An induction culture medium and induction method using marigold flowers as explants

Abstract

This invention provides an induction culture medium and method for using marigold anthers as explants. The induction culture medium is based on MS solid medium containing agar, with specific amounts of sucrose, tryptophan, phenylalanine, arginine, casein amino acids, anhydrous asparagine, 6-benzylpurine, naphthaleneacetic acid, silver nitrate, pyridoxine hydrochloride, thiamine hydrochloride, nicotinic acid, D-biotin, folic acid, ascorbic acid, and coconut juice added. This invention also provides a method for induction culture using this induction culture medium. This invention can greatly improve the callus induction rate of marigolds, significantly increasing the number of regenerated seedlings, and provides technical guidance for the cultivation of haploid marigolds using anthers.

Description

Technical Field

[0001] This invention relates to the field of tissue culture technology, and in particular to an induction culture medium and induction method using marigold flowers as explants. Background Technology

[0002] Marigold (Tageteserecta L.) is an annual herbaceous plant belonging to the genus *Tageteserecta* in the family Asteraceae. Marigolds have large, brightly colored flowers and are widely used in landscaping. Furthermore, marigold flowers contain higher levels of lutein than many other plants. Lutein extracted from marigold flowers plays an important role in regulating animal nutrition metabolism, growth performance, meat quality, and preventing animal diseases, and has been widely used for coloring poultry skin, shanks, and egg yolks.

[0003] Currently, marigold breeding mainly adopts conventional field breeding methods, requiring 4-5 generations to select pure lines. Marigold anther culture technology utilizes anthers that have developed to a certain stage, culturing them in vitro on an artificial culture medium. Under specific induction conditions, these anthers undergo multiple divisions to form embryoids or callus tissue, which then differentiate into plants. A series of homozygous germplasm can be obtained in just 80 days, from which haploid germplasm can be screened, providing methodological guidance for obtaining double haploids.

[0004] Previous studies on marigold tissue culture have mostly used stem segments and leaves as explants for marigold plant regeneration. Tang Daocheng of Qinghai University published "The Effects of Growth Regulators on Marigold Anther Culture," which studied the effects of different growth regulators on callus induction and organ differentiation during marigold anther culture, but there are no reports of successfully obtaining regenerated seedlings. The India Agricultural Research Institute published "Optimizing Protocol for Successful Development of Haploids in Marigold (Tagetes spp.) Through In Vitro Androgenesis," which reported a technique for obtaining haploids from marigold anther culture using marigold anthers as explants, but this method has not yet been used to successfully obtain regenerated marigold seedlings. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides an induction culture medium and induction method using marigold flowers as explants.

[0006] In a first aspect, the present invention provides an induction culture medium using marigold flowers as explants. The induction culture medium is based on MS solid medium containing agar, supplemented with 10-15 g / L sucrose, 15-20 mg / L tryptophan, 10-15 mg / L phenylalanine, 17-22 mg / L arginine, 150-200 mg / L casein amino acids, 18-23 mg / L anhydrous asparagine, 2-2.5 mg / L 6-benzylpurine (6-BA), 0.25-1 mg / L naphthaleneacetic acid (NAA), 1-3 mg / L silver nitrate, 1-3 mg / L pyridoxine hydrochloride, 1-2 mg / L thiamine hydrochloride, 1-2 mg / L niacin, 0.01-0.05 mg / L D-biotin, 0.01-0.03 mg / L folic acid, 0.1-0.5 mg / L ascorbic acid, and 50-100 ml / L coconut juice.

[0007] The MS solid culture medium consists of: potassium nitrate 1900 mg / L, ammonium nitrate 1650 mg / L, potassium dihydrogen phosphate 170 mg / L, magnesium sulfate 370 mg / L, calcium chloride 440 mg / L, potassium iodide 0.83 mg / L, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L, disodium EDTA 37.3 mg / L, ferrous sulfate 27.8 mg / L, inositol 100 mg / L, glycine 2 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxine hydrochloride 0.5 mg / L, nicotinic acid 0.5 mg / L, sucrose 30 g / L, and agar 7 g / L.

[0008] Furthermore, the pH of the induction medium is 5.6-6.0.

[0009] In some embodiments of the present invention, the coconut juice is the original juice of a mature coconut.

[0010] Secondly, the present invention provides a method for inducing marigold flower styrax.

[0011] The induction method provided by the present invention includes: after obtaining marigold anthers, placing the anthers in the induction culture medium described in the present invention, first culturing them in the dark for 20-30 days under the conditions of temperature 23-25℃ and humidity 80%-90%, and then transferring the anthers to the differentiation culture medium for culturing with 16 hours of light and 8 hours of darkness per day until regenerated seedlings are generated.

[0012] The method for inducing marigold anther culture of the present invention first adopts 20-30 days of dark culture, which helps to induce the development of androgen and promotes the induction of haploid anthers.

[0013] In some embodiments of the present invention, the differentiation culture medium is composed of: MS solid medium containing agar as the base medium, with 1-2 g / L of silver nitrate added; and its pH is 5.6-6.0.

[0014] In some embodiments of the present invention, obtaining marigold anthers includes collecting, pre-treating and disinfecting flower buds, as well as inoculating the anthers.

[0015] In some embodiments of the present invention, the collection and pretreatment of the flower buds includes: selecting healthy, disease-free marigold buds at the top, collecting buds with a length of 12-21 mm between 9:00 and 10:00 in the morning, placing them in a beaker with two layers of moistened filter paper at the bottom, and pretreating them in a refrigerator at 4°C for 7-10 days.

[0016] In some embodiments of the present invention, the inoculation of the anthers includes: peeling the tubular florets from the center of the flower buds, selecting anthers with anther cells in the late mononuclear to early binucleate stage, and placing them in an induction culture medium.

[0017] The marigolds to which the induction method of the present invention is applicable should have tubular flowers.

[0018] This invention controls key conditions (induction culture medium composition, number of days of bud pretreatment, number of days of dark culture, etc.) to achieve an embryogenic callus induction rate of over 68% and a regeneration plant rate of not less than 13%.

[0019] In some embodiments of the present invention, the marigold flower induction method includes the following steps:

[0020] (1) Collection and pretreatment of flower buds: Select healthy and disease-free marigold flower buds grown in artificial climate chambers. Collect flower buds with a length of 12-21 mm at 9:00-10:00 am and place them in beakers with two layers of moistened filter paper at the bottom. Pretreat them in a refrigerator at 4℃ for 7-10 days.

[0021] (2) Disinfection of flower buds: Disinfect them in a 75% ethanol solution for 1 minute, then rinse them 3 times with sterile water to completely remove any residual ethanol.

[0022] (3) Inoculation of anthers: Place the flower buds on sterile filter paper, peel off the tubular florets in the middle of the flower buds. At this time, the florets contain 5 anthers. Measure the length of the florets with vernier calipers, select anthers with anther cells in the late mononuclear to early binucleate stage, and place them in induction culture medium.

[0023] (4) Anther induction culture: The culture medium containing anthers is placed in an artificial climate chamber for dark culture for 20-30 days. The temperature of the artificial climate chamber is 23-25℃ and the humidity is 80%-90%. After 20-30 days, all anthers are transferred to differentiation culture medium and placed in an artificial climate chamber for 16 hours of light and 8 hours of darkness per day until regenerated seedlings are generated.

[0024] This invention provides an induction culture medium and method using marigold anthers as explants. By selecting a specific marigold anther induction culture medium and adding amino acids and vitamins, the regeneration rate of anther-induced seedlings can be effectively improved. Furthermore, the marigold anther induction culture method of this invention, through a preliminary 20-30 day dark culture, helps induce androgen development, promoting haploid induction in the anthers. In summary, this invention can significantly improve the callus induction rate of marigolds, greatly increasing the number of regenerated seedlings, and provides technical guidance for the cultivation of marigold haploids using anthers. Attached Figure Description

[0025] Figure 1 Images of anthers (left) and embryogenic callus (right). Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0027] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0028] In the following examples, the embryogenic callus induction rate = (number of induced embryogenic callus / number of anthers) × 100%;

[0029] Regenerated plant induction rate = (number of regenerated seedlings / number of embryogenic callus) * 100%.

[0030] In the following examples, the MS solid culture medium containing agar was composed of: potassium nitrate 1900 mg / L, ammonium nitrate 1650 mg / L, potassium dihydrogen phosphate 170 mg / L, magnesium sulfate 370 mg / L, calcium chloride 440 mg / L, potassium iodide 0.83 mg / L, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L, disodium EDTA 37.3 mg / L, ferrous sulfate 27.8 mg / L, inositol 100 mg / L, glycine 2 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxine hydrochloride 0.5 mg / L, nicotinic acid 0.5 mg / L, sucrose 30 g / L, and agar 7 g / L.

[0031] Example 1

[0032] This embodiment provides an induction culture medium using marigold flowers as explants. The formulation is as follows: MS solid medium containing agar is used as the basic medium, and the following are added: sucrose 13g / L, tryptophan 17mg / L, phenylalanine 12mg / L, arginine 20mg / L, casein amino acids 175mg / L, anhydrous asparagine 20mg / L, 6-benzylpurine 2.25mg / L, naphthaleneacetic acid 0.5mg / L, silver nitrate 2mg / L, pyridoxine hydrochloride 2mg / L, thiamine hydrochloride 1.5mg / L, niacin 1.5mg / L, D-biotin 0.03mg / L, folic acid 0.02mg / L, ascorbic acid 0.25mg / L, and coconut juice 75ml / L, and the pH is adjusted to 5.8.

[0033] This embodiment also provides a method for inducing medicinal properties in marigold flowers, including the following steps:

[0034] (1) Collection and pretreatment of flower buds: Using commercially available marigold F1 as material, seedlings were sown and raised in an artificial climate chamber at a temperature of 23-25℃ and a humidity of 80%-90%. Healthy marigold flower buds were selected, and flower buds with a length of 12-21mm were collected between 9:00 and 10:00 am. At this time, the number of anthers in the flower buds was large, and the number of microspores in the late uninucleate to early binucleate stage was also large. Then, the buds were placed in beakers with two layers of moistened filter paper at the bottom and placed in a refrigerator at 4℃ for 10 days.

[0035] (2) Disinfection of flower buds: After pretreatment, the flower buds are first disinfected with 75% ethanol solution for 1 minute, and then soaked in sterile water 3 times for 2 minutes each time to completely remove residual ethanol.

[0036] (3) Anther inoculation: The tubular florets in the center of the F1 marigold buds are removed. Each floret contains 5 anthers. The length of the floret is measured with calipers, and florets with a length of 1.8-2.3 mm are selected. Using two pointed medical forceps, the floret is longitudinally cut open, and 5 anthers are removed, taking care not to damage them. The anthers are placed in the aforementioned induction medium, with 15 anthers per medium. The culture dish size is 10cm × 10cm.

[0037] (4) Anther induction culture: The induction medium containing anthers was placed in an artificial climate chamber and cultured in the dark for 20 days. The temperature of the artificial climate chamber was 23-25℃ and the humidity was 80%-90%. After 20 days, the yellow, granular embryogenic callus tissue was transferred to the differentiation medium and placed in an artificial climate chamber for 16 hours of light and 8 hours of darkness per day until regenerated seedlings were generated. Figure 1 Images of anthers (left) and embryogenic callus (right).

[0038] The differentiation medium was MS solid medium containing agar as the base medium, with 2 g / L silver nitrate solution added and the pH adjusted to 5.8.

[0039] Example 2

[0040] This embodiment provides an induction culture medium using marigold flowers as explants. The formulation is as follows: MS solid medium containing agar is used as the basic medium, and the following are added: sucrose 10 g / L, tryptophan 15 mg / L, phenylalanine 10 mg / L, arginine 17 mg / L, casein amino acids 150 mg / L, anhydrous asparagine 18 mg / L, 6-benzylpurine 2 mg / L, naphthaleneacetic acid 0.25 mg / L, silver nitrate 1 mg / L, pyridoxine hydrochloride 1 mg / L, thiamine hydrochloride 1 mg / L, niacin 1 mg / L, D-biotin 0.01 mg / L, folic acid 0.01 mg / L, ascorbic acid 0.1 mg / L, and coconut juice 50 ml / L, and the pH is adjusted to 5.8.

[0041] This embodiment also refers to the method of Example 1, and provides a method for inducing marigold anthers using the above-mentioned induction culture medium.

[0042] Example 3

[0043] This embodiment provides an induction culture medium using marigold flowers as explants. The formulation is as follows: MS solid medium containing agar is used as the basic medium, and the following are added: sucrose 15 g / L, tryptophan 20 mg / L, phenylalanine 15 mg / L, arginine 22 mg / L, casein amino acids 200 mg / L, anhydrous asparagine 23 mg / L, 6-benzylpurine 2.5 mg / L, naphthaleneacetic acid 1 mg / L, silver nitrate 3 mg / L, pyridoxine hydrochloride 3 mg / L, thiamine hydrochloride 2 mg / L, niacin 2 mg / L, D-biotin 0.05 mg / L, folic acid 0.03 mg / L, ascorbic acid 0.5 mg / L, and coconut juice 100 ml / L, and the pH is adjusted to 5.8.

[0044] This embodiment also refers to the method of Example 1, and provides a method for inducing marigold anthers using the above-mentioned induction culture medium.

[0045] The number of anthers, embryogenic callus, and regenerated plants in each embodiment were counted. The embryogenic callus induction rate and regeneration rate were obtained according to the calculation formula. The results are shown in Table 1.

[0046] Table 1. Embryogenic callus induction rate and regenerated plant rate in Examples 1-3

[0047] Number of anthers Embryogenic callus induction rate Regeneration rate Example 1 270 81％ 18％ Example 2 300 73％ 15％ Example 3 285 70％ 14％

[0048] Comparative Example 1

[0049] Compared to Example 1, the concentration of ascorbic acid in the induction medium of this comparative example was changed. Specifically, the ascorbic acid concentrations were 0 mg / L, 0.25 mg / L, 0.5 mg / L, 0.6 mg / L, and 1 mg / L, respectively. The other components of the induction medium were the same as those in Example 1. The induction rate of embryogenic callus and the regeneration rate were compared, and the results are shown in Table 2.

[0050] Table 2. Embryogenic callus induction rate and regeneration rate at different ascorbic acid concentrations.

[0051] Ascorbic acid concentration (mg / L) Number of anthers Embryogenic callus induction rate Regeneration rate 0 150 10％ 1％ 0.25 (Example 1) 270 81％ 18％ 0.5 225 68％ 13％ 0.6 195 30％ 4％ 1 195 0％ 0％

[0052] Comparative Example 2

[0053] Compared to Example 1, the concentration of casein amino acids in the induction medium of this comparative example was changed. Specifically, the casein amino acid concentrations were 0 mg / L, 125 mg / L, 175 mg / L, 200 mg / L, 250 mg / L, and 350 mg / L, respectively. The other components of the induction medium were the same as those in Example 1. The induction rate of embryogenic callus and the regeneration rate were compared, and the results are shown in Table 3.

[0054] Table 3. Embryogenic callus induction rate and regeneration rate at different casein amino acid concentrations.

[0055] Casein amino acid concentration (mg / L) Number of anthers Embryogenic callus induction rate Regeneration rate 0 180 11％ 3％ 125 200 35％ 5％ 175 (Example 1) 270 81％ 18％ 200 180 71％ 15％ 250 210 40％ 7％ 350 270 15％ 1％

[0056] Comparative Example 3

[0057] Compared to Example 1, this comparative example changed the number of days of flower bud pretreatment. Specifically, the pretreatment days were 0 days, 7 days, 10 days, 15 days, and 30 days. The embryogenic callus induction rate and regeneration plant rate were compared, and the results are shown in Table 4.

[0058] Table 4. Embryogenic callus induction rate and regenerated plant rate of flower buds after different pretreatment days.

[0059] Flower bud pretreatment days (d) Number of anthers Embryogenic callus induction rate Regeneration rate 0 180 4％ 0％ 7 225 60％ 13％ 10 (Example 1) 270 81％ 18％ 15 210 42％ 8％ 30 270 5％ 0％

[0060] Comparative Example 4

[0061] Compared to Example 1, this comparative example changed the number of days of dark culture of anthers. Specifically, the number of days of dark culture were 0 days, 10 days, 20 days, 25 days, 30 days, and 40 days. The embryogenic callus induction rate and regeneration plant rate were compared, and the results are shown in Table 5.

[0062] Table 5. Embryogenic callus induction rate and regeneration rate of anthers under different dark culture days.

[0063] Days of dark culture of anthers (d) Number of anthers Embryogenic callus induction rate Regeneration rate 0 285 4％ 0％ 10 300 68％ 6％ 20 (Example 1) 270 81％ 18％ 25 315 76％ 15％ 30 245 72％ 13％ 40 345 15％ 1％

[0064] Comparative Example 5

[0065] Compared to Example 1, this comparative example used MS medium containing agar, with only KT and NAA added; specifically, the KT concentration was 2 mg / L and the NAA concentration was 0.3 mg / L. The results showed that the embryogenic callus induction rate in this comparative example was 30%, and the regeneration rate was 3%.

[0066] Comparative Example 6

[0067] Compared to Example 1, this comparative example was prepared by mixing gellan gum 2.5 g / L, sucrose 45 g / L, and MS solid medium without agar, and then adding tryptophan 25 mg / L, phenylalanine 17 mg / L, arginine 21 mg / L, anhydrous asparagine 22 mg / L, 6-benzylpurine 2 mg / L, naphthaleneacetic acid 0.5 mg / L, silver nitrate 2 mg / L, pyridoxine hydrochloride 2 mg / L, thiamine hydrochloride 1.5 mg / L, niacin 1.5 mg / L, D-biotin 0.02 mg / L, folic acid 0.02 mg / L, ascorbic acid 0.2 mg / L, and coconut juice 75 ml / L, and adjusting the pH to 5.8.

[0068] The results showed that the embryogenic callus induction rate in this comparative study was 10%, and the regeneration rate of plants was 1%.

[0069] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0070] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "specific implementation," or "some specific implementations," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An induction culture medium using marigold anthers as explants, characterized in that, The induction medium is based on MS solid medium containing agar, supplemented with sucrose 10-15 g / L, tryptophan 15-20 mg / L, phenylalanine 10-15 mg / L, arginine 17-22 mg / L, casein amino acids 150-200 mg / L, anhydrous asparagine 18-23 mg / L, 6-benzylpurine 2-2.5 mg / L, naphthaleneacetic acid 0.25-1 mg / L, silver nitrate 1-3 mg / L, pyridoxine hydrochloride 1-3 mg / L, thiamine hydrochloride 1-2 mg / L, niacin 1-2 mg / L, D-biotin 0.01-0.05 mg / L, folic acid 0.01-0.03 mg / L, ascorbic acid 0.1-0.5 mg / L, and coconut juice 50-100 ml / L. The MS solid culture medium consists of: potassium nitrate 1900 mg / L, ammonium nitrate 1650 mg / L, potassium dihydrogen phosphate 170 mg / L, magnesium sulfate 370 mg / L, calcium chloride 440 mg / L, potassium iodide 0.83 mg / L, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L, disodium EDTA 37.3 mg / L, ferrous sulfate 27.8 mg / L, inositol 100 mg / L, glycine 2 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxine hydrochloride 0.5 mg / L, nicotinic acid 0.5 mg / L, sucrose 30 g / L, and agar 7 g / L.

2. The induction culture medium using marigold anthers as explants according to claim 1, characterized in that, The pH of the induction medium is 5.6-6.

0.

3. The induction culture medium using marigold anthers as explants according to claim 1, characterized in that, The coconut juice is the original juice of a mature coconut.

4. A method for inducing medicinal properties in marigold flowers, characterized in that, include: After obtaining the marigold anthers, place the anthers in the induction culture medium described in any one of claims 1-3, and first culture them in the dark for 20-30 days at a temperature of 23-25℃ and a humidity of 80%-90%. Then, transfer the anthers to the differentiation culture medium and culture them in the dark for 16 hours of light and 8 hours of light per day until regenerated seedlings are generated. The process of obtaining marigold anthers includes the collection, pretreatment and disinfection of flower buds, as well as the inoculation of anthers. The collection and pretreatment of the flower buds include: selecting healthy and disease-free marigold flower buds from the top, collecting flower buds with a length of 12-21 mm between 9:00 and 10:00 in the morning, placing them in a beaker with two layers of moistened filter paper at the bottom, and pretreating them in a refrigerator at 4°C for 7-10 days. The differentiation medium consists of MS solid medium containing agar as the base medium, with 1-2 g / L of silver nitrate added; its pH is 5.6-6.

0.

5. The method for inducing marigold anthers according to claim 4, characterized in that, The inoculation of the anthers includes: peeling off the tubular florets from the center of the flower buds, selecting anthers with anther cells in the late mononuclear to early binucleate stage, and placing them in an induction culture medium.

6. The method for inducing marigold anthers according to claim 4 or 5, characterized in that, The induction method is applicable to marigolds containing tubular florets.

7. The method for inducing marigold anthers according to claim 6, characterized in that, The induction method achieves an embryogenic callus induction rate of over 68% and a regeneration rate of no less than 13%.

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