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Callus induction and plant Regeneration of cordia myxa L. via tissue culture system

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IJOPILS. APR 2016; 4(5):18-29
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International Journal of Pharmacy and Integrated Life Sciences
“Where improvisation meets innovation”
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RESEARCH ARTICLE ISSN: 2320 - 0782 V4-(I5) PG(18-29)
Callus induction and plant Regeneration of cordia myxa L. via
tissue culture system
Alaa Jabbar Taha*1
1. College of Science, Al-Mustansiriya University, Baghdad, Iraq
ABSTRACT
In the current study Leaves cordia myxa cultured on MS medium containing a different
concentration of NAA, BA (0, 0.5, 1, 1.5, 2 mg / l). The highest percentage of Callus induction
96% at a concentration of 1mg / l NAA and 1.5 mg / l BA and had maintenance Callus on this
medium, fresh and dry weight arrived to the highest rates in this combination as it reached 479,
95.30 mg respectively. For production of shoot branches, callus cultured on media containing 2mg
/ l BA and o.5 mg / l NAAwhich had significance as the number of branches reach to 8.93 branch,
branches rooting in the media equipped with IAA at1.5 mg / lconcentration which gave the highest
rate of the number of roots 6.28 root and 5.73 cm for the length of the roots. The highest percentage
acclimatization of plantlets reached 81% when culture on one part of river sand to two part of
peatmoos.
KEYWORDS: Cordia myxa L., Callus induction, Regeneration, Acclimization.
Article received on: 19/03/2016 Article accepted on: 19/04/2016
Corresponding Author: Alaa Jabbar Taha
Address : College of Science, Al-Mustansiriya University, Baghdad, Iraq
Email : alaataha1966@yahoo.com
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INTRODUCTION
Larsa or Assyrian Plum Cordia myxa L. belongs to Boraginaceae family of plants, which contain
more than 148 genera and 2470 species. Plants of this family are spread in temperate and tropical
regions of the Mediterranean areas (1) in Iraq are grown widely in the province of Basra. The fruits
are slimy mucilaginous pulp eaten fresh. Plant flowers to a white and yellow fruit color becomes
darker more mature, viscous liquid inside a transparent gum Diabetes is one of the excellent fruits
because they contain most of the vitamins (2).
Enables (3) to get the highest percentage Callus induction when cultured internode of
Heliotropium indicum on MS medium equipped with 2 mg / l of BAP and 3 mg / l NAA, (3)
succeed to obtain shoot branches when culture node and apical shoot on MS medium containing 1
mg / l of BAP where stimulate formation buds vegetative of both and reached the composition of
branches to 78% percent and reached the ratio to 92% when adding the Kin at 1mg / l and the
number of shoot branched reached to 54 branch .
The explant node of Cordia myxa cultured on MS medium added 4 mg / l of Kin and 0.01 mg / l
NAA has given shoot branches (4), and separated these branches and cultured on MS medium
containing 2 mg / l NAA, 1 mg / l IBA plus 750 mg / l of charcoal for rooting, the media gave a
higher percentage of rooting significantly. Able (5) to obtain the highest percentage of Callus
induction by cultured leaf explant of Echium amoenum on MS medium provider BAP + NAA at
concentration 1mg / l for both the regulator, and this MS medium supplemented with kin had given
the highest rate of regeneration from Callus induced of cultured leaves explant of Echium amoeum,
(6) reach to get the highest percentage of Callus which induced from the node explant of
sericostoma pauciflorum cultured on MS medium was equipped with 2,4-D at 1.5 mg / l and
reached 90%, Callus induced on MS medium equipped IAA at the concentration of 3mg /l, which
gave the highest rate of shoot branches and this rooted on MS medium equipped with IAA
concentration at 1.5mg/l, which gave the highest rate of the number of roots/plantlet.
MATERIALS AND METHODS:
Research conducted in the laboratory of plant tissue culture / Biology Department / College of
Science / Al-Mustansiriyah University. Young tree of cordia myxa taken from nurseries in
Baghdad one year old and the explant took to be cultured on the medium.
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Attended the MS solid medium (7) at laboratory by dissolving all the salt, then add the sucrose
concentration of 30 gm / l and growth regulators NAA, BA at the concentration (0, 0.5, 1, 1.5, 2
mg / l) and adjust the PH to 6.5 to 8.5 by adding a few drops of Hcl or NaoH, Normality 1N to
reach the required number, then add agar at concentration of 7 gm / l to the above ingredients then
completed by distilled water to 1L. Mix ingredients using a hot plate magneticsterrer to melt the
nutrient media components, and sterilized the medium at a degree 121Co Autoclave and pressure
1.04 Kg / cm2 for 20 mint. The explant includs (leaves and juvenile stems) and washed with
running water for 15 minutes and immersed with alcohol ethyl concentration at 95% for a period
of 30 second and washed with water sterile distilled for a period of 10 mint and the explant
immersed with Sodium hypochlorite at the concentration 2% for 10 mint then washed explant with
sterile distilled water for several times to remove the effects of sterilization solution and all steps
were done under a Laminar air flow. explant were cultured on MS medium containing a
combination of growth regulators concentration (0, 0.5, 1, 1.5, 2 mg / l) for each of the BA, NAA
at 10 replications and each repeater contain 3 explant for each parts leaf and stem to calculate the
percentage of Callus induction for each vegetarian part. Cultures were incubated in the incubator
at a temperature of 25 ± 1 Cº for 4 weeks and then were measured fresh and dry weight of Callus.
Callus induced from leaf transfer to the medium of callus maintenance containing 1.5 mg / l BA,
1mg / l NAA, which gave the highest percentage of Callus induction. Callus induction was sub
culture on this medium for four weeks and incubated in the same incubator 25±1Cº in light
conditions. Callus induction was transferred to Regenerated media which was MS medium
provided with BA concentration (0, 1, 2, 3, 4 mg / l) and NAA concentration (0, 0.5, 1 mg / l). We
cultured 4 pieces of Callus in each bottle and repeated 10 replicate for each combination and placed
in constant condition light and at a temperature of 25 ± 1 Cº. The shoots branches were rooting on
MS medium containing a different concentration of IAA (0, 0.5, 1, 1.5, 2 mg / l) and incubated at
the same conditions referred to previously and at a rate of 3 shoots / vial and every treatment repeat
5 replicate.
For acclimatization the rooted plantlet were taken out and tenderly in tap water to remove all traces
of the medium and treated with pesticide Benlate for 10 second at 1 ml / l concentration, after that
plantlets were planted in plastic pots on different mixtures of river sand and peatmoos different
proportions (0:1, 1:0, 1:1, 1:2) and by 5 replicates per treatment and placed in culture room under
the intensity of illumination 1000 lux for 16 hours a day and covered in plastic transparent to
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maintain the high rate of humidity , plantlet watering from tap water and raise them cover gradually
and finally lift, estimation the percentage of success rate of plantlet adapted after 4 weeks. All the
experiment were conducted in using the Randomized Complete Bloke Design (RCBD) and
averages were compared by multiple border Duncan test at the level of 5% probability (8).
RESULTS AND DISCUSSION
Effect of different concentration (BA, NAA) in the percentage of Callus induction:
The effect of a combination of growth regulators BA, NAA concentration (0, 0.5, 1, 1.5, 2 mg/l)
in Callus response of both the stem and leaves, this combination is the best in Callus induction of
the leaves,(Fig. 1) but the stem response was very weak for Callus induction, So been excluded
from subsequent experiments, may be the reason of that the stem cells differentiated and slow
division and their need for longer in order to divide and grow (9) or to the type of vegetation and
the source of explant totipotency in plant parts and different cells in the preparation capable of
division in these parts cells as well as the internal content of the hormones (10.11).
Leaves are as the best explant in Callus induction within this combination of growth regulators.
the results of Table (1) shows the concentration of BA significant effect in the percentage of Callus
induction, the highest value significantly reached to 60.4% at a concentration of 1.5 mg / l, while
the lowest rate arrived, a significantly 4.4% at a concentration of 0.0 mg / l. The concentration of
NAA affected significantly of Callus induction percentage, the highest percentage ratio recorded
44.4% at a concentration 1 mg / l and the lowest percentage significantly was 14.6% at a
concentration of 0.0 mg / l.
Table (1): the effect of various concentrations of BA, NAA mg/l and the combination in the
callus percentage of leaf cordia myxa After 4 weeks in MS medium.
NAA
BA
0
0.5
1
1.5
2
Average
0
on
on
on
12 lm
10 lm
4.4 e
0.5
on
7 mn
10 lm
30 ghi
25 hij
14.4 d
1
16 kl
23 ijk
39 def
56 c
40 de
34.8 c
1.5
25 hij
78 b
96 a
60 c
43 d
60.4 a
2
32 fgh
60 c
77 b
35 efg
20 jk
44.8 b
Average
14.6 d
33.6 bc
44.4 a
38.4 ab
27.6 c
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Fig. (1): callus derived from leaf explant of cordia myxa on MS medium
As for the combination between the concentration of BA, NAA in the percentage of Callus
induction, the results in (Table 1) show the highest percentage of Callus induction significantly
96% at a concentration of 1.5 mg / l BA, 1mg / l NAA, the lowest Callus induction rate was 7% at
a concentration 0.5 mg / l for each of the BA, NAA. Callus induction at a concentration of 0.0 mg
/ l BA and the concentration of 0, 0.5, 1 mg/l NAA was zero also did not Callus induction in
treatment at 0.5 mg/l BA, 0.0 mg/l NAA. The combination of BA concentration 1.5 mg/l and NAA
concentration of 1mg/l was Callus maintenance callus medium, it was friable and color Off White.
Through the results shows that increasing the concentration of BA, NAA led to a decrease in the
percentage of Callus induction may be due to the high concentration led to reduce the rate of cell
division (12) or the addition of BA, NAA to the medium culture at concentration higher than the
ideal level has played a role a negative impact on the work of the enzymes responsible when
building a cell wall which affects the mechanical properties and the impact on cell division and
the formation of Callus (13). Perhaps the difference in plant parts planted response in Callus
induction back because of auxin attributed to cytokinins additives may be due to different internal
content of these parts of the interior of hormones and this in turn affects the attainment of the ideal
focus in Callus induction of auxin to cytokinins or both when added to the culture medium (14).
The effect of different concentration of BA, NAA in fresh and dry weight of Callus:-
The results (Table 2, 3) showed the concentration of BA, NAA had significant effect for both fresh
and dry weight, the highest rate of weight significantly reached (281.8, 42.12 mg) for each of the
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fresh and dry weight respectively at a concentration of 1.5 mg/l NAA and the concentration of BA
behavior the same as it was, the highest rate significant for both fresh and dry weight (337.6, 60.90
mg) at 1.5 mg/l of BA concentration respectively. The combination between the concentration of
BA, NAA significant effect on the rate of both fresh and dry weight, the highest rate reached to
(479, 95.30 mg) respectively at a concentration of 1mg/l NAA, 1.5 mg/l BA. The lower rate (115,
7.90 mg) at the treatment 0.5 mg/l BA, NAA for both fresh and dry weight, respectively.
Table (2): the effect of different concentration of BA, NAA mg/l and the combination in the
fresh weight of callus after 4 weeks on MS medium.
NAA
BA
0
0.5
1
1.5
2
Average
0
om
om
om
183 hgi
161 ij
68.8 d
0.5
om
115 l
153 jk
264 f
194 gh
145.2 c
1
127 kl
166 hij
288 ef
318 cd
276 cf
235 b
1.5
200 g
361 b
479 a
350 b
298 de
337.6
2
270 ef
297 de
337 be
294 de
189 ghi
277.4 b
Average
131 e
187 d
251.4 b
281.8 a
223 c
Table (3): the effect of different concentration of BA, NAA mg/l and the combination in the dry
weight of callus after 4 weeks on MS medium.
NAA
BA
0
0.5
1
1.5
2
Average
0
on
on
on
17.02 j
10.16 lm
5.43 e
0.5
on
7.90 m
12.98 kl
24.63 h
18.55 j
12.81 d
1
9.66 m
13.79 k
43.15 f
57.49 cd
35.11 g
31.84 c
1.5
21.76 i
71.03 b
95.30 a
60.22 c
56.20 d
60.90 a
2
32.40 g
52.60 e
59.40 c
51.25 e
19.10 ij
42.95 b
Average
12.76 c
29.06 b
42.16 a
42.12 a
27.82 b
Through the results it shows that the increase of BA, NAA concentration lead to the less fresh
and dry weight for Callus, The reason for this is due to the higher concentration may lead to reduce
the rate of cell division (12). The addition of growth regulators concentration higher than the ideal
level may adversely affect the work of the enzymes responsible for cell wall construction which
affects the mechanical properties and its effect on cell division and the formation of Callus.
Increasing of the concentration of BA until they reach the ideal concentration lead to increase fresh
and dry weight for Callus, possibly due to the effect of cytokinins in increased cell division
especially merstamic cell and this in turn leads to increased volume of different tissues of plant
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members, whether they are connected to plants mother or separated from it and culture among
sterile medium(15), good growth of Callus happen in the culture medium due to the physiological
balance between auxin and cytokinins and increase the concentration of any of them over the other
negatively affect the growth of Callus (16).
The effect of the concentration of BA, NAA and the combination between them in the
number of shoots branches.
The results (Table 4 and Fig. 2) showed the effect of BA, NAA concentration on the number of
shoots branches, the highest average of the number of shoot branches a significant rate (6.09)
branches at 2 mg/l concentration of BA and was the lowest rate significantly of the number of
shoot branches (1.06) at 0.0 mg/l BA concentration. The highest rate significantly of the number
of shoots branches (5.07) at a concentration 0.5 mg/l of NAA while the lowest rate 2.32 branch at
0.0 mg/l concentration of NAA. The combination between the concentration of BA, NAA
significantly effect in increasing the number of shoots branches, the highest rate significantly of
the number of branches 8.93 at 2 mg/l concentration of BA and 0.5 mg/l NAA, and the lowest rate
of the number of branches reached 1.15 at 0.0 mg/l concentration of BA and 1 mg/l NAA, and the
number of shoot branches rate to 0 at the control treatment.
Table (4): the effect of different concentration BA, NAA and the combination in the average
number of branches from callus Cordia myxa in MS media.
NAA
BA
0
0.5
1
Average
0
of
2.03 def
1.15 f
1.06 d
1
3.66 c
5.17 b
3.03 cd
3.95 b
2
3.27 c
8.93 a
6.08 b
6.09 a
3
2.88 cde
5.50 b
3.56 c
3.98 b
4
1.84 ef
3.75 c
1.18 f
2.24 c
Average
2.32 c
5.07 a
3.00 b
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Fig. (2): Shoot branches induction from callus of Cordia myxa on MS medium after 4 weeks.
Perhaps the reason for the low rate of number of shoot branches to increase the concentration of
BA that may lead to accumulation in the explant as well as the contents of it and thus reaches to
the level of inhibitory and thus lower the number of branches and this is consistent with what he
referred to (17) in that the high concentration of cytokinins in media for the ideal limit could lead
to reduce the number of shoot and growth on the explant of Plectranthus brabatus. They noted
(18, 19) they noted that the viability of differentiation returning to several factors, including the
source of explant genetic and medium. And (20) observed the role of growth regulators in the
medium and source of explant and the type of plant to stimulate differentiation process. They
proved (21) the importance of the combination between auxin and cytokinins stimulate branches
of the plant Cordia myxa and this is attributable to the role played by the balance between the
growth regulator to determining the differentiation histological pattern and composition of the
members in vitro.
The existence of high concentrations of cytokinins and low concentration of auxin in the culture
medium to the formation of buds to unfold the shoot branches (22). (23) Showed that auxin works
on genes that stimulate the cytokinins control of gene expression, and gene expression products
play a fundamental role in biological processes such as cell division and chloroplast development
and metabolism of nutrients elements. The lack of control treatment in the formation of branches
may be response due to the lower inner content cytokinins and auxin for explant.
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The effect of the concentration of IAA in the number and lengths of roots: -
The shoot branches resulting from Callus Cordia myxa not be rooting which couldnt to transport
to soil so were cultured on MS medium of rooting containing different concentration of IAA. Table
5 and Fig. 3 show the concentration of 1.5 mg/l IAA has given the highest average number of roots
and a significant (6.28 ) root/branch while control did not response of rooting. Also root length
was affected by a concentration of IAA, the highest rate significantly of root length was 5.73 cm
at a concentration of 1.5 mg / l IAA, compare to the other the treatment. Increase the concentration
of IAA lead to reduce the number of roots and length a significantly. It is clear from these results
that the auxin role in rooting process as the content of shoots grown from a auxin an important role
in stimulating the root primordial from the base of shoot branches culture. Division of root
primordial depends on the concentration of total internal auxin Endogenous or added Exogenous
to the culture media so, the physiological effect of auxin representing an increase of cell division
and transfers the differentiation cells to merstamic cell and thus merstamic cell consists roots (24).
The decrease rate of the number and length of roots to increase the concentration of IAA has led
to an increase in the ideal concentration causing inhibition of root primordial process and thereby
reducing the number of root growth.
Table (5): the effect of different concentration IAA in the number of the roots and lengths rate (cm) in MS
media.
Concentration of IAA mg/l
The number of roots
Root length (cm)
0
Od
Od
0.5
2.70 c
2.03 c
1
3.96 b
3.45 b
1.5
6.28 a
5.73 a
2
3.87 b
2.90 b
Fig. (3): plantlet rooted on MS medium supplemented with IAA after 4 weeks.
Acclimatization:- The agriculture medium have an effect on the success rate of acclimatization
plantlet (table 6 and fig. 4) increased success rate to 81% when plantlet transferred to agriculture
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medium consist of river sand and peatmoos 1:2 v/v ratio while the lowest rate of plantlet
acclimatization success 13% in the medium of peatmoos only. The reason for the high success of
the acclimatization of the plantlet in the agriculture medium contain river sand and peatmoos 1:2
v/v ratio because the ability of peatmoos to retain moisture and nutrients,which allowing the spread
and growth of roots and good to drain water, The reason for the decline in the success rate of
acclimatization of plantlet when grown in peatmoos only, may be due to poor ventilation and
retaining water by much more than the need for the plantlet, which led to their death (25).
Table (6): effect of the kind cultured media on the percentage of the plantlets success
acclimatization after 4 weeks of culture.
river sand : peatmoos
The percentage of success acclimatization
0 : 1
13
1 : 0
25
1: 1
64
1 : 2
81
Fig. (4): plantlet acclimatization in culture room condition
CONCLUSION:
It had been found that leaves best explant for callus induction compared with the stem, and the
Addition of growth regulator BA and NAA at the concentration 1.5, 1 mg / l, respectively, on MS
medium to give the highest percentage of Callus induction and the highest rate of fresh and dry
weight under light condition. The combination of BA concentration of 2mg/l and 0.5 mg / l of
NAA gave the highest rate of the number of shoots per callus. In MS medium containing the 1.5
mg / l from IAA superiority on giving the highest rate of number and length of roots significantly.
Agriculture medium contain river sand and peat moose 1:2 (V/V) ratio superiority in the rate of
plantlets acclimatization success.
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