COMPOSITIONS AND METHODS FOR POLLEN STORAGE AND PRESERVATION

Abstract

Liquid compositions for preserving and storing pollen, especially maize pollen, include pollen suspensions in solutions with particular pH ranges. Methods for preserving and storing pollen, especially maize pollen, include suspending pollen in solutions with particular pH ranges and storing them at particular temperatures.

Claims

1. A liquid composition comprising: a. greater than 50% water, b. viable maize pollen, c. at least one plant nutrient, and d. greater than 0.5 molarity of an osmotic potential enhancing compound; wherein the liquid composition has a pH greater than 8.

2. The liquid composition of claim 1, wherein the at least one plant nutrient comprises at least one micronutrient.

3. The liquid composition of claim 2, wherein the at least one micronutrient is boron.

4. The liquid composition of claim 1, wherein the plant nutrient comprises at least one macronutrient.

5. The liquid composition of claim 4, wherein the at least one macronutrient comprises calcium.

6. The liquid composition of claim 1, wherein the osmotic potential enhancing compound comprises betaine, and optionally, sucrose.

7. The liquid composition of claim 6, wherein the concentration of the betaine in the osmotic potential enhancing compound is equal or greater than 500 mM.

8. The liquid composition of claim 6, wherein the concentration of the betaine in the osmotic potential enhancing compound is equal or greater than 1000 mM.

9. The liquid composition of claim 1, wherein the osmotic potential enhancing compound comprises phosphate-buffered saline (PBS) at a concentration equal or greater than 0.25×, based on a 20× PBS stock adjusted to a PH of 9.

10. The liquid composition of claim 1, comprising boric acid, calcium chloride, and at least 500 mM betaine.

11. The liquid composition of claim 1, comprising boric acid, calcium chloride, and at least 1000 mM betaine.

12. The liquid composition of claim 1, wherein the liquid composition has a pH greater than or equal to 9.

13. The liquid composition of claim 12, wherein the at least one plant nutrient comprises at least one micronutrient.

14. The liquid composition of claim 13, wherein the at least one micronutrient is boron.

15. The liquid composition of claim 12, wherein the plant nutrient comprises at least one macronutrient.

16. The liquid composition of claim 15, wherein the at least one macronutrient comprises calcium.

17. The liquid composition of claim 12, wherein the osmotic potential enhancing compound comprises betaine, and optionally, sucrose.

18. The liquid composition of claim 17, wherein the concentration of the betaine in the osmotic potential enhancing compound is equal or greater than 500 mM.

19. The liquid composition of claim 17, wherein the concentration of the betaine in the osmotic potential enhancing compound is equal or greater than 1000 mM.

20. The liquid composition of claim 12, wherein the osmotic potential enhancing compound comprises phosphate-buffered saline (PBS) at a concentration equal or greater than 0.25×, based on a 20× PBS stock adjusted to a PH of 9.

21. The liquid composition of claim 12, comprising boric acid, calcium chloride, and at least 500 mM betaine.

22. The liquid composition of claim 12, comprising boric acid, calcium chloride, and at least 1000 mM betaine.

23. A method of storing maize pollen in an aqueous solution comprising collecting maize pollen and storing the collected maize pollen in an aqueous solution, the aqueous solution comprising greater than 50% water and greater than 0.5 molarity of an osmotic potential enhancing compound, wherein the liquid composition has a pH greater than 8.

24. The method of claim 23, wherein the pollen is collected from a shedding maize anther and then placed in the aqueous solution.

25. The method of claim 23, wherein the aqueous solution further comprises calcium chloride, boric acid, and betaine.

26. The method of claim 25, wherein the aqueous solution further comprises phosphate buffered saline.

27. The method of claim 23, wherein at least one pollen encapsulating agent is added to the aqueous solution.

28. The method of claim 27, wherein the pollen encapsulating agent is oil.

29. The method of claim 27, wherein the pollen encapsulating agent is honey phenolic acids with flavonoids.

30. A method of pollinating a maize plant, comprising collecting maize pollen and storing the collected maize pollen in an aqueous solution, the aqueous solution comprising greater than 50% water and greater than 0.5 molarity of an osmotic potential enhancing compound, and applying the solution to the silk of the same or a different maize plant.

31. The method of claim 30, wherein the aqueous solution is sprayed onto the silk of the same or a different maize plant.

32. The method of claim 30, wherein the aqueous solution further comprises a surfactant.

33. The method of claim 32, wherein the surfactant is a poloxamer.

Description

EXAMPLES

Example 1: Storage of Maize Pollen in Liquid Media Environment—pH Effects

[0016] This example assesses the impact of pH on stabilizing maize pollen in liquid environments.

[0017] Solutions adjusted to pH 7.0, 8.0, and 9.0 using HCl and KOH were made from aliquots of sterile distilled water. Pollen was collected from actively shedding maize tassels and added to each solution. Pollen was stored at room temperature (21° C.) for up to 7 days and observed periodically using an inverted microscope.

[0018] Increasing pH to 9.0 showed increased pollen stability and lowered cell lysis compared to pH 7.0 or pH 8.0, which showed widespread lysis of the pollen grains (Table 1).

TABLE-US-00001 TABLE 1 Comparison of pollen grains stored in water adjusted to pH of 7.0, 8.0, and 9.0 after 7 days Adjusted Water pH Intact pollen grains pH 7.0 Widespread lysis pH 8.0 Widespread lysis pH 9.0 ~90-95% intact
Higher pH therefore has a stabilizing effect on maize pollen and prevented lysis in a liquid environment.

Example 2: Storage of Maize Pollen in Liquid Media Environment—Effects of Media Components and Plant Nutrients

[0019] This example assesses the effects of components of liquid media on pollen stability. Three aqueous media bases were prepared: sterile distilled water, 0.6 M mannitol, and 326C media. The pH was adjusted to 9.0 for all three media. Media recipes are set forth below.

[0020] Pollen was collected from actively shedding maize tassels and added to each of the media solutions described above. Pollen was stored at room temperature (21° C.) for up to 7 days and observed periodically using an inverted microscope.

[0021] Sterile water showed some cell lysis, and 0.6 M mannitol showed more wide-spread cell lysis. 326C media showed greater stability and less lysis compared to the other two medias (Table 2).

TABLE-US-00002 TABLE 2 Intact pollen grain comparison of pollen stored in water, 0.6M mannitol, and 326C media adjusted to pH 9.0 Media Intact pollen grains Water ~80-85% intact 0.6M Mannitol Widespread lysis 326C >95% intact

[0022] Next, the effects of micronutrients such as Boron and Macronutrients such as Calcium in the 326C media were determined by preparing 326C without Boron and Calcium (326C-S). Aliquots of both the complete 326C and 326C-S were prepared and adjusted to pH of 6.0, 7.0, 8.0, and 9.0. Pollen was collected as above and stored at room temperature. Micronutrients are nutrients needed in small amounts for normal or optimum plant growth, which include iron (Fe), boron (B), chlorine (Cl), manganese (Mn), zinc (Zn), copper (Cu), molybdenum (Mo), nickel (Ni). Macronutrients are needed in larger amounts than micronutrients, and include nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), sulfur (S), magnesium (Mg), carbon (C), oxygen (O), and hydrogen (H).

[0023] When comparing the complete 326C media against the 326C-S media, widespread cell lysis with the 326C-S media was observed, while the pollen stored in complete 326C remained intact (Table 3).

TABLE-US-00003 TABLE 3 Intact pollen grain comparison of pollen stored in 326C media and 326C-S (salts removed) media adjusted to pH of 6.0, 7.0, pH 8.0, and pH 9.0 Media Intact pollen grains 326C, pH 6.0 Widespread lysis 326C, pH 7.0 ~30-35% intact 326C, pH 8.0 ~80-85% intact 326C, pH 9.0 ~90-95% intact 326C-S, pH 6.0 Widespread lysis 326C-S, pH 7.0 Widespread lysis 326C-S, pH 8.0 Widespread lysis 326C-S, pH 9.0 Widespread lysis

[0024] Osmotic potential by itself is insufficient to maintain pollen stability. The presence of salts (e.g., Calcium and Boron) is important for maintaining the stability of the pollen.

Example 3: Storage of Maize Pollen in Liquid Media Environment—Effects of Media Additives

[0025] This example assesses additives to liquid media on pollen viability.

[0026] The 326C media described in the previous example was prepared with four concentrations of the osmoprotectant compound Betaine: no Betaine, 100 mM Betaine, 500 mM Betaine, and 1000 mM Betaine. Aliquots of each was prepared and adjusted to pH 6.0, 7.0, 8.0, and 9.0 for a total of sixteen solutions (four Betaine concentrations, each at four different pHs).

[0027] Pollen was collected from actively shedding maize tassels and added to each of the twelve media. Pollen was stored at 4° C. and observed microscopically. Viability was assayed using Fluorescein Diacetate (FDA) viability staining. Viability observations were taken at 8 and 15 days after being placed in storage.

[0028] Increased FDA fluorescence was shown as both pH and Betaine concentration increase, with highest viability fluorescence present at pH 9.0 and 1000 mM Betaine. These parameters also showed high viability at both 8 and 15 days of storage (Table 4 and Table 5).

TABLE-US-00004 TABLE 4 Percentage of pollen grains showing fluorescence from FDA staining after storage in liquid media for eight days at 4 C. Media FDA Fluorescence 326C, pH 6.0, No Betaine     0% 326C, pH 6.0, 100 mM Betaine  5-10% 326C, pH 6.0, 500 mM Betaine 15-20% 326C, pH 6.0, 1000 mM Betaine 30-35% 326C, pH 7.0, No Betaine   >5% 326C, pH 7.0, 100 mM Betaine  5-10% 326C, pH 7.0, 500 mM Betaine 45-50% 326C, pH 7.0, 1000 mM Betaine 60-65% 326C, pH 8.0, No Betaine 25-30% 326C, pH 8.0, 100 mM Betaine 45-50% 326C, pH 8.0, 500 mM Betaine 80-85% 326C, pH 8.0, 1000 mM Betaine 90-95% 326C, pH 9.0, No Betaine 20-25% 326C, pH 9.0, 100 mM Betaine  5-10% 326C, pH 9.0, 500 mM Betaine 85-90% 326C, pH 9.0, 1000 mM Betaine 90-95%

TABLE-US-00005 TABLE 5 Percentage of pollen grains showing fluorescence from FDA staining after storage in liquid media for fifteen days at 4 C. Media FDA Fluorescence 326C, pH 6.0, No Betaine     0% 326C, pH 6.0, 100 mM Betaine     0% 326C, pH 6.0, 500 mM Betaine     0% 326C, pH 6.0, 1000 mM Betaine   >5% 326C, pH 7.0, No Betaine     0% 326C, pH 7.0, 100 mM Betaine     0% 326C, pH 7.0, 500 mM Betaine   >5% 326C, pH 7.0, 1000 mM Betaine  5-10% 326C, pH 8.0, No Betaine   >5% 326C, pH 8.0, 100 mM Betaine 40-45% 326C, pH 8.0, 500 mM Betaine 35-40% 326C, pH 8.0, 1000 mM Betaine 80-85% 326C, pH 9.0, No Betaine  5-10% 326C, pH 9.0, 100 mM Betaine 10-15% 326C, pH 9.0, 500 mM Betaine 30-35% 326C, pH 9.0, 1000 mM Betaine 85-90%

[0029] This example demonstrates that additives to increase osmotic potential, such as Betaine, increase pollen viability over longer durations of time.

Example 4: PBS

[0030] The purpose of this experiment is to test additional additives for liquid storage media. For this test, phosphate-buffered saline (PBS) is added. Two stocks of 326C media were prepared, one with Betaine at a 1000 mM concentration, the other without Betaine. 20× PBS stock was added to create final PBS concentrations of 0.00×, 0.10×, 0.25×, 0.50×, 0.75×, 1.00×, 1.50×, and 2.00×. Solution pH was adjusted to 9.0.

[0031] Pollen was collected from actively shedding maize tassels and added to each media. Pollen was stored at 4 C and observed using the Lionheart FX Live Image Capture. Viability was assayed using FDA viability staining. Viability observations were taken at 4 days after being placed in storage.

TABLE-US-00006 TABLE 6 Phosphate Buffered Saline (PBS) Media FDA Fluorescence 326C, 1M Betaine, 0.00x PBS 10-15% 326C, 1M Betaine, 0.10x PBS 10-15% 326C, 1M Betaine, 0.25x PBS 10-15% 326C, 1M Betaine, 0.50x PBS  5-10% 326C, 1M Betaine, 0.75x PBS 15-20% 326C, 1M Betaine, 1.00x PBS 10-15% 326C, 1M Betaine, 1.50x PBS  5-10% 326C, 1M Betaine, 2.00x PBS  5-10% 326C, 0.00x PBS     0% 326C, 0.10x PBS     0% 326C, 0.25x PBS   >5% 326C, 0.50x PBS     0% 326C, 0.75x PBS   >5% 326C, 1.00x PBS     0% 326C, 1.50x PBS   >5% 326C, 2.00x PBS   >5%

[0032] Without Betaine, 326C with PBS showed low FDA fluorescence, though there were some fluorescent pollen above 0.25× concentration. With Betaine, we see an increase in FDA fluorescence at 0.75× concentration, and a decrease at 1.50× and above. Therefore, when used in combination with Betaine, PBS improved pollen viability.

Example 5: Germination of Maize Pollen after Storage in a Liquid Media Environment

[0033] This example confirms the ability of pollen to germinate after it has been stored in a liquid environment.

[0034] Maize pollen was stored in 326C media with 1000 mM Betaine at a pH of 9.0. Pollen was stored at 4° C. for six days. After six days, the storage media was carefully removed from the pollen and replaced with a modified germination media at room temperature to trigger pollen tube formation. Pollen was observed microscopically prior to replacement of media and two hours after replacement.

[0035] Within two hours, pollen tube formation was readily observable in samples where the storage media had been removed and replaced with germination media. Samples that did not undergo the media replacement did not show pollen tube formation. Pollen was viable stored using the developed storage media and is capable of pollen germination after removal from storage.

Example 6: Use of Protective Compounds in Pollen Preservation

[0036] In this example, various putative protective compounds were evaluated for their efficacy in preventing pollen from bursting on germination media. Nine different chemical treatments were applied to loose pollen shed from maize plants. Pollen was soaked in chemical solution for 20 minutes and then placed on media to observe the impact on pollen viability and compared with fresh pollen without protection as control.

[0037] A liquid composition comprising 1XPBS at PH7.4, honey, olive oil, and mineral oil was cound to prevent pollen from bursting. A liquid composition comprising 50% glycols, 5% DMSO and hair spray application did not prevent pollen from bursting on media in significantly less quantities than control (germination media alone).

Example 7: Pollen Germination Assay—Effect of Sugars on Pollen Viability and Fertility

[0038] In this example, various concentrations of sucrose mannitol were added to the germination medium in order to evaluate any effects they have on pollen germination.

[0039] Four different sugar compositions (16% sucrose, 12% sucrose and 4% mannitol, 16% sucrose and 4% mannitol, and 20% sucrose and 4% mannitol) were added to germination medium, and the effects of the compositions on pollen collected from two genetically different maize plants were observed microscopically and the percentage of pollen grains that germinated and that ruptured were quantified. Some pollen grains ruptured after germinating first, which is why Table 7 below shows some rupture and germination percentages that exceeded 100%. The sugar compositions above were added to a germination medium comprising Agar 0.7%, CaCl2 2H2O 300 mg/L, and H3BO3 100 mg/L. Table 7 below reports the results.

TABLE-US-00007 TABLE 7 Effects of sugar compositions in germination media on germination. Sugar Maize Plant 1 Results Maize Plant 2 Results Composition Germination Rupture Germination Rupture 16% Sucrose 90% 30% 90% 25% 12% Sucrose 45% 45% 60% 50% 4% Mannitol 16% Sucrose 10% 25% 10% 30% 4% Mannitol 20% Sucrose  5% 10%  2% 20% 4% Mannitol

[0040] These results show a very high rate of germination success using a germination medium including 16% sucrose and no mannitol.

Media Recipes

[0041] 0.6 M Mannitol (1 L) [0042] Mannitol—109.32g [0043] L-Proline—0.146 g [0044] Adjust pH to 7.0, 8.0, and 9.0 [0045] 326C Media (1 L) [0046] Sucrose—160.0 g [0047] Calcium Chloride Dihydrate—0.3 g [0048] Boric Acid—0.1 g [0049] Adjust pH to 6.0, 7.0, 8.0, and 9.0 [0050] 326C-S Media (1 L) [0051] Sucrose—160.0 g [0052] Adjust pH to 6.0, 7.0, 8.0, and 9.0 [0053] 326C, 100 mM Betaine (1 L) [0054] Sucrose—160.0 g [0055] Calcium Chloride Dihydrate—0.3 g [0056] Boric Acid—0.1 g [0057] 5 M Betaine—20 mL [0058] Adjust pH to 6.0, 7.0, 8.0, and 9.0 [0059] 326C, 500 mM Betaine (1 L) [0060] Sucrose—160.0 g [0061] Calcium Chloride Dihydrate—0.3 g [0062] Boric Acid—0.1 g [0063] 5 M Betaine—100 mL [0064] Adjust pH to 6.0, 7.0, 8.0, and 9.0 [0065] 326C, 1000 mM Betaine (1 L) [0066] Sucrose—160.0 g [0067] Calcium Chloride Dihydrate—0.3 g [0068] Boric Acid—0.1 g [0069] 5 M Betaine—200 mL [0070] Adjust pH to 6.0, 7.0, 8.0, and 9.0 [0071] 20× PBS [0072] NaCL—160 g [0073] KCL—4 g [0074] NA2HPO4—28.8 g [0075] KH2PO4—4.8 g [0076] H2O—800 ml

Example 8: Addition of Surfactants—Germination of Maize Pollen After Storage in a Liquid Media Environment Comprising Surfactants

[0077] This example examines the use of surfactants in media to improve contact between pollen-containing liquid media and silk tissue.

[0078] Maize pollen was stored in 326C media with 1000 mM Sucrose and 500 mM Betaine at a pH of 9.0. Pollen was stored at 1° C. for a day. After this time period, germination medias comprising surfactant were added in amounts up to five times the volume of storage media. Surfactant conditions tested were 0.1% Tween 20, 0.01% Tween 20, 0.1% Pluronic F68, and 0.01% Pluronic F68 (final concentration). Pollen was observed microscopically prior to replacement of media and two hours after replacement. Pluronic F68, also known as poloxamer 188, is a non-ionic surfactant, a member of a class of polaxamers comprising non-ionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene flanked by two hydrophilic chains of polyoxyethylene.

[0079] Within two hours, pollen tube formation was observable. Pollen treated with germination media containing Pluronic F68 at both 0.01% and 0.1% concentrations showed pollen tube formation similar to germination media without surfactant. Pollen treated with germination media containing Tween 20 showed lysis but no pollen tube formation at both concentrations. Under these conditions, pollen shows sensitivity to surfactants, but Pluronic F68 was gentle enough that it did not interfere with pollen tube formation.

Example 9: Techniques used for Enhanced Fertilization of Female Maize Gamete from Pollen Stored in Liquid Media Environment Containing Surfactants

[0080] These examples outline the techniques used to improve the effectiveness of pollen application onto silk tissue from a liquid media environment for successful fertilization.

[0081] Filter and spatula: Maize pollen was stored in 326C media with 1000 mM Sucrose, 500 mM Betaine, and 0.1% Pluronic F68 (poloxamer 188) for a series of time points. The treatments were stored in a cooler with 1 C cooler packs or in a 1 C fridge. Once ready, the contents of the culture plate well was removed and placed into a 40 um filter. A kimwipe was used to wick away excess storage media. A spatula was then used to scoop up the pollen and apply it onto the silks.

[0082] Spray Bottle Head: Maize pollen was stored in 326C media with 1000 mM Sucrose, 500 mM Betaine, and 0.1% Pluronic F68 for a series of time points. The treatments were stored in a cooler with 1C cooler packs or in a 1C fridge. Once ready, the contents of the solution was drawn up through the inlet of the spray bottle head and dispersed onto the silks. Alternatives of this was to substitute the storage media for mineral oil or to add mineral oil to the storage media, mix to encapsulate the pollen, and spray onto the silks.

[0083] Brush: Maize pollen was stored in 326C media with 1000 mM Sucrose, 500 mM Betaine, and 0.1% Pluronic F68 for a series of time points. The treatments were stored in a cooler with 1C cooler packs or in a 1C fridge. Once ready, the contents of the solution was removed from the well but the pollen was maintained, light mineral oil was added, and then applied to silks using a brush or pipette. Alternatively, the contents of the well were removed, added to a filter, and then light mineral oil was added to the filter. The liquid in the filter was then used for applying pollen to the silks with a small paint brush.

[0084] Negative Control: Maize pollen was stored in water for 30 min. A filter and spatula was used to apply the pollen onto the silks.

[0085] In summary, kernel formation/seed set was obtained from pollen that was stored in the liquid media environment up to four hours. Our negative control using water as the liquid media environment showed no kernel formation/seed set after being stored for 30 minutes.

TABLE-US-00008 TABLE 8 Kernel count results of pollen in storage media using different pollen application techniques from a liquid media environment Kernel Count Kernel Count Liquid Stored Experiment Location Treatment (Fresh - Dry) (Liquid Stored) Time (min) 1 GH Filter and spatula 480 72 30 2 GH Filter and spatula 444 85 30 3 GH Filter and spatula 454 116 30 4 GH Filter and spatula 457 79 30 5 GH Filter and spatula 549 78 30 6 Lab Spray bottle 77 36 60 7 GH Pipette 487 14 30 8 GH Brush 549 23 30 9 GH Filter and spatula — 0 30 Water (Negative Control)