International Journal of Agriculture and Crop Sciences. Available online at www.ijagcs.com IJACS/2013/6-18/1279-1283 ISSN 2227-670X ©2013 IJACS Journal
In vitro Culture of the Peppermint Plant (Mentha piperita) without the use of Hormones Saeid Gharib Bolouk1*, Seyed Kamal Kazemitabar A. 2, Jafar Masoud Sinaki3 *
1 . Ms. Student in Agricultural Biotechnology, Damghan Branch , Islamic Azad University, Damghan, Iran. 2 Associated Professor of Plant Breeding and Biotechnology, Sari Agriculture Sciences and Natural Resources University (SANRU), Iran. 3 .Agronomy Department, Damghan Branch , Islamic Azad University , Damghan , Iran. .
*Corresponding Author email:
[email protected] ABSTRACT: The peppermint plant (Mentha piperita) is a medicinal plant that has many uses in hygiene and drug industries because of its many precious essences. This study was conducted at Sari Agricultural Sciences and Natural Resources (SANRU) in 2013 by planting small peppermint samples within MS in vitro media. Some morphological factors including stem length, root length, germinations per stem, number of progressive lateral roots, and also leaf number were analyzed, and results showed significant responses from the explants in this investigation (5% level). This study indicated that use of a culture media without hormones has a significant effect on the production of this medicinal plant. The best result was achieved from the explants that included end buds and lateral buds. Significant results (5% level) were achieved for the mean stem length of 3.22 ± 1 cm, mean leaf number per stem of 8 ± 1, mean leaf length of 0.5 ± 0.1 cm, and for the germination number of 2 buds in the stem’s length. Keywords: culture media, in vitro , medicinal plant ,Mentha piperita INTRODUCTION The Peppermint plant with the scientific name (Mentha piperita) is from the mint family of (Labiatae) and is an herbaceous perennial. Mint plants are so sporadic in the world that they can be found nearly anywhere, but their peak is in the Mediterranean area (Zarghari,1990-2002). This plant can be cultivated in areas with mild winters (minimum -8 degrees) and in soils with pH 5-8 (OmidBeigy, 2010). This plant spends the winter in depression and grows again in spring producing new branches (OmidBeigy, 2005). Its leaves are elliptic, cross, sharp, jagged, and slightly covered with fluff, and it grows to a height of 7-4 cm and 3-2 cm in width. The main use of peppermint is its essence (OmidBeigy, 2005) which is used in traditional medicines (Smith and Gould, 1989). Peppermint essence has anti-fungi traits (Bishos and Thronton, 1997 ; Pandey et al, 1996) and anti-microbes (Khan et al, 2003a,b ; Murray,1995; OmidBeigy 2010; Zarghari,1990-2002). Its oily essence includes menthols (30-40% or more), menthons (15-25%), methyl acetate (to 10%), and Menthoforan (less than 15%). Mentha piperita is the result of a modulation of Mentha aquatic and Mentha viridis, but one difference in these two types is that their spawn is not fertile; therefore, the two types lack the ability to sexually reproduce and are multiplied via vegetative propagation, meaning that this plant is reproduced exclusively by creeping stems that create buds and aberrant roots or by cultivating the tissue (SafaeiKhorram et al, 2008). In tissue cultivation an explant is usually placed in an artificial cultivating environment to initiate growth, and the inactive cells are differentiated and not reproduced. The explant initially suffers some changes to reach the meristematic mode during growth (Adam and Wendel, 2005). The choice of culture media or the preparation of its formulation is necessary for successful tissue cultivation. No specific cultivation area can be recommended for growing various cells; rather, changes in the cultivation area are made as a response to the variations of the explants. Under in vitro conditions, plant tissues and organs grow over the prepared artificial environments via the nutrients needed for growth. The successfulness of plant tissue cultivation is heavily influenced by the materials used in the cultivation area. Generally the cultivation area includes inorganic salts and compounds such as growth regulators, vitamins, carbohydrates, and a gel factor to freeze the cultivation area (Afsharipor, 1993;Bagheri and Safari, 2008). Sajana et al (2011) reported that using Mentha piperita explants during the branching stage, the highest number of branches was seen in those receiving a treatment of 2 milligrams per liter of BAP with a mean of 5 ± 0.69. and maximum branch height occurred in the environment which received 1 milligram per liter of BAP with a mean of 4.6 ± 0.2 centimeters. During the rooting stage, hormone treatments of 0.5-2 milligrams per liter of IAA, 0.5-2 milligrams per liter of NAA, and 0.5-2 milligrams per liter of IBA were used, and the best result was reported from the hormone treatment of 1.5 milligrams per liter of IBA with a
Intl J Agri Crop Sci. Vol., 6 (18), 1279-1283, 2013 mean of 48.5 ± 1.45 (Sujana and Naidu. 2011). In their study on the tissue plant of menthe piperita in node explants, SonandaKumar et al (2003) observed the best branching stage result from the hormone treatment of 4.44 micromoles BA and 2.32 micromoles KIN with a mean of 4.1. During the rooting stage, the best result was observed in the cultivation area which included hormone 2.69 micromoles NAA with a mean of 4.1 (Sunandakumari et al., 2003). Qnty et al (2003) reported that they used node explants and shoot tips in the tissue cultivation of Mentha piperita, and in the branching stage they used the samples in an MS environment including hormone treatments of 0.5-2 milligrams per liter BAP, 0.5-2 milligrams per liter KIN, and 0.25-1 milligrams per liter ZEATIN. Their best result was reported for the node explants in the cultivation area including 1 milligrams per liter BAP with a mean of 49.80 ± 1.71. Maximum height with a mean of 4.69 ± 0.08 centimeters was reported from the hormone treatment of 0.5 milligrams per liter KIN (Ghanti et al., 2003). The goal of the current study is to analyze tissue cultivation of the peppermint plant and various morphological factors within a glass environment. METHODS AND MATERIALS This study was conducted in 2013 at Sari Agricultural Sciences and Natural Resources. The peppermint plant samples (Mentha piperita) were prepared from the university’s farm and used for tissue cultivation after disinfection. In this study the Murashig and Skoog (MS) culture media was used for cultivating various plant organs, direct regeneration, and rooting (Vikrant et al, 2006). Plants were disinfected after the samples were transferred from the farm to the lab by washing for 5 minutes with running water to eliminate any dust. Afterwards, they were placed in dish soap for 15 minutes, again washed with running water for 5 minutes, and then sent to the cultivation module. The entire disinfection process of the samples was done beneath a hood in which the explants were placed in 1% Binomial + 3 drops of Tween in 100 cc of water for 1 minute, then washed with distilled water for 1 minute. Next, samples were set adrift in sodium hypochlorite (5%) for 7 minutes and washed again with distilled water for 30 seconds. After that, they were set adrift in 70% alcohol for 5 seconds, washed 3 times with distilled water for 3 minutes each time. The explants were then placed over a sterile filter paper and dried, and disinfection and sterilization was complete. The explants were chosen from lateral buds, terminal buds, stems, leaves, petioles, and roots (Bagheri and Azadi, 2002; Sujana and Naidu, 2011). Using a scalpel and forceps, the explants were cut into 0.5 cm parts and cultivated within 15 cm test tubes in a hormone-free MS environment. The lids of the cultivation dishes were blocked with Teflon, and the samples were transferred to an incubator set at a temperature of 25 ± 0.2°C. Lighting conditions were 16 hours light and 8 hours darkness for micro propagation and complete plant production. CONCLUSIONS AND DISCUSSIONS Results of explants growth in the MS culture media showed that the use of this medium without using hormones could have a significant effect in producing this plant on the 5% level (picture 1) and could also prove to be an appropriate method for creating direct regeneration. Various morphological ratios (stem length, leaf, root length, shoot length, of lateral roots and of buds per stem) were also calculated and analyzed.
a b c Figure1. The grown peppermint samples within the MS culture media
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Intl J Agri Crop Sci. Vol., 6 (18), 1279-1283, 2013 Sajana et al (2011) worked on regeneration from the shoot tip and bud explants of Mentha piperita. They stated that maximum regeneration speed was achieved in the MS culture media which included 1.5 milligrams per liter BAP alongside 0.1 milligrams per liter NAA. They also observed that the stems grew exactly from where the cells were cut. They said the maximum stem number was observed in the environment including BAP and NAA. For means of rooting, the samples were transferred to an environment including 1.5 milligrams per liter IBA. Sajana et al (2011) achieved maximum stems from the BAP hormone treatment with a mean of 5 ± 0.69 and maximum stem height with a mean of 4.6 ± 0.2. SonandaKumar et al (2003) achieved best results at the branching stage from the BA and KIN hormone treatment with a mean of 4.1. Qnty et al (2003) achieved best results from the bud samples within the BAP hormone treatment with a mean of 49.8 ± 1.71 and maximum height with a mean of 4.69 ± 0.08 centimeters from the KIN hormone treatment. For stem length, sample 12 had the least length at 1.5 centimeters, and sample 7 had the maximum stem length with a height of 4.5 centimeters. The best result was achieved among sprout samples in the MS culture media with a stem length of 3.22 ± 1 centimeters. 5.00
4.50
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4.00 3.50 3.00
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Figure2. Stem length
Sajana et al (2011) achieved their best results at the rooting stage from the IBA hormone treatment with a mean of (48.5 ± 1.45). SonandaKumar et al (2003) achieved their best results at the rooting stage from the NAA hormone treatment with a mean of 11.1. Among the root length samples, 3 had the least root length at 0.2 centimeters, and sample 8 had the most with 3.5 centimeters.
Average root length(cm)
4
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2.2 1.7
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Figure3. Mean Root length
For leaf number per stem, samples 3, 9, and 12 had the least number of leaves per stem with 6 leaves, and sample 7 had the highest number with 12 leaves per stem. The best result was achieved in the bud samples within the MS culture media for the leaf number per stem with a mean of 8 ± 1 leaves.
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14
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Number leaf
12 10 8
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Figure 3. Number of Leaf Production
Samples 1, 2, 3, and 6 had the least number of lateral roots with only 1, and samples 5 and 9 had the most with 7 lateral roots.
number of lateral roots
8
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Figure 4. Lateral Roots
Samples 5, 10, and 12 had the least number of buds per stem with only 1, and samples 13 and 14 had the most 4. Best results were achieved with a mean of 2 buds per stem.
Number of buds per stem
4.5
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Figure 5. Buds per Stem
For mean leaf length, samples 6, 9, and 12 had the least length with 0.3 centimeters, and sample 2 had the most with a leaf length of 0.7 centimeters. The best result was achieved from the bud samples within the MS cultivation area with a mean of 0.5 ± 0.1 centimeters.
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Average leaf length(cm)
0.8 0.7 0.6
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Figure 6. Mean Leaf Length
In this study, the best results for inner glass cultivation of the peppermint plant were obtained in the use of lateral and end buds as explants. No hormones were used in the cultivation area for this plant’s growth, but most other researchers report the use of plant hormones for the induction of main organs, i.e. roots, stem, and leaves. Based on the data, we can conclude that there is a direct connection between stem length and leaf number. As stem length increases, leaf number within the cultivation area also increases. Table1. BA BAP IAA IBA NAA
Benzyladenine Benzylaminopurine Indole3-acetic acid Indole3-butric acid 1-Naphthaleneacetic acid
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