TY - JOUR
T1 - Development of an Agrobacterium-mediated transformation method for Taxus suspension cultures
AU - Wilson, Sarah A.
AU - Keen, Patricia
AU - McKee, Michelle C.
AU - Raia, Nicole
AU - Van Eck, Joyce
AU - Roberts, Susan C.
N1 - Publisher Copyright:
© 2018, The Society for In Vitro Biology.
PY - 2018/2/1
Y1 - 2018/2/1
N2 - The FDA-approved anti-cancer compound paclitaxel is currently produced commercially by Taxus plant cell suspension cultures. One major limitation to the use of plant cell culture as a production platform is the low and variable product yields. Therefore, methods to increase and stabilize paclitaxel production are necessary to ensure product security, especially as the demand for paclitaxel continues to rise. Although a stable transformation method for Taxus suspension cultures has been developed, stable transformant yields are low (around 1% of experiments) and the method does not translate to the Taxus cuspidata Siebold and Zucc. and Taxus canadensis Marshall cell lines utilized in this study. Therefore, a new method for Agrobacterium-mediated transformation of Taxus callus and suspension cultures was developed through identification of the optimal Agrobacterium strain, inclusion of an anti-necrotic cocktail (silver nitrate, cysteine, and ascorbic acid) and increased recovery time for cells after cocultivation, the time following infection with Agrobacterium tumefaciens. Application of the increased recovery time to transformation of T. cuspidata line PO93XC resulted in 200 calluses staining positive for GUS. Additionally, two transgenic lines have been maintained with stable transgene expression for over 5 yr. This method represents an improvement over existing transformation methods for Taxus cultures and can be applied for future metabolic engineering efforts.
AB - The FDA-approved anti-cancer compound paclitaxel is currently produced commercially by Taxus plant cell suspension cultures. One major limitation to the use of plant cell culture as a production platform is the low and variable product yields. Therefore, methods to increase and stabilize paclitaxel production are necessary to ensure product security, especially as the demand for paclitaxel continues to rise. Although a stable transformation method for Taxus suspension cultures has been developed, stable transformant yields are low (around 1% of experiments) and the method does not translate to the Taxus cuspidata Siebold and Zucc. and Taxus canadensis Marshall cell lines utilized in this study. Therefore, a new method for Agrobacterium-mediated transformation of Taxus callus and suspension cultures was developed through identification of the optimal Agrobacterium strain, inclusion of an anti-necrotic cocktail (silver nitrate, cysteine, and ascorbic acid) and increased recovery time for cells after cocultivation, the time following infection with Agrobacterium tumefaciens. Application of the increased recovery time to transformation of T. cuspidata line PO93XC resulted in 200 calluses staining positive for GUS. Additionally, two transgenic lines have been maintained with stable transgene expression for over 5 yr. This method represents an improvement over existing transformation methods for Taxus cultures and can be applied for future metabolic engineering efforts.
KW - Agrobacterium
KW - Stable transformation
KW - Taxus
KW - β-glucuronidase
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U2 - 10.1007/s11627-017-9876-8
DO - 10.1007/s11627-017-9876-8
M3 - Article
AN - SCOPUS:85040686184
SN - 1054-5476
VL - 54
SP - 36
EP - 44
JO - In Vitro Cellular and Developmental Biology - Plant
JF - In Vitro Cellular and Developmental Biology - Plant
IS - 1
ER -