اثر رقم و تيمار تنظيم كننده رشد گياهي بر باززايي درون شيشه‌اي ليليوم با استفاده از ريزنمونه‌هاي TCL

Effect of Cultivar and Plant Growth Regulators on Invitro Regeneration of Lilium Spp. Utilizing Thin Cell Layer Explants

لیلیوم به عنوان چهارمین گل شاخه بریده دنیا مطرح است و روش‌ كشت بافت، روش مناسبی را برای تكثیر این گیاه زینتی فراهم كرده است. در این پژوهش به منظور بررسی عوامل مؤثر بر باززایی لیلیوم از پیازك‌های تولید شده در شرایط درون شیشه‌ای دو آزمایش جداگانه انجام گرفت. در مرحله اول، اثر ریزنمونه‌های با لایه سلولی نازك (TCL) با ضخامت‌های 1، 3 و 5 میلی‌متر در محیط كشت پایه موراشیگ و اسكوگ (MS) حاوی تركیب‌های تنظیم كننده رشد گیاهی BA، 2ip و Kin با غلظت‌ 1 میلی‌گرم در لیتر در تركیب با 5/0 میلی‌گرم در لیتر NAA بررسی شد. نتایج این آزمایش نشان داد كه در هر سه تیمارتنظیم كننده رشد گیاهی، بیشترین میزان اندام زایی در ریزنمونه‌هایی با ضخامت 3 میلی‌متر بدست آمد. در واقع نتایج برهمكنش اندازه ریزنمونه‌ با تنظیم كننده رشد گیاهی موجود در محیط كشت نشان داد درصد باززایی در ریزنمونه¬های با ضخامت 3 میلی‌متر بهتر افزایش داشته است. بیشترین تعداد برگ و وزن خشك گیاهچه‌ها در محیط كشت حاوی BA مشاهده شد كه نسبت به سایر تیمارها تفاوت معنی‌داری داشت. در آزمایش دوم به منظور بررسی اثر رقم و نوع تنظیم كننده رشد گیاهی سیتوكینین، ریزنمونه‌های TCL ارقام Robina، Donato، Nymph، Lessoto و Roxana با ضخامت 3 میلی‌متر در محیط ‌كشت MS حاوی تركیبات تنظیم كننده رشد گیاهی مختلف (BA، Kin، 2ip و TDZ) با غلظت 1 میلی‌گرم در لیتر در تركیب با 5/0 میلی‌گرم در لیتر NAA كشت شدند. نتایج نشان داد كه رقم به عنوان یكی از عوامل تأثیرگذار بر اندام زایی بود. بیشترین تعداد ریشه، وزن خشك، تعداد پیازك و طول گیاهچه در رقم Roxanaمشاهده شد. بررسی درصد باززایی و شاخه‌زایی در ریزنمونه‌ها نشان داد كه ارقام و هورمون‌های مختلف بر میزان باززایی و ریشه‌زایی مؤثر بوده و رقم Nymph در حضور TDZ نسبت به سایر ارقام و تنظیم كننده های رشد گیاهی كمترین باززایی و ریشه‌زایی را نشان داد. همچنین مقایسه وزن خشك در تیمارهای مختلف نشان داد كه وزن خشك گیاهچه‌های رقم Roxana بطور معنی‌داری نسبت به سایر تیمارها افزایش یافته بود.

Introduction: Lily (Lilium spp.) is a genus of herbaceous flowering plants, which consisted of many beautiful ornamental species with large prominent flowers. Most species are native to the Northern hemisphere temperate, though their range extends into the Northern subtropics. Some specific hybrids of Lilium spp. have been developed as cut flower in controlled conditions and in some cases can be grown as pot plant. Propagation rate of lily in natural clonal propagation methods is very low and one year produces of 1-2 bulblets per bulb scale. There is also possibility of disease transmission; so that, tissue culture techniques has provided an efficient method for its micropropagation. Materials and Methods: In this study, two separate experiments under In vitro conditions the bulblet regeneration from thin cell layer (TCL) explants of Lilium spp. was investigated. In the first experiment, after two months the effect of TCL explants with 1, 3 and 5 mm thickness on MS medium containing 1 mg/l BA, 2ip and kin in combination with 0.5 mg/l NAA on regeneration parameters were assayed. In the second experiment to determine the effect of cultivar and cytokinin types, 3mm thickness TCL explants of five cultivars (Robina, Donato, Nymph, Lessoto and Roxana) were tested on MS medium containing different plant growth regulator (PGR) compounds including BA, kin, 2ip and TDZ at concentration of 1 mg/l in combination with 0.5 mg/l NAA. The regeneration parameters were assayed after four months. In all experiments, the medium was adjusted to pH 5.8 and autoclaved at 121°C for 15 min. All cultures were incubated at 25 ± 2°C with a 16 h photoperiod under cool white flourescent lights (30 µmol/m2). Results and Discussion: According to the first experiment results, plant growth regulator of BA in all of surveyed parameters except root number was better than other PGRs and explants with 3 mm thickness was the best in all of parameters. The interaction of PGR and explants was significant, however maximum bulblet regeneration was observed in TCL explants with 3 mm thickness in all of PGR treatments (100%). While 1 mm thickness TCL in 2ip and 1 and 5 mm thickness TCL in Kin had the least regeneration percentage. Results revealed that the interaction of explants and medium is a key factor for suitable establishment, regeneration and growth of TCLs. Bulb dormancy is one of the limiting factors in regeneration of bulbous crop species. It seems under In vitro condition explants size and PGR combination of media especially cytokinin affected on breaking of dormancy. Maximum number of leaves and dry weight of bulblets in medium containing BA was significantly higher compared with other treatments. Most of studies confirmed the positive effect of BA on regeneration of lily. The function of cytokinin in plant promoted cell division and differentiation, which lead to growth and maintaining cells in meristematic status. Result of second experiment showed that cultivar was one of the effective factors on regeneration trait. Oriental lily cultivar "Roxana " had the highest number of roots, bulblets, dry weight and length of plantlets and "Nymph " cultivar showed the lowest percentage of regeneration, dry weight, length of plantlets and rooting obtained. In all of cultivars BA induced more organogenesis percentage and plantlet dry weight, while TDZ induced more rooting percentage.The interaction of cultivar and PGR treatments on percentage of regenerated bulblets and rooting were significant. "Nymph " cultivar had minimum percentage of regeneration and rooting in medium containing TDZ and Kin. Furthermore, "Roxana " cultivar in medium containing BA showed the best dry weight comparison to other treatments. Conclusion Lily has widely used in the floral industry as a cut flower or potted plant. In recent years, tissue culture was developed as reliable and highly effective method to overcome its limitations of vegetative propagation. The most advantage of this method is high multiplication rate and disease free propagation. In this study, bulblet regeneration of lilium Spp. from TCL explants under in vitro condition was considered as a highly efficient procedure for its micropropagation. With optimization of TCL system some parameters such as exogenously applied plant growth regulators, cultivar, explants types were investigated. Favorable conditions for bulblet regeneration were achieved with 3 mm thickness TCLs in MS medium containing 1 mg/l BA with 0.5 mg/l NAA. This protocol can be used for rapid micropropagation of many cultivars.