Induced Mutation in Callus Cell Lines of Daucus carota L.
K. Ruby Mathew
S. Sundara Rajan
Daucus carota L. is an important vegetable food crop with high nutritive value. The changing climate, pollution, loss of vegetation has affected the crop quality and there is a need to improve the variety. So the present investigation was taken with an aim to induce mutation in callus cell lines for crop improvement. Callus induction from stem and leaf explants of in vitro grown Daucus carota L. plantlets was observed on 1X MS medium with 3% sucrose , 1% agar fortified with 2 mg L-1 2,4-D. Mutation was induced in Daucus carota L callus cell lines by sodium azide and UV radiations. Callus cell lines grown on media containing 200 μM of sodium azide gave the best result with callus multiplication. Callus exposed at different time intervals under UV radiation showed multiplication of callus in all the exposures. Shoot induction was achieved for both the mutants on 2 mg L-1 BAP containing medium. The physiological estimation revealed that protein content (0.460±0.04 mg mL-1) in 6 min UV exposed cell lines and carbohydrate content (0.820±0.08 mg mL-1) in 4 min UV exposed cell lines are high when compared to control cell lines, sodium azide and other UV mutated cell lines. Mutation was confirmed through DNA fingerprinting.
Received: August 01, 2012;
Accepted: February 22, 2013;
Published: June 21, 2013
Daucus carota L. is an important member of family Apiaceae, it is valued
as food mainly because it is rich source of the fat-soluble hydrocarbon, carotene
(C40H56) the β form of which is the precursor of
Vitamin A. The pigment from the roots are used for coloring butter and other
food materials. It is used in treating kidney related diseases, dropsy, as nervine
tonic, aphrodisiac stimulant and carminative (Pant and Manandhar,
2007). Genetic variability is fundamental to successful breeding programs
in vegetatively and sexually propagated plants. This variation can occur naturally
or can be induced through mutations, using physical, biological or chemical
mutagens and has attracted the interest of plant breeders from many decades.
Mutations have helped to produce many cultivars with improved economic value
(Broertjes and van Harten, 1988) and study of genetics
and plant developmental phenomena (Bulk et al., 1990;
Bertagne-Sagnard et al., 1996). Mutation breeding
as an alternative method to conventional plant breeding as a source of increasing
variability and confer specific crop improvement without much altering its acceptable
phenotype (Ojomo et al., 1979; Mensah
and Obadoni, 2007). Physically and chemically induced mutation is a method
to create genetic variation resulting in improved varieties with better characteristics
(Wongpiyasatid et al., 2000; Arulbalachandran
et al., 2009). Sodium azide is a chemical mutagen and is considered
as one of the most powerful mutagen in plant (Mostafa, 2011).
The successful utilization of sodium azide to generate genetic variability in
plant breeding has been reported in Helianthus annuus L. (Mostafa,
2011) and other crops (Akhaury et al., 1996,
Kulthe and Kothekar, 2011). It has been reported that
up to 10 folds increases in the frequency of 5-methantryptophane resistant carrot
cell increase up to 10 fold in presence of UV light (Kolb
et al., 2001). The variation at the genetic level can be checked
by adapting the DNA fingerprinting by using random markers (Piccioni
et al., 1997). The present study was aimed to study the effect of
sodium azide and UV mutation on callus cell lines and regeneration of Daucus
carota plantlets and observe its effect through physiological estimation
and to analyze mutation at the genetic level through DNA fingerprinting.
MATERIALS AND METHODS
The present study was conducted during the period from January 2012 to May 2012 at Genohelix Biolabs - A division of CASB, Jain University, Chamarajpet, Bangalore, Karnataka, India.
The chemicals which were used in the study are of A.R. grade from Nice, sd fine , Qualigens, Hi-media, Spectrochem, Colloids and Loba chemie.
Daucus carota L. seeds were collected from Lal Bagh botanical garden
to carry out the experiment. Collected seeds were taken in a conical flask and
rinsed with tap water for 5-6 times and rinsed once with distilled water and
surface sterilized using liquid detergent 2% (v/v) Savlon, 6-8 drops of Tween-20
for 15 min, rinsed with 70% ethanol for 30 sec, disinfected with 0.05% (w/v)
HgCl2 for 6 min and rinsed in sterile water several times to remove
the traces of HgCl2. The sterilized seeds were taken on a petri plate
and were inoculated on 1X MS (Murashige and Skoog, 1962)
media with 3% sucrose and 1% agar. In each bottle 5 seeds were inoculated for
germination and plantlet development. The cultures were maintained in the culture
room at a temperature of 25±2°C, light intensity of 1000 LUX, relative
humidity between 50-60%, under photo-periodic regime for 16 h light and 8 h
dark cycle. The seeds germinated and the plantlets were developed after 22 days
The in vitro grown plants of Daucus carota were taken on a petridish in the LAF and different parts of the plant were excised and inoculated on 1X MS media with 3% sucrose and 1% agar with different concentrations and combinations of growth hormones like 2,4-D and Kinetin. The cultures were incubated in the culture room under controlled conditions. Culture media inoculated with different in vitro grown plant parts showed faster and better response with callus formation on 1X MS basal medium with 3% sucrose fortified with 2 mg L-1 2,4-D. The developed callus from in vitro grown plantlets were inoculated on 1X MS media with 3% sucrose fortified with 2 mg L-1 2,4-D with different concentrations of sodium azide (200, 400, 600, 800 and 1000 μM) and were incubated for callus multiplication for 15 days.
Callus developed from in vitro grown plant were exposed to UV light for different time intervals (2-8 min) and inoculated on 1X MS media containing 3% sucrose fortified with 2 mg L-1 2,4-D and incubated for callus multiplication for 15 days. After 15 days of culture mutated callus cell lines were inoculated on 1X MS media containing 3% sucrose fortified with 2 mg L-1 BAP for shoot induction and cultures were maintained under controlled culture conditions and data were collected.
Estimation of protein: One gram of developed plantlets was weighed,
ground with 5 mL of 0.2 M phosphate buffer (pH 7) and filtered using Whatmans
No. 1 filter paper. The filtrates were subjected to protein estimation by Lowry
et al. (1951) with absorbance measured at 660 nm.
Estimation of total carbohydrate: One hundred milligram of developed
plantlets was hydrolyzed with 2.5 N HCl by incubating in boiling water bath
for 15 min and filtered using Whatmans No. 1 filter paper.
|| List of RAPD Primers
The filtrates were estimated for total carbohydrate content by Anthrone method
(Hedge and Hofreiter, 1962) with absorbance measured
at 630 nm.
Isolation of genomic DNA from Callus (CTAB DNA extraction): The in
vitro grown plantlets from mutated callus cell lines from sodium azide and
UV mutation were used for genomic DNA extraction by Cetyltrimethylammonium bromide
(CTAB) method (Doyle and Doyle, 1990). The DNA quality
was assessed by examination on a 0.8% agarose gel stained with ethidium bromide.
RAPD amplification: RAPD amplification was performed in a volume of
25 μL that contained: 50 ng of template DNA, 2.5 mM each of dNTPs (Chromus
Biotech Pvt. Ltd), 5 pM of primer (Sigma- Aldrich) (Table 1),
10x Taq assay buffer (10 mM Tris-HCl, pH -8.8, 500 mM KCl, 15 mM MgCl2,
0.1% gelatin, 0.05% Tween 20 and 0.05% Nonidet P 40), 1 unit of Taq DNA
polymerase (Chromus Biotech Pvt. Ltd) and sterilized water. Amplification was
performed in a thermal cycler (MJ Research Inc.). Initial denaturation was performed
at 94°C for 3 min before beginning the cycling protocol followed by 1 min
at 92°C, 2 min at 36°C and 2 min at 72°C. A total of 40 cycles were
performed. The cycling was terminated with a final extension at 72°C for
6 min. The amplification products were analyzed on 1.2% agarose gels with low
range molecular weight marker. The gel was stained with ethidium bromide, visualized
under ultraviolet light and documented using HEROLAB gel documentation system.
RESULTS AND DISCUSSION
Callus induction from stem and leaf explants of in vitro grown plantlets
was observed on 1X MS medium with 3% sucrose, 1% agar fortified with 2 mg L-1
2,4-D (Fig. 1a) when compared to other concentrations and
combinations of 2,4-D and Kinetin. From all the different concentrations of
sodium azide containing medium multiplication of callus was only achieved on
1xMS 3% sucrose, 1% agar fortified with 2 mg L-1 2,4-D containing
200 μM of sodium azide.
||(a) Well developed callus on 1X MS medium with 2 mg L-1
2,4-D, (b) Shoot induction from the 4 min UV mutated callus cell line on
1X MS medium fortified with 2 mg L-1 BAP and (c) Well developed
Development of shoot and root from callus was achieved on 2 mg L-1
BAP containing medium after 24 day of incubation. The physiological estimation
revealed that protein (0.160±0.04 mg mL-1) and carbohydrate
(0.720±0.06 mg mL-1) contents in the cell lines (Fig.
2, 3). Callus exposed at different time intervals under
UV radiation showed multiplication of callus in all the exposures. Development
of shoot and root from callus was achieved on mg L-1 BAP containing
medium after 24 day of incubation (Fig. 1b, c).
The physiological estimation revealed that protein content (0.460±0.04
mg mL-1) in 6 min exposed cell lines and carbohydrate content (0.820±0.08
mg mL-1) in 4 min exposed cell lines which is high when compared
to control cell lines and other sodium azide and UV exposure cell lines (Fig.
2, 3). Mutation was confirmed through DNA fingerprinting
using RAPD markers. From all the 9 primers used OPK 01 gave best result with
thick bands and amplification of the same when compared to other primers used,
which shows strong sign of dominant expression of those bands which in turn
alter the genetic character and stability, which shows the variation in banding
pattern in mutated samples compared to control cell lines (Fig.
Daucus carota L. is an important vegetable crop with high nutritional
value. The changing climate, pollution, loss of vegetation, has affected the
crop quality and there is a need to improve the variety. The present study was
started with the aim to improve the cell lines through sodium azide and UV radiation
induced mutation. In plant tissue culture, media plays a very important role
for the regeneration. In the present investigation MS medium was used for the
growth and development of Daucus carota based on the previous literatures
(Pant and Manandhar, 2007; Tavares
et al., 2010). Callus induction was well achieved on 2 mg L-1
2, 4 D when compared to other concentrations and combinations of growth regulators
which coincides with the earlier findings (Yau et al.,
2008). Callus was exposed to UV radiation and sodium azide treatment with
different time intervals and concentrations, respectively. Callus exposed to
6 min UV radiation has shown an increase in the protein content when compared
to the other durations of exposure to the UV radiation. Sodium azide with 200
μM has shown no increase in protein content when compared to the normal
cell lines. The concentration of sodium azide above 200 μM was lethal for
the cell line which wont agrees with the previous studies (Kulthe
and Kothekar, 2011; Mostafa, 2011).
||Effect of sodium azide and UV mutation on Protein content
in cell lines
||Effect of sodium azide and UV mutation on carbohydrate content
in cell lines
||RAPD amplification profile of mutated cell lines of Daucus
carota L., 1: Control, 2: Sodium azide 200 μM, 3: UV 2 min, 4:
UV 4 min, 5: UV 6 min, 6: UV 8 min, NC: Negative control
Higher the concentration of sodium azide decrease in the callus growth was
supported by the results of Khan et al. (2004,
2006), Sinha and Lai (2007)
and Al-Gawwad and Makka (2009). Exposure to UV for 4 min
and sodium azide with 200 μM has helped in increase in carbohydrate content
when compared to the normal cell lines. UV radiation above 6 min has not supported
the proper callus development which wont agrees with the findings of Ehsanpour
and Razavizadeh (2005) in callus of Medicago sativa. The increase
in protein and carbohydrate content has been supported with the DNA fingerprinting
result with polymorphic banding pattern.
The results indicated that by sodium azide and UV mutation it was able to achieve the improvement of the cell lines of Daucus carota L. by positive mutation with the increase in the protein and carbohydrate content. Mutagenic treatments increase the genetic variability, which can be utilized for selection and improvement of the vegetable crops.
We acknowledge Dr. R. Chenraj Jain, President, Jain University Trust., Dr. N Sundararajan, Vice Chancellor, Jain University., Prof. K.S. Shantamani, Chief Mentor, Jain University and Dr. S Sundara Rajan, Director, CASB- Jain University, Bangalore for providing financial assistance, the necessary laboratory facilities and support. We also thank the staff at Genohelix Biolabs for technical support.
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