In Vitro Mature Embryo Germination of Pecan (Carya illinoensis)

Introduction: The walnut family (Juglandaceae) consists of approximately 60 species of deciduous trees is native of the American continents, Europe, and Asia. Pecan (Carya illinoensis) is belonged to the Juglandaceae family and is one of the most valuable nut products all over the world. Embryo culture techniques for plant breeding as well as basic studies in physiology and biochemistry are widely used. The low percentage of germination and the long propagation cycle and the need for stratification treatments from three to six months are the most important barriers to the development of high yielding cultivars through hybridization. Plant regeneration methods from embryo culture in vitro allows overcoming the barriers of hybridization, as well as obtaining higher and faster multiplication rate of plants of an elite genotype.Materials and Methods: In this experiment, an adapted native genotype of pecan in Gorgan city, Golestan province, Iran was selected. The mature fruits were harvested after five months of pollination. They were immediately transferred to the laboratory. For cold pretreatment, nuts packed in a paper bag and stored in 4-5ºC for 15 days. The effect of two types of culture medium, growth regulators and seed pretreatment (15 days at 4-5 °C) on germination of mature embryos of pecan has been determined. Murashige and Skoog (MS) and Woody Plant Medium (WPM) and IBA (0 and 1 mgl-1), BAP (0, 1 and 2 mgl-1) and GA3 (0 and 1 mgl-1) media were used to embryo rescue evaluation. The data obtained were statistically analyzed in completely randomized block design (RCBD). Each treatment was replicated at least third, and each replicate consisted of two zygotic embryos. Means of germination period, percent of seed germination, root and shoot length and leaf number in different media and various PGRs combination were compared based on LSD at p ≤0.05.Results and Discussion: The results showed, although cold pretreatment for 15 days had no effect on germination period, root length and number of leave but also, effect on germination percentage and shoot length. There are some different hypothesis about the effect of cold pretreatment on embryo germination between researchers. Some researchers believed that, there is low efficiency in embryo germination in lack of cold pretreatment and GA3. Cold pretreatment or GA3 reduce the ABA level and promote embryos germination. The others reported poor germination for somatic embryos when they treated with GA3 and cold pretreatments. Pearce et al. (1987) reported that GA3 and substrate of GA3 can be increased during the chilling process as ABA levels decrease. Furthermore, application of exogenous GA3 induces germination. Tang et al. (2000) reported that somatic embryos germination poorly happened in cold condition and addition of GA3 did not change the poor germination. Kaur et al. (2006) and Peyghamzadeh and Kazemitabar (2010), reported that the embryo germination in Juglans regia L. was higher when GA3 and cold pretreatments were simultaneously applied as compared to those when applied separately. In this experiment, media has no effect on embryo germination period but, could effect on other parameters. As the results showed, MS media showed the maximum percentage of germination, root and shoot length and number of leave in both condition (with and without cold pretreatment). In this experiment root length of germinated pecan embryo was higher in MS medium. Mapelli et al. (2001) reported that seed germination resulted in marked changes in the metabolism of free amino acids in walnut cotyledons. About 52% of the total free amino acids in one-month-old seedlings was present in the cotyledons and about 26% was in the taproot. The concentration of free amino acids in the taproot was similar to that in the embryonic axis, and greater than that in the cotyledons. T11 (1mg/l-1 IBA, 1mg/l-1BAP and 1mg/l-1 GA3) and T12 (1mg/l-1 IBA, 2mg/l-1BAP and 1mg/l-1 GA3) treatments were the highest in germination percentages in both treatment (with and without cold pretreatment). There was no significant differences between 1 mgl-1 and 2 mgl-1 of BAP.Conclusion: Pecan as like walnut, is considered to be one of the most recalcitrant species in vitro. It is necessary to determine the optimal culture conditions to establish it for shortening time in seed propagation. This seedling could be applied as primary material for breeding programs, grafting and physiology study. The best growth of micro plant achieved in MS medium with 1 mgl-1IBA, 1 mgl-1GA3 and 2 mgl-1BAP.