Influence of Nano Formulation on Augmenting the Seed Quality in Groundnut (Arachis hypogaea L.)

Experiment was carried out during 2017 at Department of Nano Science and Technology and Department of Seed Science and Technology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India. Freshly harvested and genetically pure seeds of groundnut TMV 13 were obtained from Oilseeds Research Station, Tindivanam. The seeds were dried, cleaned, size graded using round perforated sieve (24/64) and tested for initial seed quality under laboratory conditions. Hormones (gibberellic acid), polymer (Carboxymethyl cellulose) and surfactant (Tween 80) were purchased from M/s. Sigma Aldrich Chemicals Private Limited, Bangalore, India. These seeds, hormones and chemicals constituted as study materials for the present investigation.
 
Conventional emulsions of CMC based GA₃ loaded, using surfactant Tween 80 were prepared by adopting organic solvent displacement technique (Solans et al., 2005). To prepare 100 ml of 100 ppm GA₃ loaded formulation, CMC, GA₃ and Tween 80 were mixed at the ratio of 79: 20: 1. Conventional emulsion CMC based GA₃ loaded (CMC 1% + GA₃ 100 ppm + Tween 80 1%), prepared by organic solvent displacement method were subjected to high energy homogenization where the pressure ranged from 10000 bar to 40000 bar with an interval of 10000 bar (Sharma et al., 2014). Then, the homogenized nano formulation were used for seed coating at four different doses as T₀- Control, T₁- 5 ml of nanoformulation kg^-1, T₂ - 10 ml nanoformulation kg^-1, T₃-15 ml nanoformulation kg^-1, T₄- 20 ml nanoformulation kg^-1 and seeds was evaluated for physiological and biochemical parameters with five replication in each treatments.

The following observations were observed:
 
Imbibition rate
 
Twenty-five coated seeds from each replication were taken and placed in petri plates inner layered (two layer) with moist germination paper and the Petri dishes were kept at room temperature for 24 h. Increase in seed weight was measured every two hours. The imbibed seeds were removed and wiped out with tissue paper to remove the adhering water droplets and were weighed for their final weight on a top pan balance. Based on the difference between the weights, the imbibition rate was calculated and the mean was expressed in percentage. The data observed were analysed in factorial completely randomized block design (Sundaralingam, 2005).
 
Speed of germination (Maguire, 1962)
 
The germinated seedlings from each replications of treatments were counted daily from 1^st day onwards up to 10^th day of sowing. From the number of seeds germinated on each day, the speed of germination was calculated using the following formula and the result was expressed in number:
  Where
X₁- Number of seeds germinated at first count.
X₂- Number of seeds germinated at second count.
X_n- Per cent germination on n^th day.
Y₁- Number of days from sowing to first count.
Y₂- Number of days from sowing to second count.
Y_n- Number of days from sowing to n^th count.
 
Germination (%)
 
The germination per cent was observed from the site of experiment in four replications to test 400 seeds as per (ISTA, 2015) rules. The germination rate measures the time course of the seed germination. At the end of the 25^th day the final count was taken and the number of normal seedlings were recorded: Root length (cm)
 
At the time of final count, ten normal seedlings were randomly selected from each replication and root length of each seedling was measured. The measure from the point of attachment of the seed to the tip of the primary root was taken as root length.
 
Shoot length (cm)
 
The same seedlings used to measure root length were used for measuring the shoot length and was measured from the point of attachment to the tip of the leaf.
 
Dry matter production (g per seedlings^-10)
 
The seedlings selected for the root and shoot length were placed in a paper cover, shade dried for 24 h and were dried at 80°C for 16 ±1 h in a hot air oven. Then they were cooled in a desiccator, weighed and expressed as mg 10 seedlings^-1.
 
Vigour index
 
Vigour index were computed using the following formula and the mean values were expressed as a whole number (Abdulbaki and Anderson, 1973): α-amylase activity (mg maltose min^-1)
 
The seeds from each of the treatments and replications were germinated adopting top of the paper method upto radical protrusion. From the pre-germinated seeds 500 mg was weighed and homogenized in 1.8 ml of cold 0.02 M sodium phosphate buffer (pH 6.0) and centrifuged at 20,000 rpm for 20 min. to extract the enzymes. To the 0.1 ml of enzyme extract, one ml 0.067 per cent starch solution was added. The reaction was stopped after 10 min. of incubation at 25°C by addition of one ml of iodine HCl solution (60 mg KI and 6 mg I2 dissolved in 100 ml of 0.05 NHCl). The colour change was measured at 620 nm using a double beam spectrophotometer 2205. The amylase activity was calculated by adopting the following formula and the mean values were expressed in mg maltose min^-1 (Paul et al., 1970):                                                                                                                                               
Lipase activity (meq min^-1g of sample^-1)

A. Preparation of enzyme extract
 
Two grams of pre germinated seeds from each replications of treatments was taken, ground and homogenized with twice the volume of ice-cold acetone. Then powder was washed successively with acetone, acetone:ether (1:1) and ether, dried and stored in refrigerator till used. Extract was obtained by centrifuging 1 g of powder in 20 ml of ice-cold water at 15, 000 rpm for 10 min.
 
B. Substrate
 
Two milli litre of clear vegetable oil (pH 7.0), 25 ml of water and100 mg of bile salt (Sodium taurocholate) was added and stirred well till a formulation was formed. Further, two gram of gum Arabic was added to hasten emulsification.
 
Five milli litre phosphate buffer (pH 7.0) was added to 20 ml of substrate taken in a 500 ml beaker and the contents were stirred slowly using magnetic stirrer cum hot plate maintained at a temperature of 35°C and then the pH was adjusted to 7.0. To the mixture, 0.5 ml of enzyme extract was added and the pH was recorded immediately and the timer was set on. When pH dropped by 0.2 unit, 0.1 N NaOH was added to bring the pH to the initial value and the titration was continued for 30 min. The enzyme activity was calculated from the amount of alkali consumed using the following formula and expressed in meq min^-1g of sample^-1 (Sadasivam and Manickam, 2008):
 

The results obtained from all treatments had an effective difference in all the parameters due to hydrophilic polymer nano formulation coating.

Imbibition rate
 
Imbibition rate was significantly influenced by CMC based GA3 loaded nano formulation coating with different concentrations over a period of 12 h of imbibition. The maximum imbibition rate of 74.40 % was observed in seeds coated with 15 ml kg^-1 at 12 h which had no significant difference with 10 ml kg^-1 (73.80 per cent). The minimum was recorded in control seeds (67.00 %) at 12 h of imbibition (Fig 1). Higher rate of imbibition rate was due to nano formulation coating, polymer having a capability of water absorption even under very low moisture content. It induces the fast activation of hydrolysing enzymes which are involved in seed germination process. Fastest absorption of water also hastens the speed of germination and better seedling establishments, which was supported with the following research findings (Koornneef et al., 2002; Subash et al., 2014 and Sakthivel, 2016).


 
Speed of germination
 
Significant difference was observed in speed of emergence due to CMC based GA₃ loaded nano formulation coating, due to fastest absorption of water, it influences the germination speed. The highest value of 5.5 was observed in 15 ml kg^-1 while the control recorded the lowest value (3.2) (Fig 1, Table 1). Metabolic process repair and build-up of metabolites occurred during seed in vigouration may be the plausible reason for higher germination (Chen and Arora, 2013; Jisha et al., 2013).


 
Germination (%)
 
Germination per cent was significantly influenced by different concentrations of GA₃ loaded nano formulation. Among the different concentrations, 15 ml kg^-1 of seed (T₃) recorded the maximum germination 87 per cent which was on par with 10 ml kg^-1 of seed (T₂), which recorded 85 per cent of germination. The untreated control (T₀) registered the lowest germination 76 per cent (Table 1). The improvement in germination of CMC coated seeds may be attributed to the activation of cells and enhancement of mitochondrial activity which is closely associated with the formation of energy compounds and vital biomolecules that are required during the early phase of germination (Clayton, 1988; Ghiyasi et al., 2008 and Jisha et al., 2013). Devishri (2012) who has reported that carboxylmethyl cellulose coating had improvedgermination in cotton by increasing the imbibition and water for a longer period of time.
 
Root length (cm)
 
Root length of germinated seeds was significantly differed due to nanoformulation coating. Seeds coated with nano formulation at 15 ml kg^-1 of seed (T₃) had produced the longest root length (9.4 cm) followed by 10 ml kg^-1 of seed coating, which had produced the root length of 8.9 cm whereas control has produced root length of having 7.9 cm (Table 1). Naruka et al.,  (2000) and Amanullah et al., (2008) also observed that same result as increased root length and secondary roots.
 
Shoot length (cm)
 
Shoot length of germinated seeds was significantly differed due to nanoformulation coating. Among the different concentrations, seeds coated with nanoformulation at 15 ml kg^-1 of seed (T₃) have produced the longest shoot length (15.3 cm) followed by 10 ml kg^-1, which has produced the shoot length of 14.7 cm whereas untreated seeds showed shoot length of 11.8 cm. (Table 1). The result was on par with the following findings application of nano particles at low concentrations improves the cell division and elongation in shoot growth (Ali et al., 2019 and Asl et al., 2019).
 
Vigour index
 
The results of vigour index showed significant difference due to CMC based GA₃ loaded nano formulation seed coating. Formulation at the rate of 15 ml kg^-1 of seed (T₃) had recorded the maximum vigour index of 2142, which was followed by 10 ml kg^-1 of seeds (2006). Untreated seeds have recorded the minimum (1497) vigour index (Table 1). Devishri (2012) who has reported that carboxylmethyl cellulose coating had improved seedling vigour in cotton by retaining water for a longer period of time.
 
Dry matter production (g per seedlings^-10)
 
Dry matter production was significantly differed in respect to the different concentration of nanoformulations coating. The seeds coated with CMC based GA₃ loaded nanoformulation at 15 ml kg^-1 of seed recorded the highest dry matter content of 3.451 g per 10 seedlings. Seeds coated with formulation at 10 ml kg^-1 of seed had registered the dry matter production of 3.177 g per 10 seedlings. The control seeds recorded the lowest dry matter production of 2.748 g per 10 seedlings (Table 1). The above result of dry matter accumulation was supported with the following findings (Valenciano et al., 2010; Usman et al., 2014).
 
α-amylase activity
 
Seed coating with nano formulation had significant impact on α-amylase activity. Maximum amount of α-amylase activity was measured in seeds coated with CMC based GA₃ loaded nanoformulation at the rate 15 ml kg^-1 (T₃) (14.83 mg maltose min^-1) followed by 10 ml kg^-1 (T₂) (14.26 mg maltose min^-1). Minimum value of (11.82 mg maltose min^-1) was registered in uncoated seeds (Table 2). GA₃ triggered the hydrolytic enzymes which helps in early seed germination and growth (Damaries et al., 2019).
 


Lipase activity
 
Lipase activity was influenced by different concentrations of GA₃ loaded formulation. Among the different concentrations, seeds coated with CMC based GA₃ loaded nano formulation at the rate 15 ml kg^-1 (T₃) was found to be recorded the maximum lipase activity of 1.481 meq min^-1g of sample^-1, which had no significant difference with 10 ml kg^-1 (T₂) (1.416 meq min^-1g of sample^-1). Untreated control had the lowest lipase activity of 0.895 meq min^-1g of sample^-1 (Table 2). Ido and Susno (1990) and Appleford and Lenton (1997) reported the same results of increased hydrolytic enzymes in seeds by nano formulation coating.

Moreover, the hydrolytic enzymes α-amylase and lipase activities were more in seeds coated with 15 ml kg^-1 of seeds that helped in increased germination and seedling vigour. In addition to that, the increased rate of germination, seedling vigour and hydrolytic enzymes activity was due to faster uptake of water through carboxymethyl cellulose coated seeds by hydrophilic nature of coated material (Fig 2).

It is concluded that seeds coated with CMC based GA3 loaded nano formulation, at the rate 15 ml kg^-1 of seed recorded higher rate of imbibition (48%), speed of emergence (5.5), germination (87%), seedling length (24.7 cm), vigour index (2142) and dry matter production  (3.451) with optimum coating efficiency. It was followed by 10 ml kg^-1. Hence, these two concentrations were screened and it can be used for improving seed quality in groundnut for getting better seedling quality.

We acknowledge our grateful thanks to the Department of Nano Science and Technology, TNAU, Coimbatore for financial support for analytical works.