Method for propagation of tubers of nephrolepis auriculata by slicing

Abstract

The invention relates to the technical field of plant tissue culture and asexual propagation, and particularly discloses a tuber section propagation method for nephrolepis auriculata, which comprises the steps of endophyte selective enrichment culture, section preparation, synergistic induction culture, acclimatization and transplantation and the like. Wherein the formula composition of the rooting and germination culture medium is associated with the carbon source selection of the endophyte growth-promoting culture medium; according to the method, a staged plant-microorganism collaborative culture system based on carbon source adaptation is established, so that the biological function of tuber endophyte flora is effectively utilized, and the breeding efficiency and the seedling quality are remarkably improved; according to the method, the biological stability of a culture system is enhanced while the exogenous hormone dependence is reduced, an innovative and efficient solution is provided for solving the problems of high pollution rate, insufficient seedling robustness, large transplanting survival rate fluctuation and the like in traditional tuber propagation, and the method is suitable for large-scale and standardized production.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture and asexual reproduction technology, specifically a method for propagating Nephrolepis tuber slices. Background Technology

[0002] Nephrolepis cordifolia, a fern valued for both its ornamental and ecological aspects, relies primarily on asexual reproduction for large-scale propagation. Tuber division is a key technique for obtaining a large number of genetically consistent seedlings. Traditional tuber propagation methods typically involve directly planting the divided tubers in a substrate or using a culture medium containing exogenous plant growth regulators for in vitro induction to promote germination and rooting. The core of these methods lies in using growth regulators to directly stimulate the dedifferentiation and redifferentiation of plant tissues.

[0003] However, the endophytic microbial communities widely present in plants, especially in vegetative organs such as tubers, have a close symbiotic relationship with the host plant in terms of nutrient absorption, disease resistance, and stress tolerance. Current propagation techniques often overlook this natural biological resource. Conventional in vitro culture methods, primarily relying on direct stimulation with high concentrations of exogenous hormones, while effective in initiating tissue differentiation, are susceptible to contamination by other microorganisms. Furthermore, the resulting seedlings often suffer from poor root development and physiological fragility, leading to unstable transplant survival rates and failing to meet the demands of efficient, high-quality, and stable industrial production. How to effectively utilize and guide the plant-endophytic microbial symbiotic system to replace or optimize single chemical hormone regulation, thereby achieving more stable and efficient tuber propagation, has become a technical problem to be solved in this field. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for propagating Nephrolepis tuber slices, thereby resolving the issues of high contamination rates, inconsistent seedling quality, and unstable transplant survival rates in the prior art due to excessive reliance on exogenous hormone stimulation.

[0005] A method for propagating Nephrolepis tuber slices includes the following steps:

[0006] S1. Selective enrichment culture of endophytic bacteria: The surface-sterilized tubers of Nephrolepis cordifolia were placed in an endophytic bacteria growth-promoting medium with a specific single carbon source as the main carbon source and without any exogenous plant growth regulators, and cultured in the dark at 22-26℃ for 5-10 days to obtain tubers enriched with endophytic bacteria.

[0007] S2. Slice preparation: Cut the endophytic bacteria-enriched tuber into thin slices with buds;

[0008] S3. Synergistic induction culture: The slices are inoculated into a rooting and sprouting medium containing plant growth regulators for culture; the weight concentration ratio (C:PGR) of total carbon source to plant growth regulator in the rooting and sprouting medium is (80-120):1.

[0009] Preferably, the specific single carbon source is inulin or trehalose.

[0010] Preferably, the plant growth regulator includes naphthaleneacetic acid and 6-benzylaminopurine.

[0011] Preferably, step S3 includes:

[0012] S31. First stage of synergistic induction: Dark culture at 22-26℃ for 7-14 days;

[0013] S32, Second stage of germination and growth: Cultivate at 18-22℃, light intensity of 2000-3000 Lux, and 10-12 hours of light per day.

[0014] Preferably, in step S1, the surface disinfection includes: immersing in ethanol with a volume concentration of 70-75% for 30-60 seconds, immersing in mercuric chloride solution with a mass concentration of 0.1% for 8-12 minutes, and then rinsing with sterile water 3-5 times.

[0015] Preferably, in step S2, before cutting, the endophytic bacteria-enriched tuber is further immersed in an antioxidant solution containing 0.1–0.5 g / L ascorbic acid or 0.5–2.0 g / L polyvinylpyrrolidone for 10–20 minutes.

[0016] Preferably, in step S2, the thickness of the sheet is 3 to 5 mm, and each sheet contains at least one complete bud.

[0017] Preferably, after step S3, the method further includes:

[0018] S4. Hardening off and transplanting: When the seedlings have 3-5 true leaves and reach a height of 4-6 cm, gradually uncover the covering of the culture container for hardening off for 5-7 days, and then transplant them into a seedling substrate made of peat moss, perlite and vermiculite in a volume ratio of (3-4):1:1.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] By constructing a selective endophytic bacteria enrichment system and designing a synergistic induction medium with a specific ratio of carbon source to plant growth regulator based on the characteristics of enriched carbon source, a plant-microbe synergistic growth system with close correlation between the preceding and following steps was established, which achieved the improvement of microbial community stability and the significant enhancement of tissue differentiation induction efficiency during the tuber slice propagation process.

[0021] This method effectively improves the germination and rooting efficiency of tuber slices, promotes the robust growth and morphological development of seedlings, significantly enhances the resistance of the culture system to contamination by miscellaneous bacteria, and improves the stability and success rate of the entire propagation process. The obtained seedlings have better physiological state and adaptability, laying a good foundation for subsequent transplanting survival and large-scale production. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the method for propagating Nephrolepis tuber slices according to the present invention. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] like Figure 1 As shown:

[0025] Example 1: This invention provides a method for propagating Nephrolepis tuber slices, comprising the following steps:

[0026] S1. Selective enrichment culture of endophytic bacteria:

[0027] Healthy and plump Nephrolepis tubers were selected and surface disinfected: they were soaked in 70% ethanol for 30 seconds, then in 0.1% mercuric chloride solution for 8 minutes, and then rinsed three times with sterile water and air-dried. The disinfected tubers were placed in an endophytic bacteria growth-promoting medium, which used inulin as the main carbon source and did not contain any exogenous plant growth regulators. The medium and tubers were placed in a 22°C incubator and cultured in the dark for 5 days to obtain tubers enriched with endophytic bacteria.

[0028] S2. Slice preparation:

[0029] Remove the tubers enriched with endophytic bacteria and soak them in an antioxidant solution containing 0.1 g / L ascorbic acid for 10 minutes to inhibit browning; then, use a sterile blade to cut the tubers into 3 mm thick slices, ensuring that each slice contains at least one intact bud.

[0030] S3, Synergistic Induction Culture:

[0031] The cut slices were inoculated into a rooting and sprouting medium for cultivation.

[0032] (1) The formulation of the rooting and sprouting medium is related to the carbon source of inulin used in step S1, and the weight concentration ratio (C:PGR) of the total carbon source (including residual inulin and newly added sucrose) to the plant growth regulator is 80:1; the plant growth regulator is a combination of naphthaleneacetic acid and 6-benzylaminopurine.

[0033] (2) First stage of synergistic induction: The inoculated culture was placed at 22°C and cultured in the dark for 7 days.

[0034] (3) Second stage of germination and growth: Subsequently, the cultivation conditions were adjusted to a temperature of 18℃, a light intensity of 2000 Lux, and a light exposure of 10 hours per day, and the cultivation continued until the seedlings were formed.

[0035] S4. Hardening off and transplanting:

[0036] When the seedlings have 3 true leaves and reach a height of 4 cm, gradually remove the sealing film from the culture container and harden them off for 5 days. After that, transplant the seedlings into a seedling substrate made of peat moss, perlite and vermiculite in a volume ratio of 3:1:1.

[0037] Example 2: This invention provides a method for propagating Nephrolepis tuber slices, comprising the following steps:

[0038] S1. Selective enrichment culture of endophytic bacteria:

[0039] Healthy and plump Nephrolepis tubers were selected and surface disinfected: they were soaked in 72.5% ethanol for 45 seconds and then in 0.1% mercuric chloride solution for 10 minutes, followed by rinsing with sterile water four times and air-drying. The disinfected tubers were then placed in an endophytic bacteria growth-promoting medium, which used trehalose as the main carbon source and did not contain any exogenous plant growth regulators. The medium and tubers were placed in a 24°C incubator for dark culture for 7.5 days to obtain tubers enriched with endophytic bacteria.

[0040] S2. Slice preparation:

[0041] Remove the tubers enriched with endophytic bacteria and soak them in an antioxidant solution containing 0.3 g / L ascorbic acid and 1.25 g / L polyvinylpyrrolidone for 15 minutes to inhibit browning; then, use a sterile blade to cut the tubers into 4 mm thick slices, ensuring that each slice contains at least one intact bud.

[0042] S3, Synergistic Induction Culture:

[0043] The cut slices were inoculated into a rooting and sprouting medium for cultivation.

[0044] (1) The formulation of the rooting and sprouting culture medium is related to the trehalose carbon source used in step S1, and the weight concentration ratio (C:PGR) of the total carbon source (including residual trehalose and newly added sucrose) to the plant growth regulator is 100:1; the plant growth regulator is a combination of naphthaleneacetic acid and 6-benzylaminopurine.

[0045] (2) First stage of synergistic induction: The inoculated culture was placed at 24°C and cultured in the dark for 10.5 days.

[0046] (3) Second stage of germination and growth: Subsequently, the cultivation conditions were adjusted to a temperature of 20℃, a light intensity of 2500 Lux, and 11 hours of light per day, and the cultivation continued until the seedlings were formed.

[0047] S4. Hardening off and transplanting:

[0048] When the seedlings have 4 true leaves and reach a height of 5 cm, gradually remove the sealing film from the culture container and harden them off for 6 days. After that, transplant the seedlings into a seedling substrate made of peat moss, perlite and vermiculite in a volume ratio of 3.5:1:1.

[0049] Example 3: This invention provides a method for propagating Nephrolepis tuber slices, comprising the following steps:

[0050] S1. Selective enrichment culture of endophytic bacteria:

[0051] Healthy and plump Nephrolepis tubers were selected and surface disinfected: they were soaked in 75% ethanol for 60 seconds, then in 0.1% mercuric chloride solution for 12 minutes, and then rinsed 5 times with sterile water and air-dried. The disinfected tubers were placed in an endophytic bacteria growth-promoting medium, which used inulin as the main carbon source and did not contain any exogenous plant growth regulators. The medium and tubers were placed in a 26°C incubator and cultured in the dark for 10 days to obtain tubers enriched with endophytic bacteria.

[0052] S2. Slice preparation:

[0053] Remove the tubers enriched with endophytic bacteria and soak them in an antioxidant solution containing 0.5 g / L polyvinylpyrrolidone for 20 minutes to inhibit browning; then, use a sterile blade to cut the tubers into slices 5 mm thick, ensuring that each slice contains at least one intact bud.

[0054] S3, Synergistic Induction Culture:

[0055] The cut slices were inoculated into a rooting and sprouting medium for cultivation.

[0056] (1) The formulation of the rooting and sprouting culture medium is related to the carbon source of inulin used in step S1. The weight concentration ratio (C:PGR) of the total carbon source (including residual inulin and newly added sucrose) to the plant growth regulator is 120:1. The plant growth regulator is a combination of naphthaleneacetic acid and 6-benzylaminopurine.

[0057] (2) First stage of synergistic induction: The inoculated culture was placed at 26°C and cultured in the dark for 14 days.

[0058] (3) Second stage of germination and growth: Subsequently, the cultivation conditions were adjusted to a temperature of 22℃, a light intensity of 3000 Lux, and a light exposure of 12 hours per day, and the cultivation continued until the seedlings were formed.

[0059] S4. Hardening off and transplanting:

[0060] When the seedlings have 5 true leaves and reach a height of 6 cm, gradually remove the sealing film from the culture container and harden them off for 7 days. After that, transplant the seedlings into a seedling substrate made of peat moss, perlite and vermiculite in a volume ratio of 4:1:1.

[0061] In this invention, the weight concentration of "total carbon source" in the rooting and sprouting medium described in step S3 specifically refers to the concentration of newly added soluble carbon source, which serves as the main energy source, during the preparation of the medium, excluding trace amounts of residual carbon source that may be introduced from the tubers in step S1. The "total carbon source to plant growth regulator weight ratio (C:PGR)" is calculated based on the concentration of this newly added carbon source. Commonly selected additional carbon sources include, but are not limited to, sucrose and glucose, with sucrose being preferred.

[0062] Experimental Example: Verification of the Effectiveness of the Nephrolepis tuber slice propagation method

[0063] 1. Experimental objective: To verify the technical advantages of the method of the present invention over conventional cutting / tissue culture methods in terms of propagation efficiency, seedling quality, and resistance.

[0064] 2. Experimental materials: Plant materials: healthy, mature Nephrolepis tubers of roughly the same size.

[0065] Culture medium:

[0066] Endophytic bacteria growth-promoting culture medium A: uses inulin as the main carbon source and does not contain plant growth regulators.

[0067] Endophytic bacteria growth-promoting medium B: uses trehalose as the main carbon source and does not contain plant growth regulators.

[0068] Rooting and sprouting medium A': associated with the carbon source of medium A, C:PGR=100:1.

[0069] Rooting and sprouting medium B': associated with the carbon source of medium B, C:PGR=100:1.

[0070] The standard induction medium was C: MS medium + 1.0 mg / L 6-BA + 0.2 mg / L NAA (C:PGR no specific association design).

[0071] Reagents: ethanol, mercuric chloride, ascorbic acid, polyvinylpyrrolidone, etc.

[0072] Equipment: Clean bench, light incubator, electronic balance, etc.

[0073] 3. Experimental design: A total of 6 treatment groups were set up, with each group repeated 5 times. 10 tuber slices were used in each experiment.

[0074] Experimental group 1: The method of Example 1 (inulin, C:PGR=80:1) was adopted.

[0075] Experimental group 2: The method of Example 2 (trehalose, C:PGR=100:1) was adopted.

[0076] Experimental group 3: The method of Example 3 (inulin, C:PGR=120:1) was adopted.

[0077] Comparative Example 1: No endophytic bacteria enrichment steps: After tuber disinfection, slices were directly inoculated into rooting and sprouting medium A' for culture (other conditions were the same as in experimental group 1).

[0078] Comparative Example 2: Culture medium-free design: S1 was enriched using culture medium A, but S3 was cultured using conventional induction medium C.

[0079] Comparative Example 3: Conventional tissue culture method: After sterilization and sectioning of the tubers, they were directly inoculated into conventional induction medium C for culture.

[0080] 4. Experimental Design:

[0081] All groups maintained the same basic conditions, including surface disinfection, section size (3-5 mm with buds), and culture containers, and strictly followed the procedures. The culture period lasted a total of 60 days, during which observations and records were kept.

[0082] 5. Observation indicators and records

[0083] Rooting and sprouting indicators (statistics on day 30 of culture): sprouting rate (%), average number of roots (strips / slice), average root length (cm);

[0084] Seedling growth indicators (statistics on day 60 of cultivation, before hardening): average plant height (cm), average number of true leaves (pieces), average fresh weight (g / plant).

[0085] Resistance and quality indicators (assessed on day 60 of culture):

[0086] Contamination rate (%): The proportion of slides or culture media contaminated with fungi / bacteria;

[0087] Seedling robustness index: a comprehensive score based on stem thickness, leaf color intensity, and root development (1-5 points, with 5 points being the best).

[0088] 6. Experimental Results:

[0089] Table 1: Effects of different treatments on propagation efficiency and seedling growth of Nephrolepis tuber slices

[0090] Processing group Germination rate (%) Average number of roots (stalks) Average root length (cm) Average plant height (cm) Average number of true leaves (pieces) Average fresh weight (g) Pollution rate (%) Robustness Index (1-5) Experimental group 1 86.0 4.2 3.8 5.1 3.8 0.52 4.0 4.0 Experimental group 2 94.0 5.8 4.5 5.8 4.2 0.61 2.0 4.5 Experimental group 3 88.0 4.5 4.0 5.3 4.0 0.55 3.0 4.2 Comparative Example 1 70.0 2.5 2.2 3.5 2.8 0.35 12.0 2.8 Comparative Example 2 78.0 3.0 2.8 4.0 3.2 0.40 8.0 3.2 Comparative Example 3 65.0 2.0 1.8 3.0 2.5 0.28 18.0 2.5

[0091] 7. Analysis of Experimental Results:

[0092] Overall advantages of the method of this invention:

[0093] The three experimental groups (experimental group 1, experimental group 2, and experimental group 3) were significantly better than the three comparative groups (comparative group 1, comparative group 2, and comparative group 3) in all observed indicators.

[0094] The contamination rate was significantly reduced (only 2% in experimental group 2, and 18% in comparative group 3), indicating that the enrichment of endophytic bacteria may have formed a beneficial bacterial barrier, inhibiting contamination by miscellaneous bacteria and improving production stability.

[0095] A higher seedling vigor index indicates that the seedlings obtained by this method are in better physiological condition, which is beneficial for subsequent transplanting and survival.

[0096] The embodiments of the present invention are given for the purposes of illustration and description. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Any changes, modifications, substitutions and variations made by those skilled in the art to the above embodiments within the scope of the present invention should be included within the protection scope of the present invention.

Claims

1. A method of propagating Nephthytis rhizome slices, comprising the steps of, The method comprises the following steps: S1, endophyte selective enrichment culture: placing the sterilized Nephrolepis cordifolia tubers in an endophyte growth-promoting medium with a specific single carbon source as the main carbon source and without exogenous plant growth regulators, and culturing in the dark at 22-26℃ for 5-10 days to obtain endophyte-enriched tubers; S2, slice preparation: cutting the endophyte-enriched tubers into slices with bud points; S3, synergistic induction culture: inoculating the slices into a rooting and germination medium containing plant growth regulators for culture; the weight concentration ratio of total carbon source to plant growth regulator (C:PGR) in the rooting and germination medium is (80-120):

1.

2. The method of propagating Nephthytis rhizome slices according to claim 1, wherein, The specific single carbon source is inulin or trehalose.

3. The method of propagating Nephthytis tuber slices according to claim 1 or 2, wherein, The plant growth regulators include naphthalene acetic acid and 6-benzylaminopurine.

4. The method of propagating Nephthytis cuttmg according to claim 1, wherein, Step S3 comprises: S31, first-stage synergistic induction: culturing in the dark at 22-26℃ for 7-14 days; S32, second-stage germination growth: culturing under the conditions of 18-22℃, light intensity of 2000-3000 Lux, and 10-12 hours of light per day.

5. The method of propagating Nephthytis cuttmg according to claim 1, wherein, In step S1, the surface sterilization comprises: sequentially immersing in 70-75% (volume concentration) ethanol for 30-60 seconds, immersing in 0.1% (mass concentration) mercuric chloride solution for 8-12 minutes, and then rinsing with sterile water for 3-5 times.

6. The method of propagating Nephthytis cuttmg according to claim 1, wherein, In step S2, before cutting, the endophyte-enriched tubers are further immersed in an antioxidant solution containing 0.1-0.5 g / L ascorbic acid or 0.5-2.0 g / L polyvinylpyrrolidone for 10-20 minutes.

7. The method of propagating Nephthytis cuttmg according to claim 1, wherein, In step S2, the thickness of the slices is 3-5 mm, and each slice contains at least one complete bud point.

8. The method of propagating Nephthytis cuttmg according to claim 1, wherein, After step S3, further comprising: S4, hardening-off and transplanting: when the generated seedlings have 3-5 true leaves and the plant height reaches 4-6 cm, gradually uncovering the cover of the culture container for hardening-off for 5-7 days, and then transplanting into a seedling raising substrate mixed by turf, perlite and vermiculite at a volume ratio of (3-4):1:1.

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