Alkaloids of in vitro cultures of Rhazya stricta

Dissertation

Amir Behzad Akhgari Nazarlou

Research output: ThesisDissertationMonograph

Abstract

Rhazya stricta Decne. (Apocynaceae) is a traditional medicinal plant in Middle East and South Asia. It contains a large number of terpenoid indole alkaloids (TIAs), some of which possess interesting pharmacological properties. This study was focused on biotechnological production tools of R. stricta, namely undifferentiated cell cultures, and an Agrobacterium rhizogenes-mediated transformation method to obtain hairy roots expressing heterologous genes from the early TIA pathway. As Rhazya alkaloids comprise a wide range of structures and polarities, therefore, many analytical methods were developed to investigate the alkaloid contents in in vitro cultures. Targeted and non-targeted analyses were carried out using gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy. The callus cultures accumulated lower levels of alkaloids than wild type hairy roots and adventitious roots. Surprisingly, calli derived from stems contained elevated levels of strictosidine lactam compared to other cultures. The results of transformation experiments revealed that only leaves were susceptible to Agrobacterium infection and subsequent root induction. The transformation efficiency varied from 22% to 83% depending on the gene. A total of 17 TIAs, including glycosylated alkaloids, were identified from hairy root extracts by UPLC-MS. GC-MS indicated the occurrence of 20 non-polar TIAs belonging to nine different groups. The occurrence of pleiocarpamine, fluorocarpamine, vincamine, ajmalicine, and yohimbine isomers, analysed by GCMS, and serpentine and its isomer, tetrahydrosecodinol as well as tabersonine, analysed by UPLC-MS, is reported here for the first time from R. stricta. Detailed targeted analyses by GC-MS showed that the contents of eight out of ten studied alkaloids increased as a result of elicitation by methyl jasmonate (MeJA). Multivariate analysis of NMR data showed a clear discrimination between transformed and control cultures. In conclusion, a simple and efficient gene transfer method is reported for R. stricta for the first time. New analytical methods were established which enabling comprehensive investigation of the alkaloids. These data serve as a basis for further utilization of biotechnological methods for R. stricta and its further metabolic engineering.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • University of Helsinki
Supervisors/Advisors
  • Rischer, Heiko, Supervisor
  • Yrjönen, Teijo, Supervisor, External person
  • Vuorela, Heikki, Supervisor, External person
  • Oksman-Caldentey, Kirsi-Marja, Supervisor
Award date29 May 2016
Place of PublicationEspoo
Publisher
Print ISBNs978-951-38-8299-0
Electronic ISBNs978-951-38-8300-3
Publication statusPublished - 2015
MoE publication typeG4 Doctoral dissertation (monograph)

Fingerprint

Rhazya stricta
in vitro culture
alkaloids
indole alkaloids
ultra-performance liquid chromatography
terpenoids
mass spectrometry
Rhazya
isomers
analytical methods
nuclear magnetic resonance spectroscopy
lactams
yohimbine
metabolic engineering
Rhizobium rhizogenes
South Asia
Apocynaceae
methyl jasmonate
adventitious roots
callus culture

Keywords

  • hairy roots
  • transgenic roots
  • medicinal plant
  • organ and cell culture
  • plant secondary metabolites
  • elicitation
  • chemical analysis

Cite this

Akhgari Nazarlou, A. B. (2015). Alkaloids of in vitro cultures of Rhazya stricta: Dissertation. Espoo: VTT Technical Research Centre of Finland.
Akhgari Nazarlou, Amir Behzad. / Alkaloids of in vitro cultures of Rhazya stricta : Dissertation. Espoo : VTT Technical Research Centre of Finland, 2015. 211 p.
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abstract = "Rhazya stricta Decne. (Apocynaceae) is a traditional medicinal plant in Middle East and South Asia. It contains a large number of terpenoid indole alkaloids (TIAs), some of which possess interesting pharmacological properties. This study was focused on biotechnological production tools of R. stricta, namely undifferentiated cell cultures, and an Agrobacterium rhizogenes-mediated transformation method to obtain hairy roots expressing heterologous genes from the early TIA pathway. As Rhazya alkaloids comprise a wide range of structures and polarities, therefore, many analytical methods were developed to investigate the alkaloid contents in in vitro cultures. Targeted and non-targeted analyses were carried out using gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy. The callus cultures accumulated lower levels of alkaloids than wild type hairy roots and adventitious roots. Surprisingly, calli derived from stems contained elevated levels of strictosidine lactam compared to other cultures. The results of transformation experiments revealed that only leaves were susceptible to Agrobacterium infection and subsequent root induction. The transformation efficiency varied from 22{\%} to 83{\%} depending on the gene. A total of 17 TIAs, including glycosylated alkaloids, were identified from hairy root extracts by UPLC-MS. GC-MS indicated the occurrence of 20 non-polar TIAs belonging to nine different groups. The occurrence of pleiocarpamine, fluorocarpamine, vincamine, ajmalicine, and yohimbine isomers, analysed by GCMS, and serpentine and its isomer, tetrahydrosecodinol as well as tabersonine, analysed by UPLC-MS, is reported here for the first time from R. stricta. Detailed targeted analyses by GC-MS showed that the contents of eight out of ten studied alkaloids increased as a result of elicitation by methyl jasmonate (MeJA). Multivariate analysis of NMR data showed a clear discrimination between transformed and control cultures. In conclusion, a simple and efficient gene transfer method is reported for R. stricta for the first time. New analytical methods were established which enabling comprehensive investigation of the alkaloids. These data serve as a basis for further utilization of biotechnological methods for R. stricta and its further metabolic engineering.",
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Akhgari Nazarlou, AB 2015, 'Alkaloids of in vitro cultures of Rhazya stricta: Dissertation', Doctor Degree, University of Helsinki, Espoo.

Alkaloids of in vitro cultures of Rhazya stricta : Dissertation. / Akhgari Nazarlou, Amir Behzad.

Espoo : VTT Technical Research Centre of Finland, 2015. 211 p.

Research output: ThesisDissertationMonograph

TY - THES

T1 - Alkaloids of in vitro cultures of Rhazya stricta

T2 - Dissertation

AU - Akhgari Nazarlou, Amir Behzad

N1 - BA311 190 p. + app. 18 p.

PY - 2015

Y1 - 2015

N2 - Rhazya stricta Decne. (Apocynaceae) is a traditional medicinal plant in Middle East and South Asia. It contains a large number of terpenoid indole alkaloids (TIAs), some of which possess interesting pharmacological properties. This study was focused on biotechnological production tools of R. stricta, namely undifferentiated cell cultures, and an Agrobacterium rhizogenes-mediated transformation method to obtain hairy roots expressing heterologous genes from the early TIA pathway. As Rhazya alkaloids comprise a wide range of structures and polarities, therefore, many analytical methods were developed to investigate the alkaloid contents in in vitro cultures. Targeted and non-targeted analyses were carried out using gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy. The callus cultures accumulated lower levels of alkaloids than wild type hairy roots and adventitious roots. Surprisingly, calli derived from stems contained elevated levels of strictosidine lactam compared to other cultures. The results of transformation experiments revealed that only leaves were susceptible to Agrobacterium infection and subsequent root induction. The transformation efficiency varied from 22% to 83% depending on the gene. A total of 17 TIAs, including glycosylated alkaloids, were identified from hairy root extracts by UPLC-MS. GC-MS indicated the occurrence of 20 non-polar TIAs belonging to nine different groups. The occurrence of pleiocarpamine, fluorocarpamine, vincamine, ajmalicine, and yohimbine isomers, analysed by GCMS, and serpentine and its isomer, tetrahydrosecodinol as well as tabersonine, analysed by UPLC-MS, is reported here for the first time from R. stricta. Detailed targeted analyses by GC-MS showed that the contents of eight out of ten studied alkaloids increased as a result of elicitation by methyl jasmonate (MeJA). Multivariate analysis of NMR data showed a clear discrimination between transformed and control cultures. In conclusion, a simple and efficient gene transfer method is reported for R. stricta for the first time. New analytical methods were established which enabling comprehensive investigation of the alkaloids. These data serve as a basis for further utilization of biotechnological methods for R. stricta and its further metabolic engineering.

AB - Rhazya stricta Decne. (Apocynaceae) is a traditional medicinal plant in Middle East and South Asia. It contains a large number of terpenoid indole alkaloids (TIAs), some of which possess interesting pharmacological properties. This study was focused on biotechnological production tools of R. stricta, namely undifferentiated cell cultures, and an Agrobacterium rhizogenes-mediated transformation method to obtain hairy roots expressing heterologous genes from the early TIA pathway. As Rhazya alkaloids comprise a wide range of structures and polarities, therefore, many analytical methods were developed to investigate the alkaloid contents in in vitro cultures. Targeted and non-targeted analyses were carried out using gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy. The callus cultures accumulated lower levels of alkaloids than wild type hairy roots and adventitious roots. Surprisingly, calli derived from stems contained elevated levels of strictosidine lactam compared to other cultures. The results of transformation experiments revealed that only leaves were susceptible to Agrobacterium infection and subsequent root induction. The transformation efficiency varied from 22% to 83% depending on the gene. A total of 17 TIAs, including glycosylated alkaloids, were identified from hairy root extracts by UPLC-MS. GC-MS indicated the occurrence of 20 non-polar TIAs belonging to nine different groups. The occurrence of pleiocarpamine, fluorocarpamine, vincamine, ajmalicine, and yohimbine isomers, analysed by GCMS, and serpentine and its isomer, tetrahydrosecodinol as well as tabersonine, analysed by UPLC-MS, is reported here for the first time from R. stricta. Detailed targeted analyses by GC-MS showed that the contents of eight out of ten studied alkaloids increased as a result of elicitation by methyl jasmonate (MeJA). Multivariate analysis of NMR data showed a clear discrimination between transformed and control cultures. In conclusion, a simple and efficient gene transfer method is reported for R. stricta for the first time. New analytical methods were established which enabling comprehensive investigation of the alkaloids. These data serve as a basis for further utilization of biotechnological methods for R. stricta and its further metabolic engineering.

KW - hairy roots

KW - transgenic roots

KW - medicinal plant

KW - organ and cell culture

KW - plant secondary metabolites

KW - elicitation

KW - chemical analysis

M3 - Dissertation

SN - 978-951-38-8299-0

T3 - VTT Science

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -

Akhgari Nazarlou AB. Alkaloids of in vitro cultures of Rhazya stricta: Dissertation. Espoo: VTT Technical Research Centre of Finland, 2015. 211 p.