Long-term in vitro preservation method of endangered dendrobium germplasm

Abstract

The application discloses a long-term in-vitro preservation method for endangered Dendrobium germplasm, which comprises the following two steps: a first stage of inhibiting growth medium for long-term preservation of sterile Dendrobium seeds, and a second stage of recovery growth medium for seedling induction culture; the inhibiting growth medium formula is oat 40-80 g / L+ agar 6 g / L; and the recovery growth medium formula is 1 / 2MS+ peptone 1.0 g / L+ activated carbon 1.0 g / L+ sucrose 25 g / L+ agar 6.0 g / L+ potato 50 g / L+ banana 80 g / L+ apple 15 g / L. The method can realize long-term preservation and utilization of endangered Dendrobium germplasm resources by using organic additives, preparing suitable culture medium, adjusting the pH value and the like, and has the advantages of high preservation rate, long preservation time, low cost, good germplasm traits and the like.

Description

Technical Field

[0001] This invention relates to the field of plant in vitro preservation technology, specifically to a method for long-term in vitro preservation of endangered Dendrobium germplasm. Background Technology

[0002] Dendrobium is a perennial herbaceous plant belonging to the genus Dendrobium in the family Orchidaceae. It possesses both medicinal and ornamental value, and 81 species of Dendrobium have been officially published in my country. However, due to its own inherent characteristics, natural factors, and human activities, many native Dendrobium species are on the verge of extinction. Since 1987, Dendrobium has been listed as a Class II protected plant in both the "List of Rare and Endangered Plants of the People's Republic of China" and the "Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES)". In September 2021, the National Forestry and Grassland Administration and the Ministry of Agriculture and Rural Affairs jointly issued a document announcing the "National Key Protected Wild Plants List," listing all Dendrobium species as national key protected wild plants. Among them, Dendrobium huoshanense and Dendrobium nobile are listed as Class I protected wild plants, while the other species are Class II protected wild plants. Protecting my country's Dendrobium resources has become an urgent task.

[0003] In vitro preservation of germplasm is an effective means of protecting and utilizing Dendrobium species. It not only effectively extends the preservation period, reduces subculture cycles and genetic variation, and saves costs such as land, labor, and financial resources, but also avoids the impact of various pests, diseases, and natural disasters. It is one of the important research methods for germplasm collection, breeding, and industrial production. Existing in vitro preservation methods for crops, fruit trees, and forest trees include lowering the culture temperature, adjusting the nutrient levels of the culture medium, applying osmotic compounds to increase the osmotic pressure of the culture medium, or adding growth retardants to the culture medium to delay the growth of test-tube seedlings, extend the subculture interval, and reduce the number of subcultures.

[0004] Currently, commonly used techniques for the in vitro preservation of Dendrobium include cryopreservation, inhibitor preservation, and hormone preservation. Cryopreservation requires the purchase of facilities such as low-temperature freezers or liquid nitrogen tanks, resulting in high energy consumption and maintenance costs. The use of high-osmotic regulators and growth inhibitors leads to high mortality and mutation rates in preserved Dendrobium germplasm. Even with low salt or hormone levels, Dendrobium can still proliferate, differentiate, and root normally, resulting in a high transfer frequency. These issues severely limit the long-term preservation and effective utilization of endangered Dendrobium germplasm resources. Summary of the Invention

[0005] This invention addresses the problems of existing in vitro preservation technologies, which rely on ultra-low temperature, cryogenic freezers, or liquid nitrogen tanks, resulting in high costs; frequent transfers and short subculture cycles; high labor costs; and significant damage to Dendrobium germplasm caused by growth inhibitors and high-permeability agents, leading to high mortality and mutation rates. It provides an effective long-term in vitro preservation method for endangered Dendrobium germplasm. This method utilizes organic additives, prepares suitable culture media, and adjusts pH levels to extend preservation time, reduce transfer frequency, lower preservation costs, maintain superior germplasm traits, and achieve long-term preservation and utilization of Dendrobium germplasm.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for long-term in vitro preservation of endangered Dendrobium germplasm, the method comprising the following two steps: a first stage is a growth inhibition stage for long-term preservation of sterile Dendrobium seeds using a growth inhibition medium, and a second stage is a growth restoration stage for seedling induction culture using a growth restoration medium.

[0008] The growth-inhibiting medium formulation is: 40-80 g / L oats + 6 g / L agar, with a pH of 6.1-6.3 after cooling.

[0009] The growth recovery medium formula is: 1 / 2 MS + peptone 1.0 g / L + activated carbon 1.0 g / L + sucrose 25 g / L + agar 6.0 g / L + potato 50 g / L + banana 80 g / L + apple 15 g / L. The pH value of the medium after cooling is 5.6-5.8.

[0010] As a preferred technical solution of the present invention, the preparation method of the growth inhibition culture medium is as follows: 40-80g of peeled oat grains are added to 1.2-1.3L of sterile water, boiled in a food processor for 20 minutes, blended into a paste, filtered through two layers of medical gauze to remove residue; 6g of agar is added to the oat juice, and sterile water is used to make up to 1L, and stirred well; after boiling, the pH value is adjusted to 6.3-6.5, and autoclaved.

[0011] As a preferred technical solution of the present invention, the preparation method of the recovery growth culture medium is as follows: 50g of potatoes are cooked, and 80g of ripe bananas and 15g of fresh apples are put into a food processor and stirred at room temperature for 2-4 minutes to form a paste; the potato-banana-apple paste is added to 1 / 2MS + peptone 1.0g / L + activated carbon 1.0g / L + sucrose 25g / L + agar 6.0g / L culture medium, and the volume is adjusted to 1L with sterile water; the pH value is adjusted to 5.8-6.1, and the medium is autoclaved.

[0012] As a preferred technical solution of the present invention, the culture conditions for the growth inhibition stage and the growth recovery stage are: temperature 23±2℃, light intensity 2000-3000lx, and light duration 12h / d.

[0013] As a preferred technical solution of the present invention, the high-pressure sterilization is performed at a temperature of 121°C and a pressure of 103 kPa for 20-30 minutes.

[0014] As a preferred technical solution of the present invention: the pH of the growth inhibition medium is adjusted to 6.3-6.5 using 0.1 mol / L HCl.

[0015] As a preferred technical solution of the present invention: the pH value of the recovery growth medium is adjusted to 5.8-6.1 using 0.5 mol / L NaOH.

[0016] Compared with the prior art, the advantages and beneficial effects of the present invention include:

[0017] The in vitro preservation method for endangered Dendrobium germplasm resources provided by this invention utilizes organic additives, prepares suitable culture media, and adjusts pH values ​​to achieve high germplasm germination rate, no differentiation, low mutation rate, and long transfer cycle during growth inhibition. Conversely, during growth recovery, the differentiation rate is high, recovery time is short, and the germplasm is robust. This method offers advantages such as high preservation rate, long preservation time, low cost, and maintenance of excellent germplasm traits, making it an effective method for the long-term preservation and utilization of endangered Dendrobium germplasm resources.

[0018] The present invention addresses the technical problems of existing in vitro preservation methods, which rely on facilities such as ultra-low temperature, cryogenic freezers, or liquid nitrogen tanks, resulting in high preservation costs; frequent transfers and short subculture cycles, leading to high labor costs; and the significant damage to Dendrobium germplasm caused by growth inhibitors and high-permeability agents, resulting in high mortality and mutation rates.

[0019] The method of this invention preserves endangered Dendrobium germplasm in vitro. During the growth inhibition period, the germination time is 10-12 days, the germination rate is 90-95%, the protocorm variation rate and browning rate are 0, the pseudo-protocorm color is yellowish-green, the transfer cycle is over 540 days, and the germplasm grows normally without differentiation. During the recovery period, the plant height is 2.2-3.2 cm, the stem diameter is 0.15-0.23 cm, the number of roots is 2-3, the differentiation rate is 100%, the recovery period is 18-20 days, and the survival rate is 100%. The plants grow vigorously, can fully differentiate, have well-developed root systems, recover growth in a short time, and there is no mortality. Compared with existing technologies, this method has the advantages of low transfer frequency, long preservation time, no need for experimental facilities, low cost, and preservation of excellent germplasm traits, providing an effective method for the long-term preservation and utilization of endangered Dendrobium. Attached Figure Description

[0020] Figure 1 Example 1P1: The endangered Dendrobium germplasm began to differentiate normally 45 days after restoration.

[0021] Figure 2 In Example 1P1, the endangered Dendrobium germplasm was preserved in vitro for 540 days with growth inhibition, and the germplasm was normal and showed no differentiation.

[0022] Figure 3 To compare the CK1 endangered Dendrobium germplasm, which underwent 180 days of in vitro preservation, resulting in either whitening or yellowing.

[0023] Figure 4 As a comparative example, the endangered Dendrobium officinale CK2, after being preserved in vitro for 180 days, showed less germplasm differentiation, more original bulbs, and variations in germplasm. Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. Example

[0025] A method for long-term in vitro preservation of endangered Dendrobium germplasm, comprising the following steps:

[0026] Preparation of inhibition medium: The inhibition medium formula is 40 g / L oats + 6 g / L agar (denoted as P1). The method for preparing the inhibition medium is as follows: add 40 g of hulled oat grains to 1.2 L of sterile water, boil in a food processor for 20 minutes, blend into a paste, filter through two layers of medical gauze to remove residue; add 6 g of agar to the oat juice, and make up to 1 L with sterile water, stir well; after boiling, adjust the pH to 6.3-6.5 with 0.1 mol / L HCl, autoclave (temperature 121℃, pressure 103 kPa), and ensure the pH is 6.1-6.3 after cooling.

[0027] Preparation of recovery growth medium: The recovery growth medium formula is: 1 / 2 MS + 1.0 g / L peptone + 1.0 g / L activated charcoal + 25 g / L sucrose + 6.0 g / L agar + 50 g / L potato + 80 g / L banana + 15 g / L apple. The preparation method of the recovery production medium is as follows: Cook 50 g of potato until soft, and put it into a food processor with 80 g of ripe banana and 15 g of fresh apple. Blend at room temperature for 2-4 minutes until a paste is formed. Add the potato-banana-apple paste to the 1 / 2 MS + 1.0 g / L peptone + 1.0 g / L activated charcoal + 25 g / L sucrose + 6.0 g / L agar medium, and bring the volume to 1 L with sterile water. Adjust the pH to 5.8-6.1 using 0.5 mol / L NaOH, and autoclave (temperature: 121℃, pressure: 103 kPa).

[0028] Growth inhibition treatment: The capsules of *Dendrobium nobile* and *Dendrobium dwarfum* were sterilized with 0.2% HgCl2 for 10 min. The capsules were then cut open with a sterile knife, and the seeds were evenly scattered into the inhibition medium. The bottles were capped, sealed with a plastic wrap, and stored at 23±2℃, with a light intensity of 2000-3000 lx and a photoperiod of 12 h / d. Germination days, germination rate, differentiation rate, browning rate, pseudo-corm color, and transfer cycle were recorded.

[0029] (4) Recovery growth culture: When the transfer time is reached, the original tubers of Dendrobium are re-inoculated into the recovery growth culture medium. After 3 months of recovery growth under the conditions of temperature 23±2℃, light intensity 2000-3000lx and light time 12h / d, the plant height, stem diameter and root number of P1 are measured, and the variation rate, differentiation rate, recovery growth days and survival rate are statistically analyzed. Example

[0030] A method for long-term in vitro preservation of endangered Dendrobium germplasm, comprising the following steps:

[0031] Preparation of inhibition medium: The inhibition medium formula is 60g / L oats + 6g / L agar (denoted as P2). The method for preparing the inhibition medium is as follows: add 40g of hulled oat grains to 1.2L of sterile water, boil in a food processor for 20 minutes, blend into a paste, filter through two layers of medical gauze to remove residue; add 6g of agar to the oat juice, and make up to 1L with sterile water, stir well; after boiling, adjust the pH to 6.3-6.5 with 0.1mol / L HCl, autoclave (temperature 121℃, pressure 103kPa), and ensure the pH is 6.1-6.3 after cooling.

[0032] Preparation of recovery growth medium: The recovery growth medium formula is: 1 / 2 MS + 1.0 g / L peptone + 1.0 g / L activated charcoal + 25 g / L sucrose + 6.0 g / L agar + 50 g / L potato + 80 g / L banana + 15 g / L apple. The preparation method of the recovery production medium is as follows: Cook 50 g of potato until soft, and put it into a food processor with 80 g of ripe banana and 15 g of fresh apple. Blend at room temperature for 2-4 minutes until a paste is formed. Add the potato-banana-apple paste to the 1 / 2 MS + 1.0 g / L peptone + 1.0 g / L activated charcoal + 25 g / L sucrose + 6.0 g / L agar medium, and bring the volume to 1 L with sterile water. Adjust the pH to 5.8-6.1 using 0.5 mol / L NaOH, and autoclave (temperature: 121℃, pressure: 103 kPa).

[0033] Growth inhibition treatment: The capsules of *Dendrobium nobile* and *Dendrobium dwarfum* were sterilized with 0.2% HgCl2 for 10 min. The capsules were then cut open with a sterile knife, and the seeds were evenly scattered into the inhibition medium. The bottles were capped, sealed with a plastic wrap, and stored at 23±2℃, with a light intensity of 2000-3000 lx and a photoperiod of 12 h / d. Germination days, germination rate, differentiation rate, browning rate, pseudo-corm color, and transfer cycle were recorded.

[0034] (4) Recovery growth culture: When the transfer time is reached, the original tubers of Dendrobium are re-inoculated into the recovery growth culture medium. After 3 months of recovery growth under the conditions of temperature 23±2℃, light intensity 2000-3000lx and light time 12h / d, the plant height, stem diameter and root number of P2 are measured, and the variation rate, differentiation rate, recovery growth days and survival rate are statistically analyzed. Example

[0035] A method for long-term in vitro preservation of endangered Dendrobium germplasm, comprising the following steps:

[0036] Preparation of inhibition medium: The inhibition medium formula is 80g / L oats + 6g / L agar (denoted as P3). The method for preparing the inhibition medium is as follows: add 40g of hulled oat grains to 1.2L of sterile water, boil in a food processor for 20 minutes, blend into a paste, filter through two layers of medical gauze to remove residue; add 6g of agar to the oat juice, and make up to 1L with sterile water, stir well; after boiling, adjust the pH to 6.3-6.5 with 0.1mol / L HCl, autoclave (temperature 121℃, pressure 103kPa), and ensure the pH is 6.1-6.3 after cooling.

[0037] Preparation of recovery growth medium: The recovery growth medium formula is: 1 / 2 MS + 1.0 g / L peptone + 1.0 g / L activated charcoal + 25 g / L sucrose + 6.0 g / L agar + 50 g / L potato + 80 g / L banana + 15 g / L apple. The preparation method of the recovery production medium is as follows: Cook 50 g of potato until soft, and put it into a food processor with 80 g of ripe banana and 15 g of fresh apple. Blend at room temperature for 2-4 minutes until a paste is formed. Add the potato-banana-apple paste to the 1 / 2 MS + 1.0 g / L peptone + 1.0 g / L activated charcoal + 25 g / L sucrose + 6.0 g / L agar medium, and bring the volume to 1 L with sterile water. Adjust the pH to 5.8-6.1 using 0.5 mol / L NaOH, and autoclave (temperature: 121℃, pressure: 103 kPa).

[0038] Growth inhibition treatment: The capsules of *Dendrobium nobile* and *Dendrobium dwarfum* were sterilized with 0.2% HgCl2 for 10 min. The capsules were then cut open with a sterile knife, and the seeds were evenly scattered into the inhibition medium. The bottles were capped, sealed with a plastic wrap, and stored at 23±2℃, with a light intensity of 2000-3000 lx and a photoperiod of 12 h / d. Germination days, germination rate, differentiation rate, browning rate, pseudo-corm color, and transfer cycle were recorded.

[0039] (4) Recovery growth culture: When the transfer time is reached, the original tubers of Dendrobium are re-inoculated into the recovery growth culture medium. After 3 months of recovery growth under the conditions of temperature 23±2℃, light intensity 2000-3000lx and light time 12h / d, the plant height, stem diameter and root number of P3 are measured, and the variation rate, differentiation rate, recovery growth days and survival rate are statistically analyzed.

[0040] The difference between this comparative example and Example 1 is that the inhibition medium used is 1 / 2 MS + 6.0 g / L agar (denoted as CK1).

[0041] The difference between this comparative example and Example 1 is that the inhibition medium used is 1 / 2 MS + 6.0 g / L agar + 0.5 mg / L butyryl hydrazine (denoted as CK2).

[0042] The difference between this comparative example and Example 1 is that the inhibition culture medium used is 40 g / L potato + 6 g / L agar (denoted as CK3).

[0043] The difference between this comparative example and Example 1 is that the inhibition culture medium used is formulated as follows: 40 g / L oats + 6 g / L agar + 20 g / L sucrose (denoted as CK4).

[0044] The difference between this comparative example and Example 1 is that the inhibition culture medium used is 40g / L oats + 6g / L agar (denoted as CK5), with a pH value of 5.6-5.8.

[0045] The growth inhibition and recovery of endangered Dendrobium orchids under different in vitro preservation methods in Examples 1-3 and Comparative Examples 1-5 are shown in Tables 1 and 2 and appendices, respectively. Figure 1-4 .

[0046] Table 1: Comparison of growth inhibition of endangered Dendrobium officinale by different in vitro preservation methods

[0047]

[0048] Table 1 shows that in Examples P1-P3, the germination time was 10-12 days, the germination rate was 90-95%, the protocorm variation rate and browning rate were 0, the pseudo-protocorm color was yellowish-green, the transfer period was over 540 days, the germplasm growth was normal, and there was no differentiation. In Comparative Example 1, the germination time was 7-10 days, the germination rate was 93-95%, the protocorm variation rate and browning rate were 0, the pseudo-protocorm color was green, the transfer period was 75 days, most of the protocorms differentiated, and those not transferred on time died due to whitening. In Comparative Example 2, the germination time was 28-30 days, the germination rate was 24-29%, the protocorm variation rate was 53-64%, the browning rate was 4-6%, the pseudo-protocorm color was yellow, the transfer period was 180 days, the inhibitor was highly toxic to the germplasm, a small portion differentiated, the germination and differentiation rates were low, there were many mutated protocorms, and the survival rate was low. Comparative Example 3: Germination time was 14-15 days, germination rate was low (40-60%), protocorm variation rate was 10-12%, pseudo-protocorm color was light green, and the transfer period was 360 days; otherwise, the protocorm was prone to vitrification. Comparative Example 4: Germination time was 10-12 days, germination rate was low (91-93%), protocorm differentiation rate was 30-35%, pseudo-protocorm color was light green, and the transfer period was 180 days; otherwise, the protocorm was prone to vitrification. Comparative Example 5: Germination time was 10-12 days, germination rate was low (90-92%), protocorm differentiation rate was 31-33%, pseudo-protocorm color was light green, and the transfer period was 180 days; otherwise, the protocorm gradually differentiated and turned yellow.

[0049] Table 2: Comparison of growth recovery of endangered Dendrobium under different in vitro preservation methods

[0050]

[0051] Table 2 shows that in Examples P1-P3, the plant height was 2.2-3.2 cm, the stem diameter was 0.15-0.23 cm, the number of roots was 2-3, the differentiation rate was 100%, the recovery time was 18-20 days, the plants were robust, fully differentiated, had well-developed root systems, and recovered quickly with no mortality. In Comparative Example CK1, the plant height was 2.8-3.5 cm, the stem diameter was 0.15-0.25 cm, the number of roots was 2-4, the differentiation rate was 100%, the recovery time was 7-10 days, the plants were robust, fully differentiated, had well-developed root systems, recovered quickly with the shortest time, and had a 100% survival rate. The control group CK2 had a plant height of 1.5-1.8 cm, stem diameter of 0.18-0.32 cm, 0-1 roots, a differentiation rate of 10%, a mortality rate of 85-88%, a variation rate of 2-5%, and a recovery time of 28-30 days. The plants were short and thick, with a low differentiation rate, few or no roots, a long recovery time, and a high mortality rate. The control group CK3 had a plant height of 2.1-2.6 cm, stem diameter of 0.14-0.20 cm, 2-3 roots, a differentiation rate of 100%, and a recovery time of 19-20 days. The plants were robust, fully differentiated, had well-developed root systems, a moderate recovery time, and no mortality. Comparative example CK4 had a plant height of 2.3-3.1 cm, stem diameter of 0.14-0.22 cm, 2-4 roots, a differentiation rate of 100%, and a recovery period of 19-20 days. The plants were robust, fully differentiated, had well-developed root systems, and recovered within a moderate timeframe with no mortality. Comparative example CK5 had a plant height of 2.3-3.0 cm, stem diameter of 0.13-0.24 cm, 2-4 roots, a differentiation rate of 100%, and recovered within 18-20 days. The plants were robust, fully differentiated, had well-developed root systems, and recovered within a moderate timeframe with no mortality.

[0052] In summary, the above comparative methods for inhibiting growth resulted in high germination rates or high mutation rates in endangered Dendrobium germplasm, with insignificant inhibition effects or significant toxicity to the germplasm. Most germplasm differentiated or had low differentiation rates, short transfer cycles, long recovery periods, and high mutation and mortality rates. However, the in vitro preservation method for endangered Dendrobium germplasm resources provided by this invention exhibits high germination rates, no differentiation, low mutation rates, and long transfer cycles during the growth inhibition period. Conversely, during the recovery period, high differentiation rates, short recovery times, and robust germplasm are observed. This method offers advantages such as high preservation rate, long preservation time, low cost, and maintenance of superior germplasm traits, making it an effective method for the long-term preservation and utilization of endangered Dendrobium germplasm resources.

[0053] The above description, in conjunction with specific / preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various substitutions or modifications can be made to these described embodiments without departing from the inventive concept, and all such substitutions or modifications should be considered within the scope of protection of the present invention.

Claims

1. A long-term in vitro preservation method of endangered Dendrobium germplasm, characterized by comprising the following steps: The ex situ preservation method comprises two steps: the first step is a growth inhibition stage for long-term preservation of sterile Dendrobium seeds using a growth inhibition medium, and the second step is a growth recovery stage for seedling induction culture using a growth recovery medium; the growth inhibition medium has a formula of oat 40-80 g / L+ agar 6 g / L, and the pH value of the medium after cooling is 6.1-6.3; ​ The growth recovery medium has a formula of 1 / 2MS+ peptone 1.0 g / L+ activated carbon 1.0 g / L+ sucrose 25 g / L+ agar 6.0 g / L+ potato 50 g / L+ banana 80 g / L+ apple 15 g / L, and the pH value of the medium after cooling is 5.6-5.8; The preparation method of the growth inhibition medium is as follows: 40-80 g of peeled oat kernels are added to 1.2-1.3 L of sterile water, boiled in a food processor for 20 min, beaten into a paste, filtered through two layers of medical gauze to remove residues, 6 g of agar is added to the oat juice, and the volume is adjusted to 1 L with sterile water, and stirred and mixed evenly; after boiling, the pH value is adjusted to 6.3-6.5, and high-pressure sterilization is performed; The preparation method of the growth recovery medium is as follows: 50 g of boiled potato is placed in a food processor together with 80 g of ripe banana and 15 g of fresh apple, and stirred at room temperature for 2-4 min to beat into a paste; the potato-banana-apple paste is added to 1 / 2MS+ peptone 1.0 g / L+ activated carbon 1.0 g / L+ sucrose 25 g / L+ agar 6.0 g / L medium, and the volume is adjusted to 1 L with sterile water; the pH value is adjusted to 5.8-6.1, and high-pressure sterilization is performed.

2. The long-term in vitro preservation method of the endangered Dendrobium swinhoii germplasm according to claim 1, characterized in that: The culture conditions of the growth inhibition stage and the growth recovery stage are as follows: temperature 23±2℃, light intensity 2000-3000lx, and light time 12h / d.

3. The long-term in vitro preservation method of the endangered Dendrobium swinhoii germplasm according to claim 1, characterized in that: The high-pressure sterilization is performed at a temperature of 121℃ and a pressure of 103kPa for 20-30 min.

4. The long-term in vitro preservation method of the endangered Dendrobium swinhoii germplasm according to claim 1, characterized in that: The pH value of the growth inhibition medium is adjusted to 6.3-6.5 using 0.1 mol / L HCl.

5. The long-term in vitro preservation method of the endangered Dendrobium swinhoii germplasm according to claim 1, characterized in that: The pH value of the growth recovery medium is adjusted to 5.8-6.1 using 0.5 mol / L NaOH.

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