The Effect of Different Concentrations of Growth Regulators and the Type of Explants on Embryogenesis and Rooting of German Chamomile (Matricaria chamomilla L)

Chamomile (Matricaria chamomilla L.) is one of the nine important medicinal plants found in the world. In this study, the effect of different concentrations of plant growth regulators and explants has been investigated on embryogenesis and rooting of German chamomile (Matricaria spp.). MS culture medium (Murashige Skoog., 1986) was the basal medium in this experiment. Factorial experiments were conducted in a Randomized Complete Block Design with three replications. In testing the effect of 1-Naphthaleneacetic acid (NAA) and Kinetin on the embryogenesis, factors included: (NAA) at four levels (0, 0.5, 1, 2 mg/l), Kinetin at four levels (0, 0.5, 1, 2 mg/l) and explants in three levels: Stem, Axillary buds and leaves. In investigating the effect of NAA and Kinetin on rooting, factors included NAA at four levels: (0, 0.5, 1, 2 mg/l), Kinetin at four levels: (0, 0.5, 1, 2 mg/l). Results showed that leaf explants in culture medium containing a treatment Al: (NAA: 1 +Kin: 1mg/l) and stem explants in culture medium containing a treatment A2: (NAA: 1 +Kin: 0.5 mg/l) and the Axillary bud explants in culture medium containing hormonal composition of A3: (NAA: 0.5 +Kin: 1mg/l) produced the highest embryogenic calli. In terms of rooting, the culture medium containing the treatment A4: (NAA: 1 +Kin: 0 mg/l) created the highest rooting.

Chamomile (Matricaria chamomilla L.) is one of the nine important medicinal plants in the world known by man (Salamon., 1992;Witchl., 1994). Research has shown that different parts of this plant flower (disc florets and ray florets), shoots and roots have certain chemical compounds (

Plant material and disinfection
The experiments were performed in Saravan University, Iran in 2014. The Coral genotype (2n=4x=32) was used in this study. Before the flowering stage, when the plants are 10-15 cm height, young plants with stronger structure were selected and transferred to the laboratory for the experiments. MS Medium Culture (Murashige Skoog., 1986) was a basal medium in these experiments. The culture media were added with 30 g of sucrose per liter and the required regulators were added to it according to the type of experiment and PH was set on 5.8 with the help of caustic soda and HCl 1N. Finally, to make the solid culture medium, 8 grams of agar was added to a liter of culture medium and autoclaved for 20 minutes in order to disinfect it at atmospheric pressure and a temperature of 125 °C. After autoclave, it was distributed into 9 cm Sterile Petri Dishes so that 25 mL culture medium was put into each Petri Dish. Explants disinfection for testing was so that plants were placed under running water for primary disinfection for 30 minutes after primary washing with tap water. The final disinfection under Laminar Air Flow Hood was conducted by ethanol 70 percent for 60 seconds. Then they were placed in a solution of sodium hypochlorite 1percent for 10 minutes. After these stages, they were rinsed with sterilized distilled water four times and each time for 5 minutes. Finally, explants at levels: stem, Axillary buds, and leaves were cut with a sterile razor/scalpel as long as 0.5 cm and incubated on the culture media. The required tools were sterilized with distilled water and ethanol 96 percent and wrapped in an aluminum foil and placed in the oven at 200 °C for 2 hours for disinfection.

embryogenesis and rooting tests of chamomile
With the aim of establishing sterilized explants in vitro, in order to callus production and evaluation of embryogenesis, 32 Hormonal Treatments were used in two separate tests with different concentrations and types. The experimental design used for both experiments was implemented as Factorial Experiments in Randomized Complete Block Design (RCBD) format with three replications. In testing the effect of NAA and Kinetin on the embryogenesis, factors included: (NAA) at four levels: (0, 0.5, 1, 2 mg/l, Kinetin at four levels: (0, 0.5, 1, 2 mg/l), explants at four levels: (Stem, Axillary bud and leaves). In investigating the effect of NAA and Kinetin on rooting, factors were NAA at four levels: (0, 0.5, 1, 2 mg/l) and Kinetin at four levels: (0, 0.5, 1, 2 mg/l).

results
The effect of different concentrations of growth regulators NAA and Kinetin on embryogenesis of chamomile (Matricaria chamomilla L.) explants: After moving explants (Stem, Axillary buds, and leaves) to the culture medium containing different levels of growth regulators NAA and Kinetin to produce callus and evaluate embryogenesis and rooting percentage, the traits were noted and Means were compared using Duncan's Multiple Range Test at the 5 percent level.

embryogenesis and rooting Percentage
In the table for analysis of data variance for the Embryogenesis Percentage of calluses, it was observed that the effect of different concentrations of growth regulators (NAA) and Kinetin was significant on embryogenesis of calli obtained from different explants (Table 1). It was observed that leaf explants in media containing 1  (0) Tables 2 &3. Analysis of data variance for the embryogenic callus percentage showed that growth regulators NAA and Kinetin had significant impact on embryogenesis of calli ( Table 2). So that the highest percentage of embryogenesis was achieved for NAA at level of 1mg/l with the Mean of 45.02 percent. Besides, in different levels of Kinetin, the highest percentage of embryogenic calli with the mean of 46.97 percent belongs to level of 1mg/l. Similar to previous experiment; it was observed that most calli produced roots at the same primary culture medium. Analysis of variance results for this trait is also listed in Table  2. It is observed that the studied factors did not impact on this trait significantly, so the data of this trait were not analyzed. In rooting with growth regulator NAA after 14 days of culture, Axillary bud explants with 1mg/l NAA were thickened and produced thick root-like organs. It was observed in experiments that each of explants: (stem, Axiliary buds and leaves) in various hormonal compounds induce the highest rate of embryogenic calli. So that the leaf explants in culture medium containing treatment A1: (NAA:1 + Kin: 1mg/l) (equally NAA to kinetin ratio), and stem explants in culture medium containing the treatment A2: (NAA:1 + Kin: 0.5 mg/l) (NAA/Kinetin ratio: 2) and axiliary bud explants in culture medium containing hormonal composition A3: (NAA:1 +Kin: 1mg/l) (equally NAA to kinetin ratio) produced the highest embryogenic calli. In terms of rooting, the medium containing the treatment A4: (NAA: 0.5 + Kin :0 mg/l) created the highest rooting.