Effects of phosphorus on germination and seedling growth of Cucumis sativus L . in arsenic solution culture

Arsenic (As) is a toxic metalloid which poses a serious threat to the environment and human health. Enrichment of soil with phosphorus (P) is believed to reduce the negative effects of As on plant growth. However, only a few attempts have been conducted towards finding the precise role of P. Τhis study aimed to determine the efficacy of P in reducing the negative effects of As on Cucumis sativus with respect to seed germination rate, seedling growth and biomass production of seedling. The experiment was conducted in petridish under laboratory conditions using P and As solution cultures. Seeds of C. sativus were treated with different concentrations of As (2 ppm, 5 ppm and 10 ppm), with or without addition of P (10ppm). The results revealed that, As solutions reduce the germination rate, seedling growth and biomass production of seedling in different magnitudes, based on the level of concentrations (2ppm, 5 ppm and 10 ppm) in C. sativus. However, addition of P (10 ppm) in As solution cultures (2ppm, 5ppm and 10ppm respectively) increases those parameters significantly in comparison with their corresponding As solutions. In treatments T1 (2ppm As), T3 (5ppm As) and T5 (10ppm As), seedling dry biomasses were 21.1mg, 18.3mg and 13.4mg respectively, while they were 25.6mg, 27.6mg and 20.9mg respectively in T2 (2ppm As+10ppm P), T4 (5ppm As+10ppm P) and T6 (10ppm As+10ppm P). Similar trend was also recorded for germination rate, plumule growth, seedling fresh biomass production, seedling vigor, sturdiness etc. Therefore, it can be recommended to apply P in As solution culture to reduce the negative effects of As on C. sativus growth in the laboratory, that might also be applicable in the field. Keywords— Arsenic, Cucumis sativus L, germination, phosphorus, seedling growth, sturdiness, vigor index.


INTRODUCTION
Cucumber (Cucumis sativus L.) is a widely cultivated plant in the gourd family, Cucurbitaceae.It is a creeping vine that bears cucumiform fruits that are used as vegetables [1,2] .It contains water, minerals, carbohydrates, protein, lipid, ion and vitamin in human diet.The crop is originally from South Asia, but now grown in whole over the world including Bangladesh.Cucumber can be grown on a variety of soils, but loamy soil and temperature between 18 0 C and 24 0 C is preferred for good quality fruit [2,3,4] .Arsenic (As) is a toxic element widely distributed in the environment and in organisms [5 ,6, 7, 8] .Through both natural formation and anthropogenic activities, As can enter into terrestrial and aquatic environments [9] .In some parts of Bangladesh, people irrigate their crops with Ascontaminated groundwater.Moreover, 33% of total arable land of this country is more or less contaminated with As [10] .As is a nonessential element for plants and inorganic As are highly phytotoxic.At high level of As concentrations, biomass production and yields of a variety of crops are reduced significantly [11,12] .Crops grown in As-contaminated soil or irrigated with As-contaminated water accumulate As in the seeds or grain, and this is an increasingly important problem in many parts of the world [13] because of causing significant risk to animal and human health through soil-crop transfer [14,15] .Phosphorus (P) is the chemical analogue of As and an essential element required for plant growth [16,17,18] .This plant nutrient interferes in cellular energy transfer, photosynthesis, and respiration [19] .Reduction in As uptake can be related to changes in the transport mechanisms of phosphate because P has a chemical similarity of As [20] and in plant uptake contest with As [21] .When P is used as a crop fertilizer the effect of P on the sorption/desorption of As in soil environments has received great attention [22] .Most of the previous hydroponic and soil studies on As and P interactions have shown variable results which included both increases and decreases of As effect under P addition.But very few studies have been conducted to find out the precise role of P in reducing negative effects of As specially on seed germination and seedling growth.Therefore, the objective of this study was to find out the efficacy of P in reducing the negative effects of As on C. sativus with respect to seed germination rate, seedling growth and biomass production of seedling.

Study site and period of study
The experiment was carried out in the environmental lab of the Institute of Forestry and Environmental sciences, University of Chittagong (IFESCU), Bangladesh (lies approximately at the intersection of 91 0 50´E and 22 0 30´N) (Fig. 1).The study was conducted during the month January to May, 2017, as the seeds of C. sativus are mostly available in this period in Bangladesh.

Collection of seeds
Ripe, healthy and disease free seeds were extracted from the fruits of C. sativus, dried in the sunlight and stored in airtight polybags and kept in refrigerator until use.Seeds of uniform size and color were selected to avoid nontreatment variation [23] .Before using, viability test of the seeds were also performed by using water floating method.

Experimental design and treatment combinations
Petridishes were sterilized in oven at 200 0 C overnight.In each petridish three layers of moist sterilized filter paper were placed.A Randomized Complete Block Design (RCBD) with 7 treatments and 4 replications for each treatment was adopted for this experiment.For this experiment, 28 petridishes were needed.25 seeds of C. sativus were sown in each petridish and a total seven hundred seeds were subjected to 7 different treatments.The treatment combinations used in the experiment were: T0 = Control (Hoagland's solution without P, which was also common for all other treatments); T1 = 2ppm As; T2 = 2ppm As + 10ppm P; T3 = 5ppm As; T4 = 5ppm As + 10ppm P; T5 = 10ppm As; T6 = 10ppm As + 10ppm P. Seeds were sterilized by soaking them in 0.05% Mercuric chloride solution for 1 minute followed by washing with distilled water and then drying before placement on petridishes.After sowing the seeds, all the petridishes were placed in the incubator at 25 ± 2 0 C temperature.The filter papers of the petridishes were kept wet always at the same level by applying the specific solution of As and P to the specific petridishes.

Data recording
Germination was recorded daily from the date of seed sowing to the last of germination.The seedlings were allowed to grow altogether for ten days from the time of seed sowing.After ten days, five representative seedlings from each treatment were selected for measuring 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.For recording dry weights, plumule and radical were oven dried at 70 o C for 48 hr.To assess the seedling vigor, total height (from the collar area to seedling tip) of each seedling in each petridish was measured using a ruler to the nearest 0.1 cm.Vigor index was calculated according to [24] as germination percent X seedling total length i.e. total shoot and root length.Volume index was obtained by multiplying shoot height or shoot length (cm) with the square of collar diameter (mm) 2 of the seedling [25] .Quality index was developed following [26] to quantify seedlings morphological quality.The formula for calculating quality index is as follow: where, QI is quality index, T dw is total dry weight (g), H is seedling height or plumule length (cm), D c is collar diameter (mm), S dw is shoot/plumule dry weight (g), R dw is root/radicle dry weight (g).Sturdiness was obtained by dividing or shoot/plumule length (cm) with collar diameter (cm) of the seedling.

Statistical analysis
Data related to seed germination and seedling growth attributes were analyzed statistically by using computer software SPSS ver.23.Duncan's multiple range test (DMRT) was employed to determine the statistical significance (P< 0.05) of the differences among the mean values.Significant differences were indicated by different letters in the table.

Germination percentage
Germination were 68% in T1 (2ppm As), 66% in T3 (5ppm As) and 54% in T5 (10ppm As), while they were 72% in T2 (2ppm As+10ppm P), 78% in T4 (5ppm As+10ppm P) and 60% in T6 (10ppm As+10ppm P), meaning that germination percent increased with the addition of P in corresponding As solution cultures (Table 1).(5 ppm As + 10 ppm P) at the 2 nd day followed by 37% in T5 (10 ppm P) at the same day (Figure 2).From 1 st day up to the 3 rd day, cumulative germination percent was highest in T4 (5ppm As +10 ppm P) while it was highest in T0 (Control) from 4 th day up to the 8 th day.The lowest cumulative germination was recorded in T5 (10 ppm As) (Fig. 3).

IV. DISCUSSION
In plants, As accumulates mainly in the root system, and to a lesser degree in the aboveground organs.It causes physiological changes in plants and reduces crop productivity [7,17,18,27] .As interrupt the biochemical function of cells and severely impedes different plant metabolic processes viz.photosynthesis, transpiration, respiration and other physiological functions through reacting with proteins and enzymes, and stop plant growth [28] .To alleviate As toxicity, plants must take up sufficient amount of P to balance excessive As.The plants react by increasing P accumulation as plant As increases [7,17,29] .From this study, it was found that, As solution reduces the rate of seed germination, seedling growth and biomass production of seedling in different magnitudes, based on the level of concentrations (2ppm, 5 ppm and 10 ppm) in C. sativus.However, addition of P (10 ppm) in As solution cultures (2ppm, 5ppm and 10ppm respectively) increases those parameters significantly in comparison with their corresponding As solution.Research also reported that phosphate protects As toxicity of a rice variety-Bala at a lower concentration (13.3 µM) of As in the solution culture [30] .Being a phosphate analogue, phosphate transporters act as arsenate transporters and phosphate competes with arsenate for uptake at the level of membrane transport, hence phosphate would be expected to reduce arsenate influx and this would translate into an observed increase in arsenate tolerance under phosphate addition [17,18,31,32] .Studies, especially in hydroponic environments have demonstrated that nonresistant plants can be made more resistant to arsenate by raising their P status, as the P is taken more effectively compared to arsenate [17,33,34] .Study also reported the role of phosphate in against As toxicity by increasing cytoplasmic phosphate concentration in wheat [35] .Higher phosphate concentration plays an important role to downregulation of the arsenate-phosphate plasma membrane transporter and competes with arsenate for biochemical processes where arsenate substitutes for phosphate [36] .

V.
CONCLUSION Our findings show that, As solution reduces the rate of seed germination, seedling growth and biomass production of seedling in different magnitudes, based on the level of concentrations in C. sativus.However, addition of P in As solution increases those parameters in comparison with their corresponding As solution.Hence, it can be recommended to apply P in As solution culture to reduce the negative effects of As on C. sativus growth in the laboratory that might also be applicable in the field.
Table.2: Effects of phosphorus on the fresh and dry biomasses, vigor index and volume index of Cucumis sativus in arsenicsolution culture.