Jennifer S. Jehnsen The Effects of Elicitors on Artemisia annua Growth Spring 2010 1 Effects on Artemisia annua Growth with the Addition of Gibberellic Acid, Salicylic Acid, and Methyl Jasmonate in Hydroponic Systems Jennifer Jehnsen ABSTRACT Malaria is a global health issue that will continue to spread with the increase in mosquito populations caused by the rise in global temperatures. The most effective drugs currently available for malaria treatment contain the anti-malarial compound, artemisinin, derived from Artemisia annua. To potentially increase efficiency of artemisinin production in A. annua, I investigated the effects of adding gibberellic acid, salicylic acid, and methyl jasmonate in hydroponics on the growth of intact plants. The goal of the study was to determine the optimal concentrations of each elicitor that maximizes the growth of A. annua, investigate the effects of different combinations of these elicitors on growth, and to determine the optimal exposure period of each elicitor that maximizes growth. I hypothesized that the concentrations of these chemicals that increased growth in previous studies would also yield the best results in hydroponic systems. Also, I hypothesized that the combination of all of these chemicals would yield maximum growth by inducing growth through different pathways. In terms of the optimal exposure periods, I hypothesized that the rate of growth would decrease over time and stabilize at around four weeks. My results show that the treatments containing gibberellic acid demonstrated the highest dry weights, suggesting that gibberellic acid enhances A. annua growth. In addition, the combination treatment with all three elicitors yielded the lowest dry weights. The data shows that the optimal exposure period for all elicitors and combinations was between 8 weeks and higher, except for individual salicylic acid treatments that showed the greatest growth between weeks 4 and 6. Future studies can expand on this study by investigating the relationship between A. annua growth and the addition of gibberellic acid. KEYWORDS medicinal plants, chemical elicitors, malaria, artemisinin, plant growthJennifer S. Jehnsen The Effects of Elicitors on Artemisia annua Growth Spring 2010 2 INTRODUCTION Between 300 and 500 million people are currently infected with malaria, with more than two billion at risk for infection. Experts project a continued increase in these numbers. Two million people die annually from malaria, including one million children (Davies et al. 2009). The multidrug-resistant malaria parasite, Plasmodium falciparum, is found in many tropical countries and is particularly difficult to treat. The spread of malaria is dependent on the propagation of mosquitoes that transmit the disease by carrying the Plasmodium parasites. With the estimated global temperature increases (Houghton 2005, Wagner et al. 2009), mosquito populations will increase dramatically because warmer temperatures will allow them to migrate and propagate in new areas further north (Robert et al. 2002). In addition, cycles of heat and drought followed by heavy rains will occur more frequently, which will create more bodies of water that mosquitoes can use to reproduce (Weathers et al. 1994). The increase in vectors of transmission for malaria will create devastating health crises in tropical countries, specifically in Latin America and Sub-Saharan Africa (Robert et al. 2002). Global temperature projections suggest increases in both malaria transmission and distribution, stimulating investigations on methods to decrease costs and increase efficacy of malaria treatment. Malaria treatment generally includes the use of the anti-malarial compound, artemisinin, derived from the plant Artemisia annua L. (Asteraceae), also called qinghao or sweet wormwood (Ferreira 2007, Greenwood et al. 2008). The World Health Organization currently recommends the use of artemisinin-based combination therapies (ACTs) in tropical regions where the malarial parasite has developed resistance to traditional anti-malarial drugs (Mutabingwa 2005). ACTs are the best drugs currently available for malaria treatment (Mutabingwa 2005), and drugs that contain artemisinin derivatives have almost 100% cure rates with usually only a three-day regimen (Congpuong et al. 2010). In addition to killing the Plasmodium parasite, artemisinin derivatives lower the rate at which resistance develops by reducing the survival of parasitic gametocytes. Reducing the number of viable Plasmodium gametocytes leads to fewer infectious germ cells, leading to decreased infectivity of mosquitoes and reduced parasite reproduction in the mosquito (Adjuik et al. 2004). Artemisinin derivatives kill all stages of the malaria parasite by interacting with heme in the blood and damaging the parasitic cells’ microorganelles and membranes (Balint 2001). Some examples of artemisinin-derived molecules are artesunate, arteether, artemether, and dihydro-artemisinin (DHA) (Baldi 2007). There are currently noJennifer S. Jehnsen The Effects of Elicitors on Artemisia annua Growth Spring 2010 3 known cases of parasitic resistance to artemisinin-based compounds, and resistance has not yet been successfully induced in a laboratory setting (Mutabingwa 2005). Artemisinin compounds have a particularly rapid clearance time (the rate at which a substance is removed from the blood), making them suitable for treating early cases of malaria to avoid progressions toward severe cases that lead to mortality in developing countries (Mutabingwa 2005). Therefore, increasing efficiency in artemisinin production to reduce costs of artemisinin-based medications is an important step in addressing the global spread of malaria. Increased supplies and reduced costs will facilitate increased use of ACTs and reduce rates of infection and mortality as well as infectivity of the vectors of transmission (Adjuik et al. 2004). Enhancing the production of artemisinin in A. annua has been a goal for many research groups. The low yield of artemisinin within the A. annua plant is a serious constraint on the mass production of artemisinin-based drugs (Abdin et al. 2003, Weathers 1994). The average concentration of artemisinin in A. annua is 0.01-0.8% of the plant mass, which is very low compared to other compounds of this type (Van Agtmael et al. 1999). In addition to extraction from plant tissues, artemisinin
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