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Undergraduate Category:Engineering and Technology Degree Level: Bachelors of Science Abstract ID# 1213
Developing Plant Tissue Cultures for the Production of Pharmaceuticals
1Dept.
Camille A.
2,
Martin *,
Dr. Erin
of Chemical Engineering / Bioengineering,
INTRODUCTION
GOAL
3
Cram ,
2Chemistry
Dr. Carolyn
& Chemical Biology, and
To investigate how the production of valuable pharmaceutical compounds from
Catharanthus roseus are regulated.
Figure 1. Chemical structure of the anti-cancer compounds5
Figure 2. Catharanthus roseus
The biosynthetic pathway in C. roseus consists of approximately 30 steps and
is tightly regulated by a number of transcriptions
factors. Several studies have been conducted
to determine crucial steps within the pathway
including overexpressing enzymes in plant
tissue cultures. However, these attempts were
unsuccessful in increasing alkaloid production
in C. roseus.
Figure 3. Hairy root culture (above)4
Figure 4. TIA biosynthetic pathway (right)3
The Lee-Parsons Group is investigating the transcriptional regulation of the TIA
biosynthetic pathway utilizing transgenic hairy root cultures. Hairy roots are
produced via Agrobacterium-mediated transformation. Hairy root cultures are
useful for studying the biosynthesis of TIAs in C. roseus and is scalable,
genetically stability, and can be cultivated independently of environmental
conditions.
ACKNOWLEDGEMENTS
Noreen Rizvi, PhD, and Jessica Weaver, PhD
Figure 5. Pictured left to right are the steps for developing transgenic hairy root cultures. 1. Seed sterilization and planting.
2. Engineered plasmid is inserted in the plant genome via Agrobacterium-mediated transformation during the infection step.
3. Root excision. 4. Agrobacterium elimination and selection. 5. Liquid media adaption followed by several rounds of
subculturing. 4
Waters. How Does High Performance Liquid Chromatography Work? Digital image. Waters.com. Waters, 2016. Web. 17 Mar. 2016. van der Heijden, R., et al. Current Medicinal Chemistry, 2004, 11, 607-­‐628 Goklany, S., et al., Biotechnology Progress, 2009, 25, 1289-­‐1296 Rizvi, N., et al., Journal of Plant Biotechnology, 2015, 120, 475-­‐487 Tikhomiroff, C. and Jolicoeur, M. Journal of Chromatography A, 2002, 955, 87-­‐93 Hairy root culture were extracted from ~50 mg of dried plant material
in methanol. Samples are analyzed via HPLC with a C18 column and
Empower software.
1 3
2
1 Figure 9. Chromatogram analyzed at 254 nm; peak 1: strictosidine, peak 2: ajmalicine, peak 3: serpentine
GENOMIC INTEGRATION
Figure 10. The UV spectra of ajmalicine (left) and serpentine (middle), strictosidine (right)
Figure 6. The results of the genomic integration analysis of 17 hairy root culture lines.4
Figure 7. Diagram of Agrobacterium rhizogenes used for gene transformation4
In order to ensure that the correct genes have been inserted into the DNA of C.
roseus, genomic integration was performed. The first step was to extract the
genomic DNA of each line. PCR was then used to amplify the following genes:
•  Rps9: Housekeeping gene specific to C. roseus, PCR control
•  Gfp:
Gene of interest, confirming integration of desired gene
•  HygR: Hygromycin resistance gene, crucial for root selection
•  VirD2: Virulence gene, indicative of Agrobacterium contamination
•  RolC: Hairy root control, indicative of root generated by Agrobacterium
The amplified genes were separated on a 2% agarose gel. A UV
transilluminator was used to view and confirm the expected size of the desired
PCR products. ALKALIOD PRODUCTION ANALYSIS
We study the alkaloid profile in the hairy roots by High Performance Liquid
Chromatography (HPLC). We can identify a number of alkaloids based on their
characteristic ultraviolet (UV) absorbance spectra and retention times.
Northeastern Departments of Chemical Engineering, Bioengineering,
Chemistry & Chemical Biology, and Biology
1. 
2. 
3. 
4. 
5. 
RESULTS
1 National Science Foundation – CBET AWARD NUMBER 1033889
REFERENCES
3Biology
METHODS
TRANSGENIC LINE DEVELOPMENT
Catharanthus roseus, also known as Madagascar periwinkle, is a medicinal
plant that naturally produces over 130 alkaloids including the terpenoid indole
alkaloids (TIAs), vinblastine and vincristine. Vincristine and vinblastine are
chemotherapy drugs that fight a variety of cancers, including leukemia and
Hodgkin’s disease by disrupting tumor development. The natural production of
vincristine and vinblastine by C. roseus is inefficient, requiring approximately
500 kg of plant material for 1g of isolated alkaloids. In addition to low
production, the extraction of these key compounds is extremely expensive and
strenuous. Therefore other methods of producing these compounds have been
explored.
1,2
Lee-Parsons
Figure 8. Scheme of high performance liquid chromatography1
Alkaloid concentration (mg/g DW tissue)
Victoria
1,
D’Agostino *,
3.4
3.2
3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Ajmalicine
Serpentine
Strictosidine
Figure 11. Diagram of TIAs isolated from C. roseus hairy cultures
CONCLUSIONS & FUTURE DIRECTIONS
C. roseus is known to naturally produce useful alkaloids, including the
pharmaceutical compounds vincristine and vinblastine. By modifying the
metabolic pathways of the plant, tissue cultures can be produced which
overproduce these useful alkaloids at higher production rates. In the future,
tissue cultures will be developed in which the desired target gene is
engineered, and the resulting alkaloids profiles can be monitored and quantified
(as shown above). The goal of this research is to optimize the rate of
pharmaceutical production from C. roseus.