Vol-7, Special Issue-Number2-April, 2016, pp1390-1397 http://www.bipublication.com
Research Article
Effects of EMS on Number of Internodes of the Medicinal Plant
Lemon Verbena
Zahra Maloumi
Agricultural Department, Damghan Branch, Islamic Azad University, Damghan , Iran.
*Corresponding author: Email: zahramaloumi@yahoo.com,
Tel: +989191245578
ABSTRACT:
Induced mutation is a method used in plant breeding for increasing genetic diversity. In this research, the effects of morphological changes resulting from mutation induced by the mutagenic chemical ethyl methylsulfonate (EMS) on increasing morphological diversity in the medicinal plant lemon verbena were evalu.The research project was carried out in a factorial experiment with two time levels (30 and 60 minutes) and four EMS concentrations (25, 50, 75, and 100 µM) factorial arrangement using the randomized completely design. The seedlings grown in the tissue culture medium were exposed to the mentioned concentrations of EMS. The studied morphological traits included.Results of ANOVA related to all of the mentioned traits suggest beneficial changes took place in some of the traits because of the mutations. Increased concentrations of EMS, and longer exposure time to it, caused changes in some traits such as plant height, fresh and dry weight of stems, and root length compared to the control plants. Results of ANOVA and comparison of the obtained means using Duncan’s test lead to the conclusion that , The greatest number of internodes related to the concentration of 50 M was created with treatment time of 60 minutes and the average of 7 internodes per stem.
Keywords: Lemon verbena, mutagen, tissue culture, internod , EMS.
[I]INTRODUCTION
The human has traditionally used the plants to treat the diseases so that, in the ancient books like the Bible and the ancient Hindu scriptures (Vedas), the use of some plants has been recommended to treat the diseases [1].
It is estimated that more than 10% of the thousands of known plant species have medicinal uses [2,3]. The World Health Organization has estimated that about 80% of the world’s population use medicinal plants for health care aspects of their own. Today, in industrialized communities and in many developed and developing countries, the use of traditional medicine and medicinal plants for good health
because of increased public confidence in the use of these plants is very significant.
Almost a quarter of the drugs made in the world have plant origin. Some of them have been directly extracted from the plants and some others have been modulated or synthesized based on herbal blend.
Plant tissue culture techniques have become strong tools to overcome the fundamental and applied problems of plant biology.
Therefore, using biotechnological approaches (such as tissue culture and mutation) can be very beneficial and commercially profitable in order to reproduce and enhance the genetic potential of medicinal plants as well as faster and more accurate identification of the genotypes that produce more products.
To upgrade in vitro biosynthesis of pharmaceutical metabolites, those plants are often selected that enjoy high efficiency in the production of valuable metabolites .Mutagens can substantially help in increasing this efficiency. Mutation is an effective tool for plant breeding and improvement of the agricultural products [4] . Radiation and chemical mutagens are the most common methods for creating artificial mutation in plants [5]. Plant breeding by mutation on major field crops has prompted many experts to research in this area. Accordingly, in the last 70 years, about 270 mutant varieties from 170 different plant species have been established in 60 countries in the world by mutation [6].
Mutation is a change in the genetic structure (genotype) of organisms whether this change has phenotypic manifestations or not. The mutational change is called mutation and the product of such change is called mutant. Mutation is a process of mutating that changes the cell germplasm collection and finally, creates a living being with new genetic features or in other words, mutation is a genetic change that leaves a lasting effect on the chromosome and creates a mutant.
Mutagens is a general name of the substances which artificially induce the mutation. In other words, it is a general name of the substances that the biological effects of their mutations are far more than that occur naturally or accidentally. One of the extraction method of plant active ingredients is the use of solvent. Various chemical
solvents such as ethanol, methanol, acetone, glycerin and glycol can be used [7].
According to geneticists, there are two kinds of mutation:
1. Genetic mutation: one allele related to a gene is changed and becomes another allele. Since this change happens within a gene on a chromosomal location (point), it is sometimes called point mutation.
2. Chromosomal mutation: it includes the parts of chromosome, the entire chromosome or even a set of chromosomes that often affects the order of chromosomes and genes.
Types of Mutation:
a. Natural or spontaneous mutation: natural mutation often occurs due to cosmic rays and low frequency in nature. The frequency of this kind of mutation is very low that identifying and finding the appropriate changes is usually difficult and in fact, it appears as a deterrent in breeding programs.
b. Induced mutation: an agent which creates mutation, called mutagen (mutation-causing) and using it, induced mutation can be artificially created in organisms. It is used in a form of recombination or combination and/or both of them in plant breeding.
In fact, mutation provides the bed for the emergence of genetic potential that do not occur naturally by creating new variation. Induced mutation have important contribution to identification of genetic mechanism, particularly to understanding of the structure and function of genetic materials.
Types of Mutagens: 1. Physical factors
Figure 1: Structure of mutagens
Of tens of chemical mutagens, EMS is the most common chemical mutagen for this purpose [8]. EMS (Ethyl Methyl Sulfonate) is a chemical substance that provides the connection of the carbon groups of Alkyl to the existing range in the Nucleotide molecule of DNA and causes alkylation. Because of the alkylation of DNA molecules, point mutation occurs that most of them are the changes from G to A. The use of this substance can cause a variety of mutations in living being. EMS as a factor of point mutation, causes a wide range of mutant alleles, such as the removal of efficiency, creation of efficiency, development of new traits, change in efficiency and the production of new mutants with special properties. while, the mutations created by gamma rays, often cause the elimination or addition or the growth of Nucleotide and the incidence of dysfunctional mutant and access to other types of mutants is very difficult [9]. The chemical substance of EMS creates point mutation in the genome that these mutations occur randomly and mutant plant with new phenotype may emerge. Lemon verbena (also called lemon bee brush) is a medicinal plant of the family Verbenaceae and order Lamiae’s with the scientific name of Lippia citriodora. It is called “beh-limo”, “verbon”, “aromatic lokise vervaine”, “lemon Vervaine” or “arabic tea”, in Persian it is called “lovizeh” in Arabic and it is called “verveine odorante” in French.
It is a deciduous perennial tree and, depending on weather conditions, reaches the height of 1.5-2 meters. Its stems, depending on the climate, are long, branched, and angular. The leaves are simple, rough, lance late, oblong, and 7-10 cm long with pointed tip and round base, pale green, arranged in triplets round the stems, and very fragrant [10]. It flowers are small and in a form of cup that is white from outside and blue-violet from inside and they are in a form of panicle inflorescence at the end of the stem (Figure2).
Figure 2: Morphological organs of Lemon verbena
being of great importance. requires warm air, abundant light, and high humidity, is sensitive to cold, needs light loamy soils, and pH of 5.6 is suitable for it. Fresh leaves and vegetative body of the plant contain essential oil that smells the lemon. geranial, linalool, mega geranial, Carmen phenyl oxide, and crinoline oxide the other basic constituents [10, 12, 13]. Together with vitamins A, E, and C. Limonene It also lowers blood pressure, prevents throat and larynx inflammation, stimulates bile production in the liver, is used in the perfume industry to perfume cosmetics and to make colored soaps, air fresheners, flavor ants, and is employed as a solvent in the production of resins and moisteners.
The leaves contain 0.9-1.5% Laugier essential oil. This essential oil is lighter than water, its color is bright greenish-yellow and it smells lemon. This essential oil is often mixed with citrus oils and of 100 kg of this plant’s leaves, 510 g essential oil is extracted. Depending on the time of harvesting, planting location, method of sampling, amount of extraction and type of ingredients in the leaves of lemon can be associated with fluctuations and differences. This medicinal plant is antipyretic, analgesic, anti-flatulent, and helps food digestion, is a tranquilizer, relieves stomachache and abdominal cramping, removes heart palpitations, and is a stomach tonic and It is used to relieve nerve pain and mental fatigue and to lower the body temperature. It is used to refresh the body and to relieve the symptoms of the cold [14]. Researchers have reported that lemon verbena essential oil suppresses the stimulating effects of histamine and has anti-diarrheal properties, Spasms and lytic effect of oil on ileum isolated from Guinea Pig [15]. It is also used as spice in the home for making meat, fish, jam, puree, etc. fragrant. Essence of Lemon Bee brush can be used to enhance the memory.
This plant also has antimicrobial effects due to various metabolites, especially terpenoids Vitally in its essence and in different studies, its effects in removing oral microbial flora, particularly Gram-positive bacteria and controlling diarrhea have
been referred [16]. The effect of this plant in inhibiting the growth of Escherichia coli, Mycobacterium tuberculosis and
Staphylococcus aurous has been shown in in vivo studies and possibly by destroying the cell membrane of bacterial [17].
[II] MATERIALS AND METHODS
Lemon verbena seedlings grown in vitro on MS culture medium that was free of any infections were used to conduct the experiment. Four EMS concentrations (25, 50, 75, and 100µM) and two treatment times (30 and 60 minutes) with three replications were used (with zero concentration as the control). Moreover, a 0.1M sulfate buffer with pH of 7 was required for the experiment . monopotassium phosphate (KH2OP4) and di potassium phosphate (K2OPH4) were used to prepare the buffer. To prepare the culture medium, stock solutions of macro- and micro-nutrients, vitamins, and iron- sodium were separately prepared and kept in a refrigerator at 4 C˚ [18]. After preparing the mutation environments, the micro samples were transferred to the tubes. When the treatment time determined for each treatment ( 30 or 60 minutes) was over, the seedlings were taken out of EMS solutions, washed with distilled water, and transferred to the MS culture medium that had been prepared beforehand. The temperature in the growth chamber was 25 C˚ and it was adjusted to the 16 h light/8 h darkness cycle. One week later, all treatments and replications were observed and statistical data collected to study the effects of EMS on the samples and on some of their morphological traits. All data was entered into Excel .analyzed by SPSS in the format of completely randomized design, and ANOVA and comparison of the means using Duncan’s test were performed for all experiments.
[III] RESULTS
and 60 minutes), on number of internodes were not significant at the 5% probability level. The effects of EMS concentration on this trait were significant at the 1% probability level, but the effects of the treatment times on plant height were not significant at the 5% probability level. Comparison of the means using Duncan’s test
indicated that the tallest plants (12.13 cm) were those of the 50µM concentration of EMS with the 60- minute treatment time, and that the treatments of zero and 25µM concentrations were not significantly different and could be placed in the same group (Diagram 1), (Table 1), (Figure 3).
Diagram 1: The effect of concentration on the mu,ber of internodes chart EMS
Sov Df SS MS F
concentration 2 2.949 1.475 **11.866
Time 1 0.036 0.036 0.290ns
concentration *Time 2 0.297 0.149 1.195 ns
E 12 1.491 0.124
Total 17 4.774
Ns,*,**:non significant, significant at p<0.05 and p<0.01, respectively
Table 1: Analysis of variance EMS and time interaction on number of internodes Lippia Citriodora
a- Control plant
b- EMS at 25µM and 60-minute treatment time
c- EMS at 50µM and 60-minute treatment
[IV] DISCUSSION
The effects of the EMS mutagen on various plants studied by other researchers were compared to those found in this study in order to compare the results. In EMS tests on the rice by Fotsu et al.
[19], it has been reported that there is a significant and positive correlation between the length of cluster and the number of internodes, in our study, also, it is concluded that there is a positive and significant relationship between the stem length and the number of internodes that both of them are increased by increasing the concentration.
Researchers in studied the effects of mutation induction by gamma rays on increasing diversity in some traits of second-generation gillyflower. They noted that mutation induction beneficially enhanced the extent of trait diversity in plants (reduced height in the Centum White variety and increased it in the Callum Lavender variety). These results conform to ours because increased EMS concentration and longer treatment times produced taller mutant plants, which was a suitable trait for the goals of the experiment. In a study on the effects of chemical mutagen EMS with the concentration of 140 Mm on the cultivar of Tarom rice, the role of mutagen in diversifying the agronomic traits has been studied. In studying on the plant height, the results showed that the plant height of mutagen treatment increased significantly compared to control sample and also, this trait shows the highest heritability and genetic progress.
In another research, plants treated with EMS and those not treated with it were studied employing RAPD-PCR and standard primers. It was found that differences in the bands obtained from DNA amplification in PCR of plants treated with EMS for 15 minutes were clearly visible compared to the other samples.
In their assessment of chlorophyll a and b, they concluded that the concentrations of chlorophyll measured in the various samples were not significantly different (p≥0.05).
In another study on the effect of mutations induced by chemical mutagen of ethyl methane
suffocate and relationships between traits in sainfoin in which 40 third generation mutant genotypes and 39 non-mutant genotypes were studied in terms of morphological and agronomic traits under field conditions for two years, reported that comparing the means in years showed that in the second year, the mutant families have more forage yield and leaf to stem ratio than non-mutant families.
Aswit [20] in his study on barely, reported that gamma radiation and EMS treatment have increased genetic diversity of flowering time and cluster length in the families of generation M2. In a study on the effect of combined treatment of gamma and EMS, it was reported that this treatment created the most mutation sequences compared to separate treatments of gamma and EMS, while EMS treatment had the greatest impact and efficiency compared to gamma treatment and combined treatment [21].
[V] CONCLUSION
Most morphological changes in plant traits have occurred at a concentration of 50 mM of EMS in the treatment time of 30 and 60 minutes and the use of the concentrations which are higher than 50 mM of EMS in the treatment time of 30 and 60 minutes caused the loss of tissue culture samples. Results of this research suggest the presence of diversity in morphological traits of both the mutant and the non-mutant groups. After complete establishment, the mutant plants could perform better in many of the physiological traits compared to the control plants.
These results indicate that mutant plants with more suitable performance can be produced if various concentrations of the mutagen and different treatment times are selected.
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