A tissue culture and rapid propagation method of begonia fimbristipula

Abstract

The application discloses a tissue culture and rapid propagation method of Begonia fimbristipula, and the Begonia fimbristipula is successfully tissue cultured and mass propagated by taking annual leaves of the Begonia fimbristipula as explants, and through brushing, disinfection, induction of adventitious buds, strong seedling culture, rooting culture, and seedling hardening and transplanting; wherein the fluff on the surface of the explants is removed through brushing, the problem of incomplete disinfection is solved, and the pollution rate is significantly reduced; the root protection and seedling hardening liquid is prepared by mixing the leaf extract of the Chinese silvergrass with vitamin B12 and rice vinegar, the transplanting and seedling hardening period is shortened, and the transplanting survival rate is improved. The application improves the breeding efficiency of the Begonia fimbristipula, and protects the germplasm resources of the Begonia fimbristipula.

Description

Technical Field

[0001] This invention belongs to the field of rapid propagation technology of rare plant tissue culture, specifically relating to a rapid propagation method of Begonia 'Black Peak' through tissue culture. Background Technology

[0002] Black Peak Begonia ( Begoniaferox It belongs to the Begoniaceae family ( Begoniaceae Begonia ( ) Begonia This small, terrestrial herb is found only on the limestone walls of the Nonggang National Nature Reserve in China. Its leaves have prominent black protuberances. Due to severe commercial harvesting caused by its unique leaf shape, the number of surviving individuals is low, and it has been classified as Critically Endangered (CR) by the International Union for Conservation of Nature (IUCN). It is also listed as a Class II National Key Protected Wild Plant in the 2021 National Key Protected Wild Plant List. The Black-peaked Begonia is valued for both its flowers and foliage and has significant development potential.

[0003] In recent years, research on Begonia 'Black Peak' has been relatively limited, focusing mainly on field resource surveys. In 2019, a team led by the Begonia Professional Committee of the China Wild Plant Conservation Association completed a field survey of Begonia resources, including Begonia 'Black Peak', preliminarily clarifying the distribution baseline of Begonia 'Black Peak' in the wild. Currently, research on the propagation of Begonia 'Black Peak' is essentially nonexistent, and the sustainable preservation of its living plants remains a significant challenge, severely restricting the conservation of Begonia 'Black Peak' resources. Tissue culture, as a biotechnology, is faster than propagation methods such as cuttings and grafting, and has been widely used in the field of plant propagation.

[0004] CN116849126A discloses a method for in vitro preservation of germplasm resources of Begonia 'Black Peak'. Using newly grown, incompletely unfolded leaves as explants, the method successfully achieved large-scale propagation of seedlings through explant disinfection, callus induction, callus dedifferentiation to induce adventitious buds, adventitious bud subculture, rooting and seedling cultivation, and transplanting of test-tube seedlings. However, each stage requires the preparation of specific culture media, making the process complex and increasing the risk of bacterial contamination during cultivation.

[0005] In addition, because the surface of the Black Peak Begonia is covered with a layer of fuzz, it is extremely difficult to sterilize it thoroughly during tissue culture, resulting in a high contamination rate, and the survival rate after transplanting is still at a low level. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a rapid tissue culture propagation method for Begonia 'Black Peak', which has a high propagation coefficient, a large number of roots, and a high transplant survival rate, thereby improving the propagation efficiency of Begonia 'Black Peak' and protecting its germplasm resources.

[0007] To achieve the above objectives, this invention provides a rapid tissue culture propagation method for Begonia 'Black Peak', comprising the following steps:

[0008] (1) Explant acquisition and disinfection: One-year-old leaves with good growth and no pests or diseases are used as explants. The leaves are brushed to remove the hairs and then rinsed and disinfected to obtain explants.

[0009] (2) Induction of adventitious shoots: After cutting and scratching the explants, they were inoculated into the induction medium to induce the growth of adventitious shoots;

[0010] (3) Seedling cultivation: After dividing the adventitious buds into 2-4 adventitious buds, inoculate them into the seedling cultivation medium and cultivate strong seedlings;

[0011] (4) Rooting culture: Divide the adventitious buds after the seedlings have grown strong in step (3) into individual plants, inoculate them into the rooting culture medium, and culture them to obtain rooted seedlings;

[0012] (5) Hardening and transplanting: After hardening, the rooted seedlings obtained in step (4) are rinsed and soaked in root-preserving and seedling-soothing solution, and then transplanted into the transplanting substrate.

[0013] Preferably, the rinsing in step (1) involves soaking in a 0.3% detergent solution for 15-30 minutes and then rinsing with running water for 30-60 minutes; the disinfection method involves first disinfecting with a 75% alcohol solution for 15-20 seconds, rinsing with sterile water 2-5 times, then sterilizing with 0.1% HgCl2 by shaking for 1.5-3 minutes, and rinsing with sterile water 3-5 times.

[0014] Preferably, the explant cutting method in step (2) involves removing the edge portion and then dividing the remaining explant into sections with an area of ​​2-4 cm². 2 .

[0015] Preferably, the induction medium formulation in step (2) is 1 / 2MS + 6-BA 0.8-1.0 mg / L + NAA 1.0 mg / L + 2,4-D 0.1-0.2 mg / L.

[0016] Preferably, the induction culture conditions in step (2) are as follows: callus tissue is obtained by dark culture at 25°C for 20-25 days, then adventitious buds are generated by alternating culture at a light intensity of 1000-1200 lx for 15-20 days, and finally adventitious buds are promoted by alternating culture at a light intensity of 1400-1500 lx and 27°C for 8-10 days; wherein the light intensity is 12h and the darkness is 12h when adventitious buds are generated; and the light intensity is 14h and the darkness is 10h when adventitious buds are promoted.

[0017] Preferably, the seedling culture medium in step (3) is 1 / 2MS + NAA 0.2 mg / L + IBA 0.1-0.2 mg / L; the culture conditions are 25℃ and light intensity 1800 lx, with alternating culture for 18-23 days, including 14h of light and 10h of darkness.

[0018] Preferably, the 1 / 2MS+NAA 0.2 mg / L in step (4) is cultured in the dark at 21℃ for 4-8 days, and then alternately cultured at 25℃ and light intensity of 3000 lx for 15-20 days, with 16 hours of light and 8 hours of darkness.

[0019] Preferably, the root-protecting and seedling-retreating liquid formula in step (5) consists of 1-5 parts of Agrimonia pilosa leaf extract, 1-3 parts of vitamin B12, and 3-8 parts of rice vinegar.

[0020] Preferably, the method for preparing the extract of *Dictamnus dasycarpus* leaves involves taking fresh *Dictamnus dasycarpus* leaves, crushing them, dissolving them in water, and filtering them after standing. The mass fraction of *Dictamnus dasycarpus* leaves is 0.8-1.5%.

[0021] Preferably, the hardening conditions in step (5) are a temperature of 22-26℃, a humidity of 50-60%, and a light intensity of 2300-2600 lx for 5-10 days; the transplanting substrate is composed of limestone fragments, peat soil, vermiculite, and yellow soil in a volume ratio of 3:1:1:2.

[0022] The beneficial effects of this invention are as follows:

[0023] 1. The fuzz and stains on the surface of the leaves of Begonia 'Black Peak' were brushed off, which solved the problem of incomplete disinfection of the explants and reduced the contamination rate in the subsequent cultivation process. The leaves used were one-year-old leaves, which lowered the threshold for explant selection and had the advantages of abundant quantity, no seasonal restrictions, and easy large-scale cultivation.

[0024] 2. Field surveys revealed that Begonia 'Black Peak' has a companion species, Miscanthus sinensis. Begonia 'Black Peak' thrives better in areas where Miscanthus sinensis grows. Therefore, Miscanthus sinensis leaf extract was prepared and combined with vitamin B12 and rice vinegar to create a root-protecting and seedling-retreating solution. This solution utilizes certain natural components in Miscanthus sinensis to promote the growth of Begonia 'Black Peak' and improve the transplant survival rate.

[0025] 3. Prepare a transplanting substrate by mixing limestone fragments with peat moss, vermiculite, and yellow soil to create a transplanting environment similar to the native habitat for Begonia 'Black Peak', thereby further improving the transplant survival rate of Begonia 'Black Peak'.

[0026] 4. The tissue culture process has been simplified, combining the three stages of callus induction, callus dedifferentiation induction of adventitious shoots, and adventitious shoot subculture into one stage. Only the same culture medium is used, and the two stages are controlled by adjusting the light intensity and photoperiod, which shortens the seedling cycle. Seedlings are ready for transplanting in 100 days.

[0027] 5. This invention is not limited by season, can effectively maintain the excellent quality of plants, and can be used for mass propagation, producing a large number of seedlings in a short period of time. Attached Figure Description

[0028] Figure 1 The image shows the callus tissue induced in Example 1.

[0029] Figure 2 The image shows the clustered buds induced in Example 1.

[0030] Figure 3 The image shows the clustered buds obtained from the vigorous seedling cultivation in Example 2.

[0031] Figure 4 The image shows the rooting status of the Black Peak Begonia obtained through rooting culture in Example 3.

[0032] Figure 5 Images of Begonia 'Black Peak' transplanted 7 days after Example 4. Detailed Implementation

[0033] The technical solution of the present invention will be further explained and described below with reference to the accompanying drawings and specific embodiments. It is worth noting that the following embodiments are only preferred embodiments of the present invention and should not be construed as limiting the present invention. The scope of protection of the present invention should be determined by the contents of the claims. Modifications and substitutions made by those skilled in the art to the technical solution of the present invention without creative effort all fall within the scope of protection of the present invention.

[0034] Example 1

[0035] (1) Acquisition and disinfection of explants: Select healthy and disease-free black begonias as the collection object, collect plump one-year-old leaves as explant materials, gently brush the surface of the explants with a soft brush to remove dust and some fungal stains attached to the surface of the explants, and remove the hairs on the surface of the explants, and then rinse with running water; then put the explants into a 1000ml large glass beaker, add 0.3% detergent solution, submerge the explants and soak for 20 min, and rinse with running water for 40 min after soaking; take out the rinsed explants and put them into sterilized tissue culture bottles, pour in 75% alcohol for 20s to disinfect, submerge the explants, shake the bottle continuously during the process, and rinse with sterile water 3 times after the process; after rinsing, pour in 0.1% HgCl2 to submerge the explants, sterilize for 2 min, shake continuously during the soaking process, and rinse with sterile water 5 times after the process to obtain the disinfected explants;

[0036] (2) Pretreatment of explants: Use sterile filter paper to absorb the moisture on the surface of the disinfected explants. In a sterile tray, cut off the edge of the explant leaves and discard it. Cut the remaining explants into rectangles with a side length of 2cm. Use a blade to scratch the explants to increase the chance of callus induction.

[0037] (3) Induction of adventitious shoots: Induction medium (Table 1) was prepared using plant hormones and 1 / 2 MS and introduced into culture bottles to a thickness of 1 cm. Then, the explants that were scratched in step (2) were inoculated onto the medium, sealed with a bottle cap with a breathable membrane, and cultured in the dark in a culture room at 25°C for 20 days to induce callus tissue ( Figure 1 Then, the tissue was cultured alternately under a light intensity of 1200 lx for 20 days (12h light / 12h dark alternation) to induce adventitious shoots from the callus tissue; then, it was cultured alternately under a light intensity of 1500 lx and a temperature of 27℃ for 10 days (14h light / 10h dark alternation) to promote the growth of adventitious shoots. Figure 2 When the height of the adventitious bud is ≥2cm, the induction of the adventitious bud is completed; the culture bottle specifications are 10cm in bottom diameter and 11cm in height.

[0038] Table 1. Induction medium formulation and induction results

[0039]

[0040] In this embodiment, the villi on the surface of the explant were removed, reducing the highest contamination rate to 2% on day 3, 2% on day 6, and 4% on day 9.

[0041] Comparative Example 1

[0042] The method and steps are the same as in Example 1, except that the culture conditions for inducing adventitious shoots in step (3) are changed to alternating culture at a light intensity of 2000 lx for 20 days (8h light / 16h dark alternation).

[0043] Callus tissue was induced, but the induction rate was 37%, and the callus tissue could differentiate into adventitious shoots, but the induction rate of adventitious shoots was only 29%.

[0044] Comparative Example 2

[0045] The method and steps are the same as in Example 1, except that the explant is disinfected directly without brushing.

[0046] It can induce callus and adventitious shoots, but the contamination rate reaches 34% on the 3rd day after inoculation, the highest contamination rate of 38% on the 6th day, and as high as 41% on the 9th day. It is easy for a large number of people to die, resulting in a reduction in the yield of adventitious shoots and making it difficult to scale up the performance.

[0047] Example 2

[0048] (1) Take the adventitious buds induced in Example 1 (number 1) and divide them into small clumps with 2-4 adventitious buds;

[0049] (2) Prepare seedling strengthening culture medium using different concentrations of NAA, IBA and 1 / 2MS (Table 2), and then pour the seedling strengthening culture medium into a culture bottle with a bottom diameter of 10cm and a height of 11cm, so that the thickness of the culture medium is 1cm.

[0050] (3) Inoculate one small clump obtained in step (1) into each culture bottle containing culture medium, then seal it with a bottle cap with a breathable membrane, and place it in a culture bottle at a temperature of 25℃ and a light intensity of 1800 lx for 20 days (alternating between 14h light and 10h darkness). When the height of the small clump reaches more than 4cm ( Figure 3 When the seedlings are ready, complete the cultivation of strong seedlings.

[0051] Table 2. Seedling strengthening culture medium formula and seedling strengthening results

[0052]

[0053] Comparative Example 2

[0054] The method and steps are the same as in Example 1, except that the culture bottle specifications in step (3) are changed to a bottom diameter of 8cm and a height of 9cm.

[0055] The induction rates of callus and adventitious shoots were unaffected, but the average height of adventitious shoots was only 2.5 cm after 20 days of robust seedling culture.

[0056] Example 3

[0057] (1) Take the small clump of seedlings with a height of more than 4cm that has completed the strong seedling cultivation in Example 2 and divide it into individual plants;

[0058] (2) Rooting medium was prepared using different concentrations of NAA and 1 / 2MS (Table 3), and then poured into a culture bottle with a bottom diameter of 10cm and a height of 11cm to make the medium thickness 1.2cm;

[0059] (3) The single plants obtained in step (1) are inoculated into the culture bottles obtained in step (2), with 3 plants evenly inoculated in each culture bottle in a triangular shape. The bottles are then capped with a breathable membrane and placed in a culture room at 21°C for 5 days of dark culture. Then, the bottles are cultured alternately at 25°C and 3000 lx light intensity for 18 days (16 hours of light / 8 hours of darkness alternately) to promote rooting. Figure 4 After the experiment was completed, the rooting rate was calculated and the root vigor was observed. The results are shown in Table 3.

[0060] Table 3 Rooting medium formulation and rooting results

[0061]

[0062] Table 3 shows that adding 0.1-0.2 mg / L NAA to the culture medium can increase the rooting rate to over 92% and promote vigorous root growth. However, further increasing the NAA concentration actually decreases the rooting rate, and some roots lose viability. Using commonly used AC or a combination of AC and NAA can promote rooting. The rooting rate of *Begonia 'Black Peak'* is actually lower than when NAA is used alone, and about one-third of the roots lose viability.

[0063] Comparative Example 3

[0064] The method and steps were the same as in Example 3, except that the thickness of the culture medium was adjusted to 1.5 cm, and rooting culture was completed after 23 days. Statistical analysis showed that the rooting rate was 67%, and most of the roots lost their viability.

[0065] Example 4

[0066] (1) Take the rooted plantlets in Example 3 No. 1, transfer them to the hardening room, open the bottle cap, keep the temperature in the hardening room at 22℃, the humidity at 50%, and the light intensity at 2500 lx for 7 days. After the hardening is completed, remove the plants and rinse the culture medium with clean water.

[0067] (2) Preparation of root protection and seedling recovery solution: Take 10g of fresh scutellaria leaves, crush them and put them in a 100ml volumetric flask, dilute with distilled water, let stand for 2 hours, filter and obtain scutellaria leaf extract. Then mix the scutellaria leaf extract with vitamin B12 and Hengshun rice vinegar in a certain mass ratio, and then dilute with deionized water to 1000 parts.

[0068] (3) Preparation of transplanting substrate: Mix limestone fragments, peat soil, vermiculite and yellow soil in a volume ratio of 3:1:1:2 to prepare transplanting substrate;

[0069] (4) After soaking the cleaned plants obtained in step (1) in the root-protecting and seedling-retreating solution prepared in step (2) for a period of time, they were planted in the transplanting substrate prepared in step (3). After 7 days, the seedling-retreating time, root growth and survival rate of the black begonia were counted. Figure 5 The results are shown in Table 4.

[0070] Table 4. Effects of root-protecting and seedling-establishing solution on transplanting conditions.

[0071]

[0072] Note: Root biomass is the total mass of roots and can be used to measure the amount of roots.

[0073] As shown in Table 4, treating Black Peak Begonia seedlings with a root-protecting and seedling-establishing solution can shorten the seedling establishment time from 5 days to 1-3 days and increase the survival rate from about 30% to over 50%. The formula of the root-protecting and seedling-establishing solution also has a significant impact on the seedling establishment time and survival rate. Specifically, when the mass ratio of Dictamnus dasycarpus leaf extract, vitamin B12, and rice vinegar is 3:2:5 and diluted 1000 times, soaking for 4-6 hours can shorten the seedling establishment time to 1 day and increase the survival rate to 96%-98%.

[0074] Example 5

[0075] (1) Using the rooted plants in Example 3 No. 1, harden the seedlings according to the method described in Example 4, and then soak them in the root-preserving and seedling-retreating solution in Example 4 No. 3 for 5 hours for later use.

[0076] (2) Preparation of transplanting substrate: Limestone fragments, peat soil, vermiculite perlite and yellow soil are prepared in a certain volume ratio. Then the plants treated in step (1) are transplanted. The formula of transplanting substrate and the survival rate of transplanting are shown in Table 5.

[0077] Table 5 Transplanting substrate formula and transplanting survival rate

[0078]

[0079] Table 5 shows that adding limestone fragments to the transplanting substrate can improve the transplant survival rate of Begonia 'Black Peak'. Furthermore, when the volume ratio of limestone fragments, peat moss, vermiculite, and yellow soil is 3:1:1:2, the survival rate can reach as high as 96%. Since the native habitat of Begonia 'Black Peak' is located on limestone walls, this indicates that adding limestone fragments to the transplanting substrate can create a native habitat for Begonia 'Black Peak' and promote its growth.

Claims

1. A method for rapid tissue culture propagation of Begonia 'Black Peak', characterized by: Includes the following steps: (1) Explant acquisition and disinfection: One-year-old leaves with good growth and no pests or diseases are used as explants. The leaves are brushed to remove the hairs and then rinsed and disinfected to obtain explants. (2) Induction of adventitious shoots: After cutting and scratching the explants, they were inoculated into the induction medium to induce the growth of adventitious shoots; (3) Seedling cultivation: After dividing the adventitious buds into 2-4 adventitious buds, inoculate them into the seedling cultivation medium and cultivate strong seedlings; (4) Rooting culture: Divide the adventitious buds after the seedlings in step (3) into individual plants, inoculate them into the rooting culture medium, and culture them to obtain rooted seedlings; the rooting culture medium is poured into a culture bottle with a bottom diameter of 10cm and a height of 11cm, so that the thickness of the culture medium is 1.2cm. (5) Hardening and transplanting: After hardening, the rooted seedlings obtained in step (4) are rinsed and soaked in root-preserving and seedling-establishing solution, and then transplanted into the transplanting substrate; The induction medium formulation in step (2) is 1 / 2 MS + 6-BA 0.8-1.0 mg / L + NAA 1.0 mg / L + 2,4-D 0.1-0.2 mg / L; The induction culture conditions described in step (2) are as follows: callus tissue is obtained by dark culture at 25℃ for 20-25 days, followed by adventitious buds generated by alternating culture at a light intensity of 1000-1200 lx for 15-20 days, and finally adventitious buds are promoted by alternating culture at a light intensity of 1400-1500 lx and 27℃ for 8-10 days. Among them, the light exposure is 12h and the darkness is 12h when adventitious buds are generated, and the light exposure is 14h and the darkness is 10h when adventitious buds are promoted. The seedling culture medium mentioned in step (3) is 1 / 2MS + NAA 0.2 mg / L + IBA 0.1-0.2 mg / L; the culture conditions are 25℃ and light intensity 1800 lx, with alternating culture for 18-23 days, including 14h of light and 10h of darkness; The rooting medium formula in step (4) is 1 / 2 MS + NAA 0.2 mg / L; the culture conditions are 21℃ dark culture for 4-8 days, and then alternating culture at 25℃ and light intensity of 3000 lx for 15-20 days, including 16 hours of light and 8 hours of darkness.

2. The method for rapid tissue culture propagation of Begonia 'Black Peak' according to claim 1, characterized in that: The rinsing in step (1) involves soaking in a 0.3% detergent solution for 15-30 minutes and then rinsing with running water for 30-60 minutes. The disinfection method involves first disinfecting with a 75% alcohol solution for 15-20 seconds, rinsing with sterile water 2-5 times, then sterilizing with 0.1% HgCl2 by shaking for 1.5-3 minutes, and rinsing with sterile water 3-5 times.

3. The method for rapid tissue culture propagation of Begonia 'Black Peak' according to claim 1, characterized in that: The explant cutting method described in step (2) involves removing the edge portion and then dividing the remaining explant into sections with an area of ​​2-4 cm². 2 .

4. The method for rapid tissue culture propagation of Begonia 'Black Peak' according to claim 1, characterized in that: The formula for the root-protecting and seedling-retreating liquid in step (5) is 1-5 parts of Agrimonia pilosa leaf extract, 1-3 parts of vitamin B12, and 3-8 parts of rice vinegar.

5. The method for rapid tissue culture propagation of Begonia 'Black Peak' according to claim 4, characterized in that: The method for preparing the extract of *Dictamnus dasycarpus* leaves is to take fresh *Dictamnus dasycarpus* leaves, crush them, dissolve them in water, and filter them after standing. The mass fraction of *Dictamnus dasycarpus* leaves is 0.8-1.5%.

6. The method for rapid tissue culture propagation of Begonia 'Black Peak' according to claim 1, characterized in that: The hardening conditions described in step (5) are a temperature of 22-26℃, a humidity of 50-60%, and a light intensity of 2300-2600 lx for 5-10 days; the transplanting substrate is composed of limestone fragments, peat soil, vermiculite, and yellow soil in a volume ratio of 3:1:1:2.

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