Life Science Journal 2014;11(2) http://www.lifesciencesite.com 249 In vitro Cultivation of Marjoram (Origanum majorana L.) under Influence of 2,4-D (2,4-Dichlorophenoxy Acetic Acid) as Herbicide. Yasser Hussein, Gehan Amin, ElSayed Hashem and Khaled Youssef Botany Department, Faculty of Science, Zagazig University, Egypt dryasserhussein_zu@hotmail.com Abstract: Callus induction of marjoram (Origanum majorana L., family Lamiaceae) was done by hypocotyl and seed explants which cultured on MS medium supplemented with auxin (2,4-D or NAA) at 1, 2 or 4 mg/L in combination with cytokinins (Kn or 6 BAP) at 0.2, 0.4, 0.5 or 1 mg/L. For seedling culture, the sterilized seeds were germinated on the same basal MS medium free of phytohormones but supplemented with different concentrations of 2,4-D as herbicide (6, 11, 22.5, 23.5 and 45 mg/L). Comparison of different phytohormonal combinations in terms of callus production revealed that: the 2,4-D + Kn (2+0.5 mg/L) and NAA + 6BAP (4+0.4 mg/L) were the most responsive for callus induction. A lower callus formation was obtained on medium supplemented with 2,4-D + Kn (1+1 mg/L) and NAA + 6BAP (1+1 and 1+0.5mg/L). The seed and hypocotyl calli retained high proliferation rate for two subcultures, afterwards in the 3 rd and 4 th subcultures the calli grew slower, turned brown and didn’t survive in subsequent subcultures. The highest growth rates and fresh weights of aseptic marjoram seedlings were observed on medium contained 6 and 11mg/L 2,4-D, while the lower growth rate was obtained on medium supplemented with 22.5 mg/L 2,4-D. In case of higher conc. of 2,4-D (23.7 and 45 mg/L) no growth was recorded and seedlings dead finally. From the profile of SDS-PAGE, there is no distinct variation on the proteinous bands of the treated marjoram seedlings with 6 and 11mg/L 2,4-D, in addition to control of non-treated seedlings, assuming the absence of inducing effect on gene expression. However the density of proteinous bands was increased upon 2,4-D (6 and 11mg/L) treatment as compared with the control assuming the induction of hyper amount of induced expressed protein to resist herbicide treatment. From the profile of antifungal activity, a slight fungistatic activity was observed by the aqueous extract of marjoram seedlings treated by 2,4-D at concentration of 6mg/L and 11 mg/L, comparing to untreated control. [Yasser Hussein, Gehan Amin, ElSayed Hashem and Khaled Youssef. In vitro Cultivation of Marjoram (Origanum majorana L.) under Influence of 2,4-D (2,4-Dichlorophenoxy Acetic Acid) as Herbicide. Life Sci J 2014;11(2):249-257]. (ISSN:1097-8135). http://www.lifesciencesite.com . 34 Key words: Marjoram (Origanum majorana L.), 2,4-D, herbicide, in vitro, callus, seedling, growth, protein, SDS- PAGE, antifungal. 1. Introduction: Marjoram (Origanum majorana L.) family Lamiaceae, is used as a spice and for treatment of gastrointestinal disturbances, cough and bronchial diseases. Marjoram is used in mouthwashes for oral hygiene and also applied topically to relieve symptoms of the common cold, such as nasal congestion (Bruneton, 1999). A number of the marjoram essential oil components exhibit significant antimicrobial properties when tested separately (Lambert et al., 2001). Several studies reported that methanolic extracts of marjoram had high antioxidant capacity (Hossain et al., 2011). There are a number of post-emergent grasses (weeds) growing with marjoram plant which reduce its growth and yield. Weed species are either broadleaf or grassy type. Herbicides are used to control weeds within agricultural areas, for example, 2,4- dichlorophenoxy acetic acid (2,4-D) is a systemic auxin-like selective herbicide, belonging to phenoxy acetic group and is recommended to control broadleaf weeds in cereal crops (Tomlin, 2006). In vitro cell cultures have been developed as promising alternative tool for agricultural processes in producing valuable phytochemicals (Langhansova et al., 2005; Srivastava and Srivastava, 2007). The advantage of this method is that it can ultimately provide a continuous, reliable source of natural products. Tissue culture appears to be a good alternative to conventional propagation, requiring less physical space, with high multiplication rate, without incidence of pests and diseases during cultivation, and enabling higher control of the variables involved. Thus, in the in vitro environment, with the required stimuli and appropriate conditions, different cell types express different behaviors, possibly leading to cell multiplication and differentiation into a specific tissue, characterized by a form and a function, which may lead to the regeneration of a new individual (Bhojwani and Razdan, 1996). Throughout the history of tissue culture, various kinds of culture media have been developed. However, the MS (Murashige & Skoog) medium (Murashige and Skoog, 1962) is the most widely used for the regeneration of dicots, and