A method for tissue culture and propagation of Cyclamen using ovules as explants

Abstract

The present invention discloses a tissue culture and propagation method of Cyclamen using ovules as explants, belonging to the technical field of plant tissue culture. The method includes steps of ovule inoculation, callus induction and screening, bud induction and proliferation, strong seedling culture, rooting induction, and acclimatization and transplantation. By adopting methods such as using casein hydrolysate for nutritional support, dynamic hormone regulation, bud cluster proliferation, strong seedling state rooting, combination of in-vial acclimatization and ex-vial acclimatization, and improvement of transplantation substrate, the present invention provides an effective new way to improve the survival rate of Cyclamen tissue culture inoculation and seedling emergence.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, and particularly relates to a method for tissue culture and propagation of Cyclamen using ovules as explants. Background Art

[0002] Cyclamen persicum is a bulbous flower of the genus Cyclamen in the family Primulaceae, and its origin is in the Mediterranean region such as Greece in southern Europe. Due to its peculiar flower shape, numerous varieties, long flowering period and blooming in winter, it has developed into one of the most important types of commercial potted flowers at home and abroad. Most commercial Cyclamen are hybrid F1 generations. Continuing to reproduce with seeds will result in trait segregation, and it is difficult to preserve the original traits and heterosis of the F1 generation. At present, the cultivation technology level of Cyclamen in China has approached the foreign level, but there is still no mature and perfect breeding technology system. It is still necessary to spend a huge amount of money to purchase foreign F1 generation seeds, which has become a bottleneck restricting the development of the Cyclamen industry in China.

[0003] With the continuous in-depth research on Cyclamen breeding in China, some very excellent new germplasm single plants have emerged. However, after the new excellent germplasm is propagated by seeds, the traits are prone to segregation, and the excellent traits cannot be preserved. In addition, the stamens or pistils of most new germplasms with specific flower shapes are sterile, and only asexual reproduction can be carried out. And Cyclamen cannot be asexually propagated by conventional methods such as cutting, grafting, and root division. Therefore, tissue culture has become the only way for asexual reproduction of Cyclamen.

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

[0005] In recent years, domestic scholars have conducted a large number of studies on Cyclamen tissue culture inoculation materials, culture medium screening, callus induction, organ regeneration induction, somatic embryo induction, etc. However, the existing Cyclamen tissue culture technology mostly uses leaves and tubers as explants. For example, Chinese Patent CN101112179A discloses a method for rapid propagation of Cyclamen tissue culture, which is a way for organ regeneration tissue culture and propagation of Cyclamen using leaves as explants; Chinese Patent CN103238526A discloses a method for rapidly propagating Cyclamen using tissue culture technology, and this technology is a complete set of technologies for tissue culture and propagation of Cyclamen using the corm of Cyclamen as an explant; Chinese Patent CN104982331A discloses a method for rapid propagation of Cyclamen tissue culture, and this technology is a method for tissue culture and propagation of Cyclamen using the flower stem as an explant.

[0006] For new and excellent individual plants, explants such as leaves and tubers not only have limited material collection quantity, but also have problems such as low inoculation survival rate due to difficult material disinfection, low bud differentiation rate and low seedling formation rate during organ regeneration, which become important factors restricting the success of tissue culture clones of new and excellent individual plants of Cyclamen persicum. Summary of the Invention

[0007] Aiming at the problems in the existing Cyclamen persicum tissue culture, such as few inoculation materials, high pollution rate, difficult differentiation and proliferation, and low seedling emergence survival rate, the present invention provides a tissue culture propagation method of Cyclamen persicum with ovules as explants. This method uses unfertilized ovules as explants, which have the advantages of no harm to plants, easy disinfection, large quantity and convenient material collection. It successively goes through the steps of callus induction and optimization, bud induction and differentiation proliferation, strong seedling culture, rooting induction, acclimatization and transplantation, etc. Finally, a new tissue culture technology system that is easy to succeed in inoculation, has a high seedling formation rate, good plant type and more leaves is established, which is beneficial to accelerating the asexual propagation process of Cyclamen persicum and promoting the further development of domestic Cyclamen persicum breeding and industry.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A tissue culture propagation method of Cyclamen persicum with ovules as explants, comprising the following steps:

[0010] Step 1: Ovule inoculation

[0011] Step 1-1: Take the unopened or semi-opened (unfertilized) flower buds of excellent individual plants of Cyclamen persicum 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 super clean workbench, and then place it under a dissecting microscope. Use a dissecting knife to strip the ovules from the placenta;

[0012] Step 1-2: Dip the ovules with a dissecting knife or forceps and inoculate them into the callus induction medium, and seal it;

[0013] The formula of the callus induction medium is: 1 / 2MS medium + 2,4-D 2.0 mg / L + 2-isopentenyladenine (2iP) 0.8 mg / L + casein hydrolysate 500 mg / L + sugar 30 g / L + agar 6 g / L, and the pH value is 5.8 - 6.0;

[0014] Step 2: Callus induction and screening

[0015] Place the callus induction medium inoculated with ovules in a completely dark environment at 22 - 25 °C and continuously culture it for 3 - 5 months to induce the production of callus; among them, during the callus induction culture process, screen dense, milky yellow or milky white callus and transfer it for inoculation once every 4 weeks;

[0016] Step 3: Bud induction and proliferation

[0017] Step 3-1: After the callus has been continuously cultured in the dark for 3-5 months, select dense, cream-colored or milky white callus and transfer it to a differentiation medium for bud induction culture to obtain adventitious buds;

[0018] Step 3-2: At the initial stage of adventitious bud proliferation, select dense callus with many adventitious bud balls as the proliferation organ and transfer it to a proliferation medium for proliferation culture;

[0019] Step 3-3: During the adventitious bud proliferation process, select a bud cluster with at least 3 bud points for subculture; the base of the bud cluster has callus according to the space of the proliferation medium;

[0020] Step 3-4: According to the development of the bud cluster and the callus remaining at the base of the bud cluster, continuously repeat Steps 3-2 and 3-3 to obtain more bud clusters with callus at the base;

[0021] The formulations of the differentiation medium and the proliferation medium are the same, which are: 1 / 2 MS medium + 6-benzyladenine (6-BA) 0.5-2.0 mg / L + naphthaleneacetic acid (NAA) 0.05-0.2 mg / L + casein hydrolysate 100 mg / L + sugar 30 g / L + agar 6 g / L;

[0022] Step 4: Strong seedling culture

[0023] Prune the bud clusters obtained by differentiation and proliferation in Step 3 so that the bud clusters retain more than 6 evenly distributed petioles and there is callus at the base, and then transfer the bud clusters to a strong seedling medium for 1-2 generations of strong seedling culture to obtain rootless test-tube seedlings;

[0024] The formulation of the strong seedling medium: 1 / 2 MS medium + 6-benzyladenine (6-BA) 0.05-0.2 mg / L + naphthaleneacetic acid (NAA) 0.01 mg / L + casein hydrolysate 100 mg / L + sugar 30 g / L + agar 6 g / L;

[0025] Step 5: Root induction

[0026] After the strong seedling culture is completed, prune the strong rootless test-tube seedlings so that the bud points on the bud clusters are evenly distributed and there is callus at the base of the bud clusters, and then transfer them to a rooting medium for culture for 30-45 days to obtain rooted bottle seedlings;

[0027] Step 6: Acclimatization and transplantation

[0028] Step 6-1: Place the rooted bottle seedlings under strong natural light for acclimatization for 5-7 days and then transplant them; at the same time, 2-3 days before transplantation, loosen the caps of the bottles containing the rooted bottle seedlings and expose gaps to allow the bottle seedlings to breathe for acclimatization;

[0029] Step 6-2: After the in-vitro acclimatization in the bottle is completed, take out the rooted plantlets in the bottle, wash the culture medium at their bases, and transplant them into a plug tray or a foam culture box lined with a transplant substrate; within 15 days at the initial stage of transplantation, cover with a plastic film to maintain a humidity of over 85%, and control the light intensity to be less than 6000 lux, and then gradually remove the plastic film until placed under natural conditions;

[0030] Step 6-3: After the seedlings cover the plug tray or fill the foam box, transplant them into nutrient pots and conduct routine management.

[0031] Furthermore, the excellent individual plant described in Step 1 refers to a plant that meets the breeding objectives and is robust, disease-free, and pest-free.

[0032] Furthermore, the cleaning and disinfection of the ovary on the ultra-clean workbench described in Step 1-1 means: first soak the ovary in 70% alcohol for 30 seconds for disinfection, take out the ovary after soaking, and rinse it once with sterile water; then soak it in 2% sodium hypochlorite solution for 15 - 20 minutes for disinfection, take out the ovary after soaking, and rinse it three times with sterile water, 5 minutes each time; during each soaking and disinfection, continuously shake the container containing alcohol or sodium hypochlorite solution.

[0033] Furthermore, the concentration of 6-BA in the proliferation medium described in Step 3 is dynamically adjusted based on the proliferation of bud clusters and the growth of petioles and leaves on the original basis. When there are too many young and tender buds in the bud cluster and the callus shows a transparent phenomenon, reduce the concentration of 6-BA; when there are too few bud points differentiated from the callus remaining at the base of the bud cluster and the callus shows browning, increase the concentration of 6-BA.

[0034] Furthermore, the rooting medium formula described in Step 5: 1 / 2MS medium + NAA 0.5 - 1.0 mg / L + IBA 0.5 - 1.0 mg / L.

[0035] Furthermore, the plug tray described in Step 6-2 is a 50-hole plug tray.

[0036] Furthermore, the transplant substrate described in Step 6-2 is preferably a mixed substrate prepared by compounding coconut coir and vermiculite in a mass ratio of 1:1, and 0.1 - 0.5% sodium dimethyldithiocarbamate is added to the transplant substrate.

[0037] Furthermore, the culture conditions for the bud induction and proliferation in Step 3, the strong seedling culture in Step 4, and the rooting induction culture in Step 5 are the same, which are a temperature of 18 - 25°C, a light cycle of 12 h / d, and a light intensity of 1500 - 4000 lux.

[0038] The present invention discloses a complete technical solution for tissue culture propagation of Cyclamen using unfertilized ovules as explants, providing the overall idea and specific methods for tissue culture propagation of Cyclamen, namely "initiation of ovule explants, nutritional support of casein hydrolysate, dynamic hormone regulation, bud cluster proliferation, strong seedling rooting, and in-vial and ex-vial acclimatization and transplantation", which provides an effective new way to improve the inoculation survival rate, emergence survival rate and plant type shaping of Cyclamen tissue culture. Specifically:

[0039] (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 ovules of Cyclamen are in a sterile state in the ovary, using ovules as explants is easy to obtain sterile explant materials with a very low contamination rate, overcoming the defect of easy contamination of other explants such as leaves and bulbs; and it is explored to add casein hydrolysate to the induction medium, which not only reduces the browning rate of callus, but also makes the inoculation success rate of ovules reach more than 95%;

[0040] (2) During the subculture and rooting of adventitious buds, a scheme of dynamic hormone regulation according to the growth of callus is adopted, effectively inhibiting the vitrification or browning of callus, and the induction rate of adventitious bud differentiation from callus reaches 96% - 100%;

[0041] (3) In the adventitious bud proliferation, strong seedling and rooting culture of the present invention, they are all carried out in the form of bud clusters or clustered seedlings, and an appropriate amount of callus is left at the base of the bud clusters, so that the emergence rate and rooting rate of Cyclamen can reach up to 100% at most; at the same time, during the adventitious bud proliferation process of the present invention, bud clusters with more bud points are selectively screened, and during the later strong seedling culture and acclimatization culture, the morphology of the bud clusters is appropriately trimmed, accelerating the forming speed of tissue culture seedlings, making the tissue culture seedlings have a compact, plump and symmetrical plant type and more leaves;

[0042] (4) The present invention uses a combination of in-vial and ex-vial acclimatization for acclimatization and adopts a unique transplanting substrate, making the in-vial seedlings of Cyclamen stronger and more adaptable to the later transplanting environment, with a survival rate of 95% - 100%.

[0043] 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. Description of the Drawings

[0044] Figure 1 It is the unopened ( Figure 1-1 ), or semi-opened ( Figure 1-2 ) flower buds of the excellent single plant of the hybrid offspring of the adult plant "Picotee" in Example 1 of the present invention.

[0045] Figure 2Culture and development of callus with ovules attached in Example 1; among them, Figure 2-1 is the ovule inoculated for the first time, Figure 2-2 is the preferred callus of the inoculated ovule at 4 weeks, Figure 2-3 is the preferred callus of the inoculated ovule at 8 weeks, Figure 2-4 is the preferred callus of the inoculated ovule at 12 weeks, Figure 2-5 is the preferred callus of the inoculated ovule at 16 weeks.

[0046] Figure 3 is the callus during the bud induction and proliferation culture in Example 1; among them, Figure 3-1 is the callus with adventitious bud balls formed one month after bud induction, Figure 3-2 is the bud cluster with a part of callus left at the base during the adventitious bud proliferation process.

[0047] Figure 4 is the bud cluster during the strong seedling culture in Example 1; among them, Figure 4-1 is the bud cluster with more than 6 evenly distributed petioles retained and an appropriate amount of callus left at the base, Figure 4-2 is the bud cluster for strong seedling culture in the tissue culture room, Figure 4-3 is the bud cluster with expanded leaves, showing reddish-brown or green color one month after strong seedling culture.

[0048] Figure 5 is the bud cluster during the rooting induction culture; among them, Figure 5-1 and Figure 5-2 are both bud clusters with roots grown 30 - 45 days after rooting induction.

[0049] Figure 6 is the tissue culture seedlings after acclimatization culture and transplantation; among them, Figure 6-1 is the picture of the bottle seedlings with well-developed roots during acclimatization under strong natural light; Figure 6-2 is the rooted bottle seedlings with the basal medium cleaned after acclimatization, Figure 6-3 is the tissue culture seedlings under conventional management after transplantation. Detailed implementation method

[0050] The present invention will be further explained below in conjunction with specific embodiments. Those skilled in the art will understand that the following described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. The specific conditions not specified in the embodiments are carried out according to the conventional conditions. The reagents used are all conventional products that can be obtained through commercial purchase.

[0051] Example 1

[0052] Taking the example of obtaining excellent individual plant tissue culture seedlings of Cyclamen persicum "Picotee" hybrid offspring with pink and white color, strong fragrance (FBNX) by the method of the present invention, the specific operation steps are as follows:

[0053] Step 1: Ovule inoculation

[0054] 1-1: Take the unopened or semi-opened flower buds (unfertilized) of the excellent individual plant of the Cyclamen persicum "Picotee" hybrid offspring with pink and white color, strong fragrance (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 disinfection for 30 seconds, take out the ovary after soaking, and rinse it once with sterile water; then soak it in 2% sodium hypochlorite solution for disinfection for 15-20 minutes, take out the ovary after soaking, and rinse it three times with sterile water, 5 minutes each time; during each soaking and disinfection, continuously shake the container containing alcohol or sodium hypochlorite solution;

[0055] Under a dissecting microscope, use a dissecting knife to cut the integument of the ovary from top to bottom, and gently peel the ovules from the placenta with the back of the dissecting knife at one point;

[0056] 1-2: Dip the ovules with a dissecting knife or forceps, gently place them on the pre-prepared callus induction medium, and then seal it;

[0057] The formula of the callus induction medium is: 1 / 2MS medium + 2,4-D 2.0 mg / L + 2-isopentenyladenine (2iP) 0.8 mg / L + casein hydrolysate 500 mg / L + sugar 30 g / L + agar 6 g / L, and the pH value is 5.8-6.0;

[0058] Pour the autoclaved mother bottle into 6-cm culture dishes in a laminar flow hood, 12 ml per dish, and let it cool and solidify for standby.

[0059] Step 2: Callus induction and screening

[0060] The success rate of ovule inoculation in Step 1 is over 95%. Place the callus induction medium with inoculated ovules in a completely dark environment at 22-25 °C and continuously culture for 16 weeks to induce callus ( Figure 2 ); among them, during the culture process, screen the dense, creamy yellow or milky white callus and transfer and inoculate it once every 4 weeks.

[0061] For the successfully inoculated ovules, after 4 weeks of dark culture, the callus induction rate reaches over 98%; however, after 8-12 weeks, there will be great differences in the color and proliferation rate of the callus. When subculturing, screen the dense, creamy yellow or milky white callus for optimization.

[0062] Figure 2-1 For the initially inoculated ovules,Figure 2-2 Callus preferred for 4 weeks of inoculated ovules Figure 2-3 Callus preferred for 8 weeks of inoculated ovules Figure 2-4 Callus preferred for 12 weeks of inoculated ovules Figure 2-5 Callus preferred for 16 weeks of inoculated ovules

[0063] Step 3: Bud induction and proliferation

[0064] 3-1: After culturing the callus continuously in the dark for 4 months, transfer the dense, creamy yellow or milky white callus that has been continuously screened to the differentiation medium for bud induction culture;

[0065] 3-2: After 1 month of culture, adventitious bud balls are formed ( Figure 3-1 ); Thereafter, screen the callus that is dense and has many adventitious bud balls as the proliferation organ and transfer it to the proliferation medium for continuous proliferation culture;

[0066] 3-3: During the proliferation of adventitious buds, screen the bud clusters containing at least 3 bud points for subculture; a part of the callus is left at the base of the bud cluster according to the space of the proliferation medium ( Figure 3-2 );

[0067] 3-4: According to the development of the bud clusters and the callus left at the base of the bud clusters, continuously repeat steps 3-2 and 3-3 to obtain more bud clusters with callus left at the base;

[0068] The formulas of the differentiation medium and the proliferation medium are the same, which are: 1 / 2MS medium + 6-benzylaminopurine (6-BA) 1.5 mg / L + naphthaleneacetic acid (NAA) 0.15 mg / L + casein hydrolysate 100 mg / L + sugar 30 g / L + agar 6 g / L;

[0069] Among them, the concentration of 6-BA in the proliferation medium is dynamically adjusted based on the proliferation of bud clusters and the growth of petioles and leaves on the original basis. When there are too many young buds in the bud clusters and the callus shows transparency, adjust the 6-BA concentration to 0.5 - 1.0 mg / L; when the number of bud points differentiated from the callus left at the base of the bud clusters is too small and the callus shows browning, increase the 6-BA concentration to 1.8 - 2.0 mg / L.

[0070] The differentiation rate of adventitious bud balls in the primary induction of callus in this step is 60% - 70%, and the proliferation multiple of adventitious bud balls in subculture is 1 - 3 times.

[0071] Step 4: Strong seedling culture

[0072] Properly prune the bud clusters obtained by differentiation and proliferation in step 3 so that the bud clusters retain more than 6 evenly distributed petioles and an appropriate amount of callus is left at the base ( Figure 4-1), and then transfer the bud clusters to a strong seedling medium for strong seedling culture ( Figure 4-2 ), after one month, the leaves of the bud clusters can be seen to unfold, showing reddish-brown or green ( Figure 4-3 ), and obtain rootless test-tube seedlings;

[0073] The formula of the strong seedling medium: 1 / 2 MS medium + 6-benzylaminopurine (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.

[0074] Step 5: Root induction

[0075] After the strong seedling culture is completed, trim the bud clusters of the healthy rootless test-tube seedlings so that the bud points on the bud clusters are evenly distributed, and leave an appropriate amount of callus at the base of the bud clusters. Then transfer the bud clusters to the rooting medium for culture. After two weeks, white root tips can be seen protruding from the callus at the base of the bud clusters and growing continuously. Roots are formed in 30 - 45 days, and the rooting rate is 100%, as shown in Figure 5-1 and 5 - 2;

[0076] The formula of the rooting medium: 1 / 2 MS medium + NAA 0.8 mg / L + IBA 0.8 mg / L.

[0077] Step 6: Hardening off and transplanting

[0078] 6 - 1: Place the bottle seedlings with well-developed roots on the unobstructed window sill 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 caps of the bottles containing the rooted bottle seedlings and expose a gap to allow the bottle seedlings to breathe for hardening off;

[0079] 6 - 2: After the in-bottle hardening off is completed, take out the rooted bottle seedlings, wash the medium at their bases ( Figure 6-2 ), and transplant them into a 50-hole plug tray or a foam culture box paved with a transplanting substrate (the substrate is coconut coir: vermiculite with a mass ratio of 1:1, and 0.5% sodium dimethyldithiocarbamate is added for disinfection to control diseases); among them, within 15 days at the initial stage of transplanting, cover with a plastic film to control the humidity above 85% and keep the light intensity below 6000 lux, and then gradually remove the plastic film until placed under natural conditions;

[0080] 6 - 3: When the seedlings cover the plug tray or fill the culture box, transplant them into 12-cm nutrient pots for routine management. The survival rate of the seedlings is above 95%, and it can be seen that their plant types are compact, plump, symmetrical, and there are more leaves ( Figure 6-3 ).

[0081] Comparative Example 1

[0082] Same as Example 1, the only difference is that hydrolyzed casein is not added to the callus induction and proliferation medium.

[0083] During the callus induction process, the callus is prone to browning; during the proliferation process, after culturing for more than 3 generations without adding casein hydrolysate, the number of adventitious bud balls redifferentiated from the callus significantly decreases, and the differentiation rate of adventitious bud balls is only 40%-50%.

[0084] Comparative Example 2

[0085] Same as Example 1, the difference is only that during the adventitious bud proliferation process in Step 3, the concentration of 6-BA in the proliferation medium remains constant without dynamic adjustment. For example, the concentration of 6-BA in the proliferation medium is always 2.0 mg / L, and it is found that some callus is prone to vitrification; if the concentration of 6-BA in the proliferation medium is always 0.5 mg / L, then the bud differentiation of some callus significantly decreases and the degree of browning increases.

[0086] Comparative Example 3

[0087] Same as Example 1, the difference is only that no strong seedling culture is carried out, 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%.

[0088] Comparative Example 4

[0089] Same as Example 1, the difference is only that during the strong seedling culture in Step 4, the bud cluster is cut into single buds, and it is found that only a small amount of callus is generated at its base, the differentiation rate of new buds is almost 0, and the plant is not formed.

[0090] Comparative Example 5

[0091] Same as Example 1, the difference is only that after the rooting induction in Step 5, no in-vitro acclimatization is carried out, and direct ex-vitro acclimatization and transplantation are carried out. At this time, the survival rate of the seedlings is only 50%-60%.

[0092] Comparative Example 6

[0093] Same as Example 1, the difference is only that the transplanting substrate used in Step 6-2 is a mixed substrate formed by imported peat moss, which is conventionally considered more suitable for cyclamen cultivation, and 10%-20% perlite. At this time, it is found that the survival rate of the seedlings is 60-70%.

Claims

1. A tissue culture and propagation method of Cyclamen persicum using ovules as explants, characterized in that, It includes the following steps: Step 1: Ovule inoculation Step 1-1: Take the unopened or semi-opened flower buds of the excellent individual plants of Cyclamen persicum 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 the ultra-clean workbench, then place it under the dissecting microscope, and use a dissecting knife to peel the ovules from the placenta; Step 1-2: Dip the ovules with a dissecting knife or forceps, and inoculate them into the callus induction medium, and seal it; The formula of the callus induction medium is: 1 / 2MS medium + 2,4-D 2.0 mg / L + 2iP 0.8 mg / L + casein hydrolysate 500 mg / L + sugar 30 g / L + agar 6 g / L, and the pH value is 5.8 - 6.0; Step 2: Callus induction and screening Place the callus induction medium inoculated with ovules in a completely dark environment at 22 - 25 °C and continuously culture it for 3 - 5 months to induce the production of callus; among them, during the callus induction culture process, screen the dense, milky yellow or milky white callus every 4 weeks and transfer and inoculate it once; Step 3: Bud induction and proliferation Step 3-1: After continuously culturing the callus in the dark for 3 - 5 months, screen the dense, milky yellow or milky white callus and transfer it to the differentiation medium for bud induction culture to obtain adventitious buds; Step 3-2: At the initial stage of adventitious bud proliferation, screen the callus with dense and many adventitious bud balls as the proliferation organ and transfer it to the proliferation medium for proliferation culture; Step 3-3: During the adventitious bud proliferation process, screen the bud clusters with at least 3 bud points for subculture; the base of the bud cluster has callus according to the space of the proliferation medium; Step 3-4: According to the development of the bud clusters and the callus left at the base of the bud clusters, continuously repeat steps 3-2 and 3-3 to obtain more bud clusters with callus left at the base; The formulas of the differentiation medium and the proliferation medium are the same, which are: 1 / 2MS medium + 6-BA 0.5 - 2.0 mg / L + NAA 0.05 - 0.2 mg / L + casein hydrolysate 100 mg / L + sugar 30 g / L + agar 6 g / L; Among them, the concentration of 6-BA in the proliferation medium is dynamically adjusted based on the proliferation of the bud clusters and the growth of petioles and leaves on the original basis. When there are too many young and tender buds in the bud clusters and the callus shows a transparent phenomenon, reduce the concentration of 6-BA; when there are too few bud points differentiated from the callus left at the base of the bud clusters and the callus shows browning, increase the concentration of 6-BA; Step 4: Strong seedling culture Prune the bud clusters obtained by differentiation and proliferation in step 3 so that the bud clusters retain more than 6 evenly distributed petioles and there is callus left at the base, and then transfer the bud clusters to the strong seedling medium for 1 - 2 generations of strong seedling culture to obtain rootless test-tube seedlings; The formula of the strong seedling medium: 1 / 2MS medium + 6-BA 0.05 - 0.2 mg / L + NAA 0.01 mg / L + casein hydrolysate 100 mg / L + sugar 30 g / L + agar 6 g / L; Step 5: Root induction After the strong seedling culture is completed, the healthy rootless test-tube seedlings are trimmed to make the bud points on the bud clusters evenly distributed, and callus is left at the base of the bud clusters. Then, they are transferred to the rooting medium and cultured for 30 - 45 days to obtain rooted bottle seedlings. The formula of the rooting medium: 1 / 2MS medium + NAA 0.5 - 1.0 mg / L + IBA 0.5 - 1.0 mg / L. Step 6: Hardening-off and transplanting Step 6-1: Place the rooted bottle seedlings under strong natural light for hardening-off for 5 - 7 days and then transplant them. At the same time, 2 - 3 days before transplantation, loosen the caps of the bottles containing the rooted bottle seedlings and expose a gap to allow the bottle seedlings to breathe for hardening-off. Step 6-2: After the in-bottle hardening-off is completed, take out the rooted bottle seedlings, wash the medium at their bases, and transplant them into a plug tray or foam box lined with the transplant substrate. Within 15 days at the initial stage of transplantation, cover with a plastic film to keep the humidity above 85% and control the light intensity to be less than 6000 lux, and then gradually remove the plastic film until placed under natural conditions. Step 6-3: After the seedlings cover the plug tray or fill the foam box, transplant them into nutrient pots and conduct routine management.

2. The tissue culture and propagation method of Cyclamen persicum using ovule as explant according to claim 1, characterized in that, The cleaning and disinfection of the ovary on the ultra-clean workbench in Step 1-1 means: First, immerse the ovary in 70% alcohol for disinfection for 30 seconds. After immersion, take out the ovary and rinse it once with sterile water. Then, immerse it in 2% sodium hypochlorite solution for disinfection for 15 - 20 minutes. After immersion, take out the ovary and rinse it three times with sterile water, 5 minutes each time. During each immersion disinfection, continuously shake the container containing alcohol or sodium hypochlorite solution.

3. The tissue culture and propagation method of Cyclamen persicum with ovules as explants according to claim 1, characterized in that, The transplant substrate in Step 6-2 is a mixed substrate prepared by compounding coconut coir and vermiculite in a mass ratio of 1:1, and 0.1 - 0.5% sodium fusidate is added to the transplant substrate.

4. The tissue culture and propagation method of Cyclamen persicum using ovule as explant according to claim 1, characterized in that, The culture conditions for Step 3 bud induction and proliferation, Step 4 strong seedling culture, and Step 5 rooting induction culture are the same, which are temperature 18 - 25°C, light cycle 12 h / d, and light intensity 1500 - 4000 lux.

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