Application of Random Amplified Polymorphic Analysis for the DetectionGenetic Variation in Tomato (Lycopersicon Esculentum Mill.)

M. Ibrahim, Kadhim; S. Ahmad, Narmeen; Qassar, Iyad

	Application of Random Amplified Polymorphic Analysis for the DetectionGenetic Variation in Tomato (Lycopersicon Esculentum Mill.)
Journal of Al-Nahrain University - Science
Article 1, Volume 14, Issue 1, 2011, Pages 122-133
Authors
Kadhim M. Ibrahim; Narmeen S. Ahmad; Iyad Qassar
Abstract
This study was carried out to induce variations in tissue cultures of tomato (Lycopersiconesculentum Mill.) by using physical (gamma radiation) and chemical (sodium azide) mutagens.Different plant growth regulators were tested for their potentials in callus induction, and shootand root formation. Callus initiation on hypocotyl explants was achieved on Murashige and Skoog’s(MS) medium supplemented with 0.1 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.5 mg/l 6-benzyl-aminopurine (BAP). Shoot regeneration was performed using hypocotyl explants culturedon MS medium containing 0.8 mg/l indole-3-acetic acid (IAA) and 2.0 mg/l BAP. Rooting wasachieved on MS medium supplemented with 0.4 mg/l IAA. Callus cultures and seedlings wereirradiated with gamma radiation at six doses 5, 15, 25, 35, 45 or 55 mGy from 137Cs source, ortreated with sodium azide(SA) at four concentrations 0.5, 1.0, 2.0 or 3.0 mM for 1, 3 or 6 hours.Callus fresh weights were increased after irradiation with 25 mGy gamma rays and treatment with(0.5 or 3.0) mM SA for 1 hour. Shoot regeneration was observed in all irradiated hypocotylexplants. Shoot regeneration at (15 and 35) mGy was similar to the control. Root formation wasobserved on hypocotyls irradiated with low doses (5 or 15) mGy only, while SA inhibited shoot androot regeneration. Random amplified polymorphic DNA (RAPD) technique was performed todetectgenetic mutation in extracted DNA samples. DNA was extracted from mother plant, callus tissue,irradiated calli with 15 or 45 mGy of gamma ray and calli treated with 0.5 and 3 mM SA. Twelvearbitrary 10-mer primers were used of which eight primers gave amplification products. The totalnumber of amplification products was 27 bands, of which 22 bands were monomorphic bandswhich indicates the presence of genetic mutations. One of these primers, C-02, revealedpolymorphism in callus tissues treated with 45 mGy of gamma radiation, 0.5 and 3.0 mM SA.Others primers, C-08 and C-09, revealed polymorphisms in irradiated callus tissues with 15 mGygamma radiation. RAPD was found to be effective technique to detect genetic mutations in callustissue.

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