Sodium alginate

For the preparation of immobilized or encapsulated form of nano fertilizers for treatments T3, T6, and T7, sodium alginate (Sigma Aldrich) micro-emulsion was prepared by adding 1 g of sodium alginate into 30 ml distilled water containing 3–4 drops of paraffin oil. This mixture was vigorously stirred for 40 min and then added 1.2 g of nano fertilizer from stock. This thick paste was added in a burette and droplets were allowed to fall in 1 M calcium chloride (Sigma Aldrich) solution^{22 (link)} which turned into 300 solid beads (Fig. 1) that were applied once to each plant as a single dose for three months.Figure 1

(a) Set up to prepare nano-carriers, (b) prepared nano-carriers.

Lecitase™ Ultra (LU, >10 000 U × g⁻¹) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Racemic alcohols: (E)-4-phenylbut-3-en-2-ol (1a), (E)-4-(4′-methylphenyl)but-3-en-2-ol (1b), (E)-4-(2′,5′-dimethylphenyl)but-3-en-2-ol (1c), and (E)-4-(4′-methoxyphenyl)but-3-en-2-ol (1d) were synthesized according to the procedures described earlier [61 ,62 (link),63 (link)]. Acetyl chloride (≥99%), propionyl chloride (98%), butyryl chloride (≥99%), sodium alginate, calcium chloride (≥96%), p-nitrophenyl palmitate (p-NPP), p-nitrophenol (p-NP), and polyethyleneimine (PEI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cyanogen bromide-activated agarose (Sepharose^® 4B) was purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Supelite™ DAX-8 was purchased from Supelco Analytical (Bellefonte, PA, USA). sodium alginate was purchased from Sigma-Aldrich. Other chemicals were of analytical grade.

To prepare Type 1 AOs, Type 2 AOs, and Type 3 AOs, alginate lyase (Sigma-Aldrich, A1603) was used to catalyze the cleavage of alginate. To make Type 1 AOs, 2% (w/v) of sodium alginate (Sigma-Aldrich, A0682) was prepared by dissolving sodium alginate in 100 ml of distilled water (DW) followed by incubation at 37 °C for 6 h in the presence of 20 U/mg alginate lyase in a rotary shaker at 150 rpm. After the incubation, alginate lyase in the sample was inactivated at 100 °C for 10 min and removed after centrifugation at 12,000×g for 10 min at 4 °C^{33 (link)}. Type 2 and Type 3 AOs were prepared with the same method except that alginate lyase treatment time was changed from 6 to 12 h for Type 2 AOs and 24 h for Type 3 AOs. Fractions of AOs were collected, freeze-dried, and store at − 4 °C.

For encapsulation of the OFs, 0.5% of sodium alginate (Sigma-Aldrich, St. Louis, MI, USA) solution was made of sodium alginate, and 2.0% of calcium chloride (Sigma-Aldrich, St. Louis, MI, USA) solution was made of calcium chloride. These solutions were disinfected at 120 °C for 30 min [16 (link)]. A single OF was transferred to individual drop of alginate with pipette, and then this alginate bead was placed in the encapsulation solution of calcium chloride for 2 min (Figure 1c). After encapsulation, bead was washed with MEM-α (1X) medium for CaCl₂ removal [14 (link),17 (link)]. 20 OFs were encapsulated and half of them were cultured in dishes and remaining half were cultured on chips.

In order to fabricate composite granules, the following reagents were used: sodium alginate (Sigma Aldrich, US), chondroitin sulphate sodium salt (TCI, Belgium), calcium chloride anhydrous CaCl₂ (Sigma-Aldrich, China) and the following previously synthesized powders and microgranules: HA, Ag-HA, Ga-HA, AgM and GaM.
At first, a 4% aqueous sodium alginate solution was prepared at 40 °C and chondroitin sulphate sodium salt was added to obtain a 0.5% suspension. Then, two types of composite granules were prepared.
In the first type of granules, 1 g of Ag-HA or Ga-HA (or HA) powder was added to the suspension (10 mL) and mixed vigorously, resulting in a milky, dense slurry. Meanwhile, the cross-linking solution (1.5% CaCl₂) was prepared. Finally, the slurry was added dropwise to a CaCl₂ solution, stirred using a magnetic stirrer and granules were formed. The granules obtained were left in the cross-linking agent for 10 min, rinsed with distilled water, dried in air and then lyophilized.
During the preparation of the second type of granules, pure, unsubstituted HA and AgM or GaM microgranules (ratio 1:1) were used instead of Ag-HA or Ga-HA powders. The other stages of production remained unchanged. All the obtained granules are listed in Table 6.