Agricultural Sci. J. 42(2)(Suppl.): 249-252 (2011) . . .

42(2)(): 249-252 (2554)

cell suspension
Effects of Ammonium Nitrate on Cell Growth and Production of Phenolic Compounds in Cell
Suspension Cultures of Vitis vinifera


1

1

1

Sae-Lee, N.
1
, Kerdchoechuen, O.
1
and Laohakunjit, N.
1


Abstract
Seedling shoot and root callus of Vitis vinifera cv. Ribier (Pok Dum) growth MS medium supplemented
with 6-Benzyladenine (BA) at 0.1 mg/L, sucrose 20 g/L and agar 8 g/L, were transferred into suspension
culture. The biomass accumulation and secondary metabolites productions were observed in suspension
culture with ammonium nirate (NH
4
NO
3
) at difference concentration (500, 1,000, 5,000 and 10,000 mg/L). It
was found that the greatest of dried biomass (0.433 g/L) was from V. vinifera suspension culture treated with
NH
4
NO
3
500 mg/L for 21 days. Medium supplemented with NH
4
NO
3
5,000 mg/L resulted in the highest
accumulation of phenolic contents which at 85.90 mg/100g DW. In addition, resveratrol concentration
increased to maximum at 39.21 g/g DW in liquid medium supplemented with NH
4
NO
3
at 1,000 mg/L
Keywords: Vitis vinifera, phenolics, resveratrol, suspension culture

(Vitis vinifera cv. Ribier (Pok Dum))
MS 6-Benzyladenine (BA) 0.1 20 agar 8
MS 5 500, 1,000,
5,000 10,000 21
500 0.43
5,000 phenolics 85.90
mg/100g DW 1,000
resveratrol 39.21 g/g DW
: phenolics resveratrol
Introduction
Grape cell cultures accumulate a wild range of flavonoids in common with grapes and grape seeds,
including anthocyanins, flavan-3-ols, flavonols, and stilbenes, that are biologically active (Jang et al., 1997;
Waffo-Teguo et al., 2001; Yousef et al., 2004). Polyphenols are well-known as antioxidans, veinotonics and
they may be determinant in the interest of fruit and vegetable consumption for prevention of chronic
degenerative diseases, especially against atherosclerosis and concretization (Kris-Etherton et al., 2002;
Scalbert and Williamson, 2000). Plant cell cultures are a means to study or to produce some active metabolites
such as alkaloids, triterpenes, quinines or polyphenols (Oksman-Caldentey and Inze, 2004) Cell suspension
culture cultures represent the best system of cultivation for producing secondary metabolites because fast
growth rates can be achieved. Several biotechnological advances have been developed in tissue culture that
improve secondary metabolite production such as optimization of cultural conditions, selection of high-
producing strains of lines, precursor feeding, elicitation, metabolic engineering, transformed root cultures,

1
49 . 25 10150
1
School of Bioresources and Technology, King Mongkuts University, 83 Mu 8 Tientalay Rd., Thakam, Bangkhuntein, Bangkok 10150
6 42 2 () - 2554 . 250
among others (Sarin, 2005). However, there are very few reports of using macro element to induce phenolic
increment in vitro or medium culture, in this study we have investigated the affects of different concentration of
nitrogen on cell suspension cultures of V. vinifera for biomass accumulation and phenolic production.
Materials and Methods
Plant material
Seed was separated from Vitis vinifera cv. Ribier (Pok Dum) purchased from Talad Thai Market. Surface
cleaning of grape seed was done by soaking in 75% alcohol for 30 s, then by 10% NaOCl for 10 min and 5%
NaOCl for 15 min, followed by three sequential rinses for 5 min in sterile distilled water. The seed was
subjected into solid Murashige and skoog (1962) medium (MS) and observed until root and shoot
protuberance or emergence which was about 30 days. When shoot and root were 5-6 cm length, they were cut
into 51 mm segments, and used as explants. The explant was sterile with 75% alcohol for 30 s, then in 10%
NaOCl for 10 min and 5% NaOCl for 15 min, followed by three sequential rinses for 5 min in sterile distilled
water, similar to surface cleaning of seed. The sterile pieces of shoot and root were subjected into MS medium
for initiation to callus.
Suspension culture
MS basal medium containing 0.1 BA without agar was used for suspension culture. About 200 mg callus
tissues was transferred from solidified medium into 150 conical flasks containing 50 ml of liquid medium. The
cultures were incubated on a rotary shaker operated at 120 rpm.
Macro element of cell culture
To stimulate phenolic production from cell suspension, ammonium nitrate was tested. To investigate the effects
of ammonium nitrate, cell were cultured in MS-based medium with ammonium nitrate at difference
concentration (500, 1,000, 5,000, 10,000 mg/L). Cells were collected immediately (time 0) and at 7, 14, 21, 28
days after treatment. The harvested cells on the filter paper were used for determination of dry cell weight,
phenolic content and resveratrol content.
Parameters
Analysis plant growth; the biomass = total fresh weight/initial volume (g/L)
Analysis of chemical plant; phenolic content (measurement with UV-visible spectrophotometer), resveratrol
content (measurement with high performance liquid chromatography (HPLC))
Results and Discussion
The effect of different macro elements and its varying concentrations play an important role in the
biomass accumulation and secondary metabolite production in the present study. The growth characteristics
were examined during the 28 days of culturing using dry weights. The cell lines of V. vinifera were in lag phase
for 7-14 days. Then, they were started to grow exponentially until reaching a stationary phase within 14-21
days, then a death phase due to consumption of the nutrients and lack of oxygen (Gueven and Knorr, 2011).
Cell dried weight was enhanced with increase of initial NH
4
NO
3
from 500 and 1,000 mg/L. The highest of dried
biomass from V. vinifera suspension culture after treated with NH
4
NO
3
500 mg/L and 1,000 mg/L (0.43 and
0.42 g/L, respectively) was reached at day 21 of incubation period (Figure 1). This is due to the uptake of
ammonium and nitrate ions by the cells, essential for their growth (Gueven and Knorr, 2011). Phenolic contents
were measured according to the Folin-Ciocaltue method, and expressed in gallic acid equivalents. The
kinetics of phenolic contents production by the cell suspension culture was presented in Table 1. Phenolic
contents began to increase rapidly during the early lag growth phase, reaching a maximum value at day 14
th

(85.90 mg/100g DW) after treated with ammonium nitrate 5,000 mg/L. The amounts of phenolic contents were
. 42 2 () - 2554 251
increased by 53% in the cells suspension as compared with control cultures. HPLC determination of the
resveratrol accumulation when treated with (0, 500, 1,000, 5,000 and 10,000 mg/L) was shown in Table 2. The
contents of resveratrol increased with an increasing of NH
4
NO
3
level. The resveratrol concentration increased
to maximum at day 21
th
and then gradually decreased. The best results were achieved with 5,000 mg/L (39.21
g/g DW) at day 21
th
of the culture duration. A relatively similar graduation was previously observed for the
white mulberry (Lee et al., 2010). Nitrogen sources are important for secondary product synthesis of
compounds such as alkaloids (Zhong, 2001), anthocyanins, and shikonin from cell suspension cultures (Kim
and Chang, 1990).
Summary
In conclusion, our present study on cultures of cell suspensions of V. vinifera has shown that both the
biomass and secondary metabolites accumulation were influenced by ammonium nitrate. The highest
accumulation of biomass was recorded in the medium with NH
2
NO
3
500 mg/L and the highest production of
phenolic contents was recorded in the medium with 5,000 mg/L. The highest resveratrol production of 39.21
g/g DW in liquid medium supplemented with NH
2
NO
3
at 1,000 mg/L.
Acknowledgments
This work was supported by the Higher Education Research Promotion and National Research
University Project of Thailand, Office of the Higher Education Commission, King Mongkuts University of
Technology Thonburi and Agriculture Research and Development Agency (ARDA).
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Figure 1 Growth of V. vinifera cv Pok Dum cell suspension cultures after elicitor. The cells were treated with
ammonium nitrate (NH
4
NO
3
; C Control, N1 500 mg/L, N2 1,000 mg/L, N3 5,000 mg/L, N4 10,000
mg/L)
Table 1 Effect of ammonium nitrate level on phenolic contents (mg/g DW) of the Vitis vinifera cell suspension
cultures.
NH
4
NO
3
concentration
(mg/L)
phenolic contents (mg/g) (age of cell; day)
7 day 14 day 21 day 28 day
0 42.76 39.72 37.99 30.08
500 65.55 71.91 29.64 18.88
1,000 51.98 56.52 63.34 39.89
5,000 49.94 85.90 47.63 53.73
10,000 14.50 53.36 55.65 13.62

Table 2 Effect of ammonium nitrate level on resveratrol of the Vitis vinifera cell suspension cultures measured
in HPLC and expressed in standard (g/g DW).
NH
4
NO
3
concentration
(mg/L)
resveratrol contents (g/g) (age of cell; day)
7 day 14 day 21 day 28 day
0
<0.01 <0.01 <0.01
31.43
500
<0.01 23.38 7.27 <0.01
1,000
22.45 30.86 39.21 30.99
5,000
<0.01 <0.01 19.80 <0.01
10,000
<0.01 <0.01 <0.01 <0.01

0.10
0.20
0.30
0.40
0.50
0 7 14 21 28
D
r
y


w
e
i
g
h
t

(
g
/
L
)

Days of culture
Control N1 N2 N3 N4
252