METHODS OF IN VITRO CULTURING SPHAGNUM

Abstract

A method for in vitro culturing Sphagnum is provided. The method comprises providing a vegetative fragment of Sphagnum and surface cleaning the fragment with alcohol. The method further comprises dissecting the fragment to provide a piece (106) of Sphagnum. The method further comprises initiating the piece (106) of Sphagnum in a culture medium (104).

Claims

1. A method for in vitro culturing Sphagnum, comprising: providing a vegetative fragment of Sphagnum; surface cleaning the fragment with alcohol; dissecting the fragment to provide a piece of Sphagnum; and initiating the piece of Sphagnum in a culture medium.

2. The method according to claim 1, wherein the fragment has a length of less than 10 mm.

3. The method according to claim 1, wherein the fragment is from a capitulum of Sphagnum.

4. The method according to claim 1, wherein the piece of Sphagnum has a length of less than 1 mm.

5. The method according to claim 1, wherein the surface cleaning the fragment with alcohol is performed for less than seconds.

6. The method according to claim 1, wherein the alcohol comprises between 50% and 80% alcohol by volume.

7. The method according to claim 1, wherein the alcohol comprises propanol.

8. The method according to claim 1, further comprising surface cleaning the fragment with sterile water before the surface cleaning with alcohol.

9. The method according to claim 1, further comprising surface cleaning the fragment with sterile water after the surface cleaning with alcohol.

10. The method according to claim 1, further comprising placing the piece of Sphagnum onto a sterile absorption surface before the initiating.

11. The method according to claim 1, wherein the surface cleaning does not comprise using a chlorine-based sterilant.

12. The method according to claim 1, wherein the surface cleaning does not comprise using a detergent.

13. The method according to claim 1, wherein the culture medium does not comprise sugar.

14. The method according to claim 1, wherein the culture medium comprises nutrients comprising nitrogen, phosphorus, potassium, calcium, magnesium, sodium, manganese, copper, zinc, sulfur, boron, iron, molybdenum, chlorine, cobalt, and iodine.

15. The method according to claim 1, wherein the culture medium is a solid culture medium.

16. The method according to claim 1, further comprising culturing the piece of Sphagnum in the culture medium.

17. The method according to claim 16, wherein the culturing is performed for at least 8 weeks.

18. The method according to claim 16, wherein the culturing is performed to form a gametophore.

19. The method according to claim 16, further comprising, after the culturing, transferring the Sphagnum to a second culture medium.

20. (canceled)

21. The method according to claim 19, wherein the second culture medium comprises a liquid culture medium.

22-25. (canceled)

Description

[0204] Embodiments of the disclosure are described below, by way of example only, with reference to the accompanying Figures.

[0205] FIG. 1 shows a photograph from the side of a culture vessel for in vitro culturing Sphagnum according to a first embodiment of the disclosure.

[0206] FIG. 2 shows a photograph from the side of a culture vessel for in vitro culturing Sphagnum of FIG. 1, after 6 weeks of growth.

[0207] FIG. 3 shows a photograph from the side of a culture vessel for in vitro culturing Sphagnum of FIG. 1, after 16 weeks of growth.

[0208] FIG. 4 shows a table of measured wet weights of samples of Sphagnum after 10 weeks of growth.

[0209] FIG. 5 shows a graph of measured wet weights of the results in the table of FIG. 4.

EXAMPLES

Example 1

Materials and Methods

[0210] A trial was conducted involving taking a fragment from a capitulum of in vivo Sphagnum. The fragment was surface cleaned with 70% abv propanol for 30 seconds. The fragment was then washed in sterile water for 1 minute and placed to dry on sterile absorbing paper. The fragment was then dissected by hand using a scalpel under sterile conditions to provide a piece of Sphagnum. The piece had a length less than 1 mm and was placed into a culture medium for initiation. The initiated Sphagnum on the culture medium is shown in the photograph of FIG. 1.

[0211] Referring to FIG. 1, a photograph of an apparatus 100 for culturing Sphagnum is shown as used for the present trial. The apparatus 100 comprises a culture vessel 102 in the form of a glass jar of a volume of around 300 ml. The culture vessel 102 contains a culture medium 104. A piece of Sphagnum 106 is shown arranged on the culture medium 104.

[0212] The culture medium 104 contained nutrients comprising 48.23 mg per L of nitrogen, 9.67 mg per L of phosphorus, 123.46 mg per L of potassium, 32.63 mg per L of calcium, 9.12 mg per L of magnesium, 1.32 mg per L of sodium, 5.49 mg per L of manganese, 0.02 mg per L of copper, 1.96 mg per L of zinc, 62.55 mg per L of sulfur, 1.08 mg per L of boron, 1.40 mg per L of iron, mg per L of molybdenum, 11.69 mg per L of chlorine, 0.006 mg per L of cobalt, and 0.63 mg per L of iodine. The culture medium 104 was a solid culture medium solidified with agar. There was no sugar in the culture medium 104.

[0213] The Sphagnum was then cultured for 16 weeks.

Results

[0214] FIG. 2 shows an apparatus 200 which corresponds to the apparatus 100 of FIG. 1 after a period of 6 weeks. During this time, the Sphagnum 106 has been cultured, and as shown in FIG. 2 the resultant Sphagnum 206 has grown in size.

[0215] FIG. 3 shows an apparatus 300 which corresponds to the apparatus 200 of FIG. 2 after a further period of 10 weeks (being 16 weeks after the apparatus 100 of FIG. 1). The Sphagnum 306 in FIG. 3 has significantly grown in size between FIG. 1 and FIG. 2.

[0216] This shows that the in vitro culturing techniques of surface cleaning with alcohol have been successful and contamination has been avoided.

Example 2

Materials and Methods

[0217] A trial was conducted involving taking initial samples of Sphagnum originally initiated following surface cleaning with alcohol and subsequently grown under in vitro conditions, in accordance with the present disclosure, such as in Example 1.

[0218] 12 samples of Sphagnum palustre were taken with a wet weight of 13 g1 g. The samples were then placed into a culture medium containing nutrients. The culture medium contained nutrients comprising 48.23 mg per L of nitrogen, 9.67 mg per L of phosphorus, 123.46 mg per L of potassium, 32.63 mg per L of calcium, 9.12 mg per L of magnesium, 1.32 mg per L of sodium, 5.49 mg per L of manganese, 0.02 mg per L of copper, 1.96 mg per L of zinc, 62.55 mg per L of sulfur, 1.08 mg per L of boron, 1.40 mg per L of iron, 0.10 mg per L of molybdenum, 11.69 mg per L of chlorine, 0.006 mg per L of cobalt, and 0.63 mg per L of iodine. The culture medium was a solid culture medium solidified with agar. There was no sugar in the culture medium.

[0219] The Sphagnum was then cultured for 10 weeks. After 10 weeks, the wet weights of the samples were measured.

Results

[0220] FIG. 4 shows a table of the final wet weights of the samples.

[0221] The mean weight of the 12 samples was 60.4 mg, with a standard deviation of 20.7 mg. This provided a percentage increase in weight of 365% as a result of 10 weeks of growth.

[0222] Sample 2 was identified as an anomaly as it did not grow well during the trial and showed poor growth compared to the other samples and was deemed non-viable.

[0223] Removing sample 2 from the results, the mean weight of the 11 remaining samples was 64.5 mg, with a standard deviation of 15.7 mg. This provided a percentage increase in weight of 396% as a result of 10 weeks of growth.

[0224] FIG. 5 shows a graph of the final wet weights of the samples, with sample 2 omitted from the calculation of the average.

[0225] This shows that the in vitro culturing techniques of surface cleaning with alcohol have been successful and contamination has been avoided.