مطالعه تأثير كارواكرول، تيمول، بنزيل آدنين و نفتالين استيك اسيد بر ريزازديايي گل لاله واژگون

The Effect of Carvacrol, Thymol, BA and NAA on in vitro Regeneration in Fritillaria imperialis L

تكثیر لاله واژگون از طریق كشت بافت یكی از روش¬های مناسب و سریع جهت جلوگیری از انقراض این گل به دلیل محدودیت در روش¬های مرسوم تكثیر محسوب می¬شود. این پژوهش به منظور مطالعه تأثیر غلظت¬های مختلف اسانس های گیاهی و تنظیم كننده¬های رشد بر باززایی و رشد گل لاله واژگون در قالب دو آزمایش جدا گانه در قالب طرح كاملأ تصادفی در 5 تكرار انجام شد. در آزمایش اول، تیمارهای آزمایشی شامل غلظت¬های مختلف تیمول (50، 100، 150 و 300 پی پی ام)، كارواكرول (10، 100، 500 و 1000 پی پی ام) و در آزمایش دوم تیمار های آزمایشی شامل غلظت‌های مختلف بنزیل آدنین (1، 2، 4 میلی¬گرم در لیتر) و نفتالین استیك اسید (1، 2، 4 میلی¬گرم در لیتر) بود. نتایج حاصل از تجزیه واریانس داده¬ها نشان داد كه تأثیر تیمول و كارواكرول بر قطر پیازچه، تعداد و طول ریشه، تعداد و طول برگ و تعداد و قطر كالوس در سطح احتمال 5 درصد معنی¬دار بود. مقایسه میانگین داده¬ها نیز نشان داد كه غلظت 50 پی پی ام تیمول سبب افزایش تعداد پیازچه، ریشه و برگ شد. غلظت¬های 10 و 100 پی پی ام كارواكرول نیز بیشترین تأثیر را روی شاخص¬های اندازه¬گیری شده داشت. غلظت 2 میلی¬گرم در لیتر NAA بیشترین تعداد و بالاترین قطر پیازچه را به خود اختصاص داد. همچنین بیشترین تعداد و طول ریشه از غلظت 1 میلی¬گرم در لیتر NAA و بیشترین تعداد و طول برگ نیز مربوط به تیمار 2 میلی‌گرم در لیتر بنزیل آدنین بود. این نتایج گویای تأثیر مثبت اسانس های گیاهی و تنظیم كننده¬های رشد بر باززایی و رشد گل لاله واژگون می¬باشد.

Introduction: Fritillaria imperialis L. is an ornamental and medicinal plant native to mountainous regions of Iran. This plant genetic resource is in danger of extinction, Because of grazing livestock and pest outbreaks. Therefore, micro propagation of Fritillaria through in vitro regeneration is essential for conservation and commercial production. Thymol and Carvacrol are one of the main essential oil compounds in family Lamiaceae. Material and Methods: Fritillariaimperialis L. bulbs in dormancy stage obtained from mountainous regions of Lorestan in Iran and were placed in cold room at +4 °C for 4-6 weeks. Then, Bulbs were surface-sterilized with 70% ethanol for 45 s followed by immersion in 5% (v/v) NaOCl solution for 20 min with gentle agitation, and then rinsed three times in sterile double distilled water. Present study was conducted in two separate experiments. In first experiment, effect of different concentration of Thymol and Carvacrol and in second experiment, different concentration of NAA and BA on in vitro characteristics of Fritillaria was evaluated. Explants (1× 1 cm) prepared from the lower third of scales with basal plate and were placed in MS basal medium supplemented with different concentrations of Thymol (50, 100, 150 and 300 ppm), Carvacrol (10, 100, 500 and 100 ppm), BA (1, 2 and 4 mg/l) and NAA (1, 2 and 4 mg/l).All cultures were incubated in a growth chamber at 24±2°C, and a photosynthetic photon flux of 40-60 μmol m–2 s–1 was provided by cool white fluorescent lamps with a 16-h photoperiod. This experiment wascarried out in completely randomized designs with fivereplications. Results and Discussion: Analysis of variance showed that Thymol and Carvacrol were not effective on number of new bulblets but had significant effects on bulb diameter, number and length or roots, number and length leaves and callus induction and diameter of callus obtained from scales (P < 0.05). The highest rate (3 bulblets) of bulblets formation was obtained fromMS medium supplemented with 50 ppm Thymol that showed significantly difference from other treatments. Medium containing 10 ppm Carvacrol gave the highest Bulblet formation (2.5 bulblets) between Carvacrol treatments. Investigation of rooting was done by assessment of the number and length of roots. Mean comparison of the effect of cultivar type on root number showed that the largest number of roots per explant was obtained fromMS medium containing 50 ppm Thymol. Lowest number of roots observed in mediums supplemented with 300 ppm Thymol and 100 ppm Carvacrol. The best medium for increasing the root length per explant (10.90 cm) was MS medium supplemented with 100 ppm Carvacrol, while the least increasing in root length per explant observed from culture mediums contained 300 ppm Thymol and 100 ppm Carvacrol. Also, the largest number of leave formation obtained from culture medium supplemented with 50 ppm Thymol that significantly higher than other treatments. Statistical analysis (ANOVA) of the data showed that high frequency callus induction and formation occurred in MS mediums contained 50, 100 and 150 ppm Thymol and 10 ppm Carvacrol and culture mediums supplemented with 300 ppm Thymol and 1000 ppm Carvacrol showed least callus induction. In contrast, largest callus diameter observed in culture mediums supplemented with 300 ppm Thymol and 500, 100 ppm Carvacrol. Statistical analysis of results showed that different concentrations of BA and NAA had significant effects on bulblets number and bulblets diameter (P <0.05). High frequency of bulblets formation obtained fromMS mediums containing 2 mg/l and least bulblets formed in culture medium supplemented with 4 mg/l BA. Stimart and Ascher (1978) observed highest number of bulblets formation in culture of LiliumlongiflorumL. Scales on LS medium supplemented with 0.3 mg/l NAA that correlated with our study. Also, Pierik and Steegmans (1975) explained that auxins versus Gibberellins increased number of bulb formation in Hyacinthus orientalis. Analysis of variance showed that effects of BA and NAA only was significant in root and leaf number and no significant in root and leaf length. The highest and lowest number of roots observed in MS mediums supplemented with 2 mg/l NAA and 4 mg/l BA, respectively. Pierik (1998) demonstrated that Cytokinins in low concentrations induced cell division but in high concentrations inhibited root formation. Nuth (1998) concluded that in LiliumLongiflorum medium containing NAA significantly increased root formation than PGRs free medium. Statistical analysis of results showed that different concentrations of BA and NAA had significant effects on callus induction and formation in Fritillaria imperialis L. (P <0.05). High rate of callus formation was obtained fromMS mediums supplemented with 2 and 4 mg/l NAA and 2 mg/l BA. Conclusion: Thymol and Carvacrol as additives to tissue culture medium can improve some parameters of Fritillaria imperialis L. in vitro propagation like bulblet diameter, roots and leaves number and size. In this context, Thymol showed better responses than Carvacrol and 50 ppm concentration of Thymol was more effective. Such plant compounds needs to be more considered in tissue culture mediums. Also we concluded that Auxins in comparison to Cytokinins was more effective in propagation of Fritillaria imperialis L. through in vitrobulblet formation.