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Akta Agrosia Vol. 13 No.1 hlm 77 - 81 Jan - Jun 2010 Nitrogen Dosage and Application Time of Cytokinin on Artemisia annua
L., a Traditional Antimalaria Herbal
Dosis Nitrogen dan Waktu Pemberian Sitokinin pada Tanaman Artemisia Usman Siswanto1, Yuli Widiyastuti2 dan Ruri Kustia1
1Department of Agroecotechnology University of Bengkulu Jln. Raya Kandang Limun Bengkulu 38371A 2Medicinal Plant and Traditional Medicine Research and Development Office (B2P2TO2T) ABSTRACT
Vegetative growth and artemisinin constituent may be stimulated by the application of nitrogen and cytokinin. This study aimed to determine the response of nitrogen fertilizer dosage and time of cytokinin applicationon growth and yield of Artemisia annua L. The research was undertaken at Kelobak Village, Kepahiang Regency,Bengkulu, from April to September 2009. The experiment used a Randomized Complete Block Design arrangedfactorially. The first factor was nitrogen fertilizer dosage, consisting of 0, 50, and 100 kg ha-1; and the second onewas time of cytokinin application, comprising 3, 6, 9 week after planting. Each treatment combination wasreplicated three times. Data were analyzed by analysis of variance and means were separated by orthogonalpolynomial. Thin Layer Chromatography (TLC) was employed to trace artemisinin content of leaf. The resultsindicated that there was an interaction between nitrogen fertilizer dosage and time of cytokinin application onplant height and stem diameter. Nitrogen fertilizer dosage influenced both fresh weight and dry weight of upperplant part. Time of cytokinin application did not affect all observed variables. Nitrogen fertilization 60.27 and62.49 kg ha-1 together with cytokinin application time 3 week after planting yielded both the highest plant height(102.23 cm) and the biggetst stem diameter (22.21 mm). Nitrogen fertilizer dosage 100 kg ha-1 resulted in heaviestfresh and dry weight of upper plant part, namely 421.23 g and 39.25 g, consecutively. Artemisinin was detectedin leaf.
Key words: artemisinin, cytokinin, plant height, stem diameter, fresh weight Peningkatan pertumbuhan vegetatif dan kandungan senyawa artemisinin dapat dipacu dengan pemberian nitrogen dan sitokinin. Penelitian ini bertujuan untuk mendapatkan dosis pupuk nitrogen dan waktu pemberiansitokinin terhadap pertumbuhan dan hasil Artemisia annua L. Penelitian dilaksanakan di Desa Kelobak, KabupatenKepahiang, Provinsi Bengkulu mulai bulan April sampai bulan September 2009. Percobaan menggunakanRancangan Acak Kelompok Lengkap yang disusun secara faktorial. Faktor pertama adalah dosis pupuk nitrogenyang terdiri dari 0, 50, dan 100 kg ha-1; dan faktor kedua adalah waktu pemberian sitokinin yang terdiri dari 3, 6,dan 9 minggu setelah tanam. Setiap kombinasi perlakuan diulang sebanyak tiga kali. Data dianalisis dengananalisis sidik ragam dan rata-rata dipisahkan dengan orthogonal polinomial. Untuk melacak keberadaan senyawaartemisinin digunakan Kromatografi Lapis Tipis (KLT). Hasil menunjukkan terdapat interaksi antara dosis pupuknitrogen dengan waktu pemberian sitokinin terhadap tinggi tanaman dan diameter batang. Pupuk nitrogenberpengaruh terhadap bobot segar dan bobot kering bagian atas tanaman. Waktu pemberian sitokinin tidakmemengaruhi semua parameter yang diamati. Pupuk nitrogen masing-masing 60,27 dan 62,49 kg ha-1 denganpemberian sitokinin tiga minggu setelah tanam berturut-turut menghasilkan tanaman tertinggi (102,23 cm) dandiameter batang terbesar (22,21 mm). Dosis pupuk nitrogen 100 kg ha-1 menghasilkan bobot segar dan bobotkering bagian atas tanaman, masing-masing 421,23 g dan 39,25 g. Artemisinin terdeteksi dalam daun tanamanArtemisia.
Kata kunci: artemisinin, sitokinin, tinggi tanaman, diameter batang, bobot segar Usman Siswanto, Y. Widiyastuti dan R. Kustia : Nitrogen dosage and application cytokinin time INTRODUCTION
antimalarial and anti-cancer activity. Completechemical (de novo) synthesis of artemisinin was Malaria is a global health issue that will achieved by several research groups (Avery et continue to spread with the increase in mosquito al., 1992). The procedures require several steps, and can start from different raw materials. A temperatures. Malaria is a major health problem comprehensive review on the chemistry, synthesis in many parts of the world, especially Africa, South and semisynthesis of artemisinin has been America and Southeast Asia. Between 300 and conducted. However, low yield, complexity and 500 million people are currently infected with high cost indicate that the isolation of artemisinin malaria, with more than two billion at risk for from the plant is the most economically feasible infection. Experts project a continued increase in method for its production at present (Zipper et these numbers. Two million people die annually from malaria, including one million children. In Artemisinin is the raw material needed to Indonesia, 56.3 million people out of 70.0 million population living in endemic areas are at high risk dihydroartemisinin, arteether, artemether and of malaria. In 2003, malaria spread out in 30 artesunate. The flowering stimulus appears to be Provinces covering 6,053 villages in which the perceived at the apical meristem, and that highest mortality rate went to under five-year flowering could be somewhat delayed by pinching children (Depkes, 2007; Gunawan 2000). Latest the apical meristems and providing nitrogen report indicated that there are 424 regencies as fertilization. Very little published work exists on malarial endemic regions. Malaria is an infectious the vegetative growth responses of A. annua to the specific macronutrients nitrogen, phosphorus predicament due to ineffective medication against resistant strain of Plasmodium parasite. Cerebral malaria, caused by Plasmodium falciparum, is compounds (Ferreira and Janick, 1995).
the most deadly form of malaria, is particularly Significant increase of total plant and leaf difficult to treat and results in high mortality. The dry matter (1–3 ton ha-1) was obtained in available drug, however, is slow acting, and Mississippi, USA, where a complete fertilizer resistance to chloroquine and other currently used mixture containing 100 kg N, 100 kg P and 100 kg antimalarial drugs is widespread and increasing.
K ha-1 was broadcast and worked uniformly Artemisinin, derived from Artemisia through the soil. Similarly in Tasmania, Australia, annua (family Asteraceae), which is used as an dry leaf yields of 6–12 ton ha-1 were obtained in antimalarial in traditional Chinese medicine, has experiments with a mixed fertilizer containing 60 been found effective as an antimalarial drug kg N, 60 kg P and 50 kg K ha-1 pre-drilled in bands 150 mm apart and about 50mm below seed and In the intact A. annua plant, artemisinin 75mm below transplants (Laughlin et al., 2002).
is synthesized from the mevalonate-terpenoid pathway in the glandular trichomes of the leaves, around the time of flowering, although in some and artemisinin levels are highest in the shoots cases this may be just before flowering, and in during the flowering stage One study investigating other cases during full flowering(Ferreira dan the distribution of artemisinin in five-week old A. Janick, 1995; Laughlin et al., 2002). Field annua plants found that the highest artemisinin production of A. annua is presently the only content was found in the leaves of the upper parts commercially viable method to produce artemisinin of the plant (Shukla et al., 1992).
because the synthesis of the complex molecule is uneconomic. Currently used selections reach the sesquiterpene lactone of the cadinane series. In peak in artemisinin before flowering and at the addition to a lactone group, artemisinin contains end of vegetative growth, allowing maximal an endoperoxide bridge, which is rarely found in biomass accumulation of artemisinin before secondary metabolites, and is responsible for the Akta Agrosia Vol. 13 No. 1 hlm 77 - 81 Jan - Jun 2010 Table 1. Summary of Fisher’s-Test at Pd”0.05 for the effect of nitrogen fertilizer dosage and cytokinin application time on plant height (cm), stem diameter (mm), number of bud, chlorophyll content, leaf fresh weight (g),root fresh weight (g), leaf dry weight (g), root dry weight (g).
Components of growth and yield Nitrogen (N) Cytokinin (T) N x TPlant height (cm) Note : *significant; nsnon significant.
opment is working in partnership with the WHOto increase artemisia cultivation, especially in Af- rica, and there is a pressing need to optimize village, Kepahiang regency, Bengkulu province, artemisinin production per cultivated area, which from April to September 2009. The experiment will help meet the world demand of artemisinin- based combination therapies (ACTs) without in- arranged factorially. The first factor was nitrogen truding into areas originally cultivated with sub- fertilizer dosage consisting of 0, 50, 100 kg ha-1; sistence crops used to sustain a minimal nutritional level in African countries afflicted by malaria.
application comprising 3, 6, 9 week after planting.
However, there is little published information on Each treatment combination was replicated three individual nutrient requirements for growth and times. Data were analyzed by analysis of variance increased artemisinin production (Ferreira et al., polynomial. Thin Layer Chromatography (TLC) Cytokinins are an important class of plant was employed to trace artemisinin content of leaf.
growth regulators, defined by their ability to pro-mote cell division in tissue culture in the presence RESULTS AND DISCUSSION
of auxins. Virtually all naturally occurring cytoki-nins identified to date are adenine species substi- tuted at N6 with an isoprenoid or aromatic side in A. annua has been a goal for many research chain. Cytokinins refer solely to the isoprenoid groups. The low yield of artemisinin within the A. cytokinin bases and their sugar conjugates. Cyto- annua plant is a serious constraint on the mass kinins affect many plant developmental processes production of artemisinin-based drugs. Artemisinin including cell division, cell differentiation, chloro- can also be chemically synthesized; however, its phyll senescence, and apical dominance (Astot et production is complicated and unfavorable due to al., 2000). Although exogenous application of cy- very poor yields and extremely high costs.
tokinins is mainly known for induction of sponta- Therefore, increasing yield in intact plants is a neous shoot formation in hairy root cultures, they more cost-effective production method compared can also impact growth and secondary metabolite to commercial synthesis. The following table is accumulation. Indeed, low cytokinin to auxin lev- summarizing the result of Fisher’s test indicating els have been shown to induce rapid disorganiza- the effect of nitrogen and cytokinin application on tion in hairy root cultures of C. intybus as well as A. annua growth. In the intact A. annua plant, decrease root growth and the ability of root cul- artemisinin is synthesized from the mevalonate- tures of C. intybus to produce secondary prod- terpenoid pathway in the glandular trichomes of ucts (Bais et al., 2001; Weather et al., 2005).
the leaves, and artemisinin levels are highest in Therefore, the objective of this study was to de- the shoots during the flowering stage. One study termine the response of nitrogen fertilizer and time investigating the distribution of artemisinin in five- of cytokinin application on growth and yield of Ar- week old A. annua plants found that the highest Usman Siswanto, Y. Widiyastuti dan R. Kustia : Nitrogen dosage and application cytokinin time artemisinin content was found in the leaves of the (ipt) gene should elevate cytokinin levels in plants upper parts of the plant (Shukla et al., 1992; Jaziri thereby yielding more shooty tissue and possibly artemisinin. Constitutive expression of the ipt genein A. annua was tested to gain a better understanding of the relationship between chlorophyll and cytokinins, and cytokinin function in growth, development, and production of artemisinin. This work suggested a direct correlation between shoot growth (chlorophyll content), cytokinins, and artemisinin (Sa et al., 2001; Pamela et al., 2006). The increase in plant growth with the addition of cytokinin canbe explained by the hormone’s ability to trigger growth factors and activate the defense pathway within the plant. Because of this, one of the Results indicated there was an interaction immediate effects of cytokinin treatment on A. between nitrogen fertilizer dosage and time of annua is an increase in shoot growth and height.
cytokinin application on plant height and stemdiameter. Nitrogen fertilizer dosage influenced CONCLUSION
both fresh weight and dry weight of upper plantpart. Time of cytokinin application did not affect Both the highest plant height (102.23 cm) and the biggest stem diameter (22.21 mm) were There is some work on the response of A. acquired by nitrogen fertilization 60.27 and 62.49 annua to nitrogen under field conditions, and kg ha-1 combined with cytokinin application 3 week phosphorus and potassium in tissue culture. This after planting. Nitrogen fertilizer at 100 kg ha-1 technique, if applied early enough, will cause plants resulted in heaviest fresh and dry weight of upper to branch out and potentially increase leaf biomass if the season is 4–5 months long (Ferreira dan consecutively. Qualitatively, artemisinin was Janick, 1992). Increasing N application did not significantly affect artemisinin content or yield. InMadagascar, a field crop with three plants m-2 that ACKNOWLEDGEMENT
received 97 kg ha-1 of N increased dry leafproduction from 2420 (control) to 4690 kg ha-1, while the concentration of artemisinin dropped from 1.11 to 0.87%. Also, a mean 4.7 ton ha-1 proceed. This research has been supported by increase (19%) in total fresh plant biomass, the Office of Research and Development for cultivated in densities varying from 27.8 to 111.1 thousand plants/ha, was reported with the addition of 67 kg ha-1 N, but the artemisinin concentrationwas not reported, and the total fresh plant biomass REFERNCES
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