Dendrobium officinale protocorm differentiation inhibition and rapid propagation method

Abstract

The invention discloses a dendrobium officinale protocorm differentiation inhibition and rapid propagation method. The method comprises the four steps of seed disinfection treatment, protocorm induction, protocorm domestication and protocorm propagation. According to the method, the protocorms are effectively generated by utilizing seeds, a liquid domestication mode is innovatively adopted, 1 mu M of salicylic acid is added into a liquid culture medium, differentiation of the protocorms is remarkably inhibited, the survival rate of the protocorms when the protocorms are transferred from the liquid culture medium to a solid culture medium is increased, and a large number of protocorm products are rapidly obtained through a hormone-free culture scheme; therefore, an efficient and sustainable production process is realized. In the whole cultivation process, no exogenous hormone or chemical synthetic substance is used, the method completely depends on a biological mechanism generated by natural induction and organic matter induction, and the naturalness and safety of the product are guaranteed from the source. According to the method, the production efficiency of the protocorm is improved, the production cycle of dendrobium related products is shortened, and the production cost of the protocorm is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for inhibiting differentiation and rapidly propagating protocorms of Dendrobium officinale. Technical Background

[0002] Dendrobium officinale Kimura et Migo is a perennial herbaceous plant belonging to the genus Dendrobium of the Orchidaceae family. It is a precious medicinal material unique to China. As a traditional precious Chinese medicine that is both a medicine and a food, it was listed as a "top grade" in the "Shennong Bencao Jing" as early as the "top grade", with the effects of "tonifying the five internal organs, strengthening the yin, strengthening the stomach and intestines after long-term use, and lightening the body and prolonging life". Because it is rich in polysaccharides, alkaloids, amino acids and other active ingredients, it has multiple pharmacological effects such as anti-oxidation, anti-fatigue, anti-tumor, hypoglycemic, and immune enhancement. It is widely used in traditional Chinese medicine, health foods, functional foods and other fields, and the market demand is growing year by year.

[0003] Wild Dendrobium officinale grows slowly under natural conditions, and the natural germination rate of seeds is less than 5%. Wild resources are extremely scarce. It has been listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora and included in China's National Key Protected Wild Plant List and is strictly protected. This makes artificial cultivation the main way to meet market demand.

[0004] Artificial propagation of Dendrobium officinale mainly uses tissue culture technology to obtain seedlings, combined with greenhouse facilities and mountain resources, to simulate the wild environment for large-scale planting. This method has promoted the rapid development of the Dendrobium industry, but it takes 3 to 5 years from planting to harvesting, the planting cycle is long, and the economic benefits are slow to achieve, which restricts the development of the industry.

[0005] Dendrobium protocorms are embryonic tissues generated in vitro, with the same genetic background and pharmacologically active ingredients as the source plants. Studies have shown that the polysaccharides and total amino acids contained in Dendrobium officinale protocorms are equivalent to those in wild Dendrobium officinale stems, and experimental results have confirmed that polysaccharides extracted from protocorms have good antioxidant and anti-tumor effects. According to the "Announcement on 23 "Three New Foods" Including Dendrobium Protocorms" issued in 2024, in vitro cultured Dendrobium protocorms can be used as a new type of food raw material, further promoting its industrial development.

[0006] At present, most of the research on protocorms of Dendrobium candidum is focused on seedling propagation, and the proliferation multiples are increased by adding exogenous hormones. On the one hand, the protocorms are very easy to differentiate during the growth process, and on the other hand, hormone residues or excessive levels may result, posing a safety hazard. Therefore, it is particularly urgent to develop a hormone-free culture method that inhibits the differentiation of protocorms and promotes their rapid reproduction, and to promote the efficient, low-cost, green, and safe production of protocorms. This will not only help to enhance the sustainable development of the Dendrobium industry, but also promote its wide application in multiple fields such as medicine, health care, cosmetics, and food. Summary of the invention

[0007] The purpose of the present invention is to provide a method for inhibiting differentiation and rapidly propagating protocorms of Dendrobium officinale, which can achieve large-scale, low-cost and high-survival rate protocorm propagation by adjusting the ratio of macroelements to trace elements, carbon sources, nitrogen sources, natural additives and pH value in a culture medium.

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

[0009] 1. Seed disinfection: Pick mature and pest-free yellow-green fruit pods of Dendrobium from October to November, disinfect them, and use them as explants for protocorm formation;

[0010] 2. Induction of Dendrobium protocorms: On a sterile operating table, use a sterile scalpel to cut off the top of the sterilized fruit pods to leak out the powdery seeds, clamp the fruit pods with sterile tweezers, and evenly sow the seeds in the protocorm induction medium. Culture for 40 to 60 days to obtain Dendrobium protocorms;

[0011] 3. Domestication of Dendrobium protocorms: Select the plump protocorms produced in step (2), inoculate them into liquid culture medium, culture for 30 days, and transfer them to solid culture medium and culture for 30 days;

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

[0013] Among them, in step (1), the fruit pods are disinfected by combining ethanol and chlorine dioxide. First, the freshly picked fruit pods are washed with clean water to remove surface dust, and transferred to an ultra-clean workbench. The container used for cleaning is soaked in 75% ethanol in advance. The fruit pods are placed in a beaker and poured with 75% ethanol to completely immerse them. During the period, the fruit pods are shaken continuously to ensure full contact. After disinfection for 30 seconds, they are rinsed with sterile water for 3 times, and a chlorine dioxide solution with a concentration of 250 mg / L is poured to completely immerse the fruit pods. During the period, the beaker is shaken continuously. The disinfection is carried out for 25 minutes, and the fruit pods are rinsed with sterile water for 3 to 5 times, and the fruit pods are dried.

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

[0015] Wherein, the liquid and solid culture media for the acclimation of the protocorms in step (3) are both MS culture media, with 30 g / L white sugar added, wherein the liquid acclimation culture media is added with 1 μM salicylic acid, and the solid acclimation culture media is added with 5 g / L agar, and the pH of the acclimation culture media is 5.8-6.0.

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

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

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

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

[0020] 2. The present invention adjusts the pH value of the culture medium 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. The present invention adds a liquid culture step combined with salicylic acid treatment during the protocorm acclimation process. The liquid culture medium enables the protocorms 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 the protocorms being prone to browning and dying in a liquid environment, while enhancing their stress resistance and improving their survival rate when transferred from a liquid culture medium to a solid culture medium.

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

[0023] 5. The present invention can obtain stable propagation protocorm mother plants from seeds within 2 to 3 months, and the product can be obtained in 30 to 60 days of factory production, ensuring the consistency of seed sources and genetic stability;

[0024] 6. The cultivation method of the present invention does not require the addition of hormones throughout the entire process, and can reach the edible standard test without the need for a hormone removal process, thereby avoiding the problem of hormone residues that may occur in traditional methods and ensuring the edible safety of the original corm. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Growth status of Dendrobium officinale seeds cultured in different culture media for 45 days;

[0026] Figure 2 Effects of different pH values ​​in culture medium on seed germination of Dendrobium officinale;

[0027] Figure 3 Figure 1. Growth status of protocorms after acclimation;

[0028] Figure 4 The growth status of protocorms in MS and 1 / 2MS medium;

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

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

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

[0032] Example

[0033] 1. Take the Dendrobium officinale "Mulongshan No. 3" bred by our company as the seed source, pick the fruit pods around October and November, wash off the surface dust with clean water, transfer to the clean bench, soak the container used for cleaning in 75% ethanol in advance, put the fruit pods in a beaker, pour in 75% ethanol to completely soak, shake continuously during the period to make the fruit pods fully contact, disinfect for 30 seconds, rinse with sterile water 3 times, pour in a 250mg / L chlorine dioxide solution, completely immerse the fruit pods, shake the beaker continuously during the period, disinfect for 25 minutes, rinse with sterile water 3 to 5 times, and dry the fruit pods; cut off the top with a sterile scalpel to leak out the powdered seeds, evenly sow them in the seed germination medium, set aside some seeds and drop 1% TTC solution to detect vitality; repeat the test 3 times or more, the seed survival rate is 100%, and the contamination rate is 0.

[0034] 2. For the induction of Dendrobium protocorms, the induction medium is based on MS or 1 / 2MS. Peptone or mashed potatoes, 30g / L white sugar and 5g / L agar powder are added to the medium. The pH value is adjusted to 5.8-6.0. After packaging, sterilize at 121℃ for 20min and set aside.

[0035] In a sterile operating table, use a sterile scalpel to cut off the top of the disinfected fruit pod to reveal the powdery seeds. Use sterile tweezers to clamp the fruit pod and evenly sow the seeds in the protocorm induction medium. The Dendrobium officinale seeds will turn green about 10 days after sowing, and the protocorms can be obtained after 45 to 60 days of culture. Figure 1 The growth conditions of Dendrobium officinale seeds sown in induction medium for 45 days. The germination conditions of seeds in different culture media are shown in Table 1.

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

[0037]

[0038] The induction culture medium selected in the present invention is based on MS as the basic culture medium, and 1g / L peptone, 30g / L white sugar and 5g / L agar powder are added, so as to induce the production of large and full protocorms.

[0039] In order to further explore the effect of medium pH on seed germination, S2-4 medium was used, and pH value was used as the research variable. Two treatment groups were set: one group adjusted the pH value to 5.4-5.6, and the other group adjusted the pH value to 5.8-6.0. The experiment lasted for 60 days to observe the differentiation of the protocorm. Figure 2 (In order to show the seed germination more clearly, the pictures on the right side of each treatment group are photos of the materials after being taken out of the bottle). The results showed that as the culture time was extended to 60 days, the protocorms in the culture medium with a pH value adjusted to 5.8-6.0 began to differentiate, with a differentiation rate of 22.5%. The semi-solid culture medium with a pH value adjusted to 5.4-5.6 further inhibited the differentiation of the protocorms, with a differentiation rate of 0.01%.

[0040] 3. Domestication of Dendrobium protocorms: Select large and full protocorms and inoculate them into domestication medium. The purpose is to inhibit the differentiation of protocorms by improving the culture environment and regulating the level of endogenous hormones in plants. The domestication medium is based on MS basal medium, with 30g / L white sugar added, and 5g / L agar powder added to the solid medium. The pH value is adjusted to 5.8-6.0. After packaging, sterilize at 121℃ for 20min and set aside.

[0041] In the experimental design, the culture method was used as a research variable, and a control group and a treatment group ① were set up. The control group was first cultured in a solid culture medium for 30 days, and then transferred to a solid culture medium for 30 days. The treatment group ① was first cultured in a liquid culture medium for 30 days, and then transferred to a solid culture medium for 30 days. The experimental results are as follows Figure 2 As shown, the differentiation rate of protocorms in the control group was 85%, while that in treatment group ① dropped to 11.1%, indicating that liquid culture significantly inhibited the differentiation of protocorms.

[0042] However, the experiment found that when the protocorms were transferred from liquid culture medium to solid culture medium, they would turn brown and die, and the survival rate was only 72.9%. This phenomenon may be due to the transfer of the protocorms from a moist liquid environment to a drier solid environment, or due to the increase in the osmotic pressure of the culture medium, which caused damage to the cell membrane. In order to improve the survival rate of the protocorms removed from the liquid culture medium, the formula of the liquid culture medium was further improved, with the aim of enhancing the stress resistance of the protocorms during the liquid culture process.

[0043] In the improved treatment group ②, 1 μM salicylic acid was added to the liquid culture medium. The growth status after 30 days of solid culture was as follows: Figure 2 As shown, the survival rate of protocorms increased to more than 99%. The results showed that liquid culture combined with salicylic acid treatment significantly inhibited the differentiation of protocorms on the basis of effectively improving the survival rate of protocorms.

[0044] 4. Protocorm proliferation: Select the vigorous and undifferentiated protocorms and inoculate them into the protocorm proliferation medium. Culture for 30-60 days and harvest. After the proliferation medium is prepared, adjust the pH value to 5.8-6.0, sterilize at 121℃ for 20 minutes after aliquoting, and set aside.

[0045] During the proliferation culture, the effects of the ratio of macroelements to trace elements in the basal culture medium on the proliferation of protocorms were compared. MS and 1 / 2MS were used as basal culture medium, 30 g / L sucrose and 5 g / L agar powder were added to each medium, and the protocorms of Dendrobium officinale were inoculated. The proliferation times and growth status of the protocorms were recorded during the culture process. Figure 3 As shown, after culturing for 45 days, the proliferation multiple of the protocorm in the MS medium reached 5.4 times, while the proliferation multiple of the protocorm in the 1 / 2MS medium was only 3.22 times. The MS selected as the basic medium in the present invention is more suitable for the proliferation culture of the protocorm of Dendrobium officinale.

[0046] In the process of screening the proliferation medium, MS was used as the basic medium, and 30 g / L sucrose and 5 g / L agar powder were added as the control group. The effect of peptone added to the medium as a nitrogen source on the proliferation of protocorms was studied, and 1 g / L peptone was added as the experimental group. Figure 4As shown, the statistical data of culturing for 45 days showed that the proliferation multiple of the protocorms in the control group was 4.96 times, and the proliferation multiple of the protocorms in the experimental group was 7.32 times. The addition of peptone in the proliferation medium selected by the present invention can better promote the proliferation of protocorms.

[0047] During the proliferation culture, MS was used as the basic culture medium, and 1 g / L peptone, 30 g / L sucrose and 5 g / L agar powder were added. The effect of adding organic matter such as mashed potato 50-200 g / L or banana 100 g / L in the culture medium on the proliferation of protocorms was studied. The experimental data are shown in Table 2 and Figure 5 shown.

[0048] Table 2 Effect of adding organic matter to the culture medium on the proliferation of protocorms

[0049]

[0050] The MS selected as the basic culture medium of the present invention, with the addition of 1g / L peptone and 100g / L mashed potato, has the best effect on the proliferation of protocorms, and the proliferation multiple can reach 12.81 times after culturing for 60 days, which is 2.4 times that of the control group.

[0051] In the cultivation process of Dendrobium protocorms, sucrose, as a commonly used carbon source, plays an important role in promoting plant growth and proliferation. However, in large-scale production, the high cost of sucrose restricts the production efficiency of protocorms. Therefore, exploring cheaper alternative carbon sources has important practical significance. This study took white sugar as the research object, and verified whether it could replace sucrose as a carbon source in the culture medium by evaluating the proliferation effect of protocorms, so as to achieve the goal of reducing production costs.

[0052] In this study, 30g / L sucrose was replaced with 20-30g / L white sugar in S4-5 medium, and the protocorms of Dendrobium were inoculated and cultured for 60 days, and the proliferation times were counted. The experimental results are shown in Table 3. In the group with sucrose added, the proliferation times of Dendrobium protocorms were 11.91 times the inoculation amount; while in the group with white sugar added, the proliferation times were 11.30 times the inoculation amount. The results of one-way ANOVA showed that there was no significant difference in the proliferation times between the two groups. It can be seen that in the culture process of Dendrobium protocorms, adding 20-30g / L white sugar to the culture medium can achieve a proliferation effect similar to sucrose, and at the same time can reduce the production cost by about 97%.

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

[0054]

[0055] Note: ns means there is no significant difference between the groups, P < 0.05.

[0056] The above description is only a preferred embodiment of the present invention. It should be pointed out that, for ordinary technicians in this technical field, the content of the present invention can be improved and modified without departing from the technical principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for inhibiting differentiation and rapid propagation of protocorms of Dendrobium officinale, characterized in that The following steps are involved: (1) Seed disinfection: Pick mature and pest-free yellow-green fruit pods of Dendrobium officinale from October to November, disinfect them, and use them as explants for protocorm formation; (2) Induction of Dendrobium protocorms: On a sterile operating table, use a sterile scalpel to cut off the top of the sterilized fruit pod to leak out the powdery seeds, clamp the fruit pod with sterile tweezers, and evenly sow the seeds in the protocorm induction medium. Culture for 40 to 60 days to obtain Dendrobium protocorms; (3) domestication of Dendrobium protocorms: selecting the plump protocorms produced in step (2), inoculating them into liquid culture medium, culturing for 30 days, and transferring them to solid culture medium and culturing for 30 days; (4) Protocorm proliferation: the protocorm domesticated in step (3) is inoculated into a protocorm proliferation medium, cultured for 30 to 60 days, and harvested.

2. The method for inhibiting differentiation and rapidly propagating protocorms of Dendrobium officinale according to claim 1, characterized in that: In step (1), the fruit pods are disinfected by a combination of ethanol and chlorine dioxide. First, the freshly picked fruit pods are washed with clean water to remove surface dust, and then transferred to a clean bench. The container used for cleaning is soaked in 75% ethanol in advance. The fruit pods are placed in a beaker and poured with 75% ethanol to completely immerse them. During the period, the fruit pods are shaken continuously to ensure full contact. After disinfection for 30 seconds, they are rinsed with sterile water for 3 times, and a chlorine dioxide solution with a concentration of 250 mg / L is poured to completely immerse the fruit pods. During the period, the beaker is shaken continuously. The disinfection is carried out for 25 minutes, and the fruit pods are rinsed with sterile water for 3 to 5 times, and the fruit pods are dried.

3. The method for inhibiting differentiation and rapidly propagating protocorms of Dendrobium officinale according to claim 1, characterized in that: In step (2), the protocorm induction medium is MS medium, which is added with 1 g / L peptone, 30 g / L white sugar and 5 g / L agar powder. The pH of the medium is 5.4-5.6 and is in a semi-solid state.

4. The method for inhibiting differentiation and rapidly propagating protocorms of Dendrobium officinale according to claim 1, characterized in that: In step (3), both the liquid and solid culture medium for the acclimation of the protocorms were MS culture medium, with 30 g / L of white sugar added, and 1 μ M salicylic acid, 5 g / L agar powder was added to the solid acclimation medium, and the pH of the acclimation medium was 5.8-6.

0.

5. The method for inhibiting differentiation and rapidly propagating protocorms of Dendrobium officinale according to claim 1, characterized in that: In step (4), the protocorm proliferation medium is MS medium, 1 g / L peptone and 100 g / L mashed potato, 20-30 g / L white sugar and 5 g / L agar powder are added, and the pH of the proliferation medium is 5.8-6.

0.

6. The method for inhibiting differentiation and rapidly propagating protocorms of Dendrobium officinale according to claim 1, characterized in that: The culture processes in steps (2), (3) and (4) are all carried out in a culture room, the temperature of the culture room is maintained at 21-23° C., the air humidity is maintained at 40-60%, the light intensity is 12 h / d, and the light intensity is 1000-1500 Lx.

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