Effect of Different Concentrations of Nitrogen and 2,4-D on Callus and Plantlet Production of Zamioculcas zamiifolia Engl under In vitro Condition

Zamifolia, Zamioculcas zamiifolia, belongs to the family Araceae, In vitro propagation is suggested for increase the propagation rate and disease-free production of explants because of vegetative propagation of Zamifolia. Micropropagation is one of the commercial aspects of indoor cultivation and has many advantages over conventional methods of vegetative propagation. Nitrogen is an important component in the structure of molecules and metabolic compounds in plant cells. It is also a major constituent of amino acids in the structure of proteins. Therefore, nitrogen source and its type are important factors in the application of nutrient solutions, controlling the amount of nitrogen, which is divided into two factors: concentration and type of nitrogen source. In this study, the effects of 2.4, D as plant growth regulator, different concentrations of total nitrogen and nitrate to ammonium ratios on growth of Zamifolia were investigated in separate experiments. According to the results of previous studies, the best explants for achieving full callus and seedling are petiole and main vein leaf. The leaves were rinsed with running water for 20 minutes. The leaves were then immersed in 3.5% sodium hypochlorite under laminar hood for 20 minutes. Next, they were rinsed with sterile distilled water three times and then immersed in 70% alcohol for 60 seconds. Finally, the samples were extracted from alcohol and cultured in a suitable culture medium vertically. In the first experiment, callus production from whole leaf of Zamifolia was studied in MS and 1/2 MS medium with two concentrations of 2,4-D )9.05 and 18.1 M(. In the second experiment, the effect of two concentrations of total nitrogen at 60 and 30 μM and two nitrate to ammonium ratio (1: 3 and 1: 1 ) in presence of naphthalene acetic acid (2.69 μM ) and benzyl amino purine (2.22 μM) were studied on whole plantlet production in the shortest growth period. After 16 weeks of continuous culture in the subculture medium, entire explants with tubers, roots and 1-2 expanded leaves were transferred to ex vitro condition, in peat, peat: perlite (1:1) and sand and then on a shaded greenhouse bench. Statistical analysis of the results was factorial based on completely randomized design with 20 replications, and plantlet production acclimatization test, factorial experiment based on completely randomized design with four replications, were performed and analyzed with SAS software version 9.3 The results of the first experiment showed that the largest calli were produced in MS 1.2 and 2,4-D 9.5 μM. After 16 weeks, largest embryo-like size (4.5 cm2) can be seen in the same treatment under nitrogen concentration (30 μM) and nitrate to ammonium ratio (1: 3). On the other hand, the smallest embryo-like structure size (3.5 cm2) is related to interaction of nitrogen concentration (30 μM) and nitrate to ammonium ratio (1: 1). Overall, the results of the first part of this study showed the best callus induction and growth of Zamifolia callus under culture conditions under modified M.S. 1.2 and 2, 4-D 9.5 μM. Secondly, culture medium NAA 2.69 μM + BAP 2.22 μM and modified M.S. media and nitrogen concentration of 30 μM and nitrate to ammonium ratio (1: 3). The best medium for embryo like structure growth in the early regeneration and seedling production weeks, increased leaf number in the last weeks compared to other treatments. According to this protocol, small number of leaves can be propagated to over a period of about 16 weeks to a large number of Zamifolia plants, which is comparable to other methods of reproduction of this plant, and similar studies that it has been economically justified to do this for about 30 weeks. Based on our results, we can conclude Zamifolia tissue culture can be an effective way to improve it growth conditions which can get acclimatized in ex vitro conditions on peat medium.