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Biological Control of Root-knot Nematode Meloidogyne incognita: 1-In vitro

Studies on Callus Induction from Marigold Plants Tagetes erecta L

Article · February 2008

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2 Egypt. J. Phytopathol., Vol. 36, No.1-2, pp. 19-32 (2008)

Biological Control of Root-knot Nematode

Meloidogyne incognita: 1- In vitro Studies on Callus
Induction from Marigold Plants Tagetes erecta L.
Hamida A. Osman*; A.Y. El-Gindi **; A.A. El-Kazzaz***;
H.S. Taha***; M.M.A. Yousssef*; Hoda H. Ameen* and
Asmahan M. Lashein*
* Plant Pathol. Dept., Nat. Res. Centre, Giza, Egypt.
** Dept. Zoology & Agric. Nematol., Fac. Agric., Cairo Univ., Egypt.
*** Plant Biotechnol. Dept., Nat. Res. Centre, Giza, Egypt.

A promising protocol for enhancement of callus induction from

different explants of seed, leaf, stem and root of Tagetes erecta
L. was established. The best results on callus induction were achieved
when 7.0 mg/l 2, 4-dichlorophenoxy acetic acid (D) + 10.0 mg/l
kinetin or 7.0 mg/l naphthalene acetic acid (NAA) + 10.0 mg/l
6-benzylaminopurine (BAP) were added to Murashige and Skoog,
(MS) medium with all used explants. It is interesting to notice that
dark conditions of incubation generally showed equal or better results
on callus induction with the different explants compared with calli
grown under light conditions. Also, data indicated that the growth
values (Gv) of calli derived from the different explants have gradually
increased to the fourth week of cultivation. Incubation under dark
conditions showed the best results as compared with those under light
conditions. The highest value of growth rate (Gr) was recorded at the
third week of cultivation. Culturing of leaf explants on MS medium
containing 7.0 mg /l NAA and 10.0mg/l BAP for three weeks gave the
highest growth rate as compared with other explants and incubation
under dark conditions was better than incubation under light
conditions.
Keywords: Callus induction, Meloidogyne incognita, root-knot
nematode and Tagetes erecta L.

Tagetes species, belong to family Asteraceae, were used by ancient civilization

like the Aztecs for various purposes (Neher, 1968). Dhangar et al. (1996) reported
that root extract of Tagetes erecta cv. Crackjack was significantly effective inhibitor
of larvae mobility of Meloidogyne javanica in vitro. Previous studies in Egypt have
shown the presence of about 54 genera and 160 species of phytoparasitic nematodes
associated with many cultivated plants (Ibrahim 1990, 1994 and Oteifa et al., 1970).
Thiophenes, the biocidal compounds were frequently found in Tagetes species
(Marigolds) where their accumulation changes according to the physiological state
of plants and during organogenesis (Sutfeld, 1982). Ketel (1985) reported that callus
of T. erecta rapidly formed roots and shoots in culture and contained a number of
hexane-soluble secondary metabolites, some of which corresponded with thiophenes
in their HPLC retention times and ultra violet (UV) spectra. Callus of T. patula
showed intermediate behaviour. Lately, the studies on thiophene induction in vitro
cultures was performed with the aim to select the explants having a high thiophene
induction (Ketel, 1986 & 1987; Ekes-Kretovics et al., 1993 and Rajasekaran et al.,
20 HAMIDA A. OSMAN et al.

2003). Poli et al. (1991) evaluated thiophene accumulation in leaf calli and calli
suspension cultures of T. patula. They reported that the secondary calli showed 4 to
6 fold higher fresh weight increment in 2 weeks, while the tertiary ones only
doubled their weight in the same period. Moreover, they investigated the influence
of light/dark succession on induction of thiophene. They mentioned that a
considerable increase of thiophene production (about 16.5 fold) was obtained when
growing calli under successive light/dark periods. Repeated subculturing of callus
tissues resulted in a slow decline of the thiophene yield. Ekes-Kretovics et al. (1993)
reported that various species of the genus Tagetes are well known for their
insecticidal properties. T. minuta contains large amounts of thiophenes. The optimal
conditions for the growth of callus tissue involved culture on modified Murashige
and Skoog (MS) medium supplemented with 0.1 mg/1 naphthalene acetic acid
(NAA) at 24C, 70% RH and under continuous white light (2500 photometric
unit(Lux). Rajasekaran et al. (2003) reported that the maximum value of both mass
callus and/or thiophene induction were recorded when leaf explants of T. patula
were cultured on MS medium supplemented with 2,4-dichlorophenoxy acetic acid
and kinetin at 2.0 mg/l. Benavides and Caso (1993) reported that the differences
between explant sources were apparent in terms of their competence for
differentiation and subsequent organogenesis. Leaf-derived cultures were more
responsive than the root-derived ones. Moreover, Misra and Datta (2001) reported
that high value of direct differentiation of shoot buds occurred when leaf explants of
T. erecta was used and cultured on MS medium supplemented with 14.34 µM
gibbrellic acid (GA) and 4.44 µM 6-benzylaminopurine (BAP). Biochemical studies
revealed that the plants produced were capable of synthesis of thiophene like
compounds following in vitro regeneration. Recently, El-Gengaihi et al. (2001)
reported that, the petroleum ether and chloroform extracts of the roots of T. erecta,
T. patula and T. minuta were highly potent against the reniform nematode,
Rotylenchulus reniformis. Therefore, this study was designed to investigate the in
vitro culture conditions required to enhance the callus growth derived from the
different explants of T. erecta.

Materials and Methods

Plant materials
Tagetes erecta seeds were secured from Horticulture Division, Faculty of
Agriculture, Cairo University, Giza, Egypt.
1. Establishment of sterilized Tagetes seedlings:
Seeds of T. erecta were surface sterilized by immersion in70% ethanol for 10
sec., followed by three washes with sterile distilled water. Then, these seeds were
immersed in 20% of commercial Clorax (5.25 g/l) solution containing a drop of
Tween 20 for 15 min. The sterilized seeds were germinated aseptically on 50 ml of
solidified basal MS medium (Murashige and Skoog, 1962) in 300 ml glass jars
(Fig. 1).Ten replicates, each of 10 seeds /jar, agar was added prior to autoclaving at
1.2 kg/cm2 for 20 min. The pH of this medium was adjusted to 5.8 by the addition of
0.1 N HCl or 0.1 N KOH. The jars were incubated in a growth chamber at 26±1C
and exposed to 16 h/day photoperiod at intensity of 3000 LUX from white cool light
of fluorescent lamps.

Egypt. J. Phytopathol., Vol. 36, No. 1-2 (2008)

 BIOLOGICAL CONTROL OF ROOT-KNOT NEMATODE…….. 21

2. Establishment of T. erecta callus cultures:

Aseptically grown seedlings, 60-day-old (Fig. 1-a), obtained from in vitro
germinated seeds on basal MS medium were used as sources of different explants,
i.e. leaf, stem and root as well as sterilized seeds of T. erecta. Three sections 3-4 mm
in diameter of each plant part were excised and cultured in 150 ml of glass jars
containing 25 ml of MS medium. The following media were used:
1- Solidified basal MS medium supplemented with different combinations (0, 5, 7,
10 and 12 mg/l) of both 2, 4-dichlorophenoxy acetic acid (2, 4-D) as auxin and
N-(2-furanylmethyl) 6-aminopurine (kinetin) as cytokinin.
2- Solidified basal MS medium supplemented with different combinations (0, 5, 7,
10 and 12 mg/l) of both naphthalene acetic acid (NAA) as auxin and
6-benzylamino purine (BAP) as cytokinin. Cultures of all treatments were divided
into two groups. The first group was maintained in light condition 16h/day
photoperiod at intensity of 3000 LUX from cool light fluorescent lamps for
4 weeks. The second group was maintained in dark for 4 weeks. All cultures were
incubated at 261C. For each combination of the above media, two experiments
were carried out consisting of 50 treatments of 5 replicates each under light or
dark conditions for each explant.
3. Mass callus production:
An equal weight (250 mg/jar) of callus cultures derived from seed, leaf, stem
and root explants of T. erecta were sub- cultured onto fresh MS medium which
contained the optimum concentrations from both of auxin and cytokinin (7.0 mg/1
2,4-D + 10.0 mg/l kinetin. or 7.0 mg/l NAA + 10.0 mg/l BAP). The comparative
studies were done under light or dark conditions and incubated at 261C. Fresh
weight (g/jar); Dry weight (g/jar); Dry matter content (%) were determined during
five weeks from culturing.
4- Determination of growth dynamics of callus derived from seed, leaf, stem and
root explants were determined during five weeks from culturing:
An equal weight of fresh callus (250 mg/jar) was transferred to the basal MS
medium, containing 7.0 mg/ 1 2,4-D + 10.0 mg/1 kinetin or 7.0 mg/1 NAA + 10.0
mg/1 BAP to study the growth dynamics during five weeks. For each explant, five
replicates were used. The following parameters were recorded as follow:-
A- Growth value (Gv) of callus cultures was calculated during the experiment
according to Szoke et al. (1979).
B- Growth rate (Gr) of callus cultures (mg/day) was determined weekly as described
by Dung et al. (1981). The comparative studies were done under light and dark
conditions and incubated at 261C. The obtained results were statistically
analyzed according to Snedcor and Cochran (1972).

Egypt. J. Phytopathol., Vol. 36, No. 1-2 (2008)

22 HAMIDA A. OSMAN et al.

Results

1. Establishment of callus cultures derived from different explants of T. erecta:

1.1. Effect of different combinations of 2, 4-D and kinetin on callus induction:
The effects of different combinations between 2, 4-D and kinetin on callus
induction were recorded after four weeks from culturing in Table (1). Data showed
that the best results of callus induction were achieved with all used explants when
7.0 mg/l 2, 4-D + 10.0 mg/l kinetin were added to MS medium with all used
explants. It is interesting to mention that dark conditions generally showed equal or
better results on callus induction with all used explant cultures compared with
cultures grown under light conditions.

Table 1. Effect of different combinations of 2,4-D and kinetin on callus

induction derived from different explants of Tagetes erecta under
light or dark conditions
Growth regulator (mg/l) Tagetes erecta explants
Seeds Leaf Stem Root
2,4-D Kinetin
Light* Dark** Light Dark Light Dark Light Dark
0.0 - - - - - - - -
5.0 - - - - - - - -
0.0 7.0 - - - - - - - -
10.0 - - - - - - - -
12.0 - - - - - - - -
0.0 - - - - - - - -
5.0 + + + ++ + ++ + ++
5.0 7.0 + + ++ ++ + ++ + ++
10.0 + ++ ++ ++ + ++ + ++
12.0 + + ++ ++ + ++ + ++
0.0 - - - - - - - -
5.0 + + + + + + + +
7.0 7.0 + ++ ++ ++ ++ ++ ++ ++
10.0 +++ +++ +++ +++ +++ +++ +++ +++
12.0 + ++ ++ ++ ++ ++ + ++
0.0 - - - - - - - -
5.0 + + + ++ + + + +
10.0 7.0 + + ++ ++ + + + +
10.0 + ++ ++ ++ ++ ++ ++ ++
12.0 ++ ++ ++ +++ ++ ++ ++ +++
0.0 - - - - - - - -
5.0 + + + + + + + +
12.0 7.0 + + + + + + + +
10.0 + + + + + + + +
12.0 + ++ + ++ + ++ + ++
Callus induction: - = No callus produced, + (Low)= 1-30%, ++ (Medium)= 30-60%, +++ (High)= > 60%
of callus produced from the used total jars.
* Light= 16h light. ** Dark= 24h dark.

Egypt. J. Phytopathol., Vol. 36, No. 1-2 (2008)

 BIOLOGICAL CONTROL OF ROOT-KNOT NEMATODE…….. 23

1.2. Effect of different combinations of NAA and BAP on callus induction:

The effects of supplementation of MS medium with different combinations of
NAA and BAP on callus induction derived from leaf, stem root and seed explants of
T. erecta were recorded after four weeks of culturing. Data presented in Table (2)
clearly show that the addition of 7.0 mg/l NAA in combination with 10.0 mg/l BAP
to MS medium gave the best results of callus induction with all used explants.
Generally, it was also observed that darkness showed equal or better results of callus
induction as compared with light conditions in most treatments. From the data in
Tables (1 and 2), it is indicated that among the 25 combinations of 2,4-D and
kinetin, the best results of callus induction from T. erecta explants were achieved
when 7.0 mg/l for 2,4-D in combination with 10.0 mg/l kinetin were added to the
culture medium with all explants.

Table 2. Effect of different combinations of NAA and BAP on callus induction derived
from different explants of Tagetes erecta under light or dark conditions
Growth regulator
Tagetes erecta explants
(mg/l)
Seeds Leaf Stem Root
NAA BAP
Light* Dark** Light Dark Light Dark Light Dark
0.0 - - - - - - - -
5.0 - - - - - - - -
0.0 7.0 - - - - - - - -
10.0 - - - - - - - -
12.0 - - - - - - - -
0.0 - - - - - - - -
5.0 + ++ + ++ + ++ + ++
5.0 7.0 + ++ ++ ++ ++ ++ + ++
10.0 + ++ ++ ++ ++ ++ ++ ++
12.0 + + ++ +++ ++ +++ + ++
0.0 - - - - - - - -
5.0 + + + + + + + +
7.0 7.0 ++ ++ ++ ++ ++ ++ ++ ++
10.0 +++ +++ +++ +++ +++ +++ +++ +++
12.0 ++ ++ ++ ++ ++ ++ ++ ++
0.0 - - - - - - - -
5.0 + ++ + ++ + ++ + ++
10.0 7.0 ++ ++ ++ ++ ++ ++ ++ +
10.0 ++ +++ ++ +++ ++ +++ ++ ++
12.0 ++ +++ ++ +++ ++ +++ ++ +++
0.0 - - - - - - - -
5.0 + + + + + + + +
12.0 7.0 + ++ + ++ + ++ + ++
10.0 ++ ++ ++ ++ ++ ++ ++ ++
12.0 ++ ++ ++ ++ ++ ++ ++ ++
* Callus induction as well as light and dark: As described in footnote of Table (1).

Egypt. J. Phytopathol., Vol. 36, No. 1-2 (2008)

24 HAMIDA A. OSMAN et al.

2. Mass callus production:

2.1. Effect of MS medium supplemented with 7.0 mg/l 2,4-D and 10.0 mg/l kinetin on
mass callus induction:
Data presented in Table (3) indicated that the optimum results of callus fresh
weight (g) were recorded at the end of the fourth week of culturing under both
culturing conditions. Callus fresh weights were 6.0, 5.50, 5.13 and 4.18g for callus
cultures derived from leaf, stem, seed and root explants, respectively in dark
conditions. However, they were 5.28, 5.05, 4.21 and 3.53g for leaf, stem, seed and
root callus cultures, respectively under light conditions. As presented in Table (4), at
the end of the fifth week of culturing, results showed the optimum callus dry weight
as compared with other weeks. They were 0.41, 0.39, 0.32 and 0.21g for leaf, seed,
stem and root explants, respectively, under light condition. However, it was
0.44, 0.43, 0.34 and 0.27g under dark conditions for seed, leaf, stem and root callus
cultures, respectively.

Table 3. Effect of MS medium supplemented with 7.0mg/l of 2,4-D and 10.0mg/l of

kinetin on mass callus induction from seeds, leaf, stem and root explants of
T. erecta during 5 weeks of cultivation under light or dark conditions
Weeks of cultivation
Callus growth
parameters
Explants 1st week 2nd week
Light* Dark** Light Dark
Seeds 0.75 0.04 0.860.04 1.760.06 1.9701
Fresh
Leaf 0.91 0.05 1.160.11 1.930.043 2.320.12
weight
Stem 0.57 0.02 0.840.07 1.640.061 2.040.16
(g)
Root 0.55 0.03 0.710.03 1.340.029 1.830.07
Seeds 0.015 0.001 0.0180.002 0.110.005 0.150.01
Dry
Leaf 0.02 0.002 0.040.003 0.170.04 0.1590.012
weight
Stem 0.017 0.003 0.0240.004 0.090.002 0.110.003
(g)
Root 0.0017 0.0140.003 0.080.003 0.110.003
Seeds 1.94 0.117 1.940.117 2.080.144 7.620.36
Dry matter
Leaf 2.46 0.37 2.460.37 3.150.31 6.860.52
contents
Stem 2.91 0.45 2.910.45 2.800.23 5.490.25
(%)
Root 1.5 0.29 1.50.29 1.910.29 6.050.15
3rd week 4th week 5th week
Light Dark Light Dark Light Dark
Seeds 3.740.06 4.880.04 4.210.03 5.130.07 3.140.2 5.010.1
Fresh
Leaf 4.040.05 5.440.28 5.280.04 6.00.07 5.170.05 5.840.06
weight
Stem 3.240.06 4.720.13 5.050.09 5.50.31 4.970.2 5.320.34
(g)
Root 3.070.10 3.540.07 3.530.07 4.180.04 3.360.11 4.170.08
Seeds 0.310.01 0.330.008 0.380.003 0.430.006 0.390.003 0.440.008
Dry
Leaf 0.350.01 0.380.009 0.40.005 0.420.003 0.410.005 0.430.002
weight
Stem 0.240.01 0.270.02 0.310.005 0.330.01 0.320.005 0.340.11
(g)
Root 0.160.04 0.250.03 0.190.05 0.250.03 0.210.04 0.270.02
Dry Seeds 8.250.35 6.710.17 9.010.13 8.290.21 9.390.09 8.770.2
matter Leaf 8.680.14 6.960.42 7.590.05 7.010.12 7.890.18 7.390.116
contents Stem 7.370.43 5.390.3 6.190.02 6.010.21 6.440.09 6.230.19
(%) Root 5.030.98 6.990.79 5.411.6 6.10.71 5.630.05 6.40.52
*16h light, **24h dark. Each figure represents the mean of 3 replicates,  SE= Standard error.

Egypt. J. Phytopathol., Vol. 36, No. 1-2 (2008)

 BIOLOGICAL CONTROL OF ROOT-KNOT NEMATODE…….. 25

Concerning the percentages dry matter contents, incubation under light condition
for five weeks recorded the best results, as they were 9.39, 7.89, 6.44, and 5.63% for
seeds, leaf, stem and Concerning the percentages dry matter contents, incubation
under light condition for five weeks recorded the best results, as they were 9.39,
7.89, 6.44, and 5.63% for seeds, leaf, stem and root callus cultures, respectively.
However, they were 8.77, 7.39, 6.4 and 6.23 % for seed, leaf, root and stem callus
cultures, respectively under dark conditions. From the previous data, it can be
concluded that, the dry weights of callus derived from all explants (seeds, leaf, stem
and root), incubated under dark condition for five weeks, showed the best results.
However the cultivation to the fourth or fifth weeks gave the best results of mass
callus production (in terms of fresh and dry weights, respectively) as compared with
other weeks.

2.2. Effect of MS medium supplemented with 7.0 mg/l NAA and 10.0mg/l BAP on
mass callus induction:
Data presented in Table (4) and Fig.(1 ) indicated that, the cultivation for four
weeks under dark condition gave the best results of callus fresh weight which they
were 8.62, 7.63, 6.8g and 5.82 for leaf (Fig.1-b),seed (Fig.1-c), stem (Fig.1-d) and
root callus (Fig.1-e) cultures, respectively under dark conditions. Whereas, they
were 7.64, 6.8, 5.7 and 4.6 g for leaf, seed, stem and root calli cultures, respectively
under light conditions.

Fig. 1. (A-E): Callus induction from the different explants of Tagetes erecta
under dark conditions. Aseptic seedlings (A), Leaf callus (B), Seed
callus (C), stem callus (d) and root callus (E).

Egypt. J. Phytopathol., Vol. 36, No. 1-2 (2008)

26 HAMIDA A. OSMAN et al.

Table 4. Effect of MS medium supplemented with 7.0 mg/l of NAA and

10.0 mg/l of BAP on mass callus induction from seeds, leaf, stem and
root explants of Tagetes erecta L. during 5 weeks of cultivation under
light or dark conditions
Callus Weeks of cultivation
Explants
growth 1st week 2nd week 3rd week
parameters Light * Dark** Light Dark Light Dark
Seeds 0.960.09 1.240.07 2.540.06 3.050.07 5.980.1 6.770.29
Fresh
Leaf 1.160.12 1.350.05 2.640.06 3.340.05 6.630.2 8.060.12
weight
Stem 0.840.06 1.060.05 1.880.07 2.650.12 4.140.12 5.650.3
(g)
Root 0.680.13 0.780.12 1.040.14 1.880.27 3.660.27 5.020.28
Seeds 0.030.004 0.040.003 0.140.006 0.160.006 0.430.006 0.500.03
Dry
Leaf 0.050.005 0.050.007 0.240.006 0.260.02 0.60.04 0.700.03
weight
Stem 0.020.003 0.040.009 0.130.004 0.150.008 0.320.006 0.350.008
(g)
Root 0.010.002 0.020.003 0.10.01 0.130.008 0.280.01 0.310.004
Dry Seeds 2.720.052 2.950.23 5.70.36 5.350.1 7.10.04 7.370.24
matter Leaf 3.90.02 4.080.55 9.170.15 7.910.45 9.060.62 8.680.25
contents Stem 2.310.19 3.490.67 6.960.44 5.570.54 7.620.3 6.220.25
(%) Root 1.810.19 3.290.49 9.71.58 7.160.68 7.850.84 6.140.39
4th week 5th week
Light Dark Light Dark
Seeds 6.80.26 7.630.37 6.450.28 7.040.15
Fresh
Leaf 7.640.35 8.620.28 7.150.05 7.640.16
weight
Stem 5.70.33 6.80.35 5.120.07 6.280.3
(g)
Root 4.60.31 5.820.35 3.880.3 5.120.09
Seeds 0.480.013 0.610.03 0.530.009 0.650.04
Dry
Leaf 0.690.04 0.760.03 0.730.05 0.790.03
weight
Stem 0.430.006 0.510.008 0.510.01 0.550.02
(g)
Root 0.370.008 0.310.004 0.410.02 0.490.01
Dry Seeds 7.10.21 8.020.19 8.310.27 9.230.4
matter Leaf 9.080.52 8.760.08 10.20.69 10.340.33
contents Stem 7.520.49 7.490.31 9.950.17 8.730.44
(%) Root 8.030.57 12.622.35 10.741.45 9.610.14
* As described in footnote of Table (3).

Data in Table (4) show that the best results of callus dry weights 0.79, 0.65,
0.55and 0.49g for leaf, seed, stem and root callus cultures were obtained after five
weeks of cultivation under dark condition, respectively. However, they were 0.73,
0.53, 0.51 and 0.41g for leaf, seed, stem and root explants, respectively under light
condition. Regarding the percentages dry matter contents, the incubation under light
conditions for five weeks produced the highest results, which they were 10.74, 10.2,
9.95 and 8.31% for root, leaf, stem and seeds callus cultures, respectively. Whereas
they were 10.34, 9.61, 9.23 and 8.73% for leaf, root, seed and stem cultures,
respectively, under dark conditions.

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 BIOLOGICAL CONTROL OF ROOT-KNOT NEMATODE…….. 27

3. Determination of growth dynamics of callus induction:

3.1. Growth value (Gv):
Data presented in Fig. (2 a and b) showed the effect of MS medium
supplemented with either 7.0 mg/l 2,4-D and 10.0 mg/l kinetin (Fig. 2-a) or 7.0 mg/l
NAA and 10.0 mg/l BAP (Fig.2-b) on growth values of calli derived from leaf, seed,
stem and root explants of Tagetes plants. Measurements of callus growth values
were determined weekly for five weeks of cultivation under light or dark conditions.
Calli derived from different used explants and seeds were gradually increased to the
fourth week of cultivation. The highest values of calli growth obtained from MS
medium supplemented with 7.0 mg/l 2,4-D and 10.0 mg/l kinetin were 5.75, 5.25,
4.88 and 3.93 for leaf, stem, seeds and root, respectively as shown in Fig (2-a).
Whereas, they were 8.37, 7.38, 6.55 and 4.87 for leaf, seeds, stem and root calli,
respectively when cultured on MS medium supplemented with 7.0 mg/l NAA and
10.0 mg/l BAP as shown in Fig. (2-b). Incubation under dark conditions showed the
best results as compared with light conditions.
Gr ow t h va l ue

7
6
5
4
3
2
1
0

Light Dark Light Dark Light Dark Light Dark Light Dark

1 st week 2 nd week 3 rd week 4 th week 5 th week

Seeds Leaf Stem Root

Fig. (2-a). Growth values (Gv) of calli derived from four explants of T. erecta Cultured on MS
medium supplemented with 7.0 mg/l 2,4-Dand 0.0Mg/l kinetin, under light or
dark conditions during five weeks of cultivation.
Growth value(Gv)

9
8
7
6
5
4
3
2
1
0
Light Dark Light Dark Light Dark Light Dark Light Dark

1 st week 2 nd week 3 rd week 4 th week 5 th week

Seeds Leaf Stem Root

Fig. (2-b). Growth values (Gv) of calli derived from four explants of T. erecta cultured on MS
medium supplemented with 7.0 mg/l of NAA and BAP, under light or dark
condition during five weeks of cultivation.

Egypt. J. Phytopathol., Vol. 36, No. 1-2 (2008)

28 HAMIDA A. OSMAN et al.

3.2. Growth rate (mg/day):

Data illustrated in Figs (3, a and b) revealed the effect of MS medium
supplemented with 7.0 mg/l 2,4-D and 10.0 mg/l kinetin (Fig 3-a) or 7.0 mg/l NAA
and 10.0 mg/l BAP (Fig. 3-b) on growth rates of calli derived from seeds, leaf, stem
and root explants.
As shown in Fig. (3-a), the incubation under dark conditions gave greater growth
rate than incubation under light condition. The highest value of growth rate (mg/day)
was recorded at the third week of cultivation. They were 445.71, 415.71, 382.86 and
244.29 mg/day for calli derived from leaf, seeds, stem and root cultures, respectively
under dark condition. While, they were 301.43, 282.86, 282.57 and 247.14 mg/day
for calli derived from leaf, seed, stem and root, respectively when incubated under
light conditions.
Data illustrated in Fig. (3-b) showed that the highest values of growth rate
obtained after three weeks of cultivation under dark condition were 674.29, 531.43,
448.57 and 428.57 mg/day for calli derived from leaf, seeds, root and stem,
respectively while they were 570, 491.43, 374.29 and 322.86 mg/day for leaf, seed,
root and stem, respectively when incubated under light conditions. It is interesting to
mention that, culturing of leaf explant on MS medium containing 7.0 mg/l NAA and
10.0 mg/l BAP for three weeks, gave the highest growth rate as compared with other
explants and the incubation under dark conditions was better than incubation under
light conditions.

500
Growth rate (mg/day)

450
400
350
300
250
200
150
100
50
0

Light Dark Light Dark Light Dark Light Dark Light Dark

1 St week 2 nd week 3 rd week 4 th week 5 th week

Seeds Leaf stem Root

Fig. (3-a). Growth rate (mg/day) of calli derived from different explants of
T. erecta cultured on MS medium supplemented with 7.0 mg/l 2, 4-D
and 10.0 mg/l kinetin for 5 weeks, and incubated under light or
dark conditions

Egypt. J. Phytopathol., Vol. 36, No. 1-2 (2008)

 BIOLOGICAL CONTROL OF ROOT-KNOT NEMATODE…….. 29

Growth rate (mg/day)

800
700
600
500
400
300
200
100
0
Light Dark Light Dark Light Dark Light Dark Light Dark

1 St week 2 nd week 3 rd week 4 th week 5 th week

Seeds Leaf stem Root

Fig. (3-b). Growth rate (mg/day) of calli derived from different explants of
T. erecta cultured on MS medium supplemented with 7.0 mg/l NAA
and 10.0 mg/l BAP for 5 weeks, and incubated under light and dark
conditions

Discussion

The effect of several combinations of cytokinins and auxins, added to culture

media on callus induction has been reported by several investigators (Gamborg and
Shyluk, 1981 and Torres, 1989). It has been mentioned that callus formation is due
to cell division and cell enlargement. Auxins are generally included in the culture
medium to stimulate callus production and cell growth (Moore, 1979). The
cytokinins are generally added to a culture medium aiming at promoting cell
division in callus cultures of plant tissues, and stimulating the rate of protein
synthesis in tobacco cell cultures (Skoog and Schmitz, 1972). Also, Klambt (1976)
reported that cytokinins have been shown to be required for mitosis to occur in
tobacco cells. Concerning the effect of supplementation of MS medium with
different combinations of growth regulators either NAA and BAP or 2, 4-D and
kinetin on callus induction from seed, leaf, stem and root explants of T. erecta. The
presented data indicated that the best results of callus induction were achieved when
7.0 mg/l of 2,4-D in combination with 10.0 mg/l kinetin were added to the culture
medium with all used explants. As well as, among the 25 combinations of NAA and
BAP, the best results were also obtained when 7 mg/l of NAA + 10 mg/l of BAP
were added to the culture medium with all used explants. The MS medium
supplemented with 7.0 mg/l NAA + 10.0 mg/l BAP gave the highest growth rate and
growth value of mass callus production under dark conditions. It can be concluded
that supplementation of culture medium with auxins and cytokinins increased the
mass induction of callus. This observation could be based on the fact that these
growth factors stimulate protein synthesis, cell growth and division.

Egypt. J. Phytopathol., Vol. 36, No. 1-2 (2008)

30 HAMIDA A. OSMAN et al.

The investigations so far published regarding the effect of auxins and cytokinins
on callus production seemed to be in harmony with the obtained results. Kothari and
Chandra (1986) found that the best medium for callus production from leaf explants
of T. erecta was obtained by supplementing of MS medium with 10 mg/l
BAP+2mg/l NAA + 0.5 mg/l GA3. Whereas, Belarmino et al. (1992) reported that 5
mg/l NAA and 0.2-0.5 mg/l BAP were the best concentrations for producing large
yellow calli from leaf explants. On the other hand, Ekes-Kretovics et al. (1993)
mentioned that the optimal conditions of callus growth was obtained when callus
was supplemented with 0.1 mg/l NAA However, in contrast of our obtained results
Rajasekaran et al. (2003) reported that the best medium for callus production from
leaf explants of T. patula was MS medium supplemented with both 2,4-D and
kinetin at 2 mg/l. From the present findings, it seems that the nutrition, light or dark
conditions have great effects on the mass callus and callus growth rate.

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 32 HAMIDA A. OSMAN et al.

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