Composition and method for increasing the content of glucosinolates in adult plants of the genus brassica

Abstract

The present invention relates to a composition comprising methyl jasmonate and a polysiloxane polyether for increasing the content of glucosinolates in adult plants of the genus Brassica, for example, broccoli. By means of foliar application of the composition on adult plants having a developed cuticle, a significant increase is achieved in the concentrations of glucosinolates (mainly glucoraphanin and neoglucobrassicin), in the floret of the plants, without degrading the organoleptic properties thereof.

Claims

1. A composition comprising methyl jasmonate (MeJA), a polysiloxane polyether, water and a C2-C4 alcohol, wherein: methyl jasmonate represents between 0.0015% and 0.0028% of the total volume thereof; polysiloxane polyether represents between 0.068% and 0.1272% of the total volume thereof; C2-C4 alcohol represents between 0.15% and 0.28% of the total volume thereof; and water represents the rest of the volume of the composition.

2. The composition according to claim 1, consisting of methyl jasmonate (MeJA), a polysiloxane polyether, water and a C2-C4 alcohol.

3. The composition according to claim 1, characterised in that the C2-C4 alcohol is ethanol.

4. The composition according to claim 2, characterised in that the volume consists of 0.0022% by volume of MeJA, 0.2% by volume of ethanol, 0.1% by volume of polysiloxane polyether and the rest is water to a total volume of 100%.

5. The composition according to claim 1, wherein the polysiloxane polyether is a non-ionic trisiloxane.

6. The composition according to claim 1, wherein the concentration of methyl jasmonate is between 50 and 500 molar.

7. The composition according to claim 6, wherein the concentration of methyl jasmonate is 100 molar.

8. A method for increasing the content of glucosinolates in an adult plant of the genus Brassica comprising the following steps: a) carrying out a first foliar application of the composition defined in claim 1, at the time the leaf bud appears on the plant; b) carrying out a second foliar application of the composition defined in claim 1, five days after the first application; c) carrying out a third foliar application of the composition defined in claim 1, five days after the second application.

9. The method according to claim 8, wherein the glucosinolates are selected from the list comprising glucoraphanin, glucobrassicin, methoxyglucobrassicin, neoglucobrassicin and combinations thereof.

10. The method according to claim 8, wherein the plant of the genus Brassica is selected from broccoli, cauliflower, cabbage, rapini, Brussels sprouts, swede, turnip, kohlrabi, kale and rapeseed.

11. The method according to claim 10, wherein the plant is broccoli.

12. The method according to claim 8, wherein an amount between 100 and 150 ml of the composition is applied in each of the applications.

13. The method according to claim 8, wherein the foliar application of the composition is by spray.

14. The method according to claim 8, wherein: the composition comprises 0.0022% by volume of MeJA, 0.2% by volume of ethanol, 0.1% by volume of polysiloxane polyether and water to a total volume of 100%.

15. A method to improve the defense mechanisms against herbivores and pathogens in a plant of the order Brassicales, said method comprising the use of the composition according to claim 1.

16. A method to obtain a functional food, said method comprising the use of the composition according to claim 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the basic structure of a glucosinolate.

(2) FIG. 2 shows the chemical structure of a feruloyl-indole-glucosinolate, wherein the indolic glucosinolate is neoglucobrassicin.

(3) FIG. 3 shows the photograph showing the dispersion of a drop when methyl jasmonate is added with water, Triton X-100 and MPP (modified polysiloxane polyether).

(4) FIG. 4 shows an absorption spectrum (UV-Vis) with maximum absorption peaks coinciding with a feruloyl-GLS, the arrows indicating the maximum absorption peaks at 227±3 nm (GLS) and 320±16 nm (hydroxycinnamic acids such as ferulic acid).

EXAMPLES

(5) The invention will be illustrated below by means of several tests carried out by the inventors which demonstrate the effectiveness of the composition and method of the invention.

(6) Material and Methods

(7) Field Test

(8) Seedlings of three broccoli cultivars (Brassica oleracea var. Italica) obtained from a seedbed where the seeds grew for 30 days were used. Specifically, the cultivars were Ares, Parthenon and Marathon, all of them from the SAKATA company. The experiment was carried out during the autumn/winter period at an experimental farm in the Mirador field in the municipality of San Javier (Murcia) and under a semi-arid Mediterranean climate. The average daily temperature and relative humidity were calculated from measurements taken every 10 minutes using a data logger. The humidity reached in the plot was 50/80% (day/night) and the air temperature ranged between 24/8° C. during the day and night, respectively.

(9) A total of 40 plants to be cultivated were planted, 20 for each treatment. All the seedlings were planted on the same day, under the same conditions and irrigated with fertigation [2 dS m.sup.−1 electrical conductivity (EC)] twice a day for 10 minutes for the first 45 days, and then for 15 minutes for the next 45 days.

(10) Once they reached the reproductive phase, which is when the flower bud appears (approximately 120 days after sowing the seeds in the seedbed), the treatments were applied using Composition 1 and the Control Composition and performed three times on the leaf. One at the time the flower bud appears and the following applications 5 and 10 days, respectively, after the first application. Each application was carried out by spray, applying 100-150 ml per plant. The leaves and inflorescences were collected for analysis once they reached the commercial size thereof (approximately 18 days after the appearance of the flower bud) and they were frozen in liquid nitrogen and immediately lyophilised to prevent the compounds of interest from degrading until it was time to determine the glucosinolates and other metabolites.

(11) While the treatments were applied, irrigation by fertigation was maintained under the same conditions under which it was carried out before applying the treatments.

(12) Composition 1: A solution was prepared containing 0.0022% by volume of MeJA (for a final concentration of 100 micromolar in the composition), 0.2% by volume of ethanol, 0.1% by volume of polysiloxane polyether (Break-Thru S 233 ®) and 99.6978% by volume of water.

(13) Control Composition: The control treatment consisted of applying a solution containing 0.1% by volume of polysiloxane polyether and 99.9% by volume of water.

(14) Greenhouse Test

(15) In a growth chamber, seeds of the broccoli cultivar (Brassica oleracea var. Italica) Parthenon which grew for 30 days were germinated. Subsequently, they were allowed to grow in a greenhouse for 75 days until the inflorescences reached commercial size. In this case, foliar application of the treatments indicated below was carried out by spraying between 100 and 150 ml per plant: one at the time the flower bud appears and the following applications 5 and 10 days, respectively, after the first application.

(16) Control composition: consisting of a solution containing 0.2% by volume of ethanol and 99.8% by volume of water.

(17) Surfactant composition 1 (Triton X-100): consisting of a solution containing 0.1% by volume of Triton X-100 surfactant and 99.9% by volume of water.

(18) Surfactant composition 2 (MPP): consisting of a solution containing 0.1% by volume of the modified polysiloxane polyether surfactant and 99.9% by volume of water.

(19) Elicitor composition (MeJA): consisting of a solution containing 0.0022% by volume of MeJA, 0.2% by volume of ethanol and 99.7978% by volume of water.

(20) Composition 2 (MeJA+Triton X-100): consisting of a solution containing 0.0022% by volume of MeJA, 0.2% by volume of ethanol, 0.1% by volume of Triton X-100 and 99.70% by volume of water.

(21) Composition 1 (MeJA+MPP): A solution was prepared containing 0.0022% by volume of MeJA (for a final concentration of 100 micromolar in the composition), 0.2% by volume of ethanol, 0.1% by volume of polysiloxane polyether and 99.7968% by volume of water.

(22) Subsequently, the plants were harvested and the leaves and inflorescences were frozen in liquid nitrogen and immediately lyophilised to prevent the compounds of interest from degrading until it was time to determine the glucosinolates and metabolites of interest.

(23) Determining Glucosinolates and a Feruloyl-GLS

(24) The analyses were carried out on the leaf and inflorescence at the time of collecting the commercial size. The method used for extracting glucosinolates was based on the method developed by the research group and currently in the public domain: Dominguez-Perles et al. (2010) Journal of Food Science 75, 383-392. The quantitative analysis was carried out by HPLC-DAD. The lyophilised and ground samples (100 mg) were extracted with 1 ml of 70% MeOH in a hot bath at 70° C. for 30 minutes, stirring every 5 minutes in a vortex, to optimise the extraction. The reaction was then stopped in an ice bath and the samples were centrifuged to precipitate the insoluble material, for 15 minutes, at 10,000 g and at 4° C. The supernatant was collected and the methanol was removed on a rotary evaporator with a thermostatically controlled bath at 38° C. The dry residue obtained was redissolved in ultra-pure water and filtered (00.22 μm of PVDF) for the subsequent analysis thereof.

(25) Each sample (20 μl) was analysed in high-performance liquid chromatography (HPLC) equipped with a pump for binary eluent partitioning, an inline degasser, a photodiode detector autosampler (DAD). The compounds were separated in a C18 column measuring 250×4 mm and with a 0.4 um pore diameter, in reversed phase liquid chromatography (RP-HPLC) mode. The glucosinolates were identified using the methodology already described by the research group and publicly available for multipurpose analysis of intact glucosinolates (Martinez-Sánchez et al. 2006 Postharvest Biol. Technol. 42, 86-97; Dominguez-Perles et al. (2010) Journal of Food Sci. 75, 383-392), based on the retention times of the respective analytical peaks, the maximum absorption spectra thereof in the UV-Vis range and the comparison with commercially available external standards, sinigrin for aliphatic glucosinolates and glucobrassicin for indolic glucosinolates (Baenas et al. (2012) J. Agric. Food Chemistry, 60, 11409-11420). The detection was carried out at 227 nm and the concentration of glucosinolates was expressed in μmol/g of plant tissue dry weight.

(26) The spectrum type of feruloyl-GLS (see FIGS. 2 and 4) in which a phenolic acid and an indolic glucosinolate are joined together (tentatively, neoglucobrassicin, NGB) was determined by HPLC-DAD-ESI-MS/MS fragmentation, observing the parental ion of 724 (M-H, m/z, 100%) and the resulting fragments of 499 m/z (100%), 259 m/z (10.2%, glucosinolate indicator) and 193 m/z (100%, derived feruloyl indicator).

(27) The results of applying the treatments in the two tests carried out are shown below.

(28) Field Test

(29) Table 1. Concentration of glucosinolates and a feruloyl-GLS on leaves of different cultivars of Brassica oleracea var. Italica recorded in the field test using different treatments.

(30) TABLE-US-00002 TABLE 1 mg GRA/g mg GBS/g mg MGB/g mg NGB/g mg F-GLS/g Total Variety Treatment DW DW DW DW DW GLSs Ares Control 0.59 ± 0.03 0.42 ± 0.07 0.55 ± 0.07 1.38 ± 0.11 tr 2.94 ± 0.15 Ares Composition 0.63 ± 0.04 0.56 ± 0.05 0.69 ± 0.05 1.43 ± 0.09 0.01 ± 0.001 3.31 ± 0.16 Parthenon Control 1.03 ± 0.10 1.09 ± 0.11 0.30 ± 0.05 1.17 ± 0.10 tr 3.59 ± 0.11 Parthenon Composition 1 1.11 ± 0.10 2.25 ± 0.15 0.33 ± 0.04 2.04 ± 0.18 0.01 ± 0.001 5.73 ± 0.43 Marathon Control 0.60 ± 0.10 1.09 ± 0.23 0.24 ± 0.04 0.28 ± 0.05 tr 2.21 ± 0.28 Marathon Composition 1 0.77 ± 0.05 2.61 ± 0.14 0.41 ± 0.02 1.53 ± 0.11 0.01 ± 0.002 5.32 ± 0.24 GR: Glucoraphanin GB: Glucobrassicin MGB: Methoxyglucobrassicin NGB: Neoglucobrassicin F-GLS: Feruloyl-glucosinolate Tr: concentration in trace amounts DW. Dry weight

(31) Table 2. Concentration of glucosinolates and a feruloyl-GLS in inflorescences of different cultivars of Brassica oleracea var. Italica recorded in the field test using different treatments.

(32) TABLE-US-00003 TABLE 2 mg GRA/g mg GBS/g mg MGB/g mg MGB/g mg F-GLS/g Total Variety Treatment DW DW DW DW DW GLSs Ares Control 1.15 ± 0.04 1.05 ± 0.05 0.48 ± 0.03 1.65 ± 0.08 tr 4.33 ± 0.13 Ares Composition 1.61 ± 0.26 0.77 ± 0.11 0.58 ± 0.05 2.51 ± 0.1  0.03 ± 0.01 5.50 ± 0.19 1 Parthenon Control 1.75 ± 0.04 2.12 ± 0.16 0.50 ± 0.05 1.10 ± 0.25 tr 5.48 ± 0.21 Parthenon Composition 2.02 ± 0.11 2.01 ± 0.14 0.70 ± 0.02 4.48 ± 0.06 0.25 ± 0.02 9.47 ± 0.42 1 Marathon Control 1.74 ± 0.10 1.77 ± 0.07 0.41 ± 0.02 1.14 ± 0.05 tr 5.06 ± 0.53 Marathon Composition 2.01 ± 0.16 2.11 ± 0.20 0.62 ± 0.03 4.65 ± 0.18 0.28 ± 0.03 8.68 ± 0.15 1 GR: Glucoraphanin GB: Glucobrassicin MGB: Methoxyglucobrassicin NGB: Neoglucobrassicin F-GLS: Feruloyl-glucosinolate Tr: concentration in trace amounts DW. Dry weight

(33) Tables 1 and 2 show that the foliar application of the composition of the invention (Composition 1) in adult plants that have reached the reproduction phase and have a developed cuticle causes, for plants of crops different from the Ares, Parthenon and Marathon cultivars, an obvious effect on the increase in the concentration of individual glucosinolates (GRA: Glucoraphanin, MGB: Methoxyglucobrassicin and NGB: Neoglucobrassicin), both on leaf and on inflorescence, and on the appearance of a feruloyl-GLS, also on inflorescences and on elicited leaves.

(34) Furthermore, no decrease in the organoleptic properties of the inflorescences obtained in this test were recorded.

(35) Greenhouse Test

(36) Table 3. Concentration of glucosinolates on leaves and inflorescences of the Parthenon cultivar in the greenhouse test using different treatments

(37) TABLE-US-00004 LEAF (mg/g DW) mg GRA/g mg GBS/g mg NGB/g feruloyl- Total Parthenon DW DW DW GLS/g DW GLS Control 0.37 ± 0.73 ± 0.71 ± — 1.81 ± 0.02 0.12 0.12 0.05 Triton X-100 0.35 ± 0.70 ± 0.69 ± — 1.74 ± 0.06 0.14 0.15 0.05 MPP 0.33 ± 0.68 ± 0.68 ± — 1.69 ± 0.04 0.12 0.12 0.05 MeJA 0.35 ± 0.71 ± 0.69 ± — 1.75 ± 0.06 0.12 0.14 0.05 MeJA + 0.29 ± 1.34 ± 3.41 ± — 5.05 ± Triton X-100 0.08 0.11 0.25 0.06 MeJA + 0.58 ± 1.36 ± 5.62 ± 1.68 ± 0.01 9.23 ± MPP 0.21 0.3 0.38 0.18

(38) TABLE-US-00005 INFLORESCENCE (mg/g DW) mg mg mg feruloyl- GRA/g GBS/g NGB/g GLS/g Total Parthenon DW DW DW DW GLS Control 1.74 ± 2.08 ± 1.12 ± — 4.94 ± 0.06 0.09 0.09 0.10 Triton 1.75 ± 2.16 ± 1.19 ± — 5.10 ± X-100 0.06 0.14 0.15 0.12

(39) TABLE-US-00006 INFLORESCENCE (mg/g DW) mg mg mg feruloyl- GRA/g GBS/g NGB/g GLS/g Total Parthenon DW DW DW DW GLS MPP 1.73 ± 2.08 ± 1.08 ± —  4.98 ± 0.04 0.12 0.12 0.15 MeJA 1.70 ± 2.05 ± 1.22 ± —  4.97 ± 0.10 0.10 0.12 0.25 MeJA + Triton 2.09 ± 2.31 ± 2.75 ± —  7.15 ± X-100 0.14 0.15 0.19 0.33 MeJA + MPP 2.08 ± 2.76 ± 4.83 ± 1.08 ± 10.75 ± 0.16 0.28 0.26 0.10 0.38 GR: Glucoraphanin GB: Glucobrassicin MGB: Methoxyglucobrassicin NGB: Neoglucobrassicin F-GLS: feruloyl-GLS Tr: concentration in trace amounts DW. Dry weight

(40) As can be observed in Table 3, the application of Composition 1 (MeJA+MPP) achieves increases in the concentration of glucosinolates that are substantially higher than the rest of treatments applied to inflorescences and leaves and, moreover, it is the only one that achieves the appearance of a feruloyl-GLS. Composition 2 (MeJA+Triton X-100) which also favours the increase of glucosinolates but not the increase of a feruloyl-GLS, however, achieves a much lower increase (specifically 116% with composition 1, and 44% with composition 2).

(41) Table 4.—Results of the percentage of commercial inflorescences of the Parthenon cultivarcultivar in the greenhouse test

(42) TABLE-US-00007 % of Commercial Parthenon heads Control 100 Triton X-100 81.1 MPP 100 MeJA 100 MeJA + Triton X-100 58.4 MeJA + MPP 100

(43) As can be observed in Table 4, the application of Composition 1 (MeJA+MPP) does not degrade the inflorescences, considering degradation as an irregular shape or colour alteration, which does ostensibly occur when another surfactant is used.

(44) Drop Dispersion

(45) The drop dispersion of Compositions 1 and 2 and that of elicitor Composition (MeJA) was determined under binoculars (1.6×) (FIG. 3). It can be seen that no dispersion is observed in the drop of MeJA, and that there is more dispersion in the drop of MeJA containing the Triton X-100 surfactant and total dispersion in the drop containing the MPP surfactant.

(46) The calculated dispersion area of the drop indicates that Triton X-100 disperses 4 times more than water and MPP disperses 41 times more than water and 10 times more than Triton X-100 (see FIG. 3).