Dendrobium candidum protocorm differentiation inhibition and rapid propagation method

Abstract

The application discloses a Dendrobium candidum protocorm differentiation inhibition and rapid propagation method, which comprises four steps of seed disinfection treatment, protocorm induction, protocorm domestication and protocorm proliferation. μ M salicylic acid, significantly inhibits protocorm differentiation and improves the survival rate of protocorm when it is transferred from a liquid culture medium to a solid culture medium, and a hormone-free culture scheme is used to quickly obtain a large amount of protocorm product, thereby realizing an efficient and sustainable production process. In the whole cultivation process, no exogenous hormones or chemical synthetic substances are used, and the biological mechanism generated by natural induction and organic induction is completely relied on, so that the natural and safety of the product are guaranteed from the source. The application improves the production efficiency of the protocorm, shortens the production cycle of the Dendrobium-related product and reduces the production cost of the protocorm.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant tissue culture, and particularly relates to a Dendrobium officinale protocorm differentiation inhibition and rapid propagation method. TECHNICAL BACKGROUND

[0002] Dendrobium officinale Kimura et Migo belongs to the perennial herbaceous plant of Orchidaceae Dendrobium, and is a precious Chinese medicinal material. As a traditional precious Chinese medicine with the same origin of food and medicine, it was listed as "the top product" in the Shennong Herbal Classic, and has the effects of "tonifying five zang organs, strengthening yin, nourishing the stomach and intestines, and prolonging life". Due to its rich active ingredients such as polysaccharides, alkaloids and amino acids, it has various pharmacological effects such as antioxidant, anti-fatigue, anti-tumor, hypoglycemic and immune enhancement, and is widely used in traditional Chinese medicine, health food and functional food fields, and the market demand is increasing year by year.

[0003] Wild Dendrobium officinale grows slowly under natural conditions, and the natural seed germination rate is less than 5%. Wild resources are extremely scarce, and have been listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora, and included in the national key protected wild plant list in China, which is strictly protected. This makes artificial cultivation become the main way to meet market demand.

[0004] Artificial propagation of Dendrobium officinale is mainly obtained by tissue culture technology, combined with greenhouse facilities and mountain resources, and large-scale planting is carried out by simulating wild environment. This method promotes the rapid development of Dendrobium industry, but it takes 3-5 years from planting to harvesting, the planting cycle is long, the economic benefit is slow to realize, and the development of the industry is restricted.

[0005] Dendrobium protocorm is an embryonic tissue generated in in vitro culture, which has the same genetic background and pharmacological active ingredients as the plant source. Studies have shown that the polysaccharides and total amino acids contained in Dendrobium officinale protocorm are equivalent to those in wild Dendrobium officinale stems, and experimental results have confirmed that the polysaccharides extracted from protocorm have good antioxidant and anti-tumor effects. According to the Announcement on 23 kinds of "three new foods" such as Dendrobium protocorm issued in 2024, in vitro cultured Dendrobium protocorm can be used as a new type of food raw material, which further promotes the industrialization development.

[0006] At present, the research on Dendrobium candidum protocorms is mostly focused on seedling propagation, and the proliferation rate is improved by adding exogenous hormones. On the one hand, the protocorms are prone to differentiation during growth, and on the other hand, the hormones may cause residues or exceed the standard, which may pose a safety hazard. Therefore, it is particularly urgent to develop a hormone-free culture method that can inhibit protocorm differentiation and promote rapid propagation, and promote the efficient, low-cost, green and safe production of protocorms. This not only helps to improve the sustainable development of the Dendrobium industry, but also promotes its wide application in the fields of medicine, health care, cosmetics and food. SUMMARY

[0007] The purpose of the present application is to provide a Dendrobium candidum protocorm differentiation inhibition and rapid propagation method, which realizes large-scale, low-cost and high survival rate protocorm propagation by adjusting the ratio of macroelements and microelements, carbon source, nitrogen source, natural additives and pH value in the culture medium.

[0008] The specific implementation steps of the present application are as follows:

[0009] 1. Seed disinfection treatment: pick up the mature and disease-free yellow-green fruit pods of Dendrobium candidum in October and November, and disinfect the fruit pods as explants for protocorm formation;

[0010] 2. Dendrobium candidum protocorm induction: in a sterile operating table, use a sterile scalpel to cut off the top of the disinfected fruit pod and release the powdery seeds, and use sterile tweezers to hold the fruit pod and evenly spread the seeds in the protocorm induction medium, and culture for 40-60 days to obtain Dendrobium candidum protocorms;

[0011] 3. Dendrobium candidum protocorm domestication: select the full protocorms produced in step (2) and inoculate them into liquid medium for 30 days, and then transfer them to solid medium for 30 days;

[0012] 4. Protocorm proliferation: inoculate the domesticated protocorms in step (3) into protocorm proliferation medium and culture for 30-60 days, and then harvest.

[0013] In step (1), the fruit pod disinfection adopts a combination of ethanol and chlorine dioxide. First, wash the fresh fruit pods picked up with clean water to remove surface dust, and then transfer them to a clean bench. The container used for washing is soaked in 75% ethanol in advance. Put the fruit pods into a beaker and pour in 75% ethanol to completely soak them. Shake the beaker constantly to ensure that the fruit pods are fully contacted. After 30 seconds of disinfection, rinse the fruit pods with sterile water for 3 times. Pour in a chlorine dioxide solution with a concentration of 250 mg / L to completely immerse the fruit pods. Shake the beaker constantly during the 25-minute disinfection. Rinse the fruit pods with sterile water for 3-5 times and dry them.

[0014] The original bulb induction medium in step (2) is MS medium, 1g / L peptone, 30g / L white sugar and 5g / L agar powder are added, the pH of the medium is 5.4-5.6, and the medium is semi-solid.

[0015] The liquid and solid domestication medium of the original bulb in step (3) are both MS medium, 30g / L white sugar is added, 1 μM salicylic acid is added to the liquid domestication medium, and 5g / L agar is added to the solid domestication medium, and the pH of the domestication medium is 5.8-6.0.

[0016] The original bulb proliferation medium in step (4) is MS medium, 1g / L peptone and 100g / L potato mud are added, 20-30g / L white sugar and 5g / L agar powder are added, and the pH of the proliferation medium is 5.8-6.0.

[0017] The culture process in steps (2), (3) and (4) is carried out in a culture room, the temperature of the culture room is kept at 21-23℃, the air humidity is kept at 40-60%, the light is 12h / d, and the light intensity is 1000-1500Lx.

[0018] The beneficial technical effects of the present application are embodied in:

[0019] 1. The present application uses chlorine dioxide to disinfect Dendrobium candidum seeds, which is safe to human body and environment, has no residue, and can ensure the safety of the production products, and the disinfection method can achieve 0 pollution rate and 100% survival rate.

[0020] 2. The pH value of the culture medium is adjusted to 5.4-5.6 during seed germination, and the seeds can be effectively inhibited from differentiating into seedlings in the semi-solid culture medium.

[0021] 3. In the original bulb domestication process, a liquid culture step is added combined with salicylic acid treatment, the liquid culture medium enables the original bulb to grow in a low-oxygen environment and inhibit differentiation, the addition of salicylic acid in the liquid culture medium can avoid the problem of easy browning and death of the original bulb in the liquid environment, and at the same time, the anti-stress ability of the original bulb is enhanced, and the survival rate when the original bulb is transferred from the liquid culture medium to the solid culture medium is improved.

[0022] 4. The present application uses white sugar to replace sucrose to achieve similar culture effect, and the production cost is directly reduced by about 97%;

[0023] 5. The present application can obtain stable proliferated original bulb mother plants from seeds within 2-3 months, and the products can be obtained within 30-60 days of factory production, which ensures the consistency of seed source and genetic stability.

[0024] 6. The culture method of the present application does not add hormones throughout the whole process, and achieves the edible standard detection without the need of hormone removal process, avoids the hormone residue problem that may occur in traditional methods, and ensures the edible safety of the protocorms. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Figure of the growth status of Dendrobium candidum seeds in different culture media for 45 days;

[0026] Figure 2 Effect of different pH values in the culture medium on the germination of Dendrobium candidum seeds;

[0027] Figure 3 Figure of the growth status of the domesticated protocorms;

[0028] Figure 4 Figure of the growth status of the protocorms in MS and 1 / 2MS culture media;

[0029] Figure 5 Effect of adding protein peptone in the culture medium on the proliferation of the protocorms;

[0030] Figure 6 Growth status of the protocorms in different types and concentrations of organic matter. DETAILED DESCRIPTION

[0031] The present application will be further described below in conjunction with specific examples. The features and advantages of the present application will become more apparent with the description, but the exemplary examples are only used to illustrate the present application and do not constitute any limitation on the scope of the present application.

[0032] EMBODIMENT

[0033] 1. The Dendrobium candidum "Mulongshan No. 3" selected by the company was used as the seed source, and the fruit capsules were picked in October and November, washed with clean water to remove the surface dust, and transferred to a clean bench. The container used for washing was soaked in 75% ethanol in advance, the fruit capsules were placed in a beaker, and 75% ethanol was poured to completely soak the fruit capsules. During the process, the beaker was shaken constantly to ensure that the fruit capsules were fully contacted. After 30 seconds of disinfection, the fruit capsules were washed with sterile water for 3 times, and then 250 mg / L chlorine dioxide solution was poured to completely immerse the fruit capsules. During the process, the beaker was shaken constantly. After 25 minutes of disinfection, the fruit capsules were washed with sterile water for 3-5 times, and then dried. The top end of the fruit capsules was cut off with a sterile scalpel, and the powdery seeds were evenly scattered in the seed germination culture medium. A portion of the seeds were dropped into 1% TTC solution for detection of viability. The test was repeated for 3 times or more, the seed survival rate reached 100%, and the contamination rate was 0.

[0034] 2. The induction of Dendrobium protocorms, the induction medium is based on MS or 1 / 2MS medium, adding peptone or potato mash, adding 30 g / L white sugar and 5 g / L agar powder, pH value is adjusted to 5.8-6.0, high pressure 121℃ sterilization for 20 min after dispensing, ready for use.

[0035] In a sterile operating table, the sterilized fruit pod is cut off with a sterile scalpel, and the powdery seeds are leaked out. The seeds are evenly sown in the protocorm induction medium with sterile tweezers. The Dendrobium candidum seeds turn green about 10 days after sowing, and the protocorms are obtained after 45-60 days of culture. Figure 1 The growth conditions of Dendrobium candidum seeds sown in the induction medium for 45 days, and the germination conditions of seeds in different media are shown in Table 1.

[0036] Table 1 Growth conditions of protocorms induced by seeds in different media

[0037]

[0038] The induction medium selected in the present application is based on MS medium, adding 1 g / L peptone, 30 g / L white sugar and 5 g / L agar powder, which can induce protocorms with large and full individuals.

[0039] In order to further explore the effect of medium pH on the germination of Dendrobium candidum seeds, S2-4 medium was used, and pH value was used as a research variable, two groups were set: one group was adjusted to 5.4-5.6, and the other group was adjusted to 5.8-6.0. The experiment lasted for 60 days, and the differentiation of protocorms was observed. According to Figure 2 (In order to more clearly show the seed germination, the right picture of each treatment group is the shooting effect after the material is taken out of the bottle), the results show that as the culture time is prolonged to 60d, the protocorms in the medium with pH value of 5.8-6.0 appear differentiation, and the differentiation rate is 22.5%, and the medium with pH value of 5.4-5.6 further inhibits the differentiation of protocorms, and the differentiation rate is 0.01%.

[0040] 3. Domestication of Dendrobium protocorms, select protocorms with large and full individuals, inoculate into domestication medium, the purpose is to improve the culture environment, adjust the endogenous hormone level of plants, and achieve the effect of inhibiting the differentiation of protocorms. The domestication medium is based on MS basic medium, adding 30 g / L white sugar, and adding 5 g / L agar powder for solid medium, adjusting the pH value to 5.8-6.0, high pressure 121℃ sterilization for 20 min after dispensing, ready for use.

[0041] In the experimental design, the culture mode is taken as the research variable, and a control group and a treatment group 1 are set. The control group is first cultured in solid medium for 30 days, and then transferred to solid medium for 30 days. The treatment group 1 is first cultured in liquid medium for 30 days, and then transferred to solid medium for 30 days. The experimental results are shown in Figure 2 The protocorm differentiation rate of the control group is 85%, while the differentiation rate of the treatment group 1 is reduced to 11.1%, indicating that liquid culture significantly inhibits the differentiation of protocorms.

[0042] However, it is found in the experiment that when the protocorms are transferred from the liquid medium to the solid medium, browning and death phenomenon occurs, and the survival rate is only 72.9%. This phenomenon may be due to the fact that the protocorms are transferred from a humid liquid environment to a relatively dry solid environment, or due to the increase in osmotic pressure of the medium, resulting in damage to the cell membrane. In order to improve the survival rate of protocorms removed from the liquid medium, the formula of the liquid medium is further improved, aiming to enhance the stress resistance of protocorms during liquid culture.

[0043] In the improved treatment group 2, 1 μM salicylic acid is added to the liquid medium, and the growth condition after 30 days of solid culture is shown in Figure 2 The survival rate of protocorms is increased to more than 99%. The results show that liquid culture combined with salicylic acid treatment can effectively improve the survival rate of protocorms and significantly inhibit the differentiation of protocorms.

[0044] 4. Protocorm proliferation: select vigorous and undifferentiated protocorms and inoculate them into protocorm proliferation medium for 30-60 days for harvesting. After the preparation of the proliferation medium, the pH value is adjusted to 5.8-6.0, and the medium is sterilized at 121°C for 20 minutes under high pressure after being divided into small portions, and is ready for use.

[0045] During the proliferation culture process, the effects of the proportion of macroelements and microelements in the basic medium on protocorm proliferation are compared, and MS and 1 / 2MS are used as the basic medium, respectively, and 30 g / L sucrose and 5 g / L agar powder are added, and the protocorms of Dendrobium officinale are inoculated, and the proliferation rate and growth state of the protocorms are recorded during the culture process, as shown in Figure 3 After 45 days of culture, the protocorms in the MS medium have a proliferation rate of 5.4 times, and the protocorms in the 1 / 2MS medium have a proliferation rate of only 3.22 times, and the MS selected as the basic medium is more suitable for the proliferation culture of protocorms of Dendrobium officinale.

[0046] During the selection of the proliferation medium, MS is used as the basic medium, and 30 g / L sucrose and 5 g / L agar powder are added as the control group. The effect of adding protein peptone as a nitrogen source in the medium on protocorm proliferation is studied, and 1 g / L protein peptone is added as the experimental group. Figure 4As shown in the statistical data of the 45d culture, the protocorm proliferation multiple of the control group is 4.96 times, and the protocorm proliferation multiple of the experimental group is 7.32 times. The addition of peptone in the proliferation culture medium can better promote the protocorm proliferation.

[0047] In the proliferation culture process, MS is used as the basic culture medium, 1g / L peptone, 30g / L sucrose and 5g / L agar powder are added, the effects of adding 50-200g / L potato or 100g / L banana and other organic matters in the culture medium on the protocorm proliferation are studied, and the experimental data are shown in Table 2 and Figure 5 .

[0048] Table 2 Effects of adding organic matters in the culture medium on the protocorm proliferation

[0049]

[0050] The addition of 1g / L peptone and 100g / L potato mud in the MS basic culture medium is the best for the protocorm proliferation, and the proliferation multiple can reach 12.81 times after 60d culture, which is 2.4 times of the control group.

[0051] In the culture process of Dendrobium protocorm, sucrose as a common carbon source plays an important role in promoting plant growth and proliferation. However, in large-scale production, the cost of sucrose is relatively high, which restricts the production efficiency of protocorm. Therefore, it is of great practical significance to explore a cheaper alternative carbon source. In this study, white sugar was used as the research object, and the proliferation effect of protocorm was evaluated to verify whether it could replace sucrose as a carbon source in the culture medium to achieve the goal of reducing production cost.

[0052] In this study, 30g / L sucrose was replaced by 20-30g / L white sugar in S4-5 medium, and Dendrobium protocorm was inoculated and cultured for 60d, and the proliferation multiple was counted. The experimental results are shown in Table 3. The proliferation multiple of Dendrobium protocorm in the group with added sucrose is 11.91 times of the inoculation amount, and the proliferation multiple in the group with added white sugar is 11.30 times of the inoculation amount. The results of single factor variance analysis show that there is no significant difference in the proliferation multiple between the two groups. Therefore, in the culture process of Dendrobium protocorm, the addition of 20-30g / L white sugar in the culture medium can achieve similar proliferation effect as sucrose, and at the same time, the production cost can be reduced by about 97%.

[0053] Table 3 Effects of different carbon sources in the culture medium on the protocorm proliferation

[0054]

[0055] Note: ns indicates no significant difference between groups, P<0.05.

[0056] The above merely describes the preferred embodiments of the present application, and it should be pointed out that the content of the present application can be improved and changed in several ways without departing from the technical principles of the present application, and these improvements and changes should also be considered as the protection scope of the present application.

Claims

1. A method for inhibiting differentiation of Dendrobium candidum protocorms and rapid propagation, characterized in that The method comprises the following steps: (1) seed disinfection treatment, picking 10-11 months of Dendrobium mature and no disease and insect pests of yellow green fruit pod, disinfection treatment, as the explant of protocorm formation; (2) Dendrobium protocorm induction, in aseptic operating table, using aseptic scalpel, the top of the fruit pod is cut off, the powder seed is leaked, the seed is evenly sowed in the protocorm induction medium, and the Dendrobium protocorm is obtained after 40-60d culture; (3) Dendrobium protocorm domestication, the full protocorm produced in step (2) is selected, inoculated into liquid medium, cultured for 30d, and then transferred into solid medium for 30d culture; (4) protocorm proliferation, the domesticated protocorm in step (3) is inoculated into protocorm proliferation medium, cultured for 30-60d, and then harvested; In step (1), the fruit pod disinfection adopts the combination of ethanol and chlorine dioxide, the fresh fruit pod is washed with water to remove the surface dust, transferred to the clean bench, the container for washing is soaked in 75% ethanol in advance, the fruit pod is placed in a beaker, 75% ethanol is poured to completely soak the fruit pod, the fruit pod is continuously shaken to make full contact, washed with sterile water for 3 times after 30s disinfection, 250mg / L chlorine dioxide solution is poured to completely immerse the fruit pod, the beaker is continuously shaken during disinfection for 25min, and the fruit pod is dried after washing with sterile water for 3-5 times; In step (2), the protocorm induction medium is composed of MS medium, 1g / L protein peptone, 30g / L white sugar and 5g / L agar powder, the pH of the medium is 5.4-5.6, and the medium is in a semi-solid state; The liquid medium for the domestication of protocorms in step (3) consists of MS medium, 30 g / L white sugar and 1 μ M salicylic acid, the solid medium for the domestication of protocorms consists of MS medium, 30 g / L white sugar and 5 g / L agar powder, and the pH of the domestication medium is 5.8-6.0; In step (4), the protocorm proliferation medium is composed of MS medium, 1g / L protein peptone, 100g / L potato mud, 20-30g / L white sugar and 5g / L agar powder, and the pH of the proliferation medium is 5.8-6.

0.

2. The method for inhibiting differentiation of Dendrobium candidum protocorm and rapid propagation according to claim 1, characterized in that: In steps (2), (3) and (4), the culture is carried out in a culture room, the temperature of the culture room is kept at 21-23℃, the air humidity is kept at 40-60%, the light illumination is 12h / d, and the light intensity is 1000-1500Lx.

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