2.1. Study Site and Period of Study The experiment was carried out at the environmental lab of the Institute of Forestry and Environmental Sciences (910 50´E and 220 30´N), University of Chittagong (IFESCU), Bangladesh. It was conducted between January and May 2017 as the seeds of B. hispida are mostly available in this period in Bangladesh.
2.2. Collection of Seeds Healthy and disease-free seeds were extracted from ripen B. hispida fruits. Seeds were dried in the sunlight before storage in airtight polybags which were then kept at refrigerator until use. Seeds of uniform sizes and colors were selected to avoid non-treatment variations (Bonner, 1987).
2.3. Preparation of Hoagland’s and other Solutions Hoagland’s nutrient solution was prepared as stated in Hoagland and Arnon (1950). The solution comprised of KNO3, 0.5 g L-1 ; Ca(NO3). 4H2O, 1.2 g L-1 ; MgSO4. 7H2O, 0.5 g L-1 ; H3BO3, 2.8 mg L-1 ; ZnSO4, 0.2 mg L-1 ; CuSO4, 0.05 mg L-1 ; NH4NO3, 0.08 mg L-1 ; MnCl2. 4H2O, 1.8 mg L-1 ; Na2MoO4. 2H2O, 0.12 mg L-1 ; FeEDTA, 0.02 g L-1 ; in a volume of 1 L. P was added as 0.07 g L-1 of KH2PO4 for P treatment and As was added as 0.042 g L-1 of Na2HAsO4. 7H2O for As treatment. The stock solutions were diluted as required for various treatments. The pH (6.0) of the stock and diluted solutions was adjusted with 1M HCl and 1M NaOH for all the treatments. Hoagland’s solution without P was common for all the treatments including control.
2.4. Experimental Design and Treatment Combinations Petri dishes were sterilized by keeping overnight at 200 0 C in convection oven. In each petri dish, three layers of moist sterilized filter paper were placed. A Randomized Complete Block Design (RCBD) with 7 treatments with 5 replications was adopted for this experiment. A total of 35 petri dishes were needed for this experiment. The treatment combinations used in the experiment were:
T0 = Control (Hoagland’s solution without P)
T1 = 2 ppm As (Hoagland’s solution without P + 2 ppm As)
T2 = 2 ppm As + 10 ppm P (Hoagland’s solution with 10 ppm P + 2 ppm As)
T3 = 5 ppm As (Hoagland’s solution without P + 5 ppm As)
T4 = 5 ppm As + 10 ppm P (Hoagland’s solution with 10 ppm P + 5 ppm As)
T5 = 10 ppm As (Hoagland’s solution without P + 10 ppm As)
T6 = 10 ppm As + 10 ppm P (Hoagland’s solution with 10 ppm P + 10 ppm As)
Seeds were soaked in 0.05% Mercuric chloride solution for 1 minute for sterilization followed by washing with distilled water and drying before sowing them on petri dishes. In each petri dish, 20 seeds of B. hispida were sown and a total of seven hundred seeds were subjected to 7 different treatments. After sowing the seeds, all the petri dishes were placed at ambient temperature and exposed to natural daylight in the laboratory. The filter papers of the petri dishes were kept constantly wet at the same level by applying the specific solution of As and P to the specific petri dishes.
2.5. Data Recording Germination was recorded daily from the date of seed sowing to the last date of germination. The seedlings were allowed to grow for 15 days from the time of seed sowing. After 15 days, 10 representative seedlings from each treatment were selected to measure growth parameters. The recorded parameters were plumule and radical lengths, collar diameter, fresh weights of plumule and radicle, dry weights of plumule and radicle, number of lateral roots. Plumule and radicle were oven dried at 75 0 C for 48 hr to record dry weights. Total height from the collar area to seedling tip of each seedling in each petridish was measured to the nearest 0.1 cm by using a ruler. Vigor index was calculated according to Abdul-Baki and Anderson (1973) as germination percent × mean total (plumule and radical) length. Volume index was obtained by multiplying plumule length (cm) with the square of collar diameter (mm)2 of the seedlings (Hatchell, 1985). Sturdiness was obtained by dividing plumule length (cm) with collar diameter (cm) of the seedling. The quality index (QI), as developed by Dickson et al., (1960) to quantify seedling morphological quality was calculated as follows:
QI = Tdw / (H/Dc + P dw/ R dw )
Where, QI = Quality index, Tdw= Total dry weight (g), H = Plumule height (cm), Dc= Collar diameter (mm), Pdw= Plumule dry weight (g), Rdw= Radicle dry weight (g).
2.6. Statistical Analysis SPSS ver. 23 was used for statistical analysis of data related to seed germination and seedling growth attributes. The statistical significance of the differences among the mean values was ascertained by Duncan’s multiple range test (DMRT). Different letters in the table indicates significant differences.