Cyclamen tissue culture propagation method with ovules as explants

Abstract

The invention discloses a cyclamen tissue culture propagation method taking ovules as explants, and belongs to the technical field of plant tissue culture. The method comprises the steps of ovule inoculation, callus induction and screening, bud induction and proliferation, strong seedling culture, rooting induction and acclimatization and transplantation. According to the method, casein hydrolysate nutrition supporting, dynamic hormone regulation and control, bud cluster-shaped proliferation, strong seedling rooting, combination of in-bottle seedling hardening and out-bottle seedling hardening, transplanting matrix improvement and other methods are adopted, and an effective new way is provided for improving the cyclamen tissue culture inoculation survival rate and the seedling emergence survival rate.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a cyclamen tissue culture propagation method using ovules as explants. Background Art

[0002] Cyclamen persicum is a bulbous flower of the Primulaceae family, native to Greece and other Mediterranean regions in southern Europe. Due to its unique flower shape, wide variety, long flowering period and winter blooming, it has developed into one of the most important types of commercial potted flowers at home and abroad. Most commercial cyclamen are hybrid F1 generation. If they continue to reproduce with seeds, the characteristics will separate, and it is difficult to preserve the original characteristics and hybrid advantages of the F1 generation. At present, the cultivation technology level of cyclamen in my country is close to that of foreign countries, but there is no mature and complete breeding technology system. It is still necessary to spend huge amounts of money to purchase foreign F1 generation seeds, which has become a bottleneck restricting the development of my country's cyclamen industry.

[0003] With the continuous deepening of cyclamen breeding research in China, some very excellent new germplasm plants have emerged. However, the new excellent germplasm is prone to separation of traits after seed propagation, and the excellent traits cannot be preserved. In addition, most new germplasms with special flower shapes have aborted stamens or pistils and can only be propagated asexually. Cyclamen cannot be propagated asexually by conventional methods such as cuttings, grafting, and root division. Therefore, tissue culture has become the only way to propagate cyclamen asexually.

[0004] The most successful country in the field of Cyclamen tissue culture is Japan, which has been conducting research on Cyclamen tissue culture propagation technology since the 1980s. Currently, Cyclamen tissue culture rapid propagation technology has been commercialized, but its propagation technology is a trade secret and has never been made public. Germany, the United States, Belgium and other countries have also reported on the research of Cyclamen wild species and hybrids tissue culture, but tissue culture technology is only used as a scientific research method and has not been applied in production.

[0005] In recent years, domestic scholars have conducted extensive research on cyclamen tissue culture inoculation materials, culture medium screening, callus induction, organ regeneration induction, somatic embryo induction, etc. However, existing cyclamen tissue culture techniques mostly use leaves and tubers as explants, such as Chinese patent CN101112179A discloses a cyclamen tissue culture rapid propagation method, which is a way to use leaves as explants for cyclamen organ regeneration tissue culture propagation; Chinese patent CN103238526A discloses a method for rapid propagation of cyclamen using tissue culture technology, which is a complete set of technology for tissue culture propagation using cyclamen corms as explants; Chinese patent CN104982331A discloses a cyclamen tissue culture rapid propagation method, which is a method for cyclamen tissue culture propagation using flower stems as explants.

[0006] For new and superior individual plants, not only is the number of explants such as leaves and tubers limited, but the difficulty in sterilizing the materials leads to a low inoculation survival rate. In the process of organ regeneration, there are problems such as low bud differentiation rate and low seedling rate, which have become important factors restricting the success of tissue culture asexual lines of new and superior individual plants of Cyclamen. Summary of the invention

[0007] In view of the problems of less inoculation materials, high contamination rate, difficulty in differentiation and proliferation, and low survival rate of seedlings in cyclamen tissue culture in the prior art, the present invention provides a cyclamen tissue culture propagation method using ovules as explants. The method uses unfertilized ovules as explants, has the advantages of no harm to plants, easy disinfection, large quantity, and convenient material collection, and sequentially undergoes callus induction and optimization, bud induction and differentiation and proliferation, seedling cultivation, rooting induction, seedling hardening and transplanting, and finally establishes a new tissue culture technology system that is easy to inoculate successfully, has a high seedling rate, good plant shape, and a large number of leaves, which is conducive to accelerating the asexual reproduction process of cyclamen and promoting the further development of domestic cyclamen breeding and industry.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions: A cyclamen tissue culture propagation method using ovules as explants comprises the following steps: Step 1: Ovule inoculation Step 1-1: Take the unopened or half-opened (unfertilized) flower buds of the excellent individual cyclamen to be propagated, remove the petals and leave the ovary; rinse the ovary with running tap water for more than 2 hours, clean and disinfect it on a clean bench, place it under a dissecting microscope, and use a scalpel to peel off the ovules from the placenta; Step 1-2: Use a scalpel or forceps to pick up the ovules, place them in the callus induction medium, and seal them; The formula of the callus induction medium is: 1 / 2MS medium + 2,4D 2.0 mg / L + 2-isopentyl adenine (2iP) 0.8 mg / L + hydrolyzed casein 500 mg / L + sugar 30 g / L + agar 6 g / L, pH value is 5.8-6.0; Step 2: Callus induction and screening The callus induction medium with ovules is placed in a completely dark environment at 22-25°C for 3-5 months to induce callus tissue to be produced; during the callus induction culture process, dense, creamy yellow or creamy white callus tissue is selected and inoculated once every 4 weeks; Step 3: Shoot induction and proliferation Step 3-1: After continuous dark culture of callus tissue for 3-5 months, dense, creamy yellow or creamy white callus tissue is selected and transferred to differentiation medium for bud induction culture to obtain adventitious buds; Step 3-2: In the early stage of adventitious bud proliferation, dense callus with many adventitious bud balls is selected as the proliferation organ and transferred to the proliferation medium for proliferation culture; Step 3-3: During the adventitious bud proliferation process, a bud cluster containing at least three buds is selected for subculture; callus tissue is left at the base of the bud cluster according to the space of the proliferation culture medium; Step 3-4: According to the development of the bud cluster and the callus tissue left at the base of the bud cluster, repeat steps 3-2 and 3-3 to obtain more bud clusters with callus tissue at the base; The differentiation medium and the proliferation medium have the same formula, which is: 1 / 2MS medium + 6-benzylpurine (6-BA) 0.5-2.0 mg / L + naphthaleneacetic acid (NAA) 0.05-0.2 mg / L + hydrolyzed casein 100 mg / L + sugar 30 g / L + agar 6 g / L; Step 4: Seedling cultivation The bud clusters obtained by differentiation and proliferation in step 3 are pruned so that the bud clusters retain more than 6 evenly distributed petioles and callus tissue is left at the base, and then the bud clusters are transferred to seedling medium for 1-2 generations of seedling culture to obtain rootless test tube seedlings; The seedling medium formula is: 1 / 2MS medium + 6-benzylpurine (6-BA) 0.05-0.2 mg / L + naphthaleneacetic acid (NAA) 0.01 mg / L + hydrolyzed casein 100 mg / L + sugar 30 g / L + agar 6 g / L; Step 5: Rooting Induction After the strong seedling cultivation is completed, the robust rootless test tube seedlings are pruned to make the bud points on the bud clusters evenly distributed, leaving callus tissue at the base of the bud clusters, and then transferred to the rooting medium for cultivation for 30-45 days to obtain rooted bottle seedlings; Step 6: Hardening and transplanting Step 6-1: Place the rooted seedlings under strong natural light for 5-7 days before transplanting; at the same time, 2-3 days before transplanting, loosen the bottle caps containing the rooted seedlings and leave a gap to allow the seedlings to breathe and harden; Step 6-2: After the seedlings are hardened in the bottle, take out the rooted seedlings, wash the base culture medium, and transplant them into a plug tray or foam culture box covered with transplanting medium; within the first 15 days of transplanting, cover with plastic film to maintain humidity above 85%, and control the light intensity to less than 6000 lux, then gradually remove the plastic film until it is placed under natural conditions; Step 6-3: After the seedlings fill the hole tray or the foam box, transplant them into the nutrient pots and carry out routine management.

[0009] Furthermore, the excellent individual plants in step 1 refer to plants that meet the breeding goals and are healthy, disease-free and insect-free.

[0010] Furthermore, the cleaning and disinfection of the ovary on the clean bench described in step 1-1 refers to: first, the ovary is immersed in 70% alcohol for disinfection for 30 seconds, and after the immersion is completed, the ovary is taken out and rinsed with sterile water; then, it is immersed in 2% sodium hypochlorite solution for disinfection for 15-20 minutes, and after the immersion is completed, the ovary is taken out and rinsed with sterile water three times, each time for 5 minutes; during each immersion and disinfection, the container containing alcohol or sodium chlorate solution is continuously shaken.

[0011] Furthermore, the concentration of 6-BA in the proliferation medium described in step 3 is dynamically adjusted on its original basis according to the proliferation of the bud cluster and the growth of petioles and leaves. When there are too many young buds in the bud cluster and the callus tissue becomes transparent, the concentration of 6-BA is reduced; when there are too few callus differentiation bud points left at the base of the bud cluster and the callus tissue becomes brown, the concentration of 6-BA is increased.

[0012] Furthermore, the rooting medium formula in step 5 is: 1 / 2MS medium + NAA 0.5-1.0 mg / L + IBA 0.5-1.0 mg / L.

[0013] Furthermore, the cell tray in step 6-2 is a 50-cell cell tray.

[0014] Furthermore, the transplanting medium in step 6-2 is preferably a mixed medium of coconut bran and vermiculite in a mass ratio of 1:1, and 0.1-0.5% sodium sulfamethoxam is added to the transplanting medium.

[0015] Furthermore, the culture conditions of the above-mentioned step 3, bud induction and proliferation, step 4, seedling culture and step 5, root induction culture are the same, namely, temperature 18-25°C, photoperiod 12h / d, light intensity 1500-4000lux.

[0016] The present invention discloses a complete technical solution for tissue culture propagation of cyclamen using unfertilized ovules as explants, provides an overall idea and specific method for tissue culture propagation of cyclamen including "ovule explant initiation, hydrolyzed casein nutritional support, dynamic hormone regulation, bud cluster proliferation, strong seedling rooting, and seedling hardening and transplanting inside and outside the bottle", and provides an effective new approach to improve the survival rate of cyclamen tissue culture inoculation and seedling survival rate and plant type shaping. Specifically: (1) The present invention uses unfertilized ovules as explant materials, which have the advantages of no harm to plants, large quantity, and convenient material collection, and can obtain more explant inoculation materials at the same time; because the cyclamen ovules are in a sterile state in the ovary, it is easy to obtain sterile explant materials by using ovules as explants, and the contamination rate is extremely low, which overcomes the defect of using other explants such as leaves and bulbs that are easy to be contaminated; and exploring the addition of hydrolyzed casein to the induction culture medium, not only the browning rate of callus tissue is reduced, but also the success rate of ovule inoculation reaches more than 95%; (2) During the subculture and rooting process of adventitious buds, a program of dynamic hormone regulation was adopted according to the growth of callus tissue, which effectively inhibited the vitrification or browning of callus tissue, and the callus tissue induced bud differentiation rate reached 96%~100%; (3) The present invention uses bud clusters or clustered seedlings in the process of adventitious bud proliferation, seedling hardening and rooting culture, and ensures that an appropriate amount of callus tissue is left at the base of the bud cluster, so that the cyclamen seedling emergence rate and rooting rate can reach up to 100%; at the same time, in the process of adventitious bud proliferation, the present invention selectively screens bud clusters with a large number of bud points, and in the later seedling hardening culture and seedling hardening culture, the bud clusters are properly pruned, which speeds up the formation of tissue culture seedlings, making the tissue culture seedlings compact, plump, symmetrical, and with a large number of leaves; (4) The present invention adopts a combination of in-bottle acclimatization and out-bottle acclimatization to carry out acclimatization, and uses a unique transplanting matrix, so that the cyclamen bottle seedlings are more robust and easier to adapt to the later transplanting environment, and the survival rate reaches 95% to 100%.

[0017] The application of the technical solution of the present invention will significantly improve the production efficiency of cyclamen tissue culture seedlings and provide a scientific basis for the commercial production of cyclamen tissue culture seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The unopened ( Figure 1-1 ) or half-open ( Figure 1-2 )'s buds.

[0019] Figure 2 The culture and development of callus tissue inoculated with ovules in Example 1; wherein, Figure 2-1 For the first inoculation of ovules, Figure 2-2 For inoculation of ovules 4 weeks ago, the preferred callus tissue Figure 2-3 For inoculation of ovules 8 weeks the preferred callus tissue, Figure 2-4 For inoculation of ovules 12 weeks the preferred callus tissue, Figure 2-5 Callus tissue is preferred for inoculation of ovules at 16 weeks.

[0020] Figure 3 The callus tissue in the bud induction and proliferation culture process of Example 1; wherein, Figure 3-1 After one month of bud induction, callus tissue with adventitious bud balls was formed. Figure 3-2 During the process of adventitious bud proliferation, a part of the callus tissue bud cluster is left at the base.

[0021] Figure 4 The bud clusters in the seedling cultivation process of Example 1, wherein: Figure 4-1To retain more than 6 evenly distributed petioles and a proper amount of callus buds at the base, Figure 4-2 For the bud clusters that are used for seedling cultivation in the tissue culture room, Figure 4-3 After one month of cultivation for strong seedlings, the leaves unfold and appear as reddish-brown or green buds.

[0022] Figure 5 It is a bud cluster in the process of root induction culture, among which, Figure 5-1 and Figure 5-2 All of them were induced to root for 30-45 days and produced rooted buds.

[0023] Figure 6 For seedling hardening and tissue culture seedlings after transplanting, among them, Figure 6-1 Pictures of bottle seedlings that have grown roots being hardened under strong natural light; Figure 6-2 After the seedling hardening is completed, the base culture medium has been cleaned and the rooted bottle seedlings have been Figure 6-3 These are tissue culture seedlings under routine management after transplanting. DETAILED DESCRIPTION

[0024] The present invention will be further explained below in conjunction with specific embodiments. It will be appreciated by those skilled in the art that the embodiments described below are only a part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The specific conditions not indicated in the embodiments are carried out under normal conditions. The reagents used are all conventional products that can be purchased commercially.

[0025] Example 1 Taking the method of the present invention as an example to obtain the excellent single-plant pink and white fragrant (FBNX) tissue culture seedlings of the hybrid offspring of Cyclamen "Piquet", the specific operation steps are as follows: Step 1: Ovule inoculation 1-1: Take the unopened or half-opened buds (unfertilized) of the excellent single plant of the hybrid offspring of "Pico" (FBNX) Figure 1 ), remove the petals and leave the ovary; rinse the ovary with running tap water for 3 hours, soak the ovary in 70% alcohol for 30 seconds, take out the ovary after soaking, and rinse it with sterile water; soak it in 2% sodium hypochlorite solution for 15-20 minutes, take out the ovary after soaking, and rinse it with sterile water three times, 5 minutes each time; shake the container containing alcohol or sodium chlorate solution continuously during each soaking; Under the dissecting microscope, use a scalpel to cut the ovary skin from top to bottom, and use the back of the scalpel to gently peel the ovule from the placenta from one point; 1-2: Use a scalpel or forceps to pick up the ovules, gently place them on the prepared callus induction medium, and then seal it; The formula of the callus induction medium is: 1 / 2MS medium + 2,4D 2.0 mg / L + 2-isopentyl adenine (2iP) 0.8 mg / L + hydrolyzed casein 500 mg / L + sugar 30 g / L + agar 6 g / L, pH value is 5.8-6.0; The mother bottle after high-pressure sterilization is divided into 6 cm culture dishes in an ultra-clean workbench, each dish contains 12 ml, and cooled until solidified for later use.

[0026] Step 2: Callus induction and screening Step 1: The success rate of ovule inoculation is above 95%. The callus induction medium with ovules is placed in a completely dark environment at 22-25℃ for 16 weeks to induce callus ( Figure 2 ); wherein, during the culture process, dense, creamy yellow or creamy white callus tissue is selected and inoculated once every 4 weeks.

[0027] After 4 weeks of dark culture, the callus induction rate of successfully inoculated ovules reached over 98%; however, after 8 to 12 weeks, the color of the callus and the speed of proliferation would vary greatly. During subculture, dense, creamy yellow or creamy white callus tissue should be selected for optimization.

[0028] Figure 2-1 For the first inoculation of ovules, Figure 2-2 For inoculation of ovules 4 weeks ago, the preferred callus tissue Figure 2-3 For inoculation of ovules 8 weeks the preferred callus tissue, Figure 2-4 For inoculation of ovules 12 weeks the preferred callus tissue, Figure 2-5 Callus tissue is preferred for inoculation of ovules at 16 weeks.

[0029] Step 3: Shoot induction and proliferation 3-1: After 4 months of continuous dark culture of callus tissue, the dense, creamy yellow or creamy white callus tissue that has been continuously selected is transferred to differentiation medium for bud induction culture; 3-2: After 1 month of culture, adventitious buds are formed ( Figure 3-1 ); thereafter, dense callus tissue with many adventitious bud balls is selected as a proliferation organ and transferred to a proliferation medium for further proliferation culture; 3-3: During the adventitious bud proliferation process, a bud cluster containing at least 3 buds is selected for subculture; a portion of callus tissue is left at the base of the bud cluster according to the space of the proliferation medium ( Figure 3-2 ); 3-4: According to the development of the bud cluster and the callus tissue left at the base of the bud cluster, repeat steps 3-2 and 3-3 to obtain more bud clusters with callus tissue left at the base; The differentiation medium and the proliferation medium have the same formula, which is: 1 / 2MS medium + 6-benzylpurine (6-BA) 1.5 mg / L + naphthaleneacetic acid (NAA) 0.15 mg / L + hydrolyzed casein 100 mg / L + sugar 30 g / L + agar 6 g / L; The concentration of 6-BA in the proliferation medium is dynamically adjusted on the original basis according to the proliferation of bud clusters and the growth of petioles and leaves. When there are too many young buds in the bud cluster and the callus tissue becomes transparent, the concentration of 6-BA is adjusted to 0.5-1.0 mg / L; when there are too few callus differentiation bud points left at the base of the bud cluster and the callus tissue becomes brown, the concentration of 6-BA is increased to 1.8-2.0 mg / L.

[0030] The differentiation rate of adventitious bud balls induced by the initial callus in this step is 60%-70%, and the proliferation rate of adventitious bud balls in subculture is 1-3 times.

[0031] Step 4: Seedling cultivation The bud clusters obtained by differentiation and proliferation in step 3 were properly pruned so that the bud clusters retained more than 6 evenly distributed petioles and an appropriate amount of callus tissue was left at the base ( Figure 4-1 ), and then transfer the bud clusters to the seedling medium for seedling culture ( Figure 4-2 ), one month later, the leaves of the buds can be seen unfolding, and they are reddish brown or green ( Figure 4-3 ), and obtain rootless test tube seedlings; The seedling medium formula is: 1 / 2MS medium + 6-benzylpurine (6-BA) 0.2 mg / L + naphthaleneacetic acid (NAA) 0.01 mg / L + hydrolyzed casein 100 mg / L + sugar 30 g / L + agar 6 g / L.

[0032] Step 5: Rooting Induction After the cultivation of strong seedlings is completed, the bud clusters of the robust rootless test tube seedlings are pruned to make the bud points on the bud clusters evenly distributed, leaving an appropriate amount of callus tissue at the base of the bud clusters, and then the bud clusters are transferred to the rooting medium for cultivation. After two weeks, white root tips can be seen at the base of the bud clusters protruding from the callus tissue and growing continuously. The root system is formed in 30-45 days, and the rooting rate is 100%. Figure 5-1 As shown in 5-2; The rooting medium formula is: 1 / 2MS medium + NAA 0.8 mg / L + IBA 0.8 mg / L.

[0033] Step 6: Hardening and transplanting 6-1: Place the bottle seedlings with well-grown roots on an unobstructed windowsill in the tissue culture room and expose them to strong natural light for 5-7 days ( Figure 6-1 ) for transplanting; 2-3 days before transplanting, loosen the bottle caps containing the rooted seedlings and leave a gap to allow the seedlings to ventilate for seedling hardening; 6-2: After the seedlings are hardened in the bottle, take out the rooted seedlings and wash the base culture medium ( Figure 6-2 ), transplanted to a 50-cell tray or foam culture box covered with a transplanting matrix (the matrix is ​​coconut bran: vermiculite mass ratio of 1:1, and 0.5% sodium dimethoate is added for disinfection to control pathogens); in the first 15 days of transplanting, cover with plastic film to control humidity above 85%, and keep the light intensity below 6000 lux, then gradually remove the plastic film until it is placed under natural conditions; 6-3: After the seedlings fill the plug tray or the incubator, transplant them to a 12 cm nutrient pot and carry out routine management. The survival rate of the seedlings is over 95%. It can be seen that the plant is compact, plump, and symmetrical, with a large number of leaves ( Figure 6-3 ).

[0034] Comparative Example 1 Same as Example 1, except that no hydrolyzed casein was added to the callus induction and proliferation medium.

[0035] During the callus induction process, the callus is prone to browning; during the proliferation process, after more than 3 generations of culture without the addition of hydrolyzed casein, the number of adventitious buds differentiated from the callus was significantly reduced, and the differentiation rate of adventitious buds was only 40%-50%.

[0036] Comparative Example 2 The same as Example 1, except that, in the process of adventitious bud proliferation in step 3, the concentration of 6-BA in the proliferation medium remained unchanged without dynamic adjustment. If the concentration of 6-BA in the proliferation medium was always 2.0 mg / L, it was found that some callus tissues were prone to vitrification; if the concentration of 6-BA in the proliferation medium was always 0.5 mg / L, the bud differentiation of some callus tissues was significantly reduced and the browning degree increased.

[0037] Comparative Example 3 The same as Example 1, the only difference is that no seedling culture is performed, and the tissue culture seedlings after bud induction and proliferation are directly subjected to rooting culture. At this time, the rooting rate is only 20%-50%.

[0038] Comparative Example 4 The same as Example 1, the only difference is that, during the seedling cultivation in step 4, the bud cluster is cut into single buds, and it is found that only a small amount of callus tissue is generated at the base thereof, the new bud differentiation rate is almost 0, and the plant is not formed.

[0039] Comparative Example 5 The same as Example 1, the only difference is that after the rooting induction in step 5 is completed, the seedlings are directly hardened outside the bottle and transplanted without being hardened in the bottle. At this time, the survival rate of the seedlings is only 50%-60%.

[0040] Comparative Example 6 The same as Example 1, except that the transplanting medium used in step 6-2 is a mixed medium formed by imported peat and 10%-20% perlite, which is conventionally considered to be more suitable for cyclamen cultivation. At this time, it was found that the survival rate of the seedlings was 60-70%.

Claims

1. A method for tissue culture propagation of cyclamen using ovules as explants, It is characterized in that The steps include: Step 1: Ovule inoculation Step 1-1: Take the unopened or half-opened buds of the excellent individual cyclamen to be propagated, remove the petals and leave the ovary; rinse the ovary with running tap water for more than 2 hours, clean and disinfect it on a clean bench, place it under a dissecting microscope, and use a scalpel to peel off the ovule from the placenta; Step 1-2: Use a scalpel or forceps to pick up the ovules, place them in the callus induction medium, and seal them; The formula of the callus induction medium is: 1 / 2MS medium + 2,4D 2.0mg / L + 2iP 0.8mg / L + hydrolyzed casein 500mg / L + sugar 30g / L + agar 6g / L, pH value is 5.8-6.0; Step 2: Callus induction and screening The callus induction medium with ovules is placed in a completely dark environment at 22-25°C for 3-5 months to induce callus tissue to be produced; during the callus induction culture process, dense, creamy yellow or creamy white callus tissue is selected and inoculated once every 4 weeks; Step 3: Shoot induction and proliferation Step 3-1: After continuous dark culture of callus tissue for 3-5 months, dense, creamy yellow or creamy white callus tissue is selected and transferred to differentiation medium for bud induction culture to obtain adventitious buds; Step 3-2: In the early stage of adventitious bud proliferation, dense callus with many adventitious bud balls is selected as the proliferation organ and transferred to the proliferation medium for proliferation culture; Step 3-3: During the adventitious bud proliferation process, a bud cluster containing at least three buds is selected for subculture; callus tissue is left at the base of the bud cluster according to the space of the proliferation culture medium; Step 3-4: According to the development of the bud cluster and the callus tissue left at the base of the bud cluster, steps 3-2 and 3-3 are repeated continuously to obtain more bud clusters with callus tissue left at the base; The differentiation medium and the proliferation medium have the same formula, which is: 1 / 2MS medium + 6-BA 0.5-2.0 mg / L + NAA 0.05-0.2 mg / L + hydrolyzed casein 100 mg / L + sugar 30 g / L + agar 6 g / L; Step 4: Seedling cultivation The bud clusters obtained by differentiation and proliferation in step 3 are pruned so that the bud clusters retain more than 6 evenly distributed petioles and callus tissue is left at the base, and then the bud clusters are transferred to seedling medium for 1-2 generations of seedling culture to obtain rootless test tube seedlings; The seedling medium formula is: 1 / 2MS medium + 6-BA 0.05-0.2mg / L + NAA 0.01mg / L + hydrolyzed casein 100mg / L + sugar 30g / L + agar 6g / L; Step 5: Rooting Induction After the strong seedling cultivation is completed, the robust rootless test tube seedlings are pruned to make the bud points on the bud clusters evenly distributed, leaving callus tissue at the base of the bud clusters, and then transferred to the rooting medium for cultivation for 30-45 days to obtain rooted bottle seedlings; Step 6: Hardening and transplanting Step 6-1: Place the rooted seedlings under strong natural light for 5-7 days before transplanting; at the same time, 2-3 days before transplanting, loosen the bottle caps containing the rooted seedlings and leave a gap to allow the seedlings to breathe and harden; Step 6-2: After the seedlings are hardened in the bottle, take out the rooted seedlings, wash the base culture medium, and transplant them into a plug tray or foam box covered with transplanting medium; within the first 15 days of transplanting, cover with plastic film to maintain humidity above 85%, and control the light intensity to less than 6000 lux, then gradually remove the plastic film until it is placed under natural conditions; Step 6-3: After the seedlings fill the hole tray or the foam box, transplant them into the nutrient pots and carry out routine management.

2. The method for tissue culture propagation of cyclamen using ovules as explants according to claim 1, It is characterized in that The cleaning and disinfection of the ovary on the clean bench described in step 1-1 refers to: first, the ovary is immersed in 70% alcohol for disinfection for 30 seconds, and after the immersion is completed, the ovary is taken out and rinsed with sterile water; then, it is immersed in 2% sodium hypochlorite solution for disinfection for 15-20 minutes, and after the immersion is completed, the ovary is taken out and rinsed with sterile water three times, each time for 5 minutes; during each immersion disinfection, the container containing alcohol or sodium chlorate solution is continuously shaken.

3. The method for tissue culture propagation of cyclamen using ovules as explants according to claim 1, It is characterized in that The concentration of 6-BA in the proliferation medium described in step 3 is dynamically adjusted on its original basis according to the proliferation of the bud cluster and the growth of the petiole and leaf blade. When there are too many young buds in the bud cluster and the callus tissue becomes transparent, the concentration of 6-BA is reduced; when there are too few callus differentiation bud points left at the base of the bud cluster and the callus tissue becomes brown, the concentration of 6-BA is increased.

4. The method for tissue culture propagation of cyclamen using ovules as explants according to claim 1, It is characterized in that The rooting medium formula described in step 5 is: 1 / 2MS medium + NAA 0.5-1.0 mg / L + IBA 0.5-1.0 mg / L.

5. The method for tissue culture propagation of cyclamen using ovules as explants according to claim 1, It is characterized in that The transplanting matrix in step 6-2 is a mixed matrix of coconut bran and vermiculite in a mass ratio of 1:1, and 0.1-0.5% sodium dimethoate is added to the transplanting matrix.

6. The method for tissue culture propagation of cyclamen using ovules as explants according to claim 1, It is characterized in that The culture conditions of step 3 bud induction and proliferation, step 4 seedling culture and step 5 root induction culture are the same, namely, temperature 18-25°C, photoperiod 12h / d, light intensity 1500-4000lux.

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