کالوس زایی و باززایی از لوبیا (phaseolus vulgaris l) با استفاده از ریزنمونه ی لایه ی سلولی نازک

در مورد لوبیا هنوز یک پروتوکل بهینه باززایی وجود ندارد و این خود یک چالش بزرگ جهت استفاده از روش انتقال ژن برای اصلاح لوبیا محسوب می شود. در این تحقیق دو آزمایش برای بررسی کالوس‌زایی و باززائی گیاه لوبیا مورد بررسی قرار گرفت. در آزمایش اول ریزنمونه های محور جنینی شش رقم لوبیا بعد از 20 ساعت خیساندن جدا و در سه تیمار هورمونی شامل 1 (11µm) bap و (57.0 µm) iaa، 2 (4.44µm) bap و (27.2µm) tdz و 3 (4.44µm) bap کشت شدند. بیشترین میزان تولید کالوس مربوط به تیمار هورمونی 2 در رقم های ناز، ازنا، لاین 8 و الیگودرز و تیمار هورمونی 3 در تمامی رقمها به جز لاین 4 بوده است. کالوس های تولید شده در این آزمایش قادر به باززایی نشدند. در آزمایش دو،م بذور استریل در پیش تیمار هورمونی (tdz 0 µm و tdz 10µm) جوانه زده و بعد از 14 روز ریزنمونه های لایه ی سلولی نازک عرضی به ضخامت mm03.5/0 از اپی کوتیل جدا و به محیط msb5 با تیمارهای هورمونی (10µm) tdz و (10µm) bap انتقال یافتند. بیشترین میزان کالوس زایی با پیش تیمار (10µm) tdz و تیمار هورمونی (10µm) bap و (10µm) tdz در لاین 4 و الیگودرز بدست آمد. بیشترین میزان باززایی با تیمار هورمونی (10µm) tdz در ارقام ناز، ازنا، لاین 8 و الیگودرز به دست آمد. نتایج این تحقیق نشان داد که ریزنمونه لایه سلولی نازک تحت تاثیر هورمونهای tdz و bap بهتر از ریزنمونه محور جنینی قادر به باززایی می باشد. همچنین هورمون tdz برای باززایی لوبیا موثرتر از bap است. تاثیر پیش تیمار با tdz نیز متغیر و وابسته به ژنوتیپ است

Thin Cell Layer; Embryonic axis; Callogenesis; Regeneration; Phaseolus vulgaris

Introduction Common bean (Phaseolus vulgaris L.) is the most important species of the legume. The common bean is a very important source of vegetable protein, especially in those regions of the world in which animal proteins are scarce. However, breeding cannot add certain genes that do not exist naturally in the P. vulgaris gene pool. Due to this limitation of plant breeding, new trait improvement approaches such as interspecific horizontal gene transfer via genetic engineering need to be utilized in order to complement the limitations encountered by conventional breeding of this crop. Still, there is not a optimized protocol for beans regeneration and this is a big challenge for bean breeding through genetic engineering Material and methods In this study, two experiments were done to evaluate callus induction and regeneration in bean. Seeds of P. vulgaris were washed thoroughly with distilled water and tween20 (10%). Subsequently they were surface sterilized with 70% ethanol for 2 min followed by sodium hypochloride (6%) for 15 min. After five rinses with sterile distilled water, they were allowed to germinate aseptically on half strength Murashige and Skoog (MS) medium. In the first experiment, induction of callus and regeneration were investigated in six common bean cultivars with embryonic axis explants under the influence of hormones TDZ, BAP and IAA . The Sterile seeds were soaked for 20 hours, Embryogenic axes were isolated from seeds and cultured in MS medium with different regulator hormones including BAP (11µM) and IAA (0/57µM), BAP (44/4µM) and TDZ (2/27µM), BAP (44/4µM). In the second experiment. sterile seeds germinated under the pretreatments medium (free TDZ, 10µM TDZ) and then, after 14 days, Transverse thin cell layer explants (with 0.30.5 mm Thick) were excised from epicotyls. For induction of regeneration, explants were transferred to solid MS medium supplemented with 20 g/lit sucrose, B5 vitamins and concentration of TDZ (10µM) and BAP (10µM). After 14 days, TDZ concentration reduced the amount of 1μM in the TDZ (10µM) treatment and BAP (10µM) treatment remained intact. After 28 days, all samples were transferred to medium MSB5+ BAP (10µM) + AgNO3 (10 µM). After 48 days, all samples were transferred to MSB5+ BAP (1µM) + GA3 (3 µM) + AgNO3 (10 µM) and after 62 days, all samples were transferred to MSB5+ NAA (1µM) + AgNO3 (10 µM). During this period the callogenesis and regeneration rate were recorded. Results and Discussion The results of first experiment showed that the highest callus production (100%) related to hormonal treatments BAP (44/4µM) and TDZ (2/27µM) in Naz, Azna, Line 8 and Aligoodarz cultivars and hormonal treatment BAP (44/4µM) in all of cultivars except Line 4. Based on the resulte, callus induction in all varieties (except Line 4) were same in various hormonal treatments. In Embryogenic axes explant, The impact of hormones BAP and TDZ on callus induction was much better IAA. Second experiment data showed that most callus induction in the pretreatment of the seedlings by TDZ (10µM), treatment by TDZ (10µM) and BAP (10µM) in Line 4 cultivar and Aligudarz was in next rank. Most regeneration achieved in the pretreatment TDZ (10µM) and treatment by TDZ (10µM) in Azna, Naz, Line 8 and Aligudarz cultivars. The regenration of Line 4 was weak despite the good callogenesis. from the 6 cultivars, Aligudarz showed better results in callogenesis and regeneration. It can be concluded that TDZ is better than BAP for regeneration in tTCLS explants of bean. In both experiments, the positive effect of silver nitrate on the reduction of phenolic compounds were observed. Based on previous studies , the combination of BAP and AgNO3 at equimolar concentrations(10µM) significantly raised the number of developed shoots, but Unfortunately in our study the same result was not observed and the effect of AgNO3 on shoot development was weak. Conclusion Unlike previous studies, cytokinin hormones used in this study, did not lead to direct regeneration of bean .The results showed that high concentrations of the cytokinin hormone is lead to callus induction in beans. It was observed that a prolonged exposure to this TDZ concentration had an inhibitory effect on further development of shoots and by reducing the concentrations of TDZ, regeneration will occurs. The results also showed that transverse thin cell layers explants from epicotyl are able to regenerate under the influence of cytokinin hormone better than embryonic axis explants. Regeneration severly is affected by genotype, maybe that apos;s why Still, there is not a optimized protocol for beans regeneration