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Artemisia annua L.: Polyploidy and NIRS, two tools to improve breeding efficiency

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Abstract

Breeding a new cultivar needs 5 to 15 years according to the species and the breeding objectives from bioprospection up to cultivar registration. This is a very long time for companies developing and trading plant based products. To react more quickly to the requirements of the stakeholders, methods to accelerate the breeding procedures have to be taken into account. Among different possibilities, polyploidy induction and rapid methods to measure target traits with near infrared spectroscopy (NIRS) were tested on Artemisia annua L. Tetraploid progenies were compared to the cultivar Apollon. These tests showed no significant differences between the tetraploid plants and the cultivar Apollon for the artemisinin content, as well for the leaf and the artemisin yield. The determination of artemisinin in powder of Artemisia annua using a hand-held NIRS device showed accurate results in predicting artemininin contents. Root mean square error values of cross-validation and prediction of 0.1 % were calculated, in both cases.
6th International Symposium Breeding Research on Medicinal and Aromatic Plants, BREEDMAP 6, Quedlinburg, Germany, June 19-23, 2016
Julius-Kühn-Archiv, 453, 2016 15
ASL 2: Artemisia annua L.: Polyploidy and NIRS, two tools to improve breeding
efficiency
Mélanie Quennoz1, Xavier Simonnet1, Cedric Camps2, Christoph Carlen1,2
1 Mediplant, Conthey, Switzerland, email: christoph.carlen@agroscope.admin.ch
2 Agroscope, Institute for Plant Production Sciences, Conthey, Switzerland
DOI 10.5073/jka.2016.453.003
Abstract
Breeding a new cultivar needs 5 to 15 years according to the species and the breeding objectives
from bioprospection up to cultivar registration. This is a very long time for companies developing
and trading plant based products. To react more quickly to the requirements of the stakeholders,
methods to accelerate the breeding procedures have to be taken into account. Among different
possibilities, polyploidy induction and rapid methods to measure target traits with near infrared
spectroscopy (NIRS) were tested on Artemisia annua L.
Tetraploid progenies were compared to the cultivar Apollon. These tests showed no significant
differences between the tetraploid plants and the cultivar Apollon for the artemisinin content, as
well for the leaf and the artemisin yield.
The determination of artemisinin in powder of Artemisia annua using a hand-held NIRS device
showed accurate results in predicting artemininin contents. Root mean square error values of
cross-validation and prediction of 0.1 % were calculated, in both cases.
Keywords: Artemisinin, Artemisia annua, breeding, NIRS, polyploidy
Introduction
Artemisia annua L. is an important medicinal plant for the production of antimalarial drugs based
on artemisinin. Artemisinin, a sesquiterpene lactone endoperoxid isolated from the leaves of A.
annua, is a highly potent antimalarial compound, which is also efficient against multidrug-
resistant strains of Plasmodium falciparum (ALIN, 1997). With the support of the WHO the
artemisinin-based combination therapies (ACTs) became the first line of treatment against malaria.
Despite the research of new technologies, the extraction from A. annua leaves remains the only
source of artemisinin. Only the distribution of cultivars with a high artemisinin production poten-
tial allows making this new culture attractive and this way answering to the increasing demand for
low cost artemisinin (FERREIRA et al., 2005). The breeding work conducted by Mediplant since 1989
allowed the development of hybrids with artemisinin contents in the leaves with up to 2 %
(SIMONNET et al., 2008). To increase the breeding efficiency, induction of polyploidy and the NIRS-
method were tested on Artemisia annua.
Materials and Methods
Polyploidiy: The increase of chromosomes sets per cell can be artificially induced by applying the
molecule colchicine, which leads to a doubling of the chromosome number. To improve the basis
for breeding, such a polyploidy induction was made on in-vitro plantles and seeds of A. annua.
Different concentration (0 to 2 % cholchicin) and different durations (6 to 48 h) were used. The
surviving tetraploid plants were used as parental lines to get seeds. The tetraploid plants from this
seed were compared in the field with 3 replications (RBC) and 18 plants per replication with the
cultivar Apollon.
NIRS: This rapid, low-cost method was developed to determin artemisinin content in dry powder
of A. annua leaves (Camps et al., 2011). A calibration set of 60 samples and a validation set of 40
samples of A. annua hybrids with artemisinin contents varying between 0.7 and 1.6 % were used.
The NIRS device was a handheld Phazir 1018 (Thermo Scientific, Wilmington, MA, USA).
6th International Symposium Breeding Research on Medicinal and Aromatic Plants, BREEDMAP 6, Quedlinburg, Germany, June 19-23, 2016
Results
Polyploidy: The use of colchizin at different concentration and durations allowed to get some
tetraploid A. annua plants. The crossing of these tetraploide plants was not very successful. Only
one combination of two parental lines gave seeds. The progenies of both paretal lines were tested
individually in comparision with the cultivar Apollon. These tests showed no significant differences
between the tetraploid plants and the cultivar Apollon of the artemisinin content in the leaves, as
well as of the leaf and the artemisin yield (Tab. 1).
Tab. 1 Artemisinin content in the leaves and leaf yield of 3 hybrids of Artemisia annua. Values in brackets
indicate the standard deviation (±sd).
Hybrids ds
Artemisinin content
in dried leaves
Dried leaf yield
(%) (±sd)
(g per m2) (±sd)
Cultivar Apollon
1.33 (±0.07)
4.46 (±0.03)
Tetraploide A
Tetraploide B
1.37 (±0.03)
1.50 (±0.08)
4.56 (±0.29)
4.89(±0.41)
NIRS: The development of this method is crucial in order to analyse a large number of samples
corresponding to hundreds of hybrids in a breeding program. The results obatained showed the
feasibility of artemisinin quantification by uing NIRS (Fig. 1). Root mean square error values of
cross-validation and of prediction of 0.1 % were calculated, in both cases. The accuracy of the
prediction is about 0.1 % in a fully external validation. This threshold of precision has been judged
as correct for application of the method in a breeding program.
Fig. 1 Actual values (thin layer chromatography) versus predicted values (NIRS) with the partial least squares
regressions (Camps et al., 2011).
References
ALIN, M.H., 1997. In vitro susceptibility of Tanzanian wild isolates of Plasmodium falciparum to artemisinin, chloroquine,
sulfadoxine/pyrimethamine and mefloquine. Parasitol. 114:503-506.
CAMPS, C., TOUSSIROT, M., QUENNOZ, M., SIMONNET, X., 2011. Determination of artemisinin and moisture content of Artemisia annua
L. dry powder using a hand-held near infrared spec troscopy device. J Near Infrared Spectrosc 19:191198.
FERREIRA, J.F.S., LAUGHLIN, J.C., DELABAYS N., MAGALHAES, P.M., 2005. Cultivation and genetics of Artemisia annua L. for increased
production of the antimalarial artemisinin. Plant Genetic Ressources 3:206-229.
SIMONNET, X., QUENNOZ, M., CARLEN , C., 2008. New Artemisia annua hybrids w ith high artemisinin content. Ac ta Hort 769:371-373.
16 Julius-Kühn-Archiv, 453, 2016
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