International journal of Agronomy and Plant Production. Vol., 3 (6), 196-201, 2012 Available online at http:// www.ijappjournal.

com ISSN 2051-1914 2012 VictorQuest Publications

Assessment and determination of optimum concentration of streptomycin and kanamycin as selective agents in peppermint (Mentha piperita L.) transformation
Shirin Bahrampour , Majid Shokrpour , BahramBaghban
1 2* 3

1- Former MSc. student, Faculty of Agriculture, Tabriz University 2- Dept. of Horticulture, College of Agriculture and Natural resources, University of Tehran 3- Dept. of Agronomy, Faculty of Agriculture, Tabriz University *Corresponding Author Email: shokrpour@ut.ac.ir Abstract Antibiotics as selective agents play a remarked role in development of plant transformation technologies. In this research, the effect of streptomycin and kanamycin antibiotics as selective agents was studied. Plant samples were provided from greenhouse and in vitro cultures. The experiments were analyzed in a split plot in time based on completely randomized block design. Moreover, for quantification of the variation due to the effect of the antibiotic on plant samples, chlorophyll a, b and carotenoid were measured. Results of analysis of variance (ANOVA) showed a significant concentrationday interaction at 0.01 level. The concentrations would be caused whiteness of apexes and lateral buds and finally, plant death. Kanamycin concentration of 100 ppm was in shoot and bud samples caused whiteness of apexes and lateral buds during 30 to 45 days and finally plant death. The concentration of 50 ppm over 45 days resulted in death of leaf samples. The concentration of 250 ppm was the optimal dose. Analysis of variance of different concentrations was significant at 0.01 level for photosynthetic characters including chlorophyll a, b, total chlorophyll, chlorophyll ratio a/b and carotenoid. Changes in mean values of these treatments for all the pigments showed a decreasing trend.In conclusion, streptomycin concentration of 250 ppm and kanamycin concentrations of 50-100 ppm were the optimal doses to select non-transformed peppermint tissues. The results of this study revealed that the optimum doses of the antibiotics would had high efficiency in selection of non-transformed peppermint tissues which may be proposed in transformation researches. Keywords: kanamycin, peppermint, selective agent, streptomycin and transformation. Introduction Peppermint, Mentha piperita L., from the Lamiaceae family is a herbaceous and perennialplant that is important in terms of medicinal and economical aspects (Omidbaigi, 1998). Regarding to significant pharmaceutical effects, it is grown in many countries to produce essential oil and its various products. The first place for cultivation area of peppermintbelongs to United States with 51 million hectares (worth of 5.5 billion dollars) (Veronese et al, 2001). Peppermint breeding by conventional methods is difficult because of being sterile (Kransyanski et al, 1998; Veronese et al, 2001).Therefore, use of biotechnological approaches to increase its essential oil has been remarked. All plant transformation systems include one of the techniques ofcloned DNA transfer to plant living cells, identification or selection of cells containing the DNA inside the genome (nuclear or chloroplastic) and also regeneration or recovering fully developed plants from transformed cell. Since,small numbers of cells are just transformed, recovering chance of transformed lines are very low without making selection (Miki and McHugh, 2004). The most stable transformation methods apply markers that provide survival of transformed cells under selection system condition (Bowen, 1993). Selective agent may be antibiotics, herbicides, drugs or similar metabolites, a phytohormone precursor and

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or carbon source (Haldrap et al, 1998). Antibiotics play an important role as selective compounds in the development of plant transformation technology. In culture medium containing antibiotics, plant tissue show chlorosis due to lack of chlorophyll synthesis which eliminates growth and death of cells finally (Brasileri and Arago, 2001, Yu et al, 2003, and Miki and Mc Hugh, 2004). Kransyanskiet al (1999) used 20 mg/l (20 ppm) of kanamycin as selective agent in order to transform peppermint by limonene synthase gene. They suggested that the higher concentrations of kanamycin (25 to 100 ppm) strongly prevented thebranch regeneration. In order to peppermint transformation with antisense copy of menthoforan synthase wereapplied 50 ppm of kanamycin to select transformed plants (Mahmoud and croteau, 2001). Because of crucial role of selection systems in gene transfer protocols and regard to medicinal importance of peppermint, this study was run to assess antibiotics of streptomycin and kanamycin effects on the plant and to determine the most appropriate concentration of them for removing non transformed tissues. Materials and methods Plant material:the branch explants were prepared from greenhouse grown plants. In attention to significant of in vitro plant tissues in determination of optimum dose of selective agents, some samples were surface sterilized and cultured under in vitro conditions. Treatments: the explants included branches and leaves. The four nodes branches with same sizes were surface sterilizedand put in test tubes containing MS liquid medium without sucrose and with concentrations of 0,250 and 500 ppm of streptomycin and concentrations of 0, 50 and 100 ppm of kanamycin. The leaf samplesafter surface sterilizing were cutand divided into two parts, then the explants were placed inpetri dishes containing semi solidMSmediumwithout sucrose. The Concentrations were same as test of branches. The tests were evaluated in two replications.To evaluate the antibiotics effects on pigments changes of the samples, chlorophyll isolation from the leaves were done. DMSO (dimethyl sulfoxide)was applied as solvent. Then, to determine contents of chlorophyll a and b and carotenoids, absorbance spectra at wave lengths of 645, 663 and 480 nm were measured by spectrophotometer (Arnon, 1949; Sairam et al, 2002; Anon., 2006). Statistical analysis: changes in all the cultured explants in the different media were scanned by digital camera (Nikon corp., Coolpix 6 model) from the first day till the full effect of the treatments.At the end of the experiment, the image ofthe samples, were ordered in based on the studied days and ranked by changes range. The changes were as discoloring, whitening and death of the samples. The change range was ranked from 1 to 5. So the 1st and the highest ranks were assigned to the healthy plants and the final status of plants, respectively. Analysis of variance was done based on a split plot in time with the basic design of CRD. Variance analysis of data for photosynthetic pigments was done based on completely randomized design with 2 replications. All mean comparisons were carried out using Tukey at 5% probability level. Results and discussion Streptomycin experiment:anova results showed significant effect of doseday interaction for both antibiotics of streptomycin and kanamycin. In the branch explants affected by streptomycin, the changes were started from 20th day with rooting in control treatment and with discoloring and non-rootingof apical buds in treatments of 250 and 500 ppm doses (fig. 1A). Maximum difference among control and the streptomycin doses obtained in 40th daythat led to discoloring apical and new lateral buds and eventually plant death. In leaf explants were obtained same results (fig. 1 B, 2). But in the apical bud explants, discoloring in MS medium containing streptomycin were started from 10th day with rooting in control treatment. Difference in seedling colorsinfluenced by different concentrations of streptomycin was quite obviousin days of 15 to 25th after planting as increase in the concentration was along to more intense whiteness, too (fig.1C). But 30 days after planting, all concentrations were caused to uniform whiteness of the samples. Response of in vitro explants was begun within 5 daysafter culture initiation, also after 30 days, the apical buds were become completely white (fig.1 D, 3 ).

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Figure 1. Changes trends of different explants (A. branch B. leaves C. apical buds from greenhouse plants D. apical buds from in vitro plants) affected by streptomycin

Figure 2.discoloring of apical buds, death of leaves and non-rooting of explants in MS media containing streptomycin.A. 0 B. 250 and C. 500 ppm

Figure 3. The different doses of streptomycin (left to right: 0, 250, 500 and 1000 ppm) inhibited length growth of internodes and rooting in apical buds

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Kanamycin experiment:Changes of branch explants cultured under different concentrations of kanamycin were as death of terminal buds, new lateral buds and eventually death of the explants.The changes were visible in the start of 25 days after planting. The concentration of 100 ppm caused to death of th explants from 35 day (fig. 4 A, B). In this experiment, the rooting only occurred in the control. The rooting in control treatment was found within 15 days, also increasing length and volume of roots occurred gradually. th Butno rooting was obtained in any kanamycin doses even till day of 45 . In Leaf and apical bud explants that cultured in petri dishes, negative effect of kanamycin was observed in dose of 100 ppm from day of 15 after plantingthat was appeared as necrosis and gradual death of explants (fig. 4 C, D). Kanamycin effect on apical buds of greenhouse grown plants were observed as rooting in controland lack of rooting in other doses within 15 days after culture initiation. Significant difference was obtained among dose of 300 ppm and other treatments within 20 to 45 days after culturing. Gradual process of concentration effect in the samples during the evaluation period was quite evident as the apical buds of seedlings in 50 ppm andconcentrations of 100, 200 and 300 were discolored and white, respectively. On the other hand, the seedlings were normal stem growth under control condition whilestop or reduce in internode growth was seen in samples containing kanamycin.

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Figure 4. Changes trends of different explants (A. branch B. leaves C. apical buds from greenhouse plants D. apical buds from in vitro plants) affected by kanamycin Changes in photosynthetic pigments: Significant effect of streptomycin on chlorophyll a, b, total chlorophyll, chlorophyll a to b ratio and carotenoids was found in in vitro explants. Mean comparisons of the traits showed significant difference between control and other concentrations (Table 1). While it was obtained no significant difference among the concentrations of 250, 500 and 1000 ppm for total chlorophyll and carotenoids. Changes in mean values for all the pigments of the treatments suggest reducing them is proportional to the concentration of streptomycin. Streptomycin treatments showed significant difference for all traits except chlorophyll b in greenhouse explants. Similar in vitro explants, significant difference between control and other concentrationswas observed (Table 2). It was found considerable variation for photosynthetic characteristics

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among kanamycin doses in both greenhouse and in vitro explants. Kanamycin reduced the amount of chlorophyll a, b, total chlorophyll and carotenoids. The amount of chlorophyll in greenhouse samples at all concentrations of kanamycin had no significant differences (Table 4). While concentrations of 100, 200 and 300 ppm had no significant differences for the all pigments in the in vitro explants (Table 3). The in vitro explants due to their fragile and tender cellular tissues responded quickly to kanamycin concentrations and showed a decrease in photosynthetic parameters. Table 1. Mean of pigments values in explants from in vitro plantsunder different doses of streptomycin Streptomycin Chlorophyll Total Chlorophyll Chlorophyll a carotenoid dose (ppm) b chlorophyll a/b 0 1.42a 0.48a 1.91a 0.50a 2.97a 250 0.23b 0.13b 0.36b 0.16b 1.85b 500 0.17c 0.13b 0.30b 0.12bc 1.33b 1000 0.07d 0.06c 0.13c 0.08c 1.33b Table 2. Mean of pigments values in explants fromgreen house plants under different doses of streptomycin Streptomycin dose Total Chlorophyll a carotenoid Chlorophyll a/b (ppm) chlorophyll 0 2.31a 3.14a 1.18a 2.82ab 250 0.57b 1.28b 0.71b 1.30a 500 0.69ab 1.02b 0.45c 2.10ab 1000 0.08b 0.09c 0.07d 5.85b Table 3. Mean of pigments values in explants from in vitro plants under different doses of kanamycin Kanamycin Chlorophyll Chlorophyll Total Chlorophyll carotenoid dose (ppm) a b chlorophyll a/b 0 1.75a 0.52a 2.27a 1.07a 3.40a 50 1.05b 0.46a 1.52b 0.58b 2.35ab 100 0.18c 0.14b 0.32c 0.12c 1.38bc 200 0.06c 0.04b 0.10c 0.04c 1.72ac 300 0.01c 0.05b 0.03c 0.02c 0.26c Table 4. Mean of pigments values in explants from green house plants under different doses of kanamycin Kanamycin Chlorophyll Chlorophyll Total carotenoid dose (ppm) a b chlorophyll 0 1.62a 0.69a 2.31a 0.88a 50 0.52b 0.43ab 0.95b 0.24bc 100 0.53b 0.22b 0.75c 0.32b 200 0.27bc 0.13b 0.39d 0.17bc 300 0.08c 0.09b 0.09e 0.07c Regarding to the obtained results from the antibiotics, kanamycin and streptomycin, effects on peppermint, it could be predicted that the antibiotics caused to whiteness and eventually death of tissues. However, it is considerable that no rooting produced even in low concentrations of both selective agents. Other thing that may be pointed is that of correspondence among values of discoloring and dose of the antibiotics. As, more doses were caused to more discoloring that led to death of tissues at last. This was also confirmed from the results of photosynthetic pigments. Streptomycin is irreversibly bound to30 S ribosomal subunits within the cell and inhibits protein synthesis (Springer et al, 2001). The mechanism of kanamycin action is, binding to 30S subunit of ribosomal RNA and inhibition of moving ribosomes which is followed by cessation of protein synthesis ( Malekzadeh and Malekzadeh, 1996). So it seems that elevation of their concentrations result in inhibition of protein and enzymes synthesis involved in photosynthesis and thus reduces photosynthetic pigments. Decrease in plant pigments such as chlorophyll and carotenoids occurs also by affecting environmental stresses so that changes of the pigments are used as determining parameters of tolerant varieties. Sairam et al (2002) showed that the decrease in chlorophyll and carotenoids in wheat leaves was proportional to the severity of salinity. Lack of change in Chlorophyll a to b ratio may be considered a measure of complete activity of photosynthetic systems. Bertamini and Nedouchezhian (2003) stated that no change of chlorophyll a to b ratio in young and older leaves of grape shows the complete activity of

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photosynthetic system in young leaves.The light-harvesting complex of chlorophyll a/b, the most abundant subunit of the light harvesting in photosynthetic systems of higher plants is that of chlorophyll a, b and carotenoids has been formed. Apoprotein of this complex with the pigments increasesusable optical spectrum for photosynthesis and thus increases the efficiency of photosynthesis (Ahmadi and Siosemardeh, 2001). Finally, according to the streptomycin results,it is proposed a concentration of 250 ppm for different types of peppermint explants. However, to achieve faster response can also be used higher concentrations such as 500 and 1000 ppm. The minimum appropriate concentration of kanamycin for affecting branches or seedlings is 100 ppm that will result in plant death at least during45 days. But for the rapid selection one can be used the doses of more than 100 ppm. It must be looked that transformed plant have to obtain ability of resistance to the antibioticsand due to the experiment goal one may be used kanamycin or streptomycin to peppermint transformation. References Ahmadi A, Siosemardeh A, 2001. Crop physiology. Tehran University Publications. (in Persian) Anonymous (2006) Measurement of relative amount of chlorophyll a, chlorophyll b and carotene in plant tissues.http://www-saps.plastscl.cam.ac.uk/osmoweb/chloro2.htm. Arnon DI, 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol., 24:1-15. Bertamini M, Nedunchezhian N, 2003. Photoinhibition of photosynthesis in mature and young leaves of grapevine (Vitisvinifera L.). Plant Sci., 164:635-644. Bowen BA, 1993. Markers for plant gene transfer. In: S. Kung (ed.). Transgenic plants, engineering and utilization. Vol. (1). Academic Press, New York. Brasileri ACM, Aragao FJL, 2001. Marker genes for in vitro selection of transgenic plants. J. Plant Biotech., 3:113-121. Farsi M, Zolala J, 2003. Principles of biotechnology. Ferdowsi university publications.Mashhad. (in Persian) Haldrup A, Petersen SG, Okkels FT (1988) Positive selection: a plant selection principle based on xylose isomerase, an enzyme used in the food industry. Plant Cell Rep., 18:76-81. Krasnyanski S, Ball TM, Sink KC (1998) Somatic hybridization in mint: identification and characterization of Menthe piperita(+) M. spicata hybrid plants. TAG, 96:683-687. Krasnyanski S, May RA, Loskutov A, Ball TM, Sink KC, 1999. Transformation of the limonene synthase gene into peppermint (Mentha piperita L.) and preliminary studies on the essential oil profiles of single transgenic plants. TAG, 99: 676-682. Mahmoud SS, Croteau RB, 2001. Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase. PNAS., USA, 98:8915-8920. Malekzadeh F, Malekzadeh S, 1996. Antibiotics and their mechanisms. Aghigh publisher inc., (in Persian) Miki B, McHugh S (2004) Selectable marker genes in transgenic plants: applications, alternatives and biosafety. J. Biotech., 107:193-232. Omidbaigi R, 1998. Approaches of production and processing in medicinal plants.Tarrahannashr. Vol.(2). (inpersian) Sairam RK, Rao KV, Srivastava GC, 2002. Differential response of wheat genotypes to long term salinity stress in relation to oxidative stress, antioxidant activity and osmolyte concentration. Plant Sci., 163:1037-1046. Springer B, Kidan YG, Prammananan T, Ellrott K, Bottger EC, Sander P, 2001. Mechanisms of streptomycin resistance: selection of mutations in the 16S rRNA gene conferring resistance. Antimicrobial Agents and Chemotherapy, 45:2877-2884. Veronese P, Li X, Niu X, Weller SC, Bressan RA, Hasegawa PM, 2001. Bioengineering mint crop improvement. Plant CellTissue and Organ Cultures, 64:133-144. Yu T, Yeh S, and yang J, 2003.Comparison of the effects of kanamycin and gentamicin on regeneration of papaya from root tissue. Plant Cell and Tissue Culture, 74:169-178.

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