توسعه روش مؤثر باززايي و ترانسفورماسيون توتون از طريق بهينه سازي غلظت تنظيم كننده هاي رشد و ساكارز

Development of an Efficient Regeneration and Transformation Method for Nicothiana tabacum L. through the Optimization of Growth Regulators and Sucrose Concentration

گیاه توتون یك بیوراكتور بسیار كارا به منظور تولید پروتئین¬های نوتركیب می¬باشد، لذا در این پروژه تحقیقاتی علاوه بر بهینه¬سازی سیستم كشت بافت این گیاه، فرآیند مناسب انتقال ژن به آن نیز مورد بررسی قرار گرفت. بدین منظور اثرات غلظت¬های مختلف ساكارز و 4 تركیب متفاوت هورمونی (BAP و NAA) روی القا كالوس، شاخه¬زایی مستقیم و ریشه¬زایی در قالب طرح كامل تصادفی به صورت فاكتوریل و با سه تكرار مورد آزمایش قرار گرفت. حساسیت ریزنمونه¬های توتون به آنتی¬بیوتیك كانامایسین با كشت ریزنمونه¬ها روی محیط انتخابی دارای غلظت¬های مختلف كانامایسین ارزیابی شد. برای انتقال ژن از اگروباكتریوم (Agrobacterium tumefacien) سویه GV3101 حاوی پلاسمید pBI121 استفاده شد و روش واكنش زنجیره‌ای پلیمراز (PCR) جهت بررسی گیاهان تراریخته مورد استفاده قرار گرفت. نتایج تجزیه واریانس و مقایسه میانگین نشان داد كه بالاترین میزان القا كالوس با استفاده از محیط كشت M1 (حاوی 1/0 میلی¬گرم در لیتر NAA و 1 میلی¬گرم در لیتر BAP) با غلظت 15 گرم در لیتر ساكارز حاصل شد. در صورتی‌كه تعداد بالای شاخه¬زایی مستقیم در محیط كشت M1 با غلظت 30 گرم در لیتر ساكارز بدست آمد. بیشترین فراوانی ریشه¬زایی نیز توسط 1/0 میلی¬گرم در لیتر NAA و 60 گرم در لیتر ساكارز حاصل شد. غلظت 50 میلی¬گرم در لیتر كانامایسین بطور كامل از باززایی نمونه¬های غیرتراریخته ممانعت كرد و بنابراین از این غلظت در محیط كشت انتخابگر استفاده شد. در نهایت، قطعهbp 798 مربوط به ژن nptII در ژنوم گیاهان تراریخته توتون تأیید شد و كارایی تراریختگی با استفاده از روش اگروباكتریوم بیش از 95 درصد محاسبه گردید. تكنیك باززایی مستقیم و انتقال ژن مطرح شده در این تحقیق جهت وارد كردن ژن¬های خارجی مختلف به ژنوم گیاه توتون بسیار كارا و مؤثر می¬باشد.

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.