Tissue culture method of wild Tibetan cymbidium hookerianum

Abstract

The invention provides a wild Tibet cymbidium tissue culture method which comprises the following steps: picking wild Tibet cymbidium capsules, cleaning with clear water, wiping with an ethanol solution, soaking with the ethanol solution, disinfecting with mercury bichloride, and soaking and washing with sterile water to obtain disinfected capsules; preparing capsule slices, placing the capsule slices above a sowing culture medium, knocking the edges of the capsule slices, dropwise adding sterile water, and shaking until seeds are dispersed on the surface of the culture medium and germinate and grow into protocorms; inoculating into a proliferation culture medium until rhizomes grow out, and carrying out differentiation culture. According to the method disclosed by the invention, the germination period of the wild cymbidium tigrinum seeds is shortened to less than 5 months, the germination rate is high, the rhizomes are thick, strong and full, the differentiation capability is strong, the speed is high, and the quality is high.

Description

Technical Field

[0001] This invention belongs to the field of tissue culture technology of Tibetan tiger orchid, specifically relating to a tissue culture method for wild Tibetan tiger orchid. Background Technology

[0002] *Cymbidium tracyanum* L. Castler, commonly known as Tibetan Tiger Orchid, is an epiphytic plant belonging to the genus *Cymbidium* in the family Orchidaceae. Its pseudobulbs are elliptic-ovate or oblong-narrowly ovate, 5-11 cm long and 2-5 cm wide, mostly enclosed within the leaf sheaths. *Cymbidium tracyanum* has a relatively small distribution in China, found only in southwestern Guizhou, Yunnan, northwestern Guangxi, and southeastern Tibet, and its population is quite small. The flowers are large and brightly colored, reaching 10-12 cm in diameter, making them highly ornamental. Furthermore, the seeds and pseudobulbs of *Cymbidium tracyanum* are used medicinally to treat tuberculosis, pneumonia, tracheitis, bronchitis, cough, and fractures, demonstrating significant medicinal value. In addition, *Cymbidium tracyanum* can serve as a germplasm resource for cultivating new orchid varieties and lines, possessing important scientific research value. Currently, most Tibetan tiger orchids are still propagated using the traditional division method, which results in an extremely low propagation coefficient and limits the number of plants that can be cultivated. Conventional aseptic sowing methods also present problems such as difficulty in germination, low germination rate, and long germination cycle (6-8 months), which seriously hinder the development and utilization of Tibetan tiger orchids. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a tissue culture method for wild Tibetan tiger orchid in response to the shortcomings of the prior art. This method results in a short germination time and a large germination volume of wild Tibetan tiger orchid seeds, a short protocorm proliferation time, and a germination rate of up to 90%. The germinated rhizomes are thick and full with strong differentiation ability, which significantly improves the quality and survival rate of seedlings and effectively reduces the production cost of Tibetan tiger orchid tissue culture.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a tissue culture method for wild Tibetan tiger orchid, the method being as follows: S1. Select healthy wild Tibetan tiger head orchid capsules that are 7-8 months after pollination and have a slightly yellow surface. After picking the capsules, wash the surface of the capsules with clean water to remove impurities, and then wipe the surface of the capsules with a 75% ethanol solution to obtain the wiped capsules. S2. Place the wiped capsules obtained in S1 in a clean bench, soak them in a 75% ethanol solution for 30 seconds, then surface disinfect them with 0.1% mercuric chloride for 15 minutes, and rinse them three times with sterile water for 2 minutes each time. After rinsing, use sterile filter paper to absorb the water to obtain the disinfected capsules. S3. Use a sterilized blade to cut off both ends of the sterilized capsule obtained in S2, then cut the capsule horizontally and slice it into thin slices with a spacing of 1cm to obtain a circular capsule slice with a thickness of 1cm. S4. Place the capsule slices obtained in S3 on top of the sowing medium. Use sterile tweezers to gently tap the edge of the capsule slices to make the seeds fall onto the surface of the sowing medium. Then add sterile water to the sowing medium and gently shake it to spread the seeds evenly on the surface of the sowing medium. Place the tissue culture bottle on the tissue culture rack for cultivation until the seeds germinate and grow into protocorms. The seeding medium was prepared by adding the following raw materials to MS medium at a final concentration, adjusting the pH to 5.6 with hydrochloric acid, and then sterilizing at 120°C for 20 minutes: 6-BA 0.6 mg / L, agar 7 g / L, sucrose 30 g / L, and activated carbon 0.2 g / L. S5. The protocorms obtained in S4 are inoculated into a proliferation and differentiation medium for proliferation culture until the protocorms differentiate into buds, thus obtaining budded protocorms; The proliferation and differentiation medium was prepared by adding the following raw materials to 1 / 2 MS medium at the final concentration, adjusting the pH to 5.4 with hydrochloric acid, and then sterilizing at 120°C for 20 min: NAA 0.2 mg / L, 6-BA 0.4 mg / L, 7 g / L agar, and 30 g / L sucrose. S6. The budding protocorms obtained in S5 were rooted on a rooting medium to obtain wild Tibetan tiger-head orchid seedlings. The rooting medium was prepared by adding the following raw materials to 1 / 2 MS medium at a final concentration, adjusting the pH to 5.4 with hydrochloric acid, and then sterilizing at 120°C for 20 minutes: NAA 2.0 mg / L, 6-BA 1.0 mg / L, 7 g / L agar, 30 g / L sucrose, and 1.0 g / L activated carbon.

[0005] Preferably, the conditions for cultivation in S4, proliferation cultivation in S5, and rooting cultivation in S6 are as follows: temperature 23℃±2℃, alternating light and dark treatment daily, light intensity 1800 Lux, and 10 hours of light per day.

[0006] Compared with the prior art, the present invention has the following advantages: 1. This invention can shorten the differentiation cycle of wild Tibetan tiger orchid from the conventional 6-8 months to 5 months. In addition, the seed germination rate of wild Tibetan tiger orchid in this invention is higher and the browning rate is lower than that of conventional seed germination media. The seed germination time is 60 days, which is shorter than that of conventional media.

[0007] 2. In this invention, the time from the sprouting of the protocorm to the growth of rhizomes in the proliferation medium is 60 days, which is shorter than the time for sprouting and rhizome growth in conventional medium.

[0008] 3. The seeds of Tibetan Tiger Orchid have a short germination time and a large germination rate. The protocorm proliferation time is short and the germination rate reaches 90%. The germinated rhizomes are thick and full with strong differentiation ability, which significantly improves the quality and survival rate of seedlings and effectively reduces the production cost of Tibetan Tiger Orchid tissue culture.

[0009] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0010] Figure 1 This is a comparison diagram of the sowing culture medium of the present invention and the conventional culture medium after two months of sowing in Example 1 of the present invention.

[0011] Figure 2 This is a comparison diagram of the sowing culture medium of the present invention and the conventional culture medium after 5 months of sowing in Example 1 of the present invention. Detailed Implementation

[0012] Example 1

[0013] The tissue culture method for wild Tibetan tiger orchids in this embodiment is as follows: S1. Select healthy wild Tibetan tiger head orchid capsules that are 7 to 8 months after pollination (8 months after pollination in this example) and have a slightly yellow capsule surface. After picking the capsules, wash the capsule surface with clean water to remove impurities, and then wipe the capsule surface with a 75% ethanol solution to obtain the wiped capsules. In this embodiment, the wild Tibetan Tiger Head Orchid is the native species of Tibetan Tiger Head Orchid; S2. Place the wiped capsules obtained in S1 in a clean bench, soak them in a 75% ethanol solution for 30 seconds, then surface disinfect them with 0.1% mercuric chloride for 15 minutes, and rinse them three times with sterile water for 2 minutes each time. After rinsing, use sterile filter paper to absorb the water to obtain the disinfected capsules. S3. Use a sterilized blade to cut off both ends of the sterilized capsule obtained in S2, then cut the capsule horizontally and slice it into thin slices with a spacing of 1cm to obtain a circular capsule slice with a thickness of 1cm. S4. Place the capsule slices obtained in S3 on top of the sowing medium. Gently tap the edge of the capsule slices with sterilized tweezers to allow the seeds to fall onto the surface of the sowing medium. Then, add sterile water to the sowing medium and gently shake to evenly spread the seeds on the surface. Place the tissue culture bottle on a tissue culture rack and culture for 60 days. The seeds will germinate and grow into green protocorms, which will cluster together tightly. In this embodiment, the seed germination rate can reach 90%. The seeding medium described in this example was prepared by adding the following raw materials to MS medium at the final concentration, adjusting the pH to 5.6 with hydrochloric acid, and then sterilizing at 120°C for 20 minutes: 6-BA 0.6 mg / L, agar 7 g / L, sucrose 30 g / L, and activated carbon 0.2 g / L. In this example, the MS medium was commercially available from Beijing Solarbio Science & Technology Co., Ltd. S5. After inoculating the protocorms obtained in S4 into the proliferation and differentiation medium for 25 days, the protocorms differentiated into buds and produced white villous rhizoids. New protocorms were also produced, and the protocorms clustered together to obtain budding protocorms. The proliferation and differentiation medium was prepared by adding the following raw materials to 1 / 2 MS medium at the final concentration, adjusting the pH to 5.4 with hydrochloric acid, and then sterilizing at 120°C for 20 min: NAA 0.2 mg / L, 6-BA 0.4 mg / L, 7 g / L agar, and 30 g / L sucrose. In this embodiment, the 1 / 2MS culture medium was commercially available from Beijing Solarbio Science & Technology Co., Ltd. S6. After the budding protocorms obtained in S5 were rooted in the rooting medium for 30 days, green rhizomes grew and the root system was well developed. Most of the seedlings were over 2cm tall, and wild Tibetan tiger head orchid seedlings were obtained.

[0014] The rooting medium was prepared by adding the following raw materials to 1 / 2 MS medium at a final concentration, adjusting the pH to 5.4 with hydrochloric acid, and then sterilizing at 120°C for 20 minutes: NAA 2.0 mg / L, 6-BA 1.0 mg / L, 7 g / L agar, 30 g / L sucrose, and 1.0 g / L activated carbon.

[0015] The conditions for cultivation in S4, proliferation culture in S5, and rooting culture in S6 were as follows: temperature 23℃±2℃, alternating light and dark treatment daily, light intensity 1800 Lux, and 10 hours of light per day.

[0016] Figure 1 The image shows a comparison between the sowing medium and the conventional medium in this embodiment, taken 2 months after sowing (i.e., 60 days of cultivation using the sowing medium). The left side shows the conventional medium, where no germination was observed; the right side shows the sowing medium used in step S4 of this embodiment, where germination has occurred.

[0017] Figure 1The formula for the conventional culture medium on the left side of the middle section is: MS + 0.2 mg / L NAA + 0.4 mg / L 6-BA + 7 g / L agar + 30 g / L sucrose, adjusted to pH 5.6. That is, add the following final concentrations of raw materials to the MS medium and adjust the pH to 5.6: NAA 0.2 mg / L, 6-BA 0.4 mg / L, agar 7 g / L, sucrose 30 g / L.

[0018] Figure 2 The image shows a comparison between the sowing medium and the conventional medium in this embodiment after 4 months of sowing in the medium of step S4. The right side shows the rhizomes turning green and differentiating leaf buds after 4 months of culture in the sowing medium of this embodiment. The left side shows the conventional medium, where the rhizomes have grown into protocorms, with a small number of small leaf buds growing out, but not obviously, and no rhizomes have sprouted.

[0019] Figure 2 The formula for the conventional sowing medium on the right side of the middle section is: MS + NAA 0.2 mg / L + 6-BA 0.4 ml / L + 7 g / L agar + 30 g / L sucrose, with the pH adjusted to 5.4.

[0020] (i) In the sowing experiment in step S4 of this embodiment, a screening experiment of the sowing culture medium was also conducted. The results are shown in Table 1.

[0021] A total of five sowing culture media were used in the experiment, namely: Culture medium No. 1 (i.e., the sowing culture medium in this example) is: MS + 0.6 mg / L 6-BA + 7 g / L agar + 30 g / L sucrose + 0.2 g / L activated charcoal; Culture medium No. 2 is: MS + 0.8 mg / L 6-BA + 7 g / L agar + 30 g / L sucrose + 0.2 g / L activated carbon; Culture medium No. 3 is: MS + 0.2 mg / L NAA + 0.6 mg / L 6-BA + 7 g / L agar + 30 g / L sucrose + 0.2 g / L activated charcoal; Culture medium No. 4 is: MS + 0.2 mg / L NAA + 0.8 mg / L 6-BA + 7 g / L agar + 30 g / L sucrose + 0.2 g / L activated charcoal; Culture medium No. 5 is: MS + NAA 0.2 mg / L + 6-BA 0.4 ml / L + 7 g / L agar + 30 g / L sucrose.

[0022] The pH of all culture media, from No. 1 to No. 5, was adjusted to 5.6.

[0023] Table 1 Results of the Tibetan Tiger Orchid Sowing Experiment Table 1 shows that the germination time of *Cymbidium goeringii* in the sowing medium of this invention is 60 days (until the original bulb grows), and the germination rate is 90%. Compared with other sowing media, the sowing medium used in this embodiment has a higher germination rate and a shorter germination time.

[0024] (ii) In this embodiment, the proliferation experiment in step S5 also included a screening experiment for the proliferation culture medium. The results are shown in Table 2.

[0025] A total of five proliferation culture media were used in the experiment, namely: Culture medium a is: 1 / 3 MS + NAA 0.3 mg / L + 6-BA 0.5 mg / L + 7 g / L agar + 30 g / L sucrose, adjust the pH to 5.4; Culture medium b (i.e., the proliferation medium in this example) is: 1 / 2 MS + NAA 0.2 mg / L + 6-BA 0.4 mg / L + 7 g / L agar + 30 g / L sucrose, adjust pH to 5.4; Culture medium c is: MS solution: NAA 0.3 mg / L + 6-BA 0.5 mg / L + 7 g / L agar + 30 g / L sucrose; adjust pH to 5.4. Culture medium d is: 1 / 3 MS + NAA 0.3 mg / L + 6-BA 0.5 mg / L + 7 g / L agar + 30 g / L sucrose, adjust the pH to 5.4; Culture medium e is: 1 / 2 MS + NAA 0.3 mg / L + 6-BA 0.5 mg / L + 7 g / L agar + 30 g / L sucrose, adjust the pH to 5.4; The MS and 1 / 2MS culture media consisted of the same composition as in this example. The 1 / 3MS was commercially available from Beijing Solarbio Science & Technology Co., Ltd.

[0026] Table 2 Results of the Proliferation and Differentiation Experiment of *Cymbidium goeringii* in Tibet As shown in Table 2, the Tibetan Tiger Orchid in this embodiment has the shortest budding time in the proliferation medium. From the original bulb to the top leaf bud, the budding time is 25 days, the budding rate is 90%, and the average bud height has reached 1.4 cm. It can be seen that the Tibetan Tiger Orchid cultivated in the proliferation medium of this invention has a high germination rate, and the plants are robust and of high quality.

[0027] (iii) In the rooting experiment in step S6 of this example, the rooting culture medium was also screened. The results are shown in Table 3.

[0028] Three rooting media were used in the experiment: Culture medium a (i.e., the rooting medium in this example) is: 1 / 2 MS + NAA 2.0 mg / L + 6-BA 1.0 mg / L + 7 g / L agar + 30 g / L sucrose + 1.0 g / L activated charcoal, adjust the pH to 5.4; Culture medium b is: 1 / 2 MS + NAA 1.5 mg / L + 6-BA 1.0 mg / L + 7 g / L agar + 30 g / L sucrose, adjust the pH to 5.4; Culture medium c is: 1 / 2 MS + NAA 1.0 mg / L + 6-BA 1.0 mg / L + 7 g / L agar + 30 g / L sucrose, adjust the pH to 5.4; Table 3 Results of the rooting experiment of *Cymbidium goeringii* in Tibet As shown in Table 3, the rooting medium in this example has the best effect. The Tibetan Tiger Orchid seedlings have an average of 4 roots per plant after 30 days, and the average root length has exceeded 3cm. It can be seen that the Tibetan Tiger Orchid cultured in the rooting medium of this invention has a well-developed and robust root system.

[0029] The aseptic tissue culture method for wild Tibetan Tiger Orchid in this embodiment greatly improves the problems of difficult aseptic sowing and germination of Tibetan Tiger Orchid, low germination rate and long germination cycle. It can also improve the quality of germinated protocorms of Tibetan Tiger Orchid, shortening the period from sowing seeds to rhizomes exceeding 3cm in length from 6-8 months in conventional sowing to less than 5 months. Furthermore, the germinated rhizomes are thick and full, with strong differentiation ability, fast growth rate and high quality.

[0030] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for tissue culture of wild Tibetan tiger orchid, characterized in that, The method is as follows: S1. Select healthy wild Tibetan tiger-head orchid capsules that are 7-8 months after pollination, wash them with clean water, and then wipe the surface of the capsules with a 75% ethanol solution to obtain the wiped capsules. S2. Soak the wiped capsules obtained in S1 in a 75% ethanol solution for 30 seconds, then disinfect them with 0.1% mercuric chloride for 15 minutes, and rinse them three times with sterile water for 2 minutes each time. After rinsing, dry them with sterile filter paper to obtain the disinfected capsules. S3. Cut off both ends of the sterilized capsule obtained in S2, then cut the capsule horizontally to prepare a capsule slice with a thickness of 1cm. S4. Place the capsule slices obtained in S3 above the sowing medium, tap the edge of the capsule slices to make the seeds fall onto the surface of the sowing medium, then add sterile water to the sowing medium and shake to disperse the seeds on the surface of the sowing medium, and culture until the seeds germinate and grow into protocorms. The seeding medium was prepared by adding the following raw materials to MS medium at a final concentration, adjusting the pH to 5.6 with hydrochloric acid, and then sterilizing at 120°C for 20 minutes: 6-BA 0.6 mg / L, agar 7 g / L, sucrose 30 g / L, and activated carbon 0.2 g / L. S5. The protocorms obtained in S4 are inoculated into a proliferation and differentiation medium for proliferation culture until the protocorms differentiate into buds, thus obtaining budded protocorms; The proliferation and differentiation medium was prepared by adding the following raw materials to 1 / 2 MS medium at the final concentration, adjusting the pH to 5.4 with hydrochloric acid, and then sterilizing at 120°C for 20 min: NAA 0.2 mg / L, 6-BA 0.4 mg / L, 7 g / L agar, and 30 g / L sucrose. S6. The budding protocorms obtained in S5 were rooted on a rooting medium to obtain wild Tibetan tiger-head orchid seedlings. The rooting medium was prepared by adding the following raw materials to 1 / 2 MS medium at a final concentration, adjusting the pH to 5.4 with hydrochloric acid, and then sterilizing at 120°C for 20 minutes: NAA 2.0 mg / L, 6-BA 1.0 mg / L, 7 g / L agar, 30 g / L sucrose, and 1.0 g / L activated carbon.

2. The method for tissue culture of wild Tibetan tiger orchid according to claim 1, characterized in that, The conditions for culture in S4, proliferation culture in S5, and rooting culture in S6 were as follows: temperature 23℃±2℃, alternating light and dark treatment daily, light intensity 1800 Lux, and 10 hours of light per day.

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