Proceedings of International Workshop on Biotechnology in Agriculture

HYDROPONICS AS A MODEL TECHNOLOGY

FOR PRODUCING PLANT ROOT SECONDARY METABOLITES
Tran Thi Le Minh1,2, Vu Thi Dao2, Mignard Benoît2,3, Biteau Flore2,3, Fèvre Jean-Paul2,3,
Boitel-Conti Michèle4, Guckert Armand2, Bui Cach Tuyen1, Bourgaud Frédéric2,3 and Gontier Eric3,4.
1
Nong Lam University, Linh Trung ward - Thu Duc District - Ho Chi Minh City, Vietnam
2
UMR ENSAIA-INRA 1121 Agronomie et Environnement, 2 Av. Forêt de Haye, F-54500 Vandoeuvre les Nancy, France
3
Plant Advanced Technologies SAS, 24-30, rue Lionnois, Incubateur Lorrain, 54000 Nancy-France.
4
Laboratoire Androgenèse et Biotechnologie, Université de Picardie Jules Verne, Faculté des Sciences,
33 rue St Leu, 80039 Amiens, France
Correspondence to: Gontier E. (email: Eric.Gontier@ensaia.inpl-nancy.fr)

ABSTRACT Based on biotechnological approaches, and in

The wide diversity of plant secondary metabolites is
largely used for the production of various pharmaceutical
compounds and more specifically for anticancer or anti
viral drugs. Such molecules are generally extracted from
biomass of wild or domesticated plants. In vitro cell,
tissue or organ culture has also been employed as a
possible alternative to produce such industrial
compounds. As a cross point of these technologies,
hydroponics has been used to grow entire plant in non
sterile conditions and to force root secondary metabolite
exudation in order to collect these compounds from the
nutrient solution in which the plant has grown. Various
physical and chemical treatments can be applied in this
goal. In this communication we studied the efficiency of
a detergent (Tween20) and demonstrate that
Hyoscyamine and scopolamine can be obtained from
living Datura innoxia Mill. Plants.
Keywords: Datura, alkaloids, precursors, Tween20,
hydropony
INTRODUCTION
Plants have been the source of food, fragrances,
dyes, and medicine from time immemorial. In some case,
these plants are a slow growth, rare and/or endangered.
For example, taxol is an anticancer compound produced
and accumulated in the bark of Taxus species. One
150 years old tree contains only 300mg of taxol when
the tree is sacrified with bark harvest. From this point of
view production limits arise and cells and tissue cultures,
and bioreactor technologies have been developed in
order to increase the plant biomass and metabolites
content (Boitel et al., 1995). With these biotechnologies,
the natural compounds may be extracted from the plant
materials or can be recovered from the culture medium
(Gontier et al., 2002; Morard, 1995; Sreevalli, 2004; Tran
et al., 2004; Vu et al., 2006). Nevertheless, despite good
research results, only three large scale bioreactor
processes have been industrially established for the
production of plant metabolites (respectivelly shikonin,
berberine, and ginsenosides). The limits of such
technology are linked to the high costs, the lack of
metabolites in cells or tissues (Payne et al., 1991) and
also to the preservation of axenic conditions.
order to find new ways for the production of plant
secondary metabolites we considered hydroponic as
a system comparable to bioreactors. It is known that
hydroponics can lead to high biomass production and
this culture method is largely employed for legumes
and flowers growth (Boitel et al., 2000)). Thus we
wanted to determine if it is possible to cultivate
medicinal plants in hydroponics so as to make them
grow, produce and then release secondary
metabolites in the nutrient solution without losing their
viability ?
In the present work, Datura innoxia Mill. was used
as a model plant producing tropane alkaloids. Culture
conditions, over production of hyoscyamine and
scopolamine and their release into the nutrient solution
were favoured using different treatments with
precursors (phenylalanine and ornithine) and/or a
detergent (Tween20) in the different experimentations
reported below.
MATERIALS AND METHODS
Plant culture and permeabilization
D.innoxia Mill. seeds were offered by professor
Hammer (Genbank, Gatersleben, Germany). For the
hydroponic experiment, 60 seedlings sown in soil 2
months before were transfered to container 60x40cm
(depth 15cm) with 12 L of MS nutrient solution [macro-
micro-elements and iron [no sugar and vitamins
added], pH: 5,8 (Murashige and Skoog, 1962)]. The
plants were placed on a polystyrene raft (59x39cm;
2cm thickness) floating on the liquid phase. The roots
were blocked in holes (diameter: 3cm), with pieces of
polystyrene allowing the roots to stay in the nutrient
solution (Picture A and B). Air was bubbled into the
nutrient solution. Twenty four days after (Picture C),
precursors (phenylalanine and ornithine 84mg/L each)
were added to the nutrient solution. One day later,
3% of Tween20 was added (365,3mL) into the 12L of
nutrient solution. After 24h of such a permeabilization,
the nutrient solution was harvested, the roots were
rinsed (tap water) and the culture was continued on
fresh MS solution. One week later, these plants were
permeabilized again after addition of 1g/L of the

188 Nong Lam University Ho Chi Minh City, October 20-21, 2006
Proceedings of International Workshop on Biotechnology in Agriculture
precursors. This culture was maintained in hydropony
for 18 weeks. Before each new step of procedure (first
precursor addition, first Tween20 addition, first nutrient
solution harvest), three plants and 1mL of the nutrient
solution were collected for biomass, hyoscyamine and
scopolamine (H&S) assays.
Plants cultivated in soil, in greenhouse conditions
were also analysed for H&S content.
Statistical analysis
Datas were analysed using the Statbox Pro
variance procedure. Means were classified according
to the Newman-Keuls test. Data not followed by the
same letters are significantly different (P>0,05).
RESULT AND DISCUSSION
After the first 24 days of culture, plants biomass
reached 2Kg (Fig. 1, Picture C). The precursors (P1)
were added in the nutrient solution for one day. Then
after, the plants were permeabilized with Tween20 3%
(Tw1) for 24 hours. The plant roots were then washed
with tap water and the culture was recycled with fresh
nutrient solution (R1). The procedure of
permeabilisation were realized the second time one
week later (P2, Tw2, R2). The treatment of Tween20
induced a leaf fall and, correlatively, a decrease of
the plant fresh weight.

2500

2000
Fresh weight (g)

1500

1000

500

0
0 10 20 30 40 50
Days of culture

Figure 1. Plant growth curve of Datura innoxia Mill. cultivated in hydroponic system (P: addition the
precursors, Tw: 24h permeabilization with Tween20 3%, R: roots washed with tap water
and recycled with fresh nutrient solution)

7 H+S nut.sol. (mg)

H+S plant (mg)
6
H&S (mg/plant)

0
Soil Hyd control Hyd prec Hyd prec+tween

Figure 2. Mean Hyoscyamine and Scopolamine content in plant and nutrient solution (except for plant in soil)
for plants in soil and hydroponic plants (Hyd: hydropony, prec: precursors, nut.sol. nutrient solution)

Nong Lam University Ho Chi Minh City, October 20-21, 2006 189
 Proceedings of International Workshop on Biotechnology in Agriculture

Picture A. Datura innoxia Mill. plants in the hydroponic system after initiation of the culture

Picture B. Datura innoxia Mill. roots in the hydroponic system after initiation of the culture

Picture C. Hydroponic twenty four days Datura innoxia Mill. culture

The plants H&S content was analysed for 3
replicates of each treatment step (Figure 2). Plants
in soil were also analysed for comparison with
hydroponic plants. Figure 2 shows that hydroponic
plants biosynthesise more alkaloids than plants in
soil. Moreover, alkaloids can be found in the nutrient
solution of hydroponic system. The tropane alkaloids
concentration increases after precursor addition (Hyd
prec) and more largely after permeabilization.
Contrarily, in the permeabilized plant (Hyd
prec+tween), a decrease of H&S level is correlated
with alkaloid release into the nutrient solution. The
total amount of H&S is more than two time higher in
the system after precursor addition and further
permeabilization. More specifically, the H&S amount
in the nutrient solution is higher than the H&S quantity

190 Nong Lam University Ho Chi Minh City, October 20-21, 2006
Proceedings of International Workshop on Biotechnology in Agriculture
present in the 24 days old biomass. Thus, if one would
have sacrificed these 24 days old plants for extracting
tropane alkaloids, he would have obtain less
compound than the one who apply precursor and
permeabilization in order to collect H&S in the nutrient
solution. In this case, the plants are still viable. They
can grow again, synthetise new compounds and they
can be treated again in order to make them exudate
molecules in the nutrient solution.
CONCLUSION
This technology has been called “Plant Milking
Technology (PMT)” because it consists of forcing root
exudation in order to collect the liquid nutrient solution.
An obvious analogy to cow growing and milking thus
clearly appera.
With PMT, plants can grow in the hydroponic
system and an increase of the secondary metabolites
synthesis can be obtained through addition of
precursors. Hydroponic plants release their
compounds without loose of viability. This work has
lead to a continuation of this study and more
specifically on precursors biotransformation and
trapping of the secondary metabolites released in the
nutrient solution.
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