پديد آورندگان :
سياه منصور، شيوا دانشگاه لرستان - دانشكده كشاورزي - گروه زراعت و اصلاح نباتات , اسماعيلي، احمد دانشگاه لرستان - دانشكده كشاورزي - گروه زراعت و اصلاح نباتات , نظريان فيروزآبادي، فرهاد دانشگاه لرستان - دانشكده كشاورزي - گروه زراعت و اصلاح نباتات
كليدواژه :
اگروباكتريوم رايزوژنز , تراريختگي , زيست توده , زيستي , غير زيستي
چكيده فارسي :
گياه خشخاش (Papaver somniferum L.)، از مهم‌ترين گياهان در صنعت دارويي جهان و منشأ توليد آلكالوئيدها مي‌باشد. يكي از تكنيك‌هاي توليد آلكالوئيد، استفاده از كشت ريشه مويين مي‌باشد؛ امّا در بيشتر مواقع توليد آلكالوئيدها در مقياس تجاري كم است و براي افزايش آن‌ها، نياز به تحريك توليد با روش‌هاي مختلفي از جمله استفاده از محرك‌هاي زيستي و غيرزيستي است. در اين پژوهش، به منظور افزايش زيست‌توده ريشه‌هاي مويين، اثر 4 نوع محرك (ساليسيليك اسيد، نيترات نقره، سولفات مس و عصاره مخمّر) به‌صورت آزمايش‌هاي جداگانه و در قالب طرح كاملاً تصادفي با سه تكرار مورد مطالعه قرار گرفت. جهت انجام اين آزمايش‌ها، ريشه‌هاي مويين اوليه توليد شده در نهايت درون ارلن‌هاي حاوي 30 ميلي‌ليتر محيط كشت ½MSاضافه شدند و هر ارلن به عنوان يك تكرار در نظر گرفته شد. تأييد تراريختگي ريشه‌هاي مويين با تكثير اختصاصي ژن‌هاي rolC، انجام شد و باندي با اندازه‌اي مطابق انتظار، روي ژل الكتروفورز مشاهده گرديد. نتايج نشان داد كه غلظت 5/0 ميلي‌گرم در ميلي‌ليتر از محرك عصاره مخمّر بيش‌ترين تأثير را بر صفات مورفولوژيكي داشت. تجزيه و تحليل داده‌ها نشان داد كه محرك‌هاي زيستي، نقش مؤثرتري نسبت به تيمارهاي غيرزيستي دارند و غلظت‌هاي بالاي محرك‌ها، اعم از زيستي و غيرزيستي، بر زيست‌توده ريشه‌هاي مويين، تأثير منفي دارند.
چكيده لاتين :
Background and Objectives
As a source of alkaloids, Poppy (Papaver somniferum L.), is one of the most important plants in the field of pharmaceutical industry. Various methods including use of elicitors, adding precursor, optimizing culture, culture of hairy roots and its metabolites engineering are used to increase the production of secondary metabolites in plant cell cultures. In recent years, cultivation of hairy roots, to the production of valuable metabolites in a number of species of medicinal plants, has taken on commercial scale; but in most cases, this technique produces low amount of alkaloid in commercial scale. Hence, it is needed to optimize the production rate using biological and non-biological elicitors.
Materials and Methods
This experiment was conducted in 2015 in biotechnology laboratory of Lorestan University, Khorramabad, Iran. Sterile explants were prepared from seedling and were then inoculated with Agrobacterium. Inoculated explants were kept in a dark condition for 2 days and then washed and transferred to glasses containing½ MS medium complemented with cefotaxime. Finally, the glasses were kept in tissue culture room until production of hairy roots. The produced hairy roots were used as explants for elicitors' experiments in flask containers. In this study, in order to increase biomass of hairy roots, 4 types of elicitor treatments (salicylic acid, silver nitrate, copper sulphate and yeast extract) were evaluated in separate experiments based on a completely randomized design with 3 replicates.
Results
Confirmation of transgenic hairy roots was tested by PCR using the rolC gene primers to ensure the removal of bacteria from the produced roots. Results of PCR products were separated by electrophoresis and expected fragments were observed in gel. The results showed that the concentration of 5.0 mg/mL of yeast extract elicitor had the most impact on morphological traits. Totally, results showed that biotic elicitors had better effects in comparison to abiotic elicitors. Also, high concentrations of elicitors in all biotic and abiotic cases had a negative impact on biomass of hairy roots.
Discussion
It was shown that biotic elicitors had better effects than abiotic elicitors. The remarkable result of this study was that the high concentration of elicitors had negative effects on biomass hairy root production in poppies plants. It seems that bio-active compounds elicitors induce the plant responses but high concentration of biotic and abiotic elicitors may cause cell death and browning of hairy root tissues.
