بررسی توانایی سویه های مختلف Agrobacterium Rhizogenes در القای ریشه مویین و تولید متابولیت های ثانویه در گیاه ریحان (Ocimum Basilicum L.)

ریحان(ocimum basilicum l.) حاوی مقادیر بالایی از ترکیبات فنلی و ترکیبات فعال زیستی با نقش آنتی‌اکسیدانی است. سیستم کشت ریشه‌ مویین˓ روشی مناسب برای تولید ترکیبات ثانویه دارویی می‌باشد. جهت بررسی توانایی سویه‌های باکتری agrobacterium rhizogenes در القای ریشه مویین و تولید متابولیت های ثانویه˓ گیاهچه های ریحان در محیط کشت ms1.2 کشت شدند. پس از 45 روز˓ ریزنمونه‌های برگ˓ ساقه و نواحی گره توسط چهار سویه باکتری (atcc-15834, a4, msu, r1000) تلقیح شدند و ریشه مویین را در زمان‌های متفاوت القاء کردند. پس از 14 روز˓ریشه‌های مویین تنها در نواحی گره ظاهر شدند ولی ریزنمونه‌های برگ و ساقه پاسخی به تلقیح نشان ندادند. ریشه های تولید شده به محیط ms مایع منتقل شدند و پس از 60 روز˓ ویژگی‌های رشد و میزان فنل کل بررسی گردید. dna‌ ژنومی از ریشه‌های مویین و غیرمویین استخراج و ماهیت القای ریشه های مویین با استفاده از آغازگرهای اختصاصی ژن rol c در واکنش زنجیره ای پلیمراز (pcr) تایید شد. درصد القای ریشه مویین به طور معنی‌داری تحت تاثیر سویه باکتری قرار داشت. بالاترین درصد القای ریشه (68.1 درصد)˓ بیشترین تعداد ریشه مویین در هر ریزنمونه (4.8 عدد) و بیشترین طول ریشه (1.8 سانتی‌متر) به سویه atcc-15834 تعلق داشت. بالاترین وزن خشک (103.2 میلی‌گرم) و میزان فنل کل (312 میلی گرم گالیک اسید بر گرم ماده خشک) به همین سویه مربوط بود که افزایش 4.6 برابری نسبت به شاهد غیرمویین داشت. نتایج این مطالعه نشان داد که سویه atcc-15834 را می توان به عنوان یک سویه موثر در القا و رشد ریشه‌‎های مویین و نیز تولید متابولیت ثانویه در ریحان معرفی کرد.

Influence of Different Agrobacterium rhizogenes Strains on Hairy Roots Induction and Secondary Metabolites Production in Ocimum basilicum L.

Introduction: Agrobacterium rhizogenes hairy roots induction is used for secondary metabolite production in plants. A. rhizogenes is a genus of gramnegative soil bacteria belonging to the Rhizobiaceae family that causes hairy roots at the site of infection. Hairy roots have various advantages, including high growth rate, more genetic stability than the callus and suspension cultures, growing well on hormonefree media that have been reported effective for producing high levels of secondary metabolites. Basil (Ocimum basilicum) is a popular herb with important economical applications in food, cosmetic, and pharmaceutical industry. It is a digestive stimulant with anticarcinogenic, antibacterial, and anticonvulsant properties. The main phenolics reported in basil plants are in the classes of phenolic acids and flavonoids, some of which have human health benefits. This study was designed to develop hairy root culture from O. basilicum using different of A. rhizogenes strains for the production of total phenols and introduce the best strain of A. rhizogenes to induce hairy root and growth and production of total phenol.Materials and Methods: Different A. rhizogenes strains (ATCC15834, A4, MSU, and R1000) were studied to investigate their effects for the transformation and production of secondary metabolites in O. basilicum. Therefore, shoot and leaf explants and nodes of the seedlings were used for Agrobacteriummediated transformation. These explants were inoculated with four A. rhizogenes strains and transferred to ½ MS medium. About four weeks after cultivation with A. rhizogenes, hairy roots were excised from the seedlings and subcultured to fresh medium MS liquid culture containing 500 mg/l cefotaxime. After 60 days of inoculation, various parameters, including dry weight, infection percentage, number of hairy roots per explant, and total phenol contents were measured. The growth rate and phenolic contents of the transformed hairy roots were compared with normal ones. Total genomic DNA was isolated from nontransgenic and transgenic hairy root lines using the Cetyl Trimethyl Ammonium Bromide (CTAB) method. Isolated genomic DNA was used to detect the rolC gene through polymerase chain reaction (PCR) analysis. The PCR using specific primers for rolC of TDNA and virD2 was used to confirm the nature of resulted transgenic hairy roots.Results and Discussion: Selecting efficient A. rhizogenes strains, as well as the type of explants, are crucial factors for hairy root induction. All used A. rhizogenes strains were able to produce hairy roots. Hairy roots appeared on the nodes at the point of injection, but were not forming on the shoot and leaf explants. So, the choice of the plant material is crucial for successful transformation with A. rhizogenes and usually, transformation of young tissues gives the best results. The transgenic status of the hairy roots was confirmed using PCR with rolC and virD specific forward and reverse primers. All lines showed the presence of 612 bp rolC amplified products, indicating the integration of TDNA of A. rhizogenes and O. basilicum. Hairy roots could synthesize phenolic compounds, which was significantly higher in hairy roots than nontransformed control. Four hairy root lines were independently evaluated for their content and these lines showed variation in total phenolic contents, with the highest amount (312 mgGAE/ g DW) in hairy roots induced by ATCC15834 strain and the lowest amount (113.2 mgGAE/ g DW) in hairy roots induced by R1000 strain. The results showed that the strain ATCC15834 caused the highest infection percentage (68.1%) along with the highest number of hairy roots (4.8) per explant and root length (1.8 cm). The growth rate and phenolics production were investigated in each hairy root of O. basilicum from infection by four different A. rhizogenes strains. The highest growth rate (103.2 mg DW) and production of total phenol (312 mg/g DW) were found in ATCC15834. The growth rate of transformed hairy roots was more than that of normal ones.Total phenol contents in all hairy roots were also increased significantly compared with nontransformed control plants (4.6 times in hairy roots induced by A. rhizogenes strain ATCC15834). ATCC15834 has been reported as the most widely used A. rhizogenes strain owing to its strong induction ability, and the variation in hairy root induction could be due to disparity in the virulence of different A. rhizogenes strains.Conclusion: The hairy roots of O. basilicum had shown promising results in terms of significant yield of phenolic contents and had the potential for being scaledup further for phenol production. It could be concluded that A. rhizogenes strains had different abilities in hairy roots induction. Therefore, the selection of an effective A. rhizogenes strain for the production of transformed root cultures is important, highly dependent on the plant species, and must be determined in future experiments.