Effect of genotype, explant type and 2,4-D on Cell dedifferentiation and
callus induction in cumin (Cuminum cyminum) medicinal plant
Abstract
Cumin (Cuminum cyminum L.) as a member of the Apiaceae family is one of the most important
medicinal plants in the world. An experiment was conducted for evaluation of callus induction
optimization in cumin accessions (Shahdad, Koohbanan, Badrood and Afghanistan). Factorial
experiment based on completely randomized design (CRD) with three replications was
conducted on MS medium supplemented with different concentrations of 2,4-D (0, 0.5, 1 and 2
mg/l) plus 0.1 mg/l Kinetin in different explants (Root, Shoot, Leaf, Embryo and Seed) of cumin
accessions. In this experiment the evaluated traits include days to callus induction (DCI), callus
induction percentage (CIP) and callus growth rate (CGR). Statistical analysis showed that seed
(as the latest) and root (as the earliest) explants require to 54 and 11 days in order to callus
induction initiation respectively. Results showed that accession, explant, accession × explant and
2,4-D × explant interactions were statistically significant effects (P<0.01) on callus induction
percentage and callus growth rate traits. Furthermore, 2,4-D had a high significant effect on
callus induction percentage. According to results of this study, root explant is an appropriate
explant for large production of callus in some accessions of Cuminum cyminum. Also,
Koohbanan accession produced callus in minimum time and Afghanistan accession produced
high percentage of callus.
Keywords: 2,4-D, Callus growth rate, Days to callus induction, Seed explant
Abbreviations:
2,4-D = 2,4-dichlorophenoxyacetic acid
PGR = Plant Growth Regulator
MS = Murashige and Skoog
CDI = Days to Callus Induction
CIP = callus induction percentage
CGR = callus growth rate
Introduction
Cumin (Cuminum cyminum Linn) is a commercial plant which is valued for its aroma and its
medicinal and therapeutic properties. Although India was the primary exporter of cumin seeds
and cumin oil in the world until recently, but comparatively Turkey and Iran are providing stiff
competition now. India, Turkey, Syria, China, the US, Iran, Indonesia, Sudan, Egypt, Morocco,
Algeria, and Libya are the leading producers of cumin in the world. Iran produce 15,000 to
20,000 tons of cumin seeds and stand third in the leading producer’s list (1).
Callus culture plays a special role in plant biotechnology area for producing medicinal
compounds in large-scale from plants (2). Also, callus masses derived from plant tissues can
sometimes produce high amount of secondary metabolites (3). Callus production from roots,
shoots and leaves are generally applied to determine the conditions required by the explants to
survive and grow, exploit products coming from primary and secondary metabolism, study cell
development and obtain cell suspension in propagation (2).
Callus culture of cumin has been previously reported by some researchers using leaf ((4-7),
embryo ((8-10) hypocotyl and cotyledon (5, 6) but there is no comparative study on callus
induction of this plant in various explants specially root and seed explants.
Jha, Roy (11) published the first study on the callus induction of cumin. They used the hypocotyl
and leaf explants in B5 medium complemented with various PGRs. Beiki, Mafavi-fard (12)
studied callus induction of three Iranian landraces with the use of embryo explant.
In spite of cumin importance in numerous industries such as medical industry, there are limited
studies on rapid and efficient callus induction in the world. Therefore, present experiment was
conducted in order to achieve the various objectives such as large-scale production of callus to
generate somaclonal variation, application in genetic engineering strategies and extraction of
valuable materials in this medicinal plant.
Material and Methods
The present experiment was conducted in Medicinal Plants Tissue Culture Lab., Agriculture and
Natural Resources Campus, Razi University in 2012.
Plant material
The experiments were conducted on four Cuminum cyminum seeds which were collected from
Shahdad, Koohbanan, Badrood (tree regions of Iran) and Afghanistan. The seeds were surface
disinfested following the procedure previously described (7).
One month after seed sowing, when the astatically grown seedlings were about 5 cm in height,
different explants include the root, shoot and leaf were prepared (Figure 1).
The sterilized seeds were culture directly on MS medium supplemented with different 2,4-D
concentrations. The mature zygotic embryo explant were prepared following Ebrahimie, Naghavi
(9) procedure (Figure 2).
Statistical analysis
All experiments were repeated three times. Before statistical analysis to normalise the
distribution, the callus induction percentage (CIP) was transformed by a root square
transformation (y=√(x + 0.5). Three-factor analysis of variance were performed to examine the
effect of the genotype, explant type and 2,4-D on callus induction characters in cumin. The
differences between means were compared using the LSD at a significance level of 0.05.
Studied factors were cumin accessions (Shahdad, Koohbanan, Badrood and Afghanistan), 2,4-D
concentrations (0, 0.5, 1 and 2 mg/l) plus 0.1 mg/l Kinetin and different explants of cumin
accessions (Root, Shoot, Leaf, Mature embryo and Seed).
The evaluated traits including days to callus induction (DCI), callus induction percentage (CIP)
and callus growth rate (CGR) were calculated as follow:
Days to callus induction (DCI):
Number of days from explant cultivation until callus initiation was recorded and used as a trait in
the comparison of treatments.
Callus induction percentage (CIP):
The callus induction percentage was calculated as the number of explants with callus divided by
the total number of explants and multiplying by 100.
Callus growth rate (CGR):
The callus growth rate (millimeter per day) was calculated five weeks after explant cultivation,
according to following equation [1]:
𝐶𝐺𝑅 =
(
𝐶𝐷1
𝐶𝐷2−𝐶𝐷1
𝐶𝐷3−𝐶𝐷2
𝐶𝐷4−𝐶𝐷3
𝐶𝐷5−𝐶𝐷4
)+(
)+(
)+(
)+(
)
7
7
7
7
7
4
[1]
Where CD1-CD5 are the callus diameter of explants after 7, 14, 21, 28 and 35 days, respectively.
Callus diameter (CD) for each callus explant was measured as following procedure:
CD = √𝐿𝑒𝑛𝑔𝑡ℎ 𝑜𝑓 𝑐𝑎𝑙𝑙𝑢𝑠 × 𝑊𝑖𝑑𝑡ℎ 𝑜𝑓 𝑐𝑎𝑙𝑙𝑢𝑠
[2]
Due to delay in callus production of seed explant and discordance with other explants, the CGR
was not calculated for seed.
Results and Discussion
The efficient callus production method was optimised in various explants of four local
accessions of cumin using MS medium supplemented with 2,4-D concentrations. The analysis of
variance (ANOVA) for some callus induction characteristics showed that genotype and explant
type had a significant effects on DCI, CIP and CGR traits at 0.01 level of probability and also,
2,4-D had a significant effects on DCI and CIP traits (Table 1). These observations imply that
2,4-D concentration can changed the callus induction but cannot change the callus growth speed.
Days to callus induction (DCI):
Callus induction were initiated in Koohbanan landrace at minimum time (Figure 3). Thus, this
genotype can be used in large scale production of callus at minimum time.
Although the time required for callus induction is an important traits in callus production in
plants, but this trait is not considered in the most of experiments. It is clear that lower value of
this trait is better. Because callus can be produced in shorter time and it is would be
commercially valuable.
So far, no report has been published in related to DCI characteristics of the callus induction of
cumin in detail, although some reports have generally cited the time to callus induction (8, 11).
In general, higher levels of 2,4-D (videlicet 1 and 2 mg/l) accelerated the callus induction in
cumin accessions (Figure 4).
The callus was induced more rapidly in root explant (about 11 days), where it occurred latest
(about 54 days) in seed explant (Figure 5).
It can be reported that shoot explant produced the more proper callus for plant regeneration
(Figure 6), because vitrification was not observed and callus culture was not very bright. Some
calli of these experiment were regenerated and reported previously (13).
It seems that callus was induces from mature embryo of the seed explant (Figure 7). In spite of
technically easiness of callus production from seed, due to later callus induction in seed, it’s
recommended the using of mature embryo instead of seed explant for sooner callus production.
Results of interaction effects analysis (Figure 8) revealed the responses of treatments in details,
so that roots of Koohbanan landrace produced callus in the minimum time (about 8 days).
As shown in figure 9, in roots of cumin accessions on MS medium supplemented with 2 mg/l
2,4-D callus was induced in the lowest time (about 10 days).
Callus induction percentage (CIP):
Callus induction percentage is one of the most important traits of callus induction that reported in
most callus experiments. Afghanistan accession produced the most percentage of callus in
comparison with other accessions (Figure 10).
Tested levels of 2,4-D had a same effect on CIP trait, which had statistically significant effect
with control (Figure 11).
The seeds of cumin accessions showed the lowest CIP, which root and shoot explants produced
the most callus induction percentage (Figure 12).
Roots and shoots in all of the genotypes and also leafs of Koohbanan, Badrood and Afghanistan
genotypes produced the most CIP (Figure 13).
Mean comparisons for CIP in various explants and different 2,4-D concentrations showed that
different levels of 2,4-D (0.5, 1 and 2 mg/l) produced the most CIP in root and shoot explants,
but produced the lowest in seed (Figure 14).
Callus growth rate (CGR):
The results shown in Figure 15 indicate that Koohbanan, Badrood and Afghanistan genotypes
had the highest CGR with an average of 0.276, 0.268 and 0.277 mm/day, respectively, and
Shahdad had the least amount of CGR (about 0.214 mm/day).
The highest and lowest calculated CGR with an average of 0.318 and 0.210 mm/day was
obtained in root and embryo explants, respectively (Figure 16). This result illustrated that root
explant is an appropriate explant for large production of callus, because produced callus rapidly
and had the highest callus growth rate.
As seen in Figure 17, callus growth rate is a genotype-dependent process. According to this
figure, the highest CGR was observed in Afghanistan and Koohbanan accessions (0.378 and
0.359 mm/day, respectively).
The results of explant×2,4-D interaction revealed that the highest CGR obtained in root explants
(in all of the 2,4-D concentrations) and also shoot explant on MS medium plus 1 mg/l 2,4-D.
Conclusion
Study on callus induction in cumin is very useful applications, such as somaclonal variation,
plant regeneration, suspension culture and secondary metabolites studies. Though Koohbanan
accession produced callus in minimum time, but the high percentage of callus was observed in
Afghanistan accession. According to results of this study, root explant is an appropriate explant
for large production of callus in some accessions of Cuminum cyminum.
Declaration of interest statement
The authors report no conflicts of interest. The authors alone are responsible for the content and
writing of this article. The Razi University, Iran, has financially supported this research project.
Appendices
References
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Organ Culture. 2007;90(3):293-311.
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12.
13.
Kahrizi D, Soorni J. Study on shoot regeneration and somatic embryogenesis in
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Figure 1. Cumin seedling with a height of 6 cm
suitable for the preparation of root, shoot and leaf
explants
Figure 2. Mature zigotic embryo with a length of
2 mm (left) and drive embryo explant (right)
30
c
c
days to cullus induction
25
b
a
20
15
10
5
0
Figure 3. Mean comparisons of days to cullus induction in
cumin accessions
24
b
23.5
days to cullus induction
23
22.5
22
21.5
a
21
a
20.5
20
19.5
19
0.5
1
2
2,4-D (mg/l)
Figure 4. Mean comparisons of days to cullus induction in 2,4-D
concentrations
60
d
days to cullus induction
50
40
30
20
a
b
c
b
10
0
Root
Shoot
Leaf
Embryo
Seed
Figure 5. Mean comparisons of days to cullus induction in different explants
of cumin
Figure 6. Calli of shoot explant on MS
medium supemented with 0.5 mg/l 2,4-D in
Shahdad accession
Figure 7. Calli of seed explant on MS medium supemented with 0.5 mg/l 2,4-D in
Shahdad accession after 50 days
65
i
i
55
days to cullus induction
h
h
45
Shahdad
Koohbanan
35
Badrood
Afghanistan
25
g
15
de
fg
ef
ef
ef
cde
ab
a
ab
Root
Shoot
bcd
cde
de
abc
ef
de
5
Leaf
Embryo
Seed
Figure 8. Mean comparisons of days to cullus induction in different explants and accessions
of cumin
65
g
days to cullus induction
55
f
f
2,4-D
(mg/l)
45
0.5
35
1
2
25
e
15
bcd
ab ab a
cde de
ab ab
bc bc ab
5
Root
Shoot
Leaf
Embryo
Seed
Figure 9. Mean comparisons of days to cullus induction in different explants and 2,4-D
concentrations in cumin
60
a
callus induction percentage
b
50
b
b
40
30
20
10
0
Figure 10. Mean comparisons of callus induction
percentage in different accessions of cumin
callus induction percentage
80
70
60
50
40
30
20
10
0
a
a
a
0.5
1
2,4-D (mg/l)
2
b
0
Figure 11. Mean comparisons of callus induction percentage
in different 2,4-D concentrations
80
a
callus induction percentage
70
a
60
b
50
c
40
30
20
d
10
0
Root
Shoot
Leaf
Embryo
Seed
Figure 12. Mean comparisons of callus induction
percentage in different explants of cumin
80
a a a
a a
callus induction percentage
70
a
a
a
a ab
60
a
Shahdad
50
c
bc
40
c
bc
Koohbanan
c
Badrood
c
30
Afghanistan
d
20
e e
10
0
Root
Shoot
Leaf
Embryo
Seed
Figure 13. Mean comparisons of callus induction percentage in different explants and
accessions of cumin
120
a
callus induction percentage
100
a a
ab ab
abc
80
cd bc cd
0
60
de
e
0.5
1
e
2
40
f f
20
g
g
g
g
f
g
0
Root
Shoot
Leaf
Embryo
Seed
Figure 14. Mean comparisons of callus induction percentage in different explants
and 2,4-D concentrations in cumin
callus growth rate (mm/day)
0.3
a
0.25
a
a
b
0.2
0.15
0.1
0.05
0
Figure 15. Mean comparisons of callus growth rate in
different accessions of cumin
callus growth rate (mm/day)
0.35
a
0.3
b
c
0.25
d
0.2
0.15
0.1
0.05
0
Root
Shoot
Leaf
Embryo
Figure 16. Mean comparisons of callus growth rate in different
explants of cumin
0.4
a
ab
bc
cd cd
0.3
de
0.25
def e-h
e-h
hi
def
f-i
ghi ghi hi
i
0.2
Shahdad
Koohbanan
Badrood
0.15
Afghanistan
0.1
0.05
0
Root
Shoot
Leaf
Embryo
Figure 17. Mean comparisons of callus growth rate in different explants and
accessions of cumin
0.35
a ab ab
b ab
0.3
callus growth rate (mm/day)
callus growth rate (mm/day)
0.35
b
c
0.25
c
d
0.2
cd cd cd
2,4-D
(mg/l)
0.5
0.15
1
0.1
2
0.05
0
Root
Shoot
Leaf
Embryo
Figure 18. Mean comparisons of callus growth rate in different explants and 2,4D concentrations in cumin
Table 1. Analysis of variation for days to cullus induction, callus induction percentage and
callus growth rate in Cuminum Cyminum L.
days to cullus
callus induction
induction
percentage
d
Mean
callus growth rate
Mean
d
Mean
df
Source of variations
f
squares
squares
f
squares
Genotype (G)
3
443.10**
3
8.70**
3
0.032558**
2,4-D
2
140.85**
3
721.91**
2
0.001427 ns
Explant type (E)
4
12018.16**
4
165.38**
3
0.077247**
6
ns
0.003808
G × 2,4-D
5.69
9
2.76
ns
6
ns
1
G×E
1
**
28.41
2
2,4-D × E
7.99**
9
0.012505**
19.50**
6
0.010824**
1
0.002165
2
8
1
65.29**
2
2
G × 2,4-D × E
3
16.58 ns
4
2.49 ns
6
1
Error
1
10.79
20
ns
8
9
1.75
60
0.002104
6
Coefficient of
15.09 %
22.40 %
17.73 %
variation
ns, * and ** means no significant, significant at 0.05 and 0.01 level of probability