The genus Celosia consists of about 60 species belongs to the family
Amaranthaceae. It is a native plant in subtropical and temperate zones of Africa,
South America and South East Asia. Celosia argentea is a cultivated annual
plant that is mainly used for planting flowerbeds in different types of gardens.
Celosia grown under full light and warm conditions, reach heights up to 71 cm,
tolerate a wide range of soil conditions. C. argentea is a well known
medicinal plant for treating dysentery, diarrhea, acute abdominal pain, inflamed
stomach, skin eruptions and exhibited antibacterial activity against, Bacillus
subtilis, Salmonella typhi, Escherichia coli, Agrobacterium
tumefaciens and Mycobacterium tuberculosis. Celosia is also
one of the main sources of natural pigments used in several industries and the
seeds were used in bird-feed for poultry production (Eid
et al., 2006).
The curative properties of C. argentea are due to the presence of various
complex chemical substances of different composition which occur as secondary
metabolites. They are grouped as alkaloids, glycosides, flavonoids, saponins,
tannins, carbohydrate (Patel et al., 2010).
Alkaloids play some metabolic role and control development in living system
(Edeoga and Eriata, 2001). They are also involved in
protective function in animals and are used as medicine especially the steroidal
alkaloids (Stevens et al., 1992). Alkaloids are
known to exhibit marked physiological activity when administered to animals
(Okwu, 2004). Pure isolated plant alkaloids and their
synthetic derivatives are used as basic medicinal agents for analgesic antispasmodic
and bactericidal effects (Stray, 1998). Phenolic compounds
may be the reason for the theraputic, antiseptic, antifungal or bactericidal
properties of the plants (Osuagwu et al., 2007).
Chemical mutagenesis is a simple approach to create mutation in plants for
their improvement of potential agronomic traits. Mutation methodology has been
used to produce many cultivars with improved economic value and to study the
genetics and plant developmental phenomena (Aruna et
Dimethyl Sulphate (DMS) is a chemical compound with the formula (CH3O)2SO2
which is monofunctional alkylating agents that have been shown to induce mutations,
chromosomal aberrations and other genetic alterations in a diversity of organisms.
As an alkylating agent DMS is a typical SN2 agent attacking predominantly
nitrogen sites in nucleic acids (Hoffman, 1980).
Inter Simple Sequence Repeat (ISSR) markers have been used with success to
identify the mutants and study the genetic diversity of different medicinal
plant species and crops (Shafie et al., 2009;
Farajpour et al., 2011).
This investigation was carried out to study the effect of dimethyl sulphate
on the growth and some phytochemical compositions of Celosia argentea,
to produce a new pattern of vegetative and flowering growth and identify them
using ISSR marker.
MATERIALS AND METHODS
The study was carried out at the Nursery of Floricultural and Ornamental Plants,
Faculty of Agriculture, Alexandria University, Alexandria, Egypt during 2 successive
generations of 2011 and 2012.
Seeds of Celosia argentea var. spicata were sown on April 20th
2011 in 20 cm clay pots containing a mixture of clay: sand (1:1 v/v). After
two months the seedlings were transplanted into clay pots containing the soil
mixture of clay and sand (3:1 v/v). Then plants were treated with dimethyl sulphate
solutions (0, 1000, 2000, 3000 and 4000 ppm) as a soil drench (10 mL for each
pot). Seeds for the M2-generation were sown on April 9th, 2012. The
procedure of sowing and transplanting were made likewise the first generation.
All plants of the different treatments were examined daily to search for variation
in the vegetative and flowering growth.
The experimental layout was a randomized complete block design containing three
replications (Steel and Torrie, 1980). Each replication
contained five treatments and every treatment consisted of five plants.
||Plant height (cm)
||No. of leaves plant-1
||Stem diameter (cm) at the stem base and above the soil surface
||Leaf area (cm2): The weight in g of one cm2
of leaves was calculated as an average from two leaves taken from the 7th
node of two plants, one leaf was taken from the main stem of each plant
as a sample. The leaf area was then expressed as the average mean weight
of a leaf divided by the mean weight of one cm2
||Flowering date: Flowering date was calculated as days from seed
sowing date to showing colour of inflorescences
||Length of the inflorescence (cm)
||Chlorophyll content: Total chlorophyll (SPAD units) was determined
in the leaves at the flowering stage with SPAD apparatus as described by
||Anthocyanin determination in the leaves: The procedure of Fuleki
and Francis (1968) was used to determine the anthocyanin content in
||Anthocyanin determination in the inflorescence: The determination
was done as mentioned by the leaves
||Alkaloids determination in the leaves: The quantity of alkaloids
in the sample was determined using Osuagwu et al.
(2007) method. Five gram of the powdered sample was extracted with 10
mL of petroleum ether. The petroleum ether was removed using aspirator.
One gram of the extract was suspended in 10 mL of double distilled water
and the pH was adjusted to 7.6. After shaking for 1 h, the suspension was
centrifuged. One milliliter of the supernatant was diluted to 50 mL with
phosphate buffer. The absorbance was measured spectrophotometrically at
580 nm wave length
||Phenols determination in the leaves: The quantity of the phenols
was determined as the procedure stated by Mostafa and
||Induction of variations: All changes in the vegetative and flowering
growth were recorded
ISSR analysis was carried out to identify the mutants. The identification and
fingerprinting of the mutants was carried out at the Department of Nucleic Acid
Research, Mubarak City for Scientific Research and Technology Application, New
Borg El-Arab, Egypt.
DNA isolation and ISSR analysis: Genomic DNA was extracted from 1 g
of leaf tissue using Biospin Plant Genomic DNA Extraction Kit (BioFlux, China).
Ten anchored ISSR primers were used (Table 1). PCR was performed
in reaction volume of 25 μL using 12 ng of DNA of each sample, 25 pmol
of each primer, 5X Taq DNA polymerase buffer (promega ) including MgCl2,
0.2 mM dNTPs and 0.5 U μL-1 Taq DNA polymerase (promega ).
||ISSR primers, sequence, size of amplified fragment (bp), used
to analyze genetic relationships among the control and treated plants of
Celosia argentea var. spicata
ISSR amplification was carried out using Gen Amp PCR system 9700 Thermal cycling
programmed with 5 min at 95°C for initial denaturation, followed by 40 cycle
of 1 min at 95°C , 1 min at 45°C, 1 min at 72°C and a final extension
at 10 min at 72°C. The amplified DNA fragments were separated on 2% agarose
gel, stained with ethidium bromide, visualized on a UV Transilluminator and
photographed by Gel Documentation system.
ISSR bands were scored as present (1) or absent (0) to form a binary matrix.
Cluster analysis was conducted based on genetic similarity estimates using the
unweighted pair-group method arithmetic average (UPGMA) procedure in NTSYSpc
version 2.1 software package (Rohlf, 2000) in order to
deduce genetic relationships among the control and the mutants.
RESULTS AND DISCUSSION
All concentrations of dimethyl sulphate decreased significantly the plant height
in the M1-generation as shown in Table 2, while
the concentration of 2000 ppm increased significantly the plant height in the
M2 generation compared to the control (62.2 and 50.3, respectively).
These results are in agreement with the finding of Aliyu
and Adamu (2007) and Roychowdhury and Tah (2011).
Plants treated with high concentrations of dimethyl sulphate 3000 and 4000
ppm gave the highest number of leaves in both generations compared to the control
(201.7, 122.1 and 104.6 for M1-generation and 200.3, 121.5 and 104.0
for M2 generation, respectively).
The concentration of 4000 ppm DMS produced the largest leaf area in both generations
(20.08 and 19.24 cm2 for M1and M2, respectively)
compared to control (17.7 and 17.17 cm2).
The stimulative effect of dimethyl sulphate might be attributed to cell division
rates as well as an activation of growth hormones, e.g., auxin (Zaka
et al., 2004; Joshi et al., 2011).
All concentrations of dimethyl sulphate decreased significantly stem diameter
and length of the inflorescences in both generations as shown in Table
2, while insignificant differences were found in plants treated with 1000
ppm with respect to inflorescences length in the M1 generation compared
to control (9.0 and 10.8, respectively). Low concentration of DMS induced some
stimulation effect on plant growth, while the higher concentrations resulted
in an inhibiting effect as found by further studies (Mostafa,
2009; Mostafa and Alhamd, 2011; Roychowdhury
and Tah, 2011). This inhibition effect can be due to physiological damage
produced cumulatively by increased chemical mutagen concentrations.
||Effect of dimethyl sulphate concentrations on plant height
(cm), number of leaves, leaf area (cm2), stem diameter (cm),
flowering date (days) and length of the inflorescences (cm)
|Values in the same column not followed by the same letter
are significantly different at the 5% level of probability *, **Significant
at p = 0.05 and 0.01, respectively
||Effect of dimethyl sulphate concentrations on chlorophyll
content, concentration of the anthocyanin in the leaves and inflorescences,
concentration of the alkaloids and phenols
|Values in the same column not followed by the same letter
are significantly different at the 5% level of probability NS: Not significant,
*, **Significant at p = 0.05 and 0.01, respectively
Reduced growth due to higher doses was explained differently by different
workers. It may be attributed to one or more of the following reasons: The increase
in destruction on growth inhibitors, drop in the auxin level or inhibition of
auxin synthesis and decline of assimilation mechanism as reported by Roychowdhury
and Tah (2011).
Concerning to flowering date, significant differences were obtained among the
treatments in both generations. In general all treatments delayed flowering
in both generations as shown in Table 2. This result agrees
with the results of Mostafa and Alhamd (2011) and Gad
The data shown in Table 3 indicated that, chlorophyll content
was decreased gradually with increasing the concentration of dimethyl sulphate
in the M2-generation. The concentration of 1000 ppm DMS decreased
significantly chlorophyll content in the M1-generation compared with
the control (27.9 and 37.7, respectively). But insignificant differences were
found between the other treatments compared to the control.
These slightly decrease in chlorophyll content with high concentrations of
dimethyl sulphate was supported by the results of Pandey
et al. (2012) where they found chlorophyll damage caused by various
physical and chemical mutagens.
The concentrations of 3000, 4000 and 1000 ppm. DMS increased significantly
the concentration of anthocyanin in the leaves in the M1 generation
compared to control (38.9,37.3,36.8 and 25.7 mg mL-1) as shown in
Table 3. In the M2 generation, the concentrations
of 2000, 3000 and 1000 ppm. DMS increased significantly the concentration of
anthocyanin in the leaves compared to control (41.9, 41.4, 37.1 and 34.9 mg
All treatments increased significantly the concentration of anthocyanin in
the inflorescences in both generations except that of the 2000 ppm. DMS (168.6
mg mL-1) compared to control (166.0 mg mL-1) in the M1-
The results of the alkaloids concentration for both generations are presented
in Table 3. The results showed that the concentrations of
1000, 2000 and 3000 ppm did not differ significantly from the control in the
M1-generation, while the concentration of 4000 ppm gave the lowest
value (0.199) and differed significantly from the control (0.324). For M2
generation, the concentrations of 1000, 2000 and 3000 ppm DMS increased significantly
the alkaloids concentration compared to the control (2.758, 1.478, 1.494 and
An increase in the alkaloids concentration was found in the second generation
with comparable to first generation with using 1000, 2000 and 3000 ppm dimethyl
sulphate, this may be due to a recessive mutant which was not indicated in the
M1 generation. Mutations are mostly recessive and they cannot be
selected until the second generation as reported by Toker
et al. (2007). Similar results were reported by Gad
No significant differences were obtained among the treatments in the M1-generation
with respect to the phenols concentration. In the M2 generation,
the concentration of 3000 ppm DMS increased significantly the phenols concentration
compared to the control (1.621 and 1.492 mg mL-1, respectively).
The treatments of 2000, 3000 and 4000 in the first generation and 1000, 2000
and 3000 ppm in the second generation produced changes in the leaf form as shown
in Fig. 1 and 2. These changes of leaf form
or shape may be due to chromosomal disturbances. Also these changes could be
referred to the layer rearrangement as a result of the chemical mutagens effect
(El-Nashar, 2006; Mostafa, 2009).
||Changes in the leaf form in the M1-generation as
a result of the treatment with dimethyl sulphate, (a) Control, (b) 2000,
(c) 3000 and (d) 4000 ppm
||Changes in the leaf form in the M2-generation as
a result of the treatments with dimethyl sulphate, (a) Control, (b) 1000,
(c) 2000 and (d) 3000 ppm
The treatment of 3000 ppm produced one plant with greater growth (Taller and
thicker branches) than the control as shown in Fig. 3. The
stimulatory effect of the mutagen may be attributed to the increase in the rate
of cell division or cell elongation as reported by Joshi
et al. (2011).
Dwarfed plants were obtained from the plants treated with 2000, 3000 and 4000
ppm in both generations as shown in Fig. 4. This dwarfed growth
may be due to physiological damage resulted in the alteration from normal to
dwarf growth. Joshi et al. (2011) explained the
dwarfed growth to auxin destruction, changes in ascorbic acid content and physiological
and biochemical disturbances.
||Plant growth in M2-generation as a result of the
treatment with dimethyl sulphate at 3000 ppm, (a) Control and (b) plant
with greater growth
||Dwarfed plants obtained in the M2-generation as
a result of the treatments with dimethyl sulphate (a) Control, (b) 2000,
(c) 3000 and (d) 4000 ppm
Many changes of inflorescences shape were found after the treatments of 2000,
3000 and 4000 ppm in the M2-generation as shown in Fig.
5 and 6. These abnormalities are inflated of inflorescence,
splitting inflorescence apex and later splitting. Splitting the inflorescence
may be due to a gene mutant resulted in floral meristem being replaced with
meristems that have some or all of the characteristics of inflorescence. In
this case there is a failure, or delay in the production of flowers and a proliferation
of inflorescence-like structures in their place as reported by Coen
and Carpenter (1993).
Plants with many inflorescences produced at the main stem directly from the
lateral bud without slightly branches were found in the M2-generation
after the treatment of 3000 ppm as shown in Fig. 7.
Two plants having green vegetative growth (branches and leaves) with yellow
nodes were found in the M1-generation after the treatment of 3000
ppm as shown in Fig. 8. Lacks of anthocyanin pigments in the
vegetative growth mutant was transmitted to the M2- generation.
||Changes in the inflorescence form in the M2-generation
as a result of the treatments with dimethyl sulphate, (a) Control, (b) 2000
and (c) 3000 ppm
||Changes in the inflorescence form (a) Splitting apex and (b)
Bases of the inflorescences in the M2-generation as a result
of the treatment with dimethyl sulphate at 4000 ppm
||Plants with many inflorescences at the main stem in the M2-generation
as a result of the treatment with dimethyl sulphate, (a) Control and (b)
||Plants with green vegetative growth and with yellow nods in
the M2-generation as a result of the treatment with dimethyl
sulphate at 3000 ppm
Five mutants were used to identify them from the control (Table
4) using Inter Simple Sequence Repeat (ISSR) markers.
The genetic relationship among control and mutants were evaluated using ISSR
assay. Ten ISSR primers amplified a total of 74 bands, 31 of them were polymorphic.
The percentage of polymorphism of the amplified products was 41.8% (Table
5). The size of amplified bands ranged from 100-1000 bp (Fig.
9). Primer ISSR4 did not yield clear bands and primer ISSR5 did not produced
polymorphic band patterns.
||ISSR patterns of Celosia argentea var. spicata
generated by primer ISSR (1-10). Lane 1: Control, lanes 2-6: The mutants
plants produced by dimethyl sulphate and M: 100 bp DNA ladder, respectively
|| Description of the mutants
||Number of amplified and polymorphic bands and percentage of
primer polymorphism used to analyze genetic relationships among the control
and mutants of Celosia argentea var. spicata produced by ISSR
marker using ten primers
||Dendrograme constructed on the basis of ISSR profile for the
control and 5 mutants of Celosia argentea var. spicata using
ten ISSR primer
The largest number of polymorphic bands was nine bands with the primer ISSR1,
while primers: ISSR3, ISSR6 and ISSR9 generated low number of polymorphic bands
(two bands) as shown in Table 5. UPGMA dendrogram program
which constructed with coefficient of similar matrix (Table 6)
based on ISSR data classified the samples into two large clusters: Cluster I
and II. The control sample was grouped in cluster I and the other five different
morphological characters are were grouped in cluster II (Fig.
10). These results referred to that those morphological characters genetically
different from the control.
|| Genetic similarity of DNA among five mutants and control
plants of Celosia argentea produced by dimethyl sulphate treatments
These variation came from mutagenic effect of dimethyl sulphate treatments
as an alkylating agents which led to induce chromosome damage, chromosom aberrations
and base pair substitution, resulting in amino acid change which change the
function of proteins (Khan and Tyagi, 2009).
It is quite evident that, dimethyl sulphate could be suitable for inducing
genetic variability in the natural gene pool of Celosia. It is also appropriate
to induce valuable mutants. It can be concluded that also, ISSR marker can be
used for the identification and fingerprinting the mutants of Celosia argentea.