Method for medium-short-term preservation and growth recovery of pinellia ternate

Abstract

The invention discloses a method for medium-short-term preservation and growth recovery of pinellia ternate. The method comprises the steps of material culture and disinfection, second-generation test-tube plantlet and second-generation tuber culture, medium-short-term preservation of third-generation tubers, growth recovery and propagation expansion. The Pinellia ternate medium-short term preservation technology can be applied to Pinellia ternate germplasm resource preservation, compared with field preservation, the Pinellia ternate medium-short term preservation technology can greatly save manpower and material resources, is easy and convenient to operate and not prone to being attacked by natural disasters, has the advantages of being small in occupied space, low in preservation cost, long in preservation time, stable in heredity and the like, and Pinellia ternate germplasm can be preserved in a medium-short term.

Description

Technical Field

[0001] The invention belongs to the technical field of crop breeding, and in particular relates to a method for medium- and short-term preservation and growth recovery of pinellia. Background Art

[0002] The development of natural medicines in various countries around the world has brought heavy pressure to the protection and sustainable use of medicinal plant resources. The protection and sustainable use of germplasm resources are hot issues in the world today. Medicinal plant germplasm resources are the material basis for medical and health care, agricultural production and national economic development. It is related to human survival and social development. It occupies an important position in the sustainable development of agriculture. my country has a vast territory and the richest medicinal plant germplasm resources in the world. However, due to various reasons, some important resources are decreasing and disappearing at an alarming rate. Therefore, actively exploring germplasm resource preservation technology is of great significance to preventing the depletion of important resources and promoting the development of medicinal plant breeding and biotechnology. Pinellia [ Pinellia ternate (Thunb.) Beri.] is a plant of the Araceae family, a perennial herb of the genus Pinellia, with tubers used as medicine. It has the effects of drying dampness and resolving phlegm, relieving adverse reactions and stopping vomiting, and eliminating lumps and dispersing knots. It is a commonly used bulk Chinese medicinal material in my country, with a history of more than 2,000 years of medicinal use. Nearly 800 Chinese medicine companies in my country use Pinellia in nearly 558 prescriptions, and the frequency of use of Pinellia ranks 22nd. In recent years, the market demand for Pinellia in my country is about 6,000 tons, but the amount of wild and domesticated Pinellia can only meet 1 / 3 of the market demand, resulting in a long-term high price of Pinellia and a shortage of supply. Gansu Province is one of the main production areas of Pinellia, accounting for 72% of the country's production. Xihe County, Gansu Province is known as the "Hometown of Pinellia in China". However, due to the drive for utilization, people have uncontrolled excavation of wild resources, resulting in the almost extinction of wild germplasm. In field cultivation, Pinellia ternata mainly relies on tubers for asexual reproduction, and most of the seed stems are brought in from other places, which leads to the gathering of foreign diseases and pests during the production process, high field incidence, serious yield reduction, confusion of germplasm resources, and unstable quality, which seriously affects and limits the quality and yield of Pinellia ternata, and the planting cost has been rising year by year.

[0003] The germplasm resources of asexually propagated crops are mainly preserved in the field and in vitro. Field preservation requires a lot of land and human resources, has high preservation costs, and is easily attacked by various natural disasters during the preservation process. Delayed growth preservation in in vitro preservation is to place these explants under growth inhibition conditions to make them grow slowly or not grow, so as to achieve the purpose of medium-term or long-term preservation. The use of in vitro preservation technology to establish a test tube gene bank requires less space, which can greatly save manpower, material resources and land; the preservation period can be protected from external pests, bacteria and viruses, and avoid degradation and extinction caused by pathogens; germplasm can be directly distributed or exchanged internationally without quarantine; it has a high reproduction coefficient, and if necessary, the preserved materials can be used as core raw materials for mass reproduction. The use of this technology provides a fundamental guarantee for germplasm preservation. At present, there are many examples of delayed growth preservation of asexually propagated crops. For example, this technology is used to safely and effectively preserve germplasm in dendrobium, golden thread lotus, chrysanthemum, white schisandra, potato, sweet potato, sugarcane, orchid, etc.

[0004] There are many reports on the tissue culture research of Pinellia ternata, but there are no reports on the delayed growth preservation of its intact test tube tubers. Summary of the invention

[0005] The invention aims to provide a method for medium- to short-term preservation and growth recovery of Pinellia ternata, so as to provide technical support for the preservation and sustainable utilization of Pinellia ternata germplasm resources.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is: A method for short-term storage and growth recovery of pinellia ternata, comprising the following steps: Step 1: Material culture and disinfection: The Pinellia ternata seed stems were sown in a flower pot to germinate and grow to obtain the Pinellia ternata plants. The common petioles of the Pinellia ternata plants were removed and treated with 75% ethanol for 10 seconds. The common petioles were taken out and placed in 0.1% mercuric chloride with 1 drop of Tween 80 for 5 minutes. The common petioles were taken out and placed in 2% sodium hypochlorite for 8 minutes. Finally, the common petioles were taken out and rinsed with sterile water for 3 times. Step 2: Second-generation test tube seedlings and second-generation tuber culture: The petiole obtained in step 1 is cut into small segments of 0.5-2 cm, inoculated into a medium of 1 / 2MS+3 wt % sucrose+0.8 wt % agar, pH 5.8, cultured under weak light conditions to obtain an independent and complete small tuber, transferred to light conditions to allow the small tuber to germinate into seedlings, the obtained small tuber is the first-generation tuber, and the obtained seedling is the first-generation test tube seedling, after cutting off the stems and leaves of the first-generation tuber, transferred to a fresh medium of 1 / 2MS+3wt % sucrose+0.8 wt % agar, pH 5.8, the first-generation tuber germinates into a seedling again and grows up, and the second-generation test tube seedling and the second-generation tuber are obtained; Step 3: Short- to medium-term storage of third-generation tubers: The second-generation tubers were transferred to culture bottles filled with 1 / 2MS fresh solid culture medium, cultured in dark conditions until the buds reached 2 cm in height, and then transferred to light conditions to grow into complete plants, thereby obtaining third-generation tubers and third-generation test tube seedlings. When the stems and leaves withered and the tubers were in a dormant state, the third-generation tubers and culture bottles were placed in the dark and stored at room temperature for 450 days; When it is necessary to preserve the Pinellia germplasm resources in the short to medium term, repeat steps 2 to 3 with the obtained complete plants; Step 4: Resume growth: After 450 days, add 10 ml of 1 / 2MS + 3 wt % sucrose, pH 5.8 liquid culture medium to the culture bottle, culture at 25°C in the dark, and when the tubers germinate to a bud length of 2-4 cm, switch to 12 h light and 12 h dark alternation to obtain complete plants, with a light intensity of 1000-2000 lux; Step 5: Expand reproduction: The petioles of the intact plants obtained in step 4 were cut into small segments of 0.5-2 cm, and inoculated into 1 / 2MS+3 wt % sucrose+0.8 wt % agar, pH 5.8 culture medium to obtain an independent and intact small tuber and germinate it into a seedling.

[0007] In order to further implement the present invention, the weak light condition in step 2 is 500-1000 lux.

[0008] In order to further realize the present invention, the lighting conditions in step three are lighting intensity 1000-2000LUX and lighting duration 12-16h / d.

[0009] Compared with the prior art, the present invention has the following beneficial effects: The medium- and short-term preservation technology of Pinellia ternata established by the present invention can be applied to the preservation of Pinellia ternata germplasm resources. Compared with field preservation, this technology can greatly save manpower and material resources, is easy to operate, and is not easily affected by natural disasters. It has many advantages such as small space occupation, low preservation cost, long preservation time, and genetic stability. It can preserve Pinellia ternata germplasm in the medium and short term.

[0010] There is research on using callus and protocorm as preservation materials, using low temperature combined with high osmotic pressure or growth inhibitor preservation technology to preserve Pinellia germplasm resources in vitro, but the preservation cost is high, easily mutated, and the preservation survival rate is not high. The test material used in the present invention is the independent and complete test tube tuber of Pinellia, and the tuber is induced from one end of petiole by the minimal medium without adding technical hormones, and 1 independent and complete bulbil will be produced at the base of the petiole of Pinellia under natural conditions, and the occurrence of test tube tuber is very similar to it, therefore, compared to the callus, protocorm and clustered tuber induced under high concentration hormone conditions, the independent and complete tuber used in the present invention has very high genetic stability, which has been proved by morphology and molecular level and histological research, and the survival rate after preservation is as high as 93.33%. In addition, the existing technology uses low temperature conditions or illumination conditions to preserve materials, but because of delayed growth preservation up to 1-2 years, this means that a large amount of electric energy will be consumed to control temperature or provide illumination. The present invention adopts early stage illumination to form seedlings, and after the seedlings wither, they are transferred to dark room temperature conditions to achieve the purpose of short-term and medium-term storage, which can greatly save electric energy. The complete tubers of the pinellia tuber preserved in vitro do not need to be transferred, and only 1 / 2MS liquid culture medium needs to be added thereto to germinate and form seedlings, thereby obtaining a large number of pinellia tuber seedlings. Therefore, the present invention not only has a high survival rate, but also has high genetic stability and is simple to operate, and saves energy. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 These are photos of tubers of different generations in Example 1 of the present invention after drying and storage for 450 days, wherein A is the fifth generation, B is the fourth generation, C is the third generation, D is the second generation, and E is the first generation; Figure 2 This is a photo of the growth status of the third-generation tubers in Example 1 of the present invention when they germinated into seedlings 21 days after being stored for 450 days; Figure 3 This is a photo of the growth status of the fourth-generation tuber in Example 1 of the present invention when it germinated into a seedling 21 days after being stored for 450 days; Figure 4 The following are photos of the expanded propagation of the regenerated plants after the third-generation tubers were stored at room temperature in Example 1 of the present invention (AD indicates parallel experiments); Figure 5 The ISSR and RAPD detection of molecular genetic stability amplification maps of regenerated plants after three generations of tubers stored at room temperature in Example 1 of the present invention, wherein A is the amplification map of ISSR primer 835, and B is the amplification map of RAPD primer Em13; Figure 6 The tubers and culture medium states after different treatments in Example 2 of the present invention are shown; A is the mature tubers after low-temperature storage, B is the first-generation test tube seedlings after low-temperature storage, and C is the high osmotic pressure combined with low-temperature storage; Figure 7The growth of plants after recovery culture after different treatments of delayed growth in Experimental Examples 2 and 3 of the present invention, wherein A is mature tubers stored at low temperature, B is test tube seedlings stored at low temperature, C is stored at 3% sucrose, D is stored at 6% sucrose, E is stored at 9% sucrose, F is stored at 3% sucrose + 2% mannitol, G is stored at 3% sucrose + 4% mannitol, H is stored at 3% sucrose + 6% mannitol, I is stored at 6% sucrose + 2% mannitol, and J is stored at 9% sucrose + 2% mannitol before recovery culture; Figure 8 The polysaccharide positioning of the longitudinal section of the tuber of Pinellia ternata in Experimental Example 3 of the present invention, wherein A is a 3% sucrose leaf primordium, B is a 3% sucrose apical meristem, C is a 3% sucrose tuber middle, D is a 9% sucrose leaf primordium, E is a 9% sucrose apical meristem, F is a 9% sucrose tuber middle, LP is a leaf primordium, AM is an apical meristem, Sg is a starch granule, MC is a mucus cell, the arrow is a crystal cluster bundle contained in the mucus cell, Ve is a vessel, and Vb is a vascular bundle; Fig. 9 The polysaccharide positioning of the longitudinal section of the tuber of Pinellia ternata in Experimental Example 3 of the present invention, wherein A is a 3% sucrose + 6% mannitol leaf primordium, B is a 3% sucrose + 6% mannitol apical meristem, C is a 3% sucrose + 6% mannitol tuber middle, D is a 9% sucrose + 2% mannitol leaf primordium, E is a 9% sucrose + 2% mannitol apical meristem, F is a 9% sucrose + 2% mannitol tuber middle, LP is a leaf primordium, AM is an apical meristem, Sg is a starch granule, MC is a mucus cell, the arrow is a crystal cluster bundle contained in the mucus cell, Ve is a vessel, and Vb is a vascular bundle; Fig.10 The polysaccharide positioning of the longitudinal section of the tuber of Pinellia ternata in Experimental Example 3 of the present invention, wherein A is the leaf primordium after preservation with 3% sucrose, B is the middle part of the tuber after preservation with 3% sucrose, C is the dead plant after recovery with 3% sucrose, D is the dead plant after recovery with 3% sucrose, E is the surviving plant after recovery with 3% sucrose, F is the surviving plant after recovery with 3% sucrose, LP is the leaf primordium, AM is the apical meristem, Sg is the starch granule, and MC is the mucus cell; Fig.11 The RAPD detection results in Experimental Example 3 of the present invention, wherein A is the map of 9% sucrose amplified by primer Em2, B is the map of 3% sucrose + 6% mannitol amplified by primer Em2, C is the map of 9% sucrose amplified by primer Lect7, and D is the map of 3% sucrose + 6% mannitol amplified by primer Lect7; Fig.12 These are the ISSR detection results in Experimental Example 3 of the present invention, wherein A is the map of 9% sucrose amplified by primer 881, B is the map of 3% sucrose + 6% mannitol amplified by primer 881, C is the map of 9% sucrose amplified by primer 854, and D is the map of 3% sucrose + 6% mannitol amplified by primer 854. DETAILED DESCRIPTION

[0012] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0013] A method for short-term storage and growth recovery of pinellia ternata, comprising the following steps: Step 1: Material culture and disinfection: The Pinellia ternata seed stems were sown in a flower pot to germinate and grow to obtain the Pinellia ternata plants. The common petioles of the Pinellia ternata plants were removed and treated with 75% ethanol for 10 seconds. The common petioles were taken out and placed in 0.1% mercuric chloride with 1 drop of Tween 80 for 5 minutes. The common petioles were taken out and placed in 2% sodium hypochlorite for 8 minutes. Finally, the common petioles were taken out and rinsed with sterile water for 3 times. Step 2: Second-generation test tube seedlings and second-generation tuber culture: The petiole obtained in step 1 is cut into small segments of 0.5-2 cm, inoculated into a medium of 1 / 2MS+3 wt % sucrose+0.8 wt % agar, pH 5.8, cultured under weak light conditions of 500-1000 lux to obtain an independent and complete small tuber, transferred to light conditions to allow the small tuber to germinate into seedlings, the obtained small tuber is the first-generation tuber, and the obtained seedling is the first-generation test tube seedling, after cutting off the stems and leaves of the first-generation tuber, transferred to a fresh medium of 1 / 2MS+3wt % sucrose+0.8 wt % agar, pH 5.8, the first-generation tuber germinates into seedlings again and grows up, and the second-generation test tube seedlings and second-generation tubers are obtained; Step 3: Short- to medium-term storage of third-generation tubers: The second-generation tubers were transferred to a culture bottle filled with 1 / 2MS fresh solid culture medium, cultured in the dark until the buds were 2 cm high, and then transferred to light conditions to grow into complete plants. The light conditions were light intensity of 1000-2000LUX and light duration of 12-16h / d to obtain third-generation tubers and third-generation test tube seedlings. When the stems and leaves withered and the tubers were in a dormant state, the third-generation tubers and the culture bottles were placed in the dark at room temperature for 450 days. When it is necessary to preserve the Pinellia germplasm resources in the short to medium term, repeat steps 2 to 3 with the obtained complete plants; Step 4: Resume growth: After 450 days, add 10 ml of 1 / 2MS + 3 wt % sucrose, pH 5.8 liquid culture medium to the culture bottle, culture at 25°C in the dark, and when the tubers germinate to a bud length of 2-4 cm, switch to 12 h light and 12 h dark alternation to obtain complete plants, with a light intensity of 1000-2000 lux; Step 5: Expand reproduction: The petioles of the intact plants obtained in step 4 were cut into small segments of 0.5-2 cm, and inoculated into 1 / 2MS+3 wt % sucrose+0.8 wt % agar, pH 5.8 culture medium to obtain an independent and intact small tuber and germinate it into a seedling.

[0014] Experimental Example 1: Using Pinellia tubers stored at room temperature: 1. Material cultivation and disinfection The Pinellia ternata seed stems were sown in pots to germinate and grow to obtain the Pinellia ternata plants. The petioles were cut into small sections, treated with 75% ethanol for 10 seconds, treated with 0.1% mercuric chloride with 1 drop of Tween 80 for 5 minutes, treated with 2% sodium hypochlorite for 8 minutes, and rinsed with sterile water 3 times.

[0015] 2. Obtaining different generations of test tube tubers and test tube seedlings The obtained Pinellia ternata petiole is cut into a small section of 0.5-2 cm and inoculated into 1 / 2MS+3 wt% sucrose+0.8 wt% agar, pH5.8 medium under weak light conditions to obtain an independent and complete small tuber and germinate into a seedling, thereby obtaining a first-generation tuber and a first-generation test tube seedling. The stems and leaves are cut off, and the first-generation tuber is transferred to a fresh medium of 1 / 2MS+3% sucrose+0.8% agar, pH5.8, and the tuber germinates into a seedling again and grows up, thereby obtaining a second-generation tuber and a second-generation test tube seedling; the third-generation, fourth-generation, and fifth-generation tubers and test tube seedlings can be obtained in this way.

[0016] 3. Storage of tubers of different generations at room temperature The tubers of different generations obtained by culture were transferred to 1 / 2MS fresh solid culture medium. When the buds grew to 2 cm in height under dark conditions, they were transferred to 1000-2000 LUX and 12-16h / d light conditions to grow into complete plants. When the stems and leaves withered and the tubers were in a dormant state, the tubers and the culture bottles were placed in the dark at room temperature for 450 days.

[0017] 4. Regeneration plant culture Pour 10 ml of 1 / 2MS + 3 wt % sucrose, pH 5.8 liquid culture medium into the tube tube culture bottles of different generations after preservation under sterile operation on the clean bench, and culture them in the dark at 25°C. When the tubers germinate and the buds grow to about 2 cm, switch to the light intensity of 1000-2000 LUX and the light duration of 12-16h / d to culture the regenerated plants. After 30 days of recovery, observe and count the status after recovery culture. Figure 1 The comparison of room temperature storage effects of tubers of different generations is shown in Table 1.

[0018] After 450 days of storage, the first generation tuber culture medium lost the most water, the culture medium shrank into a thin layer, and the tubers were scattered on the culture medium. The third, fourth, and fifth generation tuber culture medium shrank into a ball, and the tubers also gathered into a ball (see Figure 2 ). As shown in Table 1, after the 1st, 3rd, 4th and 5th generation test tube tubers were stored at room temperature in the dark for 450 days, nutrient solution was directly added to the culture bottle to resume growth. The results showed that the 1st generation tubers could not survive when stored at room temperature. The reason was that the 1st generation tubers were small in size and weak in resistance and could not withstand the extreme drought. With the increase of tuber generations, the survival rate first increased and then decreased. Among them, the 3rd generation tubers had the best preservation effect and the highest seedling rate, reaching 93.33%. With the increase of generations, the seedling rate after the resumption of culture gradually decreased. The 4th and 5th generations were 58.33% and 34.44% respectively, and the number of petioles were 1.93, 1.00 and 0.48 respectively. This shows that with the increase of generations, the test tube tubers also tend to age, the seedling rate decreases, the plants are short, and the number of stems and leaves extracted also decreases (see Figure 3 and Figure 4 ). Therefore, after the second-generation tubers are inoculated in the fresh culture medium, they are cultured in the dark at 25°C. When the tubers germinate and the buds grow to about 2 cm, they are transferred to a light intensity of 1000-2000 lux and a light duration of 12-16 h / d to culture them into seedlings. The third-generation test tube seedlings and test tube tubers are obtained. After their stems and leaves wither, they are transferred to room temperature in the dark for storage, which is the best preservation method.

[0019] 5. Expansion and reproduction of regenerated plants after preservation: The petioles of the regenerated plants after the preservation of the third-generation tubers obtained in Example 1 were cut into small sections of 0.5-2 cm and inoculated into 1 / 2MS + 3 wt % sucrose + 0.8 wt % agar, pH 5.8 culture medium, and cultured according to "2" in this experimental example to obtain an independent and complete small tuber and germinate it into a seedling, thereby obtaining a large number of Pinellia test tube seedlings.

[0020] 6. Extraction of genomic DNA from regenerated plants and detection of genetic stability of regenerated plants: (1) Extraction of genomic DNA from regenerated plants: Tender leaves of 10 Pinellia test tube seedlings were randomly selected from the regenerated plants with the highest survival rate after recovery (3rd generation tubers in room temperature and darkness), and total DNA was extracted using the improved CTAB method. 2 μL of DNA was detected by 0.8% agarose gel electrophoresis, and the DNA samples were stored at -20°C for later use.

[0021] (2) RAPD and ISSR-PCR system amplification procedures: In this experiment, six primers with clear amplification bands, good polymorphism and good stability were selected from RAPD and ISSR primers for analysis of genetic stability, and the optimal annealing temperature was selected according to its theoretical annealing temperature (see Table 2). The PCR amplification system is: a total volume of 25 μL, containing 12.5 μL 2× Taq PCR Master Mix‖ (containing dye solution), 9.5 μL ddH2O, 1 μL primer, and 2 μL DNA mixed evenly.

[0022] Amplification program: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 50.2-60.6°C for 45 s, extension at 72°C for 2 min, 35 cycles, extension at 72°C for 7 min, and storage at 4°C.

[0023] (3) PCR product detection: After amplification, 5 μL of amplified product was electrophoresed on a 1.5% agarose gel. 2 μL of green fluorescent nucleic acid dye was added to every 80 mL. The electrophoresis buffer was 0.5×TBE. DL 2000 Marker was used as the molecular weight standard. The voltage was 110 V and the electrophoresis was performed for 90 min. After the electrophoresis, images were taken on a gel imager and photographs were taken.

[0024] (4) ISSR and RAPD detection of genetic stability of regenerated plants after three generations of test tube tube storage: The RAPD and ISSR techniques were used to detect the DNA of 10 regenerated plants of the third generation of P. ternata tubers stored at room temperature. A total of 570 electrophoretic patterns were amplified, and no variant bands were generated (see Figure 5 ), so the method of the present invention can maintain the genetic stability of Pinellia ternata, and is a simple and efficient medium- and short-term preservation technology for Pinellia ternata.

[0025] Experimental Example 2: Using the low temperature storage of Pinellia tubers: 1. Low temperature storage of mature tubers The petiole segments of the test tube seedlings of about 1 cm were inoculated in 1 / 2MS medium. After the tubers matured (bud tips grew), they were placed in a 4°C refrigerator together with the original medium for 360 days, and the status of the tubers was counted every 40 days. After storage, the tubers were restored in 1 / 2MS medium, and the germination and seedling status of the tubers were counted after 20 days of culture.

[0026] 2. First generation test tube seedlings (with tubers) stored at low temperature The petiole segments of the test tube seedlings of about 1 cm were inoculated into 1 / 2MS culture medium. After the tubers became seedlings (first generation test tube seedlings), they were placed in a 4°C refrigerator together with the original culture medium for 360 days. The subsequent operations were the same as "1" in this experimental example.

[0027] 3. Restoration of culture conditions and observation indicators After the storage, the tubers were transferred to 1 / 2MS culture medium and placed in an artificial climate box at 25°C for recovery culture, with a light duration of 12h / d and a light intensity of about 2000lx.

[0028] During the low-temperature storage of mature tubers, the number of tubers with sprouting buds, the sprouting rate, the number of rooted tubers, the rooting rate, the number of tubers with seedlings, the tuber status, and the seedling rate and petiole number after storage were observed and counted; during the low-temperature storage of the first-generation test tube seedlings (with tubers), the number of fresh petioles, the number of dried petioles, the petiole drying rate, and the seedling rate and petiole number after storage were observed and counted.

[0029] 4. Data statistics and analysis This experiment adopted a single-factor experimental design, with 5 replicates for each treatment and 6 tubers or petiole segments for each replicate. Excel software was used for data statistics, and IBM SPSS26.0 Duncan's new multiple range method was used for variance analysis.

[0030] (1) Effect of preservation of mature tubers under low temperature environment After the tubers were cultivated and matured, they were stored at 4℃ together with the original culture medium. It was found that the tuber growth rate was very slow (see Table 3). There was no sprouting of the bud tips within 40 days. The sprouting rate was 10% after 80 days of storage. The sprouting bud tips were thin and dull in color. After 160 days of storage, the sprouting rate reached 26.7%, and the state of the stem tips was the same as at 80 days. In addition, the stem tips that sprouted in the early stage were partially dried up and did not grow normally into seedlings. At this time, the culture medium was slightly shrunk. After 240 days of storage, the sprouted stem tips were all dried up, and no new stem tips were produced. At this time, the culture medium was relatively shrunk (see Figure 6 After the storage, the tubers were restored and cultured. After one week of culture, no stem tip sprouted. After one month, 36.7% of the tubers sprouted into seedlings, and each tuber had an average of 3.27 petioles (see Figure 7 Middle A).

[0031] (2) Effect of cryopreservation of first generation test tube seedlings (with tubers) The first generation of Pinellia ternata test tube seedlings (with tubers) were stored at low temperature, and it was found that the petiole drying rate was very slow in the early stage of storage (see Table 4). The petiole drying rate in the first 40 days was 7.06%, indicating that the test tube seedlings had strong cold resistance. At this time, the culture medium was at a lower temperature and the water loss rate was slow, so it could still provide nutrients and water for the test tube seedlings. However, as the storage time increased, the culture medium gradually dried up and the petiole drying rate accelerated. After 120 days of storage, the petiole drying rate increased rapidly to 45.88%. After 200 days of storage, the petioles had all dried up. In addition, because the water loss of the culture medium was relatively serious at this time (see Figure 6 After the storage, the tubers were restored and cultured. After one week of culture, the sprouting rate of the stem tip was 33.30%. After one month, 50.00% of the tubers germinated into seedlings, and each tuber had an average of 2.2 petioles (see Figure 7 Middle B).

[0032] Experimental Example 3: Using low temperature + high osmotic pressure to store the tubers of Pinellia ternata: 1. Materials The petiole segment of the test tube seedling of about 1 cm is planted in 1 / 2MS culture medium to form spherical tubers and grow bud tips, which are mature tubers.

[0033] 2. High osmotic pressure combined with low temperature to preserve mature tubers The mature tubers were inoculated into 1 / 2MS medium containing different concentrations of sucrose (3%, 6%, 9%) and mannitol (0%, 2%, 4%, 6%), and stored together with the original medium in a 4°C refrigerator for 360 days. The status of the tubers was counted every 40 days. After storage, the tubers were restored in 1 / 2MS medium, and the germination and seedling status of the tubers were counted after 20 days of culture.

[0034] 3. Observation and determination of morphological indicators of regenerated plants The tubers after delayed growth preservation were inoculated on the recovery medium and grown into seedlings under the conditions of 25℃, light intensity of 1500lx and light time of 12h / d. After recovery culture, the seedling rate, petiole number / tuber, leaf shape, leaf color and average petiole length of different treatments were counted.

[0035] 4. Histochemical localization of starch granules Paraffin tissue sectioning technique and modified periodic acid-Schiff reagent were used for preparation and polysaccharide staining, and the samples were observed and photographed under a microscope. The differences in starch quantity and distribution, the number of mucilage cells, and the number of intracellular crystal clusters were compared between plants preserved with 3% sucrose and regenerated plants, and between tubers with high survival rate and tubers with low survival rate (3% sucrose and 9% sucrose, 3% sucrose + 6% mannitol and 9% sucrose + 2% mannitol) after recovery.

[0036] 5. Extraction and detection of genomic DNA from regenerated plants The young leaves of 10 Pinellia test tube seedlings were randomly selected from the two treatments with the highest survival rate after recovery growth. The total DNA was extracted by the improved CTAB method. 2 μL of DNA was detected by 0.8% agarose gel electrophoresis. The DNA samples were stored at -20°C for future use.

[0037] 6. Detection of genetic stability of regenerated plants (1) RAPD and ISSR-PCR amplification procedures In this experiment, 6 primers with clear amplification bands, good polymorphism and good stability were selected from 12 RAPD and ISSR primers for analysis of genetic stability, and the optimal annealing temperature was selected based on its theoretical annealing temperature (see Table 5). The PCR amplification system was: a total volume of 25 μL, containing 12.5 μL 2× Taq PCR Master Mix (containing dye) solution, 9.5 μL ddH 2 O, 1 μL primer, and 2 μL DNA were mixed evenly.

[0038] Amplification program: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 50.2-60.6°C for 45 s, extension at 72°C for 2 min, 35 cycles, extension at 72°C for 7 min, and storage at 4°C.

[0039] (2) PCR product detection After amplification, 5 μL of amplified product was electrophoresed on a 1.5% agarose gel. 2 μL of green fluorescent nucleic acid dye was added to every 80 mL. The electrophoresis buffer was 0.5×TBE. DL 2000 Marker was used as the molecular weight standard. The voltage was 110 V and the electrophoresis was performed for 90 min. After the electrophoresis, images were taken on a gel imager and photographs were taken.

[0040] 7. Data statistics and analysis This experiment adopted a single-factor experimental design, with 5 replicates for each treatment and 6 tubers for each replicate. Data were statistically analyzed using Excel software, and variance analysis was performed using IBM SPSS26.0 Duncan's new multiple range method. The electrophoresis bands of the amplified DNA were manually identified, with the bands in the electrophoresis being marked as "1" and the absence of the bands being marked as "0". The increase or loss of bands between the same treatments was considered a variant band.

[0041] 8. Results and Analysis (1) Growth of regenerated plants after preservation using different growth-delaying methods The in vitro tubers were inoculated on the culture medium after 360 days of storage and recovered at 25℃. The results showed that among the different sucrose concentration treatments, the seedling rate was the highest with 9% sucrose and the lowest with 3% sucrose. However, the plant height of 9% sucrose was significantly lower than that of 3% sucrose (see Figure 7 C and E), it may be that with the increase of sucrose concentration, the osmotic pressure of the culture medium increased, and the tuber was hindered from absorbing water and nutrients, thus inhibiting the growth of the plant. In the treatment of 3% sucrose and different concentrations of mannitol added at the same time, the seedling rate increased with the increase of mannitol concentration, among which the seedling rate of 6% mannitol was significantly higher than that of other concentrations. This is because the combination of sucrose and high-concentration mannitol maintained a balance with each other without forming osmotic stress, which improved the seedling rate of the regenerated plants, but the specific mechanism remains to be studied. In order to further explore the effect of sucrose on the preservation effect, different concentrations of sucrose were added to the same 2% mannitol culture medium. It can be seen from Table 6 that the seedling rate of 9% sucrose + 2% mannitol was significantly lower than that of other treatments, and the preservation effect was not good when high-concentration sucrose and mannitol were combined. It may be that high-concentration sucrose and mannitol formed double osmosis, further inhibiting the growth of the plant (see Figure 7 In J), too high osmotic pressure is toxic to plants. In summary, the two treatments of 9% sucrose and 3% sucrose + 6% mannitol in the test tube seedlings after preservation have high seedling rates and good preservation effects. They are suitable methods for delaying the growth and preservation of in vitro tubers of Pinellia ternata. Compared with the normal growing plants, the morphological performance of the regenerated plants is consistent, as shown in Table 6.

[0042] (2) Histological and starch granule changes of in vitro tubers before and after storage A. Comparison of tubers with high survival rate after recovery culture (9% sucrose, 3% sucrose + 6% mannitol) and tubers with low survival rate (3% sucrose, 9% sucrose + 2% sucrose) Depend on Figure 8 As shown in Table 7, in the 3% sucrose treatment, there were fewer leaf primordia, more starch granules, starch granules in the apical meristem, more mucilage cells in the middle of the tuber, and more crystal clusters. Compared with 9%, 3% contained more starch granules; while the 9% sucrose tubers had more leaf primordia, which were pointed, with a small amount of starch granules in the leaf primordia cells, more crystal clusters in the leaf primordia, no starch granules in the apical meristem, fewer mucilage cells, and more vessels. In summary, the starch granule content of plants recovered under 9% sucrose was less, which may be due to the high concentration of sucrose increasing the osmotic pressure of the culture medium, forming a serious injury stress, effectively inhibiting its recovery growth, so the starch granule content was less; but the number of leaf primordia was more, which was related to the large number of stems and leaves in its morphological statistics. After recovery culture with 3% sucrose, it was found that its plant height was significantly higher than that of 9% sucrose, which may be due to the large number of starch granules and vigorous plant growth.

[0043] Note: “+++” indicates the largest number of starch granules and mucus cells; “++” indicates the average content of starch granules and mucus cells; “+” indicates the smallest number of starch granules and mucus cells; “-” indicates the absence of starch granules and mucus cells, the same below By Fig. 9 As shown in Table 8, in the regenerated plants with the best preservation effect of 3% sucrose + 6% mannitol, there were more leaf primordia, no starch grains, and fewer crystal clusters; there were no starch grains in the apical meristem, fewer mucilage cells, fewer crystal clusters, more starch grains in the middle of the tuber, and fewer vascular bundles; while in 9% sucrose + 2% mannitol: there were more crystal clusters and starch grains in the leaf primordia, starch grains in the apical meristem, more crystal clusters in mucilage cells, and more vascular bundles. Overall, 3% sucrose + 6% mannitol contained more starch grains. In summary, the starch granule content was basically consistent with the survival rate. 3% sucrose + 6% mannitol had a high survival rate, good growth status, and high starch granule content; while high concentrations of sucrose and mannitol combined treatment formed a double stress, and the hyperosmotic environment inhibited its absorption of nutrients and water, which hindered its growth, so the starch granule content of 9% sucrose + 2% mannitol was less.

[0044] B. Comparison of starch distribution in tubers after storage and resumption of growth with 3% sucrose like Fig.10 Table 9 shows that the tubers preserved with 3% sucrose and the dead tubers after recovery have fewer leaf primordia, less starch granules, no crystal clusters, and no vascular bundles. This may be due to the long storage time, which consumes a lot of nutrients to preserve vitality, resulting in a decrease in starch granule content. The regenerated plants recovered with 3% sucrose have the highest starch granule content. The surviving tubers have obtained good nutrients on the new culture medium, so they have more starch granules, a wide distribution of mucilage cells, and scattered vascular bundles.

[0045] 1.4 Analysis of genetic stability of regenerated plants at the molecular level 1.4.1 RAPD detection RAPD detection was performed on the test tube seedlings after being stored in the two treatments with higher survival rates, 9% sucrose and 3% sucrose + 6% mannitol. The three selected primers were used for amplification. The results showed that the three primers produced a total of 420 RAPD bands in 20 samples, with an average of 140 bands amplified by each primer. There was no loss or increase of markers in the two treatments using the selected primers (see Fig.11 ), indicating that this method can maintain the genetic stability of Pinellia ternata.

[0046] 1.4.2 ISSR detection ISSR amplification was performed on the genomic DNA treated above after delayed growth preservation. Using the extracted samples as templates, three primers 881, 835 and 854 were selected for ISSR-PCR amplification. A total of 720 bands were obtained, and no interfering bands were generated (see Fig.12 ), no somaclonal variation was detected.

[0047] RAPD and ISSR techniques were used to detect the DNA of 20 plants of Pinellia ternata after delayed growth and preservation. A total of 1140 electrophoresis patterns were amplified, and no variant bands were produced, indicating that this method can maintain the genetic stability of Pinellia ternata.

[0048] Summarize: Experimental Example 1: Storage of Pinellia tubers at room temperature: The second generation tubers were transferred to 1 / 2MS fresh solid culture medium, and when the buds grew to 2 cm in height under dark conditions, they were transferred to 1000-2000 LUX and 12-16h / d light conditions to grow into complete plants. When the stems and leaves withered and the tubers were dormant, the tubers and culture bottles were placed in the dark and room temperature for 450 days. After the growth was restored, the survival rate was 93.33%.

[0049] Experimental Example 2: Preservation of Pinellia tubers at low temperature: (1) Cryopreservation of mature tubers: The petiole segments of test tube seedlings of about 1 cm in diameter were inoculated in 1 / 2MS culture medium. After the mature tubers (bud tips grew), they were placed in a 4°C refrigerator together with the original culture medium for 360 days. After the growth resumed, the survival rate was only 36.7%.

[0050] (2) Low temperature storage of first-generation test tube seedlings (with tubers): After inducing mature tubers, they were allowed to germinate and become first-generation test tube seedlings. They were then placed in a 4°C refrigerator together with the original culture medium for 360 days. After they resumed growth, their survival rate was only 50.00%.

[0051] Experimental Example 3 is to preserve the tubers of Pinellia ternata by low temperature + high osmotic pressure: The mature tubers (with shoot tips) were transferred to 1 / 2MS medium containing different concentrations of sucrose (3%, 6%, 9%) and mannitol (0%, 2%, 4%, 6%) and stored in a 4°C refrigerator for 360 days. After the growth resumed, the survival rate of the 3% sucrose + 6% mannitol high osmotic pressure treatment was as high as 96.67%.

[0052] From the experimental results, the survival rates of mature tubers preserved at low temperature and first-generation test tube seedlings (with tubers) preserved at low temperature in Experimental Example 2 were 36.7% and 50%, respectively, and the preservation effect was not ideal. In Experimental Example 3, the survival rate of 3% sucrose + 6% mannitol high osmotic pressure treatment in the preservation of Pinellia tubers at low temperature combined with high osmotic pressure was as high as 96.67%, and the survival rate of the third-generation tubers preserved at room temperature was as high as 93.33%, and the preservation effect was very good. However, considering the cost of preservation, low temperature combined with high osmotic pressure preservation requires the use of low temperature equipment, and the long-term operation of low temperature equipment consumes a lot of electricity. In addition, this method also requires the addition of some drugs. In Experimental Example 1, the three-generation tubers can be stored at room temperature without any equipment and can be stored under dark conditions at room temperature. From the perspective of storage time, the low-temperature storage is only the storage result of 360 days. The three-generation tubers still have a very high survival rate after 450 days of storage, and no transfer is required when resuming growth. Only 1 / 2MS liquid culture medium needs to be added to them to germinate and form seedlings. Therefore, using the method of Experimental Example 1, the three-generation tubers stored at room temperature not only have a high survival rate, high genetic stability and simple operation, but also save energy.

Claims

1. A method for short-term storage and recovery of Pinellia ternata, characterized in that The steps include: Step 1: Material culture and disinfection: The Pinellia ternata seed stems were sown in a flower pot to germinate and grow to obtain the Pinellia ternata plants. The common petioles of the Pinellia ternata plants were removed and treated with 75% ethanol for 10 seconds. The common petioles were taken out and placed in 0.1% mercuric chloride with 1 drop of Tween 80 for 5 minutes. The common petioles were taken out and placed in 2% sodium hypochlorite for 8 minutes. Finally, the common petioles were taken out and rinsed with sterile water for 3 times. Step 2: Second-generation test tube seedlings and second-generation tuber culture: The petiole obtained in step 1 is cut into small segments of 0.5-2 cm, inoculated into a medium of 1 / 2MS+3 wt % sucrose+0.8 wt % agar, pH 5.8, cultured under weak light conditions to obtain an independent and complete small tuber, transferred to light conditions to allow the small tuber to germinate into seedlings, the obtained small tuber is the first-generation tuber, and the obtained seedling is the first-generation test tube seedling, after cutting off the stems and leaves of the first-generation tuber, transferred to a fresh medium of 1 / 2MS+3 wt % sucrose+0.8 wt % agar, pH 5.8, the first-generation tuber germinates into a seedling again and grows up, to obtain a second-generation test tube seedling and a second-generation tuber; Step 3: Short- to medium-term storage of third-generation tubers: The second-generation tubers were transferred to culture bottles filled with 1 / 2MS fresh solid culture medium, cultured in dark conditions until the buds reached 2 cm in height, and then transferred to light conditions to grow into complete plants, thereby obtaining third-generation tubers and third-generation test tube seedlings. When the stems and leaves withered and the tubers were in a dormant state, the third-generation tubers and culture bottles were placed in the dark and stored at room temperature for 450 days; When it is necessary to preserve the Pinellia germplasm resources in the short to medium term, repeat steps 2 to 3 with the obtained complete plants; Step 4: Resume growth: After 450 days, add 10 ml of 1 / 2MS + 3 wt % sucrose, pH 5.8 liquid culture medium to the culture bottle, culture at 25°C in the dark, and when the tubers germinate to a bud length of 2-4 cm, switch to 12 h light and 12 h dark alternation to obtain complete plants, with a light intensity of 1000-2000 lux; Step 5: Expand reproduction: The petioles of the intact plants obtained in step 4 were cut into small segments of 0.5-2 cm, and inoculated into 1 / 2MS+3 wt % sucrose+0.8 wt % agar, pH 5.8 culture medium to obtain an independent and intact small tuber and germinate it into a seedling.

2. The method for short-term preservation and growth recovery of Pinellia ternata germplasm resources as claimed in claim 1, characterized in that: The weak light condition in step 2 is 500-1000 lux.

3. The method for short-term storage and growth recovery of Pinellia ternata as claimed in claim 1 or 2, characterized in that: The lighting conditions described in step 3 are a light intensity of 1000-2000LUX and a lighting duration of 12-16h / d.

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