A cultivation method for seedlings of regenerated Hippeastrum plants and its application

Abstract

The present invention belongs to the technical field of plant tissue culture, and particularly relates to a cultivation method and application of seedlings of regenerated plants of Hippeastrum rutilum. The cultivation method includes: selecting Hippeastrum rutilum flower buds as explants, inducing the culture of Hippeastrum rutilum callus, culturing the callus for redifferentiation, or directly performing redifferentiation culture after cutting the explants into pieces; after 30-40 days of redifferentiation culture, adventitious buds are formed, and seedlings are generated after 50-80 days of culture. The present invention selects relatively active red flower buds as explants to cultivate Hippeastrum rutilum. The survival rate of the seedlings of the regenerated plants of Hippeastrum rutilum obtained by culturing using the method provided by the present invention is as high as 95.3%, and the induction rate is over 96.2%. By culturing callus blocks with a surface area of only 1 cm<supgt;2< / supgt;, 17-25 Hippeastrum rutilum bulbs can be differentiated and proliferated. It can not only obtain a large number of Hippeastrum rutilum seedlings in a relatively short time, but also protect the mother bulbs from being damaged, providing a simple and reliable method for the propagation and protection of Hippeastrum rutilum.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant tissue culture, and particularly relates to a cultivation method and application of Hippeastrum vittatum regenerated plant seedlings. Background Art

[0002] Hippeastrum vittatum, also known as Hippeastrum hybridum and Amaryllis vittata, belongs to the genus Hippeastrum of the monocotyledon subclass Amaryllidaceae, and is a perennial ornamental bulbous flower plant with bulbs. It is native to Central and South America. During the two or three decades after the 19th century, the breeding process of Hippeastrum vittatum was greatly promoted, forming many excellent varieties. The Netherlands is the world center for the production and breeding of Hippeastrum vittatum, and many excellent horticultural varieties have also been cultivated in countries such as Australia, the United States, Peru, South Africa, and Japan. It was introduced into China in the early 20th century, and a large number of new varieties began to enter China. China has also developed rapidly in recent years.

[0003] The natural propagation rate of Hippeastrum vittatum bulbs is very low, and most varieties hardly produce daughter bulbs. Bulb cutting and wounding methods have been used for Hippeastrum vittatum propagation at home and abroad, but the operation procedures of bulb cutting and wounding methods are cumbersome, the cost is high, the rooting rate is low, the growth of cuttings is weak, and the seedling formation rate is low, making it difficult to meet the growing market demand. Therefore, establishing a high-quality and efficient bulb propagation technology system is the key to improving the quality of Hippeastrum vittatum bulbs.

[0004] In vitro culture technology has been widely used in the large-scale production of high-quality planting materials. Adventitious buds have also been induced by in vitro culture of Hippeastrum vittatum bulb scales. However, when using bulb scales as explants, the microbial content inside and outside the bulb scales is relatively high, and the plants are seriously contaminated during in vitro culture; moreover, the mortality rate and browning rate are relatively high when using bulb scales as explants; in addition, bulb scales are very precious, and their seedlings are expensive. The conventional scale cutting propagation speed is slow and requires a large number of bulbs; moreover, the bulbs of newly bred Hippeastrum vittatum varieties are scarce, and it is very easy to fail when the bulbs of newly bred Hippeastrum vittatum varieties are cut into scales for propagation, resulting in the disappearance of newly bred Hippeastrum vittatum varieties. Therefore, there is not much advantage in Hippeastrum vittatum scale cutting, and there is an urgent need to provide a new cultivation method for regenerated plant seedlings to improve the propagation speed and survival rate of Hippeastrum vittatum. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a cultivation method and application of Hippeastrum vittatum regenerated plant seedlings, which can obtain a large number of Hippeastrum vittatum seedlings in a relatively short time with a high survival rate, providing a new method for Hippeastrum vittatum propagation and protection.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a cultivation method of Hippeastrum vittatum regenerated plant seedlings, including any one of the following methods:

[0008] (1) Using sterilized Hippeastrum flower buds as explants, inoculate the explants into a callus induction medium for induction culture to obtain callus;

[0009] Cut the callus into pieces and then inoculate them into a redifferentiation induction medium, and conduct callus redifferentiation culture under alternating dark and light conditions to obtain regenerated plant seedlings;

[0010] (2) Using sterilized Hippeastrum flower buds as explants, cut the explants into pieces and then inoculate them into a redifferentiation induction medium, and conduct callus redifferentiation culture under alternating dark and light conditions to obtain regenerated plant seedlings;

[0011] The callus induction medium is based on one of MS medium, WPM medium or B5 medium, and also includes: 6-BA 1 - 4 mg / L, 2,4-D 1 - 5 mg / L and activated carbon 1.1 - 1.9 g / L;

[0012] The redifferentiation induction medium is based on one of MS medium, WPM medium or B5 medium, and also includes: 6-BA 1 - 4 mg / L, NAA 0.05 - 0.4 mg / L, KT 1 - 4 mg / L and activated carbon 1.1 - 1.9 g / L.

[0013] Preferably, the temperature for the induction culture and callus redifferentiation culture is 20 - 30 °C, and the humidity is 60% - 90%.

[0014] Preferably, during the induction culture, it also includes culturing under dark conditions for 5 - 9 d first, and then culturing under alternating dark and light conditions.

[0015] Preferably, in the alternating dark and light conditions, the light duration per 24 h is 10 - 14 h, and the light intensity is 2000 - 4000 lx.

[0016] Preferably, take the cross-sectional structure part of the Hippeastrum flower bud as the explant, and the cross-sectional structure part is the cross-sectional structure part at the connection point of the petal and the receptacle.

[0017] Preferably, the thickness of the cross-section of the Hippeastrum flower bud is 0.1 - 0.3 cm.

[0018] Preferably, the sterilization method is seed soaking sterilization.

[0019] Preferably, the sterilization includes the following steps: first sterilize with 75% alcohol for 30 - 180 s; then sterilize with 1% mercuric chloride for 60 - 120 min.

[0020] The present invention provides an application of the above-mentioned method for culturing seedlings of regenerated Hippeastrum plants in culturing seedlings of regenerated Hippeastrum plants.

[0021] Beneficial effects: The present invention provides a method for culturing seedlings of regenerated Hippeastrum plants, which selects Hippeastrum flower buds as explants, induces the culture of Hippeastrum callus, redifferentiates the callus, or directly conducts redifferentiation culture after cutting the explants into pieces; after 30-40 days of redifferentiation culture, adventitious buds are formed, and seedlings are generated after 50-80 days of culture. The Hippeastrum seedlings cultivated by the method provided by the present invention have a survival rate as high as 95.3%, an induction rate as high as over 96.2%, a dry matter accumulation amount of up to 46.8 g, the leaves of the seedlings are dark green, grow vigorously, the leaves are wide, and the diameter of the bulb is 5-10 mm. The present invention selects relatively active red flower buds as explants to cultivate Hippeastrum, overcomes the limitation problem of Hippeastrum in vitro materials, and at the same time overcomes the problems of too high cost, serious pollution, high mortality and browning rate when using bulbs as explants. The newly bred Hippeastrum varieties are scarce in bulbs, and a large number of bulbs can be obtained by in vitro culture of flower buds, overcoming the problem of the disappearance of all resources caused by the failure of bulb scale propagation. The present invention uses the culture method of Hippeastrum flower bud callus regeneration. By culturing a callus block with a surface area of only 1 cm 2 , it can be differentiated and proliferated into 17-25 Hippeastrum bulbs. It can not only obtain a large number of Hippeastrum seedlings in a relatively short time, but also directly differentiate the Hippeastrum flower bud explants to obtain Hippeastrum bulbs, further shortening the cultivation time. Moreover, using flower buds as explants can protect the mother bulbs from damage, providing a simple and reliable method for Hippeastrum propagation and protection. Description of the Drawings

[0022] Figure 1 It is a picture of a Hippeastrum flower scape;

[0023] Figure 2 It is a picture of a Hippeastrum flower bud;

[0024] Figure 3 It is a longitudinal section picture of a Hippeastrum flower bud;

[0025] Figure 4 It is a cross-section picture of a Hippeastrum flower bud;

[0026] Figure 5 It is a picture of a cross-section explant of a Hippeastrum flower bud inoculated into a tissue culture bottle of a callus induction medium;

[0027] Figure 6 It is a picture of a cross-section explant of a Hippeastrum flower bud after 1 week of callus induction culture;

[0028] Figure 7 It is a picture of a cross-section explant of a Hippeastrum flower bud after 50 days of callus induction culture;

[0029] Figure 8 Figure of the callus redifferentiation induction culture of Hippeastrum hybridum flower buds for 35 days;

[0030] Figure 9a Figure inside the bottle of the callus redifferentiation induction culture of Hippeastrum hybridum flower buds for 65 days;

[0031] Figure 9b Figure outside the bottle of the callus redifferentiation induction culture of Hippeastrum hybridum flower buds for 65 days;

[0032] Figure 9c Figure after pruning the leaves of 9b;

[0033] Figure 10 Figure of the transverse explant of Hippeastrum hybridum flower buds inoculated on the callus redifferentiation induction medium for 35 days;

[0034] Figure 11 Figure of the transverse explant of Hippeastrum hybridum flower buds inoculated on the callus redifferentiation induction medium for 75 days;

[0035] Figure 12 For Figure 11 Figure of the subculture inoculation of plant tissue into the redifferentiation induction medium bottle and continued culture for 55 days. Specific implementation method

[0036] The present invention provides a method for culturing Hippeastrum hybridum regenerated plant seedlings, including any one of the following methods:

[0037] (1) Using the disinfected Hippeastrum hybridum flower buds as explants, inoculating the explants into the callus medium for induction culture to obtain callus;

[0038] Cutting the callus and inoculating it into the redifferentiation induction medium, and performing callus redifferentiation culture under the alternating conditions of darkness and light to obtain regenerated plant seedlings;

[0039] (2) Using the disinfected Hippeastrum hybridum flower buds as explants, cutting the explants and inoculating them into the redifferentiation induction medium, and performing callus redifferentiation culture under the alternating conditions of darkness and light to obtain regenerated plant seedlings;

[0040] The callus induction medium is based on one of MS medium, WPM medium or B5 medium, and further includes: 6-BA 1 - 4 mg / L, 2,4-D 1 - 5 mg / L and activated carbon 1.1 - 1.9 g / L;

[0041] The redifferentiation induction medium uses one of MS medium, WPM medium or B5 medium as a basic medium, and further comprises: 1-4 mg / L 6-BA, 0.05-0.4 mg / L NAA, 1-4 mg / L KT and 1.1-1.9 g / L activated carbon.

[0042] The present invention preferably cultivates the Amaryllis bulbs first, until the Amaryllis has flower stalks and the flower stalks have flowers. The present invention has no special limitation on the source of Amaryllis, and conventional commercial products can be used. The diameter of the Amaryllis bulbs of the present invention is preferably 5 to 10 cm, more preferably 6 to 8 cm; the cultivation time of the Amaryllis bulbs is preferably 25 to 40 days, more preferably 28 to 32 days. The present invention utilizes the method of in vitro regeneration of plants from Amaryllis flower organs and the totipotency of plant cells to differentiate plants that are completely identical to the maternal genes.

[0043] The present invention preferably uses the cross-section structure of the amaryllis bud as the explant, and the cross-section structure is the cross-section structure of the petal and the receptacle connection point. The cross-section structure is mainly used to intercept the connection point of the petal and the receptacle. This part has strong vitality and is easy to grow and induce callus and adventitious buds. The thickness of the cross-section structure of the amaryllis bud of the present invention is preferably 0.1 to 0.3 cm, more preferably 0.2 cm. The resources of the flower bud are insufficient, and the thickness is too large to waste materials, and the thickness is too small to intercept the connection point of the petal and the receptacle.

[0044] The present invention preferably sterilizes the explant, and the sterilization method is preferably seed soaking sterilization.

[0045] The disinfection in the present invention preferably includes the following steps: first disinfection with 75% alcohol, the time for the first disinfection being 30 - 180 s, more preferably 50 - 90 s, and most preferably 70 s; second disinfection with 1% mercuric chloride, the time for the second disinfection preferably being 60 - 120 min, more preferably 80 - 100 min, and most preferably 90 min; alcohol immersion disinfection can preferably complete surface disinfection, and mercuric chloride can disinfect the interior of plants. 1% mercuric chloride causes little harm to plants and can kill fungi and bacteria. After the first disinfection and the second disinfection in the present invention, it preferably further includes rinsing with sterile water, the number of rinsing times preferably being 2 - 6 times, more preferably 3 - 5 times. After disinfection in the present invention, it is preferably to pour out the mercuric chloride in the beaker, collect the mercuric chloride and the waste water together, and perform harmless treatment after the experiment to avoid polluting water sources and soil. While performing the second disinfection in the present invention, it also includes disinfecting the operation space, the operation space preferably being a laminar flow hood; the disinfection preferably includes ultraviolet lamp disinfection for 15 - 30 min, more preferably 18 - 25 min, and most preferably 20 min; during the period when the ultraviolet lamp in the laminar flow hood is turned on, the operator should leave the laminar flow hood to avoid skin burns and eye damage caused by ultraviolet rays. After the ultraviolet lamp disinfection in the present invention, it further includes air exhaust, the time for the air exhaust preferably being 5 - 15 min, more preferably 10 min; air exhaust can discharge the harmful gases in the laminar flow hood to avoid harming the respiratory tract. During the disinfection in the present invention, stirring is also accompanied, the stirring preferably being carried out during the non-ultraviolet disinfection period, the time for the stirring preferably being 5 - 40 min, more preferably 10 - 30 min. In the present invention, it is preferably to dry the disinfected explants, and the drying method preferably is to blot the moisture with sterile qualitative filter paper.

[0046] In the present invention, it is preferably to inoculate the explants with blotted moisture into a callus induction medium for induction culture to obtain callus, and the explants are preferably cut into pieces for culture. The callus induction medium in the present invention uses one of MS medium, WPM medium or B5 medium as the basal medium, more preferably MS medium; the callus induction medium preferably further includes: 6 - BA 1 - 4 mg / L, 2,4 - D 1 - 5 mg / L and activated carbon 1.1 - 1.9 g / L; more preferably includes: 6 - BA 3.5 mg / L, 2,4 - D 2.5 mg / L and activated carbon 1.5 g / L. The activated carbon in the present invention is preferably sieved, and the sieve mesh number is preferably 300 - 400 meshes. Activated carbon can prevent the browning and oxidation of the medium, and at the same time can promote rooting. When the mesh number of activated carbon is small, it is easy to precipitate and cannot be evenly distributed in the medium, affecting the experimental results.

[0047] The temperature of the induction culture in the present invention is preferably 20 - 30°C, more preferably 23 - 27°C, and most preferably 25°C; the humidity of the induction culture is preferably 60% - 90%, more preferably 70% - 80%, and most preferably 75%. When performing the induction culture in the present invention, it preferably includes culturing for 5 - 9 days, more preferably 6 - 8 days, and most preferably 7 days under dark conditions first; then culturing under alternating dark and light conditions. In the alternating dark and light conditions in the present invention, the light duration per 24 hours is 10 - 14 hours, more preferably 11 - 13 hours, and most preferably 12 hours; the intensity of the light is preferably 2000 - 4000 lx, more preferably 2500 - 3500 lx, and most preferably 3000 lx. After the explants in the present invention are subjected to induction culture for 40 - 60 days, callus is obtained. For the cross-sectional structure part of the Hippeastrum flower bud in the present invention, the wound can heal after about one week of dark culture, and then the light condition is changed, that is, culturing under alternating dark and light conditions. Under this condition, the callus formed during the culture has a faster redifferentiation rate and more bud primordia are generated.

[0048] Preferably, the obtained callus in the present invention is cut into pieces and then inoculated into a redifferentiation induction medium, and the callus is redifferentiated and cultured under alternating dark and light conditions to obtain regenerated plant seedlings. The surface area of the cut pieces in the present invention is preferably 0.8 - 1.5 cm 2 , more preferably 1 cm 2 ; the thickness is preferably 0.1 - 0.6 cm, more preferably 0.3 cm. The redifferentiation induction medium in the present invention is based on one of MS medium, WPM medium or B5 medium, more preferably MS medium; the redifferentiation induction medium also preferably includes: 6 - BA 1 - 4 mg / L, NAA 0.05 - 0.4 mg / L, KT 1 - 4 mg / L and activated carbon 1.1 - 1.9 g / L; more preferably includes: 6 - BA 3.5 mg / L, NAA 0.15 mg / L, KT 2.5 mg / L and activated carbon 1.5 g / L. The activated carbon in the present invention is preferably sieved, and the sieve mesh number is preferably 300 - 400 mesh, further preferably 350 mesh.

[0049] The temperature of the callus redifferentiation culture in the present invention is 20 - 30°C, more preferably 23 - 27°C, and most preferably 25°C; the humidity is 60% - 90%, more preferably 70% - 80%, and most preferably 75%. In the alternating dark and light conditions in the present invention, the light duration per 24 hours is 10 - 14 hours, more preferably 11 - 13 hours, and most preferably 12 hours; the intensity of the light is preferably 2000 - 4000 lx, more preferably 2500 - 3500 lx, and most preferably 3000 lx. After the callus in the present invention is subjected to redifferentiation culture for 30 - 40 days, adventitious buds are formed, and seedlings are generated after culturing for 50 - 80 days.

[0050] The present invention can directly use disinfected Hippeastrum flower buds as explants for redifferentiation culture, that is, cut the explants into pieces and inoculate them into the redifferentiation induction medium, and carry out callus redifferentiation culture under alternating dark and light conditions to obtain regenerated plant seedlings.

[0051] The present invention also preferably cuts the explants after disinfection and blotting to dry, inoculates them into the redifferentiation induction medium, and carries out callus redifferentiation culture under alternating dark and light conditions to obtain regenerated plant seedlings. The redifferentiation induction culture of the explants in the present invention is the same as the above process of callus redifferentiation culture, which will not be elaborated here. After 30 to 40 days of redifferentiation culture of the explants in the present invention, bud seedlings are generated, and after 60 to 90 days of culture, rootless seedlings grow. Then, they are continuously inoculated into the redifferentiation induction medium and cultured for 50 to 60 days to obtain rooted seedlings.

[0052] The present invention provides the application of the above-mentioned method for culturing Hippeastrum regenerated plant seedlings in culturing Hippeastrum regenerated plant seedlings. The present invention uses Hippeastrum flower buds as explants to culture Hippeastrum regenerated plant seedlings. By culturing callus blocks with a surface area of about 1 cm 2 , it can be differentiated and proliferated into 17 to 25 Hippeastrum bulbs, which can not only obtain a large number of Hippeastrum seedlings in a short time, but also protect the mother bulbs from damage, providing a new method for Hippeastrum propagation and protection.

[0053] In order to further illustrate the present invention, the following describes in detail a method for culturing Hippeastrum regenerated plant seedlings and its application provided by the present invention with reference to the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0054] Example 1

[0055] A method for culturing Hippeastrum regenerated plant seedlings comprises the following steps:

[0056] Plant Hippeastrum bulbs with a diameter of 8 cm in nutrient soil and water them thoroughly; after about one month, flower stalks will emerge; wait for the flower stalks of Hippeastrum to emerge and the flowers on the flower stalks to appear (as Figure 1 ).

[0057] Place the callus induction medium, sterile water, prepared 75% alcohol, 1% mercuric chloride solution, 500 mL beaker, sterilized qualitative filter paper, sterilized culture dish, scalpel, forceps, sterilizer in the ultra-clean workbench for standby;

[0058] Cut off the flowers on the flower stalks to obtain flower buds (as Figure 2)As explants, clean them thoroughly with dishwashing liquid and rinse with running water for 20 minutes. Then, evenly spray the floral bud explants with an alcohol spray bottle for simple disinfection, wash twice with sterile water, and place them in a petri dish inside a laminar flow hood for later use. Next, use forceps to put the floral bud explants into a 500 mL beaker, add 75% alcohol for disinfection for 70 seconds, and continuously stir with a glass rod at the same time. Rinse 3 times with sterile water and pour out the waste liquid in the beaker. Add 1% mercuric chloride and soak for disinfection for 40 minutes. Stir with a glass rod, turn on the ultraviolet lamp to sterilize the inside of the laminar flow hood. After 20 minutes of ultraviolet air disinfection of the laminar flow hood, turn off the ultraviolet lamp, turn on the exhaust fan, turn on the sterilizer, and exhaust for 10 minutes. Stir with a glass rod for 20 minutes, pour out the mercuric chloride in the beaker, and rinse 5 times with the prepared sterile water. Collect the mercuric chloride and waste water together and perform harmless treatment after the experiment. Blot the moisture of the disinfected floral bud explants with sterilized qualitative filter paper.

[0059] For the blotted floral bud explants with moisture removed, take the part at the connection point of the petal and the receptacle (the specific position is shown in Figure 3 ) and cut it into the cross-sectional structure part of the floral bud (such as Figure 4 ), and the thickness of the cross-sectional structure part is 0.2 cm.

[0060] Inoculate the cross-sectional structure part of the floral bud into a tissue culture bottle filled with callus induction medium; the callus induction medium is MS medium, and the medium also only contains a combination of 3.5 mg / L of 6-BA (6-benzylaminopurine), 2.5 mg / L of 2,4-D (2,4-dichlorophenoxyacetic acid), and 1.5 g / L of AC (activated carbon); the AC passes through a 350-mesh sieve. Place it in an incubator at room temperature of 25°C, humidity of 75%, and culture in the dark for one week. Then place it in an incubator at room temperature of 25°C, humidity of 75%, with 12 h of 3000 lx light culture, and 12 h of dark culture every 24 h, and culture for 20 d (such as Figure 5 ), culture for 30 d (such as Figure 6 ), and after 50 d of culture, obtain callus (such as Figure 7 ).

[0061] Cut the callus into 1 cm 3 small square callus and inoculate it into a tissue culture bottle of the regeneration induction medium; the regeneration induction medium contains 3.5 mg / L of 6-BA (6-benzylaminopurine), 0.15 mg / L of NAA (naphthaleneacetic acid), 2.5 mg / L of KT (kinetin), and 1.5 g / L of AC (activated carbon); place it in an incubator at room temperature of 25°C, humidity of 75%, with 12 h of 3000 lx light culture and 12 h of dark culture, and after 35 d of culture, such as Figure 8 , and after 65 d of culture, such as Figure 9a , 9b , 9c.

[0062] Example 2

[0063] The cross-sectional structure part of the flower bud of the same strain of Hippeastrum vittatum as in Example 1 (such as Figure 4 ) was inoculated into a bottle of redifferentiation induction medium, placed at room temperature of 25°C, humidity of 75%, 12h of 3000lx light, and after culturing for 35d, as Figure 10 , after culturing for 75d, as Figure 11 , and Figure 11 the plant tissue was subcultured and inoculated into a bottle of redifferentiation induction medium and continued to be cultured for 55d, as Figure 12 .

[0064] Example 3

[0065] It was carried out in the same manner as in Example 1, except that the callus induction medium was replaced with one containing: 2mg / L of 6-BA, 3.5mg / L of 2,4-D, 1.5g / L of AC (activated carbon).

[0066] Example 4

[0067] It was carried out in the same manner as in Example 1, except that the callus redifferentiation induction medium was replaced with one containing: 2mg / L of 6-BA, 0.2mg / L of NAA, 3mg / L of KT, 1.5g / L of AC (activated carbon).

[0068] Example 5

[0069] It was carried out in the same manner as in Example 1, except that the callus induction medium MS medium was replaced with WPM medium.

[0070] Example 6

[0071] It was carried out in the same manner as in Example 1, except that the callus induction medium MS medium was replaced with B5 medium.

[0072] Comparative Example 1

[0073] It was carried out in the same manner as in Example 1, except that the disinfection method of the explant was replaced with soaking in 75% alcohol for 30s, rinsing 3 times with sterile water, and then using 1% mercuric chloride for disinfection for 15min.

[0074] Comparative Example 2

[0075] It was carried out in the same manner as in Example 1, except that the disinfection method of the explant was replaced with soaking in 75% alcohol for 190s, rinsing 3 times with sterile water, and then using 1% mercuric chloride for disinfection for 80min.

[0076] Test Example 1

[0077] After 20 days of culture, the contamination rate and survival rate of the explants were detected. That is, the state of the explants was observed with the naked eye and the data results were recorded. Six tests were carried out, and the calculation was performed according to the following formula and the average value was obtained.

[0078] Contamination rate (%) = Number of contaminated explants / Total number of inoculated explants × 100%

[0079] Survival rate (%) = Number of surviving explants / Total number of inoculated explants × 100%

[0080] Examples 1, Comparative Examples 1 and 2 were compared to obtain the effects of different disinfection methods on the explants. The results are shown in Table 1.

[0081] Table 1 Effects of different disinfection methods on explants

[0082] Pollution rate (%) Survival rate (%) Example 1 5.3 95.3 Comparative Example 1 100 0 Comparative Example 2 1.3 23.3

[0083] It can be seen from Table 1 that the disinfection contamination rate of Example 1 is the lowest, the survival rate is the highest, and the effect is the best.

[0084] Comparative Example 3

[0085] It was carried out in the same way as in Example 1, except that the callus induction medium was replaced with: MS medium, 0.5 mg / L of 6-BA, 0.5 mg / L of 2,4-D, 1.5 g / L of AC (activated carbon).

[0086] Comparative Example 4

[0087] It was carried out in the same way as in Example 1, except that the callus induction medium was replaced with one containing: MS medium, 6 mg / L of 6-BA, 8 mg / L of 2,4-D, 1.5 g / L of AC (activated carbon).

[0088] Test Example 2

[0089] After 50 days of culture, the induction rate of the explants was detected. That is, the state of the explants was observed with the naked eye and the data results were recorded. Six tests were carried out, and the calculation was performed according to the following formula and the average value was obtained.

[0090] Induction rate (%) = Number of explants with induced callus / Total number of inoculated explants × 100%

[0091] Examples 1 and 3 and Comparative Examples 3 and 4 were compared to obtain the effects of different medium formulations on callus induction. The results are shown in Table 2.

[0092] Table 2 Effects of different medium formulations on callus induction

[0093] Induction rate (%) Example 1 100 Example 3 96.3 Comparative Example 3 15.2 Comparative Example 4 30.3

[0094] As can be seen from Table 2, the callus induction rate of the formulation in Example 1 can reach 100%, that in Example 3 can reach 96.3%, while the induction rates of Comparative Examples 3 and 4 are only 15.2% and 30.3% respectively. It can be seen that the induction rate of the examples is significantly better than that of the comparative examples.

[0095] Comparative Example 5

[0096] It was carried out in the same manner as in Example 1, except that the callus redifferentiation induction medium was replaced with one containing: 0.5 mg / L of 6-BA, 0.02 mg / L of NAA, 0.5 mg / L of KT, and 1.5 g / L of AC (activated carbon).

[0097] Comparative Example 6

[0098] It was carried out in the same manner as in Example 1, except that the callus redifferentiation induction medium was replaced with one containing: 6 mg / L of 6-BA, 2 mg / L of NAA, 6 mg / L of KT, and 1.5 g / L of AC (activated carbon).

[0099] Test Example 3

[0100] The induction rate of the plants after 35 days of redifferentiation induction culture was detected using the method of Test Example 2, and the number of seedlings emerging per bottle (plants) was recorded by visual observation. Six tests were carried out and the average value was calculated.

[0101] Examples 1 and 4 were compared with Comparative Examples 5 and 6 to obtain the influence of different culture medium formulations on the redifferentiation induction of callus. The results are shown in Table 3.

[0102] Table 3 Influence of different culture medium formulations on the redifferentiation induction of callus

[0103] Induction rate (%) Number of seedlings per bottle (pcs) Example 1 100 56.8 Example 5 96.2 39.3 Comparative Example 5 12.6 2.5 Comparative Example 6 32.8 10.2

[0104] As can be seen from Table 3, the callus induction rate of the formulation in Example 1 can reach 100%, that in Example 5 can reach 96.2%. The average number of seedlings emerging per bottle in Example 1 can reach 56.8 plants, and that in Example 5 can reach 39.3 plants on average. It can be seen that both the induction rate and the number of seedlings emerging per bottle in the examples are significantly higher than those in the comparative examples.

[0105] Comparative Example 7

[0106] It was carried out in the same manner as in Example 1, except that the light intensity was replaced with 0.

[0107] Comparative Example 8

[0108] It was carried out in the same manner as in Example 1, except that the light intensity was replaced with 500 lx.

[0109] Comparative Example 9

[0110] It was carried out in the same manner as in Example 1, except that the light intensity was replaced with 4500 lx.

[0111] Test Example 4

[0112] The plants cultured for 80 days were weighed, and six tests were carried out and the average value was obtained; the state of the seedlings was observed with the naked eye and the data results were recorded.

[0113] Example 1 and Comparative Examples 7-9 were compared to obtain the influence of different light intensities on the growth of Hippeastrum vittatum, and the results are shown in Table 4.

[0114] Table 4 Influence of different light intensities on the growth of Hippeastrum vittatum

[0115]

[0116] As can be seen from Table 4, different light intensities have different effects on Hippeastrum vittatum. In Example 1, the biomass per bottle was 46.8 g, the leaves of the seedlings were dark green, the growth was vigorous, the leaves were wide, the diameter of the bulb was 5-10 mm, and the growth situation was the best; under dark conditions, the dry matter accumulation per bottle was less, the seedlings had no leaves, and the explants were yellowish-white; when the light intensity was 500 lx, the leaves of the seedlings were yellowish-green, the leaves were small, and the diameter of the bulb was 1.0-3 mm; in Comparative Example 9, due to too strong light, there were red dots on the leaves of the seedlings, the burning was obvious, the growth was weak, and the diameter of the bulb was 3-4 mm. It can be seen that under the light intensity conditions of Example 1, the growth of Hippeastrum vittatum seedlings is the best.

[0117] Comparative Example 10

[0118] It was carried out in the same manner as in Example 1, except that the addition amount of AC (activated carbon) was replaced with 0.

[0119] Comparative Example 11

[0120] It was carried out in the same manner as in Example 1, except that the addition amount of AC (activated carbon) was replaced with 0.5 g / L.

[0121] Comparative Example 12

[0122] It was carried out in the same manner as in Example 1, except that the addition amount of AC (activated carbon) was replaced with 1.0 g / L.

[0123] Comparative Example 13

[0124] It was carried out in the same manner as in Example 1, except that the addition amount of AC (activated carbon) was replaced with 2.0 g / L.

[0125] Test Example 5

[0126] After culturing for 80 days, the browning rate of the seedlings was detected, that is, the state of the seedlings was observed with the naked eye and the data results were recorded. Six tests were carried out, and the calculation was carried out according to the following formula and the average value was obtained.

[0127] Browning rate (%) = Number of browning explants / Total number of inoculated explants × 100%

[0128] Examples 1 and Comparative Examples 10 - 13 were compared to obtain the effects of the activated carbon addition amount on the seedlings and the culture medium. The results are shown in Table 5.

[0129] Table 5 Effects of Activated Carbon Addition Amount on Seedlings and Culture Medium

[0130] Browning rate (%) Medium state Example 1 7.3 Solid Comparative Example 10 94.2 Solid Comparative Example 11 72.6 Solid Comparative Example 12 30.4 Solid Comparative Example 13 7.3 Flowing paste

[0131] As can be seen from Table 5, in Example 1, the browning rate of the seedlings with 1.5 g / L of activated carbon added was 7.3%, and the browning rate was low. In Comparative Examples 10 - 12, as the activated carbon addition amount increased, the browning rate of the seedlings gradually decreased. However, when the activated carbon addition amount increased to 2.0 g / L, the culture medium was a flowing paste with insufficient hardness, which affected the experimental operation.

[0132] Therefore, it can be seen that a cultivation method for seedlings of regenerated Hippeastrum plants provided by the present application uses Hippeastrum flower buds as explants, and the growth of the cultivated seedlings of regenerated Hippeastrum plants is good. It can not only obtain a large number of Hippeastrum seedlings in a short time, but also protect the mother bulbs from damage, providing a new method for the propagation and protection of Hippeastrum.

[0133] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for culturing seedlings of regenerated Hippeastrum plants, characterized in that, it includes any one of the following methods: (1) Using disinfected Hippeastrum flower buds as explants, inoculating the explants into a callus medium for induction culture to obtain callus; taking the cross-sectional structure part of the Hippeastrum flower bud as the explant, and the cross-sectional structure part is the cross-sectional structure part at the connection point of the petal and the receptacle; The disinfection method is seed soaking disinfection; the disinfection includes the following steps: first disinfecting with 75% alcohol for 30 - 180 s; second disinfecting with 1% mercuric chloride for 60 - 120 min; Cutting the callus into pieces and then inoculating them into a redifferentiation induction medium, and carrying out callus redifferentiation culture under alternating dark and light conditions to obtain seedlings of regenerated plants; (2) Using disinfected Hippeastrum flower buds as explants, cutting the explants into pieces and then inoculating them into a redifferentiation induction medium, and carrying out callus redifferentiation culture under alternating dark and light conditions to obtain seedlings of regenerated plants; The callus induction medium is based on MS medium or WPM medium, and further includes: 6 - BA 1 - 4 mg / L, 2,4 - D 1 - 5 mg / L, and activated carbon 1.1 - 1.9 g / L; The redifferentiation induction medium is based on MS medium or WPM medium, and further includes: 6 - BA 1 - 4 mg / L, NAA 0.05 - 0.4 mg / L, KT 1 - 4 mg / L, and activated carbon 1.1 - 1.9 g / L.

2. The culturing method according to claim 1, characterized in that, the temperature for the induction culture and the callus redifferentiation culture is 20 - 30 °C, and the humidity is 60% - 90%.

3. The culturing method according to claim 1, characterized in that, during the induction culture, it also includes culturing under dark conditions for 5 - 9 d first, and then culturing under alternating dark and light conditions.

4. The culturing method according to claim 1 or 3, characterized in that, in the alternating dark and light conditions, the light duration per 24 h is 10 - 14 h, and the light intensity is 2000 - 4000 lx.

5. The culturing method according to claim 1, characterized in that, the thickness of the cross-section of the Hippeastrum flower bud is 0.1 - 0.3 cm.

6. Application of the culturing method for seedlings of regenerated Hippeastrum plants according to any one of claims 1 - 5 in culturing seedlings of regenerated Hippeastrum plants.

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