Vol-7, Special Issue3-April, 2016, pp1031-1037 http://www.bipublication.com
Research Article
Study of Callus Formation, Regeneration, and Possibility of Mucilage
Production in the Medicinal Herb Flax
Sahar Karimi1 and Leya Shooshtary*2
1Department of Plant Breeding, Kermanshah Branch,
Islamic Azad University, Kermanshah, Iran.
2Department of Plant Breeding, Kermanshah Branch,
Islamic Azad University, Kermanshah, Iran.
*Corresponding author: Email:L_shooshtary@yahoo.com,
Tel: +98-8337243181 ; Fax: +98-8337243194
ABSTRACT:
In this research, the responses of roots and leaves of seedlings produced from in vitro germination of flax seeds were studied. For this purpose, a factorial experiment including culture media with 10 experimental treatments each with three replications that consisted of combinations of micro samples and hormone was carried out using the randomized complete block design to study the extent of callus formation. Three hormonal combinations were used to prepare the culture media for the callus regeneration phase, and the MSFUL was employed as the base culture medium. Results of data analysis showed the most suitable culture medium for callus formation in flax was culture medium number 4(1 mg/L NAA and 2 mg/L BAP). Regeneration results indicated culture medium number 2 enjoyed the maximum callus growth rate, and regeneration culture media numbers 1 and 3 had the same callus growth rates. Culture medium number 2 was suitable for regeneration, callus formation, and callus size, while culture medium number 3 was more suitable in relation to the quantities of produced Flavonols and Flavonoids, and culture medium number 1 with respect to the amount of produced mucilage and harvest index
Keywords: Flax (Linumussitatissimum), callus formation, regeneration, mucilage, hormone
[I] INTRODUCTION
Linumussitatissimum is one of the most important herbal medicine in pharmacology, cosmetics industry. Linumussitatissimum is herbaceous, annual, belonging to flax, Geraniaceae, this plant is originated in westMediterranean [4]. The flax seed is used in pharmacology and paste is provided from the powdered seeds of flax. By plant tissue culture, it can be a good ground to keep the mother genotypes in nature as valuable sources of Germplasm[12]. One of the most important feature of using tissue culture to proliferate the plant include rapid proliferation of best cultivars and mother plants, the increase of production capacity at short time, non-pathogenic
Mucilages are biopolymers with high molecular weight with great range of physicochemical properties highly applied in pharmacology, industry, cosmetics and medicine. This material is synthesized naturally in normal growth of plants [7]. Mostly, the main source of them is seed but is found in fruit, leaf, flower and other plant organs [12].
[II] MATERIALS AND METHODS
This test was conducted in herbal medicine tissue culture laboratory of Islamic Azad University of Kermanshah. The applied micro samples were root and leaf provided from the plants of nucleation of flax seedling under glass condition. To disinfect the seeds, they were washed for 30s with Ethanol (70% v/v) and then for 20 minutes with sodium hypochlorite 2.5% treatment and finally three times with sterile distilled water under laminar hood. If sterile condition is not applied, culture medium is contaminated rapidly [3]. Microorganisms including bacteria and fungus grown on substrate change the chemical environment rapidly. Suitable capping and autoclaving tubes, bottles in tissue culture can eliminate these microorganisms [8].
The medium can be disinfected totally at temperature 120 for 20min at pressure 15 bars in autoclave or it can be disinfected by sodium hypochlorite for 10-30 minutes [10]. Suitable pH of culture medium is of great importance. Acid or alkaline pH affects the food absorption in tissue culture [2]. Thus, the culture medium should have pH of 5, 6-6. For seed nucleation, after their disinfection, the seeds are cultured in petri dish in MS medium as one of the most suitable media of plant tissue culture and include three types of highly applied food, low applied food and vitamin without hormone [11].
2.1. Experimental treatments and statistical design
In Callus Induction stage, the effect of two micro sample factors (at two levels) and hormone combination (at five levels) was studied asfactorial test (with 10 experimental treatments)
in totally random design with three replications on flax callus induction. Some properties including callus induction, wet weight, dry weight, callus diameter, average callus growth, callus water content, mean diameter of callus, Mucilage percent, Mucilage harvest index and Mucilage yield [9]. As shown in Table 1, hormone combination in callus induction is as follows:
Media code
Hormone combination 2,4-D
(mg/l)
BAP
(mg/l) NAA(mg/l)
A 2 1 0
B 0 1 1
C 0 1 2
D 0 2 1
E 1 0.5 0
Table 1- Hormone composition in callus induction
2.2. Callus induction
In callus induction stage, 5microsamples were placed in each culture medium and for each treatment (combination of hormone and micro sample), three replications were considered. Then, the cultured samples were transferred to growth chamber for callus induction at temperature 25 and 16-hour light and 8 hours darkness photoperiod to perform dedifferentiation. As shown in Figure 1, callus induction of samples was observed after 30 days of micro samples culture.
After 6 weeks, the entire dish was full of callus. During this period, the properties including callus induction percent, callus diameter, average growth velocity of callus, dry and wet weight of callus,
relative water content of callus,
Mucilagepercentage, Harvest index of Mucilage andMucilage yield were registered. After taking notes of experimental data, to evaluate the effect of studied treatments, the attributes were analyzed as factorial test at totally random design using SPSS software. Duncan multi-range test was used to compare the mean of treatments. Then the charts were plotted in Excel and the results were
F igure 1- Callus induction, 30 days after culture
[III] RESULTSANDDISCUSSION
The results of variance analysis of callus percent, wet and dry weight, callus diameter, average growth of callus, relative water content of callus, mean diameter of callus, Mucilage percentage, Mucilage harvest index and Mucilage yield, Flavonols and Flavonoid after thirty days at Murashige and Skoog culture medium showed that there was a significant difference between the studied media in terms of Flavonols and
Flavonoid at the level 1%. There was a significant difference between wet weight and Mucilage yield at the level 5%. The effect of medium on callus induction percent, wet weight, dry weight, and callus diameter, average growth of callus, and relative water content of callus, the mean diameter
of callus, Mucilage percentage and
Mucilage harvest was not significant. Indeed, different hormones had no significant difference at different media and this showed that genetic potential of these genotypes in production of different callus at various hormone levels had not significant difference.
3.1. The comparison of the mean callus induction and callus size at 5 different media The results of the comparison of the properties mean by Duncan method in Table 2 showed that callus induction percent for most of flax samples in different media was 100%. The comparison of the genotype mean for callus size showed that the highest size of callus was dedicated to medium 2 with the mean 19.78 and the lowest value was dedicated to medium 3 with value 14.93.
F la v o n o id F la v o n o ls H ar v es t in d ex M u ci la g e y ie ld W at e r re la ti v e co n te n t M u ci la g e D ry w ei g h t o f ca ll u s W et w ei g h t o f ca ll u s C al lu s in d u ct io n p er ce n t C al lu s d ia m et er M ed iu m 8 1 .4 1
B 12
2
.6
D 1.1
3 7 A B 3 4 1 .4
B 21
.7
5
A 17
.1
5
A 18
.3
5
A 43
8 .3 A B 1 0 0
A 15
.0 7 B 1 7 5 .7 6
C 12
2
.6
E 0.6
5
B 34
1
B 21
.7
A 10
.7
3
A 34
.9
8
A 75
6
.7
A 10
0
A 19
.7 8 A 2 8 7 .1 2
A 12
8
A 2.8
5
A 46
7
.2
B 24
.4
6
A 35
.5
5
A 12
.9
7
A 33
6
B 96
.6
7
A 14
.9 3 B 3 6 4 .3 6
D 12
4
C 1.3
8 2 A B 1 3 3 8
A 21
.9
A 37
.5
8
A 76
.9
8
A 76
5
A 10
0
A 16
.8 6 A B 4 5 8 .7 9
E 125
.7
B 1.0
4 3 A B 3 4 3 .1
B 22.7
8
A 15.9
7
A 20 A 500 AB 96.6
7
A 15.4 B 5
Table 2- The comparison of the mean of callus properties at 5 different media
Mucilage
The comparison of the mean genotype for Mucilage in five different media shows that the highest mucilage of callus (37.58mg) is dedicated to medium 4 and the lowest value (10.73mg) is dedicated to medium 2. The comparison of the mean genotype for mucilage harvest index in five different media hassignificant difference and the highest mean harvest index of mucilage of callus (2.85mg) is dedicated to medium 3 and the lowest value (0.65mg) is dedicated to medium 2. As shown in Chart 1, the mean mucilage yield at different media is compared and we can say BAP, NAA hormones had high effect on Mucilage yield of flax and 2, 4-D hormone has low effect compared to two other hormones in Mucilage yield of flax.
Chart 1- The comparison of the mean yield of Mucilage at different media
3.3. The comparison of the mean attributes of callus induction in two different micro samples The results of comparison of the mean based on callus percent, wet weight, dry weight, callus diameter, average growth of callus, relative water content of callus, the mean diameter of callus, Mucilage percent, Mucilage yield, Flavonol and Flavonoid showed that leaf microsample had the highest mean in terms of these properties. The mean of Mucilage harvest index in root was more than that of leaf. Based on the results of Table 3, leaf samples of flax had better response to tissue culture and root micro sample had weak callus induction in terms of properties and the chart of callus diameter and wet weight of two micro
samples in five different media of callus induction showed that leaf micro sample had highest mean compared to root micro sample.
Micro sample Properties Root
Leaf
Callus diameter 14.13
18.68
Flavonols 119.84
125.38
Flavonoid 71.2
75.77
Callus induction percent 97
100
Mucilage value 20.3
26.5
Wet weight of callus 322
797
Dry weight of callus 14.5
50.8
Mucilage harvest index 1.47
1.35
Mucilage yield 353.44
778.98
Relative water content 20.4
24.67
Table 3- The comparison of the mean of callus properties in two different micro samples
As shown in Chart 2 regarding the comparison of the mean of flavonoid in different media and two micro samples, among the micro samples of root, medium 1 (1mg/ I BAP, 2mg/I 2, 4-D) had the highest effect on flavonoid of callus. Among the micro samples of flax leaf, medium 3(2mg/ I NAA, 1mg/I BAP) had the highest mean of flavonoid. We can introduce medium 3 for micros maple of leaf and medium 1 for root micro sample as the optimal medium to increase flavonoid value. The lowest value of flavonoid was dedicated to Medium 5 of two micro samples.
Chart 2- The comparison of the man Flavonoid at different media and two micro samples
and rot had no difference at different media. The highest value of Flavonol was dedicated to leaf micro sample at medium 1 and the lowest Flavonol to root micro sample at medium 2.
Chart 3- The comparison of the mean Flavonol at different media and two micro samples
The relationship between callus induction properties at 5 different mediaThe results of correlation analysis of measured properties of
callus in callus induction at five different callus media are shown in Table 4. The correlation coefficients between callus diameter and Flavonol (-0.916), between Mucilage yield and dry weight of callus (0.918) and between Mucilage harvest index and relative water content (0.904) are significant at the level 5%(P>0.5). This shows that these properties are correlated genetically to each other and there is a negative and significant correlation between callus diameter and Flavonol. The high correlation coefficient between callus diameter and Flavonol based on millimeter diameter per day shows that the higher the diameter of callus, the lower the flavonol in callus, the correlation between two properties of mucilage yield and dry weight of callus, mucilage yield index and relative water con tent are positive and significant. By the increase of relative water content, Mucilage harvest index is increased and by increase of dry weight of callus, Mucilage yield is increased.
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Callus diameter
1
Flavonol
0
.9
1
6
-*
*
1
Flavonoid
0
.1
3
0
-
0
.0
2
3
-
1
Callus induction percent
0
.5
5
8
*
*
0
.6
5
5
-*
*
0
.0
4
0
Mucilage value 0 .3 8 4 -* 0 .6 5 0 * * 0 .1 1 7 0 .1 7 6 - 1
Wet weight of callus
0 .8 3 3 * * 0 .6 7 1 -* * 0 .5 0 2 -* * 0 .6 6 7 * * 0 .1 1 6 - 1
Dry weight of callus
0 .4 1 8 * 0 .1 7 2 - 0 .4 8 5 -* 0 .5 6 6 * * 0 .4 2 4 * 0 .8 1 6 * * 1
Mucilage harvest index
0 .5 7 7 -* * 0 .7 0 8 * * 0 .5 4 1 * * 0 .5 7 5 -* * 0 .7 4 9 * * 0 .6 4 7 -* * 0 .2 5 9 - 1 Mucilage yield 0 .0 7 4 0 .2 0 5 0 .3 6 2 -* 0 .3 3 8 * 0 .7 3 3 * * 0 .5 2 6 * * 0 .9 1 8 * * 0 .1 0 1 1
Relative water content
0 .5 4 1 -* * 0 .6 8 1 * * 0 .3 5 9 * 0 .8 5 4 -* * 0 .5 1 5 * * 0 .6 9 9 -* * 0 .4 6 2 -* 0 .9 0 4 * * 0 .1 4 9 - 1
Table 4- The correlation coefficients of callus induction at five different media
3.4. The relationship between regeneration properties at three different media
The results of correlation analysis of the properties of callus in callus regeneration stage in three different regeneration media are shown in Table 5. The results of correlation analysis showed that the highest correlation between dry weight of callus and Flavonoid (-1.00) and this correlation is negative and significant, it means that by increase of dry weight, flavonoid is reduced. The lowest correlation is regarding the correlation between callus percent and mucilage yield (0.059).
C a ll u s d ia me te r F la v o n o l F la v o n o id C a ll u s in d u ct io n p e rc en t M u c il a g e v a lu e C al lu s w e t w e ig h t C al lu s d ry w e ig h t M u ci la g e h ar v e st in d ex M u c il ag e y ie ld Callus diameter 1 Flavonol 0 .8 9 3 -* * 1 Flavonoid 0 .4 0 3 -* 0 .7 7 2 * * 1
Callus induction percent
0 .1 3 9 - 0 .3 2 2 -* 0 .8 5 0 -* * 1 Mucilage value 0 .2 9 1 -* 0 .1 7 1 - 0 .7 5 9 -* * 0 .9 8 8 * * 1
Wet weight of callus
0 .9 9 4 * * 0 .8 3 8 -* * 0 .3 0 0 -* 0 .2 4 7 -* 0 .3 9 4 -* 1
Dry weight of callus
Mucilage harvest index
0
.7
9
2
*
*
0
.4
3
2
-*
0
.2
4
0
*
0
.7
1
5
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*
0
.8
1
5
-*
*
0
.8
5
4
*
*
0
.2
6
8
-*
1
Mucilage yield
0
.9
8
0
*
*
0
.9
6
4
-*
*
0
.5
7
5
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*
0
.0
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.0
9
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3
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0
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5
1
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5
6
*
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Table 5-Correation coefficients of regeneration properties at three different media
[IV] CONCLUSION
The best medium for callus induction of flax is dedicated to medium 4(1mg/I NAA, 2mg/I BAP) and BAP, NAA hormones have high impact on callus induction of most of properties of flax and 2, 4D hormone has less effect compared to two other hormones. In callus of these media (1mg/I 2,4-D+0.5mg/I BAP, 2mg/I NAA+1mg/I BAP, 1mg/I NAA+1mg/1 BAP), rooting was defined and flax leaf micro samples showed better response to tissue culture and root micro sample in terms of callus induction had weak response. In mutual effect of micro sample and medium, the micro samples provided from flax leaf compared to root micro sample in most properties showed better reaction compared to different media of callus culture. Among different media with different hormone concentrations, medium 1, 3 in terms of properties mean had superiority compared to other media. According to regeneration results, medium No. 2 had the highest trend of callus growth and callus growth was similar at regeneration media 1, 3. In terms of callus regeneration and callus size, Medium 2 is good. In terms of Flavonol and Flavonoid production, regeneration medium 3 and in terms of Mucilage, harvest index, regeneration medium 1 was good.
ACKNOWLEDGEMENT
This paper is supported by Islamic Azad University of Kermanshah branch.
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