Document Type : Research Article

Authors

1 Ferdowsi University of Mashhad

2 Mashhad University of Medical Sciences

3 Birjand university

Abstract

Introduction: Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition and widely used to produce clones of a plant in a method known as micropropagation. Plant research often involves growing new plants in a controlled environment. These may be plants that we have genetically altered in some way or may be plants of which we need many copies all exactly alike. These things can be accomplished through tissue culture of small tissue pieces from the plant of interest. These small pieces may come from a single mother plant or they may be the result of genetic transformation of single plant cells which are then encouraged to grow and to ultimately develop into a whole plant. Tissue culture techniques are often used for commercial production of plants as well as for plant research. Tobacco (Nicotiana tabacum L.) is one of the most important model plants used in the physiologic, genetic and tissue culture studies. The manipulation of tobacco genetic structure requires an efficient technique of gene transferring and regeneration. Whereas, the tobacco plant is a very effective bioreactor in the production of recombinant proteins, in this research we optimized the best tissue culture system and also, genetic transformation process of this plant.
Materials and Methods: Our plant tissue culture protocols, Include helpful information for Murashige and Skoog media, plant growth regulators, plant growth hormones, plant transformation systems, and other products for plant tissue culture. For this purpose, different concentrations of sucrose and 4 combinations of growth regulators (BAP and NAA) on callus induction, direct shoot regeneration and rooting were examined in a factorial experiment based on completely randomized design with 3 replications. The sensitivity of tobacco explants to kanamycin was examined through the cultivation of them on the selective medium with different concentrations of antibiotic. For genetic transformation, agrobacterium tumifacious (GV3101) harboring plasmid pBI121 was used and the transgenic plants were confirmed by PCR analysis.
Results and Discussion: The results of variance analysis and the means comparison showed that the best medium for callus induction was M1 (0.1 mg/l NAA and 1 mg/l BAP) with 15 g/l sucrose in the leaf explants, while the most direct shoot regeneration rate was obtained on the M1 medium with 30 g/l sucrose concentration. High-frequency of rooting was also influenced by 0/1 mg/l NAA and 60 g/l sucrose. So, supplementing the medium with NAA and BAP at different concentrations facilitated induction of multiple shoots from explants. NAA was proved to be the best and the number of shoots increased with increase in the concentration up to (0.1 mg/l), and exceeding this concentration resulted in decline in percent response as well as number of shoots was recorded shoot regeneration. The concentration of BAP was further increased a linear increase in the number of shoots was observed up to an optimal level (1 mg/l). Beyond the optimal concentration (1 mg/l), a decrease in the response as well as number of shoots was recorded due to profuse basal callusing. The effect of cytokinins on multiple shoot regeneration, higher concentrations of NAA found to be inhibitory for shoot regeneration because of huge callusing which hampered the growth and development of new shoots. Also different concentrations of sucrose have a different effect on the shoots and callus. The concentration of sucrose had significant effect on direct shoot regeneration. The main effect of sucrose concentration, concentration of 30 grams per liter, compared with a concentration of 15 grams per liter had the highest direct shoot regeneration. Concentration of 50 mg/l kanamycin could completely prevent the regeneration of untransformed explants so was used in the selective culture medium. Subsequently, the presence of nptII gene (798 bp) in the transgenic plants was confirmed and the transformation efficiency obtained by using the agrobacterium-mediated transformation was more than 95%.
Conclusions In present research, an efficient in vitro regeneration protocol has been developed for tobacco, where different factors including the age of the explant and plant growth regulators were optimized for maximum propagation of tobacco. The results showed that regeneration and transformation method described here is highly efficient and fast for the introduction of any foreign gene directly in tobacco plant.

Keywords

1- Ali G., Hadi F., Ali Z., Tariq M., and Khan M.A. 2007. Callus Induction and in vitro Complete Plant Regeneration of Different Cultivars of Tobacco (Nicotiana tabacum L.) on Media of Different Hormonal Concentrations. Biotechnology, 6: 561-566.
2- Babbar S.B., Jain R., and Walia N. 2005. Guar gum as a gelling agent for plant tissue culture media. In Vitro Cellular & Developmental Biology – Plant, 41(3):258–261.
3- Ekrum G. 2001. Insertion of an antimicrobial gene into Agrobacterium and its further use in transforming tobacco. Turkish Journal of Botany, 25:169-175.
4- Gangopadhyay G., Das S., Mitra S.K., Poddar R., Modak B.K., and Mukherjee K. 2002. Enhanced rate of multiplication and rooting through the use of coir in aseptic liquid culture media. Plant Cell, Tissue and Organ Culture, 68: 301–310.
5- Habibi-Pirkoohi M., Malekzadeh-Shafaroudi S., Marashi H., Moshtaghi N., Nassiri M.R., and Zibaee S. 2014. Transient Expression of Foot and Mouth Disease Virus (FMDV) Coat Protein in Tobacco (Nicotiana tabacom) via Agroinfiltration. Iranian Journal of Biotechnology, 12: 28-35.
6- Joshi N. 2009. In Vitro Growth and Shoot Multiplication in Nicotiana tabacum L-influence of gelling agent and carbon source. International Journal of Plant Developmental Biology, Global Science Books.
7- Klems M., Balla J., Machackova I., Eder J., and Prochazka S. 2000. The uptake and metabolism of 3H-benzylaminopurine in tobacco (Nicotiana tabacum L.) and cucumber (Cucumis sativus L.) explants. Plant Growth Regulation, 31(3):135–142.
8- Leuba V., and LeTourneau D. 1990. Auxin activity of phenylacetic acid in tissue culture. Journal of Plant Growth Regulation, 9:71-76.
9- Linsmaier E.M., and Skoog F. 1965. Organic Growth Factor Requirements of Tobacco Tissue Cultures. Physiologia Plantarum, 18:100-127.
10- Lucyszyn N., Quoirin M., Homma M.M., and Sierakowski M.R. 2007. Agar/galactomannan gels applied to shoot regeneration from tobacco leaves. Biologia Plantarum, 51(1):173–176.
11- Mangat B.S., and JANJUA S. 1987. Cyclic Nucleotides and In Vitro Plant Cultures: Induction of organogenesis in tobacco (Nicothiana tabacum) callus cultures. Journal of Experimental Botany, 38 (12):2059-2067.
12- Miao-Miao Y., Chan X., Chun-Hwan K., Yeong-Cheol U., Amadou Apho B., and De-Ping G. 2009. Effects of explant type, culture media and growth regulators on callus inductionand plant regeneration of Chinese jiaotou (Allium chinense). Scientia Horticulturae, 123:124-128.
13- Mohan Pathi K., Tula S., and Tuteja N. 2013. High frequency regeneration via direct somatic embryogenesis and efficient Agrobacterium- mediated genetic transformation of tobacco. Plant Signaling & Behavior, 8:6.
14- Murashige T., and Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiology Journal. 15: 473–497.
15- Nhut D.T., Aswath C.R., Teixeira da Silva J.A., Le B.V., Thorpe T., and Tran Thanh Van K. 2003. Tobacco Thin Cell Layer Morphogenesis. Thin Cell Layer Culture System: Regeneration and Transformation Applications, 17-63.
16- Nugroho H., and Verpoorte R. 2002. Secondary metabolism in tobacco. Plant Cell, Tissue and Organ Culture, 68:105-125.
17- Ozel C.A., Khawar K.M., and Arslan O. 2008. A comparison of the gelling of isubgol, agar and gelrite on in vitro shoot regeneration and rooting of variety Samsun of tobacco (Nicotiana tabacum L.). Scientia Horticulturae, 117(2):174–181.
18- Parc G., Rembur J., Rech P., and Chriqui D. 2007. In vitro culture of tobacco callus on medium containing peptone and phytate leads to growth improvement and higher genetic stability. Plant Cell Reports, 26(2):145-52.
19- Ramage C.M., and Williams R.R. 2002. Inorganic nitrogen requirements during shoot organogenesis in tobacco leaf discs. Journal of Experimental Botany, 53(373):1437-43.
20- SAS. 2001. SAS/STAT User’s Guide (8.02) SAS Institute Inc., Cary, NC, USA.
21- Smykalova I., Smirous P., Kubosiova M., Gasmanova N., and Griga M. 2009. Doubled haploid production via anther culture in annual, winter type of caraway (Carum carvi L.). Acta Physiologiae Plantarum, 31:21-31.
22- Stolarz A., Macewicz J., and Lorz H. 1991. Direct somatic embryogenesis and plant regeneration from leaf explants of Nicotiana tabacum L. Journal of Plant Physiology, 137:347-357.
23- Taravat E., Sohrabi M., and Zebarjadi A. 2012. Optimization and gene transformation of Tobacco. Proceedings of 7th national biotechnology congress of I.R. Iran. (in Persian with English abstract).
24- Teixeira da Silva J.A. 2002. Polyamines in the regulation of chrysanthemum and tobacco in vitro morphogenic pathways. Propagation of Ornamental Plants, 2: 9-15.
25- Teixeira da Silva J.A. 2005. Simple multiplication and effective genetic transformation (four methods) of in vitro-grown tobacco by stem thin cell layers. Plant Science, 169: 1046–1058.
26- Teixeira da Silva J.A., and Fukai S. 2001. The impact of carbenicillin, cefotaxime and vancomycinon chrysanthemum and tobacco TCL morphogenesisand Agrobacterium growth. Journal of Applied Horticulture, 3(1):3-12.
27- Tran Thanh Van M., Dien N.T., and Chlyah A. 1974. Regulation of organogenesis in small explants of superficial tissue of Nicotiana tabacum L. Planta, 119(2):149-59.
28- Van den Ende G., Croes A.F., Kemp A., and Barendse G.W.M. 1984a. Development of flower buds in thin-layer cultures of stalk tissue from tobacco: Role of hormones in different stages. Physiologia Plantarum, 61: 114–118.
29- Van den Ende G., Croes A.F., Kemp A., Barendse G.W.M., and Kroh M. 1984b. Floral morphogenesis in thin-layer tissue cultures of Nicotiana tabacum. Physiologia Plantarum, 62: 83–88.
30- Yanjie C. 2007. Callus induction and plant regeneration From leaf explants of tobacco. Available at http://nhjy.hzau.edu.cn/kech/xbgc/sy/PDF/ChaoYanjie.pdf.
31- Zhan-sheng P., Ji-yao C., and Huai-xin C. 1989. The regulation effect of the light and hormones on morphogenesis in tobacco leaf tissue cultures. Acta biologiae experimentalis Sinica, 22 (3):279-285.
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