اثر فتوپريود بر جنين‌زايي ‌رويشي در اندام‌هاي مختلف نخود (Cicer arietinum L.)

Effect of photoperiod on somatic embryogenesis in different organs of chickpea (Cicer arietinum L.)

این مطالعه با هدف بررسی قابلیت جنین‌زایی‌رویشی اندام‌های برگ، اپی‌كوتیل و هیپوكوتیل دو رقم نخود "پیروز " و "كاكا " روی محیط‌كشت پایۀ موراشیگ و اسكوگ(MS) در شرایط تاریكی و روشنایی انجام شد. جهت ایجاد كالوس‌جنین‌زا، از محیط‌كشت MS حاوی تنظیم‌كننده‌های رشد، 2،4-دی‌كلرو¬استیك‌اسید (2,4-D) و نفتالین‌استیك‌اسید(NAA) هر كدام در غلظت‌های 2، 3، 4 و 5 میلی‌گرم در لیتر و همچنین تیدیازورون(TDZ) و پیكلورام در غلظت‌های 1، 5/1، 2و 5/2‌ میلی‌گرم در لیتر استفاده شد. صفات مورد‌مطالعه عبارت بودند از: كالوس‌زایی، جنین‌زایی، فراوانی جنین‌های كروی‌شكل، قلبی‌شكل و لپه‌ای و درصد جنین‌های نمو‌یافته در هر ریز‌نمونه. داده‌ها بر اساس روش آماری فاكتوریل در قالب طرح كاملاً تصادفی تجزیه و تحلیل گردیدند. نتایج نشان‌داد اكسین‌ها نسبت به سیتوكنین‌ها روی كالوس‌زایی تأثیر بیشتری داشتند. بیشترین فراوانی جنین‌زایی در محیط‌كشت 2‌میلی‌گرم در لیتر 2,4-D از ریزنمونه هیپوكوتیل در رقم كاكا در شرایط تاریكی مداوم حاصل شد. برای توسعه و بلوغ جنین، كالوس‌های دارای جنین‌های كروی به محیط‌كشت پایۀ MS حاوی تنظیم‌كننده‌های رشد مختلف انتقال داده‌شدند. فراوانی جنین‌زایی و شرایط تاریكی بیش‌تر از شرایط نوری بود. بالاترین درصد توسعه جنین كروی به جنین قلبی و سپس به لپه‌ای از ریزنمونه برگ رقم كاكا در محیط‌كشت حاوی تنظیم‌كننده‌رشد 5/0‌میلی‌گرم در لیتر BA + 5/0‌میلی‌گرم در لیتر2,4-D حاصل شد. غلظت 2‌میلی‌گرم در لیتر 2,4-D در هر دو شرایط نوری و تاریكی بالاترین جنین‌زایی را ایجاد نمود و همراه با افزایش غلظت2,4-D فراوانی جنین‌زایی كاهش یافت.

Introduction Somatic embryogenesis is an efficient platform for the generation of transgenic plants and synthetic seeds (Kiran et al., 2005). Somatic embryo’s growth and development were influenced by different factors, including photoperiod, genotype as well as acidity, plant growth regulators (PGRs) and nutrient content of tissue culture media (May & Trigiano, 1991). Darkness is one of the important and vital affecting variables on somatic embryogenesis (Angoshtari et al., 2009). In some studies, positive effects of darkness on some plant species have been reported. This study was carried out to investigate the potential of somatic embryogenesis from leaf, hypocotyl and epicotyl explants of two chickpea cultivars (Piruz and Kaka) on basal Murashige and Skoog (MS) medium in darkness and light conditions. Materials and Methods In this study, hypocotyl, epicotyl and young leaf segments of two chickpea cultivars “Piruz” and “Kaka” were used as explant. Surface sterilization was performed by soaking chickpea seeds in 96% ethanol solution for 60 Seconds and immediate immersion in 2% sodium hypochlorite solution for 15 minutes. Following rinsing three times in sterile double-distilled water, the seeds were aseptically cultured on free-PGRs ½ MS medium (Murashige & Skoog, 1962). After 3-4 days, explants were excised from germinated seedlings and implanted on MS medium supplemented with different concentration of 2,4-D and NAA (2, 3, 4 and 5 mg/l) as well as different levels of TDZ and Picloram (1, 1.5, 2 and 2.5 mg/l) to the initiation of embryogenic callus. To produce globular embryos, 16 hormonal treatments were then incubated at 25±1 ºC under continuous darkness and photoperiod (16-h light and 8-h darkness) at a light intensity of 36 µmol m–2 s–1. In order to develop the embryogenesis, another three treatments were used under similar condition. For embryonic callus induction the following characteristics were studied: embryogenesis frequency, the frequency of globular, heart shape and cotyledonary embryos. Regenerated plantlets from mature embryos were washed thoroughly with sterile water and then transplanted to plug tray and fertilized with Hogland solution two times per week. Plants were transferred to the pot and kept under greenhouse condition. Plants were finally transferred to open-field condition. This study was conducted as the factorial design based on completely a randomized design with five replicate (jar) and four shoots per each jar. The collected data were analyzed by SAS ver. 9.1 software. Mean comparisons were carried out by Duncan test at the 1% level of significancy. Results and Discussion The results showed that auxins were more effective than cytokinins in terms of callus induction. The highest frequency of embryogenesis was achieved with hypocotyl explants in 2 mg/l 2,4-D in Kaka cultivar under constant darkness. For the development of embryos, callus with globular embryo were transferred to MS medium supplemented with different PGRs. The frequency of embryogenesis was higher in dark condition than that of in the light condition. During callogenesis, plant species require different physical (light and temperature) conditions e.g. some plant produces more callus in darkness while in the other species, callus induction and proliferation took place in a normal photoperiod (Suhasini et al., 1994). It probably depends on the genetic structure of the plant. The highest percentage of globular embryo development of the heart shape embryo and then to cotyledonary embryo was obtained from Kaka leaf explants growing on medium supplemented with 0.5 mg/l BA + 0.5 mg/l of 2,4-D. 2¬mg/l of 2,4-D in both dark and light conditions was induced the highest rate of embryogenesis. For the initiation of embryogenesis, 2, 4-D play an undeniable role because of this synthetic auxin can lead to overexpression of different genes during stress as well as genes involved in initiation of somatic embryogenesis (Kitamiya et al., 2000; Quiroz-Figueroa, 2006). 2,4-D by its strong auxin activity, can influence metabolism of other phytohormones which in turn affect somatic embryogenesis directly or indirectly (Quiroz-Figueroa, 2006). A prerequisite to induce somatic embryo is a reorganization of cell physiology, metabolism and gene expression (Fehret et al., 2002). In this study, with increasing concentrations of the 2,4-D, embryogenesis was reduced. Additionally increasing of 2,4-D concentration could reduce frequency of embryogenesis. Conclusion Somatic embryo induction depends on various factors including cultivar, type, concentration and combination of PGRs as well as explant type. By comparison to the light condition, darkness is in favor of somatic embryo biogenesis. Between two cultivars, Kaka showed better ability in formation of somatic embryos. Response of a plant tissue or specific organ in somatic embryogenesis process is the outcome of endogenous hormones and exogenous growth regulators, and this response can be physiologically varied depending on the type of explant.