Effect of Ulva lactuca L. Seaweed Biostimulant on Seed germination, Growth, and some Biochemical properties of Vigna radiata L.

The effect of green marine biostimulant Ulva lactuca evaluated as Biofertilizer to improve growth of Vigna radiata L. Seeds waspresoaked with different concentration seaweed extract of Ulva lactuca such as 0.2%, 0.5%, 0.7%, 1.0%, 1.5%, 2.0% and control (without treatment) in different time period such as 0h, 1h, 2h, 3h, 4h, 5h, 6h, 12h and 24h. In Petri dish, after 5 days plant growth parameter like % seed germination, root length, shoot length, seedling length, seed vigour index, seed stamina index, No. of lateral roots, wet weight, dry weight, % moisture contentwere observed. After 40, 50 and 60 days some growth parameter like No. of branches per plant, No. of leaves per plant, total height of the plant, No. of flower per plant, No. of pod per plant, length of the pod, No. of seed per pod and leaf area were observed in field trial. In this study, after 50 days Bio-chemical parameter of chlorophyll-a, chlorophyll-b and total chlorophyll, carotenoid, protein, total soluble sugar and reducing sugar measured in plant material. Better result of plant growth observed in higher concentration such as 1.5% & 2.0% of seaweed extract of U. lactuca in Vigna radiata L. and bio-chemical parameter observed in lower concentration such as 0.2% and 0.7%of seaweed extract, respectively. Some biochemical parameter like protein,reducing sugar and carotenoid were decrease the amount of concentration compared to control. Keywords— Bio-chemical parameter, Growth parameters, Ulva lactuca, Vigna radiata.


INTRODUCTION
India has a huge coastline of more than 7000km which possesses a large diversity of marine seaweeds. India, the Gujarat state covers a total coastline of 1600km, which is the longest among all the states of the country, and it covers almost 21.9% of the Indian coastline of India. Port Okha coast, which is known for its luxuriant growth of a diverse assemblage of seaweeds on Saurashtra coast, is found to have abundant quantities of seaweeds being drifted and washed ashore every year. Port Okha is located at 22 o 28' N latitude and 69 o 05' E longitude and is situated within the opening of Gulf of Kachchh, has a flat rocky intertidal belt, with many tide pools, gullies and crevices. Marine algae found attached to the bottom on solid substrates such as rocks, pebbles, shells, dead corals and plants. Seaweed has great importance, rich in vitamins, minerals, proteins, carbohydrates, amino acids, lipids etc. Due to the richness of bioactive constituents, it revealed many applications such as antioxidant, anticancer, antimalarial, pharmacological and cosmeceutical, activities. Approximately, 271 genera and 1153 species of marine algae, including forms and varieties have been enumerated till date from the Indian waters. Seaweed extracts are soluble, non-toxic and nonpolluting extracts contribute to the uptake of more nutrients from the soil. Seaweeds extract as organic biostimulant is fast becoming accepted practice in agriculture and horticulture due to its beneficial effects

Collection of Seaweed:
The green seaweed sampleUlva lactuca Linnaeus belonging to Chlorophyceae family were collected during 2020-2021 at Okha coast is situated at 22 0 28'7.3272 N & 69 0 4'11.3664 E in the "Gulf of Kutch" on the northwestern most part of Saurashtra in Gujarat, India. After collection of seaweeds, its washed immediately with seawater to remove other debris, other epiphytes and sand particles. Then it was transferred in laboratory and washed thoroughly with simple water up to 3 to 4 times for removal of extra salts on the surface.

Preparation of Seaweed Liquid Fertilizer (SLF)
The seaweed of Ulva lactuca was shade dried followed by oven drying at 60 o C for 3-5 hours according to the species. The dried sample was grounded with blender to get fine powder and it was stored for future use. These fine powder 10g was weighed on electronic balance, 100ml distilled water was added. The mixture was incubated for One day (24h). Thereafter, the extract was filtered through What-man No. 1 filter paper (Bhosle et al. 1975). The collected filtrate was stored in refrigerator (0-20 o C). The obtained filtrate was considered as 100%.

Collection of Vegetable Seeds:
The Vigna radiata L. seed collected from Vasundhara Agro Agency, Bhavnagar, Gujarat during 2020-21.

Seed Soaking:
The seeds surface was sterilized with 0.1% HgCl2 up to 1-2 minutes and washed help of distilled water. Seeds was presoaked with different concentration seaweed extract of Ulva lactuca such as 0.2%, 0.5%, 0.7%, 1.0%, 1.5%, 2.0% and control (without treatment) in different time period such as 0h, 1h, 2h, 3h, 4h, 5h, 6h, 12h and 24h at room temperature for better germination and early growth. After 5 days all the vegetative parameter was observed.

Seed Germination
Germination was observed daily over a period of 5

Preparation field for further analysis:
All healthy and specific time selected Vigna radiata L. seed primed in each concentration of seaweed extract and sawing in enough space in each field. Water is sprinkle after sawing seed for maintaining the moisture content.

Physicochemical Analysis of Seaweed Extract:
All Physicochemical parameters were analyzed by Handheld Multiparameter meters -Aquasol Digital (Testing Equipment) Model No. AM-AL-01

Biochemical Analysis:
After 50 days biochemical parameter of chlorophyll-a, chlorophyll-b, total chlorophyll, carotenoidwere observed. Protein was determined by Lowry method, Total Soluble Sugar was determined by Anthrone meyhod, reducing sugar was determined by Nelson-Somogyi's method.

Germination and growth parameters of Vigna radiata L. seedling
Vigna radiata L. showed 80% germination in the control and 88.88% in the 1.0% and 2.0% seaweed biostimulant treated seeds. A minimum of 81.11% germination was observed at 0.2% seaweed biostimulant when compared to all other concentrations of treatment (Table 1 to 7). Germination occurred in all treatments in the 2 nd day. The effect of seaweed extract showed significantly highest seed germination than control (Table 1 to 7). The maximum seedling length of 18.30cm was recorded when the plants applied with 1.5% of Seaweed biostimulants in 12h time duration (Table 5), and minimum seedling length was recorded in 0.2% (13.2cm) of Seaweed biostimulants in 2h time period (Table 1). A maximum total plant height of 28 cm was recorded applied with 2.0% (4h) of seaweed biostimulants and minimum in 1.5% (12h) (18cm). Also, the plants applied with 2.0% (4h) seaweed biostimulants increased the No. of branches, No. of leaves, length of the pod and leaf area. A maximum seed vigour index of 1830 in 1.5% seaweed biostimulants.

Physicochemical properties of seaweed extract
The values of P H in Ulva lactuca 2.0% (Table 11)                Control

Biochemical properties of Vigna radiata L. treated with seaweed extract Ulva lactuca
Maximum concentration of 0.2371mg/g dry weight of chlorophyll a, 0.5491mg/gm dry weight of chlorophyll b and 0.5451mg/gm dry weight of total chlorophyll was recorded at 0.5% of U. lactuca seaweed extract treatment ( Figure 1). Their decreases more than 43%, 54.87% and 53.47% respectively, when compared to control. Carotenoid also continue decreases amount of all seaweed extract concentration ( Figure 2). The maximum concentration of 3.8475mg/gm dry weight of protein was recorded at 1.5% seaweed extract in U. lactuca (Figure 3). A maximum concentration of 7.9677 µg/ml dry weight of total soluble sugar was recorded at 0.2% U. lactuca seaweed extract treatment ( Figure 4). In total soluble sugar amount of concentration was high compared to control. Maximum concentration of 11.4245 µg/ml dry weight of reducing sugar was recorded at 0.7% of U. lactuca seaweed extract treatment ( Figure 5). Their decrease 62.72% compared to control.

IV. DISCUSSION
The Vigna radiata L. seeds soaked with higher concentrations of the seaweed biostimulant showed higher rates of germination, while the lower concentrations of the extracts inhibited the germination. The value of seaweed as fertilizer is not from the nitrogen, phosphorous, potash and organic matter but from trace element and metabolites similar to plants growth regulators (Booth, 1969). Seaweed liquid fertilizer was found superior than chemical fertilizer because of the presence of high level of organic matter (Aitken and Senn, 1965). The increased germination percentage at high concentrations may be due to the presence of some growth promoting substances such as IAA and IBA, Gibberellins (A&B), cytokinins, micronutrients (Fe, Cu, Zn, Co, Mo, Mn, Ni), vitamins and amino acids (Challen and Hemingway, 1965). The increased seed germination percentage at high concentrations may be due to the presence of growth promoting substances (Jennings and Yulloch, 1965 (Dhargalkar and Untawale, 1983). Additionally, it is known that higher concentrations of (1.0%) the Ulva lactuca liquid extract can inhibit the germination of mung bean (Castellanos-Barriga, 2017). In this study, total chlorophyll contents highest in 0.5% concentration in U. lactuca seaweed biostimulants and lowest in 1.5% seaweed biostimulants in U. lactuca. Protein, total soluble sugar and reducing sugar was highest in 1.5%, 0.2% and 0.7% concentrations respectively, and lowest in 0.7% and1.0% respectively. Vigna catajung when treated with 10% Caulerpa racemose extract contained highest protein and amino acid contents (Anantharaj and Venkatesalu, 2001). The amount of protein content was found highest the shoot system under the treatment of Enteromorpha and Jania extract (El-Sheekh and El-Saied, 1999).

V. CONCLUSION
The results obtained in this experiment showed this presoaking experiment seed with seaweed extract for improve and increase seed germination and plant growth of selected plant of Vigna radiata L. the study showed that there is significant effect of treatments with seaweed extract from Ulva lactuca on bio-chemical parameter of chlorophyll a, b & total, carotenoid, protein, total soluble sugar and reducing sugar. The seed priming is a new and unique method for improvement of healthy and fast seed germination. Seaweed biostimulants as a Bio-primer material and this Bio-priming method is a organic, very easy, very cheap, easily available and eco-friendly. Based on this experiment, green algae of Ulva lactuca on giving a result of in a plant of Vigna radiata L. higher growth are