Centella asiatica polyploidy seedling factory production method based on micro-organ cutting-water planting combination

Abstract

The invention discloses a centella asiatica polyploidy seedling factory production method based on micro-organ cutting-water culture combination, which comprises the following steps: (1) selecting tetraploid centella asiatica aseptic seedlings as materials, transplanting rooting seedlings of the tetraploid centella asiatica aseptic seedlings into nutrient soil for culture, and cutting growing stolons into stem segments with axillary buds; (2) micro-organ cutting culture: cutting the stem sections treated in the step (1) on an improved MS solid culture medium for culture; simultaneously germinating new buds and new roots; transferring the seedlings into an improved MS liquid culture medium for water culture, transferring the obtained seedlings to outdoor for seedling hardening, and then performing field transplanting and field planting. The invention provides an efficient, economic and large-scale propagation scheme for new germplasm after genetic improvement of centella asiatica, so that large-scale industrialized production can be realized in a short time, and the method has a wide industrial application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant polyploid induction and cultivation, and particularly relates to a method for industrialized production of Centella asiatica polyploid seedlings based on the combined use of micro-organ cutting and hydroponics. Background Art

[0002] Centella asiatica (L.) Urban is a perennial herb widely distributed in tropical and subtropical regions of the world. Centella asiatica has important medicinal, edible and cosmetic values. Its extracts are rich in various active ingredients such as triterpenoid saponins and flavonoid compounds, and have effects such as anti-inflammatory, antibacterial, antioxidant and promoting wound healing. Centella asiatica has been used in traditional medicine to treat various diseases such as skin ulcers, burns, eczema, varicose veins, etc. Modern research shows that Centella asiatica extracts also have effects such as anti-aging, anti-tumor and nerve protection, and have broad application prospects. The traditional Centella asiatica varieties are small in size and low in yield, making it difficult to meet the market demand. Through means such as artificial selection or genetic engineering, new varieties of Centella asiatica with larger size and higher yield can be cultivated. The selection of tetraploid Centella asiatica can significantly increase the yield and economic benefits of Centella asiatica, meet the market demand for natural medicines and functional foods, and also provide strong support for the development of the Centella asiatica industry.

[0003] Based on the above research background, the applicant has successfully cultivated a new germplasm of tetraploid Centella asiatica with high quality, high yield and high content through polyploid breeding technology. However, how to achieve large-scale propagation of this new germplasm in a short period is the key challenge currently faced. Although tissue culture technology has the potential to rapidly propagate Centella asiatica, its high cost, strict technical requirements and low transplanting survival rate make it difficult to achieve large-scale production in a short period. The traditional cutting and ramet propagation methods of Centella asiatica are mainly carried out in soil or substrates. Not only are they easily invaded by pests and diseases, resulting in the rotting of bud-bearing stem segments, but also the rooting situation is difficult to observe, seriously restricting the propagation efficiency. For example: Ramet propagation is usually carried out in spring and autumn, and the mother plant is divided into small plants with roots, and the survival rate is about 90%. The growth cycle is short, but the propagation amount is limited; cutting propagation (selecting strong stem segments) requires maintaining humidity and a semi-shady environment, and rooting takes 2-4 weeks, and the survival rate is about 70%-80%, but the management requirements are relatively high. The above cutting and ramet propagation methods are not suitable for large-scale propagation with only a few polyploid seedlings, and the number of explants is too small.

[0004] The present invention aims to expand the cultivation of snow grass with buds in a semi-sterile environment through the technique of in vitro micro-organ organ cutting culture. Compared with the traditional method of culturing aseptic seedlings in tissue culture, it aims to accelerate the propagation of polyploid seedlings of snow grass by precisely regulating the formula of the in vitro micro-organ organ cutting liquid medium and the hydroponic conditions. The inorganic salt concentration of the MS medium (such as nitrates, ammonium salts, calcium, and magnesium) is much higher than that of the conventional hydroponic liquid medium, which may cause osmotic stress or salt damage to the roots of hydroponic plants. Long-term use may inhibit growth and even cause death. Therefore, the present invention mainly modifies the MS medium and combines the techniques of in vitro micro-organ organ cutting and hydroponics to establish an efficient polyploid snow grass breeding system and rapidly achieve the large-scale production of polyploid seedlings of snow grass. Summary of the Invention

[0005] To meet the urgent need for large-scale propagation of new polyploid germplasms of snow grass in the short term, the main object of the present invention is to innovatively propose a factory seedling raising method combining micro-organ organ cutting and hydroponics. By precisely preparing the in vitro micro-organ organ cutting liquid medium, this method significantly shortens the rooting time and greatly improves the rooting rate and transplanting survival rate. At the same time, the hydroponic technique is innovatively introduced, enabling the plant roots to be fully immersed in the liquid medium, providing comprehensive and balanced nutritional support for their growth and development, and effectively accelerating the seedling propagation process. The present invention provides an efficient, economical, and scalable propagation scheme for the new germplasms after genetic improvement of snow grass, enabling large-scale factory production in the short term and having broad industrial application prospects.

[0006] A factory production method for polyploid seedlings of snow grass based on the combined use of micro-organ organ cutting and hydroponics, comprising the following steps:

[0007] (1) Selection of explants

[0008] Select aseptic seedlings of tetraploid snow grass as the material, transplant their rooted seedlings into nutrient soil for cultivation, and cut the growing stolons into stem segments with axillary buds;

[0009] (2) Micro-organ organ cutting culture

[0010] Micro-organ organ cutting culture: Insert the stem segments processed in step (1) into the modified MS solid medium for cultivation; simultaneously germinate new buds and new roots; transfer them to the modified MS liquid medium for hydroponics, and transplant the obtained seedlings to the outdoor for hardening and then to the field for transplanting and planting.

[0011] Furthermore,

[0012] Step (1) The nutrient soil includes cutting peat coconut coir seedling-raising soil. First, add water and stir to moisten it. Transplant the tetraploid seedlings well, and culture them under the conditions of a temperature of 28±2°C, a humidity of 60-70%, a light intensity of 2000-2500 Lx, and a light time of 10-12 h / d.

[0013] In step (1), the rooted seedlings are transplanted into the nutrient soil and cultured for 1.5-2 months. Select the robust stolons and cut them into stem segments with axillary buds.

[0014] In step (1), cut into stem segments with axillary buds that are 1-3 cm long.

[0015] Step (2) Prepare a solid medium of 3 liters of modified MS liquid medium + 3.0 g / L of agar, and then insert 45-55 stem segments processed in step (1) into the medium, with the insertion depth being 0.5-1 cm.

[0016] The solid medium with the inserted stem segments is cultured for 3-7 days under the conditions of a temperature of 28±2°C, a humidity of 60-70%, a light intensity of 2000-2500 Lx, and a light time of 10-12 h / d, and new buds and new roots germinate simultaneously; after continuing to culture for 10 days, transfer it to the modified MS liquid medium for hydroponics, with 45-55 seedlings / 3 L of medium. During hydroponics, the roots are immersed in the medium, and the liquid medium is supplemented once every 5-7 days, with 1 L supplemented each time; the hydroponics temperature is 28±2°C, the humidity is 60-70%, the light intensity is 2000-2500 Lx, and the light time is 10-12 h / d. After 30-60 days of hydroponics, complete seedlings are obtained; move the hydroponics container to a shaded and ventilated place outdoors for acclimatization for 5-7 days. During the acclimatization period, gradually increase the light intensity and ventilation volume to make it adapt to the natural environment; after the plants grow stably, carry out field transplanting and planting.

[0017] In step (2), after 30-60 days of hydroponics, complete seedlings are obtained; then continue to cut the obtained stolons into stem segments with axillary buds that are 1-3 cm long and repeat step (2) for rolling production.

[0018] Furthermore,

[0019] In the solid medium and liquid medium of step (2), add no more than 1.0 mg / L of CCC.

[0020] The modified MS medium of the present invention replaces NH 4 NO 3 1650 mg / L with 40 mg / L, and replaces KNO 3 1900 mg / L with K 2 SO 4 40 mg / L, and replaces KH 2 PO 3 170 mg / L with NaH2 PO 3 10 mg / L, MgSO 4 .7H 2 O 370mg / L is replaced with 40mg / L, CaCl 2 .2H 2 O 440mg / L is replaced with 40mg / L; other MS components remain unchanged.

[0021] Furthermore, the pH of the culture medium is 5.8 - 6.0.

[0022] Compared with the existing tissue culture technology, the "factory production method of Centella asiatica polyploid seedlings by combined micro - organ cutting and hydroponics" of the present invention has the following 4 main advantages:

[0023] (1) The production cycle of the present invention is significantly shortened: Using the existing tissue culture technology: the technical route of explant selection - aseptic seedling culture - multiple shoot proliferation - rooting culture - indoor transplanting and acclimatization - field cultivation, it takes about 20 - 24 months to achieve large - scale factory production of seedlings. While the present invention uses the technical route of micro - organ cutting - with complete roots and leaves - hydroponic propagation - field cultivation, and only 12 - 15 months are needed to obtain a large number of complete plants for large - scale factory breeding. The production cycle is greatly shortened, and the production efficiency is significantly improved.

[0024] (2) The present invention has a faster rooting speed (3 - 7d) and a higher rooting rate (100%): By finely regulating the formula of the liquid medium for micro - organ cutting outside the bottle, the present invention can make the stem segments germinate new buds and new roots simultaneously within 3 - 7 days, significantly shortening the rooting time and increasing the rooting rate.

[0025] (3) The present invention has a higher transplanting survival rate (100%): Combining with hydroponic technology, the present invention immerses the plant roots in the liquid medium, provides various nutrient elements required for plant growth, accelerates the large - scale propagation of seedlings, and significantly improves the transplanting survival rate.

[0026] (4) The present invention is easier to operate and has a lower cost: The present invention optimizes the tissue culture process, reduces multiple steps such as multiple shoot proliferation and rooting, simplifies the operation, and thus reduces the labor and material costs.

[0027] All in all, through the organic combination of micro - organ cutting and hydroponic technology, the present invention realizes the rapid, efficient and low - cost factory production of Centella asiatica polyploid seedlings. Brief Description of the Drawings

[0028] Figure 1 : Growth conditions of tissue culture propagation control and micro - organ cutting propagation of tetraploid Centella asiatica new germplasm;

[0029] Among them: a is a new germplasm of tetraploid centella asiatica with stable genetic characteristics; b is about 15 days of potted tetraploid centella asiatica new germplasm; c is about 30 days of potted tetraploid centella asiatica new germplasm; d is about 60 days of potted tetraploid centella asiatica new germplasm; e is a stem segment with axillary buds cut into 1 - 3 cm; f and g are about 7 days of aseptic inoculation culture; h is about 25 days of aseptic inoculation culture; i is about 30 days of the 3rd subculture proliferation culture of aseptic seedlings; j and k are about 15 days of rooting culture on the MS + 6 - BA 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + agar 6.5 g / L medium; l is about 30 days of transplanted culture of rooted seedlings; m is about 5 months of transplanted and field - planted culture of tissue - cultured seedlings; n is 0 day of in vitro rooting culture of stem segments on solid medium (A); o is 0 day of in vitro rooting culture of stem segments on solid medium (B); p is 7 days of in vitro rooting culture of stem segments on solid medium (A); q is 7 days of in vitro rooting culture of stem segments on solid medium (B); r is the growth of roots and leaves in 7 days of in vitro rooting culture of stem segments on solid medium (A); s is the growth of roots and leaves in 7 days of in vitro rooting culture of stem segments on solid medium (B).

[0030] Figure 2 Growth of micro - organ cutting and hydroponic seedlings of new tetraploid centella asiatica germplasm;

[0031] Among them: a is 0 day of hydroponic culture of in vitro rooted seedlings on solid medium (A) transferred to liquid medium (A); b is 0 day of hydroponic culture of in vitro rooted seedlings on solid medium (B) transferred to liquid medium (B); c is about 15 days of hydroponic culture of in vitro rooted seedlings on solid medium (A) transferred to liquid medium (A); d is about 15 days of hydroponic culture of in vitro rooted seedlings on solid medium (B) transferred to liquid medium (B); e and g are the root and leaf conditions of in vitro rooted seedlings on solid medium (A) transferred to liquid medium (A) for about 15 days; f and h are the root and leaf conditions of in vitro rooted seedlings on solid medium (B) transferred to liquid medium (B) for about 15 days; i is the root and leaf condition of in vitro rooted seedlings on solid medium (A) transferred to liquid medium (A) for 30 days; j is the root and leaf condition of in vitro rooted seedlings on solid medium (B) transferred to liquid medium (B) for 30 days; k is the growth condition of in vitro rooted seedlings on solid medium (A) transferred to liquid medium (A) for about 60 days; l is the growth condition of in vitro rooted seedlings on solid medium (B) transferred to liquid medium (B) for 60 days.

[0032] Figure 3 Factory - scale production of polyploid seedlings of centella asiatica by combined use of micro - organ cutting and hydroponics;

[0033] Wherein: a is that stolons of the new germplasm of Centella asiatica with four times the volume are cut into stem segments with axillary buds of 1-3 cm and inserted into (A) a solid medium; b is that rooting occurs outside the bottle of the (A) solid medium for about 7 days; c-d are the growth conditions of the seedlings rooted outside the bottle of the (A) solid medium transferred to the (A) liquid medium for hydroponics for about 30 days; e-i are the growth conditions of the seedlings rooted outside the bottle of the (A) solid medium transferred to the (A) liquid medium for hydroponics for about 60 days; j is that the micro-organ cutting hydroponic seedlings are transplanted to the field for about 30 days; k is that the micro-organ cutting hydroponic seedlings grow in the field for about 5 months. Detailed implementation mode

[0034] The following is further to illustrate the present invention in combination with the detailed implementation mode, but the methods and the scope of protection described in the present invention are not limited thereto:

[0035] Example 1:

[0036] (1) Material and method

[0037] (1) Selection of explants

[0038] Sterile seedlings of Centella asiatica with stable genetic characteristics are selected as materials, and their rooted seedlings are transplanted into nutrient soil. The general-purpose cutting peat coconut coir seedling soil of Guangzhou Shengsheng Agricultural Seedling Substrate is selected. First, tap water is added and stirred until it is moist (the water content is about 30%). The tetraploid seedlings are transplanted well and placed in an artificial climate chamber for cultivation. The temperature is maintained at 28±2°C, the humidity is about 60%, the light intensity is 2000 Lx, and the light time is 12 h / d per day. After culturing for about 60 days, healthy stolons are selected and cut into stem segments with axillary buds of 1-3 cm ( Figure 1 a-e).

[0039] (2) Factory production method of polyploid seedlings of Centella asiatica by combining micro-organ cutting and hydroponics

[0040] Tissue culture propagation was used as the control group: The stem segments processed in step (1) were disinfected with 0.1% mercuric chloride for 12 min, washed 4 - 5 times with sterile water, and placed on a medium of MS + 30 g / L sucrose + 6.5 g / L agar for culture. 2 - 3 explants were inoculated in each bottle, with a total of 50 inoculated. They were cultured under weak light conditions in the culture room, maintaining a temperature of 24 ± 2 °C. New buds germinated after 10 - 15 d of culture. After continuing to culture for about 15 d, they were cultured on the same medium for 3 - 4 months, and then transferred to the preferred proliferation medium. The preferred proliferation medium was MS + 1.0 mg / L 6 - BA + 0.05 mg / L NAA + 30 g / L sucrose + 6.5 g / L agar, and cultured in the proliferation medium for 25 - 30 d; The robust proliferated seedlings were selected and separated into single plants, and transferred to the rooting medium. The preferred rooting medium was: MS + 0.5 mg / L 6 - BA + 0.5 mg / L NAA + 30 g / L sucrose + 6.5 g / L agar. They were cultured on the rooting medium for about 15 d to induce 10 - 15 roots; The rooted seedlings were transferred to a transplanting container with a top glass and placed in an artificial climate chamber for culture, maintaining a temperature of 28 ± 2 °C, a humidity of about 60%, a light intensity of 2000 Lx, and a light time of 12 h / d per day. When new leaves grew after 25 - 30 d, they were considered to have survived; The survived seedlings were transplanted outdoors for acclimatization for 5 - 7 d. During the acclimatization period, the light intensity and ventilation volume were gradually increased to adapt to the natural environment, and then transplanted to the field for conventional management. Seedling production cycle: Using the technical route of explant culture (1.5 - 2 months) - establishment of aseptic seedling regeneration system (4 - 5 months) - proliferation of clustered buds (6 - 7 months) - rooting (1.5 - 2.5 months) - indoor transplantation and acclimatization (2 - 3.5 months) - field cultivation (5 - 6 months), large-scale industrialized production of seedlings was achieved, and the cycle was about 20 - 24 months.

[0041] Micro-organ organ cutting culture of the present invention: The stem segments processed in step (1) are respectively placed on the culture media of (A) improved MS + 3.0 g / L agar and (B) improved MS + 0.5 mg / L CCC + 3.0 g / L agar. Select a white pot as the transplanting container, weigh 9 g of agar, add a small amount of prepared solution A or B, boil for 2 - 3 minutes, make up the volume to 3 liters, pour it into the pot and cool. Then insert the stem segments processed in step (1) onto the culture medium (the insertion depth is 0.5 - 1 cm), insert about 50 explants in each pot, place them in an artificial climate chamber for cultivation, keep the temperature at 28 ± 2 °C, the humidity at about 60%, the light intensity at 2000 Lx, and the light time at 12 h / d. After culturing for 3 - 7 days, new buds and new roots germinate simultaneously. After continuing to culture for about 10 days, transfer them to solution A or B for hydroponics correspondingly, with 45 - 55 explants / 3 L of culture medium, and supplement the liquid culture medium once every 5 - 7 days (1 L each time). The culture medium should just submerge the roots, place them in an artificial climate chamber for cultivation, keep the temperature at 28 ± 2 °C, the humidity at about 60%, the light intensity at 2000 Lx, and the light time at 12 h / d. After 30 - 60 days of hydroponics, well - growing seedlings are obtained. Then use the above - mentioned method to carry out rolling production on the obtained stolons. After about 5 - 6 months, a large number of seedlings are obtained. Move them to the outdoor for acclimatization for 5 - 7 days. During the acclimatization period, gradually increase the light intensity and ventilation volume to adapt to the natural environment. After the plants grow stably, carry out field transplanting and planting, and strengthen field management to ensure the normal growth of the plants. Seedling production cycle: Using the technical route of explant culture (1.5 - 2 months) - micro - organ cutting (0.5 - 1 month) - hydroponic propagation (5 - 6 months) - field cultivation (5 - 6 months), large - scale factory - like production of seedlings can be achieved in 12 - 15 months.

[0042] (II) Results and Analysis

[0043] 2.1 Propagation by tissue culture method

[0044] 2.1.1 Establishment of aseptic regeneration system and optimization of hormone concentration in culture medium

[0045] Select pot seedlings cultured in nutrient soil for 1.5 - 2 months, cut the healthy stolons into stem segments with axillary buds of 1 - 3 cm, after aseptic disinfection, inoculate them on the culture medium of MS + 0.5 - 2.0 mg / L 6 - BA + 0.05 - 0.5 mg / L NAA + 30 g / L sucrose + 6.5 g / L agar, and the culture cycle is 25 - 30 days per generation. The experimental results are shown in Table 1. Culture media with different hormone ratios can all induce the production of a large number of seedlings. Under the low - concentration combination of 6 - BA and NAA, new buds begin to germinate in about 7 days, the elongation speed increases after culturing for about 15 days, and roots are formed simultaneously in some plants ( Figure 1h). As the concentration of 6-BA increased, the germination time of new buds extended to more than 10 days. After three consecutive subculture times, cluster buds and callus gradually formed at the base.

[0046] According to the results analysis in Table 1, it was found that the optimal medium ratio could be determined according to experimental requirements:

[0047] Optimal germination medium: MS + 6-BA 0.5 mg / L + NAA 0.05 mg / L + sucrose 30 g / L + agar 6.5 g / L. The germination time of new buds was about 7 days, as shown in Figure 1 f - g.

[0048] Optimal proliferation medium: MS + 6-BA 1.0 mg / L + NAA 0.05 mg / L + sucrose 30 g / L + agar 6.5 g / L. The proliferation coefficient was 6, as shown in Figure 1 i.

[0049] Optimal rooting medium: MS + 6-BA 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + agar 6.5 g / L. The rooting rate was 100%, as shown in Figure 1 j - k.

[0050] On the above medium, 50 explants were inoculated, and a total of 36 sterile seedlings were obtained.

[0051] Six stem segments of the sterile seedlings grown on the 6 media were inoculated respectively, and after three consecutive subculture times, a total of 121 seedlings were obtained. The whole process from inducing axillary bud germination of stem segments to forming cluster buds took 4 - 5 months.

[0052] Table 1 Effects of 6-BA and NAA on inducing axillary bud seedling formation of Centella asiatica

[0053]

[0054] 2.1.2 Large-scale production of tissue-cultured seedlings

[0055] The 121 seedlings obtained from the culture in Section 2.1.1 were divided into clusters at 1 - 5 plants / cluster (a total of 40 clusters), and transferred to the MS + 6-BA 1.0 mg / L + NAA 0.05 mg / L + sucrose 30 g / L + agar 6.5 g / L proliferation medium. The culture conditions were the same as those described above. The culture and subculture inoculation time on the proliferation medium was 35 - 45 days, and the growth state of the seedlings was as shown in Figure 1 i. The cycle of each subculture was 35 - 45 days per generation, and each cluster of seedlings could be further divided into 3 clusters. Calculated according to a 5% contamination rate, the number of seedlings in each subculture was as follows:

[0056] The first subculture: 40 × 3 × 95% ≈ 114 clusters

[0057] The second subculture: 114×3×95% ≈ 325 clusters

[0058] The third subculture: 325×3×95% ≈ 926 clusters

[0059] The fourth subculture: 926×3×95% ≈ 2639 clusters

[0060] The fifth subculture: 2639×3×95% ≈ 7521 clusters

[0061] The total cost of 5 subcultures is 6 - 7 months.

[0062] Select 5000 - 6000 robust and effective seedlings with a height of 3 - 5 cm from 7521 clusters of seedlings, transfer them to the rooting medium of MS + 6 - BA 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + agar 6.5 g / L, and culture for about 15 days. Each seedling forms 10 - 15 roots, and the rooting rate reaches 100% ( Figure 1 j - k). Divide the cluster seedlings into single plants and transfer them to the rooting medium for culture, which takes a total of 1.5 - 2.5 months. The remaining small seedlings are continued to be transferred to the subculture medium for preservation. Transplant the rooted bottle seedlings into the nutrient soil and place them in an artificial climate chamber for culture, maintaining a temperature of 28 ± 2°C, a humidity of about 60%, a light intensity of 2000 Lx, and a daily light of 12 h / d. One month after transplantation, when new leaves grow, it indicates successful transplantation ( Figure 1 l). Then transfer the surviving seedlings cultivated in the greenhouse for about 2 months to a shady outdoor environment for acclimatization for 5 - 7 days, and gradually transfer them to the light conditions according to the actual situation to make them adapt to the natural environment. This stage takes 2 - 3.5 months. After the plants grow stably, carry out field transplantation and planting, and strengthen field management to ensure the normal growth of the plants. It is also necessary to carry out field cultivation for 5 - 6 months to obtain the first batch of tissue - cultured seedlings ( Figure 1 m). Through the technical route of explant culture (1.5 - 2 months) - establishment of aseptic seedling regeneration system (4 - 5 months) - multiplication of cluster buds (6 - 7 months) - rooting (1.5 - 2.5 months) - indoor transplantation and acclimatization (2 - 3.5 months) - field cultivation (5 - 6 months), large - scale industrial production of seedlings is realized, and the whole cycle is about 20 - 24 months.

[0063] 2.2 Micro - organ cutting culture

[0064] (1) Select potted seedlings cultured in nutrient soil for 1.5 - 2 months, cut the healthy stolons into stem segments with axillary buds of 1 - 3 cm, and directly inoculate them without disinfection onto the solid media of A: improved MS + agar 3.0 g / L and B: improved MS + CCC 0.5 mg / L + agar 3.0 g / L. The improved MS medium is shown in Table 2.

[0065] Specific method: Select a white pot as the transplanting container. Weigh 9 g of agar, add it to about 1.5 L of the prepared Solution A or Solution B, boil for 2 - 3 minutes, make up the volume to 3 L, and pour it into the pot to cool. Then cut the treated stem segments and insert them onto the culture medium ( Figure 1 n - o). Insert about 50 explants into each pot, place them in an artificial climate chamber for cultivation, maintain the temperature at 28 ± 2 °C, the humidity at about 60%, the light intensity at 2000 Lx, and the daily light duration at 12 h / d. Cultivate on the (A) solid medium for 7 days, and new buds and new roots will germinate simultaneously ( Figure 1 p). After cultivating on the (B) solid medium for about 7 days, new buds and new roots will germinate simultaneously ( Figure 1 q). Continue to cultivate for 7 - 10 days for subsequent hydroponics. From the results, the bud emergence speed, root length, and overall growth of the seedlings on the (A) solid medium are all better than those on the (B) solid medium ( Figure 1 r - s), and this stage takes 0.5 - 1 month.

[0066] Transfer the seedlings with complete roots and leaves cultivated on the above - mentioned solid medium into the corresponding (A) or (B) liquid medium for hydroponics, maintain the temperature at 28 ± 2 °C, the humidity at about 60%, the light intensity at 2000 Lx, and the daily light duration at 12 h / d; hydroponics 50 plants in each of the two media, a total of 100 plants ( Figure 1 a - b). During the cultivation process, add the corresponding liquid medium (1 L each time) every 5 - 7 days. After 30 - 60 days of hydroponics, obtain seedlings with lush growth and well - developed roots. The growth of the seedlings cultivated in the (A) liquid medium is significantly better than that in the (B) liquid medium ( Figure 2 c - l). Cut the first batch of stolons obtained into 712 stem segments with axillary buds, each 1 - 3 cm long. Calculate the multiplication coefficient: 712 ÷ 100 explants = 7.12.

[0067] Table 2 Comparison of the components of MS and modified MS

[0068]

[0069] (2) Industrialized production of Centella asiatica polyploid seedlings by combining micro - organ cutting and hydroponics

[0070] Place the 712 stem segments with axillary buds obtained above on the (A) modified MS + 3.0 g / L agar medium ( Figure 3 a). Insert 45 - 55 explants into each pot, and the cultivation conditions are the same as described above. After cultivating on the (A) solid medium for about 7 days, the growth state of the seedlings is as shown in ( Figure 3 b), continue to cultivate for 7 days and then transfer them to the liquid medium for hydroponics; after 30 days of liquid cultivation, it is as shown in ( Figure 3 c - d); after cultivating for about 60 days, the growth state of the seedlings is as shown in (Figure 3 As shown in e-i), approximately 712 × 7.12 ≈ 5069 seedlings with lush growth and well-developed roots were obtained. After three rounds of rolling production, which took 5 - 6 months, approximately 5069 rooted seedlings were obtained. These rooted seedlings were transferred to a shaded outdoor area for 5 - 7 days of acclimatization, and gradually transferred to light conditions according to the actual situation to enable them to adapt to the natural environment. After the plants grew stably, they were transplanted and planted in the field ( Figure 3 j), and field management was strengthened to ensure the normal growth of the plants. Approximately 5 - 6 months after field cultivation, the first batch of micro-cutting seedlings could be obtained ( Figure 3 k). Using the technical route of explant culture (1.5 - 2 months) - micro-organ organ cutting (0.5 - 1 month) - hydroponic propagation (5 - 6 months) - field cultivation (5 - 6 months), large-scale industrialized production of seedlings can be achieved in 12 - 15 months.

Claims

1. A method for the factory production of polyploid Centella asiatica seedlings based on micro-organ cutting-hydroponics, characterized in that: The following steps are involved: (1) Explant selection Select sterile seedlings of four times the volume of snow grass as materials, transplant the rooted seedlings into nutrient soil for cultivation, and cut the growing runners into stem segments with axillary buds; (2) Micro-organ cutting culture Micro-organ cutting culture: the stem segments treated in step (1) are cut and cultured on a modified MS solid culture medium; new shoots and new roots are germinated at the same time; the stem segments are transferred to a modified MS liquid culture medium for hydroponics, and the obtained seedlings are moved outdoors for seedling hardening and then transplanted into the field.

2. The method according to claim 1, characterized in that: Step (1) the nutrient soil comprises peat coconut husk seedling soil for cuttings, water is first added to stir and moisten, tetraploid seedlings are transplanted, and culture is carried out under the conditions of maintaining a temperature of 28±2°C, a humidity of 60-70%, a light intensity of 2000-2500Lx, and a light duration of 10-12h / d.

3. The method according to claim 1 or 2, characterized in that: Step (1) The rooted seedlings are transplanted into nutrient soil and cultured for 1.5-2 months, and healthy runners are selected and cut into stem segments with axillary buds.

4. The method according to claim 1, 2 or 3, characterized in that: Step (1) Cut into 1-3 cm stem segments with axillary buds.

5. The method according to claim 1, characterized in that: Step (2) prepare 3 liters of modified MS liquid medium + 3.0 g / L agar solid medium, and then insert 45-55 stem segments processed in step (1) into the medium at a depth of 0.5-1 cm.

6. The method according to claim 1 or 5, characterized in that: Step (2) The solid culture medium of the stem segments after cutting is cultured for 3-7 days under the conditions of temperature of 28±2°C, humidity of 60-70%, light intensity of 2000-2500Lx, and light duration of 10-12h / d, and new shoots and new roots are germinated at the same time; after further culture for 7-10 days, the stem segments are transferred to a modified MS liquid culture medium for hydroponic culture, with 45-55 seedlings / 3L culture medium, the roots are immersed in the culture medium during hydroponic culture, and the liquid culture medium is supplemented every 5-7 days, with 1L supplemented each time; the hydroponic culture temperature is 28±2°C, the humidity is 60-70%, the light intensity is 2000-2500Lx, and the light duration is 10-12h / d, and after hydroponic culture for 30-60 days, mature seedlings are obtained; the hydroponic container is moved to a cool and ventilated place outdoors for seedling hardening for 5-7 days, and the light intensity and ventilation volume are gradually increased during the seedling hardening period to adapt the seedlings to the natural environment; after the plant growth is stable, the plant is transplanted and planted in the field.

7. The method according to claim 1, 5 or 6, characterized in that: After 30-60 days of hydroponics in step (2), mature seedlings are obtained; the obtained runners are further cut into 1-3 cm stem segments with axillary buds and step (2) is repeated for rolling production.

8. The method according to any one of claims 1 to 7, characterized in that: The amount of CCC added to the solid culture medium and liquid culture medium in step (2) does not exceed 1.0 mg / L.

9. The method according to any one of claims 1 to 8, characterized in that: The improved MS medium is to replace NH4NO3 1650mg / L with 40mg / L, KNO3 1900mg / L with K2SO4 40mg / L, KH2PO3 170mg / L with NaH2PO3 10mg / L, MgSO4.7H2O 370mg / L with 40mg / L, and CaCl2.2H2O 440mg / L with 40mg / L in the MS medium; other MS components remain unchanged.

10. The method according to any one of claims 1 to 9, characterized in that: The pH of the culture medium was 5.8-6.0.

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