Hemerocallis fulva doubling method

Abstract

The invention belongs to the technical field of agricultural breeding, and particularly relates to a day lily doubling method. Through a ploidy breeding method, a group culture seedling induced by an explant is used for treatment, a hemerocallis fulva hybridized seed sowing seedling is directly subjected to double induction or a hemerocallis fulva field seedling is treated, so that the hemerocallis fulva is doubled. The result of the embodiment shows that the success rate of the hemerocallis fulva doubling method is 100%, the doubling efficiency is high, the seedling loss is small, and a powerful technical guarantee is provided for hemerocallis fulva ploidy breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural breeding, and in particular relates to a method for doubling daylilies. Background Art

[0002] Hemerocallis belongs to the Asphodeloides family. It is a famous ornamental and edible flower, a perennial root plant. It is a plant for viewing leaves in spring and autumn and flowers in summer. It has strong adaptability, low maintenance cost, and is easy to manage and reproduce. There are many varieties, long flowering period, various flower shapes, and rich colors. It can be said that it has both color and shape, and combines many advantages in one. It is a rare good material in landscaping. Today, when the calls for ecological civilization construction and low-carbon environmental protection are getting louder and louder, the large-flowered daylily will undoubtedly become the first choice for building energy-saving gardens and ecological civilization cities.

[0003] Daylilies can not only be used in flower beds, flower borders, roadsides, lawns, woods, grass slopes, etc. to create natural landscapes, but can also be used as cut flowers and potted flowers to beautify homes, and have broad application prospects.

[0004] Plant breeding techniques include conventional hybrid breeding, mutation breeding, ploidy breeding and gene editing. my country has a very rich wild species of daylilies. There are 11 species and some variants of the 14 species of daylilies in my country. The use of native species is very important in daylily breeding. Although foreign species are already very rich, some wild species have not been fully utilized. In daylily breeding, the vast majority of wild species are diploid, with small flowers, and most of their offspring also have small flowers. In addition, many large-flowered daylily horticultural varieties are polyploid and do not bear fruit when hybridized with the original species.

[0005] Therefore, there is an urgent need to develop a method for efficiently doubling daylilies. Summary of the invention

[0006] The purpose of the present invention is to provide a method for doubling daylilies. The method provided by the present invention not only has a high success rate, but also has a high doubling efficiency and less seedling loss.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a method for doubling daylilies, which specifically comprises the following steps:

[0009] S1. Clean the roots of daylily seedlings and keep the root length at 2-6 cm.

[0010] S2. Induce polyploidy in the cleaned seedlings with colchicine aqueous solution.

[0011] Preferably, the seedlings are tissue culture seedlings of new varieties or hybrid varieties of daylilies, sowing seedlings or field seedlings of hybrid offspring of daylilies.

[0012] Preferably, the colchicine aqueous solution is a 0.05% to 0.1% aqueous solution prepared by mixing colchicine with 70% alcohol.

[0013] More preferably, the cultivation process of the tissue culture seedlings of the new variety or hybrid new variety of daylily is as follows: collect the new variety or hybrid new variety, cut off the upper leaves and keep 5 cm long of the stem, culture it in a primary culture medium, culture it for 2 to 3 months, take the inflorescence that has sprouted or the whole plant as an explant, induce the explant, and after successful induction, carry out proliferation culture and rooting culture in the primary culture medium, and after hardening the seedlings, plant them in a plug tray and culture them until they grow 4 to 6 leaves for use.

[0014] More preferably, the primary culture medium formula is MS culture medium, 1.0-2.0 mg / L 6-benzylaminoadenine, 0.1-0.2 mg / L naphthaleneacetic acid, 30 g / L sucrose and 6 g / L agar.

[0015] More preferably, the explant induction process is to wash with detergent, rinse with running water for 30 minutes, wash with distilled water for 3 to 4 times, take out the material and place it on sterile dry filter paper, absorb the surface moisture and place it on a clean bench; disinfect the surface with 75% alcohol for 30 seconds, then sterilize in a 0.1% mercuric chloride solution with a mass volume percentage concentration for 10 to 12 minutes or a 0.2% sodium hypochlorite solution for 12 to 15 minutes, rinse with sterile water 4 to 5 times, each time for 4 to 5 minutes, and place it in an inoculation tray; cut off the browned part with a sterilized blade; cut the pedicel into 1 cm segments as explant material and inoculate it into the primary culture medium for culture.

[0016] More preferably, the substances used in the proliferation culture process include MS culture medium, 1.0-3.0 mg / L of 6-benzylaminoadenine, 0.05-1.0 mg / L of naphthaleneacetic acid, 30 g / L of sucrose and 6 g / L of agar; the substances used in the rooting culture process include 1 / 2MS culture medium, 0.1-0.5 mg / L of indoleacetic acid, 30 g / L of sucrose and 6 g / L of agar.

[0017] More preferably, the seedling hardening refers to placing the rooted seedlings in a greenhouse for 3 days, washing the rooted seedlings, soaking them in a 1000-fold concentration of the pesticide oxazolidinone, taking them out after 5 minutes to shake off the water, spreading them flat to dry, and planting them in plug trays after drying until they grow 4 to 6 leaves.

[0018] Preferably, the cultivation process of the seedlings of the hybrid offspring of the daylily is as follows: harvest the daylily fruits when they are ripe and just cracked one month after hybridization, store them in a refrigerator at 4°C for one month after harvesting and then ripen them, take out the seeds from the fruits and soak them at high temperature before germination, keep the germination temperature at 25°C, wash them 1 to 2 times a day, sow them in plug trays when they are white, and wait for the seedlings to grow to 4 to 6 leaves for use.

[0019] Preferably, the cultivation process of the field seedlings is as follows: dig out the daylily seedlings in the field, remove the upper leaves and retain 5 cm of the stem, retain 5 cm of the root system, divide them into small clumps with 1 to 2 buds, and transplant them in a square tray. The substrate is made of peat and coconut bran in a ratio of 5:3. Cultivate until small buds grow on the side of the seedlings and grow to 4 to 6 leaves for later use.

[0020] Beneficial effects of the present invention:

[0021] 1. The present invention selects tissue culture seedlings of new hybrid varieties of daylilies or tissue culture seedlings of new varieties introduced, seeding seedlings of hybrid offspring or field seedlings, performs colchicine immersion and culture treatment on the seedlings obtained in various aspects, and doubles the daylilies, thereby providing a strong technical guarantee for the promotion and application of daylily ploidy breeding technology.

[0022] 2. The present invention has low cost, simple operation, high induction efficiency, small seedling loss, and an induction success rate of up to 100%. The application of this method can successfully promote the evolution of daylily varieties by effectively doubling daylilies, and set off a climax in daylily breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0024] Figure 1 To double the breeding flow chart;

[0025] Figure 2 This is a picture of the new leaves of the self-bred variety 'Shili Hongzhuang' before doubling the tissue culture seedlings;

[0026] Figure 3 This is a state diagram of new leaves of the plant after doubling the tissue culture seedlings of the independently bred variety 'Shili Hongzhuang' using the method of the present invention;

[0027] Figure 4 This is a picture of the state of the seeds collected from the female parent when they turn white;

[0028] Figure 5 This is a comparison of the offspring of the seed-sown seedlings of the female parent before and after doubling; the three pictures in the upper row of Figure A are pictures of the state before doubling, and the three pictures in the lower row are pictures of the state after doubling; the left side of Figure B is the picture of the state before doubling, and the right side is the picture of the state after doubling;

[0029] Figure 6 This is a picture of the state of Hemerocallis fulva seedlings before doubling after being transplanted in the field;

[0030] Figure 7This is a picture of the state of the daylily seedlings (Hemerocallisfulva) after they were doubled after being transplanted in the field;

[0031] Figure 8 This is the cotton ball drip method at the field growth point. It is a comparison picture of one plant in the same clump that was doubled and one plant that was not doubled. The leaves on the left are the leaves that were not doubled, and the leaves on the right are the leaves that were doubled. DETAILED DESCRIPTION

[0032] The invention provides a method for doubling daylilies, which uses colchicine immersion culture treatment on hybrid new variety tissue culture seedlings of daylilies or introduced new variety tissue culture seedlings, hybrid offspring sowing seedlings or field seedlings to achieve rapid doubling of daylilies.

[0033] The invention discloses a method for doubling daylilies, which comprises the following method.

[0034] On the one hand, the present invention can collect explants from the bred new daylily hybrid variety or the introduced new daylily variety for explant induction, and obtain seedlings for use after cultivation.

[0035] (1) Before collecting explants, remove the dead leaves at the base of the daylily variety, cut off the upper leaves and keep 5 cm in length, plant them in pots, and culture them in a greenhouse for at least 2 to 3 months. The inflorescence of the shoots can be used as an explant for induction, or the whole plant can be directly used as an explant. The primary culture medium formula is MS + 6-benzylaminoadenine 1.0-2.0 mg / L + naphthaleneacetic acid 0.1-0.2 mg / L + sucrose 30 g / L + agar 6 g / L.

[0036] The explant induction process is to wash with detergent, rinse with running water for 30 minutes, and wash with distilled water for 3 to 4 times. The material is taken out and placed on sterile dry filter paper, and the surface moisture is absorbed and placed on a clean bench; the surface is disinfected with 75% alcohol for 30 seconds, and then sterilized in a 0.1% mercuric chloride solution with a mass volume percentage concentration for 10 to 12 minutes or a 0.2% sodium hypochlorite solution for 12 to 15 minutes, rinsed with sterile water 4 to 5 times, each time for 4 to 5 minutes, and placed in an inoculation tray; the browned part of the outside is cut off with a sterilized blade; the pedicels are cut into 1 cm segments as explant materials and inoculated into the primary culture medium for culture.

[0037] (2) after successful induction, proliferation culture and rooting culture are performed; the substances used in the proliferation culture process include MS culture medium, 1.0-3.0 mg / L 6-benzylaminoadenine, 0.05-1.0 mg / L naphthaleneacetic acid, 30 g / L sucrose and 6 g / L agar; the substances used in the rooting culture process include 1 / 2 MS culture medium, 0.1-0.5 mg / L indoleacetic acid, 30 g / L sucrose and 6 g / L agar;

[0038] (3) After hardening the rooted seedlings, plant them in a plug tray and set them aside when they grow to 4 to 6 leaves; this means that after placing the rooted seedling tissue culture bottle in the greenhouse for 3 days, take out the rooted seedlings, remove the culture medium, wash them in clean water, and then soak them in a 1000-fold concentration of the pesticide cypermethrin. After 5 minutes, take them out and shake off the water, spread them flat to dry, and then plant them in a 50-hole plug tray with a substrate ratio of peat: coconut bran of 5:3. Water them thoroughly and cover them with a small arch shed to keep them moist. After the root balls are formed, remove the arch shed. When they grow to 4 to 6 leaves, the induction experiment can be carried out.

[0039] (4) Take the seedlings out of the plug tray and clean the roots. If the roots are too long, cut them short to keep a root length of 2-6 cm. Soak the cleaned seedlings in a container filled with colchicine aqueous solution, ensuring that the liquid level is 0.5-1 cm higher than the growth point of the plant, and record the variety and date; it means that colchicine is dissolved in 70% alcohol to make a 0.05%-0.1% aqueous solution, and the colchicine is placed in a glass container, and the seedlings are immersed in the solution, ensuring that the liquid level is 0.5-1 cm higher than the growth point of the plant. The container is partially covered with a black plastic bag to prevent the solution from being exposed to light, and the variety and date are recorded.

[0040] On the other hand, the invention uses daylily varieties to be hybridized, and the fruits are harvested after they are ripe, sown in a plug tray, and are used when they grow to 4 to 6 leaves.

[0041] (1) Hybridization of daylilies. The fruits mature one month after hybridization and are harvested when the fruits just crack. After harvesting, they are stored in a refrigerator at 4°C for about one month for post-ripening. The seeds are taken out from the fruits and soaked at high temperature for germination. The temperature is maintained at 25°C during the germination process. They are washed 1 to 2 times a day and sown in 50-hole trays when the seeds are white. The substrate ratio is peat: coconut bran 5:3. Water thoroughly to keep the substrate moist. When the seedlings emerge and grow to 4 to 6 leaves, the induction experiment can be carried out.

[0042] (2) Take the seedlings out of the plug tray and clean the roots. If the roots are too long, cut them short to keep a root length of 2-6 cm. Soak the cleaned seedlings in a container filled with colchicine aqueous solution, ensuring that the liquid level is 0.5-1 cm higher than the growth point of the plant, and record the variety and date; it means that colchicine is dissolved in 70% alcohol to make a 0.05%-0.1% aqueous solution, the colchicine is placed in a glass container, the seedlings are immersed in the solution, ensuring that the liquid level is 0.5-1 cm higher than the growth point of the plant, and the container is partially covered with a black plastic bag to prevent the solution from being exposed to light, and the variety and date are recorded.

[0043] Another aspect of the present invention is to directly conduct induction experiments using daylilies in the field.

[0044] (1) The seedlings are dug out, divided into small clusters with 1 to 2 buds, and transplanted in a square tray. When the small buds on the side grow to 4 to 6 leaves, they can be used for later use. This means that daylily seedlings in the field are dug out, the upper leaves are removed and 5 cm is retained, the longer roots are cut off, and 5 cm of roots are retained. The seeds are divided into small clusters with 1 to 2 buds and transplanted in a square tray at a high density. The substrate is made of peat and coconut coir in a ratio of 5:3. The substrate is watered thoroughly to keep it moist. When the small buds on the side grow to 4 to 6 leaves, the induction experiment can be carried out.

[0045] (2) Take the seedlings out of the plug tray and clean the roots. If the roots are too long, cut them short to keep a root length of 2-6 cm. Soak the cleaned seedlings in a container filled with colchicine aqueous solution, ensuring that the liquid level is 0.5-1 cm higher than the growth point of the plant, and record the variety and date; it means that colchicine is dissolved in 70% alcohol to make a 0.05%-0.1% aqueous solution, the colchicine is placed in a glass container, the seedlings are immersed in the solution, ensuring that the liquid level is 0.5-1 cm higher than the growth point of the plant, and the container is partially covered with a black plastic bag to prevent the solution from being exposed to light, and the variety and date are recorded.

[0046] The seedlings of the present invention are soaked in colchicine for 7 to 10 days, then taken out for washing. After washing, the seedlings are soaked in a container filled with clean water. New buds at the growing points are observed. If the new buds swell and become obviously fat, it means that the induction is successful. When the buds at the growing points of more than half of the seedlings become fat, they can be rinsed, which generally takes 7 to 10 days. At this time, the leaves also begin to turn yellow due to the toxicity of colchicine. The seedlings are taken out for washing, and after washing, they are immersed in clean water again for cultivation, and all the roots are immersed in the clean water.

[0047] After soaking in clean water for 2 to 3 days, take out the seedlings and plant them in flowerpots with a diameter of 10 cm. The flowerpots are placed on a bracket and cultivated in a greenhouse or shed. The substrate used is a mixture of peat and coconut coir in a ratio of 5:3. Water thoroughly and keep the soil moist.

[0048] After the new leaves at the growing point grow out, it can be clearly identified whether the doubling is successful. After the successful doubling seedlings form a root ball, they can be planted in the field; it means waiting for the swollen new leaves at the growing point to slowly sprout, the doubled new leaves to slowly unfold, and the growing point to continuously produce fat new leaves. After the new leaves unfold, they can be compared with the leaves of the control seedlings that have not been treated with colchicine, or the new and old leaves on the same plant. It can be confirmed whether the doubling is successful. After the root ball is formed, it can be planted in the field.

[0049] Furthermore, you can wait until flowering to confirm the doubling effect, or you can take leaves and control plant leaves and use flow cytometry ploidy detection or compression method chromosome ploidy detection. It means that the diameter of the doubled daylily flower will be larger than the original variety, and the thickness of the scape will also be thicker than the original variety, so it can be finally confirmed whether the doubling is successful. You can also take the leaves of the doubled seedlings and the leaves of the control seedlings that have not been treated with colchicine to measure the genome size using flow cytometry, and compare the genome sizes of the two leaves to determine whether the doubling is successful. You can also take the root tips of the doubled seedlings and the root tips of the control seedlings that have not been treated with colchicine and use the compression method to detect the number of chromosomes to determine whether the doubling is successful.

[0050] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0051] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0052] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0053] Example 1

[0054] A method for rapid doubling of daylilies, the specific doubling breeding flow chart is as follows Figure 1 shown.

[0055] The inflorescence of the authorized new daylily variety 'Shili Hongzhuang' was collected as an explant for induction. The induction process was as follows: after washing with detergent, rinse with running water for 30 minutes, and wash with distilled water for 3 to 4 times, the material was taken out and placed on sterile dry filter paper, and the surface moisture was absorbed and placed on a clean bench; the surface was disinfected with 75% alcohol for 30 seconds, and then sterilized in a 0.1% mass volume percentage concentration of mercuric chloride solution for 12 minutes, rinsed with sterile water 4 to 5 times, each time for 5 minutes, and placed in an inoculation tray; the browned part of the outside was cut off with a sterilized blade; the pedicels were cut into 1 cm segments as explant materials and inoculated into the primary culture medium, the formula of the primary culture medium was MS + 6-benzylaminoadenine 2.0 mg / L + naphthaleneacetic acid 0.2 mg / L + sucrose 30 g / L + agar 6 g / L.

[0056] After successful induction, proliferation culture and rooting culture were carried out. After the rooted seedlings were placed in the greenhouse for 3 days, the rooted seedlings were taken out, the culture medium was removed, and washed in clean water, and then immersed in 1000 times the concentration of cypermethrin. After 5 minutes, they were taken out and the water was shaken off. They were spread flat to dry and planted in 50-hole trays after drying. The substrate ratio was peat: coconut bran 5:3. They were watered thoroughly and a small arch shed was covered to keep them moist. After the root balls were formed, the arch shed was removed. When they grew to 4 to 6 leaves, the second induction experiment could be carried out.

[0057] Pull out the seedlings and clean the roots. If the roots are too long, cut them short to keep 3 cm in length. Dissolve colchicine in 70% alcohol to make a 0.05% to 0.1% aqueous solution. Put the colchicine in a glass container and immerse the seedlings in the colchicine aqueous solution. Make sure the liquid level is 1 cm higher than the growth point of the plant. Cover the container with a black plastic bag to prevent the solution from being exposed to light. Record the date.

[0058] After soaking for 7 days, take out the seedlings for cleaning. After cleaning, soak the seedlings in a container filled with clean water. After 7 days, it was found that the new shoots at more than half of the growth points were swollen and obviously fatter. Take out the seedlings for cleaning. After cleaning, immerse them in clean water again for cultivation, and immerse all the roots in clean water.

[0059] After soaking in clean water for 2 days, take out the seedlings and plant them in a 10cm diameter pot. The pots are placed on a bracket and cultured in a greenhouse or shed. When new leaves grow out of the growth point of the seedlings, and the leaves are very wide, you can clearly identify that the doubling is successful. Wait until the seedlings are out of the pot and have formed a root ball before planting in the field.

[0060] The experimental results are as follows Figure 2-3 It shows that the new leaves of the plants that have successfully doubled are significantly wider than those of the control seedlings that have not been treated with colchicine.

[0061] The leaves and control plant leaves were taken for ploidy detection using a flow cytometer. The detection results show that the present invention doubles the excellent rare varieties, changes the flower size, and uses the tissue culture seedlings induced by explants to achieve variety doubling, with a success rate of 100%, high doubling efficiency, and low seedling loss, providing a strong technical guarantee for the improvement of the flower size of new and excellent daylily varieties.

[0062] Example 2

[0063] A method for rapid doubling of daylilies, the specific doubling breeding flow chart is as follows Figure 1 shown.

[0064] Hybridize between daylily varieties. The fruit matures one month after hybridization and is harvested when the fruit just cracks. After harvesting, it is stored in a refrigerator at 4°C for about one month for after-ripening. The seeds are taken out of the fruit and soaked at high temperature for germination. The germination process is maintained at 25°C and washed 1 to 2 times a day. When the dew is white, the seeds are sown in a 50-hole plug tray. The substrate ratio is peat: coconut bran 5:3. Water thoroughly to keep the substrate moist. When the seedlings emerge and grow to 4 to 6 leaves, the induction experiment can be carried out. The seed status at the time of white dew is as shown in the figure Figure 4 shown.

[0065] Pull out the seedlings and clean the roots. If the roots are too long, cut them short to keep 3 cm of root length. Dissolve colchicine in 70% alcohol to make a 0.05% to 0.1% aqueous solution. Put the colchicine aqueous solution in a glass container and immerse the seedlings in the solution. Make sure the liquid level is 1 cm higher than the growth point of the plant. Cover the container with a black plastic bag to prevent the solution from being exposed to light. Record the date.

[0066] After soaking for 7 days, take out the seedlings for cleaning. After cleaning, soak the seedlings in a container filled with clean water. After 7 days, it was found that the new shoots at more than half of the growth points were swollen and obviously fatter. Take out the seedlings for cleaning. After cleaning, immerse them in clean water again for cultivation, and immerse all the roots in clean water.

[0067] After soaking in clean water for 2 days, take out the seedlings and plant them in a 10cm diameter pot, which is placed on a bracket and cultured in a greenhouse or shed. When new leaves grow out of the growth point and the leaves are very wide, you can clearly identify the doubling success. Wait until the seedlings are out of the pot and have formed a root ball before planting in the field.

[0068] The comparison results of the offspring of the seed-sown seedlings of the edible daylily variety No. 3 as the mother before and after doubling are as follows Figure 5 As shown, the new leaves of the plants that successfully doubled were significantly wider than those of the control seedlings that were not treated with colchicine.

[0069] The leaves and the leaves of the control plants were tested for ploidy using a flow cytometer. The experimental results showed that the present invention can directly perform doubling induction on the hybrid seed seedlings of daylily to achieve variety doubling, with a success rate of 100%, high doubling efficiency and low seedling loss, providing a strong technical guarantee for daylily ploidy breeding.

[0070] This embodiment is to directly perform doubling induction on daylily hybrid seed seedlings, with a success rate of 100%, high doubling efficiency, and small seedling loss. The hybrid progeny of traditional breeding is directly doubled, and then variety selection is performed, which innovates the daylily breeding method.

[0071] Example 3

[0072] A method for rapid doubling of daylilies, the specific doubling breeding flow chart is as follows Figure 1shown.

[0073] Dig out the daylily seedlings (Hemerocallisfulva) in the field, remove the upper leaves and leave 5 cm, cut off the longer roots, leave 5 cm of roots, divide them into small clumps with 1 to 2 buds, and transplant them in a square tray with a high density. The substrate is made of peat and coconut coir in a ratio of 5:3. Water thoroughly to keep the substrate moist. When small buds grow on the sides and grow to 4 to 6 leaves, you can conduct an induction experiment.

[0074] Pull out the seedlings and clean the roots. If the roots are too long, cut them short to keep 3 cm in length. Dissolve colchicine in 70% alcohol to make a 0.05% to 0.1% aqueous solution. Put the colchicine in a glass container and immerse the seedlings in the solution. Make sure the liquid level is 1 cm above the growth point of the plant. Cover the container with a black plastic bag to prevent the solution from being exposed to light. Record the date.

[0075] After soaking for 7 days, take out the seedlings for cleaning. After cleaning, soak the seedlings in a container filled with clean water. After 7 days, it was found that the new shoots at more than half of the growth points were swollen and obviously fatter. Take out the seedlings for cleaning. After cleaning, immerse them in clean water again for cultivation, and immerse all the roots in clean water.

[0076] After soaking in clean water for 2 days, take out the seedlings and plant them in a 10cm diameter pot, which is placed on a bracket and cultured in a greenhouse or shed. When new leaves grow out of the growth point and the leaves are very wide, you can clearly identify the doubling success. Wait until the seedlings are out of the pot and have formed a root ball before planting in the field.

[0077] Comparison of the field seedlings of Hemerocallis fulva before and after doubling after transplanting, all the bases of the growing points have grown fat new buds, cracking the bases of the original leaves, and the leaves turn yellow due to the poison of the pesticide. Figure 6-7 As shown. The new leaves of the plants that have successfully doubled are significantly wider than those of the control seedlings that have not been treated with colchicine. The leaves and leaves of the control plants can also be taken for ploidy detection using flow cytometry, and the same results can be obtained.

[0078] This embodiment is for any daylily field seedlings, with a success rate of 100%, high doubling efficiency, and low seedling loss. It can achieve doubling of any field stock variety, improve flower size, and create more large-flowered varieties of existing daylily varieties.

[0079] Example 4 Verification Test

[0080] The present invention was used to double three other tissue culture daylily varieties, one hybrid combination offspring, and one field daylily variety, and polyploid plants were successfully obtained. The invention has an extremely high success rate and a very high induction efficiency.

[0081] Comparative Example 1

[0082] Using the induction method of the present invention, the daylily seed soaking method, the tissue culture seedling layered induction method in the culture medium, the field seedling growth point cotton ball drip method, the tissue culture plug seedling cotton ball drip method and the spraying method were repeatedly tested and compared, as shown in Table 1:

[0083] Table 1 Comparison of various methods of ploidy breeding in daylilies

[0084]

[0085] Through the above table and Figure 8 As shown, the cotton ball drip method at the field growth point, one plant of the same clump is doubled and one plant is not doubled for comparison, but the success rate is low, the seedlings are seriously damaged, and it is suitable for varieties with abundant seedlings. It can be seen that the doubling method of the present invention has better doubling effect, the highest success rate, the highest doubling efficiency, and simple operation compared with the other 5 doubling experiment methods, and is suitable for all daylily varieties.

[0086] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for doubling daylilies, characterized in that: The specific steps include: S1. Clean the roots of daylily seedlings and keep the root length at 2-6 cm; S2. Induce polyploidy in the cleaned seedlings with colchicine aqueous solution.

2. The method according to claim 1, characterized in that: The seedlings are tissue culture seedlings of new varieties or hybrid new varieties of daylilies, sowing seedlings or field seedlings of hybrid offspring of daylilies.

3. The method according to claim 1, characterized in that: The colchicine aqueous solution is a 0.05% to 0.1% aqueous solution prepared by mixing colchicine with 70% alcohol.

4. The method according to claim 2, characterized in that: The culture process of the tissue culture seedlings of the new variety or hybrid new variety of daylily is as follows: the new variety or hybrid new variety is collected, the upper leaves are cut off and the stem is retained with a length of 5 cm, and the seedlings are cultured in a primary culture medium for 2 to 3 months, the inflorescence of the shoots or the whole plant is taken as an explant, the explant is induced, and after the induction is successful, the explant is cultured for proliferation and rooting in the primary culture medium, and after hardening, the seedlings are planted in a plug tray for culture until they grow to 4 to 6 leaves for later use.

5. The method according to claim 4, characterized in that: The explant induction process is as follows: the explant is washed, the surface water is dried, disinfected and sterilized, the external browned part is cut off after being rinsed, and the pedicels are cut into 1 cm sections and inoculated into the primary culture medium for culture.

6. The method according to claim 4, characterized in that: The formula of the primary culture medium is MS culture medium, 1.0-2.0 mg / L of 6-benzylaminoadenine, 0.1-0.2 mg / L of naphthaleneacetic acid, 30 g / L of sucrose and 6 g / L of agar.

7. The method according to claim 4, characterized in that: The substances used in the proliferation culture process include MS culture medium, 1.0-3.0 mg / L of 6-benzylaminoadenine, 0.05-1.0 mg / L of naphthaleneacetic acid, 30 g / L of sucrose and 6 g / L of agar; the substances used in the rooting culture process include 1 / 2 MS culture medium, 0.1-0.5 mg / L of indoleacetic acid, 30 g / L of sucrose and 6 g / L of agar.

8. The method according to claim 4, characterized in that: The seedling hardening refers to placing the rooted seedlings in a greenhouse for 3 days, washing the rooted seedlings, soaking them in a 1000-fold concentration of the pesticide oxazolidinone, taking them out after 5 minutes to shake off the water, spreading them flat to dry, and planting them in a plug tray after drying until they grow 4 to 6 leaves.

9. The method according to claim 2, characterized in that: The cultivation process of the seedlings of the hybrid offspring of daylily is as follows: harvesting the daylily fruits when they are ripe and just cracked one month after hybridization, storing them in a refrigerator at 4°C for one month after harvesting and then ripening them, taking out the seeds from the fruits and soaking them at high temperature and then germinating them, maintaining the germination process at 25°C, washing them 1 to 2 times a day, sowing them in plug trays when they are white, and culturing them until the seedlings grow to 4 to 6 leaves for use.

10. The method according to claim 2, characterized in that: The cultivation process of the field seedlings is as follows: dig out daylily seedlings in the field, remove the upper leaves, retain 5 cm of the stem, retain 5 cm of the root system, divide them into small clusters with 1 to 2 buds, and transplant them in a square tray. The substrate is mixed with peat and coconut bran in a ratio of 5:

3. Cultivate until small buds grow on the side of the seedlings and grow to 4 to 6 leaves for later use.

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