Establishment of transgenic Rhazya stricta hairy roots to modulate terpenoid indole alkaloid production

Amir Akhgari, Teijo Yrjönen, Into Laakso, Heikki Vuorela, Kirsi-Marja Oksman-Caldentey, Heiko Rischer

Research output: Contribution to journalArticleScientificpeer-review

7 Citations (Scopus)

Abstract

Key message: Transgenic hairy roots of R. stricta were developed for investigation of alkaloid accumulations. The contents of five identified alkaloids, including serpentine as a new compound, increased compared to non-transformed roots .Abstract: Rhazya stricta Decne. is a rich source of pharmacologically active terpenoid indole alkaloids (TIAs). In order to study TIA production and enable metabolic engineering, we established hairy root cultures of R. stricta by co-cultivating cotyledon, hypocotyl, leaf, and shoot explants with wild-type Agrobacterium rhizogenes strain LBA 9402 and A. rhizogenes carrying the pK2WG7-gusA binary vector. Hairy roots initiated from the leaf explants 2 to 8 weeks. Transformation was confirmed by polymerase chain reaction and in case of GUS clones with GUS staining assay. Transformation efficiency was 74 and 83 % for wild-type and GUS hairy root clones, respectively. Alkaloid accumulation was monitored by HPLC, and identification was achieved by UPLC-MS analysis. The influence of light (16 h photoperiod versus total darkness) and media composition (modified Gamborg B5 medium versus Woody Plant Medium) on the production of TIAs were investigated. Compared to non-transformed roots, wild-type hairy roots accumulated significantly higher amounts of five alkaloids. GUS hairy roots contained higher amounts two of alkaloids compared to non-transformed roots. Light conditions had a marked effect on the accumulation of five alkaloids whereas the composition of media only affected the accumulation of two alkaloids. By successfully establishing R. stricta hairy root clones, the potential of transgenic hairy root systems in modulating TIA production was confirmed.
Original languageEnglish
Pages (from-to)1939-1952
JournalPlant Cell Reports
Volume34
Issue number11
DOIs
Publication statusPublished - 2015
MoE publication typeA1 Journal article-refereed

Fingerprint

Rhazya stricta
indole alkaloids
terpenoids
genetically modified organisms
alkaloids
Rhizobium rhizogenes
clones
explants
metabolic engineering
woody plants
hypocotyls
root systems
cotyledons
leaves

Keywords

  • agrobacterium rhizogenes
  • GUS reporter gene
  • medicinal plant
  • Rhazya stricta (Apocynaceae)
  • terpenoid indole alkaloid (TIA)
  • transgenic hairy roots

Cite this

Akhgari, Amir ; Yrjönen, Teijo ; Laakso, Into ; Vuorela, Heikki ; Oksman-Caldentey, Kirsi-Marja ; Rischer, Heiko. / Establishment of transgenic Rhazya stricta hairy roots to modulate terpenoid indole alkaloid production. In: Plant Cell Reports. 2015 ; Vol. 34, No. 11. pp. 1939-1952.
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abstract = "Key message: Transgenic hairy roots of R. stricta were developed for investigation of alkaloid accumulations. The contents of five identified alkaloids, including serpentine as a new compound, increased compared to non-transformed roots .Abstract: Rhazya stricta Decne. is a rich source of pharmacologically active terpenoid indole alkaloids (TIAs). In order to study TIA production and enable metabolic engineering, we established hairy root cultures of R. stricta by co-cultivating cotyledon, hypocotyl, leaf, and shoot explants with wild-type Agrobacterium rhizogenes strain LBA 9402 and A. rhizogenes carrying the pK2WG7-gusA binary vector. Hairy roots initiated from the leaf explants 2 to 8 weeks. Transformation was confirmed by polymerase chain reaction and in case of GUS clones with GUS staining assay. Transformation efficiency was 74 and 83 {\%} for wild-type and GUS hairy root clones, respectively. Alkaloid accumulation was monitored by HPLC, and identification was achieved by UPLC-MS analysis. The influence of light (16 h photoperiod versus total darkness) and media composition (modified Gamborg B5 medium versus Woody Plant Medium) on the production of TIAs were investigated. Compared to non-transformed roots, wild-type hairy roots accumulated significantly higher amounts of five alkaloids. GUS hairy roots contained higher amounts two of alkaloids compared to non-transformed roots. Light conditions had a marked effect on the accumulation of five alkaloids whereas the composition of media only affected the accumulation of two alkaloids. By successfully establishing R. stricta hairy root clones, the potential of transgenic hairy root systems in modulating TIA production was confirmed.",
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author = "Amir Akhgari and Teijo Yrj{\"o}nen and Into Laakso and Heikki Vuorela and Kirsi-Marja Oksman-Caldentey and Heiko Rischer",
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Establishment of transgenic Rhazya stricta hairy roots to modulate terpenoid indole alkaloid production. / Akhgari, Amir; Yrjönen, Teijo; Laakso, Into; Vuorela, Heikki; Oksman-Caldentey, Kirsi-Marja; Rischer, Heiko.

In: Plant Cell Reports, Vol. 34, No. 11, 2015, p. 1939-1952.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Establishment of transgenic Rhazya stricta hairy roots to modulate terpenoid indole alkaloid production

AU - Akhgari, Amir

AU - Yrjönen, Teijo

AU - Laakso, Into

AU - Vuorela, Heikki

AU - Oksman-Caldentey, Kirsi-Marja

AU - Rischer, Heiko

PY - 2015

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N2 - Key message: Transgenic hairy roots of R. stricta were developed for investigation of alkaloid accumulations. The contents of five identified alkaloids, including serpentine as a new compound, increased compared to non-transformed roots .Abstract: Rhazya stricta Decne. is a rich source of pharmacologically active terpenoid indole alkaloids (TIAs). In order to study TIA production and enable metabolic engineering, we established hairy root cultures of R. stricta by co-cultivating cotyledon, hypocotyl, leaf, and shoot explants with wild-type Agrobacterium rhizogenes strain LBA 9402 and A. rhizogenes carrying the pK2WG7-gusA binary vector. Hairy roots initiated from the leaf explants 2 to 8 weeks. Transformation was confirmed by polymerase chain reaction and in case of GUS clones with GUS staining assay. Transformation efficiency was 74 and 83 % for wild-type and GUS hairy root clones, respectively. Alkaloid accumulation was monitored by HPLC, and identification was achieved by UPLC-MS analysis. The influence of light (16 h photoperiod versus total darkness) and media composition (modified Gamborg B5 medium versus Woody Plant Medium) on the production of TIAs were investigated. Compared to non-transformed roots, wild-type hairy roots accumulated significantly higher amounts of five alkaloids. GUS hairy roots contained higher amounts two of alkaloids compared to non-transformed roots. Light conditions had a marked effect on the accumulation of five alkaloids whereas the composition of media only affected the accumulation of two alkaloids. By successfully establishing R. stricta hairy root clones, the potential of transgenic hairy root systems in modulating TIA production was confirmed.

AB - Key message: Transgenic hairy roots of R. stricta were developed for investigation of alkaloid accumulations. The contents of five identified alkaloids, including serpentine as a new compound, increased compared to non-transformed roots .Abstract: Rhazya stricta Decne. is a rich source of pharmacologically active terpenoid indole alkaloids (TIAs). In order to study TIA production and enable metabolic engineering, we established hairy root cultures of R. stricta by co-cultivating cotyledon, hypocotyl, leaf, and shoot explants with wild-type Agrobacterium rhizogenes strain LBA 9402 and A. rhizogenes carrying the pK2WG7-gusA binary vector. Hairy roots initiated from the leaf explants 2 to 8 weeks. Transformation was confirmed by polymerase chain reaction and in case of GUS clones with GUS staining assay. Transformation efficiency was 74 and 83 % for wild-type and GUS hairy root clones, respectively. Alkaloid accumulation was monitored by HPLC, and identification was achieved by UPLC-MS analysis. The influence of light (16 h photoperiod versus total darkness) and media composition (modified Gamborg B5 medium versus Woody Plant Medium) on the production of TIAs were investigated. Compared to non-transformed roots, wild-type hairy roots accumulated significantly higher amounts of five alkaloids. GUS hairy roots contained higher amounts two of alkaloids compared to non-transformed roots. Light conditions had a marked effect on the accumulation of five alkaloids whereas the composition of media only affected the accumulation of two alkaloids. By successfully establishing R. stricta hairy root clones, the potential of transgenic hairy root systems in modulating TIA production was confirmed.

KW - agrobacterium rhizogenes

KW - GUS reporter gene

KW - medicinal plant

KW - Rhazya stricta (Apocynaceae)

KW - terpenoid indole alkaloid (TIA)

KW - transgenic hairy roots

U2 - 10.1007/s00299-015-1841-6

DO - 10.1007/s00299-015-1841-6

M3 - Article

VL - 34

SP - 1939

EP - 1952

JO - Plant Cell Reports

JF - Plant Cell Reports

SN - 0721-7714

IS - 11

ER -