TY - ABST

T1 - Transformation of Rhodiola rosea with rol-genes from Agrobacterium rhizogenes to enhance the production of bioactive compounds and analyses of key genes in their pathways

AU - Stofberg, Jimmi

AU - Antypa, Natalia-Meropi

AU - Müller, Renate

AU - Lütken, Henrik Vlk

PY - 2015

Y1 - 2015

N2 - Abstract Rhodiola rosea known as roseroot contains the secondary metabolites salidroside and rosavinoids. These composites are bioactive and plant products containing these compounds have been used for centuries to alleviate depression and stimulate the memory. Exploitation of the plant has led to decreasing wild populations and climate changes threaten the plant further. Hence, alternatives are needed to circumvent this development. The objective of this study is to increase the content of bioactive compounds of R. rosea in planta. Transformation with root oncogenic loci (rol) genes from Agrobacterium rhizogenes leads to development of hairy roots (HRs) at the infection site. Transformed HRs have for several plant resulted in a higher contents of secondary metabolites compared to untransformed wild types. The method comprises clonal propagation of in vitro and in vivo material followed by A. rhizogenes-mediated transformation of leaf explants to obtain HRs. It consists of two steps: a) growth of HRs in bioreactors and b) regeneration of entire plants derived from nodular tissue. Another part of the study involves gene expression analyses of key genes in the biosynthetic pathway of salidroside and the rosavinoids. This will be approached e.g. by gene expression analyses of the phenylalanine ammonia-lyase gene (PALrs1). PALrs1 is involved in the salidroside biosynthetic pathway catalysing its precursors.Leaves from R. rosea were sterilized in 70 % (v/v) ethanol for 1 min. and 1.4 % (v/v) NaOCl and 0.03 % (v/v) tween20 for 5 min. Leaf cuttings were then inoculated with A. rhizogenes strain ATCC4305 pRiA4 for more approximately 30 min. The inoculated leaf cuttings were then co-cultivated with the bacteria for 2 days in darkness, washed in MilliQ water with 10 g l-1 timentin and transferred to a 1x MS medium containing 100 mg l-1 timentin. After this explants were monitored for development of HRs. Approximately 60 % of the transformed explants developed HRs compared to 27 % for the untreated controls. Our preliminary data showed that the transformed HR formation peaked between 5 and 10 weeks after inoculation with 65 HRs. HRs from control explants peaked with 16 HRs after 3 weeks after inoculation. The putatively transformed explants also had a perpendicular increase between the 1st and 2nd weeks after inoculation. The HRs will then be isolated and subjected to various hormone treatments comprising auxins and cytokinins to stimulate further root growth and nodule formation for regeneration.

AB - Abstract Rhodiola rosea known as roseroot contains the secondary metabolites salidroside and rosavinoids. These composites are bioactive and plant products containing these compounds have been used for centuries to alleviate depression and stimulate the memory. Exploitation of the plant has led to decreasing wild populations and climate changes threaten the plant further. Hence, alternatives are needed to circumvent this development. The objective of this study is to increase the content of bioactive compounds of R. rosea in planta. Transformation with root oncogenic loci (rol) genes from Agrobacterium rhizogenes leads to development of hairy roots (HRs) at the infection site. Transformed HRs have for several plant resulted in a higher contents of secondary metabolites compared to untransformed wild types. The method comprises clonal propagation of in vitro and in vivo material followed by A. rhizogenes-mediated transformation of leaf explants to obtain HRs. It consists of two steps: a) growth of HRs in bioreactors and b) regeneration of entire plants derived from nodular tissue. Another part of the study involves gene expression analyses of key genes in the biosynthetic pathway of salidroside and the rosavinoids. This will be approached e.g. by gene expression analyses of the phenylalanine ammonia-lyase gene (PALrs1). PALrs1 is involved in the salidroside biosynthetic pathway catalysing its precursors.Leaves from R. rosea were sterilized in 70 % (v/v) ethanol for 1 min. and 1.4 % (v/v) NaOCl and 0.03 % (v/v) tween20 for 5 min. Leaf cuttings were then inoculated with A. rhizogenes strain ATCC4305 pRiA4 for more approximately 30 min. The inoculated leaf cuttings were then co-cultivated with the bacteria for 2 days in darkness, washed in MilliQ water with 10 g l-1 timentin and transferred to a 1x MS medium containing 100 mg l-1 timentin. After this explants were monitored for development of HRs. Approximately 60 % of the transformed explants developed HRs compared to 27 % for the untreated controls. Our preliminary data showed that the transformed HR formation peaked between 5 and 10 weeks after inoculation with 65 HRs. HRs from control explants peaked with 16 HRs after 3 weeks after inoculation. The putatively transformed explants also had a perpendicular increase between the 1st and 2nd weeks after inoculation. The HRs will then be isolated and subjected to various hormone treatments comprising auxins and cytokinins to stimulate further root growth and nodule formation for regeneration.

M3 - Conference abstract for conference

Y2 - 16 March 2015 through 19 March 2015

ER -