Tissue culture method using thin-layer cells of polygonatum cyrtonema and application thereof

Abstract

The present application belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture method using thin-layer cells of Smilax bracteata and application thereof. The present application sequentially carries out pre-culture, induction culture and seedling culture on the Smilax bracteata explant slice with a thickness of 1 mm, and obtains the Smilax bracteata seedlings after 25 days by controlling the culture conditions of the induction culture and the seedling culture, the emergence rate reaches 76.1%, and the Smilax bracteata tissue culture seedlings grow well after being transplanted to the seedling raising substrate, and the plant survival rate reaches more than 90%. The tissue culture method greatly shortens the seedling raising time of the Smilax bracteata, and has high seedling rate and survival rate.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a tissue culture method using thin-layer cells of Polygonatum yunnanense and its application. Background Technology

[0002] *Polygonatum kingianum* Coll. et Hemsl., a perennial herb belonging to the genus *Polygonatum* in the family Liliaceae, is mainly distributed in Yunnan Province and Guangxi Zhuang Autonomous Region. It grows in shady and damp forests and thickets at altitudes of 2000–3900 m. Its rhizomes, seeds, and other organs can all be used as explants. Due to over-exploitation, wild resources of *Polygonatum kingianum* are becoming increasingly scarce. Traditional propagation methods for *Polygonatum kingianum* include seed propagation, rhizome propagation, and tissue culture propagation. However, seed and rhizome propagation have drawbacks such as long cycles, high material consumption, and low survival rates, failing to meet market demand. Tissue culture can solve this problem, offering a shorter culture cycle, higher propagation rate, and being unaffected by seasons. Therefore, using tissue culture for rapid propagation of *Polygonatum kingianum* is one of the effective methods to address resource shortages.

[0003] The tissue culture technology of Polygonatum yunnanense, which has emerged in recent years, has effectively solved the problems of low seed germination rate, long seedling growth cycle, low root and rhizome propagation coefficient, and high cost faced by seed propagation and rhizome propagation methods. However, most of these methods are complex and still suffer from slow propagation speed and low survival rate. For example, Chinese patent application number 201510420973.5, entitled "A Tissue Culture Method for One-Step Seedling Transformation of Polygonatum yunnanense", involves selecting 80% mature fruits and placing them in a 4℃ refrigerator for 7 days. The treated seeds are then inoculated into culture bottles containing MS+BA 0.5mg / L medium and cultured at a temperature of 25℃±2℃, a photoperiod of 12-14h / day, and a light intensity of 150lx until the Polygonatum yunnanense seeds germinate into 2 true leaves. At this point, the culture bottles are placed in a culture environment of 25℃±2℃, a photoperiod of 12h / day, and a light intensity of 1500lx to continue culturing until rooting occurs. The Yunnan Polygonatum tissue culture method has a seedling growth time of up to 90-99 days from seed inoculation. Summary of the Invention

[0004] The purpose of this invention is to provide a solution that overcomes the shortcomings of the prior art, shortens the seedling cultivation time of Polygonatum yunnanense, and improves the survival rate and seedling establishment rate of tissue culture seedlings of Polygonatum yunnanense.

[0005] To achieve the above objectives, the present invention provides a method for tissue culture of Polygonatum yunnanense, comprising the following steps:

[0006] The explant sections of Polygonatum yunnanense were pre-cultured using germination medium. The adventitious buds obtained from the pre-culture were cut into single buds and then transferred to induction medium for induction culture to generate new adventitious buds.

[0007] New adventitious buds were transferred to a new germination medium for seedling culture to obtain tissue culture seedlings of Polygonatum yunnanense.

[0008] The thickness of the explant section of Polygonatum yunnanense was 1 mm.

[0009] The pre-culture temperature is 23-27℃, the light intensity is 1500-2000 lx, and the daily light duration is 16 hours.

[0010] The induction culture was conducted at a temperature of 23–27°C, a light intensity of 1500–2000 lx, and a daily light duration of 16 hours.

[0011] The seedling culture was conducted at a temperature of 23–27°C, a light intensity of 1500–2000 lx, and a daily light duration of 16 hours.

[0012] The induction medium is based on MS medium and includes the following components at the following concentrations: IBA 1 mg / L, sucrose 20 g / L, plant gel 3 g / L and activated carbon 1 g / L.

[0013] The germination medium is based on MS medium, without plant growth regulators, and includes the following components at the following concentrations: 20 g / L sucrose, 3 g / L plant gel and 1 g / L activated carbon.

[0014] Preferably, the induction medium further includes 6-BA 1 mg / L.

[0015] Preferably, the preparation of the *Polygonatum yunnanense* explant slices includes: transverse cutting along the direction perpendicular to the main growth point to obtain the *Polygonatum yunnanense* explant slices.

[0016] Preferably, the explant of Polygonatum yunnanense comprises a Polygonatum yunnanense bulb after removing the seeds, plumules and radicles.

[0017] Preferably, the disinfected seeds of Polygonatum yunnanense are germinated to obtain the Polygonatum yunnanense bulbs.

[0018] Preferably, the germination culture conditions are: dark culture at 25°C.

[0019] Preferably, the disinfection method includes: disinfecting with a 75% ethanol solution for 30 seconds, followed by disinfecting with a 2% NaDCC solution for 20 minutes.

[0020] This invention also provides the application of the tissue culture method described above in the cultivation of Polygonatum yunnanense.

[0021] Preferably, the application includes: transplanting the tissue culture seedlings of Polygonatum yunnanensis obtained by the tissue culture method described above into a seedling substrate for culture.

[0022] Preferably, the seedling substrate includes humus, red soil, and perlite;

[0023] The mass ratio of the humus, red soil and perlite is 1:1:1.

[0024] Beneficial effects:

[0025] This invention involves sequentially pre-culturing, inducing, and cultivating seedlings from 1mm thick explant sections of *Polygonatum yunnanense* (containing a thin layer of cells). By controlling the culture conditions during these stages, new adventitious buds are generated after 15-20 days of induction culture, with a budding rate of 76%. After two months of continued seedling cultivation, healthy *Polygonatum yunnanense* seedlings are obtained. These tissue-cultured seedlings grow well after being transplanted into a seedling substrate, with a survival rate exceeding 90%. This tissue culture method significantly shortens the seedling cultivation time for *Polygonatum yunnanense*, resulting in a high seedling survival rate. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0027] Figure 1 The bulbous stem formed after culturing *Polygonatum yunnanense* seeds for 20 days in step 1 of Example 1; scale bar 1 mm;

[0028] Figure 2 This is a schematic diagram of the cross-cutting process in step 2 of Example 1;

[0029] Figure 3 This is a schematic diagram of the thin-layer sectioning culture dish in step 2 of Example 1;

[0030] Figure 4 The adventitious buds induced by thin-layer sections in step 2 of Example 1;

[0031] Figure 5 This refers to the adventitious bud formation of plants in step 2 of Example 1;

[0032] Figure 6 The seedlings of *Polygonatum yunnanense* formed by seed germination in step 2 of Example 1;

[0033] Figure 7 For example, in step 3 of step 1, plants were induced for 20 days using induction medium 3;

[0034] Figure 8 The image shows a *Polygonatum yunnanense* plant after 30 days of transplanting and cultivation, as shown in Example 2. Detailed Implementation

[0035] This invention provides a method for tissue culture of Polygonatum yunnanense, comprising the following steps:

[0036] The explant sections of Polygonatum yunnanense were pre-cultured using germination medium. The adventitious buds obtained from the pre-culture were cut into single buds and then transferred to induction medium for induction culture to generate new adventitious buds.

[0037] New adventitious buds were transferred to a new germination medium for seedling culture to obtain tissue culture seedlings of Polygonatum yunnanense.

[0038] The thickness of the explant section of Polygonatum yunnanense was 1 mm.

[0039] The pre-culture temperature is 23-27℃, the light intensity is 1500-2000 lx, and the daily light duration is 16 hours.

[0040] The induction culture was conducted at a temperature of 23–27°C, a light intensity of 1500–2000 lx, and a daily light duration of 16 hours.

[0041] The seedling culture was conducted at a temperature of 23–27°C, a light intensity of 1500–2000 lx, and a daily light duration of 16 hours.

[0042] The induction medium is based on MS medium and includes the following components at the following concentrations: IBA 1 mg / L, sucrose 20 g / L, plant gel 3 g / L and activated carbon 1 g / L.

[0043] The germination medium is based on MS medium, without plant growth regulators, and includes the following components at the following concentrations: 20 g / L sucrose, 3 g / L plant gel and 1 g / L activated carbon.

[0044] This invention utilizes a germination medium to pre-culture explant sections of *Polygonatum yunnanense* to obtain adventitious shoots. In this invention, the thickness of the sections is 1 mm. The preparation of the *Polygonatum yunnanense* explant sections preferably includes: transverse cutting along the direction perpendicular to the principal growth point to obtain the *Polygonatum yunnanense* explant sections. Preferably, this invention involves transverse cutting along the direction perpendicular to the principal growth point under aseptic conditions to obtain the *Polygonatum yunnanense* explant sections. This invention uses *Polygonatum yunnanense* explant sections as explants, limits the thickness of the sections, and performs induction culture. This method has a wide range of material sources, requires less hormone, has a shorter growth cycle, produces a large area of ​​thin slices, and has a smaller number of cells. During culture, the explants can fully contact the culture medium, improving transformation efficiency and reducing the occurrence of chimeras.

[0045] In this invention, the explants of *Polygonatum yunnanense* preferably comprise *Polygonatum yunnanense* corms after the seeds, plumules, and radicles have been removed. Using *Polygonatum yunnanense* corms after the seeds, plumules, and radicles have been removed as explant material further shortens the seedling time and increases the seedling survival rate. Preferably, the *Polygonatum yunnanense* seeds are germinated to obtain the *Polygonatum yunnanense* corms. The disinfection method preferably includes: disinfection with a 75% ethanol solution for 30 seconds, followed by disinfection with a 2% NaDCC solution for 20 minutes. Preferably, before disinfection, the *Polygonatum yunnanense* seeds are soaked in 100 mg / L gibberellin for 24 hours.

[0046] In this invention, the pre-culture temperature is 23–27°C, preferably 25°C; the pre-culture light intensity is 1500–2000 lx, preferably 1600–1800 lx; ​​and the daily light duration for pre-culture is 16 hours. The pre-culture time is preferably 30 days.

[0047] After obtaining adventitious buds, the present invention cuts the adventitious buds into single buds and transfers them to an induction medium for induction culture to generate new adventitious buds. In the present invention, the temperature of the induction culture is 23-27℃, preferably 25℃; the light intensity of the induction culture is 1500-2000 lx, preferably 1600-1800 lx; ​​and the daily light duration of the induction culture is 16 hours. The induction culture time of the present invention is preferably 15-20 days. The induction medium of the present invention comprises the following components at the following concentrations: 20 g / L sucrose, 3 g / L plant gel, 1 g / L activated carbon, and the balance MS medium. The induction medium of the present invention preferably also includes 1 mg / L 6-BA.

[0048] After obtaining new adventitious buds, the present invention transfers these new adventitious buds to a germination medium for seedling cultivation. In the present invention, the temperature for seedling cultivation is 23–27°C, preferably 25°C; the light intensity for seedling cultivation is 1500–2000 lx, preferably 1600–1800 lx; ​​and the daily light duration for seedling cultivation is 16 hours. The preferred seedling cultivation period is 2 months. The composition of the germination medium in the present invention has been described above and will not be repeated here. When conducting seedling cultivation in the present invention, it is preferable to transfer a single bud to the germination medium for seedling cultivation.

[0049] This invention involves sequentially pre-culturing, inducing, and cultivating 1mm thick *Polygonatum yunnanense* explant sections under strictly controlled culture conditions. After approximately 10 days of induction culture, adventitious buds begin to appear on the explants, and the number of adventitious buds increases with the duration of culture. After 25 days of induction culture, 3-6 adventitious buds appear on a single explant section, and some of these buds begin to develop into complete plants. After 15 days of cultivation to cultivate the adventitious buds into seedlings, *Polygonatum yunnanense* tissue culture seedlings are obtained. The tissue culture method provided by this invention requires only 25 days to obtain *Polygonatum yunnanense* tissue culture seedlings, with a seedling survival rate of 76%. Furthermore, the transplanted seedlings grow well in a seedling substrate, with a survival rate exceeding 90%.

[0050] This invention also provides the application of the above-mentioned tissue culture method for *Polygonatum yunnanense* in the cultivation of *Polygonatum yunnanense*. In this invention, the application preferably includes: transplanting the *Polygonatum yunnanense* tissue culture seedlings obtained by the tissue culture method described above into a seedling substrate for cultivation. The seedling substrate of this invention preferably includes humus, red soil, and perlite; the mass ratio of the humus, red soil, and perlite is preferably 1:1:1. This invention preferably conducts the cultivation in a greenhouse; the cultivation time is preferably 7 days. The tissue culture method provided by this invention can obtain well-grown *Polygonatum yunnanense* tissue culture seedlings in a short time, with a high seedling survival rate. The obtained *Polygonatum yunnanense* tissue culture seedlings grow well when transplanted into the substrate, with a plant survival rate of over 90%.

[0051] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a method for tissue culture using thin-layer cells of Polygonatum yunnanensis and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0052] The culture media used in the examples and comparative examples are as follows:

[0053] Germination medium (denoted as MS): 20 g / L sucrose + 3 g / L plant gel + 1 g / L activated carbon + the remainder MS medium, pH adjusted to 5.8;

[0054] Induction medium 1 (denoted as MS+IBA): IBA 1 mg / L + 20 g / L sucrose + 3 g / L plant gel + 1 g / L activated carbon + the remainder MS medium, pH adjusted to 5.8;

[0055] Induction medium 2 (denoted as MS+6-BA): 6-BA 1 mg / L + 20 g / L sucrose + 3 g / L plant gel + 1 g / L activated carbon + the remainder MS medium, pH adjusted to 5.8;

[0056] Induction medium 3 (denoted as MS+IBA+6-BA): IBA 1 mg / L + 6-BA 1 mg / L + 20 g / L sucrose + 3 g / L plant gel + 1 g / L activated carbon + the remainder MS medium, pH adjusted to 5.8;

[0057] Example 1

[0058] 1. Seed-germinating bulbs

[0059] After cleaning, the seeds of *Polygonatum yunnanense* were soaked in 100 mg / L gibberellin for 24 hours. Then, they were placed in a sterile operating table and shaken with 75% alcohol for 30 seconds. After rinsing with sterile water 3-4 times, they were disinfected with 2% NaDCC for 20 minutes. The waste liquid was discarded, and the seeds were rinsed with sterile water 3-4 times. The surface moisture was then blotted dry with filter paper. The seeds were then inoculated into germination medium and cultured in the dark at 25°C. After 20 days, the seeds germinated into bulbous structures. Figure 1 (Scale bar is 1mm).

[0060] 2. Adventitious bud culture of bulb-like stems

[0061] Take the round corms of *Polygonatum yunnanense* obtained in step 1 and perform thin-layer sectioning under sterile conditions using a stereomicroscope: remove the seeds, plumules, and radicles of each round corm using sterile forceps and a scalpel, and make transverse cuts (downwards) along the direction perpendicular to the main growth point, making transverse cuts at points numbered 1, 2, 3, and 4 respectively. Figure 2 Each cormoid was transversely sliced ​​into four thin slices approximately 1 mm thick. These slices were then laid flat on germination medium (composed of 20 g / L sucrose + 3 g / L plant gel + 1 g / L activated charcoal + the remainder MS medium, pH adjusted to 5.8) and cultured under the following conditions: temperature 25℃, light intensity 1500–2000 lx, 16 h light per day. A total of 21 corms were processed in this step, each cormoid being cut into four slices, for a total of 84 slices. These slices were placed in germination medium, 12 slices per dish, for a total of seven dishes. Figure 3 ).

[0062] 3. After culturing the thin-layer sections for 10 days in step 2, buds begin to appear. After culturing the thin-layer sections for 25 days (i.e., continuing to culture for 15 days after the appearance of buds), 3-6 adventitious buds appear on a single thin-layer section, and some of the adventitious buds on the thin-layer cells begin to develop into complete plants. Figure 4 After culturing thin-layer sections for 55 days (i.e., continuing culture for 45 days after the appearance of buds, or 30 days for adventitious buds), and then continuing culture for another 30 days, 32 seedlings were produced. During the tissue culture seedling culture period, new adventitious buds appeared simultaneously, and some results are shown below. Figure 5 As shown;

[0063] Adventitious buds that failed to develop into seedlings after 30 days of culture in step 2 were cut into single buds under a stereomicroscope and placed in different induction media (induction medium 1, induction medium 2, and induction medium 3) for induction culture. Ten single buds were placed per bottle for each induction medium, and five replicates were set for each medium. Culture conditions were: temperature 25℃, light intensity 1500–2000 lx, 16 h light per day. After 15 days of culture, the culture progress was observed, and the results are shown in Table 1. The plants cultured in the germination medium were as follows... Figure 6 As shown, plants induced for 20 days using induction medium 3 are as follows: Figure 7 As shown.

[0064] Table 1. Sprouting of adventitious buds under different treatments

[0065]

[0066] As shown in Table 1, all three different hormone ratios in the culture medium affected the differentiation of adventitious buds in Polygonatum yunnanense. The IBA and 6-BA+IBA combination media had a stronger effect on the differentiation of adventitious buds in thin layers of Polygonatum yunnanense than the medium using 6-BA alone. Among them, 6-BA+IBA had the best effect on the differentiation of adventitious buds, with the highest thin-layer budding rate of 76%, which was 15.15% and 22.58% higher than that of IBA and 6-BA alone, respectively.

[0067] 4. After 20 days of induction in the induction medium of step 3, the seedlings obtained in step 2 (i.e., the adventitious bud seedlings obtained after 30 days of culture in step 2) were transferred to tissue culture bottles containing germination medium for separate culture. The culture conditions were: temperature 25℃, light intensity 1500-2000 lx, and light exposure 16h per day. After 2 months of culture, the growth characteristics of the plants were analyzed, including parameters such as root system, bulb diameter, and plant height. The results are shown in Table 2.

[0068] Table 2. Growth traits of plants under different treatments

[0069] Different plant sources Number of roots Bulb size (cm) Number of leaves (blades) Plant height (cm) Step 2: Seed germination directly produces seedlings. 4.67±0.58a 0.69±0.13a 2.08±0.38b 9.11±1.07a Induction medium 1 7.67±2.19a 0.58±0.05a 7.50±0.66a 6.67±0.88b Induction medium 2 5.11±0.84b 0.39±0.03b 2.83±0.76a 8.23±1.07a Induction medium 3 8.56±2.14a 0.63±0.09a 3.58±2.32a 7.78±0.69a

[0070] Note: Different lowercase letters in the table indicate significant differences (p < 0.05).

[0071] Table 2 shows that there were significant differences in the growth of *Polygonatum yunnanensis* plants between direct seeding from seed and the culture media containing the three different hormone combinations. In terms of root number, the MS+6-BA+IBA (induction medium 3) and MS+IBA (induction medium 1) media produced the most roots. Corm size was largest in both direct seeding from seed and MS+6-BA+IBA (induction medium 3) media. Leaf number was highest in MS+IBA (induction medium 1) media. Plant height was higher in MS and MS+6-BA media. Considering root number, corm size, leaf number, and plant height, the MS+6-BA+IBA (induction medium 3) medium showed the best culture effect.

[0072] Example 2

[0073] Tissue culture seedlings with a height of 5-7cm and good growth were selected from Example 1. The seedlings were hardened off for 7 days at room temperature with the bottle caps opened. The seedlings were then taken out, and the culture medium attached to the roots was cleaned off with tap water. They were then transplanted into a substrate of sterilized humus soil: red soil: perlite = 1:1:1. The survival rate of the regenerated plants was counted after 30 days.

[0074] Plant survival rate (%) = (Number of surviving plants / Number of transplanted plants) × 100

[0075] The results showed that the survival rate of tissue culture seedlings obtained by induction culture in medium 3 was over 90%, and the growth of some plants was as follows: Figure 8 As shown, the survival rate of tissue culture seedlings obtained by induction culture in induction medium 1 or 2 reached over 85%.

[0076] As can be seen from the above, the tissue culture method provided by this invention can quickly produce seedlings with a high seedling survival rate. The tissue culture seedlings of Polygonatum yunnanense grow well when transplanted into the substrate, and the survival rate of the plants is over 90%.

[0077] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A tissue culture method of Polygonatum franchetii, characterized by, It comprises the following steps: Pre-culturing the explant slices of Disporum megalonyx by using a germination medium, cutting the adventitious buds obtained by pre-culturing into single buds, and then transferring the single buds to an induction medium for induction culture to generate new adventitious buds; the explant of Disporum megalonyx is a Disporum megalonyx bulbil after removing seeds, plumules and radicles; The preparation of the explant slices of Disporum megalonyx comprises: transversely cutting along the vertical tangent direction of the main growth point to obtain the explant slices of Disporum megalonyx; Transferring the new adventitious buds to a new germination medium for seedling culture to obtain Disporum megalonyx tissue culture seedlings; The thickness of the explant slices of Disporum megalonyx is 1mm; The pre-culturing temperature is 23-27℃, the light intensity is 1500-2000lx, and the light time per day is 16h; The induction culture temperature is 23-27℃, the light intensity is 1500-2000lx, and the light time per day is 16h; The seedling culture temperature is 23-27℃, the light intensity is 1500-2000lx, and the light time per day is 16h; The induction medium takes MS medium as a basic medium, and further comprises the following components with the following concentrations: IBA 1mg / L, 6-BA 1mg / L, sucrose 20g / L, plant gel 3g / L and activated carbon 1g / L; The germination medium takes MS medium as a basic medium, and further comprises the following components with the following concentrations: sucrose 20g / L, plant gel 3g / L and activated carbon 1g / L.

2. The tissue culture method according to claim 1, characterized by, The germination culture of the disinfected Disporum megalonyx seeds obtains the Disporum megalonyx bulbil.

3. The tissue culture method according to claim 2, characterized in that, The germination culture condition is 25℃ dark culture.

4. The tissue culture method according to claim 2, characterized by, The disinfection method comprises: 75% ethanol solution disinfection for 30s, and then 2% NaDCC disinfection for 20min.

5. The use of the tissue culture method in claim 1-4 in Disporum megalonyx planting.

6. Use according to claim 5, characterized in that, The use comprises: transplanting the Disporum megalonyx tissue culture seedlings obtained by the tissue culture method in claim 1-4 into a seedling raising substrate for culture.

7. Use according to claim 6, characterized in that, The seedling raising substrate comprises humus soil, red soil and perlite; The mass ratio of the humus soil, red soil and perlite is 1:1:1.

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