بررسي كالزايي و باززايي گياه دارويي كاسني (L.Cichoriumintybus) با استفاده از ريز نمونه‌هاي برگ و دمبرگ

Studies on Callus Induction and Regeneration of Medicinal Plant Chicory (Cichorium intybus L.) from Leaf and Petiole Explants

كاسنی (Cichorium intybus L.) گیاه دارویی مهمی می‌باشد كه به دلیل داشتن انواع مختلف تركیبات دارویی حائز اهمیت می‌باشد. این تحقیق به منظور بهینه‌سازی كالوس‌زایی و باززایی گیاه كاسنی با استفاده از ریز نمونه‌های برگ و دمبرگ مورد مطالعه قرار گرفت. آزمایش به صورت فاكتوریل در قالب طرح كاملاً تصادفی انجام گردید.در این آزمایش از غلظت‌های مختلف نفتالین استیك اسید (NAA) (0، 1/0،3/0، 6/0، 1 میلی‌گرم در لیتر)، بنزیل آدنین (BA) (0، 1/0، 5/0، 1، 5/1 میلی‌گرم در لیتر) و كینتین (KIN)(0، 5/0، 1، 5/1 میلی‌گرم در لیتر) استفاده گردید. صفات در‌صد كالوس‌زایی، در‌صد باززایی، تعداد نوساقه‌های باززایی شده در هر ریز نمونه بررسی شد. در تركیب هورمونی نفتالین استیك اسیدو بنزیل آدنین در هر دو ریز نمونه برگ و دمبرگ در همه تركیب‌های هورمونی كالوس‌زایی مشاهده شد. در تركیب هورمونی نفتالین استیك اسید و كینتین بیشترین درصد كالوس‌زایی در ریز نمونه برگ در محیط كشت حاوی 3/0 میلی‌گرم در لیتر نفتالین استیك اسید در تركیب با هورمون كینتین با غلظت 1 و یا 5/1 میلی گرم در لیتر مشاهده شد و در ریز نمونه دمبرگ بیشترین درصد كالوس‌زایی در محیط كشت حاوی 3/0 میلی‌گرم در لیتر نفتالین استیك اسید و 1 میلی‌گرم در لیتر كینتینحاصل شد. در تركیب هورمونی نفتالین استیك اسیدو بنزیل آدنین در هر دو ریز نمونه برگ و دمبرگ بیشترین باززایی از لحاظ درصد و تعداد نوساقه در محیط كشت موراشیگ و اسكوگ (MS) حاوی 3/0 میلی‌گرم در لیتر نفتالین استیك اسید و 1/0 میلی‌گرم در لیتر بنزیل آدنینبدست آمد. در تركیب هورمونی نفتالین استیك اسید وكینتیندر ریز نمونه برگ بیشترین باززایی از لحاظ درصد و تعداد نوساقه در محیط كشت موراشیگ و اسكوگحاوی 1/0 میلی‌گرم در لیتر نفتالین استیك اسید و 1 میلی‌گرم در لیتر كینتین و در ریز نمونه دمبرگ در محیط كشت موراشیگ و اسكوگ حاوی 3/0 میلی‌گرم در لیترنفتالین استیك اسید و 5/0 میلی‌گرم در لیتر كینتین بدست آمد.

Introduction: Chicory (Cichorium intybus L.) belongs to Asteraceae family is commonly known as witloof chicory. The leaves and the roots of this medicinal plant are edible and commonly used as salad. Some varieties are also cultivated as coffee substitute after roasting the roots. All parts of the plant contain these volatile oils, with the majority of the toxic components concentrated in the plant 's root. In folk medicine, the plant is used for the treatment of diarrhea, spleen enlargement, fever, and vomiting. Antihepatotoxic activity on damaged rat’s liver sections and anti-bacterial activity of this crop has been recently reported. In vitro regeneration from leaf explants with various hormonal combinations has been reported previously. Moreover, in vitro regeneration of Chicory from cotyledon explants using different combinations of plant growth regulators has been studied. Also, a protocol for the regeneration of plantlets from leaf and petiole explants of witloof chicory has been developed. The aim of the present investigation was optimization of callus induction and shoot regeneration from leaf and petiole tissues of Chicory (Esfahan genotype). Materials and Methods: In this investigation, Esfahan genotype was used for callus induction and direct shoot regeneration. Seeds were first washed with running tap water for 30 min then seeds were surface sterilized by dipping in 70% ethanol for 90 s and rinsed with sterile distilled water, followed by immersing in 5% sodium hypochlorite solution for 25 min and thereafter rinsed for 30 min with sterile distilled water. The basal medium used in this investigation was MS. For shoot regeneration, leaf and petiole explants (5 mm segments) were excised from 4-week-old sterile seedlings and cultured on MS medium containing different combinations of NAA / BA and KIN / BA in two separate experiments. Experiments were performed factorial based on completely randomized design. Cultures were incubated at 25° C ± 2 with a 16/8 hour (day/night) photoperiod and an irradiance of 1500 LUX using Sylvania cool white fluorescent tubes. The percentage of callus induction, shoot regeneration and the number of regenerated shoots were calculated for the leaf and petiole explants. Data was subjected for analysis of variance and means were compared in 5% level with Duncan’s multiple range tests. Results and Discussion: Explants cultured on medium containing either no plant growth regulators (control) or cytokines alone produced no callus. However, after 2 weeks, other concentrations of NAA and BA indicated callus formation from leaf and petiole explants in all hormone combinations. In leaf explants, the highest callus induction were obtained in the medium containing 0.3 mg l-1 NAA with 1 mg l-1 KIN and 0.3 mg l-1 NAA with 1.5 mg l-1 KIN (81.25%). Leaf and petiole explants cultured on medium containing no plant growth regulators (control treatment) and medium containing NAA produced no shoots. The combination of 0.3 mg l-1 NAA and 0.1 mg l-1 BA was the best treatment tested. This treatment produced 2.7 shoots per explant at 71% shoot regeneration frequency in leaf explant and 2.73 shoots per explant at 73% shoot regeneration frequency in petiole explants. The results also showed that the highest percentage of regeneration and the highest number of regenerated shoots were obtained in the medium containing 0.1 mg l-1 NAA and 1 mg l-1 KIN in leaf explants (65.6% regeneration and 1.37 shoots per explant, respectively). The highest number of regenerated shoots was obtained in the medium containing 0.3 mg l-1 NAA and 0.5 mg l-1 KIN in petiole explants (40.6% regeneration and 0.5 shoot per explants, respectively. Shoot regeneration requires plant cells to undergo dedifferentiation which is known to be affected by not only exogenous plant growth regulators but also endogenous content of the hormones. Different tissues may have different levels of endogenous hormones and, therefore, the type of explant source would have a critical impact on the regeneration success. In our study, when leaf and petiole explants were compared, it was clear that leaf explants were much more productive for regeneration than petiole explants. Conclusion: Callus induction and shoot regeneration are in vitro tissue culture methods. Plant growth regulators and types of explant are the most important factors for callus induction and shoot regeneration phases. Therefore, optimization of these factors is essential to establish a high frequency of callus induction, shoot regeneration and gene transfer to this plant.