بهينه سازي انتقال ژن به عدس (Lens culinaris Medik) با استفاده از آگروباكتريوم

Optimization of genetic transformation in Lentil (Lens culinaris Medik.) using Agrobacterium tumefaciens

عدس از جمله حبوبات مهم در ايران است كه با توجه به وقوع تنش هاي مختلف در مناطق كشت آن در كشور، اصلاح آن جهت افزايش تحمل به تنش هاي زيستي و غيرزيستي از اهميت بسزايي برخوردار است. در اين زمينه، كارآيي استفاده از فناوري هاي نوين از جمله كشت اين ويترو و انتقال ژن، قابل توجه مي باشد. در اين مطالعه بهينه سازي انتقال ژن به عدس با استفاده از ژن گزارشگر gus با واسطه اگروباكتريوم توميفاشينس انجام شد. در اين پژوهش اثر عواملي مانند نوع ريزنمونه، آنتي بيوتيك مورد استفاده، غلظت باكتري، مدت زمان كشت مشترك، تاثير استوسرينگون، بر موفقيت تراريزش بررسي شدند. نتايج پژوهش نشان داد كه ريزنمونه لپه شاخه دارشده روي محيط كشت داراي سيتوكنين پس از حذف سرشاخه ها يك ريزنمونه خوب براي فرايند انتقال ژن مي باشد. غلظت پنج ميلي گرم بر ليتر هيگرومايسين بهترين محيط انتخابي براي شاخه هاي تراريخته شناخته شد. غلظت چهار ميلي ليتر از كشت شبانه باكتري در 25 ميلي ليتر محيط كشت مايع و مدت زمان 72 ساعت كشت مشترك شرايط مناسب را براي انتقال ژن فراهم كرد. استفاده از استوسرينگون بر نتايج بي تاثير بود. پس از كشت مشترك، شاخه زايي ريزنمونه ها روي محيط كشت انتخابي، ريشه زايي، سازگاري و انتقال به گلخانه انجام شد. آزمون هيستوشيميايي gus در مرحله قبل از ريشه زايي بيان ژن gus را تاييد نمود. پس از انتقال گياهچه ها به گلخانه تاييد حضور ژن gus با استفاده از واكنش زنجيره اي پليمراز(PCR) روي نمونه هاي برگي انجام شد. اميد است، نتايج اين پژوهش براي اصلاح عدس از طريق مهندسي ژنتيك و همچنين استفاده از عدس به عنوان ميزبان براي توليد تركيبات دارويي مفيد واقع شود.

Lentil (Lens culinaris Medik.) may have been one of the first agricultural crops grown more than 8,500 years ago and belongs to genus Lens from family leguminous and it is diploid (2n=14). Lentil is a seed propagating, self-pollinating crop originating from Near East. Lentil contains high percentage of protein, and has high nutrient value and easy digestibility. Genetic engineering has high potential to improve tolerance of lentil against biotic and abiotic stresses. Genetic engineering may also have a role in eliminating anti-nutritional factors and improving the nutritional quality of lentil proteins. Lentil can also be employed as host to produce drug using gene transformation. To date only a few reports are available on attempts to transform lentil. In this research we attempted to optimize conditions of gene transformation in lentil. Materials & MethodsSeeds of lentil (Lens Culinaris Medik) variety of Gachsaran (ILL6212) were used in present investigation, collected from Shirvan Agriculture Research Center, northeast of Iran. The Explants of Cotyledon with slight part of Embryo Axes (CEA) were prepared as described in Zakertavallaie et al, (2011) study. The explants were kept on Shoot Induction Media (SIM) including MS media supplemented with 7.5µM 2ip, 4 µM Kin and 2 µM TDZ for 8 days. Then explants containing shoots sub cultured on MS media for another 7 days. 15 days explants containing shoots were used for co-cultivation with Agrobacterium. Agrobacterium tumifaciens strain C58 containing binary vector of pCAMBIA1301 was used for transformation experiments. This plasmid contains a reporter gene gus and a selectable gene hptΙΙ, into T-DNA for selection of putative transgenic shoots and selectable marker gene nptΙΙ to select transformed bacteria in backbone sequence. Suspension of agrobacterium in 1/2 MS with 4 concentration of OD: 0.13, OD: 0.18, OD: 0.22 and OD: 0.26 was compared to use in co-cultivation media. Explants containing shoots were used for co-cultivation. There was compared two type of preparation of explants to use in co-cultivation. About some of explants, only tip of the shoots removed using scalpel but about some of them almost of shoots removed. All of explants were submerged in Agrobacterium suspension for 2 second and were co-cultured on shoot induction media. Cu-cultivated explants containing shoots were cultured on MS media containing 250mg/lit cefotaxim for 7 days. In this stage shoots transferred to elongation media including MS supplemented with 4µM 2ip and 1µM Kin containing 3 mg/L hygromycine to select transgenic shoots and 200 mg/lit cefotaxim to kill bacteria. The hygromycine was increased with each subculture at 7 days intervals up to 5 mg/lit and the non-health and non-green shoots were discarded in each subculture. Elongation of shoots, rooting, hardening and glasshouse growth was done according to Zakertavallaie et al, (2011) protocol. Gus assay carried out before root induction using Hay et al. (2004) protocol and Blue dot were observed under microscope. DNA was extracted from leaves of both non transgenic and putative transgenic plants using modified CTAB protocol. DNA was subjected to Polymerase change reaction using the hyg and gus primers with. PCR was carried out with following condition: PCR reaction mix of 25 µl contained 2 µl genomic DNA (100ng/µl), 2.5µl 10X buffer, 2 µl MgCl2 (50mM), 0.5 µl dNTP (10Mm) and 0.5 µl Taq DNA polymerase (5u/µl). The concentration of primers were also 100 pmol/ µl. in PCR amplification DNA denatured at 94ºC for 5 min then followed by 35 cycles with 1 min at 94ºC, I min at annealing temperature (for gus gene at 58 ºC and for hpt gene at 59 ºC), 1 min at 72 ºC. final extension was carried out at 72 ºC for 10 min. PCR product was run on 1.2% agarose gel and observed under UV after staining with ethidium bromide. Results & DiscussionExplants after shoot induction were prepared for co-cultivation. Co-cultivation period was 72 hours. Concentration of Agrobacterium was important. Acetoceringone has no effect on success of gene transformation. Concentrations of 3 to 5 mg/lit hygromycine were suitable for selection media in shooting stage. Gus assay confirmed gene transformation in putative transgenic shoots. Also PCR reaction confirmed existence of gus and hpt genes in plants. ConclusionThe employed protocol in this research can be used for gene transformation of lentil using another gene to improve it about other treats such as enhancement of tolerance to biotic and abiotic stresses.