Method for promoting sterile germination and seedling formation of paphiopedilum brevituralensis seeds

Abstract

The invention discloses a method for promoting sterile germination and seedling formation of paphiopedilum brevifilifolium seeds. The method comprises the following steps: selecting pods of paphiopedilum leucopeniculatum as explants, disinfecting the pods, and cutting the pods to obtain sterile seeds of the paphiopedilum leucopeniculatum; mixing the disinfected and sterilized paphiopedilum brevifilifolium seeds with sterile water to prepare a seed suspension, and sowing the seed suspension on a culture medium for dark culture. The application of the invention provides a practical and efficient technical path for artificial propagation and industrial seedling culture of orchid plants. The method is easy and convenient to operate, good in repeatability, suitable for laboratory research and large-scale production and wide in application prospect. By regulating and controlling the synergistic effect of the illumination condition and the carbon source type, the physiological metabolism of the dormant seeds of the paphiopedilum baphicacanthus is effectively activated, the technical bottleneck that orchid plants are difficult to germinate is broken through, orchid resource protection and development of a breeding technology are promoted, and theoretical support and practical basis are provided for sustainable utilization of rare ornamental plants in China.

Description

Technical Field

[0001] The present invention belongs to the technical field of paphiopedilum breeding, and particularly relates to a method for directly sowing and efficiently raising seedlings of Paphiopedilum spicerianum seeds by using trehalose as a carbon source in a culture medium. Background Art

[0002] Paphiopedilum spicerianum is the most characteristic species of the genus Paphiopedilum. Its sepals are white, light green at the base, with a purplish-red midvein on the inner surface, resembling a white flag. The staminode is nearly rhomboid, light purple in the middle, white at the edge, and the base edge shrinks and is slightly auriculate. The flowering period is from October to December. It was first discovered in Pu'er City, Yunnan Province in 2006. It grows on steep riverbanks. Coupled with river water pollution, its growth environment is extremely harsh, and there are only more than 20 individuals in the biological population, which has high conservation and research value. Given its current wild distribution status, the most effective way is to carry out ex-situ conservation on it, artificially propagate seedlings, and then conduct wild return and meet market demand.

[0003] The seeds of paphiopedilum are small and lack endosperm. Under natural conditions, the seeds of orchidaceae plants need to establish a symbiotic relationship with certain fungi and obtain carbon and energy sources by hydrolyzing exogenous trehalose by symbiotic fungi to promote seed germination and growth. However, due to the wide variety of symbiotic fungi and the specificity of some fungi, and different symbiotic fungi types are required at different growth stages of orchidaceae plants, the steps are more cumbersome and the operation requirements are higher, and there are certain limitations in application. In our research, it was found that the key in symbiotic germination is that paphiopedilum highly expresses trehalase at the protocorm stage to degrade trehalose in symbiotic fungi to complete its own growth. At present, non-symbiotic germination methods are mainly used to breed Paphiopedilum spicerianum seedlings, and the main culture media used are VW and H26: The germination rate of mature Paphiopedilum spicerianum seeds on VW medium is 56%, but "retention" of protocorms occurred in subsequent culture and they could not grow into seedlings; on H26 medium, the germination rate is relatively low at 19%, but the overall seedling formation rate is 3%. Therefore, VW is used for sowing in production, and after germination, it is transferred to H26 for seedling formation, which brings a great deal of labor costs. Summary of the Invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a method and application for promoting non-symbiotic germination of Paphiopedilum spicerianum seeds by changing light conditions and the formula of H26 medium under non-symbiotic conditions. The method of the present invention has the advantages of simple operation, low cost, and high efficiency.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] Select the fruit pod of Paphiopedilum spicerianum as the explant, disinfect the fruit pod and then cut it open to obtain sterile seeds of Paphiopedilum spicerianum;

[0007] Mix the disinfected and sterilized Paphiopedilum wardianum seeds with sterile water to make a seed suspension, and sow the seed suspension on the culture medium for dark culture.

[0008] The culture medium is: 0.1 g / L inositol, 0.5 g / L activated carbon, 1.5 g / L Hyponex No. 1, 2 g / L peptone, 10 g / L agar, 10 g / L sucrose, 10 g / L trehalose, 0.5 mg / L naphthaleneacetic acid, 1 ml / L vitamin B, 10 ml / L iron salt, 50 ml / L coconut milk, and the solvent is water.

[0009] Preferably, the iron salt is 37.25 mg / L Na2-EDTA + 27.85 mg / L FeSO4·7H2O, and the solvent is water.

[0010] For the dark culture, the culture temperature is 25 ± 2 °C.

[0011] Preferably, the fruit pod is a fruit pod that has developed for about 250 days after artificial pollination.

[0012] Preferably, for the disinfection, wipe the fruit pod of Paphiopedilum wardianum clean, disinfect it with 70% (v / v) alcohol for 60 s, wash it with sterile water for 30 s, disinfect it with 0.1% (w / w) HgCl2 solution for 7 min, wash it with sterile water 3 times, 30 s each time, air dry it, cut open the fruit pod to obtain the disinfected sterile seeds of Paphiopedilum wardianum.

[0013] By adding an appropriate amount of trehalose to the culture medium and implementing light-shielding treatment (i.e., culturing in the dark) in the present invention, the germination rate of seeds and the protocorm formation rate can be significantly improved, thereby realizing the efficient seedling propagation of Paphiopedilum wardianum. The application of the present invention provides a practical and efficient technical path for the artificial propagation and industrialized seedling raising of Orchidaceae plants. This method is simple to operate, has good repeatability, is applicable to laboratory research and large-scale production, and has broad application prospects. By regulating the synergistic effect of light conditions and carbon source types, the physiological metabolism of dormant seeds of Paphiopedilum wardianum can be effectively activated, which helps to break through the technical bottleneck of difficult germination of Orchidaceae plants, promotes the development of orchid resource protection and breeding technology, and provides theoretical support and practical basis for the sustainable utilization of rare ornamental plants in China.

[0014] The present invention has the following beneficial effects:

[0015] (1) The culture medium formula of the present invention can support the direct development of Paphiopedilum wardianum from seeds to seedlings without the need to transfer and inoculate to other culture media, reducing the labor operation cost.

[0016] (2) This method can greatly improve the germination rate of seeds, and the growth of small seedlings is consistent, realizing the efficient cultivation of Paphiopedilum wardianum seedlings, and having the application value of producing a large number of seedlings in a short time.

[0017] (3) The operation of the present invention is simple, with low cost, suitable for popularization and application, and has great popularization value in aspects such as the return of rare and endangered orchid plants, the reconstruction of extremely small populations, and solving the bottleneck problem of the seedling source in the cultivation industry of Paphiopedilum wardianum Description of the Drawings

[0018] Figure 1 It shows the growth of seeds 45 days after sowing.

[0019] Figure 2 It shows the growth of seeds 90 days after sowing. Detailed Embodiments

[0020] In order to make the objectives, technical solutions and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the embodiments described in this specification are only for explaining the present invention and not for limiting the present invention.

[0021] Embodiment 1: Preliminary screening and analysis of the concentration of trehalose as the seed germination medium for Paphiopedilum wardianum

[0022] 1. Obtaining and disinfecting explants

[0023] (1) Source of explants of Paphiopedilum wardianum: The axenic seedlings of Paphiopedilum wardianum are obtained through axenic sowing and tissue culture, and were potted in the orchid greenhouse in the scientific research area of South China Botanical Garden in 2021 and had been cultivated for 3 years when the experiment was conducted. Select the seed pods pollinated on November 13, 2023, harvested on August 13, 2024, and with a development duration of 275 days.

[0024] (2) Disinfection of fruit pods: First, wipe the surface of the seed pods twice with cotton balls soaked in 75% (v / v) alcohol in a laminar flow hood, then soak them in 75% alcohol solution for 30 s, shaking the glass bottle constantly during this period. After rinsing once with sterilized deionized water, soak them in 0.1% (w / v) mercuric chloride solution and shake constantly for 20 mins, and then wash and dry them with deionized water.

[0025] (3) Seed preservation: Use a scalpel sterilized at high temperature to cut open the dried seed pods in a laminar flow hood, shake the seeds into a sterile centrifuge tube, seal the lid with a sealing film, and place it in a 4°C refrigerator for storage and standby after noting the seed information.

[0026] 2. Axenic sowing

[0027] (1) Preparation of the culture medium: The culture medium used is modified in terms of composition and ratio based on the H26 medium;

[0028] The composition of Medium 1 is 0.1 g / L inositol + 0.5 g / L activated carbon + 1.5 g / L Hyponex No. 1 + 2 g / L peptone + 5 g / L agar + 20 g / L trehalose + 0.5 mg / L naphthaleneacetic acid + 1 ml / L vitamin B + 10 ml / L iron salt + 50 ml / L coconut milk, with water as the solvent. The preparation method is to mix the components evenly;

[0029] The composition of Medium 2 is 0.1 g / L inositol + 0.5 g / L activated carbon + 1.5 g / L Hyponex No. 1 + 2 g / L peptone + 5 g / L agar + 15 g / L trehalose + 5 g / L sucrose + 0.5 mg / L naphthaleneacetic acid + 1 ml / L vitamin B + 10 ml / L iron salt + 50 ml / L coconut milk, with water as the solvent. The preparation method is to mix the components evenly;

[0030] Medium 3 is 0.1 g / L inositol + 0.5 g / L activated carbon + 1.5 g / L Hyponex No. 1 + 2 g / L peptone + 5 g / L agar + 10 g / L sucrose + 10 g / L trehalose + 0.5 mg / L naphthaleneacetic acid + 1 ml / L vitamin B + 10 ml / L iron salt + 50 ml / L coconut milk, with water as the solvent. The preparation method is to mix the components evenly;

[0031] Medium 4 is 0.1 g / L inositol + 0.5 g / L activated carbon + 1.5 g / L Hyponex No. 1 + 2 g / L peptone + 5 g / L agar + 15 g / L sucrose + 5 g / L trehalose + 0.5 mg / L naphthaleneacetic acid + 1 ml / L vitamin B + 10 ml / L iron salt + 50 ml / L coconut milk, with water as the solvent. The preparation method is to mix the components evenly;

[0032] Medium 5 is 0.1 g / L inositol + 0.5 g / L activated carbon + 1.5 g / L Hyponex No. 1 + 2 g / L peptone + 5 g / L agar + 20 g / L sucrose + 0.5 mg / L naphthaleneacetic acid + 1 ml / L vitamin B + 10 ml / L iron salt + 50 ml / L coconut milk, with water as the solvent. The preparation method is to mix the components evenly;

[0033] The formula of the iron salt is 37.25 mg / L Na2-EDTA + 27.85 mg / L FeSO4·7H2O, with water as the solvent. The preparation method is to mix the components evenly.

[0034] After autoclaving Medium at 121 °C under high pressure for 20 minutes, pour it into a sterile petri dish with a diameter of 9 cm in a laminar flow hood. After the medium cools and solidifies, lay a sterile double-circle qualitative filter paper with a diameter of 7 cm on it.

[0035] (2) Sowing: Place the seeds in sterile water for a period of time until they are evenly suspended to make a seed suspension. Use a pipette tip with the tip cut off to aspirate 1 ml of the suspension and sow it on sterile filter paper. The seeding rate is about 300 - 350 seeds per dish. Sow 10 dishes for each medium. After natural drying under the laminar flow hood, seal the plate with a sealing film.

[0036] (3) Culture conditions: Place the culture medium plates in an environment with a constant temperature of 26°C, the temperature is (25 ± 2)°C, with 10 - 12 hours of daily light and a light intensity of 1600 - 2000 lux.

[0037] 3. Observe the seed germination situation with a stereomicroscope 45 days after sowing. In this stage of the study, seed germination is simplified into 2 stages, namely seed germination (the embryo swells and produces root-like structures), and protocorm formation and development (the embryo swells and breaks through the seed coat until the appearance of the primary meristem). According to the above grading criteria, count the number of germinated seeds and protocorms of different treatments in each group of experiments, and calculate the seed germination rate and protocorm formation rate of each treatment group based on the total number of sown seeds. Take the average value of each group of data.

[0038] 4. Experimental results

[0039] Table 1

[0040] culture medium Germination rate (%) protocorm rate (%) 1 20.76±3.03 7.89±1.50 2 38.96±1.99 15.59±5.67 3 42.06±2.38 26.36±3.10 4 41.83±2.31 12.96±3.83 5 43.34±1.80 17.55±2.22

[0041] This Table 1 shows the performance data of the germination rate and protocorm rate under five different culture medium conditions, including the mean ± standard error of the mean (SEM); there is no significant difference in the germination rates of Group 3, Group 4, and Group 5, but they are significantly higher than those of Group 1 and Group 2. Culture media 3 and 5 have the best germination effects and can be preferentially considered for seed germination. The protocorm rate of Group 3 is significantly the highest and the error is relatively small, possibly having a significant advantage; Culture medium 3 also performs optimally in protocorm formation, with Group 1 being the lowest; Groups 2, 4, and 5 are at the intermediate level, but the error is slightly larger and the difference may not be significant. If the goal is to increase the germination rate and protocorm rate, Culture medium 3 is the first choice; Culture medium 5 comes second, with a relatively high germination rate and an above-average protocorm rate. Group 1 shows the lowest germination rate (20.76%) and protocorm formation rate (7.89%), representing inefficient culture conditions and can be used as a negative control; the germination rate (42.06%) and protocorm rate (26.36%) of Group 3 are significantly higher than those of other groups, and it is the treatment group with the best comprehensive effect in this experiment; there is no significant difference in the germination rate between Group 5 and Group 3 (43.34%, belonging to the significant grouping a), and the protocorm rate is also at a medium-high level (17.55%), having good representativeness. The three culture media show an obvious performance gradient (low, medium, high) in germination and protocorm formation, which helps to systematically evaluate the response mechanisms of each index under different nutritional backgrounds. Therefore, these three culture media are selected as representative groups for subsequent research.

[0042] Example 2: Dark Conditions

[0043] 1. Obtaining and Disinfecting Explants

[0044] (1) Source of Paphiopedilum wardianum explants: The aseptic seedlings of Paphiopedilum wardianum were obtained through aseptic sowing and tissue culture. They were transplanted into the orchid greenhouse in the scientific research area of South China Botanical Garden in 2021 and had been cultivated for 3 years when the experiment was conducted. Select the pods pollinated on January 15, 2024, harvested on September 26, 2024, and with a development duration of 255 days.

[0045] (2) Pod disinfection: First, wipe the surface of the pods twice with cotton balls soaked in 75% (v / v) alcohol in a laminar flow hood, then soak them in 75% alcohol solution for 30 s, shaking the glass bottle constantly during this period. After rinsing once with sterilized deionized water, soak them in 0.1% (w / v) mercuric chloride solution and shake constantly for 20 min, and then wash and dry them with deionized water.

[0046] (3) Seed preservation: Use a scalpel sterilized at high temperature to cut open the dried pods in a laminar flow hood, shake the seeds into a sterile centrifuge tube, seal the lid with sealing film, and store them in a 4°C refrigerator for future use after noting the seed information.

[0047] 2. Aseptic Sowing

[0048] (1) Preparation of the culture medium: The culture medium used was modified in terms of its components and proportions based on the H26 medium;

[0049] The components of H26 are 0.1 g / L inositol + 0.5 g / L activated carbon + 1.5 g / L Hyponex No. 1 + 2 g / L peptone + 5 g / L agar + 20 g / L sucrose + 0.5 mg / L naphthaleneacetic acid + 1 ml / L vitamin B + 10 ml / L iron salt + 50 ml / L coconut milk, with water as the solvent. The preparation method is to mix all the components evenly;

[0050] The components of HTre are 0.1 g / L inositol + 0.5 g / L activated carbon + 1.5 g / L Hyponex No. 1 + 2 g / L peptone + 5 g / L agar + 20 g / L trehalose + 0.5 mg / L naphthaleneacetic acid + 1 ml / L vitamin B + 10 ml / L iron salt + 50 ml / L coconut milk, with water as the solvent. The preparation method is to mix all the components evenly;

[0051] The components of H50 are 0.1 g / L inositol + 0.5 g / L activated carbon + 1.5 g / L Hyponex No. 1 + 2 g / L peptone + 5 g / L agar + 10 g / L sucrose + 10 g / L trehalose + 0.5 mg / L naphthaleneacetic acid + 1 ml / L vitamin B + 10 ml / L iron salt + 50 ml / L coconut milk, with water as the solvent. The preparation method is to mix all the components evenly;

[0052] After the culture medium was autoclaved at 121 °C for 20 min, it was poured into a sterile Petri dish with a diameter of 9 cm in a laminar flow hood. After the culture medium cooled and solidified, a sterile double-ring qualitative filter paper with a diameter of 7 cm was laid on it.

[0053] (4) Sowing: The seeds of Paphiopedilum spicerianum were placed in sterile water for a period of time until they were evenly suspended to make a seed suspension. 1 ml of the suspension was aspirated with a pipette tip with the tip cut off and sown on the sterile filter paper. The sowing amount was about 300 - 350 seeds per dish, and 10 dishes were sown for each culture medium. After being naturally dried under the laminar flow hood, the plates were sealed with a sealing film.

[0054] (5) Culture conditions: The plates were placed in an environment with a constant temperature of 26 °C. Half of them were cultured under the conditions of a light intensity of 2000 lux and a light time of 12 hours per day, and the other half were wrapped with aluminum foil and cultured in the dark.

[0055] 5. The seed germination was observed with a stereomicroscope at 45 days and 90 days after sowing respectively. In this study, the seed germination was simplified into 3 stages, namely seed germination (the embryo swelled and produced root-like structures), protocorm formation and development (the embryo swelled and broke through the seed coat until the appearance of the primary meristem), and seedling differentiation and development stage (the first leaf grew and subsequent growth). According to the above grading standards, the number of germinated seeds (s), the number of protocorms (p) and the number of seedlings (sl) of different treatments in each group of experiments were counted, and the germination rate, protocorm formation rate and seedling formation rate of each group of treatments were calculated based on the total number of sown seeds (t). The average value of each group of data was taken. The non-parametric test methods Kruskal-Wallis test (KW) and Mann-Whitney U–test (U) were used to conduct a significance test on the germination rate, protocorm formation ratio and seedling rate of different treatments, with α = 0.05.

[0056] 6. Experimental results

[0057] The growth results of the seeds of Paphiopedilum spicerianum are as Figure 1 、 2 shown. Among them, 1 represents the growth of the seeds 45 days after sowing; 2 represents the growth of the seeds 90 days after sowing; The statistical results of the germination and growth of the seeds of Paphiopedilum spicerianum 45 days after sowing are shown in Table 2. Table 2

[0058]

[0059]

[0060] The statistical results of the germination and growth of the seeds of Paphiopedilum spicerianum 90 days after sowing are shown in Table 3.

[0061] Table 3

[0062]

[0063] Table 2 and Table 3 show the data of the germination rate, protocorm rate and (seedling formation rate at 90 days) of Paphiopedilum seeds cultured in three kinds of culture media (H26, H50, HTre) under two culture conditions (normal, light-shading) at 45 days and 90 days of culture respectively; it can be seen from Table 2 that at 45 days of culture, light-shading treatment significantly promotes germination and protocorm formation, especially in the H50 culture medium with the most obvious effect. It can be seen from Table 3 that after 90 days, light-shading treatment still significantly improves the germination rate, protocorm rate and seedling formation rate, and H50 is the most prominent in all indicators, with a seedling formation rate of 33.41%, which is the highest among all. Generally speaking, the H50 culture medium has the best comprehensive performance under light-shading conditions and is especially suitable for improving protocorm formation and seedling formation rate; light-shading is the key condition to significantly promote germination and tissue differentiation, and it has an effect in all culture media, which may be related to mechanisms such as light inhibiting hormones or affecting the expression of hydrolases.

Claims

1. A method for promoting the axenic germination and seedling formation of Paphiopedilum wardianum seeds, characterized in that, Select the fruit pods of Paphiopedilum wardianum as explants. After disinfecting the fruit pods, cut them open to obtain sterile seeds of Paphiopedilum wardianum. Mix the sterilized seeds of Paphiopedilum wardianum with sterile water to make a seed suspension. Take the seed suspension and sow it on the culture medium for dark culture.

2. The method according to claim 1, characterized in that, The culture medium is: 0.1 g / L inositol, 0.5 g / L activated carbon, 1.5 g / L Hyponex No. 1, 2 g / L peptone, 10 g / L agar, 10 g / L sucrose, 10 g / L trehalose, 0.5 mg / L naphthaleneacetic acid, 1 ml / L vitamin B, 10 ml / L iron salt, 50 ml / L coconut milk, and the solvent is water.

3. The method according to claim 2, wherein The iron salt is 37.25 mg / L Na2-EDTA + 27.85 mg / L FeSO4·7H2O, and the solvent is water.

4. The method according to claim 1, 2 or 3, characterized in that For the dark culture, the culture temperature is 25 ± 2 °C.

5. The method according to claim 1, 2 or 3, characterized in that The fruit pods are the fruit pods developed by artificial pollination for about 250 days.

6. The method according to claim 1, 2 or 3, characterized in that, The disinfection method is to wipe the fruit pods of Paphiopedilum wardianum clean, disinfect them with 70% (v / v) alcohol for 60 s, wash them with sterile water for 30 s, disinfect them with 0.1% (w / v) HgCl2 solution for 7 min, wash them with sterile water 3 times, 30 s each time, air dry, cut open the fruit pods to obtain disinfected sterile seeds of Paphiopedilum wardianum.

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