Rapid tissue propagation method of zantedeschia aethiopica and application

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a calla lily tissue rapid propagation method and application, 75% alcohol, hydrogen peroxide, a saturated bleaching powder solution and a 0.01% mercury bichloride solution are organically matched, the pollution rate and the disinfection death rate can be reduced to the maximum extent on the premise that explants are not damaged, and the method is suitable for large-scale popularization and application. By optimizing a culture medium formula and culture conditions, the primary induction rate and the multiplication coefficient of callalily tissue culture seedlings are remarkably increased, the primary induction rate reaches 100%, the multiplication coefficient is 5.8 or above, the breeding cycle is shortened, efficient breeding is achieved, and by adjusting the variety and the concentration of a growth regulator in a rooting culture medium, the yield of callalily is increased. The rooting rate reaches 92% or above, the root system is robust, and the transplanting survival rate of the tissue culture seedlings reaches 95% or above by optimizing the seedling hardening and transplanting matrix.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, and specifically to a method for rapid propagation of calla lily tissues and its application. Background Art

[0002] Calla lily (scientific name: Zantedeschia aethiopica (L.) Spreng.), also known as arrowhead flower, water taro, arum lily, flower taro, etc., is a perennial robust herbaceous plant of the genus Zantedeschia in the family Araceae. It has tubers; the leaves are basal, the leaf blades are relatively thick, green, heart-shaped sagittate or sagittate, entire, without patches; the inflorescence stalk is smooth; the spathe is 10 - 25 cm long, the tube part is short, yellow; the limb is slightly reclined, acute or acuminate, with a conical tip, bright white, sometimes with a green tint. The spadix is cylindrical, yellow: the female inflorescence is 1 - 2.5 cm long; the male inflorescence is 5 - 6.5 cm long; the berries are short ovoid, light yellow, with persistent styles; the seeds are obovoid spherical. The flowering period is from February to March, and the fruits mature in August - September. Calla lily prefers a warm climate, is not cold - tolerant, not heat - tolerant, slightly shade - tolerant, likes humidity, is not drought - tolerant, and prefers loose, fertile, humus - rich clay loam.

[0003] There are usually two methods for the propagation of calla lily. The traditional method is tuber meristem division, which has the disadvantages of slow propagation, low survival rate, long production cycle, easy variation, and small propagation coefficient. Another method is tissue culture rapid propagation technology. However, since different varieties require different culture media in the induction, subculture multiplication, and rooting stages, each flower color variety needs a special culture medium suitable for its own rapid propagation requirements.

[0004] Publication number: CN103229717A, Title: A Method for Improving the Proliferation Rate of Cluster Buds in Tissue Culture of Zantedeschia hybrida. The steps include: explant disinfection, bud induction culture, 60Coγ ray radiation treatment, rooting culture, and acclimatization and transplantation. This method is not practical because it uses 60Coγ ray radiation treatment. Publication number: CN104041407A, Title: A Rapid Propagation Method for Tissue Culture of Deep Purple Zantedeschia hybrida. Healthy and plump main buds or lateral buds of deep purple Zantedeschia hybrida are collected as explants, disinfected in sodium hypochlorite solution, and induced to produce adventitious buds on the induction medium MS + 6-BA 0.5 - 1.5 mg / L + NAA 0.1 - 0.3 mg / L. The induced adventitious buds are subcultured and propagated on the subculture medium MS + 6-BA 2.0 - 3.0 mg / L + NAA 0.2 - 0.3 mg / L. The small buds with a height of more than 2 cm are transferred to the rooting and strong seedling medium MS + 6-BA 0.5 - 1.0 mg / L + NAA 0.3 - 0.5 mg / L for rooting culture and strong seedling cultivation. The light intensity is 2000 lux, and the temperature is 25 °C. Finally, the acclimatized plants are transplanted to the greenhouse; the success rate of primary induction of this method is 100%, the induction rate of subculture is 100%, and the survival rate of acclimatization and transplantation after rooting is 95%. Publication number: CN107667866A, Title: A Cultivation Method for Tissue Culture Seedlings of Zantedeschia hybrida. The steps include: (1) Selecting the main bud or lateral bud of Zantedeschia hybrida as the explant, disinfecting it with 65% ethanol by mass, rinsing it with sterile water for 1 - 2 minutes, and then placing it in a sodium hypochlorite solution with a mass fraction of 15 - 25% for 5 minutes, and rinsing it with sterile water for 4 - 6 minutes to obtain the explant; (2) Culturing the explant in the induction medium until adventitious buds are formed; (3) Transferring the adventitious buds to the subculture medium for subculture and propagation; (4) When the small buds on the subculture medium grow to more than 2 cm, transferring them to the rooting and strong seedling medium for rooting culture and strong seedling cultivation; (5) When the tissue culture seedlings on the rooting and strong seedling medium grow to 2 - 3 leaves, acclimatizing them for 4 - 7 days, and transplanting them when the tissue culture seedlings have grown 3 - 5 leaves after acclimatization. The germination rate of tissue culture seedlings is 95.3 - 96.1%, and the cycle is 41 - 45 days. Publication number: CN118765789A, Title: A Tissue Culture Medium for Regeneration of Zantedeschia hybrida and a Regeneration Tissue Culture Method. The medium formula is: Induction medium: MS, 0.5 - 4.0 mg / L 6-BA, 30 g / L sucrose, and 6.5 - 7.0 g / L agar; Strong bud medium: MS, 1.0 - 2.5 mg / L 6-BA, 0 - 0.5 mg / L NAA, 0.5 - 2.0 mg / L carbon nanotubes, 40 - 60 g / L sucrose, and 6.5 - 7.0 g / L agar; The bulblet formation and rooting medium is based on the MS medium, and also includes: 0.5 - 2.0 mg / L NAA, 0.5 - 2.0 mg / L carbon nanotubes, 40 - 60 g / L sucrose, and 6.5 - 7.0 g / L agar.The inducing material is a sterile young bulblet thin slice with a length and width of 0.3 - 0.8 cm and a thickness of 0.1 - 0.2 cm. The inducing rate of buds by this method is 53.33 - 70.00%. The publication number is: CN119498201A, and the name is: Culture medium combination and method for constructing a regeneration system of Zantedeschia hybrida using tubers. Using the sterile and budless tubers of Zantedeschia hybrida at a specific growth stage as explants, and cooperating with specific induction medium, proliferation medium and differentiation medium to obtain cluster buds; the inducing rate of callus is 80%, the proliferation multiple is 4.5, the differentiation coefficient of callus is 48, and the rooting rate of seedlings is 93.33%. Zhao Runzhou et al. (Zhao Runzhou, Li Guirong, Yang Pengming. Optimization of in vitro rapid propagation system of Zantedeschia hybrida [J]. Chinese Agricultural Science Bulletin, 2009(24):4. DOI: CNKI:SUN:ZNTB.0.2009 - 24 - 064.) disclosed the optimization of the in vitro rapid propagation system of Zantedeschia hybrida. During the implementation of this method, the disinfection pollution rate is relatively high, browning occurs during primary culture, and the vitrification stripping rate is high, and the explants grow weakly. Summary of the Invention

[0005] The object of the present invention is to provide a method for rapid tissue propagation of Zantedeschia and its application in view of the problems existing in the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is: A method for rapid tissue propagation of Zantedeschia, the steps include:

[0007] (1) Selection and cleaning of explants: Select the young leaves or tubers of healthy Zantedeschia plants as explants, rinse with tap water for 1 - 2 h, then dip a soft - bristle toothbrush in a saturated soap solution and brush the surface of the explants 5 - 10 times, and finally rinse with tap water until there is no obvious soap solution residue;

[0008] (2) Disinfection of explants: Cut the cleaned explants into small segments 2 - 3 cm long, soak them in 75% alcohol for 30 - 40 s in sequence, rinse with sterile water 3 - 4 times, disinfect with 80% hydrogen peroxide for 30 - 40 min, rinse with sterile water 5 - 6 times, then disinfect with a saturated bleaching powder solution for 15 - 20 min, rinse with sterile water 3 - 4 times, and finally disinfect with 0.1% mercuric chloride for 6 - 7 min, rinse with sterile water 7 - 8 times;

[0009] (3) Primary culture: Inoculate the disinfected explants into the primary culture medium, place them in the dark at a temperature of 25 ± 2 °C for 10 - 15 days, and then place them at a temperature of 25 ± 2 °C, with 8 h of light per day and a light intensity of 2000 - 3000 lx for light - dark alternating culture for 50 - 60 days;

[0010] The composition of the primary medium is: MS + NAA 0.05 - 0.8 mg / L + CPPU 1.0 - 1.2 mg / L + TDZ 1.0 - 1.5 mg / L;

[0011] (4) Subculture and proliferation culture: The clustered buds obtained from the primary culture are cut into single buds and transferred to the subculture and proliferation medium, placed at a temperature of 25 ± 2 °C, illuminated for 10 h per day, with a light intensity of 2000 - 3000 lx, and cultured under light - dark alternation for 50 - 60 days;

[0012] The composition of the subculture and proliferation medium is: MS + NAA 0.02 - 0.05 mg / L + TDZ 2.0 - 2.5 mg / L;

[0013] (5) Rooting culture: The robust buds obtained from the proliferation culture are transferred to the rooting medium, placed in the dark at a temperature of 25 ± 2 °C for 10 - 15 days, and then placed at a temperature of 25 ± 2 °C, illuminated for 8 h per day, with a light intensity of 2000 - 3000 lx, and cultured under light - dark alternation for 50 - 60 days;

[0014] The composition of the rooting medium is: 1 / 2MS + 6 - BA 3.0 - 4.0 mg / L + NAA 0.5 - 0.6 mg / L + IBA 0.2 - 0.4 mg / L + AC 0.1 - 0.3 mg / L;

[0015] (6) Acclimatization: The rooted seedlings obtained are planted in a greenhouse with a 75% shading rate. The substrate is a mixed substrate of peat soil, perlite and garden soil, and conventional management is carried out;

[0016] (7) Transplanting: After 3 - 4 months of acclimatization, they are transplanted into the field and conventional management is carried out.

[0017] Furthermore, in step (2), the washed explants are cut into small segments 2 cm long, soaked in 75% alcohol for 35 s, rinsed 4 times with sterile water, disinfected with 80% hydrogen peroxide for 35 min, rinsed 6 times with sterile water, then disinfected with saturated bleaching powder solution for 17 min, rinsed 4 times with sterile water, and finally disinfected with 0.1% mercuric chloride for 6 min, rinsed 8 times with sterile water.

[0018] Furthermore, in step (3), the composition of the primary medium is: MS + NAA 0.2 mg / L + CPPU 1.1 mg / L + TDZ 1.3 mg / L.

[0019] Furthermore, in step (4), the composition of the subculture and proliferation medium is: MS + NAA 0.03 mg / L + TDZ 2.3 mg / L.

[0020] Further, in step (5), the rooting medium consists of: 1 / 2MS + 6-BA 3.5 mg / L + NAA 0.5 mg / L + IBA 0.3 mg / L + AC 0.2 mg / L.

[0021] Further, in step (6), the mass ratio of peat soil, perlite and garden soil is 5:1:2.

[0022] The beneficial technical effects of the present invention are as follows:

[0023] 1. The disinfection method of the present invention uses a combination of 75% alcohol, hydrogen peroxide, saturated bleaching powder solution and 0.01% mercuric chloride solution. Without damaging the explants, it can reduce the contamination rate and disinfection mortality rate to the greatest extent, improve the success rate of establishing the aseptic system of calla lilies, and significantly improve the primary induction rate and proliferation coefficient of calla lily tissue culture seedlings by optimizing the culture medium formula and culture conditions. The primary induction rate reaches 100%, and the proliferation coefficient is above 5.8, shortening the propagation cycle and achieving efficient propagation.

[0024] 2. The present invention uses leaves or tubers as explants and combines strict disinfection procedures, effectively reducing the pathogen carrying rate and obtaining healthy disease-free tissue culture seedlings, which can significantly reduce the relatively high incidence of root rot common in the existing ramet propagation technology.

[0025] 3. By adjusting the types and concentrations of growth regulators in the rooting medium, the present invention enables the rooting rate to reach over 92%, and the roots are strong and healthy. By optimizing the acclimatization and transplanting substrates, the transplanting survival rate of tissue culture seedlings reaches over 95%.

[0026] 4. The present invention uses conventional culture medium components, simplifies the operation steps, reduces the production cost, is suitable for large-scale production, and its tissue culture seedlings can stably inherit the excellent traits of the female parent, ensuring variety consistency. Description of the Drawings

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0028] Figure 1 It is a photo of the proliferation culture result of Example 1 of the present invention.

[0029] Figure 2 It is a photo of the proliferation culture result of Example 2 of the present invention.

[0030] Figure 3It is a photo of the proliferation culture result of Example 3 of the present invention.

[0031] Figure 4 It is a photo of the proliferation culture result of Comparative Example 1 of the present invention.

[0032] Figure 5 It is a photo of the proliferation culture result of Comparative Example 2 of the present invention.

[0033] Figure 6 It is a photo of the proliferation culture result of Comparative Example 3 of the present invention. Detailed implementation manners

[0034] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0035] Example 1

[0036] A method for rapid tissue propagation of calla lilies, the steps include:

[0037] (1) Selection and cleaning of explants: Select young leaves or tubers of healthy calla lily plants as explants, rinse with tap water for 1 h, then dip a soft-bristle toothbrush in a saturated soap solution, brush the surface of the explants 5 times, and finally rinse with tap water until there is no obvious soap solution residue.

[0038] (2) Disinfection of explants: Cut the cleaned explants into 2-cm-long segments, soak them in 75% alcohol for 30 s, rinse with sterile water 3 times, disinfect them with 80% hydrogen peroxide for 30 min, rinse with sterile water 5 times, then disinfect them with a saturated bleach solution for 15 min, rinse with sterile water 3 times, and finally disinfect them with 0.1% mercuric chloride for 6 min, rinse with sterile water 7 times.

[0039] (3) Primary culture: Inoculate the disinfected explants into the primary culture medium (the composition is: MS + NAA 0.05 mg / L + CPPU 1.0 mg / L + TDZ 1.0 mg / L), place them in the dark at a temperature of 25 ± 2 °C for 10 days, and then place them at a temperature of 25 ± 2 °C, with 8 h of light per day and a light intensity of 2000 lx for light-dark alternating culture for 50 days.

[0040] (4) Subculture proliferation culture: Cut the clustered buds obtained from the primary culture into single buds and transfer them to the subculture proliferation medium (the composition is: MS + NAA 0.02 mg / L + TDZ 2.0 mg / L), place them at a temperature of 25 ± 2 °C, with 10 h of light per day and a light intensity of 2000 lx for light-dark alternating culture for 50 days. The proliferation culture result is as Figure 1as shown

[0041] (5)Rooting culture: Transfer the robust buds obtained from the proliferation culture to the rooting medium (composition: 1 / 2MS + 6-BA 3.0 mg / L + NAA 0.5 mg / L + IBA 0.2 mg / L + AC 0.1 mg / L), place them in the dark at a temperature of 25±2°C for 10 days, and then place them at a temperature of 25±2°C, with 8 hours of light per day and a light intensity of 2000 lx, and culture them alternately in light and dark for 50 days.

[0042] (6)Acclimatization: Plant the seedlings after rooting culture in a greenhouse with a shading rate of 75%. The substrate is a mixed substrate of peat soil, perlite and garden soil (5:1:2), and carry out routine management.

[0043] (7)Transplanting: After 4 months of acclimatization, transplant them into the field and carry out routine management.

[0044] Example 2

[0045] A rapid tissue propagation method for calla lilies, the steps include:

[0046] (1)Explant selection and cleaning: Select the young leaves or tubers of healthy calla lily plants as explants, rinse them with tap water for 2 hours, then use a soft-bristled toothbrush dipped in saturated soapy solution to brush the surface of the explants 10 times, and finally rinse them with tap water until there is no obvious soapy solution residue;

[0047] (2)Explant disinfection: Cut the cleaned explants into 3-cm-long segments, soak them in 75% alcohol for 40 s, rinse them with sterile water 4 times, disinfect them with 80% hydrogen peroxide for 40 min, rinse them with sterile water 6 times, then disinfect them with saturated bleaching powder solution for 20 min, rinse them with sterile water 4 times, and finally disinfect them with 0.1% mercuric chloride for 7 min, and rinse them with sterile water 8 times;

[0048] (3)Primary culture: Inoculate the disinfected explants into the primary medium (composition: MS + NAA 0.8 mg / L + CPPU 1.2 mg / L + TDZ 1.5 mg / L), place them in the dark at a temperature of 25±2°C for 15 days, and then place them at a temperature of 25±2°C, with 8 hours of light per day and a light intensity of 3000 lx, and culture them alternately in light and dark for 60 days.

[0049] (4)Subculture proliferation culture: Cut the cluster buds obtained from the primary culture into single buds and transfer them to the subculture proliferation medium (composition: MS + NAA 0.05 mg / L + TDZ 2.5 mg / L), place them at a temperature of 25±2°C, with 10 hours of light per day and a light intensity of 3000 lx, and culture them alternately in light and dark for 60 days. The proliferation culture results are as Figure 2 as shown

[0050] (5) Rooting culture: Transfer the robust shoots obtained from the proliferation culture to the rooting medium (composition: 1 / 2MS + 6-BA 4.0 mg / L + NAA 0.6 mg / L + IBA 0.4 mg / L + AC 0.3 mg / L), and place them in the dark at a temperature of 25 ± 2°C for 15 days. Then, place them at a temperature of 25 ± 2°C, with 8 hours of light per day and a light intensity of 3000 lx, and culture them under alternating light and dark conditions for 60 days.

[0051] (6) Domestication: Plant the seedlings after rooting culture in a greenhouse with a 75% shading rate. The substrate is a mixed substrate of peat soil, perlite, and garden soil (5:1:2), and conduct routine management.

[0052] (7) Transplanting: After 4 months of domestication, transplant them into the field and conduct routine management.

[0053] Example 3

[0054] A rapid tissue propagation method for calla lilies, the steps include:

[0055] (1) Selection and cleaning of explants: Select the young leaves or tubers of healthy calla lily plants as explants, rinse them with tap water for 1.5 h, then dip a soft-bristle toothbrush in a saturated soap solution and brush the surface of the explants 8 times, and finally rinse with tap water until there is no obvious soap residue.

[0056] (2) Disinfection of explants: Cut the cleaned explants into 2-cm-long segments, soak them in 75% alcohol for 35 s, rinse them 4 times with sterile water, disinfect them with 80% hydrogen peroxide for 35 min, rinse them 6 times with sterile water, then disinfect them with a saturated bleaching powder solution for 17 min, rinse them 4 times with sterile water, and finally disinfect them with 0.1% mercuric chloride for 6 min and rinse them 8 times with sterile water.

[0057] (3) Primary culture: Inoculate the disinfected explants into the primary medium (composition: MS + NAA 0.2 mg / L + CPPU 1.1 mg / L + TDZ 1.3 mg / L), place them in the dark at a temperature of 25 ± 2°C for 13 days, then place them at a temperature of 25 ± 2°C, with 8 hours of light per day and a light intensity of 2500 lx, and culture them under alternating light and dark conditions for 55 days.

[0058] (4) Subculture and proliferation culture: Cut the clustered shoots obtained from the primary culture into single shoots and transfer them to the subculture and proliferation medium (composition: MS + NAA 0.03 mg / L + TDZ 2.3 mg / L), place them at a temperature of 25 ± 2°C, with 10 hours of light per day and a light intensity of 2500 lx, and culture them under alternating light and dark conditions for 55 days. The results of the proliferation culture are as Figure 3 shown.

[0059] (5) Rooting culture: Transfer the robust buds obtained from the proliferation culture to the rooting medium (composition: 1 / 2MS + 6-BA 3.5 mg / L + NAA 0.5 mg / L + IBA 0.3 mg / L + AC 0.2 mg / L), and place them in the dark at a temperature of 25±2°C for 13 days. Then, place them at a temperature of 25±2°C, with 8 hours of light per day and a light intensity of 2500 lx, and culture them alternately in light and dark for 55 days.

[0060] (6) Acclimatization: Plant the seedlings after rooting culture in a greenhouse with a shading rate of 75%. The substrate is a mixed substrate of peat soil, perlite and garden soil (5:1:2), and carry out routine management.

[0061] (7) Transplanting: After 4 months of acclimatization, transplant them into the field and carry out routine management.

[0062] Comparative Example 1

[0063] Operate according to Example 1 of the method for rapid tissue culture and propagation of deep purple calla lily with the publication number CN104041407A and the name: A method for rapid tissue culture and propagation of deep purple calla lily. The results of proliferation culture are as Figure 4 shown.

[0064] Comparative Example 2

[0065] Operate according to Example 1 of the method for culturing calla lily tissue culture seedlings with the publication number: CN107667866A and the name: A method for culturing calla lily tissue culture seedlings. The results of proliferation culture are as Figure 5 shown.

[0066] Comparative Example 3

[0067] Operate according to the formula disclosed in the publication number: CN 118765789A and the name: A tissue culture medium and regeneration tissue culture method for the regeneration of colored calla lily. The results of proliferation culture are as Figure 6 shown.

[0068] Comparative Example 4

[0069] Operate according to the optimal formula disclosed by Zhao Runzhou, Li Guirong, Yang Pengming. Optimization of the in vitro rapid propagation system of colored calla lily [J]. Chinese Agricultural Science Bulletin, 2009(24):4.DOI:CNKI:SUN:ZNTB.0.2009-24-064.

[0070] The disinfection contamination rate, disinfection mortality rate, primary induction rate, proliferation coefficient, rooting rate and transplanting survival rate of Examples 1-3 and Comparative Examples 1-4 were statistically analyzed. The results are shown in Table 1 below.

[0071] Table 1 Statistical results

[0072] Disinfection contamination rate Disinfection mortality rate Primary induction rate Proliferation coefficient Rooting rate Transplanting survival rate Example 1 12.4% 1.3% 100% 6.3 93.4% 96.8% Example 2 13.1% 1.7% 100% 5.8 94.1% 98.1% Example 3 12.8% 1.2% 100% 6.1 92.8% 95.9% Comparative example 1 34.6% 10.3% 84.6% 3.2 90.6% 90.3% Comparative example 2 32.7% 21.0% 81.3% 2.7 89.4% 84.6% Comparative example 3 46.3% 16.4% 70.0% 3.9 93.1% 83.2% Comparative example 4 32.4% 23.6% 96.9% 13.1 100% 93.5%

[0073] As can be seen from Table 1, in Examples 1-3, the disinfection contamination rate and the disinfection mortality rate are both lower than those in Comparative Examples 1-4, the primary induction rate and the transplanting survival rate are both higher than those in Comparative Examples 1-4, the multiplication coefficient is higher than that in Comparative Examples 1-3, and the rooting rate is higher than that in Comparative Examples 1-2; although in Comparative Example 4, the multiplication coefficient and the rooting rate are higher than those in Examples 1-3, there are still phenomena of explant browning and a high vitrification ratio during the primary culture in Comparative Example 4, which ultimately results in relatively weak explants.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that: the present invention can still be modified or equivalently replaced, and any modification or partial replacement without departing from the spirit and scope of the present invention should be covered by the scope of the claims of the present invention.

Claims

1. A rapid propagation method for calla lily tissue, characterized in that , The steps include: (1) Explant selection and cleaning: Select the young leaves or tubers of healthy calla lily plants as explants, rinse them with tap water for 1 - 2 h, then dip a soft-bristle toothbrush in a saturated soap solution and brush the surface of the explants 5 - 10 times, and finally rinse with tap water until there is no obvious soap residue; (2) Explant disinfection: Cut the cleaned explants into small segments 2 - 3 cm long, soak them in 75% alcohol for 30 - 40 s in sequence, rinse with sterile water 3 - 4 times, disinfect them with 80% hydrogen peroxide for 30 - 40 min, rinse with sterile water 5 - 6 times, then disinfect them with a saturated bleaching powder solution for 15 - 20 min, rinse with sterile water 3 - 4 times, and finally disinfect them with 0.1% mercuric chloride for 6 - 7 min, rinse with sterile water 7 - 8 times; (3) Primary culture: Inoculate the disinfected explants into the primary culture medium, place them in the dark at a temperature of 25 ± 2 °C for 10 - 15 days, and then place them at a temperature of 25 ± 2 °C, with 8 h of light per day and a light intensity of 2000 - 3000 lx for 50 - 60 days of light-dark alternating culture; The composition of the primary culture medium is: MS + NAA 0.05 - 0.8 mg / L + CPPU 1.0 - 1.2 mg / L + TDZ 1.0 - 1.5 mg / L; (4) Subculture and proliferation culture: Cut the clustered buds obtained from the primary culture into single buds and transfer them to the subculture proliferation medium, place them at a temperature of 25 ± 2 °C, with 10 h of light per day and a light intensity of 2000 - 3000 lx for 50 - 60 days of light-dark alternating culture; The composition of the subculture proliferation medium is: MS + NAA 0.02 - 0.05 mg / L + TDZ 2.0 - 2.5 mg / L; (5) Rooting culture: Transfer the strong buds obtained from the proliferation culture to the rooting medium, place them in the dark at a temperature of 25 ± 2 °C for 10 - 15 days, and then place them at a temperature of 25 ± 2 °C, with 8 h of light per day and a light intensity of 2000 - 3000 lx for 50 - 60 days of light-dark alternating culture; The composition of the rooting medium is: 1 / 2MS + 6-BA 3.0 - 4.0 mg / L + NAA 0.5 - 0.6 mg / L + IBA 0.2 - 0.4 mg / L + AC 0.1 - 0.3 mg / L; (6) Acclimatization: Plant the rooted seedlings in a greenhouse with a 75% shading rate, and use a mixed substrate of peat soil, perlite and garden soil for routine management; (7) Transplanting: After 3 - 4 months of acclimatization, transplant them into the field for routine management.

2. The method according to claim 1, wherein In step (2), the cleaned explants are cut into small segments 2 cm long, soaked in 75% alcohol for 35 s in sequence, rinsed with sterile water 4 times, disinfected with 80% hydrogen peroxide for 35 min, rinsed with sterile water 6 times, then disinfected with a saturated bleaching powder solution for 17 min, rinsed with sterile water 4 times, and finally disinfected with 0.1% mercuric chloride for 6 min, rinsed with sterile water 8 times.

3. The method according to claim 1, characterized in that: In step (3), the composition of the medium is: MS + NAA 0.2 mg / L + CPPU 1.1 mg / L + TDZ 1.3 mg / L.

4. The method according to claim 1, characterized in that: In step (4), the subculture multiplication medium consists of: MS + 0.03 mg / L of NAA + 2.3 mg / L of TDZ.

5. The method according to claim 1, wherein In step (5), the rooting medium consists of: 1 / 2MS + 3.5 mg / L of 6-BA + 0.5 mg / L of NAA + 0.3 mg / L of IBA + 0.2 mg / L of AC.

6. The method according to claim 1, wherein In step (6), the mass ratio of peat soil, perlite and garden soil is 5:1:

2.

7. Application of the method according to any one of claims 1-6 in the rapid tissue propagation of calla lilies.

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