Rapid tissue culture propagation method of polygonatum sibiricum

Abstract

This invention relates to a method for rapid propagation of Polygonatum sibiricum by tissue culture. The method comprises the following steps: (1) Polygonatum sibiricum seeds are subjected to low-temperature stratification, then soaked in a mixed solution of Polygonatum sibiricum and ethephon, sterilized, and inoculated into MS medium to induce seed germination. The primary rhizomes obtained are then used as explants after removing the seeds and radicles; (2) The explants are inoculated into an adventitious bud induction medium and cultured for 30 days to induce adventitious bud differentiation; the induced adventitious buds are cut into single buds and transferred to a new adventitious bud induction medium to generate new adventitious buds. The adventitious bud induction medium is an MS solid medium containing 6-BA and NAA; (3) The adventitious buds are transferred to a rooting medium and cultured in suspension for 60 days to induce rooting. The rooting medium is an MS liquid medium containing IAA, NAA and 6-BA; (4) The seedlings are hardened off and then transplanted. The method of this invention can achieve an adventitious bud induction rate of over 90%, a rooting rate of over 72%, and a transplant survival rate of over 85%. It is simple to operate, has a short propagation cycle, and a high propagation coefficient, providing reliable technical support for the industrialized seedling production of Polygonatum sibiricum.

Description

Technical Field

[0001] This invention relates to the field of tissue culture technology for medicinal and edible plants, specifically a rapid propagation method for Polygonatum sibiricum tissue culture. Background Technology

[0002] Polygonatum ( Polygonatumsibiricum Polygonatum sibiricum is a perennial herb belonging to the genus Polygonatum in the family Liliaceae. It is an important medicinal plant resource in my country, possessing the effects of tonifying qi and nourishing yin, strengthening the spleen and moistening the lungs, and benefiting the kidneys. It is widely used in the field of traditional Chinese medicine. With the continuous increase in market demand, wild resources of Polygonatum sibiricum are becoming increasingly depleted, and artificial cultivation has become an important way to meet market demand.

[0003] However, the artificial cultivation of Polygonatum faces numerous technical bottlenecks. On the one hand, traditional tuber propagation methods result in slow growth, low propagation coefficients, and low survival rates, severely restricting the scale of Polygonatum production. On the other hand, traditional seed propagation methods suffer from long seed dormancy periods, weak germination ability, and susceptibility to mutation, making it difficult to meet the large-scale demand for high-quality seedlings in factory-scale seedling production. Therefore, developing efficient and stable rapid propagation technology for Polygonatum through tissue culture is of great significance for solving the shortage of Polygonatum seedlings and promoting the sustainable development of the Polygonatum industry.

[0004] Current tissue culture techniques for Polygonatum sibiricum primarily use rhizomes and leaves as explants, which presents challenges such as difficulties in explant sterilization, high contamination rates, and unstable adventitious bud induction efficiency. Furthermore, existing techniques rarely utilize the primary rhizomes that emerge after seed germination, failing to fully leverage their advantages as explants. Therefore, there is an urgent need to develop a rapid propagation method for Polygonatum sibiricum tissue culture that offers stable explant sources, high induction efficiency, high rooting rate, and high transplant survival rate. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a rapid tissue culture propagation method for Polygonatum sibiricum. This method overcomes the deficiencies of existing Polygonatum sibiricum propagation techniques, such as slow growth, low propagation coefficient, long seed dormancy period, weak germination ability, difficulty in explant sterilization during tissue culture, and unstable induction efficiency.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A rapid propagation method for Polygonatum sibiricum by tissue culture, characterized by comprising the following steps: Step 1: The seeds of Polygonatum sibiricum after low-temperature stratification are sterilized and then germinated using germination medium to obtain explants; Step 2: Transfer the explants obtained in Step 1 into an adventitious bud induction medium to obtain adventitious buds of Polygonatum sibiricum; cut the induced adventitious buds into single buds and transfer them into a new adventitious bud induction medium to generate new adventitious buds. Step 3: Transfer the new adventitious buds obtained in Step 2 into the rooting medium for rooting culture to obtain Polygonatum tissue culture seedlings; Step 4: After hardening off the rooted tissue culture seedlings obtained in Step 3, transplant them into the substrate.

[0007] Based on the above plan, The conditions for the low-temperature stratification treatment in step 1 are as follows: mix Polygonatum seeds with moist sand and culture them at 1-10℃; The sterilization process described in step 1 is as follows: Soak the Polygonatum seeds in sodium hypochlorite solution for 0.5-3 hours, and then soak them in mercuric chloride solution for 10-30 minutes. The germination culture conditions described in step 1 are: 24-26℃, dark culture.

[0008] Based on the above plan, The conditions for the low-temperature stratification treatment in step 1 are as follows: the mass ratio of Polygonatum seeds to moist sand is 1:(2-10) (preferably 1:4), and the seeds are cultured at 2-5℃ for a period of 30 days.

[0009] The sterilization process described in step 1 is as follows: Soak the Polygonatum seeds in sodium hypochlorite solution for 0.5-2 h (more preferably 1 h), and then soak them in mercuric chloride solution for 15-25 min (more preferably 15 min).

[0010] Based on the above plan, The adventitious bud induction conditions described in step 2 are: temperature 18-30℃, alternating light (10-24) h / dark (5-12) h.

[0011] Based on the above plan, The adventitious bud induction conditions described in step 2 are: temperature 20-28℃ (more preferably 22℃), light (12-18) h / dark (6-9) h alternation (more preferably light 16 / dark 8 h alternation).

[0012] Based on the above plan, The germination medium is a solid MS medium containing sucrose, agar powder, plant growth regulator GA3 and ethephon, with a pH of 5-7 (preferably 5-6). The adventitious bud induction medium is a solid MS medium containing plant growth regulators 6-BA and NAA, with a pH of 5-7 (preferably 5-6). The rooting medium is a liquid MS medium containing plant growth regulators IAA, NAA and 6-BA, with a pH of 5-7 (preferably 5-6).

[0013] Based on the above plan, The germination medium contains 10-50 g / L of sucrose and 5-15 g / L of agar powder. The concentration of 6-BA in the adventitious bud induction medium is 1-10 mg / L, and the concentration of NAA is 0.1-0.5 mg / L. The rooting medium contains 0.1-2 mg / L of IAA, 0.1-1 mg / L of NAA, and 0.1-1 mg / L of 6-BA.

[0014] Based on the above plan, The germination medium contains 15-30 g / L sucrose (more preferably 30 g / L) and 8-12 g / L agar powder (more preferably 8 g / L). The concentration of 6-BA in the adventitious bud induction medium is 2-6 mg / L (more preferably 4 mg / L), and the concentration of NAA is 0.1-0.2 mg / L (more preferably 0.2 mg / L). The rooting medium contains 0.5-1.5 mg / L (more preferably 0.5 mg / L) IAA, 0.1-0.5 mg / L NAA (more preferably 0.2 mg / L) and 0.1-0.5 mg / L 6-BA (more preferably 0.2 mg / L).

[0015] Based on the above plan, The explants mentioned in step 2 can be treated as follows: after removing the seeds and radicles, the primary rhizomes of Polygonatum are obtained, and wounds are made on the primary rhizomes.

[0016] Based on the above plan, The transplanting process described in step 4 is as follows: select seedlings with roots 2-3 cm long and more than 3 roots for transplanting.

[0017] The rapid propagation method for Polygonatum sibiricum by tissue culture described in this invention has the following beneficial effects: (1) This invention uses the primary rhizomes formed from the germination of Polygonatum seeds as explants, through... Combined treatment with ethephon effectively breaks seed dormancy, yielding sterile explants with a stable source and low contamination rate.

[0018] (2) The present invention screened the optimal adventitious bud induction culture medium formula. Under the conditions of 4 mg / L 6-BA + 0.2 mg / L NAA, the adventitious bud induction rate can reach more than 90%, and the induction period is 30 days, which is significantly better than the existing technology.

[0019] (3) The present invention screened out the optimal rooting culture medium type and formula. In liquid MS medium containing 0.2 mg / L 6-BA + 0.5 mg / L IAA + 0.2 mg / L NAA, the rooting rate can reach more than 72%, the tissue culture seedlings have strong roots, and the induction period is 60 days.

[0020] (4) After hardening and transplanting, the survival rate of tissue culture seedlings of the present invention can reach more than 85%, which provides reliable technical support for the industrialized seedling production of Polygonatum odoratum. Attached Figure Description

[0021] The present invention includes the following figures: Figure 1 Schematic diagram of the process of obtaining explants: (AC) Seed germination of Polygonatum sibiricum, (D) Explants with seeds and radicles removed; Figure 2 Diagrams illustrating the process of inducing adventitious shoots from explants: (A) 1 day after explant inoculation; (B) 10 days after explant inoculation; (C) 20 days after explant inoculation; Figure 3 The process of inducing rooting in adventitious buds: (A) Single adventitious bud inducing rooting; (B) Day 60 of induction of rooting; (C) Enlarged view of a part after rooting. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the accompanying drawings.

[0023] Example 1: Preparation of explants (1) Seed pretreatment: The harvested Polygonatum seeds were placed in a plastic bag for fermentation for one week. After fermentation, the fruits were placed in a nylon mesh bag and rubbed and rinsed until the pericarp and pulp were completely removed. The treated seeds were spread flat on a plate and placed in a dry and ventilated place to air dry. The air-dried seeds were mixed with wet sand at a mass ratio of 1:4 and subjected to low-temperature stratification at 2℃ for 30 days. The diameter of the sand used was 0.05 mm to 0.08 mm. Water was added and fresh sand was replaced regularly, and moldy seeds were checked and removed.

[0024] (2) Explant acquisition: After stratification, the seeds were soaked in a mixed solution of 200 mg / L GA3 and 0.05% ethephon for 24 h. After soaking, the seeds were sterilized in a clean bench: soaked in 10% sodium hypochlorite for 1 h and washed three times with sterile water; then soaked in 0.1% mercuric chloride for 15 min and washed 6-8 times with sterile water. The seeds were dried with sterile filter paper and inoculated onto MS medium, with 20-25 seeds per tray. The seeds were cultured in the dark at 22℃ for 25 days to induce germination. After germination, primary rhizomes were obtained, the seeds and radicles were removed, and wounds were made on the primary rhizomes to serve as explants.

[0025] Example 2 Adventitious bud induction The explants obtained in Example 1 were inoculated into adventitious shoot induction medium for adventitious shoot induction. MS medium (containing 30 g / L sucrose, 8 g / L agar powder, pH 5.8) was used as the basal medium, with a fixed NAA concentration of 0.2 mg / L, and different concentrations of 6-BA (2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L) were added. Ten bottles were inoculated for each treatment, with 5 explants evenly inoculated in each bottle, and each treatment was set up in 3 independent replicates. The culture conditions were 24±2℃, 16 h light / 8 h dark, and cultured for 30 days.

[0026] The adventitious bud induction rate was statistically analyzed, and the results are shown in Table 1. The highest adventitious bud induction rate (90.67%) was observed at a 6-BA concentration of 4 mg / L. Seedlings showed good growth, with most leaves being dark green, more than two-thirds of the plants having fully extended leaves, and normal root and stem development. No obvious deformities or abnormal growth were observed. The lowest induction rate (less than 67%) was observed at a 6-BA concentration of 2 mg / L, with some plants exhibiting growth stagnation and bulb browning. When the 6-BA concentration increased to 5 mg / L or 6 mg / L, the induction rate decreased to 80.67% and 78.00%, respectively, and some plants showed phenotypes such as yellowing leaf tips and leaf curling. Therefore, the optimal adventitious bud induction medium is MS + 4 mg / L 6-BA + 0.2 mg / L NAA.

[0027] Table 1. Effects of different concentrations of 6-BA on adventitious shoot induction rate ; Example 3 Rooting Induction The vigorous and robust subculture seedlings with well-developed adventitious buds from Example 2 were used as culture material and transferred to rooting medium for suspension culture. MS medium (liquid medium containing 30 g / L sucrose, pH 5.8) was used as the basal medium, with a fixed 6-BA concentration of 0.2 mg / L. Nine treatments were established, consisting of different concentrations of IAA (0.5 mg / L, 1.0 mg / L, 1.5 mg / L) and NAA (0.2 mg / L, 0.4 mg / L, 0.6 mg / L). Each treatment was administered in 10 culture flasks, with one adventitious bud inoculated per flask, and each treatment was replicated three times independently. The culture conditions were 24 ± 2 °C, a 16 h light / 8 h dark photoperiod, and cultured for 60 days. Rooting rate was recorded after the culture period.

[0028] The results are shown in Table 2. In MS liquid medium containing 0.2 mg / L 6-BA, 0.5–1.5 mg / L IAA, and 0.2–0.6 mg / L NAA, adventitious buds were able to absorb nutrients and induce rooting. Treatment 1 (MS + 0.2 mg / L 6-BA + 0.5 mg / L IAA + 0.2 mg / L NAA) showed the highest rooting rate, reaching 72.22%, with good root morphology. The rooting rates of the other treatment combinations ranged from 32.22% to 57.78%. During the rooting process, rooting was observed to be slow in the first 25 days of induction culture, gradually entering a rapid rooting stage after 30 days, with the peak rooting period from 30 to 50 days, and obvious root hairs forming by day 60. Considering both rooting rate and root morphology, MS + 0.2 mg / L 6-BA + 0.5 mg / L IAA + 0.2 mg / L NAA was the optimal combination of rooting induction media.

[0029] Table 2 Effects of different hormone combinations on rooting rate ; Example 4 Seedling hardening and transplanting Take the test-tube seedlings from Example 3 that have been rooted for 60 days, select relatively robust plants with good rooting, and harden them off in open culture bottles for 3-5 days. Select plants with roots 2-3 cm long and more than 3 roots, wash off the surface culture medium with clean water, transplant them into the substrate, and culture them at 20-22℃. After 30 days, the transplant survival rate can be counted and can reach over 85%.

[0030] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

Claims

1. A tissue culture rapid propagation method of Polygonatum sibiricum, characterized in that, Includes the following steps: Step 1: Sterilize the Polygonatum seeds after low-temperature stratification using germination medium, and then carry out germination culture to obtain explants; Step 2: Transfer the explants obtained in Step 1 into an adventitious bud induction medium to obtain adventitious buds of Polygonatum sibiricum; cut the induced adventitious buds into single buds and transfer them into a new adventitious bud induction medium to generate new adventitious buds. Step 3: Transfer the new adventitious buds obtained in Step 2 into the rooting medium for rooting culture to obtain Polygonatum tissue culture seedlings; Step 4: After hardening off the rooted tissue culture seedlings obtained in Step 3, transplant them into the substrate.

2. The method for rapid propagation of Polygonatum sibiricum by tissue culture as described in claim 1, characterized in that: The conditions for the low-temperature stratification treatment in step 1 are as follows: mix Polygonatum seeds with moist sand and culture them at 1-10℃; The sterilization process described in step 1 is as follows: Soak the Polygonatum seeds in sodium hypochlorite solution for 0.5-3 hours, and then soak them in mercuric chloride solution for 10-30 minutes. The germination culture conditions described in step 1 are: 24-26℃, dark culture.

3. The method for rapid propagation of Polygonatum sibiricum by tissue culture as described in claim 2, characterized in that: The conditions for the low-temperature stratification treatment in step 1 are as follows: the mass ratio of Polygonatum seeds to moist sand is 1:(2-10), and the mixture is cultured at 2-5℃. The sterilization process described in step 1 is as follows: Soak the Polygonatum seeds in sodium hypochlorite solution for 0.5-2 hours, and then soak them in mercuric chloride solution for 15-25 minutes.

4. The method for rapid propagation of Polygonatum sibiricum by tissue culture as described in claim 1, characterized in that: The adventitious bud induction conditions described in step 2 are: temperature 18-30℃, alternating light (10-24) h / dark (5-12) h.

5. The method for rapid propagation of Polygonatum sibiricum by tissue culture as described in claim 4, characterized in that: The adventitious bud induction conditions described in step 2 are: temperature 20-28℃, alternating light (12-18) h / dark (6-9) h.

6. The method for rapid propagation of Polygonatum sibiricum by tissue culture as described in claim 1, characterized in that: The germination medium is a solid MS medium containing sucrose, agar powder, plant growth regulator GA3 and ethephon, with a pH of 5-7; The adventitious shoot induction medium is a solid MS medium containing plant growth regulators 6-BA and NAA, with a pH of 5-7; The rooting medium is a liquid MS medium containing plant growth regulators IAA, NAA and 6-BA, with a pH of 5-7.

7. The method for rapid propagation of Polygonatum sibiricum by tissue culture as described in claim 6, characterized in that: The germination medium contains 10-50 g / L of sucrose and 5-15 g / L of agar powder. The concentration of 6-BA in the adventitious shoot induction medium is 1-10 mg / L, and the concentration of NAA is 0.1-0.5 mg / L. The rooting medium contains 0.1-2 mg / L of IAA, 0.1-1 mg / L of NAA, and 0.1-1 mg / L of 6-BA.

8. The method for rapid propagation of Polygonatum sibiricum by tissue culture as described in claim 1, characterized in that: The germination medium contains 15-30 g / L of sucrose and 8-12 g / L of agar powder. The concentration of 6-BA in the adventitious shoot induction medium is 2-6 mg / L, and the concentration of NAA is 0.1-0.2 mg / L. The rooting medium contains 0.5-1.5 mg / L of IAA, 0.1-0.5 mg / L of NAA, and 0.1-0.5 mg / L of 6-BA.

9. The method for rapid propagation of Polygonatum sibiricum by tissue culture as described in claim 1, characterized in that: The explants mentioned in step 2 can be treated as follows: after removing the seeds and radicles, the primary rhizomes of Polygonatum are obtained, and wounds are made on the primary rhizomes.

10. The method for rapid propagation of Polygonatum sibiricum by tissue culture as described in claim 1, characterized in that: The transplanting process described in step 4 is as follows: select seedlings with roots 2-3 cm long and more than 3 roots for transplanting.

Images