The study of 2,4-D and 2,4,5-T effects on gene expression at early stages of embryogenesis in chickpea (Cicer arientinum L.)

Abstract

Factors affecting the induction of embryogenesis in somatic plant cells in vitro are highly variable depending on different growth regulators, explants types, culture media and stress treatments. A series of experiments were set to study the effect of some factors involve in the initiation and development of somatic embryos including genotype, growth regulator and the kind of explants in four Kabuli type chickpea genotypes (F3, Jam, Bivanig and ILC485). Mature embryos were transferred on Murashige and Skooges (MS) medium plus 3% sucrose supplemented with two kind of auxin as growth regulators in order to induce embryogenesis. The best induction media (found in the preliminary experiment) was the one supplemented with either 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) or 3 mg/l 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) in single use or in combination, to induce globular embryos. Globular shape embryos were distinguishable 6 to 7 weeks after culture. Desiccation treatments were applied by keeping the samples for 7, 14 and 20 min in laminar air flow to induce globular embryos maturity. Total RNAs were isolated using guanidium isothiocyanate method. The polymerase chain reaction amplification (PCR) reaction was performed using three one-base-anchored oligo-dT primers, in combination with eight arbitrary primers. Two genotypes of Bivanig and ILC482 were selected as explants for gene expression studies due to their highest number of globular embryos production, then 3 mg/l 2,4,5-T and 3 mg/l 2,4-D were used to produce embryogenic and non-embryogenic calli, respectively. The results showed a similar gene expression pattern in both treatments with three eminent steps. Comparison of both similar band patterns of two auxin treatments indicated that, a number of particular genes are involved in cells determination. Different growth regulators and thegenotypes created little changes in gene expression pattern through theembryogenesis process just before the globular stage, resulting that, a large number of changes and genes occur similarly in both genotypes and auxin treatments. Key words: Chickpea, embryogenesis, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), gene expression.