Supporting Information Synthesis, Characterization and Encapsulation of Novel Plant Growth Regulators (PGRs) in Biopolymer Matrices

1 University of Zagreb, Faculty of Agriculture, Department of Chemistry; Svetošimunska 25, 10000 Zagreb, Croatia; kvkahlina@agr.hr (K.V.-K.); sjuric@agr.hr (S.J.); marijan.marijan1986@gmail.com (M.M.); mvincekovic@agr.hr (M.V.) 2 Biotechnology Department, M. Auezov South-Kazakhstan University, Shymkent, pr. Tauke-Khan; mbota@list.ru (B.M) 3 Ukrainian State University of Chemical Technology, Gagarina av., Dnipro, Ukraine; swetasnegur9@gmail.com (S.V.K), prosyanykav@gmail.com (A.V.P)


Methods:
Testing of plant growth-regulating activity SI-2 Testing of auxin-and gibberellin-like activities SI-2 Toxicity determination SI-2 Field trials SI-2 Meteorological conditions of research during field trials SI-2 Fourier transform infrared spectroscopy and NMR analysis SI-4

Results and discussion:
Field trials SI-4 Molecular interactions between constituents in microcapsule formulations SI-6 NMR and C/H/N Elemental analysis SI-9 Methods: Testing of plant growth-regulating activity. Laboratory tests of the sowing properties of corn (sort Odessa 100), barley (sort Zernogradsky), winter wheat (sort Fedorovka) seeds were carried out by the method of germination in Petri dishes on filter paper [1]. Seeds were germinated in enamines aqueous solutions at concentrations 0.01, 0.001 and 0.0001 % (w/v) During germination, the following regimes were maintained: for corn 25 °C, barley 20 °C, winter wheat 20 °C. 50 pcs of barley and winter wheat seeds and 30 pcs of corn seeds were placed in each germinator. Before seed hatching, the seedlings were covered with dark paper, and then germinated during the photoperiod night: day 8:16 and temperature 22-24 °C. Plant analysis was performed on the 14th day. The repetition of the experiment was fourfold.
Seed cultivation was carried out in 500 ml chemical glasses with agar-agar. Initially, a minimal amount of water was poured into the glass to avoid dilution, and then water was added to the initial mark. For the first six days, the seeds were in complete darkness at a temperature of 22 °C. Then the growth took place at a photoperiod of 6:18. Biometric analysis of plants was carried out on day 14.
Testing of auxin-and gibberellin-like activities. A simple and reliable method for determining the auxin-like activity of any compounds is the method of direct growth of segments of coleoptiles of etiolated seedlings of oats, wheat or corn with fertilized top. The coleoptile of cereal plants is very sensitive to exogenous indole-3-acetic acid (IAA), and therefore it is a classic object for biological tests for auxin. The increase in the length of the segment after 24 h in the dark was taken as a measure of auxin-like activity. Deionized water and IAA (5 mg dm -3 ) were used as controls. The growth rate of oat coleoptiles correlates well with the level of diffuse IAA [2]. This test is practically insensitive to gibberellins and cytokinins [3].
As a highly specific test for gibberellin, either cuts of four-day-old corn seedlings with a coleoptile knot [4] or dwarf pea seedlings [5] are used. These segments were placed in Petri dishes with the test solutions and incubated for 48 or 72 hours in a dark thermostat at 26 °C. The gibberellic acid solution was used as a standard. The growth of the first leaf protruding above the cylinder of the coleoptile was measured.
Toxicity determination. Toxicity during intragastric exposure was determined after the method of Sanotykiy, 2013 [6].
Average lethal doses (LD50) for intragastric intake were established in sexually mature animals: rats -males weighing 180-240 g, female rats weighing 180-220 g, mice -males weighing 18-22 g. The substance was injected in its native form intragastrically on an empty stomach using a metal probe in compliance with the relevant rules. In total, 80 rats and 30 mice were used in the experiments.
The values of the administered doses ranged from 2000 to 8000 mg kg -1 . The obtained LD50 values for male and female rats were 5163 ± 811 and 6282±420 m1 kg -1 , respectively. The intragastric administration of PGR1 to white mice showed the LD50 value was 5350 ± 737 mg kg -1 .  Tables S1 and S2. Precipitation on the territory during all time were falling extremely unevenly. In 2018, the duration of the period without precipitation made up 264 days. The maximum daily amount of precipitation varied within 16-18 mm (Table S1).
The intensity of precipitation varied throughout the year. The greatest amount of precipitation in the growing season was observed in June, and the least -in August (Table S2). Annual precipitation was mainly due to intense precipitation in April-June 149.7 (45.5% per annum).
The average annual temperature made up 10.89 ± 0.56 ºС. The temperature range for the study period made up from -21.3 to + 37.8 ºС. The minimum temperatures were observed in January-February, and the maximum in July-August.
The greatest temperature fluctuations were observed in winter or spring. The autumn period is usually marked by a sharp and distinct drop in temperatures.
The winds were predominantly east and north-east.
The highest value of the average atmospheric pressure was observed in January-February, after which the pressure in March dropped rather sharply and reached a minimum in August.     Information on molecular interactions between enamines, calcium chloride, chitosan and sodium alginate in mixtures was obtained using FTIR. Spectra of single enamines and spectrum of their mixtures with calcium chloride, alginate or chitosan are presented in Figure S1a and S1b,c,d, respectively. Experimental vibrational frequencies and band assignments of enamines are listed in Table S9.

Molecular interactions between constituents in microcapsule formulations
Spectra of PGR1 and PGR2 exhibit the spectral characteristics for aliphatic amines and spectrum of PGR3 for secondary and tertiary amines. Both, PGR1 and PGR2 spectra show the N-H stretching in the range of 3300-3000 cm -1 with two bands corresponding to asymmetric (higher frequency) and symmetric N-H stretching of primary amines. These bands are weaker and sharper than those alcoholic O-H stretching occurring in the same region. All characteristic peaks of PGR1 are relatively less intense than those of PGR2. PGR3 exhibits a sharp peak corresponding to secondary amine at 3332 cm -1 , and peaks characteristic for tertiary amine st 1240 and 1150 cm -1 .
Analysis of single calcium chloride, sodium alginate and chitosan spectra are previously reported [7]. The characteristic peaks in the calcium chloride spectrum are at 3494, 3396, 3214, 1646, 663 cm -1 . The frequency range 3214-3494 cm -1 and the medium intensity band at 1646 cm -1 represent the bending forms of hydroxyl groups. The medium intensity peak at 663 cm -1 represents the stretching of the Ca-O bond.
Analyses of sodium alginate and chitosan FTIR spectra were in correlation with literature data [8]. Spectra of enamines with chitosan exhibit almost no shifting of characteristic bands but all characteristic peak intensities are enhanced due to reaction between the amino group of dimethyl esters of amino fumaric acid and the -OH group of chitosan as a result of different dipole moments of the groups, and the formation of saturated amides (Figure 1d). The peak intensities increase is significant in the PGR1 spectrum and much smaller in the PGR2 and PGR3 spectra.