaloids, ascorbic acid, reduced glutathione and α-tocopherol were abundance increased significantly with CLEs applications. Reactive oxygen species (ROS) decreased explaining the involvement of CLEs in induction of antioxidant enzymes catalase, ascorbate peroxidase, polyphenol oxidase, guaiacol peroxidase and glutathione-S-transferase for modifying cell oxidative status witnessed by lower lipid peroxidation that kept below the untreated plants' baseline reflected the improvement of growth and quality rather than phytotoxic effect. The promotion of wholesome-promoting secondary metabolites by CLEs was closely correlated to elevated phenylalanineammonialyase activity. The comparable efficient effect induced by all treatments might be judged by the relation between C. procera phytochemicals and C. roseus metabolism (donor-receiver relation). It is concluded that application of CLEs can be a promising approach for improving the yield and quality of plants despite using polluting fertilizers. The current investigation may provide a matrix for coming studies to seek illustration of numerous plants' response to C. procera extracts.

Project description:Somatic embryogenesis is an important and wonderful biotechnological tool used to develop whole plant from a single or a group of somatic cells. The differentiated somatic cells become totipotent stem cells by drastic reprogramming of a wide range of cellular activities, leading to the acquisition of embryogenic competence. After acquiring competence, the cells pass through globular, heart, torpedo and cotyledonary stages of embryo; however, all advanced embryos do not convert into full plant, produce adventive embryos or callus instead, thus reverses the programming. This is a big limitation in propagation of many plants. Understanding and unraveling the proteins at this 'embryo to plantlet' transition stage will help to get more numbers of plants. Thus, our study was aimed at an identification of differentially abundant proteins between two important advanced stages, i.e. cotyledonary-(T1) and maturation stage (T2) of somatic embryos in Catharanthus roseus. A total of 2949 and 3030 proteins were identified in cotyledonary and maturation stage, respectively. Of these, 1129 proteins were common to both. Several proteins were found to be differentially accumulated in two different embryo stages in which over 60 proteins were most accumulated during somatic embryo maturation time. More chlorophyll accumulation was noted at this time under the influence of gibberellic acid (GA3). Proteins like Mg-protoporphyrin IX chelatase, chlorophyll a-b-binding protein, photosystem I iron-sulfur center, photosystem II Psb, photosystem II subunit P-1, P-II domain-containing protein, RuBisCO large chain, RuBisCO small chain, RuBisCO activase, RuBisCO large subunit-binding proteins were synthesized. Some of the identified proteins are linked to chlorophyll synthesis, carbohydrate metabolism and stress. The identified proteins are categorized into different groups on the basis of their cellular location, role and other metabolic processes. Biochemical attributes like protein, sugar, proline, antioxidant enzyme (APX, SOD and CAT) activities were high in T2 as compared to T1. The proteins like peroxidases, pathogenesis-related proteins, the late-embryogenesis abundant proteins, argonaute, germin and others have been discussed in C. roseus somatic embryo maturation process.

Project description:BACKGROUND:An accurate assessment of transcription 'rate' is often desired to describe the promoter activity. In plants, isolation of transcriptionally active nuclei and their subsequent use in nuclear run-on assays has been challenging and therefore limit an accurate measurement of gene transcription 'rate'. Catharanthus roseus has emerged as a model medicinal plant as it exhibits an unsurpassed spectrum of chemodiversity, producing over 130 alkaloids through the terpenoid indole alkaloid (TIA) pathway and therefore serves as a 'molecular hub' to understand gene expression profiles. RESULTS:The protocols presented here streamline, adapt and optimize the existing methods of nuclear run-on assay for use in C. roseus. Here, we fully describe all the steps to isolate transcriptionally active nuclei from C. roseus leaves and utilize them to perform nuclear run-on transcription assay. Nuclei isolated by this method transcribed at a level consistent with their response to external stimuli, as transcription rate of TDC gene was found to be higher in response to external stimuli i.e. when seedlings were subjected to UV-B light or to methyl jasmonate (MeJA). However, the relative transcript abundance measured parallel through qRT-PCR was found to be inconsistent with the synthesis rate indicating that some post transcriptional events might have a role in transcript stability in response to stimuli. CONCLUSIONS:Our study provides an optimized, efficient and inexpensive method of isolation of intact nuclei and nuclear 'run-on' transcription assay to carry out in-situ measurement of gene transcription rate in Catharanthus roseus. This would be valuable in investigating the transcriptional and post transcriptional response of other TIA pathway genes in C. roseus. Isolated nuclei may also provide a resource that could be used for performing the chip assay as well as serve as the source of nuclear proteins for in-vitro EMSA studies. Moreover, nascent nuclear run-on transcript could be further subjected to RNA-Seq for global nuclear run-on assay (GNRO-Seq) for genome wide in-situ measurement of transcription rate of plant genes.

Dataset Information

Induction and flow cytometry identification of tetraploids from seed-derived explants through colchicine treatments in Catharanthus roseus (L.) G. Don.

Publications

Induction and flow cytometry identification of tetraploids from seed-derived explants through colchicine treatments in Catharanthus roseus (L.) G. Don.

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