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ﺑﻬﻴﻨﻪ ﺳﺎﺯﻯ ﮐﺸﺖ ﺑﺎﻓﺖ Lateriflora Scutellaria ﺑﻪ ﻣﻨﻈﻮﺭ اﻓﺰاﻳﺶ ﺗﻮﻟﻴﺪ ﻣﺘﺎﺑﻮﻟﻴﺖﻫﺎﻯ ﺛﺎﻧﻮﻳﻪ

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ﺑﻬﻴﻨﻪ ﺳﺎﺯﻯ ﮐﺸﺖ ﺑﺎﻓﺖ Lateriflora Scutellaria ﺑﻪ ﻣﻨﻈﻮﺭ اﻓﺰاﻳﺶ ﺗﻮﻟﻴﺪ ﻣﺘﺎﺑﻮﻟﻴﺖﻫﺎﻯ ﺛﺎﻧﻮﻳﻪ ﺑﻬﻴﻨﻪ ﺳﺎﺯﻯ ﮐﺸﺖ ﺑﺎﻓﺖ lateriflora Scutellaria ﺑﻪ ﻣﻨﻈﻮﺭ اﻓﺰاﻳﺶ ﺗﻮﻟﻴﺪ ﻣﺘﺎﺑﻮﻟﻴﺖﻫﺎﻯ ﺛﺎﻧﻮﻳﻪ ﻧﺎﻡ ﺩاﻧﺸﺠﻮ: ﺳﻴﻤﺎ ﺻﺪﻳﻖ ﺟﻮﻗﺎﻥ اﺳﺘﺎﺩ ﺭاﻫﻨﻤﺎ: ﻋﺬﺭا ﺻﺒﻮﺭا اﺳﺘﺎﺩ ﻣﺸﺎﻭﺭ: ﻓﺮﺡ ﮐﺮﻳﻤﻰ ﭼﮑﻴﺪﻩ: ﮔﻴﺎﻩ L. lateriflora Scutellaria ﻣﺘﻌﻠﻖ ﺑﻪ ﺧﺎﻧﻮاﺩﻩ ﻧﻌﻨﺎﺋﻴﺎﻥ ﺑﻪ ﺩﻟﻴﻞ ﻭﺟﻮﺩ ﺗﺮﮐﻴﺒﺎﺕ ﻓﻨﻮﻟﻰ ﻭ ﻓﻼﻭﻧﻮﺋﻴﺪﻯ ﺩﺭ ﻃﺐ ﺳﻨﺘﻰ ﺑﻪ ﻋﻨﻮاﻥ ﻳﮏ ﮔﻴﺎﻩ ﺩاﺭﻭﻳﻰ ﮐﺎﺭﺑﺮﺩﻫﺎﻯ ﻭﺳﻴﻌﻰ ﺩاﺷﺘﻪ اﺯﺟﻤﻠﻪ ﺩﺭ ﺩﺭﻣﺎﻥ ﺑﻴﻤﺎﺭﻯﻫﺎﻯ ﻋﺼﺒﻰ ﻭ ﮔﻮاﺭﺷﻰ ﻣﻮﺭﺩ اﺳﺘﻔﺎﺩﻩ ﻗﺮاﺭ ﮔﺮﻓﺘﻪ اﺳﺖ. اﺯ ﺁﻧﺠﺎﻳﻰ ﮐﻪ ﻣﻨﺎﺑﻊ ﻃﺒﻴﻌﻰ ﮔﻴﺎﻫﺎﻥ ﻭﺣﺸﻰ ﻣﺤﺪﻭﺩ اﺳﺖ ﺩﺭ اﻳﻦ ﺗﺤﻘﻴﻖ ﺟﻬﺖ ﺗﮑﺜﻴﺮ ﮔﻴﺎﻩ ﻭ ﺑﻬﺮﻩ ﺑﺮﺩاﺭﻯ اﺯ ﻣﺘﺎﺑﻮﻟﻴﺖﻫﺎﻯ ﺛﺎﻧﻮﻳﻪ ﺁﻥ اﺯ ﺭﻭﺵﻫﺎﻯ ﺭاﻳﺞ ﮐﺸﺖ ﺑﺎﻓﺖ اﺳﺘﻔﺎﺩﻩ ﺷﺪ. ﺑﻪ ﻣﻨﻈﻮﺭ ﺑﻬﻴﻨﻪ ﺳﺎﺯﻯ ﻣﺤﻴﻂ ﮐﺸﺖ ﮔﻴﺎﻩ اﺑﺘﺪا ﺑﺬﺭ ﺳﺘﺮﻭﻥ ﮔﻴﺎﻩ ﺩﺭ ﻣﺤﻴﻂ MS ﭘﺎﻳﻪ ﻓﺎﻗﺪ ﻫﻮﺭﻣﻮﻥ ﮐﺸﺖ ﺷﺪ. ﺳﭙﺲ ﺟﺪاﮐﺸﺖﻫﺎﻯ ﻣﻴﺎﻧﮕﺮﻩ ﺣﺎﺻﻞ اﺯ ﮔﻴﺎﻫﭽﻪﻫﺎﻯ ۶۰ ﺭﻭﺯﻩ ﺩﺭ ﻣﺤﻴﻂ MS ﭘﺎﻳﻪ ﺩاﺭاﻯ ۴ ﺗﺮﮐﻴﺐ ﻫﻮﺭﻣﻮﻧﻰ /KIN NAA/ ۲,۴-D/BAP، NAA، BAP ﻭ D/KIN-۲,۴ ﺩﺭ ﻣﺤﺪﻭﺩﻩ mg/L -۲ ۰ ﮐﺸﺖ ﮔﺮﺩﻳﺪ. ﺗﻤﺎﻡ ﺁﺯﻣﺎﻳﺶﻫﺎ ﺩﺭ ﻗﺎﻟﺐ ﻃﺮﺡ ﺁﺯﻣﺎﻳﺸﻰ ﺑﻠﻮﮎﻫﺎﻯ ﮐﺎﻣﻞ ﺗﺼﺎﺩﻓﻰ ﺑﺎ ﺣﺪاﻗﻞ ۵ ﺗﮑﺮاﺭ اﻧﺠﺎﻡ ﮔﺮﺩﻳﺪ. ﻧﺘﺎﻳﺞ ﺣﺎﺻﻞ اﺯ ﮐﺸﺖ ﺑﺎﻓﺖ ﮔﻴﺎﻩ .lateriflora S ﻧﺸﺎﻥ ﺩاﺩ ﺑﻬﺘﺮﻳﻦ ﺗﺮﮐﻴﺐ ﻫﻮﺭﻣﻮﻧﻰ ﺟﻬﺖ اﻟﻘﺎﻯ ﮐﺎﻝﺯاﻳﻰ ﺗﺮﮐﻴﺐ D/KIN-۲,۴ ﺑﻮﺩ. ﺑﻴﺸﺘﺮﻳﻦ ﺗﻌﺪاﺩ ﮐﺎﻟﻮﺱ ﺩﺭ ﻫﺮ ﺟﺪاﮐﺸﺖ ﺑﺎ ﺗﻴﻤﺎﺭ K۲D۰٫۱ ﻭ ﺑﻴﺸﺘﺮﻳﻦ ﻗﻄﺮ ﮐﺎﻟﻮﺱ ﺑﺎ ﺗﻴﻤﺎﺭ K۰٫۵D۲ ﺣﺎﺻﻞ ﺷﺪ. ﺑﻬﺘﺮﻳﻦ ﭘﺎﺳﺦ ﺷﺎﺧﻪﺯاﻳﻰ ﻭ ﺭﻳﺸﻪﺯاﻳﻰ ﺑﺎ ﺗﺮﮐﻴﺐ ﻫﻮﺭﻣﻮﻧﻰ /BAP NAA اﻟﻘﺎ ﮔﺮﺩﻳﺪ. ﺑﻴﺸﺘﺮﻳﻦ ﺳﻄﺢ ﺑﺮﮒ ﻭ ﺗﻌﺪاﺩ ﻧﻮﺷﺎﺧﻪ ﺗﺤﺖ ﺗﺎﺛﻴﺮ ﺗﻴﻤﺎﺭ B۰٫۱N۰ ﻭ ﺑﻴﺸﺘﺮﻳﻦ ﺗﻌﺪاﺩ ﺭﻳﺸﻪ ﺑﺎ ﺗﻴﻤﺎﺭ B۰N۲ ﺑﻪ ﺩﺳﺖ ﺁﻣﺪ. ﻣﺤﺘﻮاﻯ ﺗﺮﮐﻴﺒﺎﺕ ﻓﻨﻮﻟﻰ ﻭ ﻓﻼﻭﻧﻮﺋﻴﺪﻯ اﻧﺪاﻡﻫﺎﻯ ﻧﻮﭘﺪﻳﺪ ﺩﺭ ﻣﺤﻴﻂﻫﺎﻯ ﮐﺸﺖ ﭘﺮ ﺑﺎﺯﺩﻩ ﺑﻪ ﺭﻭﺵ ﺭﻧﮓ ﺳﻨﺠﻰ ﺳﻨﺠﻴﺪﻩ ﺷﺪ. ﻧﺘﺎﻳﺞ ﻧﺸﺎﻥ ﺩاﺩ ﺑﻴﺸﺘﺮﻳﻦ ﻣﺤﺘﻮاﻯ ﺗﺮﮐﻴﺒﺎﺕ ﻓﻨﻮﻟﻰ ﺩﺭ ﺭﻳﺸﻪ ﺗﺤﺖ ﺗﻴﻤﺎﺭK۱D۰٫۱ ﻭ ﺑﻴﺸﺘﺮﻳﻦ ﻣﻘﺪاﺭ ﺗﺮﮐﻴﺒﺎﺕ ﻓﻼﻭﻧﻮﺋﻴﺪﻯ ﺩﺭ ﻧﻮﺷﺎﺧﻪﻫﺎﻯ اﻟﻘﺎ ﺷﺪﻩ ﺩﺭ ﺗﻴﻤﺎﺭB۱D۰ ﺗﻌﻴﻴﻦ ﺷﺪ. ﺩﺭ ﺑﺨﺶ ﺩﻭﻡ اﻳﻦ ﭘﮋﻭﻫﺶ، ﺑﻪ ﻣﻨﻈﻮﺭ اﻓﺰاﻳﺶ ﺗﻮﻟﻴﺪ ﻣﺘﺎﺑﻮﻟﻴﺖﻫﺎﻯ ﺛﺎﻧﻮﻳﻪ ﺳﻪ ﺗﺎﺯﻥ ﻋﺼﺎﺭﻩ ﻣﺨﻤﺮ، ﺳﺎﻟﻴﺴﻴﻠﻴﮏ اﺳﻴﺪ ﻭ ﻧﻴﺘﺮاﺕ ﻧﻘﺮﻩ ﺩﺭ ﺳﻪ ﻏﻠﻈﺖ ﻣﺨﺘﻠﻒ ﺑﻪ ﻣﺤﻴﻂ ﮐﺸﺖ MS ﭘﺎﻳﻪ اﻓﺰﻭﺩﻩ ﺷﺪ ﻭ ﮐﺎﻟﻮﺱﻫﺎﻯ ﺣﺎﺻﻞ اﺯ ﺗﺮﮐﻴﺐ ﻫﻮﺭﻣﻮﻧﻰ NAA/KIN ﺭﻭﻯ ﺁﻥ ﮐﺸﺖ ﺩاﺩﻩ ﺷﺪ. ﺳﻰ ﺭﻭﺯ ﭘﺲ اﺯ ﻧﮕﻬﺪاﺭﻯ ﮐﺎﻟﻮﺱﻫﺎ ﺩﺭ ﺷﺮاﻳﻂ ﺗﺎﺭﻳﮑﻰ، ﻣﺤﺘﻮاﻯ ﺗﺮﮐﻴﺒﺎﺕ ﻓﻨﻮﻟﻰ ﻭ ﻓﻼﻭﻧﻮﺋﻴﺪﻯ ﻣﻮﺟﻮﺩ ﺩﺭ ﻋﺼﺎﺭﻩ اﺗﺎﻧﻮﻟﻰ اﻳﻦ ﮐﺎﻟﻮﺱﻫﺎ ﺳﻨﺠﻴﺪﻩ ﺷﺪ. ﻧﺘﺎﻳﺞ ﻧﺸﺎﻥ ﺩاﺩ ﮐﻪ ﻣﻘﺪاﺭ ﺗﺮﮐﻴﺒﺎﺕ ﻓﻨﻮﻟﻰ ﻭ ﻓﻼﻭﻧﻮﺋﻴﺪﻯ ﺑﺎ ﮐﺎﺭﺑﺮﺩ μM ۵۰ ﻭ ۱۰ ﺳﺎﻟﻴﺴﻴﻠﻴﮏ اﺳﻴﺪ اﻓﺰاﻳﺶ ﻳﺎﻓﺖ اﻣﺎ ﻇﺎﻫﺮ ﮐﺎﻟﻮﺱﻫﺎ ﻭ ﺭﺷﺪ ﺁﻥﻫﺎ ﺩﺭ ﻣﺤﻴﻂ ﻭاﺟﺪ ﻋﺼﺎﺭﻩ ﻣﺨﻤﺮ ﺑﻬﺘﺮ اﺯ ﺩﻭ ﺗﺎﺯﻥ ﺩﻳﮕﺮ ﺑﻮﺩ. ﺩﺭ ﺑﺨﺶ ﺁﺧﺮ اﻳﻦ ﭘﮋﻭﻫﺶ، ﺟﻬﺖ ﺗﺮاﺭﻳﺨﺖ ﻧﻤﻮﺩﻥ ﻗﻄﻌﺎﺕ ﺑﺮﮔﻰ ﻭ ﻣﻴﺎﻧﮕﺮﻩ ﮔﻴﺎﻫﭽﻪﻫﺎﻯ ﺣﺎﺻﻞ اﺯ ﮐﺸﺖ ﺑﺬﺭ .lateriflora S اﺯ ﭼﻨﺪ ﺱ ﺩاﻧﺸﮑﺪﻩ ﺯﻳﺴﺖ ﺷﻨﺎﺳﻰ ﺗﺎﺭﻳﺦ ﺩﻓﺎﻉ: ۱۳۹۵ ﺩاﻧﺸﮕﺎﻩ اﻟﺰﻫﺮا دانشگاه الزهرا )س( دانشکده علوم زیستی پایان نامه جهت اخذ درجه کارشناسی ارشد رشته علوم گیاهی گرایش فیزیولوژی گیاهی عنوان بهینه سازی کشت بافت Scutellaria lateriflora به منظور افزایش تولید متابولیتهای ثانویه استاد راهنما دکتر عذرا صبورا استاد مشاور دکتر فرح کریمی دانشجو سیما صدیق جوقان بهمن 5931 کلیه دستاوردهای این تحقیق متعلق به دانشگاه الزهرا (س) است. تقدیم هب خدایی هک آفرید و پدر و ماردم هک رد سختیاه و دشواریاهی زندگی همواره یاوری دلسوز و فداکار و ابیتشپنی محکم و مطمئن ربایم بودهاند. سپاس و ستایش خداوندی را سزاست کسوت هستی را رب اندام موزون آفرینش بپوشانید و تجلیات قدرت ﻻیتزالی را رد مظاره و آاثر طبیعت نمایان گردانید. أ با سپاس فراوان از راهنماییاه و زحمات استاد محترم و گرانقدر سرکار خانم دکتر عذرا صبورا هک از ابتدای راه و رد طی انجام این تحقیق مرا رد نگارش این ارث یاری نمودند و بدون راهنماییاه و دلسوزی ایشان اتمین این پایان انهم ممکن نبود، سپاس فراوان از سرکار خانم دکتر فرح کریمی استاد محترم مشاور هب دلیل یاری اه و راهنماییاهی بی ردیغ ایشان هک بسیاری از سختیاه را ربایم آااتتر نمودند، تح رد پایان از زحمات جناب آاقی دکتر رهنما و ااری کسانی هک رد انجام این قیق م را یاری نمودند متشکرم و از خداوند منان اﻻمت و سعادت ایشان را خواستارم. چکیده گیاه .Scutellaria lateriflora L متعلق به خانواده نعنائیان به دلیل وجود ترکیبات فنولی و فﻻونوئیدی در طب سنتی به عنوان یک گیاه دارویی کاربردهای وسیعی داشته ازجمله در درمان بیماریهای عصبی و گوارشی مورد استفاده قرار گرفته است. از آنجایی که منابع طبیعی گیاهان وحشی محدود است در این تحقیق جهت تکثیر گیاه و بهره برداری از متابولیتهای ثانویه آن از روشهای رایج کشت بافت استفاده شد. به منظور بهینه سازی محیط کشت گیاه ابتدا بذر سترون گیاه در محیط MS پایه فاقد هورمون کشت شد. سپس جداکشتهای میانگره حاصل از گیاهچههای 06 روزه در محیط MS پایه دارای 4 ترکیب هورمونی KIN /NAA ،BAP/2,4-D ، NAA /BAP و KIN/2,4-D در محدوده mg/L 2- 6 کشت گردید. تمام آزمایشها در قالب طرح آزمایشی بلوکهای کامل تصادفی با حداقل 1 تکرار انجام گردید. نتایج حاصل از کشت بافت گیاه S. lateriflora نشان داد بهترین ترکیب هورمونی جهت القای کالزایی ترکیب KIN/2,4-D بود. بیشترین تعداد کالوس در هر جداکشت با تیمار K2D0.1 و بیشترین قطر کالوس ب با تیمار K0.5D2 حاصل شد. بهترین پاسخ شاخهزایی و ریشهزایی با ترکیب هورمونی BAP /NAA القا گردید. بیشترین سطح برگ و تعداد نوشاخه تحت تاثیر تیمار B0.1N0 و بیشترین تعداد ریشه با تیمار B0N2 به دست آمد. محتوای ترکیبات فنولی و فﻻونوئیدی اندام های نوپدید در محیطهای کشت پر بازده به روش رنگ سنجی سنجیده شد. نتایج نشان داد بیشترین محتوای ترکیبات فنولی در ریشه تحت تیمار K1D0.1 و بیشترین مقدار ترکیبات فﻻونوئیدی در نوشاخههای القا شده در تیمار B1D0 تعیین شد. در بخش دوم این پژوهش، به منظور افزایش تولید متابولیتهای ثانویه سه تازن عصاره مخمّر، سالیسیلیک اسید و نیترات نقره در سه غلظت مختلف به محیط کشت MS پایه افزوده شد و کالوسهای حاصل از ترکیب هورمونی KIN/NAA روی آن کشت داده شد. سی روز پس از نگهداری کالوس ها در شرایط تاریکی، محتوای ترکیبات فنولی و فﻻونوئیدی موجود در عصاره اتانولی این کالوسها سنجیده شد. نتایج نشان داد که مقدار ترکیبات فنولی و فﻻونوئیدی با کاربرد μM 16 و 56 سالیسیلیک اسید افزایش یافت اما ظاهر کالوسها و رشد آنها در محیط واجد عصاره مخمّر بهتر از دو تازن دیگر بود. در بخش آخر این پژوهش، جهت تراریخت نمودن قطعات برگی و میانگره گیاهچههای حاصل از کشت بذر S. lateriflora از چند سویه A. rhizogenes به کمک دو روش غوطهور سازی سوسپانسیون باکتری و اسمیر استفاده شد. پس از تلقیح قطعات برگی و میانگرهها با سویههای R2 ،2656 ،ATCC15831 و A4 و نگهداری آنها در محیط واجد آنتیبیوتیک، نمونههای نکروز شده غیر تراریخت حذف و جداکشتهایی که در محیط دارای آنتیبیوتیک سفوتاکسیم و در شرایط تاریکی به مدت 54 روز زنده مانده بودند تفکیک و به محیط مناسب برای رشد ریشه های مویین منتقل شدند. نتایج حاصل نشان داد که علیرغم تکرار آزمایشات تعداد ریشههای مویین القا شده کم بود و رشد ریشههای مویین با سرعت کم صورت می گرفت و احتمال داده می شود که تراریختی پایدار نبوده یا قطعه T-DNA در جایگاه مناسب وارد نشده است. کلمات کلیدی: تازن، بشقابی آمریکایی، هورمون گیاهی، کشت بافت، Scutellaria lateriflora Agrobacterium rhizogenes ، ج د فصل اول: مقدمه 5-5- مشخصات گیاهشناسی سرده .Scutellaria lateriflora L .................................................................................. 2 2-5- ویژگی های گونه Scutellaria lateriflora........................................................................................................ 9 9-5- پراکنش گیاهان سرده Scutellaria ...................................................................................................................... 7 4-5- ترکیبات شیمیایی سرده Scutellaria ................................................................................................................... 3 1-5- ترکیبات شیمیایی گونه Scutellaria lateriflora .............................................................................................. 52 0-5-کاربردهای گیاهان سرده Scutellaria ................................................................................................................ 50 7-5- کاربردهای دارویی Scutellaria lateriflora ................................................................................................... 51 1-5- کشت بافت ........................................................................................................................................................ 53 3-5- تراریختی به کمک آگروباکتریوم ....................................................................................................................... 96 56-5- هدف پژوهش .................................................................................................................................................. 90 فصل دوم: مواد و روش ها 5-2-کشت بذر ........................................................................................................................................................... 93 2-2- آماده سازی محیط کشت و اعمال تیمار هورمونی ................................................................................................ 93 9-2- کاربرد تازن ها در کشت بافت S. lateriflora ................................................................................................... 45 4-2- استخراج و سنجش ترکیبات فنولی و فﻻونوئیدی تام ............................................................................................ 49 1-2- القای ریشه های مویین ........................................................................................................................................ 41 0-2- آنالیز آماری ....................................................................................................................................................... 47 فصل سوم: نتایج ........................................................................................................................................................... 41 5-9-نتایج حاصل از کشت بافت .................................................................................................................................. 43 2-9-نتایج حاصل از سنجش محتوای ترکیبات فنولی و فﻻونوئیدی در اندام های نوپدید تحت تاثیر تیمارهای مختلف ..... 10 9-9- نتایج حاصل از واکشت کالوس ها در محیط کشت MS واجد تازن ...................................................................... 32 4-9- نتایج حاصل از تلقیح برگ و میانگره S. lateriflora با سویه های مختلف A. rhizogenez ............................... 566 ه فصل چهارم: بحث ..................................................................................................................................................... 562 5-4-تفسیر نتایج حاصل از کشت بافت ....................................................................................................................... 569 2-4-تفسیر نتایج
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