Culture medium for germination of paphiopedilum seeds, application of culture medium and method for germinating paphiopedilum seeds

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to a culture medium for germination of paphiopedilum seeds, application of the culture medium and a method for germination of the paphiopedilum seeds, in particular to application of a broad-leaved tree fallen leaf ash water extract in the culture medium for germination of the paphiopedilum seeds. The broad-leaved tree fallen leaf ash water extract is applied to paphiopedilum seed germination, the seed germination rate can reach 83.7 + / -4.7 (%), germination of the paphiopedilum seeds is effectively promoted, and meanwhile the germination culture cost can be reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a culture medium for germinating Paphiopedilum seeds and an application thereof, and a method for germinating Paphiopedilum seeds. Background Art

[0002] Paphiopedilum, also known as the slipper orchid, has a unique flower shape and rich colors, and is highly ornamental, making it popular among the public. Currently, there are three main ways to propagate Paphiopedilum: traditional division propagation, non-symbiotic germination propagation in a sterile environment, and fungal symbiotic germination propagation.

[0003] Seed germination under sterile environment is an effective method for producing a large number of Paphiopedilum seedling individuals. The type and preparation of culture matrix are the key links to promote seed germination. The conventional culture matrix for Paphiopedilum seed germination usually uses coconut milk as an additive, such as the purple-veined Paphiopedilum non-symbiotic germination culture medium formula disclosed in CN 114557279A (public date 2022.05.31) in the prior art: 1 / 4MS+20-30g / L sucrose+7-8g / L agar powder+100-150ml / L fresh coconut milk+0.1-0.3mg / L 6-BA+0.05-0.15mg / LNAA, pH6.0-7.0, high preparation cost and different quality of raw material acquisition, resulting in differences in the effectiveness of the culture matrix, reducing the germination rate of Paphiopedilum seeds. Therefore, it is urgent to provide a culture medium for Paphiopedilum seed germination to improve the germination rate of Paphiopedilum seeds and provide technical support for Paphiopedilum seedling cultivation. Summary of the invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a culture medium for germination of Paphiopedilum seeds and its application and a method for germinating Paphiopedilum seeds. The culture medium for germination of Paphiopedilum seeds provided by the present invention can effectively promote the germination of Paphiopedilum seeds and achieve the purpose of improving the germination rate of Paphiopedilum seeds.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The invention provides application of a water extract of deciduous ash of broadleaved trees in a paphiopedilum seed germination culture medium.

[0007] The invention provides a culture medium for germination of Paphiopedilum seeds. The culture medium uses 1 / 2MS culture medium as a basic culture medium. The seed germination culture medium also includes the following components: 0.3-0.7 mg / L of 6-BA and 0.1-0.3 mg / L of NAA. The solvent of the seed germination culture medium includes a water extract of deciduous ash of broad-leaved trees.

[0008] Preferably, 1 L of the culture medium comprises: an aqueous extract of broad-leaved tree fallen leaf ash prepared with 5 to 20 g of broad-leaved tree fallen leaves.

[0009] Preferably, the fallen leaves of broad-leaved trees include at least one of the following: fallen leaves of small-leaved banyan, fallen leaves of litchi, fallen leaves of longan trees and fallen leaves of pointed-leaved Elaeocarpus.

[0010] Preferably, the pH value of the aqueous extract of deciduous tree ash is 5.8-7.0.

[0011] Preferably, the method for preparing the aqueous extract of deciduous tree ash comprises the following steps: completely burning deciduous tree leaves in a semi-enclosed container to obtain ashes; extracting the ashes with water, performing solid-liquid separation, and obtaining the aqueous extract of deciduous tree ash.

[0012] Preferably, the incineration further includes adding a combustion aid, and the combustion aid includes at least one of the following: ethanol, anhydrous ethanol and methanol with a volume concentration of 95%.

[0013] Preferably, the extraction time is not less than 1 hour.

[0014] Preferably, the extraction temperature is 20-30°C.

[0015] The present invention also provides a method for germinating Paphiopedilum seeds using the above-mentioned culture medium for Paphiopedilum seed germination, the method comprising the following steps: sowing mature seeds on the culture medium for Paphiopedilum seed germination, and culturing in a light-shaded environment at 25±2°C.

[0016] Beneficial effects: The present invention provides the use of a water extract of ashes from fallen leaves of broad-leaved trees in a culture medium for germination of Paphiopedilum seeds. The present invention utilizes a water extract of ashes from burning fallen leaves of broad-leaved trees as a solvent, and burning fallen leaves promotes biological processes such as seed germination and plant seedling growth. The present invention applies a water extract of ashes from fallen leaves of broad-leaved trees to the germination of Paphiopedilum seeds, and the seed germination rate can reach 83.7±4.7 (%), which effectively promotes the germination of Paphiopedilum seeds and can also reduce the cost of germination culture.

[0017] The present invention provides a culture medium for the germination of Paphiopedilum seeds, which uses a water extract of ashes from the burning of fallen leaves of broad-leaved trees as a solvent to replace the conventional water used to prepare the germination culture matrix of Paphiopedilum seeds. The water extract of the ashes from the burning of fallen leaves is detected and analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), and 120 major compounds are detected. The ashes produced by the burning of grass and trees show a good effect on promoting seed germination in the seedling cultivation of various crops, and the extract of fallen leaf ashes can also effectively promote the germination of Paphiopedilum seeds. The culture medium of the present invention can effectively reduce the preparation cost and the difference between batches of culture matrix preparation, and improve the germination rate of Paphiopedilum seeds, providing technical support for Paphiopedilum seedling cultivation.

[0018] The invention selects the fallen leaves of broad-leaved trees, mainly a combination of fallen leaves of small-leaved banyan, fallen leaves of litchi, fallen leaves of longan tree and fallen leaves of pointed-leaved elaeocarpus, which can effectively control the types and proportions of raw materials used for burning ash, and achieve the controllability of the differences between batches of culture medium preparation.

[0019] The present invention also provides a method for germinating Paphiopedilum seeds using the culture medium for germinating Paphiopedilum seeds. Germinating Paphiopedilum seeds according to the method of the present invention can effectively improve the germination rate of Paphiopedilum seeds and reduce the cost of germination culture. DETAILED DESCRIPTION

[0020] The present invention provides the use of a water extract of deciduous ash from broadleaf trees in a paphiopedilum seed germination medium. The present invention utilizes the biological functions of ash-like substances produced by burning plants in seed germination, promoting seedling growth, etc., to achieve the effect of improving the germination rate of paphiopedilum seeds.

[0021] The invention provides a culture medium for germination of Paphiopedilum seeds. The culture medium uses 1 / 2MS culture medium as a basic culture medium. The culture medium also includes the following components: 0.3-0.7 mg / L of 6-BA (6-benzylaminoadenine) and 0.1-0.3 mg / L of NAA (naphthaleneacetic acid); and the solvent of the culture medium includes a water extract of deciduous ash of broad-leaved trees.

[0022] Unless otherwise specified, the present invention has no special requirements for the preparation raw materials, and commercially available products known to those skilled in the art can be used.

[0023] The culture medium for Paphiopedilum seed germination described in the present invention uses 1 / 2MS culture medium as the basic culture medium, and the culture medium also includes the following components: 6-BA 0.3-0.7 mg / L and NAA 0.1-0.3 mg / L; wherein the 6-BA is more preferably 0.4-0.6 mg / L, and most preferably 0.5 mg / L; the NAA is more preferably 0.15-0.25 mg / L, and most preferably 0.2 mg / L.

[0024] The solvent of the culture medium of the present invention includes a water extract of deciduous ash of broadleaved trees. 1L of the culture medium of the present invention preferably includes: a water extract of deciduous ash of broadleaved trees prepared with 5-20g of deciduous leaves of broadleaved trees, more preferably 8-10g, most preferably 10g.

[0025] The solvent of the culture medium of the present invention includes an aqueous extract of ashes of fallen leaves of broad-leaved trees, and the fallen leaves of broad-leaved trees include at least one of the following: fallen leaves of small-leaved banyan, fallen leaves of litchi, fallen leaves of longan trees, and fallen leaves of pointed-leaved Elaeocarpus. The present invention does not limit the ratio of the fallen leaves of broad-leaved trees used. In an embodiment of the present invention, the fallen leaves of broad-leaved trees are preferably fallen leaves of small-leaved banyan and fallen leaves of litchi, and the weight ratio of the fallen leaves of small-leaved banyan and fallen leaves of litchi is preferably 1:1; or the fallen leaves of broad-leaved trees are preferably fallen leaves of longan trees and fallen leaves of litchi, and the weight ratio of the fallen leaves of longan trees and fallen leaves of litchi is preferably 1:1; or the fallen leaves of broad-leaved trees are preferably fallen leaves of pointed-leaved Elaeocarpus and fallen leaves of litchi, and the weight ratio of the fallen leaves of pointed-leaved Elaeocarpus and fallen leaves of litchi is preferably 1:1. The broad-leaved tree fallen leaves described in the present invention are preferably naturally fallen broad-leaved tree fallen leaves, that is, leaves that fall off from trees through normal physiological processes. Such leaves will transport away some of the transportable substances therein before falling off, while leaves that fall abnormally do not have the process of material transport, and therefore contain different material components from fallen leaves.

[0026] In the preparation of the aqueous extract of deciduous tree ash, the deciduous tree is preferably completely burned in a semi-enclosed container to obtain ash. The deciduous tree is preferably burned in a semi-enclosed container, and the semi-enclosed container includes a semi-enclosed metal container or a semi-enclosed glass container, and the semi-enclosed container can effectively intercept part of the smoke.

[0027] The incineration of the present invention preferably also includes the addition of a combustion aid, and the combustion aid is preferably a high-purity alcohol that burns without residue. The combustion aid of the present invention preferably includes at least one of the following: ethanol, anhydrous ethanol and methanol at a volume concentration of 95%, more preferably ethanol at a volume concentration of 95%. The mass volume ratio of the broad-leaved tree fallen leaves to the combustion aid of the present invention is preferably 8-12g:1-3mL, more preferably 9-11g:1.5-2.5mL, and most preferably 10g:2mL. The combustion aid of the present invention can completely burn lignin, cellulose or other substances containing sugar structures in the fallen leaves of broad-leaved trees, which is conducive to fully obtaining the effective ingredients in the flue gas and ash.

[0028] When the ashes are leached with water according to the present invention, it is preferred to first rinse the inner wall of the container with water to fully dissolve the effective components in the intercepted flue gas in the semi-closed container, and then mix the rinsing water with the ashes for leaching. The leaching time of the present invention is preferably not less than 1h, more preferably 1-2h, and most preferably 1h. The leaching temperature of the present invention is preferably 20-30°C, more preferably 22-28°C, and most preferably 25°C. The present invention preferably uses water to leach the ashes, and the mass volume ratio of the ashes to water is preferably 0.5-1.5g:8-12mL, more preferably 0.8-1.2g:9-11mL, and most preferably 1g:10mL. The present invention preferably performs solid-liquid separation after leaching the ashes to obtain a water extract of deciduous ashes of broad-leaved trees. The solid-liquid separation of the present invention preferably includes filtering with qualitative filter paper.

[0029] The present invention preferably adjusts the pH of the aqueous extract of deciduous tree ash to 5.8-7.0, more preferably to 7.0, before using it to prepare the culture medium. The aqueous extract of deciduous tree ash obtained in the present invention is alkaline, and the pH needs to be adjusted before preparing the culture medium to avoid metal salt ion precipitation reaction of metal salts in the culture medium.

[0030] The present invention also provides a method for germinating Paphiopedilum seeds using the above-mentioned culture medium for Paphiopedilum seed germination, the method comprising the following steps: sowing mature seeds on the prepared culture medium, and culturing in a light-shaded environment at 25±2°C, more preferably at 25°C.

[0031] To further illustrate the present invention, a culture medium for germinating Paphiopedilum seeds and its application and a method for germinating Paphiopedilum seeds provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1

[0033] Preparation of aqueous extract of ashes from fallen leaves of broad-leaved trees: 100 g (50 g each, weight ratio of 1:1) of naturally fallen leaves of small-leaved banyan and litchi were selected, placed in a semi-closed metal container, 20 mL of 95% volume concentration ethanol was added, the fallen leaves were ignited and burned until complete, the cylinder wall was moistened with 1 L of pure water, and the ashes were soaked at 25° C. for 1 h, and filtered with qualitative filter paper to obtain a clear aqueous extract of ashes from fallen leaves of broad-leaved trees for later use.

[0034] Preparation of culture medium: 100 mL of water extract of fallen ash from broad-leaved trees, adjust the pH to 7.0, use the water extract of fallen ash from broad-leaved trees to prepare 1 L of 1 / 2MS culture medium according to the conventional dosage, add 0.5 mg / L 6-BA and 0.2 mg / L NAA, adjust the pH of the culture medium to 5.8, and sterilize it for use.

[0035] Sowing: Sow mature Paphiopedilum bulingense seeds on the prepared culture medium, place in a shaded place, and cultivate at room temperature of 25±2℃.

[0036] Example 2

[0037] Preparation of aqueous extract of deciduous tree leaf ash: select 100g of naturally fallen longan tree leaves and litchi leaves (50g each, weight ratio of 1:1), place them in a semi-closed metal container, add 20mL of 95% volume concentration ethanol, ignite the leaves and burn them until complete, rinse the cylinder wall with 1L of pure water, and then soak the ashes at 25°C for 1h, filter with qualitative filter paper to obtain a clear aqueous extract of deciduous tree leaf ash, and set aside.

[0038] Preparation of culture medium: 100 mL of water extract of fallen ash from broad-leaved trees, adjust the pH to 7.0, use the water extract of fallen ash from broad-leaved trees to prepare 1 L of 1 / 2MS culture medium according to the conventional dosage, add 0.5 mg / L 6-BA and 0.2 mg / L NAA, adjust the pH of the culture medium to 5.8, and sterilize it for use.

[0039] Sowing: Sow mature Paphiopedilum bulingense seeds on the prepared culture medium, place in a shaded place, and cultivate at room temperature of 25±2℃.

[0040] Example 3

[0041] Preparation of aqueous extract of deciduous tree leaf ash: Select 80g (40g each, weight ratio of 1:1) of naturally fallen leaves of Elaeocarpus sylvestris and litchi, place them in a semi-closed metal container, add 20mL of 95% volume concentration ethanol, ignite the leaves and burn them until complete, rinse the cylinder wall with 1L of pure water, and then soak the ashes at 25°C for 1h, filter with qualitative filter paper to obtain a clear aqueous extract of deciduous tree leaf ash, and set aside.

[0042] Preparation of culture medium: 100 mL of water extract of fallen ash from broad-leaved trees, adjust the pH to 7.0, use the water extract of fallen ash from broad-leaved trees to prepare 1 L of 1 / 2MS culture medium according to the conventional dosage, add 0.5 mg / L 6-BA and 0.2 mg / L NAA, adjust the pH of the culture medium to 5.8, and sterilize it for use.

[0043] Sowing: Sow mature Paphiopedilum bulingense seeds on the prepared culture medium, place in a shaded place, and cultivate at room temperature of 25±2℃.

[0044] Comparative Example 1

[0045] The culture medium was prepared and the seeds were sown in the same manner as in Example 1, except that the aqueous extract of deciduous ash from broad-leaved trees was replaced with pure water during the preparation of the culture medium.

[0046] Test Example 1

[0047] 2 μL of the water extract of deciduous ash prepared in Example 1 was taken, and the main compounds contained in the water extract were detected by WaterACQUITY PREMIER-SYNAPTXS mass spectrometer combined with unifi software. The results are shown in Table 1.

[0048] Table 1 Main compounds in the water extract of fallen leaf burning ash

[0049]

[0050]

[0051]

[0052]

[0053] Test Example 2

[0054] Embodiments 1 to 3 and comparative example 1 were sown in parallel for 3 times under the same planting conditions, and the number of test sowings was counted after sowing; when the embryo expanded to break through the seed coat to form a protocorm, which was about 45 days, the number of seeds that germinated was counted, and the germination rate was calculated according to the following formula; the total germination rate was the average value of the germination rates of the embodiments; the results obtained are shown in Table 2.

[0055] Germination rate (%) = number of germinations (pieces) / number of seeds sown (pieces)

[0056] Table 2 Germination rate of Paphiopedilum seeds under different treatments

[0057]

[0058]

[0059] As can be seen from Table 1, the germination rate of Paphiopedilum seeds sown in the manner of Example 1 reached 83.7±4.7 (%), and the total germination rate of Examples 1 to 3 reached 80.2%. In Comparative Example 1, the aqueous extract of deciduous ash from broadleaf trees was replaced with pure water, and the germination rate of Paphiopedilum seeds was only 32.8±4.0 (%).

[0060] It can be seen that it can effectively reduce the preparation cost and the differences between batches of culture matrix preparation, and improve the germination rate of Paphiopedilum seeds, providing technical support for Paphiopedilum seedling cultivation.

[0061] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of aqueous extract of fallen leaf ash of broad-leaved trees in Paphiopedilum seed germination culture medium.

2. A culture medium for germination of Paphiopedilum seeds, characterized in that: The culture medium uses 1 / 2MS culture medium as the basic culture medium, and further comprises the following components: 0.3-0.7 mg / L 6-BA and 0.1-0.3 mg / L NAA; the solvent of the culture medium comprises a water extract of deciduous ash from broad-leaved trees.

3. The culture medium according to claim 2, characterized in that 1L of the culture medium comprises: a water extract of broad-leaved tree fallen leaf ash prepared with 5 to 20g of broad-leaved tree fallen leaves.

4. The culture medium according to claim 3, characterized in that The broad-leaved tree fallen leaves include at least one of the following: fallen leaves of small-leaved banyan, fallen leaves of litchi, fallen leaves of longan tree and fallen leaves of Elaeocarpus punctatus.

5. The culture medium according to claim 2, characterized in that The pH value of the water extract of deciduous ash from broadleaf trees is 5.8-7.

0.

6. The culture medium according to claim 2, characterized in that The method for preparing the water extract of deciduous ash of broadleaf trees comprises the following steps: completely burning deciduous leaves of broadleaf trees in a semi-enclosed container to obtain ashes; and leaching the ashes with water to obtain the water extract of deciduous ash of broadleaf trees through solid-liquid separation.

7. The culture medium according to claim 6, characterized in that The incineration also includes adding a combustion aid, and the combustion aid includes at least one of the following: ethanol, anhydrous ethanol and methanol with a volume concentration of 95%.

8. The culture medium according to claim 6, characterized in that The extraction time is not less than 1 hour.

9. The culture medium according to claim 6, characterized in that The extraction temperature is 20-30°C.

10. A method for germinating Paphiopedilum seeds using the culture medium for Paphiopedilum seeds germination according to any one of claims 2 to 9, characterized in that: The method comprises the following steps: sowing mature seeds on the culture medium for germination of Paphiopedilum seeds, and culturing in a light-shaded environment at 25±2°C.

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