Method for efficiently and rapidly propagating dioscorea opposita bulbil

Abstract

The invention discloses a method for efficiently and rapidly propagating dioscorea opposita bulbil, and belongs to the technical field of vegetative propagation of plants. According to the method, healthy disease-free bulbil harvested from the iron stick yam in the current year is used as a propagation material, and efficient and rapid propagation of the iron stick yam is achieved through four key steps of surface disinfection, adventitious bud induction pretreatment, adventitious bud induction culture and rooting and seedling culture. The method is easy and convenient to operate, short in propagation period and high in seedling rate, the adventitious bud induction rate reaches up to 89.3%, 5.4 regeneration plants can be cultivated in each bulbil on average, the problems that traditional iron stick yam is low in propagation efficiency, low in propagation coefficient and the like can be effectively solved, and the method is suitable for large-scale propagation of high-quality yam seed sources.

Description

Technical Field

[0001] This invention belongs to the field of plant asexual reproduction technology, specifically relating to a method for efficient and rapid propagation of tubers in Dioscorea opposita. Background Technology

[0002] Iron yam is a unique economic crop that can be used as medicine, vegetable, and grain. It has high nutritional and medicinal value and broad market prospects. Traditionally, iron yam is mostly propagated by cutting tubers into pieces, which has problems such as low propagation coefficient, easy degeneration of seed source, and high risk of carrying diseases and pests, which seriously restricts the large-scale development of the iron yam industry.

[0003] Bulbs, round or oval modified stems developed from lateral buds in the leaf axils of *Dioscorea opposita*, possess strong reproductive capacity, carry few viruses, and maintain the excellent agronomic traits of the parent plant, making them ideal materials for yam propagation. However, the natural dormancy period of bulbils severely limits their application in seedling cultivation. Under natural conditions, bulbils generally require a dormancy period of 4-6 months before sprouting; furthermore, a single bulbil typically germinates only one seedling, which to some extent limits the demand for large-scale propagation of superior *Dioscorea opposita* seedlings. Currently, existing research focuses mainly on breaking bulbil dormancy, with limited research on the induction of adventitious buds outside the incubator and seedling formation. Therefore, based on the selection of superior strains, it is urgent to develop a method for efficient and rapid propagation of *Dioscorea opposita* bulbils to construct an efficient bulbil seedling cultivation technology system and achieve year-round, large-scale production of superior *Dioscorea opposita* seedlings.

[0004] Chinese patent application CN102907239A discloses a breeding method for *Dioscorea opposita*, employing a combination of bulbils and tubers for propagation. The selected bulbils are disinfected and treated with nutrient solution to break their dormancy and accelerate germination. This patented method of using bulbils for propagation ensures the stable inheritance of superior morphological traits. Furthermore, by treating the bulbils, the dormancy period of yam bulbils can be shortened from the original 4-6 months to 2-3 months, allowing planting to begin earlier, from April to February. However, this patent only increases the germination rate of bulbils to 56-70%, thus requiring further improvement. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the existing technology and provide a method for efficient and rapid propagation of tubers of Chinese yam. This method can significantly improve the induction rate and seedling rate of adventitious buds of Chinese yam tubers, increase the propagation coefficient, and realize the rapid and efficient propagation of superior strains of Chinese yam.

[0006] The present invention solves the above-mentioned technical problems through the following technical means: This invention proposes a method for efficient and rapid propagation of aerial seeds in Dioscorea opposita, comprising the following steps: (1) Using tubers of Dioscorea opposita as material, after germination and disinfection, adventitious shoot induction pretreatment was carried out in a mixture of thiamethoxam (TDZ) and meta-topolin; (2) Place the germination seeds treated in step (1) in a nutrient substrate and induce adventitious buds under suitable conditions; (3) After 4 weeks of cultivation, the bulbils with adventitious buds obtained in step (2) were transplanted into a mixed nutrient substrate sprayed with NAA for rooting and seedling cultivation. After 6 weeks of light cultivation, regenerated plants of Dioscorea opposita were obtained. Meta-topolin: Its Chinese name is 3-[(9H-purine-6-ylamino)methyl]phenol, and its CAS number is 75737-38-1.

[0007] Preferably, in step (1), the tubers of the Chinese yam are healthy and plump tubers of the Chinese yam harvested in the current year that are free from pests and diseases.

[0008] Preferably, in step (1), germination refers to germination after being treated at a low temperature of 4°C for 7-10 days.

[0009] Preferably, in step (1), the disinfection refers to rinsing the germinated bulbils under running water for 10 to 20 minutes, then soaking them in tap water for 4 to 6 hours, and then immersing them in a 0.05 to 0.2% potassium permanganate solution for 5 to 10 minutes for disinfection.

[0010] Preferably, in step (1), the concentration of TDZ in the mixture is 25-200 mg / L and the concentration of meta-topolin is 100-500 mg / L.

[0011] Preferably, in step (1), the pretreatment time is 1 to 10 minutes.

[0012] Preferably, in step (2), the nutrient substrate refers to vermiculite and nutrient soil mixed in a ratio of 3-6:1-3, and soaked in advance with a solution containing 0.1-0.2% potassium permanganate.

[0013] Preferably, in step (2), the suitable conditions refer to a temperature of 25–27°C and a light intensity of 60–100 μmol m. -2 s -1 Under greenhouse conditions with a photoperiod of 14 / 10 h (light / dark), spray water once a week to ensure the substrate is thoroughly soaked, and spray 1 / 4 to 1 / 2 MS macronutrients once every 2 weeks.

[0014] Preferably, in step (3), the planting depth of the bulbils with adventitious buds is such that the bud cluster is covered with a nutrient substrate of 0.5 to 1.0 cm thickness.

[0015] Preferably, in step (3), the concentration of NAA is 100-500 mg / L.

[0016] Preferably, in step (3), the mixed nutrient substrate refers to vermiculite and Pinscher nutrient soil mixed in a ratio of 1 to 3: 1 to 6, and soaked in tap water containing 1 / 16 to 1 / 4 MS macronutrients in advance.

[0017] Preferably, in step (3), the rooting and seedling culture conditions are a temperature of 22–25℃ and a light intensity of 40–60 μmol m. -2 s -1 A greenhouse with a photoperiod of 14 / 10 h (light / dark).

[0018] The beneficial effects of this invention are as follows: 1. This invention uses the bulbils of Chinese yam as propagation material, which is not only convenient and abundant, but also allows for flexible application of the bulbils after processing, and can preserve the agronomic traits of the yam parent plant line, ensuring the genetic stability of the offspring.

[0019] 2. The method involved in this invention has the advantages of high propagation efficiency and high propagation coefficient. By directionally inducing the production of adventitious buds from bulbils, the propagation efficiency and propagation coefficient are significantly improved. The adventitious bud induction rate is as high as 89.3%, and on average, each bulbil can cultivate 5.4 regenerated plants, effectively overcoming the problem of low propagation coefficient under natural germination conditions of bulbils.

[0020] 3. The operation process of this invention is simple and easy to implement, with a short propagation cycle, making it suitable for large-scale and standardized production applications. This method provides key technical support for the germplasm preservation and large-scale propagation of superior strains of Chinese yam, and can meet the year-round demand for high-quality seedlings in industrial development.

[0021] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0022] Figure 1 This is a photograph of the tuberous seeds of Dioscorea opposita placed in a nutrient substrate after adventitious bud induction treatment in Example 2 of the present invention; Figure 2 This is a photograph of the budding points induced from the bulbils of Dioscorea opposita after 3 weeks of light cultivation in Example 2 of this invention. Figure 3 This is a photograph of the adventitious buds of Dioscorea opposita after 5 weeks of light cultivation in Example 2 of the present invention; Figure 4 This is a photograph of seedlings cultivated for 4 weeks in Example 2 of the present invention, showing the elongation of adventitious buds accompanied by the formation of new tubers and roots; Figure 5 This is a photograph of a complete regenerated plant obtained after 6 weeks of seedling cultivation in Example 2 of the present invention. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0024] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0025] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0026] Example 1: A method for efficient and rapid propagation of aerial seeds in Chinese yam is as follows: (1) Using the current year's tubers of superior yam strains collected in early November as material, after being treated at low temperature of 4℃ for at least 7 days, they were rinsed for 10 minutes under running water; then soaked in tap water for 4 hours and then disinfected in 0.05% potassium permanganate solution for 5 minutes. After absorbing the surface moisture, they were placed in an aqueous solution containing 25 mg / L TDZ and 100 mg / L meta-topolin for 1 minute for adventitious bud induction pretreatment.

[0027] (2) Place the yam bulbils that have undergone surface disinfection and adventitious bud induction pretreatment in step (2) on the surface of the mixed nutrient substrate in the seedling tray (the substrate is a mixture of vermiculite and nutrient soil in a 3:1 ratio, and has been soaked in a solution containing 0.1% potassium permanganate beforehand), cover with the seedling cover, and place in a nursery at a temperature of 25-27℃ and a light intensity of 60 μmol / m². -2 s -1Adventitious shoot induction was performed under greenhouse conditions with a photoperiod of 14 / 10 h (light / dark). Water was sprayed weekly to ensure substrate saturation, and 1 / 4 MS macronutrients were applied every two weeks. After three weeks of light cultivation, shoot primordia were induced on the surface of yam bulbils. After another two weeks of light cultivation, adventitious shoot clusters were induced on the surface of yam bulbils, with an adventitious shoot induction rate of 61.3%, averaging 3.1 adventitious shoots per bulbil.

[0028] (3) Place the yam bulbils with adventitious buds obtained after 4 weeks of light cultivation in step (3) into seedling trays containing a mixed nutrient substrate (vermiculite: Pinscher soil = 1:1) that has been pre-soaked in tap water containing 1 / 16 MS macroelements and sprayed with 50 mg / L NAA. Maintain the trays at a temperature of 22–25℃ and a light intensity of 40 μmol / m². -2 s -1 Rooting and seedling cultivation were carried out in a greenhouse under a photoperiod of 14 / 10 h (light / dark), with a 0.5 cm thick layer of nutrient substrate covering the bud clusters. Water was sprayed once a week to ensure the substrate was thoroughly soaked. After 4 weeks of cultivation, the adventitious buds of the yam bulbils elongated to about 3.0 cm, with adventitious roots and tubers forming at the base, achieving a tuber induction rate of 77.8%. After another 2 weeks of cultivation, robust, fully regenerated *Dioscorea opposita* plants were finally obtained.

[0029] Example 2: A method for efficient and rapid propagation of aerial seeds in Chinese yam is as follows: (1) Using the current year's tubers of superior yam strains collected in early November as material, after being treated at 4℃ for 10 days, they were rinsed for 10 minutes under running water; then soaked in tap water for 5 hours and then disinfected in 0.1% potassium permanganate solution for 10 minutes. After absorbing the surface moisture, they were placed in an aqueous solution containing 50 mg / L TDZ and 300 mg / L meta-topolin for 5 minutes for adventitious bud induction pretreatment.

[0030] (2) Place the yam bulbils that have undergone surface disinfection and adventitious bud induction pretreatment in step (1) on the surface of the mixed nutrient substrate in the seedling tray (the substrate is a mixture of vermiculite and nutrient soil in a 6:1 ratio, and has been soaked in a solution containing 0.2% potassium permanganate beforehand), cover with the seedling cover, and place in a nursery at a temperature of 25-27℃ and a light intensity of 80 μmol / m². -2 s -1 Adventitious bud induction treatment was carried out under greenhouse conditions with a photoperiod of 14 / 10 h (light / dark). Figure 1(As shown). Spray water once a week to ensure the substrate is thoroughly soaked, and spray with 1 / 2 MS macronutrients once every two weeks. After 3 weeks of light cultivation, shoot primordia were induced on the surface of the yam tubers (as shown). Figure 2 (As shown); After two weeks of continued light cultivation, adventitious shoot clusters were induced on the surface of the yam bulbils, with an induction rate of 90.3%, and an average of 5.4 adventitious shoots were produced per bulbil (as shown). Figure 3 (As shown).

[0031] (3) Place the bulbils from the induced adventitious buds in step (2) into seedling trays containing a mixed nutrient substrate (vermiculite: Pinscher potting soil = 2:6) that has been pre-soaked in tap water containing 1 / 8 MS macroelements and sprayed with 100 mg / L NAA. Maintain the trays at a temperature of 22–25°C and a light intensity of 50 μmol / m². -2 s -1 Rooting and seedling cultivation were carried out in a greenhouse under a photoperiod of 14 / 10 h (light / dark), with a 1.0 cm thick layer of nutrient substrate covering the bud clusters. Tap water was sprayed once a week to ensure the substrate was thoroughly soaked. After 4 weeks of cultivation, the adventitious buds of the yam bulbils elongated to 3.0–4.0 cm, with adventitious roots and tubers forming at the base (e.g., ...). Figure 4 As shown), the tuber induction rate was 93.5%; after culturing for another 2 weeks, robust and fully regenerated *Dioscorea opposita* plants were finally obtained (as shown). Figure 5 (As shown).

[0032] Example 3: This example tested the effects of different types and concentrations of exogenous inducers on the induction of adventitious shoots from *Dioscorea opposita* bulbils. *Dioscorea opposita* bulbils, after soaking and surface disinfection as in Example 2, were placed in solutions containing different types and concentrations of plant growth regulators for 5 minutes. After drying, they were transplanted into the same nutrient substrate as in Example 2 under the same greenhouse conditions for adventitious shoot induction. The study found that different types and concentrations of inducers had a significant impact on the induction of adventitious shoots from *Dioscorea opposita* bulbils. Among the different plant growth regulators tested, appropriate concentrations of meta-topolin and TDZ effectively induced the formation of adventitious shoots from *Dioscorea opposita* bulbils. Furthermore, with increasing concentration, the induction rate and number of adventitious shoots showed a trend of first increasing and then decreasing. Treatment with aqueous solutions containing 50 mg / L TDZ and 300 mg / L meta-topolin for 5 minutes was most beneficial for the induction of adventitious shoots and the subsequent seedling cultivation of *Dioscorea opposita* bulbils. After 5 weeks of light culture, the induction rate of adventitious shoots from the bulbils of Dioscorea opposita was 90.3%, with an average of 5.4 adventitious shoots produced per explant.

[0033] Table 1. Effects of different types and concentrations of plant growth regulators on adventitious shoot induction in Dioscorea opposita.

[0034] Note: Data are averages. Each treatment contained 120 explants and was repeated three times.

[0035] Example 4: This example tested the effect of exogenous NAA treatment concentration on the rooting of adventitious buds and tuber induction in yam bulbils. Bulbils from Example 2, which had induced adventitious buds, were placed in seedling trays containing a mixed nutrient substrate (vermiculite: Pinscher potting mix = 2:6) that had been pre-soaked in tap water containing 1 / 8 MS macroelements and sprayed with different concentrations (25-200 mg / L) of NAA. The trays were kept at a temperature of 22-25°C and a light intensity of 50 μmol / m². -2 s -1 Rooting and seedling cultivation were conducted in a greenhouse under a photoperiod of 14 / 10 h (light / dark), with a 1.0 cm thick nutrient substrate covering the bud clusters. A mixed nutrient substrate sprayed only with tap water served as a control group. Tap water was sprayed weekly after transplanting to ensure substrate saturation. The study found (Table 2) that spraying NAA into the substrate effectively promoted the rooting of adventitious buds and tuber formation in yam. With increasing NAA concentration, the rooting rate of adventitious buds gradually increased, and the rhizome formation rate also showed a trend of first increasing and then decreasing with increasing exogenous NAA concentration. We speculate that high concentrations of NAA, while inducing a large number of adventitious roots, inhibited rhizome formation. Among the tested NAA concentrations, the 100 mg / L NAA treatment showed the best effect, most favorable for the rooting of adventitious buds and subsequent tuber induction in yam, with a rooting rate as high as 93.5% and a tuber induction rate as high as 91.8%.

[0036] Table 2. Effects of exogenous NAA concentration on adventitious shoot rooting and tuber induction in yam tubers.

[0037] Note: Data are averages. Each treatment contained 120 explants and was repeated three times.

[0038] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for efficient and rapid propagation of aerial seeds of Dioscorea opposita, characterized in that, Includes the following steps: (1) Using tuberous seeds of Dioscorea opposita as material, after germination and disinfection, adventitious shoot induction pretreatment was carried out in a mixture of thiamethoxam and meta-topolin. (2) Place the germination seeds treated in step (1) in a nutrient substrate and induce adventitious buds under suitable conditions; (3) After 4 weeks of cultivation, the bulbils with adventitious buds obtained in step (2) were transplanted into a mixed nutrient substrate sprayed with NAA for rooting and seedling cultivation. After 6 weeks of light cultivation, regenerated plants of Dioscorea opposita were obtained. Meta-topolin: Its Chinese name is 3-[(9H-purine-6-ylamino)methyl]phenol, and its CAS number is 75737-38-1.

2. The method according to claim 1, characterized in that, In step (1), the bulbils of the iron yam are healthy and plump bulbils of the iron yam harvested in the current year that are free from diseases and pests.

3. The method according to claim 1, characterized in that, In step (1), germination refers to germination after being treated at a low temperature of 4°C for 7-10 days.

4. The method according to claim 1, characterized in that, In step (1), the disinfection refers to rinsing the germinated bulbils under running water for 10 to 20 minutes, then soaking them in tap water for 4 to 6 hours, and then immersing them in a 0.05 to 0.2% potassium permanganate solution for 5 to 10 minutes for disinfection.

5. The method according to claim 1, characterized in that, In step (1), the concentration of thiamethoxam in the mixture is 25–200 mg / L, and the concentration of meta-topolin is 100–500 mg / L; the pretreatment time is 1–10 min.

6. The method according to claim 1, characterized in that, In step (2), the nutrient substrate refers to vermiculite and nutrient soil mixed in a ratio of 3-6:1-3, and soaked in a solution containing 0.1-0.2% potassium permanganate in advance.

7. The method according to claim 1, characterized in that, In step (2), the suitable conditions refer to a temperature of 25–27°C and a light intensity of 60–100 μmol m. -2 s -1 Under greenhouse conditions with a photoperiod of 14 / 10 h (light / dark), spray water once a week to ensure the substrate is thoroughly soaked, and spray 1 / 4 to 1 / 2 MS macronutrients once every 2 weeks.

8. The method according to claim 1, characterized in that, In step (3), the planting depth of the bulbils with adventitious buds is such that the bud cluster is covered with a nutrient substrate of 0.5 to 1.0 cm thickness.

9. The method according to claim 1, characterized in that, In step (3), the concentration of NAA is 100-500 mg / L; the mixed nutrient substrate refers to vermiculite and Pinscher nutrient soil mixed in a ratio of 1-3:1-6, and soaked in tap water containing 1 / 16-1 / 4 MS macronutrients in advance.

10. The method according to claim 1, characterized in that, In step (3), the rooting and seedling culture conditions are a temperature of 22–25℃ and a light intensity of 40–60 μmol m. -2 s -1 A greenhouse with a photoperiod of 14 / 10 h (light / dark).

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