Rubus cultivation

Abstract

The present invention relates to the cultivation, preferably indoor cultivation, of Rubus chingii for increased production of rubusoside and the provision of a plant material, preferably a dried plant material, having an increased content of rubusoside.

Description

[0001] Rubus Cultivation

[0002] The present invention relates to the cultivation, preferably indoor cultivation, of Rubus chingii (also called Rubus suavissimus or Rubus chingii var. suavissimus) for increased production of rubusoside and the provision of a plant material, preferably a dried plant material, having an increased content of rubusoside. Background of the invention

[0003] Consumers generally have a strong preference for foodstuffs or indulgence foods, which have a large amount of high caloric sugar, in particular sucrose (saccharose), glucose, fructose or mixtures thereof, due to the pleasant sweetness and sweetness profile associated therewith. On the other hand, it is generally known that a large content of readily metabolizable carbohydrates causes a steep rise in blood sugar levels, leads to the formation of fat deposits and ultimately can result in health problems such as overweight, obesity, insulin resistance, age-onset diabetes and complications thereof. Another particular aggravating factor is that many of the above-mentioned carbohydrates can also have an adverse effect on dental health, as they are decomposed by specific types of bacteria in the oral cavity into lactic acid, for example, and can attack the enamel of milk teeth or adult teeth (caries).

[0004] Therefore, it has long been an objective to reduce the high caloric sugar content of food or beverage products and replace it partly or entirely by other substances that impart a sweet taste or which can positively affect the sweet taste in a low concentration without exhibiting sweet taste itself at these low concentration (taste modulators).

[0005] Rubus chingii (syn. Rubus suavissimus) is cultivated since ancient times in China, where it has been used for medical purposes and its leaves are used for preparation of a sweet tea. The active compound, rubusoside, is found predominantly in the leaves of the plant and can be extracted and used as a flavoring substance.

[0006] Rubusoside belongs to a group of compounds called steviol glycosides. It is a plant metabolite and is highly useful as natural non-caloric sweetener. Apart from that, rubusoside has further beneficial properties, such as solubilizing, antidiabetic, antiinflammatory, antiangiogenic and antiallergic properties (Zhou et al. 2021 , Industrial Crops and Products, 162m April 2021 , 113245; https: / / doi.Org / 10.1016 / j.indcrop.2021.113245).

[0007] Rubusoside is the main sweet component extracted from the leaves of R. Chingii. To this end, the plants’ approx. 3 m long tendrils including foliage are harvested and, after drying, e.g. at room temperature or under heat, subjected to rubusoside extraction. Analytical studies showed that the highest concentrations of rubusoside are found in the leaf tips (up to 8% per dry weight) and decrease with increasing age of the leaves to below 2%.

[0008] Rubusoside is also a minor steviol glycoside existing in the leaves of Stevia rebaudiana, which however predominantly comprises stevioside (and in lesser amounts rebaudioside A and rebaudioside C). Strategies to produce rubusoside through enzymatically hydrolyzing stevioside have been proposed (Zhou et al 2021 , supra). However, such strategies rely on additional enzymatic processing steps; thus, increasing the production costs.

[0009] On the other hand, efforts to extract of rubusoside directly from R. Chingii are limited by the abundance of abundance of rubusoside in the plants. Therefore, there is a great need in the art to increase the concentration of the active compound in R. chingii material, in particular leaf materiel, to lower the production costs for rubusoside and rubusoside- comprising extracts.

[0010] For Stevia rebaudiana, it was observed in in vitro cultures that a number of elicitors could affect the stevioside production in micropropagated explants (Bayraktar et al. 2016, Plant Cell Tiss Organ Cult (2016) 127:289-300.) However, rebaudioside A production remained entirely unaltered and for some elicitors (chitosan, methyl jasmonate and salicylic acid) their concentration inversely correlated with the stevioside content, leaving an unclear relationship between elicitors and steviol glycosides. Moreover, the plant hormones salicylic acid and methyl jasmonate showed pronounced adverse effects on biomass and root phenotype throughout all used concentrations, contraindicating against their use. Overall, the obtained stevioside quantities where drastically lower than in field-grown plants and as the application requires in vitro tissue culture, it is not applicable for field or indoor cultivation of whole plants suitable for commercial production.

[0011] For Rubus chingii, there is no cultivation method described, neither for cell culture of explants nor for whole plant cultivation, that may help to increase the rubusoside concentration.

[0012] It was thus a task of the present invention to provide a method for the commercial large- scale cultivation of R. chingii and provision of the plant material, in particular dried leaf material, having an increased rubusoside concentration for subsequent processing.

[0013] Description of the invention

[0014] In a first aspect, there is provided a method for increasing the rubusoside concentration and / or amount in a Rubus chingii plant, the method comprising: (i) at least one application of a composition comprising methyl jasmonate (MeJA) in a concentration of 0.05 to 50 mM; and / or (ii) at least one application of a composition comprising 0.15 to 120 mg MeJA per plant per application; to said Rubus chingii plant during cultivation.

[0015] In a second aspect, there is provided a Method for producing a Rubus chingii plant with an increased rubusoside concentration and / or amount, the method comprising a) providing a Rubus chingii plant, seedling or seed; b) cultivating said Rubus chingii plant, seedling or seed, wherein the cultivation comprises (i) at least one application of a composition comprising MeJA in a concentration of 0.05 to 50 mM; and / or (ii) at least one application of a composition comprising 0.15 to 120 mg MeJA per plant per application; to said Rubus chingii plant; c) obtaining a Rubus chingii plant with an increased rubusoside concentration and / or amount.

[0016] In a third aspect, there is provided a method for producing Rubus chingii leaf material with an increased rubusoside concentration and / or amount, the method comprising a) providing a Rubus chingii plant, seedling or seed; b) cultivating said Rubus chingii plant, seedling or seed, wherein the cultivation comprises (i) at least one application of a composition comprising MeJA in a concentration of 0.05 to 50 mM; and / or (ii) at least one application of a composition comprising 0.15 to 120 mg MeJA per plant per application; to said Rubus chingii plant; c) harvesting leaf material of said Rubus chingii plant.

[0017] It is advantageous in terms of the present invention to cultivate Rubus chingii but also hybrids thereof and targeted crosses with Rubus chingii as one of the parental plants can be cultivated with a method according to the present invention.

[0018] In certain embodiments of the third aspect, step c) is followed by: d) drying the harvested leaf material; and e) obtaining dried leaf material. The drying of leaf material may, for instance, be performed at a temperature of 20 to 110°C, preferably at a temperature of 30 to 70°C, more preferably at a temperature of 40 to 60°C. Preferably, the obtained dry plant material has a remaining water content of maximum 20 wt.-%, more preferably maximum 10 wt.-%, most preferably of maximum 5 wt.-%.

[0019] Methods for determining of the remaining water content are well known in the art. Preferably the water content is determined by Loss-on-Drying (LOD) method determined e.g. by a dry-mass balance or thermogravimetry or by Karl Fischer Titration.

[0020] In one embodiment of the third aspect, harvesting of step c) comprises harvesting of leaf material and stem material, wherein after drying of step d) the plant material is divided into leaf and stem material, preferably by a method selected from air separation, air floating or sieving, to obtain a plant material, which consists of or comprises at least 75 wt.-%, preferably at least 85 wt.-%, especially preferably at least 95 wt.-% leaf material.

[0021] The term “harvesting” in terms of the present invention means the removal of the grown plant material from the plant. Harvesting of leaf material may be performed by removing the leaves from the remaining plant and thus harvesting only leaf material. In other embodiments leaf material may be harvested together with other material, such as combination of upper stems and leaves. Preferably, only leaf material is harvested.

[0022] Generally, the term “rubusoside” describes a chemical compound (4a)-13-(p-D- glucopyranosyloxy)-kaur-16-en-18-saure-p-D-glucopyranosylester having the following chemical structure:

[0023] “Methyl jasmonate” and “MeJA” are used interchangeably herein. Methyl jasmonate is the methyl ester of jasmonic acid. It is a lipid-based plant hormone from the group of oxylipids (Avanci NC, Luche DD, Goldman GH, Goldman MH. Jasmonates are phytohormones with multiple functions, including plant defense and reproduction. Genet Mol Res. 2010 Mar 16;9(1):484-505). Methyl jasmonate according to the present invention may be (-)-methyl- (3R,7R)-jasmonate or (+)-methyl-(3S,7S)-jasmonate or mixtures thereof, also referred to as (±)-Methyl Jasmonate.

[0024] It was surprisingly found that the application of MeJA on whole R. chingii plants during cultivation leads to a dose dependent induction and increase of rubusoside without affecting biomass generation.

[0025] In one embodiment according to the first, second or third aspect, the method comprises at least one application of 0.2 to 100 mg, or 0.5 to 80 mg, or 1 to 60 mg, or 2 to 40 mg, or 3 to 35 mg MeJA per plant per application to said Rubus chingii plant during cultivation.

[0026] In preferred embodiments at least 1 mg, preferably at least 2 mg, more preferably at least 3 mg and not more than 100 mg or 80 mg, preferably not more than 60 mg or 40 mg or 30 mg, MeJA is applied per plant per application to said Rubus chingii plant during cultivation.

[0027] In one embodiment according to the first, second or third aspect, the composition comprises MeJA in a concentration of 0.05 to 50 mM, or 0,1 to 30 mM, or 0,1 to 20 mM, or 0,1 to 10 mM, or 1 mM to 10 mM. In preferred embodiments, the composition comprises MeJA in a concentration of at least 0.1 mM, preferably at least 0.5 mM or 1 mM, and at most 50 mM or 40 mM, preferably at most 30 mM or 20 mM or 10 mM.

[0028] In one embodiment according to the first, second or third aspect, about 10 to about 100 hours, or about 20 to about 90 hours, or about 30 to about 70 hours, or about 40 to about 60 hours, or about 40 to about 56 hours are between each application.

[0029] In one embodiment according to the first, second or third aspect, the Rubus chingii plant is at least 4 weeks, 5 week, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 1 months or at least 12 months old at the time of the at least one application according to the present invention, counted from the initial cultivation of a seed or cutting resulting in said Rubus chingii plant.

[0030] In certain embodiments, the Rubus chingii has a root fresh weight of at least 50 mg, at least 100 mg or at least 150 mg, optionally up to 500 mg, 600 mg or 700 mg, at the time of the at least one application according to present invention.

[0031] In certain embodiments, the Rubus chingii has developed or is beginning to develop additional shoots at its roots at the time of the at least one application according to present invention.

[0032] The MeJA application according to the present invention may be performed by foliar application and / or by adding the desired amount into the cultivation water or by providing it in gaseous form to the plant.

[0033] In preferred embodiments the according to the first, second or third aspect, the application comprises or consists of foliar application. “Foliar application” is a method in which the substance is directly applied to the leaves. It is preferred that the composition comprising MeJA is distributed over the surfaces of the leaves. Therefore, it is particularly preferred that application comprises or consist of spray application. The composition is sprayed directly onto the leaves surfaces to facilitate a homogeneous distribution. Naturally, a part of the composition will also reach other parts of the plant, in particular the upper stem, due to the nature of spray application.

[0034] In one embodiment according to the first, second or third aspect, the cultivation further comprises 1 to 20 days, or 1 to 15 days, or 1 to 10 days, or 4 to 10 days afterthe application or, if the application is performed more than once, after the last application. Forthe second aspect, a cultivation after the last application means cultivation after the last application and before the plant is obtained in step c). For the third aspect, a cultivation after the last application means cultivation after the last application and before the leaf material is harvested in step c).

[0035] It is preferred that the composition comprising MeJA is liquid, such as an aqueous solution comprising MeJA. MeJA may be solved directly in water in the desired concentration to obtain a composition comprising MeJA according to the various aspects herein. In one embodiment, MeJA is solved in distilled, deionized and / or demineralized water.

[0036] In one embodiment according to the first, second or third aspect, the composition further comprises at least one surfactant. A surfactant may be used as a spreading aid to facilitate an even wetting of plants surfaces and as a penetration aid. Any surfactant known in the art for spray application of plants, in particular a non-ionic surfactant, may be used for of the present invention. It is preferable that a surfactant used in the composition according to the present invention has little or no effect on the synthesis and / or accumulation of cell metabolites in the plant. A surfactant may for example, without being limited thereto, be BREAK-THRU® SP 133 or BREAK-THRU® S 301 .

[0037] The composition comprising MeJA according to the first, second or third aspect, may comprise one or more adjuvants, including oil-based adjuvants, such as crop oils, crop oil concentrates (CoCs), methylated seed oils (MSOs) and essential oils; fertilizers; pesticides, such as herbicides, insecticides, nematicides, molluscicides, avicides, rodenticides, bactericides, microbicides, fungicides, insect repellents and animal repellents; compatibility agents; buffering agents; water-softening agents; defoaming agents; deposition agents; and anti-drift agents.

[0038] In one embodiment according to the first, second or third aspect, the cultivation is indoor cultivation or field cultivation, preferably wherein the cultivation is indoor cultivation.

[0039] Indoor cultivation may be performed in a greenhouse comprising transparent materials for exposing the interior to sunlight or may be performed in a structure without sunlight, relying on artificial lighting alone.

[0040] Preferably cultivation is performed in a controlled environment, i.e. day and night temperatures and / or lighting and shading and / or humidity and / or CO2 levels and or / nutrient levels are monitored and regulated. A "controlled environment" refers to a specific area or setting in which various factors and conditions are regulated and maintained within predetermined limits to ensure consistency, stability, and control over certain variables. The purpose of creating a controlled environment is to minimize external influences and fluctuations that could affect, e.g., the quality of products in a way that might introduce unwanted variability or errors. Agricultural settings, like greenhouses, are controlled environments where variables like temperature, humidity, light, and CO2 levels are managed to optimize plant growth and crop production. To this end, the system includes sensors and controllers that monitor and adjust environmental conditions and nutrient delivery, ensuring optimal plant health and growth. The environmental conditions preferably to be controlled are one or more of temperature, humidity, nutrient level and light receipt. The term "light receipt" in the context of crop farming refers to the amount and quality of light that crops receive during their growth cycle. Light is one of the essential factors influencing plant growth and development, and its quantity, duration, and quality can have a significant impact on crop yields and overall plant health.

[0041] “Cultivation” in terms of the present invention means exposing of the cuttings or rooted plants to conditions under which they grow and develop more biomass. The person skilled in the art is well aware of corresponding conditions and how to achieve such conditions in order to enable biomass growth for R. Chingii plants. Cultivation of R. Chingii according to the present invention can thus be achieved in any way and under any conditions known in the art as suitable for R. Chingii cultivation.

[0042] An R. Chingii plant that is cultivated according to present invention may, for example without being limited thereto, be provided by growing an R. Chingii plant from a seed and / or by rooting by an R. Chingii cutting (root cutting or stem cutting). The term “rooting” in terms of the present invention means providing conditions, in which a head and / or shoot is able to develop roots. Once the cutting has developed roots, it is referred to as a plant. The person skilled in the art is well aware of corresponding conditions and how to achieve such conditions in order to enable root growth.

[0043] Cultivation conditions of R. chingii may be changed during cultivation. In particular, the cultivation may comprise pre-cultivation followed by different cultivation conditions for the at least one application according to the present invention.

[0044] R. chingii may be cultivated in soil, such as clay soil and / or peat soil, or may be cultivated in / on soil-free substrates, such as perlite, gravel, expanded clay aggregate and / or other inert substrate material. In preferred embodiments, the Rubus chingii plant is cultivated in soil, or is cultivated in / on a soil-free substrate for hydroponic, aeroponic and / or fogponic cultivation, at the time of the at least one application according to present invention. In one embodiment, the R. chingii plant is cultivated in / on a soil-free substrate, including perlite, for hydroponic, aeroponic and / or fogponic cultivation at the time of the at least one application according to the present invention.

[0045] Soil-free cultivation may be performed in a hydroponic, aeroponic and / or fogponic system. A “hydroponic system” is a cultivating system, wherein the plants are cultivated in a soil- free substrate with continuous exposure to a water-based nutrient solution. In an “aeroponic system” is a system, plants are suspended without soil and the roots and / or lower stems are sprayed with a water-based nutrient solution. A “fogponic” system describes a system, in which the plants are cultivated without soil, wherein the water-based nutrient solution is applied to the roots form of small particles, such as vapor.

[0046] In certain embodiments, the R. chingii plant is cultivated in a hydroponic system at the time of the at least one application according to the present invention.

[0047] Hydroponic cultivation may, for example, be performed in a static solution culture and / or in continuous-flow solution culture, such as the nutrient film technique. A nutrition film technique describes a system, wherein the planting trays are standing in a continuous cultivation water film, which is supplemented by a nutrient solution. Hydroponic cultivation may also use an ebb and flow irrigation system. In ebb and flow hydroponics, the waterbased nutrient solution is pumped into and drained from the soil-free substrate at regular intervals.

[0048] A hydroponic system may also be a passive hydroponic system, deep water culture or a run-to waste system.

[0049] Irrigation may, for example, also be achieved by surface irrigation, sprinkler irrigation, drip irrigation and / or trickle irrigation.

[0050] It is particularly preferable to supply the plants during cultivation with all essential nutrients. Soils, substrates and fertilizers for cultivation of Rubus Chingii are known to the skilled person. A preferred fertilizer is a hydroponic fertilizer. Hydroponic fertilizer, often referred to simply as "hydroponic nutrients" or "hydroponic solution," is a specialized type of fertilizer designed for use in hydroponic systems, i.e. growing plants without soil, where plants receive all of their nutrients directly from a water-based nutrient solution. Because soil is not present in hydroponic systems, plants rely entirely on the nutrient solution for their essential mineral elements and nutrients. Hydroponic fertilizers are formulated to provide these nutrients in a form that is readily available to plants grown in this soilless environment.

[0051] To ensure compatibility and effective nutrient delivery of a hydroponic fertilizer in an aeroponic system, the following considerations may be helpful: Aeroponic systems require a nutrient solution that is highly oxygenated and fine-misted for plant roots to absorb nutrients effectively. While hydroponic fertilizers are designed to be water-soluble, the concentration or dilution of the fertilizer may need to be adjusted for use in an aeroponic system. Further, maintaining the correct pH and electrical conductivity (EC) levels of the nutrient solution is important in aeroponics. Thus, the hydroponic fertilizer preferably allows for being adjusted to the desired pH and EC. Moreover, the hydroponic fertilizer preferably dissolves readily in water. A particular preferred fertilizer is a mixture of NPK 15:15:15 with magnesium nitrate enrichment.

[0052] In certain embodiments, the water-based nutrient solution may have an electrical conductivity (EC) of 1 to 3 mS / cm, or 1 to 2.5 mS / cm or 1 .2 to 2.2 mS / cm, or 1 .4 to 2 mS / cm or or 1 .6 to 1 .8 mS / cm.

[0053] In certain embodiments, the water-based nutrient solution may have a pH value of 5 to 7, or 5.3 to 6.5 or 5.5 to 6.3 or 5.8 to 6.0.

[0054] Lighting may be filtered and / or shaded sunlight exposure or filtered and / or shaded sunlight exposure with additional artificial lighting, or artificial lighting.

[0055] Artificial lighting can be generated e.g. via classical filament lamps, gas-discharge or metal discharge lamps optionally filtered to reduce UV-B radiation or LED (light emitting diodes) with various spectral widths. The global irradiation should result in at least 50 % of photosynthesis active radiation (PAR, between 380 and 780 nm) and in a photosynthetic photon flux density (PPFD) of 25 to 800, preferred 50 to 500, in particular 50 to 250 pmol photons m-2 s-1 . The distribution of the VIS part can be similar to sun light or different to sunlight with higher ratios of red and / or blue and lower intensity of yellow and green wavelengths. In certain embodiments, cultivation is performed at an air temperature of 15 to 35 °C, preferably of 15 to 30 °C, more preferably 18 to 28 °C.

[0056] In certain embodiments, cultivation is performed at a humidity ranging from 70 to 95%, preferably 75 to 90%, more preferably 80 to 85 %.

[0057] In one embodiment according to the first, second or third aspect, the increased rubusoside concentration and / or amount is an increased rubusoside concentration and / or amount in the leafs of said Rubus chingii plant, preferably wherein the rubusoside concentration and / or amount is increased at least 5%, at least 10 %, at least 15 %, at least 20 %, at least 25 %, at least 30%, at least 35 % or at least 40 %.

[0058] An increase in rubusoside concentration and / or amount according to the present invention refers to a rubusoside concentration and / or amount in a Rubus chingii plant treated by a method according to the present invention that is higher than in an identical or essentially identical Rubus chingii plant under the same or essentially the same cultivation conditions but without the at least one application of a composition comprising MeJA. As the rubusoside concentration and / or amount in a given population of Rubus chingii plants is subject to statistical variation, not every member of a plant population treated with MeJA according to the present invention may necessarily have an increased rubusoside concentration compared to each and every member of a control population that is not treated with MeJA. It is sufficient for an increase in rubusoside concentration and / or amount according to the present invention if that increase is present on average.

[0059] Percentages of rubusoside increase described herein denote a percent dry weight (%DW).

[0060] In preferred embodiments, the increased rubusoside concentration and / or amount is an increased rubusoside concentration and / or amount in plant material, preferably dried leaf material.

[0061] The drying of Rubus chingii plant material as well as the measurement of the rubusoside concentration and / or amount may be performed as disclosed herein and / or by any suitable method known in the art, wherein the drying and measurement of Rubus chingii plants with and without MeJA treatment are performed under the same or essentially the same conditions within reasonable limits of the drying and measurement techniques. In certain embodiments, the plant may additionally be exposed at least once, preferably several times, during cultivation, to artificial stress conditions such as to targeted UV light exposure. Targeted UV light exposure means a continuous or pulsed exposure of the plants with a certain quality and quantity of UV light in addition to the visible spectrum (400- 700nm), preferred UV-A (400-315 nm) light with 1 to 25 %, preferably with 2 to 10 % of the whole emission spectrum (UV plus VIS, 315 - 700 nm), especially preferably in the wavelength range of 340 to 380 nm for 1 to 100 %, preferably for 10 to 80 %, especially preferably for 50 to 100 % of the cultivation time.

[0062] In a fourth aspect, there is provided a Rubus chingii plant, or plant material thereof, preferably leaf material, obtained or obtainable in a method of the first, second or third aspect.

[0063] In preferred embodiment, the plant material comprises or consist of leaf material. It is particularly preferred, that the plant material comprises or consists of dried leaf material.

[0064] Description of Figures

[0065] Figure 1A shows rooted cuttings of Rubus chingii after approx. 4 weeks, freshly potted in soil, and Figure 1 B shows plants after an additional approx. 4 weeks.

[0066] Figure 2 shows Rubus chingii plants approx. 40 weeks after initial potting of rooted cuttings.

[0067] Figure 3A shows the rubusoside content as percent dry weight (%DW, shown on the y- axis) of sampled leaves one day after the last hormone application with the indicated concentrations. Statistically significant differences to the water control (0 mM) are indicated by asterisks (***: a < 0.001).

[0068] Figure 3B shows the rubusoside content as percent dry weight (%DW, shown on the y- axis) of sampled leaves seven days after the last hormone application with the indicated concentrations. Statistically significant differences to the water control (0 mM) are indicated by asterisks (**: a < 0.01 ; ***: a < 0.001). Examples

[0069] It was an aim of the present inventors to provide a method for increasing the rubusoside content of Rubus chingii plants, in particular in the leaves of R. Chingii, that can be applied during cultivation of whole plants and is suitable for commercial production of Rubus chingii plant material for subsequent extraction of rubusoside.

[0070] To address the object of the present invention, whole R. chingii plants were treated with different plant hormones during cultivation and the resulting rubusoside content in dried leaf material was determined.

[0071] Example 1 : Cultivation of Rubus chingii

[0072] Pre-culture:

[0073] In calendar week (CW) 20 of 2022, cuttings of R. Chingii were placed in a greenhouse of the experimental farm of the Osnabriick University of Applied Sciences. After four weeks (CW 24), approx. 160 rooted cuttings were potted in approx. 1 L clay- and peat-based soil (Klasmann-Deilmann GmbH; see Figure 1A after potting in CW 24 and Figure 1 B after 4 further weeks in CW 28). Once to twice daily, plants were watered and fertilized with liquid fertilizer Ferty3 (Planta Dungemittel GmbH, Regenstauf, Deutschland) with an ebb and flow system at a moderate electric conductivity (~1 .5 EC). Plants were trimmed for the first time after four weeks (CW 28), and again after six further weeks (CW 34).

[0074] Culture during hormone application:

[0075] After the second trimming, plants were potted into 5 L pots filled with perlite (particle size ~6 mm; Gramoflor GmbH, Fechta, Deutschland). Plants were then cultivated as follows:

[0076] 15 °C heating temperature during the day and 10 °C heating temperature during the night (as of CW 38: 5 °C day and night)

[0077] 20 °C ventilation temperature day and night shading between 35 and 100 klx with 20 % remaining opening (southeast shading, morning to midday or afternoon) sutomatic drip irrigation using a recycling, hydroponic channel system; irrigation as required, in particular high irrigation ( >3 liters per day) in the first seven days in perlite

[0078] EC of 1 .6 to 1 .8 and pH of 5.8 to 6.0

[0079] Fertilizer Ferty9Hydro (Planta Dungemittel GmbH)

[0080] VERTIMEC® PRO (Syngenta) was applied in CW 35 and Bulldock Top (NuFarm Deutschland GmbH, Kbln, Deutschland) was applied in CW 36.

[0081] Between CW 37 to CW 41 , irrigation was gradually reduced to achieve an average drain rate of < 50 %. In CW 48 irrigation was increased again and once again in CW 18 of 2023: plants were then irrigated every 4 hours or earlier if in the 4 hour interval a light integral of 100 klxh was exceeded.

[0082] Approximately 40 weeks (CW 12 of 2023) after initially potting the rooted cuttings (in CW 24 of 2022, see above) R. Chingii plants began developing main shoots (see Figure 2), which have been cut back repeatedly until the beginning of the hormone application.

[0083] Example 2: Hormone application

[0084] The hormone application started in CW 19 of 2023 and all hormones where applied three times with approx. 48 hours between applications (08th, 10thand 12thof May). Samples were taken after 1 day (13thof May) and after 7 days (19thof May). Only young, fully- developed leaves of 6 to 10 cm were used for sample extraction.

[0085] There were a total of 11 treatment groups with 14 plants randomly allocate to each group: Water control, surfactant control, and plant hormones (methyl jasmonate, salicylic acid and ethephon) each at 0.1 or 1 or 10 mM. All plant hormones were solved in distilled water and the surfactant BREAK-THRU® S 301 was added to ensure a homogeneous wetting of leaf surfaces. For each application, the hormone solution (approx. 14 ml per plant) was applied in a manually operated spray bottle.

[0086] Dried leaves were crushed into fine powder in a mill for 3 minutes at 15000 rpm (IKA Tube Mill, Staufen, Germany) and 10 mg (±5 mg) were weighed in a 2 mL Eppendorf tube. After adding 2 mL of methanol, the sample material was extracted by means of an ultrasonic bath for 30 minutes. The sample solutions were then filtered through a syringe filter (0.2 pm) and transferred to HPLC vials. The sample processing was carried out in duplicate.

[0087] Measurements were carried out using Waters Acquity UPLC system consisting of a binary pump, an autosampler, a column manager and a PDA detector (waters, Milford, MA, United States). Empower was used for instrument control, data acquisition and data evaluation. Samples were analyzed using the following chromatographic conditions: Column: Phenomenex (Torrance, CA, United States) Luna Omega Polar RP-18 50 x 2.1 mm, 1.6 pm, 100 A, column temperature: 50 °C, injection volume: 5 pL; mobile phase: A - water + 0.1 % formic acid, B - acetonitrile + 0.1 % formic acid, gradient mode: 0.00 min - 30 % B, 1 .8 min - 34 % B, 1 .81 min - 95 % B, 3.01 min - 95 % B, flow rate: 0.8 ml / min, pre-injection time: 1 min. Rubusoside was detected using a PDA detector at 210 nm and quantified using external calibration.

[0088] The treatment groups were analyzed in a post-hoc-test against the controls (Dunnett’s Table) using the glht method and the multcomp packages in R.

[0089] The surfactant control comprising only the surfactant but none of the three plant hormones did not show any difference to the water control (distilled water without any additives), confirming that the surfactant alone did not influence on the rubusoside content in the experiments (data not shown).

[0090] Application of salicylic acid or ethephon did not show any effect on the rubusoside amounts in the leaves of R. Chingii at any of the used hormone concentrations, neither after 1 day nor after seven days. In contrast, application of 1 mM or 10 mM of MeJA showed a statistically significant increase of rubusoside both after 1 day (see Figure 3A) and after 7 days (see Figure 3B). Application of 0.1 mM MeJA, led to an average increase of rubusoside concentration, which was however not statistically significant in the present experimental setting.

[0091] The experiments demonstrate that, foliar application of MeJA is an effective method to increase the rubusoside content in R. chingii plants. Moreover, it was determined that the increase of rubusoside occurs without loss of biomass, making it an excellent induction method that can easily be integrated into any cultivation regime.

Claims

Claims1 . Method for increasing the rubusoside concentration and / or amount in a Rubus chingii plant, the method comprising:(i) at least one application of a composition comprising methyl jasmonate (MeJA) in a concentration of 0.05 to 50 mM; and / or(ii) at least one application of a composition comprising 0.15 to 120 mg MeJA per plant per application; to said Rubus chingii plant during cultivation.

2. Method for producing a Rubus chingii plant with an increased rubusoside concentration and / or amount, the method comprising a) providing a Rubus chingii plant, cutting, seedling or seed; b) cultivating said Rubus chingii plant, seedling or seed, wherein the cultivation comprises(i) at least one application of a composition comprising MeJA in a concentration of 0.05 to 50 mM; and / or(ii) at least one application of a composition comprising 0.15 to 120 mg MeJA per plant per application; to said Rubus chingii plant; c) obtaining a Rubus chingii plant with an increased rubusoside concentration and / or amount.

3. Method for producing Rubus chingii leaf material with an increased rubusoside concentration and / or amount, the method comprising a) providing a Rubus chingii plant, seedling or seed;b) cultivating said Rubus chingii plant, cutting, seedling or seed, wherein the cultivation comprises(i) at least one application of a composition comprising MeJA in a concentration of 0.05 to 50 mM; and / or(ii) at least one application of a composition comprising 0.15 to 120 mg MeJA per plant per application; to said Rubus chingii plant; c) harvesting leaf material of said Rubus chingii plant.

4. Method of any of the previous claims, wherein the method comprises at least one application of 0.2 to 100 mg, or 0.5 to 80 mg, or 1 to 60 mg, or 2 to 40 mg MeJA per plant per application to said Rubus chingii plant during cultivation.

5. Method of any of the previous claims, wherein the composition comprises MeJA in a concentration of 0.05 to 50 mM, or 0,1 to 30 mM, or 0,1 to 20 mM, or 0,1 to 10 mM, or 1 mM to 10 mM.

6. Method of any of the previous claims, wherein the method comprises at least two, or at least three applications as defined in any of the previous claims.

7. Method of claim 6, wherein about 10 to about 100 hours, or about 20 to about 90 hours, or about 30 to about 70 hours, or about 40 to about 60 hours are between each application.

8. Method of any of the previous claims, wherein the Rubus chingii plant is at least 4 weeks, 5 week, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or at least 12 months old at the time of the at least one application as defined in any of the previous claims, counted from the initial cultivation of a seed or cutting resulting in said Rubus chingii plant.

9. Method of any of the previous claims, wherein the application comprises or consists of foliar application.

10. Method of claim 9, wherein the foliar application comprises or consists of spray application.

11. Method of any of the previous claim, wherein the cultivation further comprises 1 to 20 days, or 1 to 15 days, or 1 to 10 days, or 4 to 10 days after the application or, if the application is performed more than once, after the last application.

12. Method of any of the previous claims, wherein the composition further comprises at least one surfactant.

13. Method of any of the previous claims, wherein the Rubus chingii plant is cultivated in soil, or is cultivated in / on a soil-free substrate for hydroponic, aeroponic and / or fogponic cultivation, at the time of the at least one application as defined in any of the previous claims.

14. Method of any of the previous claims, wherein the increased rubusoside concentration and / or amount is an increased rubusoside concentration and / or amount in the leafs of said Rubus chingii plant, preferably wherein the rubusoside concentration and / or amount is increased at least 5%, at least 10 %, at least 15 %, at least 20 %, at least 25 %, at least 30%, at least 35 % or at least 40 %.

15. Rubus chingii plant, or plant material thereof, preferably leaf material, obtained or obtainable in a method according to any one of claims 1 to 14.

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