DANDELION RUBBER PRODUCTION BY THERMAL CYCLES IMPLEMENTATION

Abstract

The present invention concerns a method for culturing a plant, said method comprising a culture step wherein said plant is submitted to repeated thermal cycles, wherein, for each thermal cycle, cool temperature and warm temperature are alternately artificially applied to all or a part of said plant, over a period shorter than natural seasons.

The present invention also concerns a method for producing natural rubber from latex-producing plants, said method comprising the steps of (a) culturing the latex-producing plant by implementing the method of culture of the invention, (b) harvesting part or all of root part of said plant, and (c) extracting natural rubber from the root part harvested at step b).

Claims

1. Method for culturing a plant, said method comprising a culture step wherein said plant is submitted to repeated thermal cycles wherein, for each thermal cycle, cool temperature and warm temperature are alternately artificially applied to all or a part of said plant, over a period shorter than natural seasons.

2. Method according to claim 1, wherein said alternation of cool and warm temperatures is applied over a period of a day.

3. Method according to claim 1, wherein said alternation of cool and warm temperatures is applied over a period during which the plant is submitted to a daytime (solar or artificial radiation)/nighttime (no radiation) alternation.

4. Method according to claim 3, wherein the plant is daily submitted to cool temperature during the nighttime (no radiation) period and to warm temperature during the daytime (solar or artificial radiation) period.

5. Method according to claim 4, wherein the day is a 24 hour-day.

6. Method according to claim 3, wherein the photoperiodism daytime/nighttime is between 0.2 and 5.

7. Method according to claim 1, wherein the difference between the cool temperature and the warm temperature is of at least 4° C.

8. Method according to claim 1, wherein the warm temperature is strictly higher than 15° C.

9. Method according to claim 1, wherein the cool temperature is lower than or equal to 15° C.

10. Method according to claim 1, wherein the cool temperature is only applied to the root part of the plant while the aerial part of the plant remains at the warm temperature.

11. Method according to claim 1, wherein the culture step is a soil-less culture step.

12. Method according to claim 1, for optimizing production of a secondary metabolite by said plant.

13. Method according to claim 12, wherein said secondary metabolite is a polymer.

14. Method according to claim 13, wherein said polymer is cis-1,4-polyisoprene.

15. Method according to claim 1, wherein said plant is a latex-producing plant.

16. Method according to claim 15, wherein said plant is a Taraxacum kok-saghyz dandelion or a Taraxacum brevicorniculatum dandelion.

17. Method according to claim 1, wherein said culture step is implemented after a step of 8 to 15 weeks of seedling growth.

18. Method according to claim 17, wherein the culture step is implemented on a mature plant during more than 24 hours.

19. Method according to claim 1, further comprising, after the culture step, a step of harvesting the cultured plant or a part thereof.

20. A method for producing natural rubber from latex-producing plants, said method comprising the steps of: a) culturing the latex-producing plant by implementing the method of culture according to claim 15, b) harvesting part or all of the root part of said plant, and c) extracting natural rubber from the root part harvested at step b).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0086] FIG. 1: Dry root mass (in g) after 10 weeks and 16 weeks of culture with and without refrigeration of the nutrient solution.

EXAMPLE

[0087] The following strategy was used by the inventors: [0088] 1) Leading the culture under ideal conditions (in terms of light, photoperiod, temperature, etc. . . . ) for 10 weeks to promote the development of root biomass, [0089] 2) After 10 weeks, applying a temperature drop to the root system (via refrigeration of the nutrient solution) at night, in order to induce the biosynthesis of rubber and inulin degradation to sugars (fructose) usable for biosynthesis. During the day, the growing conditions permit normal plant metabolism and promote photosynthetic activity and the capture of carbon. [0090] Root-accumulated inulin during the daytime when the photosynthesis is active in leaves, may be used for rubber synthesis in cold-treated roots during the night.

Materials and Methods

Hydroponic System Design

[0091] The hydroponics system able to accommodate plants for further experiments was designed and prototyped between January and October 2018.

[0092] The roots being the part of the plant producing rubber, it was important to achieve a hydroponic system facilitating access to them. A first culture system inspired by the method DWC (Deep water culture) has therefore been developed.

[0093] “Deep water culture” involves producing plants with a minimum of substrate. Indeed, the roots of the plant are retained by a basket pot (filled with ball clay or rockwool) and soaked directly in the oxygenated nutrient solution.

[0094] A first DWC system which was developed is a tank of 400 l (60 cm in height), and a second prototype used for the experiment, was developed in a 100 l tank (35 cm in height). To this tank was added a stirring system of the nutrient solution (pump 1500 l/h). Circular bubblers of 35 cm in diameter were placed at the bottom of the tank and were fed by an air compressor (2700 l/h). A PVC plate was pierced to the diameter of the pots, each ferry welcoming between 12 and 25 plants

Refrigeration System of the Nutrient Solution

[0095] In order to reduce the temperature of the nutrient solution, a cold group (capacity 250 l) was added to the culture system. The 1500 l/h pump immersed in the tank was then connected to the cold group. A 130 μm filter was added before the cold unit to prevent the system from clogging.

[0096] The pump ran 24 h/24 h, so the nutrient solution was constantly circulating in the DWC tank. The cold unit was set using a timer on the time zone desired.

Plant Material

[0097] Taraxacum kok-saghyz plants used during the experiment were derived from seeds provided by the Ohio State University.

Refrigeration Test of the Nutrient Solution

[0098] The plants were grown hydroponically in DWC system for 4 months (16 weeks) in accordance with the culture conditions described in Table 1 below.

TABLE-US-00001 TABLE 1 Culture conditions of Taraxacum kok-saghyz in DWS hydroponics Week 1-4 Week 5-10 (culture Week 11-16 (greenhouse) room) (greenhouse) Air 18-23° C. day/ 20° C. day/15° C. 18-23° C. day/13- temperature 13-18° C. night night 18° C. night Photoperiod 16 h day/8 h night Lighting HPS + natural 4000K light LED HPS + natural light light 300-350 μmol (150-300 μmol) (150-300 μmol) Hygrometry 80-100% 70% 50-80% Nutritional Week 1: Week 5-10: Week 11-16: solutions Clear water N15/P10/K30 N15/P10/K30 unadjusted pH pH = 5.8-6.2 pH = 5.5-6 EC = 0.4 mS EC = 1-1.2 mS EC = 1 mS Week 2: N10/ Foliar application 2 Foliar application P52/K10 times per week of 1 time per week of pH = 5.5-6 biostimulant biostimulant EC = 0.6 mS enriched in enriched in Week 3-4: N10/ Magnesium Magnesium P52/K10 (Phylgreen ® Mg) (Phylgreen ® Mg) pH = 5.5-6 EC = 0.8 mS

[0099] Seeds were germinated on a honeycombed plate of rock wool covered with a thin layer of vermiculite (<1 cm). The plate was then placed under high relative humidity for 3 weeks in a greenhouse.

[0100] After 4 weeks of cultivation, the seedlings were repotted in a basket pot on clay balls and 72 plants were placed in culture chamber for 6 weeks.

[0101] After 10 weeks of culture, the roots of 12 plants were harvested, lyophilized and then evaluated for root dry mass and rubber concentration. Other plants were then moved to a greenhouse where the test of chilling the nutrient solution was performed.

[0102] For this test two batches of 7 randomly selected plants were used: the first batch was placed in a control condition, where the nutrient solution was not refrigerated (its temperature varied between 18-22° C.). The second batch was placed in refrigerated condition (the temperature of the nutrient solution was lowered to 12-13° C. overnight).

[0103] In hydroponics, the ideal temperature of the nutrient solution is between 16 and 20° C. This temperature ensures both good root development and good oxygen level of the nutrient solution. Below 15° C., plants are under stress. However, this stress does not have a lasting physiological impact if it is not prolonged or if the temperature does not fall below a certain threshold.

[0104] The plants were grown for 6 weeks, then the roots were harvested to assess the root dry mass and rubber concentration

Extraction of Sugars, Resins, and Rubber

[0105] After lyophilization, the roots were mixed and milled with a ball mill. The protocol used for the extraction of sugars, resin and rubber was an ASE method (Accelerated Solvent Extraction) disclosed in Ramirez-Cadavid et al. (2018) Industrial crops and products 122:647-656.

[0106] Extractions were performed in duplicate on each pool of individuals.

Results

Evaluation of the Root Biomass

[0107] The plants cultivated for 16 weeks had a higher dry biomass than those taken after 10 weeks of culture (see FIG. 1).

[0108] After 16 weeks, the dry root mass was 5.23 g/plant (FIG. 1) for individuals grown in control condition in the non-refrigerated nutrient solution.

[0109] Cold-processed plants tended to present lower root biomass (3.84 g/plant).

[0110] The cold applied at night did not seem to prevent the development of root biomass but the difference in biomass after 6 weeks of treatment could be explained by a slower metabolism caused by the cold.

Evaluation of the Rubber Concentration

[0111]

TABLE-US-00002 TABLE 2 Concentration of sugars, resin and rubber (mg/g dry root) Amount (mg/g dry root) Culture duration Treatment Total sugars Resin Rubber 10 weeks Control 605 ± 28 39.0 ± 1.4 7.0 ± 2.1 16 weeks Control 702 ± 18 29.5 ± 3.5 17.5 ± 7.7  Cold 650 ± 35 28.0 ± 0.2 42.0 ± 25.4

[0112] Extractions were performed in duplicate on each pool of individuals.

[0113] With the exception of the resin, the concentrations of sugars and rubber were higher after 16 culture weeks (Table 2).

[0114] Cold-grown plants had a lower sugar concentration than plants cultivated in a control condition after 16 weeks. Conversely, the plants grown in the chilled solution had a higher rubber concentration (on average 2.4 times higher). Nevertheless, after 16 weeks, the concentrations of sugars in plants grown in the chilled nutrient solution were higher than those obtained after 10 weeks of culture, which showed that bringing cold at night did not block the plant's sugar metabolism.

CONCLUSION

[0115] This example demonstrates the efficacy of cold treatment during cultivation for increasing the rubber concentration in Taraxacum kok-saghyz.