A method for tissue culture of artemisia

Abstract

This invention relates to a method for tissue culture of Artemisia species, comprising the following steps: 1-1) culturing explants of the Artemisia species in callus differentiation medium to obtain clustered shoots; 2-1) culturing the clustered shoots in clustered shoot proliferation medium to obtain unrooted seedlings; or 1-2) culturing explants of the Artemisia species in callus differentiation medium to obtain unrooted seedlings; 3) culturing the unrooted seedlings obtained in step 2-1) or step 1-2) in rooting medium to obtain rooted seedlings; 4) transplanting the rooted seedlings and culturing them to obtain complete Artemisia species plants.

Description

[0001] This invention claims priority to patent application No. CN 2024100298762, filed on January 9, 2024, entitled "A method for tissue culture of Artemisia". Technical Field

[0002] This invention relates to the field of plant tissue culture, and particularly to a method for plant tissue culture. Background Technology

[0003] Artemisia annua L., also known as bitter wormwood or yellow wormwood, is an annual herb belonging to the genus Artemisia in the family Asteraceae. It is a traditional Chinese medicine with a recorded history of over two thousand years, first appearing in the "Fifty-Two Prescriptions" from the Spring and Autumn and Warring States periods. Artemisinin, the main active ingredient in Artemisia annua, is the first-line drug for treating malaria and has been recognized by the World Health Organization as the only safe and effective new drug for treating malaria globally. Artemisia annua is widely distributed in Europe and Asia, with the largest distribution in central, eastern, and southern Europe and northern, central, and eastern Asia. There are approximately 350 species of Artemisia worldwide, with 186 species and 44 varieties in China, but only Artemisia annua contains artemisinin. In most parts of the world, the artemisinin content in Artemisia annua is less than 0.2%. In my country, the artemisinin content gradually increases from north to south. In areas north of the Qinling-Huaihe line, the artemisinin content is less than 0.6%, while in areas south of the Qinling-Huaihe line, the content is higher. Since the world's high-quality Artemisia annua resources are mainly concentrated in the areas south of the Qinling-Huaihe line in China, such as Hainan, Guizhou, Guangxi, and Hunan provinces, my country has a significant advantage in terms of resources and has become the main supplier of artemisinin raw materials.

[0004] Tissue culture technology can produce a large number of Artemisia annua seedlings in a short time. However, in the traditional Artemisia annua tissue culture process, the process of explants forming callus and then differentiating into adventitious buds is relatively long and has a long cycle. Summary of the Invention

[0005] One aspect of the present invention provides a method for tissue culture of Artemisia species, comprising the following steps:

[0006] 1-1) The explants of the Artemisia species were cultured in callus differentiation medium to obtain clustered shoots;

[0007] 2-1) The clustered buds were cultured in a clustered bud proliferation medium to obtain seedlings that had not yet developed roots;

[0008] or

[0009] 1-2) The explants of the Artemisia species were cultured in callus differentiation medium to obtain seedlings that had not yet developed roots;

[0010] 3) The unrooted seedlings described in step 2-1) or step 1-2) are cultured in a rooting medium to obtain rooted seedlings;

[0011] 4) Transplant the rooted seedlings and cultivate them to obtain complete Artemisia plants.

[0012] In one specific embodiment, the callus differentiation culture medium comprises MS liquid culture medium, a first coagulant, a first sugar, and a first hormone, wherein the pH value of the callus differentiation culture medium is 5.8 to 6.2; based on the volume of the MS liquid culture medium as 1000 mL, the amount of the first coagulant is 5 to 10 g / L, the amount of the first sugar is 25 to 35 g / L, and the amount of the first hormone is 2.2 to 2.8 mg / L.

[0013] In one specific embodiment, the first coagulant is agar, the first sugar is sucrose, and the first hormone is 6-benzylaminopurine (6-BA) and α-naphthaleneacetic acid (NAA).

[0014] In one specific embodiment, the mass ratio of 6-benzylaminopurine to α-naphthaleneacetic acid is 1:(0.08 to 0.15).

[0015] In one specific embodiment, the bud proliferation medium comprises MS liquid medium, a second solidifying agent, a second sugar, and a second hormone, wherein the pH value of the bud proliferation medium is 5.8 to 6.2; the volume of the MS liquid medium is 1000 mL, the amount of the second solidifying agent is 5 to 10 g / L, the amount of the second sugar is 25 to 35 g / L, and the amount of the second hormone is 0.55 to 0.85 mg / L.

[0016] In one specific embodiment, the second coagulant is agar, the second sugar is sucrose, and the second hormone is 6-benzylaminopurine (6-BA) and indole-3-butyric acid (IBA).

[0017] In one specific embodiment, the mass ratio of 6-benzylaminopurine to indole-3-butyric acid is (5 to 15):1.

[0018] In one specific embodiment, the rooting medium comprises MS liquid medium, a third solidifying agent, a third sugar, and a third hormone, wherein the pH of the rooting medium is 5.8 to 6.2; the volume of the MS liquid medium is 1000 mL, the amount of the third solidifying agent is 5 to 10 g / L, the amount of the third sugar is 25 to 35 g / L, and the amount of the third hormone is 0.11 to 0.22 mg / L.

[0019] In one specific embodiment, the third coagulant is agar, the third sugar is sucrose, and the third hormone is indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA).

[0020] In one specific embodiment, the mass ratio of indole-3-butyric acid to α-naphthaleneacetic acid is (5 to 20):1.

[0021] In one specific implementation, in steps 1-1), 2-1), 1-2), and 3), the cultivation conditions are independently 22 to 27°C, light intensity 1000 to 1400 Lx, and photoperiod L:D = 12:12 to 14:10.

[0022] In one specific implementation, in steps 1-1) and 1-2), the cultivation conditions are independently 23 to 27°C, light intensity 1200 Lx, and photoperiod L:D = 12:12.

[0023] In one specific implementation, in step 2-1), the cultivation conditions are 23 to 27°C, light intensity 1200 Lx, and photoperiod L:D = 14:10.

[0024] In one specific implementation, in step 3), the cultivation conditions are 22 to 26°C, light intensity 1200 Lx, and photoperiod L:D = 14:10.

[0025] In one specific embodiment, before step 1-1) or 1-2), the following step is also included:

[0026] P1) Obtain explants of the Artemisia species;

[0027] P2) Disinfect the explant to obtain disinfected explants; and optionally...

[0028] P3) Pre-culture the disinfected explants.

[0029] In one specific embodiment, in step P1), the explant is a leaf; preferably a young leaf.

[0030] In one specific embodiment, in step P3), the pre-culture medium comprises MS liquid medium, a fourth coagulant, and a fourth sugar, with a pH of 5.8 to 6.2.

[0031] In one specific embodiment, the volume of the MS liquid culture medium is 1000 mL, the amount of the fourth solidifying agent is 5 to 10 g / L, and the amount of the fourth sugar is 25 to 35 g / L.

[0032] In one specific embodiment, the fourth coagulant is agar, and the fourth sugar is sucrose.

[0033] In one specific implementation, the cells are pre-cultured in the dark for two days.

[0034] In one specific embodiment, the Artemisia species is Artemisia annua.

[0035] In one specific embodiment, the Artemisia annua is the "Haiqing No. 1" Artemisia annua.

[0036] In one specific embodiment, in step 4), the rooted seedlings are transplanted into soil or a cultivation substrate for further cultivation.

[0037] The beneficial effects of this invention are:

[0038] The tissue culture method for Artemisia annua provided by this invention can significantly shorten the period of explant formation of clustered shoots by using a modified culture medium, thereby shortening the entire tissue culture time, quickly and effectively preserving Artemisia annua plants with good traits, and enabling rapid propagation of Artemisia annua year-round.

[0039] Furthermore, this invention reduces the proliferation culture steps in the traditional two-step method (callus differentiation culture and shoot proliferation culture) for cultivating unrooted seedlings to a one-step method, which further significantly shortens the culture cycle.

[0040] The tissue culture method of the present invention can solve the technical problem that it is difficult to obtain a large number of superior seedlings in a short period of time when the demand for superior seedlings or propagation materials is small, especially when the number of selected individual plants is small, thus accelerating the artificial breeding process. Attached Figure Description

[0041] Figure 1 This is an image of callus tissue with clustered buds formed from the leaves of Artemisia annua explants in Example 1.

[0042] Figure 2 This is an image of callus differentiation forming clustered buds from the leaves of Artemisia annua explants in Example 1.

[0043] Figure 3 This is an image of the clustered buds formed through proliferation culture in Example 1.

[0044] Figure 4 This is a picture of Artemisia annua plantlets grown through rooting culture in Example 1.

[0045] Figure 5 This is a picture of a fully grown Artemisia annua plant after hardening and transplanting, as shown in Example 1. Detailed Implementation

[0046] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0047] Unless otherwise specified, all reagents used in the embodiments of this invention are commercially available.

[0048] Example 1

[0049] The tissue culture method for Artemisia annua provided in this embodiment includes the following steps:

[0050] (1) Select robust and well-formed Artemisia annua plants with good growth and cut the 2nd to 6th tender leaves from the top down as explants to obtain detached leaves. Soak the detached leaves in a 5wt% detergent solution for 10 minutes, then rinse with running water for 30 minutes and air dry naturally to obtain detached leaves cleaned with running water.

[0051] (2) Rinse the detached leaves with running water three times in sterile water, then soak them in 75% ethanol aqueous solution for 30 seconds for disinfection, and then rinse them three times with sterile water. Then soak them in 0.1wt% mercuric chloride aqueous solution for 5.5 minutes for disinfection, and then rinse them four times with sterile water. Then, under sterile conditions, use sterile filter paper to absorb excess water to obtain sterilized detached leaves. Cut open the edges of the detached leaves and inoculate them on solid culture medium (MS + 30g / L sucrose + 7g / L agar) and pre-culture them in the dark at 25±2℃ for 2 days (not included in the culture period). Observe whether they are contaminated and obtain uncontaminated explants.

[0052] (3) Uninfected explants were inoculated onto callus differentiation medium (1000 mL MS + 7.0 g / L agar + 30 g / L sucrose + hormone, pH 6.0, hormone types and dosages are shown in Table 1); several uninfected explants were inoculated into each culture dish, and then placed in the tissue culture room. The culture conditions were as follows: temperature: 25 ± 2℃, light intensity: 1200 Lx, photoperiod: 12 h light, 12 h dark. Day 0 was the day of inoculation. On day N1, clustered buds appeared in the callus tissue, as shown in Table 1. Figure 1 Day 0 is defined as the day when clustered buds appear. During the N2 day of cultivation, clustered buds with a height of 0.5 to 1 cm appear. Figure 2 .

[0053] (4) Cut the bud from the base into cubes with 1 to 2 clustered buds and transfer them to a clustered bud proliferation medium (1000 mL MS + 7.0 g / L agar + 30 g / L sucrose + hormone, pH 6.0, hormone types and dosages are shown in Table 3) for rapid propagation culture of the bud. Several clustered buds were inoculated into each culture dish. The culture conditions were as follows: temperature: 25 ± 2℃, light intensity: 1200 Lx, photoperiod: 14 h light, 10 h dark. Day 0 was the day of transfer to the clustered bud proliferation medium. After 3 days of culture, proliferated, unrooted seedlings appeared. See [Table 3]. Figure 3 .

[0054] (5) Cut individual seedlings from the base of the proliferated, unrooted seedlings and transfer them to test tubes containing adventitious rooting medium (1000 mL MS + 7.0 g / L agar + 30 g / L sucrose + hormone, pH 6.0, hormone types and dosages are shown in Table 4) for cultivation. The cultivation conditions are as follows: temperature: 25 ± 2℃, light intensity: 1200 Lx, photoperiod: 14 h light, 10 h dark. Day 0 is the day of transfer to the adventitious rooting medium. Cultivate for N4 days to obtain rooted seedlings. See Table 4. Figure 4 .

[0055] The total number of days from callus differentiation to the formation of rooted seedlings, as calculated by steps (3) to (5) above, is N1+N2+N3+N4 days.

[0056] (6) Open the cap of the test tube seedling bottle, adapt to the tissue culture room for 1 day, take out the rooted seedlings, wash off the residual culture medium attached to the roots of the rooted seedlings, plant them in a seedbed containing vermiculite: soil = 1:1, water thoroughly, spray with MS liquid culture medium diluted 10 times with water, harden the seedlings indoors, and cultivate them under the following conditions: temperature: 25±2℃, light intensity 1200Lx, photoperiod: 14h light, 10h darkness, cultivate for 20 days to obtain transplanted seedlings.

[0057] (7) Spray an appropriate amount of water on the leaves of the transplanted seedlings every day. Ventilate for 30 minutes every day for the first 3 days, then increase to all day. After 7 days (see...). Figure 5 Simply move the seedbed outdoors and carry out normal seedling cultivation and management to obtain complete Artemisia annua plants.

[0058] In the above steps, each petri dish or each test tube seed bottle is a replicate, for a total of 5 replicates.

[0059] Examples 2 to 4

[0060] The difference between Examples 2 to 4 and Example 1 lies in the hormone content in the callus differentiation culture medium, as shown in Table 1.

[0061] Everything else is the same as in Example 1.

[0062] Example 5

[0063] The tissue culture method for Artemisia annua provided in this embodiment includes the following steps:

[0064] (1) Select robust and well-formed Artemisia annua plants with good growth and cut the 2nd to 6th tender leaves from the top down as explants to obtain detached leaves. Soak the detached leaves in a 5wt% detergent solution for 10 minutes, then rinse with running water for 30 minutes and air dry naturally to obtain detached leaves cleaned with running water.

[0065] (2) Rinse the detached leaves with running water three times in sterile water, then soak them in 75% ethanol aqueous solution for 30 seconds for disinfection, and then rinse them three times with sterile water. Then soak them in 0.1wt% mercuric chloride aqueous solution for 5.5 minutes for disinfection, and then rinse them four times with sterile water. Then, under sterile conditions, use sterile filter paper to absorb excess water to obtain sterilized detached leaves. Cut open the edges of the detached leaves and inoculate them on solid culture medium (MS + 30g / L sucrose + 7g / L agar) and pre-culture them in the dark at 25±2℃ for 2 days (not included in the culture period). Observe whether they are contaminated and obtain uncontaminated explants.

[0066] (3) Uninfected explants were inoculated onto callus differentiation medium (MS + 7.0 g / L agar + 30 g / L sucrose + hormone, pH 6.0, hormone types and dosages are shown in Table 1); several uninfected explants were inoculated into each culture dish and then placed in the tissue culture room under the following conditions: temperature: 25±2℃, light intensity: 1200 Lx, photoperiod: 12 h light, 12 h dark. Day 0 was the day of inoculation. By day N1, clustered buds appeared on the callus. Day 0 was the day the clustered buds appeared. By day N2, clustered buds with a height of 0.5 to 1 cm appeared. Day 0 was the day the clustered buds appeared. By day N3, seedlings without roots appeared.

[0067] (4) Cut off individual seedlings from the base of the unrooted seedlings and transfer them to adventitious rooting medium (MS + 7.0 g / L agar + 30 g / L sucrose + hormone, pH 6.0, hormone types and dosages are shown in Table 4) in test tube seedling bottles for cultivation. The cultivation conditions are as follows: temperature: 25±2℃, light intensity 1200 Lx, photoperiod: 14 h light, 10 h dark. The day of transfer to adventitious rooting medium is day 0. Cultivate for N4 days to obtain rooted seedlings.

[0068] The statistics from steps (3) to (4) above show that the total number of days from callus differentiation to the formation of rooted seedlings is N1+N2+N3+N4 days.

[0069] (5) Open the cap of the test tube seedling bottle, adapt to the tissue culture room for 1 day, take out the rooted seedlings, wash off the residual culture medium attached to the roots of the rooted seedlings, plant them in a seedbed containing vermiculite: soil = 1:1, water thoroughly, spray with MS liquid culture medium diluted 10 times with water, harden the seedlings indoors, and cultivate them under the following conditions: temperature: 25±2℃, light intensity 1200Lx, photoperiod: 14h light, 10h darkness, cultivate for 18 days to obtain transplanted seedlings.

[0070] (6) Spray an appropriate amount of water on the leaves of the transplanted seedlings every day. For the first 3 days, ventilation can be increased from 30 minutes to 24 hours a day. After 7 days, move the seedbed outdoors and carry out normal seedling cultivation and management to obtain complete Artemisia annua plants.

[0071] In the above steps, each petri dish or each test tube seed bottle is a replicate, for a total of 5 replicates.

[0072] Comparative Examples 1 to 8

[0073] The difference between Comparative Examples 1 to 8 and Example 1 lies in the hormone content in the callus differentiation culture medium, as shown in Table 1.

[0074] Everything else is the same as in Example 1.

[0075] Comparative Example 9

[0076] The difference from Example 5 lies in the hormone content in the callus differentiation medium, where the hormone content in the callus differentiation medium is the same as that in Comparative Example 5, as detailed in Table 1.

[0077] Test Example 1

[0078] The number of explants inoculated in step (3) of Examples 1 to 5 and Comparative Examples 1 to 9, the number of callus tissues forming buds, and the number of days N1 for bud formation culture were statistically analyzed. The average values ​​of the induction rate and the number of days for bud formation were calculated. Wherein, the induction rate = average number of callus tissues forming buds per culture dish / average number of explants inoculated per dish × 100%.

[0079] The number of explants inoculated in step (3) of Examples 1 to 5 and Comparative Examples 1 to 9 and the number of days N2 for the formation of clustered shoots were statistically analyzed, and the average value was calculated.

[0080] The results are shown in Table 2. Different lowercase letters in the same column indicate significant differences at the P<0.05 level according to the t-test.

[0081] Table 1

[0082]

[0083] Table 2

[0084]

[0085] As can be seen from the data in Table 2, compared with Comparative Examples 1 to 9, the callus differentiation culture medium used in Examples 1 to 5 has a higher induction rate and a shorter time to form buds and clusters of shoots.

[0086] Examples 6 to 8

[0087] The difference between Examples 6 to 8 and Example 1 lies in the hormone content in the bud proliferation medium, as shown in Table 3.

[0088] Everything else is the same as in Example 1.

[0089] Comparative Examples 10 and 11

[0090] The difference between Comparative Examples 10 and 11 and Example 1 lies in the hormone content in the bud proliferation medium, as shown in Table 3. Everything else is the same as in Example 1.

[0091] Test Example 2

[0092] The number of unrooted seedlings and the number of cultivation days N3 formed in step (4) of Examples 1, 6 to 8, and Comparative Examples 8, 10, and 11, as well as the number of unrooted seedlings and the number of cultivation days N3 formed in step (3) of Examples 5 and Comparative Example 9, were statistically analyzed. The differentiation rate and the average number of days for forming unrooted seedlings were calculated. The differentiation rate was calculated as: (Average number of unrooted seedlings per culture dish / Average number of clustered shoots formed per culture dish) × 100%.

[0093] The results are shown in Table 3. The data in the table are the mean ± standard error. Different lowercase letters after the data in the same column indicate that the differences are significant at the P < 0.05 level according to the t-test.

[0094] Table 3

[0095]

[0096] As can be seen from the data in Table 3, the differentiation rates of Examples 1 and 6 to 8 were significantly higher than those of Comparative Examples 10 and 11, and the average N3 days were also shorter than those of Comparative Examples 10 and 11, especially for Examples 1 and 6, where the N3 was significantly shorter than that of Comparative Examples 10 and 11. Since Examples 1 and Comparative Example 5 used the same shoot proliferation culture medium and had the same culture conditions, their differentiation rates and N3 days were comparable. It should be noted that because Example 5 omitted the step of transferring to shoot proliferation culture, its average N3 days were significantly shorter than those of Examples 1 and 6 to 8, indicating that the callus differentiation culture medium of the examples, combined with omitting the transfer step, could significantly shorten the culture time. However, after Comparative Example 9 omitted the step of transferring to shoot proliferation culture, its average N3 days were actually longer than those of Comparative Example 5, indicating that the callus differentiation culture medium of Comparative Example 5, combined with omitting the transfer step, prolonged the culture time.

[0097] Examples 9 to 13

[0098] The difference between Examples 9 to 13 and Example 1 lies in the hormone content in the rooting medium, as shown in Table 4.

[0099] Everything else is the same as in Example 1.

[0100] Test Example 3

[0101] The number of rooted seedlings with adventitious roots and the number of cultivation days N4 in step (5) of Examples 1 and 9 to 12, and the number of rooted seedlings with adventitious roots and the number of cultivation days N4 in step (4) of Example 5 were statistically analyzed to calculate the average rooting rate and the average number of days to form rooted seedlings. Wherein, the rooting rate = average number of rooted seedlings per bottle / average number of non-rooted seedlings transferred per bottle × 100%.

[0102] The results are shown in Table 4. The data in the table are the mean ± standard error. Different lowercase letters after the data in the same column indicate that the differences are significant at the P < 0.05 level according to the t-test.

[0103] Table 4

[0104]

[0105] As can be seen from the data in Table 4, the rooting culture media provided by the embodiments of the present invention all have a high rooting rate, and the number of days to form rooted seedlings is within a relatively ideal range. In terms of the total number of culture days, the total culture days of Example 5 are the shortest, only 44 days, while in the preferred comparative example (comparative example 5), the culture days are the shortest, 78 days, which is significantly higher than the embodiments of the present invention. The time taken by comparative example 9 is as long as 93 days.

Claims

1. A method for tissue culture of Artemisia species, comprising the following steps: 1-2) The explants of the Artemisia species were cultured in callus differentiation medium to obtain non-rooted seedlings; the callus differentiation medium consisted of MS liquid medium, a first solidifying agent, a first sugar, and a first hormone, and the pH of the callus differentiation medium was 5.8 to 6.2; the first solidifying agent was agar, the first sugar was sucrose; the first hormone was 6-benzylaminopurine and α-naphthaleneacetic acid; based on the volume of the MS liquid medium of 1000 mL, the amount of the first solidifying agent was 5 to 10 g / L, the amount of the first sugar was 25 to 35 g / L, the amount of 6-benzylaminopurine was 2 mg / L, and the amount of α-naphthaleneacetic acid was 0.3 mg / L; 3) The unrooted seedlings from steps 1-2) are cultured in a rooting medium to obtain rooted seedlings. The rooting medium consists of MS liquid medium, a third solidifying agent, a third sugar, and a third hormone. The pH of the rooting medium is 5.8 to 6.

2. The third solidifying agent is agar, and the third sugar is sucrose. The third hormone is indole-3-butyric acid and α-naphthaleneacetic acid. The volume of the MS liquid medium is 1000 mL. The amount of the third solidifying agent is 5 to 10 g / L, the amount of the third sugar is 25 to 35 g / L, the amount of 6-benzylaminopurine is 0.2 mg / L, and the amount of α-naphthaleneacetic acid is 0.01 mg / L. 4) Transplant the rooted seedlings and cultivate them to obtain complete Artemisia plants; In steps 1-2), the cultivation conditions are 23 to 27°C, light intensity 1200 Lx, and photoperiod L:D = 12:12; In step 3), the cultivation conditions are 22 to 26°C, light intensity 1200 Lx, and photoperiod L:D = 14:10; The Artemisia species mentioned is Artemisia annua; The explant is a leaf.

2. The method according to claim 1, characterized in that, Before steps 1-2), the following steps are also included: P1) Obtain explants of the Artemisia species; P2) Disinfect the explants to obtain disinfected explants.

3. The method according to claim 2, characterized in that, Following step P2), step P3) is performed to pre-culture the sterilized explants.

4. The method according to claim 3, characterized in that, In step P3), the pre-culture medium consists of MS liquid medium, a fourth solidifying agent, and a fourth sugar; the fourth solidifying agent is agar, and the fourth sugar is sucrose.

5. The method according to claim 3, characterized in that, Pre-culture in the dark for two days.

6. The method according to claim 1, characterized in that, The Artemisia annua mentioned is the "Haiqing No. 1" Artemisia annua.

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