Culture medium and culture method of gametophytes of barbula unguiculata

Abstract

This invention relates to the field of bioengineering technology, and proposes a culture medium and method for cultivating gametophytes of *Bryophytum pumilus*. The spore culture medium for *Bryophytum pumilus* gametophytes comprises the following components: MgSO4·7H2O 250 mg / L, KH2PO4 250 mg / L, FeSO4·4H2O 12.5 mg / L, KNO3 1010 mg / L, ammonium tartrate 921 mg / L, CaCl2·2H2O 147 mg / L, and glucose 5000 mg / L. Through the above technical solution, this invention can achieve the formation and propagation of *Bryophytum pumilus* gametophytes in a short time, providing a feasible approach for the cultivation and subculturing of *Bryophytum pumilus*, and providing seed material and propagation technology for using mosses in environmental remediation and landscaping.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to the culture medium and culture method for gametophytes of *Scleroderma pulcherrima*. Background Technology

[0002] *Mammillaria septemlobus* belongs to the family Bryophyceae and the genus *Mammillaria*. The plant grows in dense clumps, forming a "moss carpet." It is dark green, turning slightly brownish-green in later stages of growth. The plant is about 2-3 cm tall, with a single or bundled stem. Leaves are clustered at the stem apex, long lanceolate, often curling into a tubular shape when dry, unfolding when moist. The leaf margins are toothed, the midrib is stout and disappears below the leaf tip, and the basal cells are long rectangular. The capsules are erect, cylindrical, with a distinct peduncle, yellowish-green, turning brown with age. The stalk is slender, about 1 cm long, and often twisted when dry. It is monoecious. It is endemic to my country, widely distributed in Heilongjiang, Hebei, Gansu, Yunnan, and Tibet.

[0003] Small-leaved sphagnum moss is widely distributed in cities and easy to collect. Its leaves have a double-layered cell structure, giving it good drought and cold resistance. It can complete its entire life cycle in the wild and readily produces capsules. It also has good water absorption and retention properties, making it a promising candidate for applications in water-absorbing and water-storing materials manufacturing, outdoor and indoor landscaping, environmental indicator systems, habitat reconstruction, restoration, and maintenance.

[0004] Because bryophytes are small plants, they are not widely used for ecological and economic purposes. Therefore, there is relatively little research on bryophyte tissue culture both domestically and internationally. The research mainly focuses on spore germination or protonema development, with less research on bryophyte propagation and subculture.

[0005] Currently, there are no reports in existing technologies regarding the propagation of *M. spp.* gametophytes in culture bottles using capsules, nor are there any research reports on soil colonization of *M. spp.* Summary of the Invention

[0006] This invention proposes a culture medium and culture method for gametophytes of *Bryophytum pulcherrima*, which can achieve the formation and propagation of a large number of gametophytes of *Bryophytum pulcherrima* in a short time and establish gametophytes of *Bryophytum pulcherrima* in soil.

[0007] The technical solution of the present invention is as follows:

[0008] The spore culture medium for gametophytes of *Bryophytum pulmonale* includes MgSO4·7H2O 200–300 mg / L, KH2PO4 200–300 mg / L, FeSO4·4H2O 10–15 mg / L, KNO3 1000–1500 mg / L, ammonium tartrate 900–1000 mg / L, CaCl2·2H2O 100–150 mg / L, and glucose 4800–5200 mg / L.

[0009] As a further technical solution, the following are included: MgSO4·7H2O 250mg / L, KH2PO4 250mg / L, FeSO4·4H2O 12.5mg / L, KNO3 1010mg / L, ammonium tartrate 921mg / L, CaCl2·2H2O 147mg / L, and glucose 5000mg / L.

[0010] As a further technical solution, the pH of the KH2PO4 is 6.5.

[0011] The proliferation medium for gametophytes of *Sphaerothorax spp.* includes components of the spore culture medium, as well as 0.5 mg / L of 2,4-D and 8 g / L of agar powder.

[0012] The method for culturing gametophytes of *Scleroderma recurvatum* includes the following steps:

[0013] S1. Inoculate the spore suspension of *Bryophytum comosum* into the spore culture medium and culture it to obtain protonema and gametophytes;

[0014] S2. The protonema and gametophytes are inoculated into the proliferation medium and cultured to obtain new gametophytes.

[0015] S3. The newly formed gametophytes are inoculated into the colonization culture medium and cultured to obtain small reverse twisted moss gametophytes.

[0016] As a further technical solution, when culturing in S1, S2, and S3, the temperature is 25±2℃, the light intensity is 2500lx, and the light duration is 16h / day.

[0017] As a further technical solution, the spore suspension in S1 is prepared by the following method: the sterilized capsules are crushed to prepare the spore suspension;

[0018] The disinfection of the capsules specifically involves rinsing with water, sterilizing with a 75% ethanol solution for 5 minutes, and rinsing with water in sequence.

[0019] As a further technical solution, the culture time in S1 is 30 days.

[0020] As a further technical solution, the culture time in S2 is 40 days.

[0021] As a further technical solution, the colonization culture medium in S3 is a moist humus soil culture medium, and the culture time is 10 days.

[0022] The subculture method for gametophytes of *Scleroderma recurvatum* includes the following steps:

[0023] S1. Inoculate the spore suspension of *Bryophytum comosum* into the spore culture medium and culture it to obtain protonema and gametophytes;

[0024] S2. The protonema and gametophytes are broken up and inoculated into a subculture medium to obtain subcultured small reverse twisted moss gametophytes.

[0025] As a further technical solution, the subculture medium is a spore culture medium or a spore culture medium with 8 g / L of agar powder added.

[0026] The working principle and beneficial effects of this invention are as follows:

[0027] 1. This invention establishes for the first time a method for culturing gametophytes of *M. spp.* using capsules, filling a gap in the existing technology; and using the culturing method of this invention, a large number of *M. spp.* gametophytes can be rapidly obtained through artificial cultivation, providing abundant materials for fields such as landscaping, rooftop greening, vertical greening, indoor greening, and the production of absorbent materials.

[0028] 2. The culture medium and culture method provided by the present invention can also realize the subculture and propagation of *Sphaeromorpha recurvata*, thereby continuously providing moss material. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Experimental Group 1

[0031] The effects of disinfection and sterilization methods were investigated, and eight examples were conducted, namely Examples 1 to 8 (the difference being the different disinfection and sterilization methods). The experimental methods are as follows:

[0032] The capsules were first rinsed with water, then sterilized, and finally rinsed with water again to release the spores, forming a spore suspension. This suspension was then inoculated into a spore culture medium and cultured in a constant temperature and light incubator at 25±2℃, light intensity of 2500lx, and light duration of 16h / day. The spore germination rate was observed and the spore contamination-free rate was calculated.

[0033] Spore contamination-free rate (%) = Number of uncontaminated spores / Total number of spores × 100%

[0034] The spore culture medium consisted of: MgSO4·7H2O 250 mg / L, KH2PO4 250 mg / L (the pH of the stock solution was adjusted to 6.5 with KOH), FeSO4·4H2O 12.5 mg / L, KNO3 1010 mg / L, ammonium tartrate 921 mg / L, trace elements (CuSO4·5H2O 0.055 mg / L, H3BO3 0.614 mg / L, CoCl2·6H2O 0.055 mg / L, Na2MoO4·2H2O 0.025 mg / L, ZnSO4·7H2O 0.055 mg / L, MnCl2·4H2O 0.389 mg / L, KI 28 mg / L), CaCl2·2H2O 147 mg / L, and glucose 5000 mg / L.

[0035] Example 1

[0036] Sterilize with a 75% volume ethanol solution prepared with 1% NaClO for 2 minutes; spore contamination rate is 20%.

[0037] Example 2

[0038] Sterilize with a 75% volume ethanol solution prepared with 1% NaClO for 3 minutes; spore contamination rate is 28%.

[0039] Example 3

[0040] Sterilize with a 75% volume ethanol solution prepared with 1% NaClO for 4 minutes; spore contamination rate is 56%.

[0041] Example 4

[0042] Sterilize with a 75% volume ethanol solution prepared with 1% NaClO for 5 minutes; spore contamination rate is 72%.

[0043] Example 5

[0044] Sterilize with 75% ethanol solution for 2 minutes; spore contamination rate is 83%.

[0045] Example 6

[0046] Sterilize with 75% ethanol solution for 3 minutes; spore contamination rate is 85%.

[0047] Example 7

[0048] Sterilize with 75% ethanol solution for 4 minutes; spore contamination rate is 92%.

[0049] Example 8

[0050] Sterilize with 75% ethanol solution for 5 minutes; spore contamination rate is 98%.

[0051] As can be seen from Examples 1-8, sterilization with 75% ethanol solution for 5 minutes resulted in the highest contamination-free rate of *Styrax spp.* capsules, reaching 98%.

[0052] Experimental Group 2

[0053] The influence of spore culture medium was investigated in three examples, namely Examples 9-11 (the difference being the composition of the spore culture medium). The experimental methods are as follows:

[0054] The capsules were first rinsed with water, then sterilized with 75% ethanol solution for 5 minutes, and finally rinsed with water. The capsules were then broken to release the spores, and a spore suspension was prepared. The suspension was then inoculated into a spore culture medium for cultivation. The cultivation conditions were: temperature 25±2℃, light intensity 2500lx, and light duration 16h / day. The growth and development of the capsules were observed regularly.

[0055] Example 9

[0056] The spore culture medium consisted of: MgSO4·7H2O 250 mg / L, KH2PO4 250 mg / L (the pH of the stock solution was adjusted to 6.5 with KOH), FeSO4·4H2O 12.5 mg / L, KNO3 1010 mg / L, ammonium tartrate 921 mg / L, CaCl2·2H2O 147 mg / L, and glucose 5000 mg / L.

[0057] Example 10

[0058] The spore culture medium consisted of: MgSO4·7H2O 250 mg / L, KH2PO4 250 mg / L (the pH of the stock solution was adjusted to 6.5 with KOH), FeSO4·4H2O 12.5 mg / L, KNO3 1010 mg / L, ammonium tartrate 921 mg / L, trace elements (CuSO4·5H2O 0.055 mg / L, H3BO3 0.614 mg / L, CoCl2·6H2O 0.055 mg / L, Na2MoO4·2H2O 0.025 mg / L, ZnSO4·7H2O 0.055 mg / L, MnCl2·4H2O 0.389 mg / L, KI 28 mg / L), CaCl2·2H2O 147 mg / L, and glucose 5000 mg / L.

[0059] Example 11

[0060] The spore culture medium consisted of: MgSO4·7H2O 250 mg / L, KH2PO4 250 mg / L (the pH of the stock solution was adjusted to 6.5 with KOH), FeSO4·4H2O 12.5 mg / L, KNO3 1010 mg / L, trace elements (CuSO4·5H2O 0.055 mg / L, H3BO3 0.614 mg / L, CoCl2·6H2O 0.055 mg / L, Na2MoO4·2H2O 0.025 mg / L, ZnSO4·7H2O 0.055 mg / L, MnCl2·4H2O 0.389 mg / L, KI 28 mg / L), CaCl2·2H2O 147 mg / L, and glucose 5000 mg / L.

[0061] Examples 9-11 were cultured in their respective spore culture media for 30 days, and their growth and development were observed as follows:

[0062] When spores were placed in the culture medium, some spores germinated in about 3 days and reached multi-stage germination in about 10 days. The germination polarity of the culture medium in Example 11 was significantly higher than that in Examples 9 and 10.

[0063] After spore germination, the protonema elongated and branched. During the 14-day culture period until gametophyte formation, the total length of the protonema growing in the culture medium of Example 9 significantly exceeded that of Examples 10-11. After 21 days, the total length of the protonema growing in the culture medium of Example 9 was more than twice that of Examples 10-11. In addition, the gametophyte development time in the culture medium of Example 9 was 2 days earlier than that in Examples 10-11, and the number of protonema branches in the culture medium was significantly greater than that in Examples 10-11.

[0064] Therefore, the gametophyte growth rate and biomass in the culture medium of Example 10 were lower than those in Example 9; the culture medium of Example 11 affected the growth of *Symplocos macrantha*, and the number of gametophytes produced was not as high as in Examples 9 and 10. The absence of trace elements in the culture medium of Example 9 actually promoted the growth of *Symplocos macrantha* protonemata and facilitated the development of protonemata into gametophytes.

[0065] Experimental Group 3

[0066] The effects of the growth medium on the experiment were investigated, and ten examples were conducted, namely Examples 12 to 21 (the difference being the composition of the growth medium). The experimental methods are as follows:

[0067] S1. Rinse the capsules with water, then sterilize them with 75% ethanol solution for 5 minutes. Finally, rinse with water, break them to release the spores, and prepare a spore suspension. Inoculate the suspension into a spore culture medium and culture for 30 days. The culture conditions are: temperature 25±2℃, light intensity 2500lx, and light duration 16h / day.

[0068] The spore culture medium consisted of: MgSO4·7H2O 250 mg / L, KH2PO4 250 mg / L (the pH of the stock solution was adjusted to 6.5 with KOH), FeSO4·4H2O 12.5 mg / L, KNO3 1010 mg / L, ammonium tartrate 921 mg / L, CaCl2·2H2O 147 mg / L, and glucose 5000 mg / L.

[0069] S2. The protonema and gametophytes obtained after S1 culture were inoculated into the proliferation medium and cultured. The culture conditions were: temperature 25±2℃, light intensity 2500lx, light duration 16h / day. Their growth and development were observed regularly.

[0070] Example 12

[0071] The proliferation medium consists of spore medium and 8 g / L agar powder.

[0072] Example 13

[0073] The proliferation medium consists of spore medium + 0.01 mg / L NAA + 8 g / L agar powder.

[0074] Example 14

[0075] The proliferation medium consists of spore medium + 0.1 mg / L NAA + 8 g / L agar powder.

[0076] Example 15

[0077] The proliferation medium consists of spore medium + 0.5 mg / L NAA + 8 g / L agar powder.

[0078] Example 16

[0079] The proliferation medium consists of spore medium + 0.01 mg / L 6-BA + 8 g / L agar powder.

[0080] Example 17

[0081] The proliferation medium consists of spore medium + 0.1 mg / L 6-BA + 8 g / L agar powder.

[0082] Example 18

[0083] The proliferation medium consists of spore medium + 0.5 mg / L 6-BA + 8 g / L agar powder.

[0084] Example 19

[0085] The proliferation medium consisted of spore medium + 0.01 mg / L 2,4-D + 8 g / L agar powder.

[0086] Example 20

[0087] The proliferation medium consisted of spore medium + 0.1 mg / L 2,4-D + 8 g / L agar powder.

[0088] Example 21

[0089] The proliferation medium consisted of spore medium + 0.5 mg / L 2,4-D + 8 g / L agar powder.

[0090] Examples 12–21 were cultured in their respective proliferation media for 40 days, and their growth and development were observed as follows:

[0091] In Example 12, gametophyte buds were observed to form in the culture medium, with protonemata and gametophytes appearing green. In Example 13, gametophyte development was significantly poor, and the growth of gametophytes and protonemata was not as good as in Example 12. In Examples 14-15, the growth of gametophytes and protonemata was not as good as in Example 13, with a large number of brown filaments and a very small number of green filaments, but the formation of rhizoids was significantly promoted. In Example 16, the growth was good, and the area of ​​derived gametophytes was significantly better than in Example 12. Example 1 Culture media 7–18 inhibited gametophyte derivation in *Bryophytum pumilum*, but the inhibitory effect was much smaller than in Examples 13–15, with a small amount of brown filaments and some green filaments. In the culture media of Examples 19–20, the gametophyte growth was good, and the gametophyte derivation area was significantly larger than that of Example 12, but its fresh weight was much lower than that of Example 12. In the culture media of Example 21, the fresh weight significantly exceeded that of Example 12, and the gametophyte derivation area was no different from that of Example 12, with the differentiation and formation of new gametophyte buds on the gametophytes.

[0092] Therefore, it can be seen that adding different concentrations of hormone treatment solution to the spore culture medium has varying degrees of effect on inducing the formation of new gametophyte plants. The growth state was worst with the addition of NAA, and the inhibitory effect on the propagation culture of *Bryophytum comosum* increased with increasing NAA concentration. During the propagation culture of *Bryophytum comosum*, the increase in derived gametophytes and biomass can be promoted by applying 0.5 mg / L 2,4-D.

[0093] Example 22

[0094] S1. Rinse the capsules with water, then sterilize them with 75% ethanol solution for 5 minutes. Finally, rinse with water, break them to release the spores, and prepare a spore suspension. Inoculate the suspension into a spore culture medium and culture for 30 days. The culture conditions are: temperature 25±2℃, light intensity 2500lx, and light duration 16h / day.

[0095] The spore culture medium consisted of: MgSO4·7H2O 250 mg / L, KH2PO4 250 mg / L (the pH of the stock solution was adjusted to 6.5 with KOH), FeSO4·4H2O 12.5 mg / L, KNO3 1010 mg / L, ammonium tartrate 921 mg / L, CaCl2·2H2O 147 mg / L, and glucose 5000 mg / L.

[0096] S2. The protonema and gametophytes obtained after S1 culture were inoculated into the proliferation medium and cultured for 40 days. The culture conditions were: temperature 25±2℃, light intensity 2500lx, and light duration 16h / day.

[0097] The proliferation medium consisted of: MgSO4·7H2O 250 mg / L, KH2PO4 250 mg / L (the pH of the stock solution was adjusted to 6.5 with KOH), FeSO4·4H2O 12.5 mg / L, KNO3 1010 mg / L, ammonium tartrate 921 mg / L, CaCl2·2H2O 147 mg / L, glucose 5000 mg / L, 2,4-D 0.5 mg / L, and agar powder 8 g / L.

[0098] S3. The newly formed gametophytes obtained in S2 were inoculated into moist humus culture medium and cultured under the following conditions: temperature 25±2℃, light intensity 2500lx, and light duration 16h / day. Their growth and development were observed.

[0099] The cultivation results are as follows:

[0100] The yellowish-green gametophytes gradually turned green after growing in humus medium for 17 days. The small reverse twisted moss, which grew in humus medium for 52 days, could still continue to branch and generate new gametophytes. The small reverse twisted moss, which grew in humus medium for 70 days, no longer produced new gametophyte branches, but had not yet shown yellowing.

[0101] The results above show that the gametophytes of *M. spp.* that are in good condition after proliferation culture can be placed in humus soil to continue growing, thereby maintaining the continuous propagation and growth of *M. spp.* material.

[0102] Experimental Group 4

[0103] The influence of the subculture medium on the subculture of *Bryophytum comosum* was investigated in two examples, Examples 23-24 (the difference being whether or not agar powder was added to the subculture medium). The experimental methods are as follows:

[0104] S1 is the same as S1 in Example 22;

[0105] S2. After grinding the protonema and gametophytes obtained from S1 culture, they were inoculated into the subculture medium and cultured. Subculture was performed every 30 days. The culture conditions were: temperature 25±2℃, light intensity 2500lx, and light duration 16h / day. Their growth and development were observed regularly.

[0106] Example 23

[0107] The subculture medium consisted of: MgSO4·7H2O 250 mg / L, KH2PO4 250 mg / L (the pH of the stock solution was adjusted to 6.5 with KOH), FeSO4·4H2O 12.5 mg / L, KNO3 1010 mg / L, ammonium tartrate 921 mg / L, CaCl2·2H2O 147 mg / L, and glucose 5000 mg / L.

[0108] Example 24

[0109] The subculture medium consisted of: MgSO4·7H2O 250 mg / L, KH2PO4 250 mg / L (the pH of the stock solution was adjusted to 6.5 with KOH), FeSO4·4H2O 12.5 mg / L, KNO3 1010 mg / L, ammonium tartrate 921 mg / L, CaCl2·2H2O 147 mg / L, glucose 5000 mg / L, and agar powder 8 g / L.

[0110] The cultivation results are as follows:

[0111] Three subculture operations were performed consecutively in the culture media of Examples 23 and 24. A significant increase in the biomass of *Mammillaria pulcherrima* was observed within 30 days of each operation. Furthermore, in the culture medium of Example 24, the protonemata derived from the gametophytes continuously elongated, and distinct buds were observed on the derived protonemata. A small portion of the derived buds could form gametophytes; most derived buds maintained this growth stage and did not form stems or leaves. Some derived protonemata extended into the air; these were called aerial derived protonemata, which were generally whitish in color and had low chlorophyll content.

[0112] The results above show that when the protonemae and gametophytes of *Bryophyta pulveratum*, which have been cultured on spore medium for about 30 days, are ground and inoculated into solid-liquid subculture medium, a large number of new gametophytes can be obtained within 120 days for propagation or preservation.

[0113] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A spore culture medium for the gametophytes of *Scleroderma purpureus*, characterized in that, It consists of the following components: MgSO4·7H2O 250mg / L, KH2PO4 250mg / L, FeSO4·4H2O 12.5mg / L, KNO3 1010mg / L, ammonium tartrate 921mg / L, CaCl2·2H2O 147mg / L, glucose 5000mg / L.

2. The spore culture medium for gametophytes of *Sphaerothorax spp.* according to claim 1, characterized in that, The pH of the KH2PO4 is 6.

5.

3. A culture medium for the proliferation of gametophytes of *Sphaerothorax spp.*, characterized in that, It is composed of the components described in any one of claims 1 to 2, 0.5 mg / L of 2,4-D, and 8 g / L of agar powder.

4. A method for culturing gametophytes of *Symplocos buergeriana*, characterized in that, Includes the following steps: S1. Inoculate the spore suspension of *Bryophytum comosum* into the spore culture medium described in any one of claims 1 to 2 and culture it to obtain protonema and gametophytes; S2. The protonema and gametophytes are inoculated into the proliferation medium described in claim 3 and cultured to obtain new gametophytes; S3. The newly formed gametophytes are inoculated into a moist humus culture medium and cultured to obtain small reverse twisted moss gametophytes.

5. The method for culturing gametophytes of *Sphaerothorax spp.* according to claim 4, characterized in that, When culturing in S1, S2, and S3, the temperature is 25±2℃, the light intensity is 2500 lx, and the light duration is 16 h / day.

6. The method for culturing gametophytes of *Sphaerothorax spp.* according to claim 4, characterized in that, The spore suspension in S1 is prepared by the following method: the sterilized capsules are crushed to prepare the spore suspension; The disinfection of the capsules specifically involves rinsing with water, sterilizing with a 75% ethanol solution for 5 minutes, and rinsing with water in sequence.

7. The method for culturing gametophytes of *Sphaerothorax spp.* according to claim 4, characterized in that, The culture time in S1 is 30 days.

8. The method for culturing gametophytes of *Sphaerothorax spp.* according to claim 4, characterized in that, The culture time in S2 is 40 days.

9. The method for culturing gametophytes of *Symplocos pulmonale* according to claim 4, characterized in that, The culture time in S3 is 10 days.