Method for inducing regeneration of Hangzhou chrysanthemum leaf adventitious buds by using potato extract

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing Hangzhou chrysanthemum leaf adventitious bud regeneration by using a potato extract, which sequentially comprises the following steps: cultivating Hangzhou chrysanthemum stem segments with buds into Hangzhou chrysanthemum aseptic seedlings with at least four leaves; the method comprises the following steps: taking down 3-4 leaves from the top of a sterile seedling of Hangzhou chrysanthemum, taking a leaf disc from each leaf close to a petiole by using a puncher, immediately inoculating the leaf disc onto an adventitious bud induction culture medium, and culturing in a plant tissue culture room; and obtaining Hangzhou chrysanthemum leaf adventitious buds. According to the method, the leaf disc adventitious bud induction time is short, the growth state is good, subsequent normal rooting, transplanting and breeding can be carried out, and a foundation is laid for successfully establishing a Hangzhou chrysanthemum genetic transformation system.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a method for inducing adventitious bud regeneration of chrysanthemum leaves by utilizing potato extract. Background Art

[0002] Chrysanthemum morifolium Ramat. is one of the famous "Eight Flavors of Zhejiang Province". It is rich in active ingredients such as chlorogenic acid, luteolin and 3,5-O-dicaffeoylquinic acid. It can be made into medicines or beverages, and exerts multiple health benefits such as anti-inflammatory, antioxidant, anti-allergic and cardiovascular protection. It has important economic value.

[0003] In recent years, with the continuous progress of biotechnology, the study of genetic transformation of medicinal chrysanthemum has become particularly important. On the one hand, genetic engineering methods can improve the genetic characteristics of medicinal chrysanthemum, enhance its stress resistance and increase its yield, so as to meet the growing market demand. On the other hand, in the biosynthesis of medicinal active ingredients, transgenic technology helps to reveal and regulate the synthesis pathways of key active ingredients (such as chlorogenic acid, luteolin, etc.), further improve the content of effective ingredients in medicinal chrysanthemum, and provide strong support for the development of efficient and safe traditional Chinese medicine products. Therefore, the rapid development of genetic transformation technology not only brings new opportunities for the breeding of medicinal chrysanthemum, but also is an important way to enhance its medicinal and economic value. In chrysanthemum, the implementation of transgenic technology mainly relies on the transformation method of leaf disc mediated by Agrobacterium tumefaciens. At present, ornamental chrysanthemum varieties such as 'Li Jin' (Hong Yan et al., 2015), 'Breeze Deep Red' (Luo Yuting et al., 2020), and 'Shenma' (Zhang Cui, 2021) have established mature transgenic operation procedures, but there are no reports on genetic transformation cases of Hangzhou chrysanthemum. Studying the adventitious bud regeneration mechanism of chrysanthemum is an important prerequisite for realizing its genetic transformation. However, due to the complexity and polyploidy of the chrysanthemum genome, it is difficult to apply a unified regeneration system to different chrysanthemum varieties.

[0004] In previous studies, axillary buds have been successfully used as explants to achieve the proliferation and rapid in vitro propagation of chrysanthemum buds (Zou Keqin et al.). However, due to the low infection efficiency of Agrobacterium on stem segments and the high efficiency of direct organ regeneration from axillary buds, the proportion of transgenic positive seedlings using stem segments as explants was low.

[0005] The references mentioned above are as follows:

[0006] Hong Yan, Zhou Qiong, Dai Silan. Optimization of the regeneration system of the cut chrysanthemum variety 'Lijin'[C] / / Ornamental Horticulture Committee of the Chinese Society of Horticulture, National Flower Engineering Technology Research Center. Research Progress of Ornamental Horticulture in China 2015. College of Landscape Architecture, Beijing Forestry University, Beijing Key Laboratory of Flower Germplasm Innovation and Molecular Breeding; 2015:6;

[0007] Luo Yuting, Huang Yiqing, Huang Qi, et al. Establishment of efficient regeneration and transformation system of selected potted chrysanthemum[J]. Jiangsu Agricultural Science, 2020, 48(11): 61-66. DOI: 10.15889 / j.issn.1002-1302.2020.11.012;

[0008] Zhang Cui. Optimization of chrysanthemum genetic transformation system and preliminary study on the establishment of 'Shenma' mutant library using gene editing technology[D]. Shandong Agricultural University, 2021.DOI:10.27277 / d.cnki.gsdnu.2021.000872;

[0009] Zou Keqin, Li Sufang, Wang Weimin, et al. A method for tissue culture and propagation of Chrysanthemum morifolium[P]. Zhejiang Province: CN201410126818.8, 2016-01-27. Summary of the invention

[0010] The technical problem to be solved by the present invention is to provide a method for regenerating adventitious buds from chrysanthemum leaves.

[0011] In order to solve the above technical problems, the present invention provides a method for inducing adventitious bud regeneration of chrysanthemum leaves using potato extract, which comprises the following steps in sequence:

[0012] 1) Explant disinfection and sterile seedling culture:

[0013] Using stem segments with buds (stem segments with 1 to 2 buds) of chrysanthemum as explant materials, cultivating chrysanthemum sterile seedlings with at least 4 leaves;

[0014] 2) Leaf disc regeneration of adventitious buds:

[0015] Remove 3 to 4 leaves from the top of the sterile chrysanthemum seedlings obtained in step 1), use a hole puncher to take a leaf disc with a diameter of 6±1 mm near the petiole of each leaf, and immediately inoculate it on the adventitious bud induction medium (adaxial side facing up), and culture it in a plant tissue culture room;

[0016] The culture conditions were: dark culture for the first week, followed by light culture for 12 h / d (i.e., 12 h of light and 12 h of darkness per day), with a light intensity of 30-40 μmol·m -2 ·s -1 The culture temperature was 24±1℃; the light culture time was 2 to 4 weeks; and adventitious buds of chrysanthemum leaves were obtained.

[0017] Note: After 2 weeks of light culture, new buds have already appeared, and new buds will continue to appear as the culture time goes on; in actual application, when the length of a single adventitious bud is ≥0.5cm, the buds can be cut for rooting.

[0018] As an improvement of the method for regenerating adventitious buds from chrysanthemum leaves of the present invention, the adventitious bud induction medium is: based on 4.43g of MS basic medium, 0.9-1.1mg of 6-benzylaminoadenine (6-BA), 0.4-0.6mg of naphthaleneacetic acid (NAA), 200-800g of potato extract corresponding to potatoes, 29-31g of white sugar are added, deionized water is added to make the volume to 1L and then evenly mixed, the pH is adjusted to 5.8±0.2, and 3.4-3.6g of plant gel is added;

[0019] The preparation method of potato extract is as follows: wash and peel the potatoes, weigh 200-800 g, cut into small pieces (2×2 cm 2 ) and then put it into 400-600 mL of deionized water, boil it for 18-22 minutes, filter it with a sieve after cooling, and the filtrate obtained is the potato extract.

[0020] Note: High pressure sterilization is required (121℃ high pressure sterilization for 20 minutes).

[0021] As a further improvement of the method for regenerating adventitious buds from chrysanthemum leaves of the present invention, the adventitious bud induction medium is: based on 4.43 g of MS basic medium, 1 mg of 6-benzylaminoadenine (6-BA), 0.5 mg of naphthaleneacetic acid (NAA), 400 g of potato extract corresponding to potatoes, 30 g of white sugar are added, deionized water is added to make the volume to 1 L and then evenly mixed, the pH is adjusted to 5.8±0.2, and 3.5 g of plant gel is added.

[0022] As a further improvement of the method for regenerating adventitious buds from chrysanthemum leaves of the present invention, step 1) is:

[0023] Select chrysanthemums with good growth (dark green leaves, strong plants, no diseases and insect pests, and vigorous growth period), cut stem segments with 1 to 2 buds (about 2 cm long) as explant materials, and then disinfect (conventional disinfection);

[0024] The disinfected stem segments were cut off from the browned part and inoculated into 1 / 2MS medium. The culture conditions were illumination culture for 12 h / d and the light intensity was 30-40 μmol·m -2 ·s -1 The culture temperature is 24±1℃; after culturing for 4 to 6 weeks, sterile seedlings of Hangzhou chrysanthemum are obtained.

[0025] Note: The disinfection is to rinse under running water for 1 to 2 times, and dry the water with filter paper; then disinfect with 75% ethanol for 1 minute, wash with sterile water 3 times, then disinfect with 0.1% mercuric chloride solution for 12 minutes, and wash with sterile water 3 times.

[0026] As a further improvement of the method for regenerating adventitious buds from chrysanthemum leaves of the present invention, the chrysanthemum sterile seedlings obtained in step 1) are used to cut stem segments with 1 to 2 buds for subculture.

[0027] That is, individual seedlings can be cut into stem segments with 1 to 2 buds (the length of the stem segment is about 2 cm) and subcultured once every 4 to 6 weeks.

[0028] The present invention also provides a method for obtaining chrysanthemum plants by using adventitious buds from chrysanthemum leaves, including rooting culture and tissue culture seedling domestication and transplantation;

[0029] 1. Rooting and cultivation

[0030] Cut each adventitious bud with a length of ≥0.5 cm obtained in step 2) to obtain a single bud;

[0031] The single buds were transferred to 1 / 2MS medium and cultured in the plant tissue culture room under the conditions of 12h / d of light and 30-40μmol·m -2 ·s -1 , the culture temperature was 24±1℃;

[0032] The culture time of this step 3) is 3 to 4 weeks;

[0033] Note: Generally, rooting will occur after 15 days of cultivation;

[0034] 2. Acclimation and transplantation of tissue culture seedlings

[0035] The rooted chrysanthemum bottle seedlings obtained in step 1 are acclimated under normal temperature and natural light conditions for 7 to 10 days, the culture medium at the roots of the seedlings is washed, and they are planted in plug trays. After planting, they are watered thoroughly, shaded, and the humidity is maintained at 75% to 90%. After being planted in the plug trays for 2 weeks, the leaves of the chrysanthemum are dark green and the stems are upright normally, and they can be subsequently planted in the field.

[0036] In vitro leaves are the preferred receptor system for plant genetic engineering. In the present invention, leaf discs are innovatively used as explants to directly induce the differentiation of adventitious buds. This method not only simplifies the operation process, effectively shortens the transformation cycle brought about by callus dedifferentiation and redifferentiation, but also makes it easier for Agrobacterium-mediated gene transformation, which is a key step in accelerating the process of transgenic breeding of chrysanthemum.

[0037] The present invention has the following technical advantages:

[0038] 1) Using leaf discs as explants, direct regeneration of adventitious buds of chrysanthemum is carried out without dedifferentiation and redifferentiation of callus tissue. The time period for inducing adventitious buds is short, and new buds can be obtained after 3 to 5 weeks of cultivation. The genetic stability of the adventitious buds obtained is good;

[0039] 2) The bud induction medium was improved, and 400g / L potato extract was preferably added, which greatly improved the differentiation efficiency of adventitious buds in chrysanthemum leaf discs.

[0040] In summary, the adventitious bud induction time of the leaf disk of the method of the present invention is short and the growth state is good, and subsequent normal rooting, transplanting and reproduction can be carried out, which lays the foundation for the successful establishment of the genetic transformation system of chrysanthemum. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0042] Figure 1 Flow chart of obtaining chrysanthemum plants by leaf disc method for regenerating adventitious buds;

[0043] Figure 2 Effects of different supplementary additives on adventitious bud regeneration from leaf discs. In the figure, CK represents no additives, TD represents the addition of potato extract, ZS represents the addition of purple sweet potato extract, and HZ represents the addition of red date extract. The concentration of the additives is 400 g / L. For detailed formulas, see Comparative Examples 1-1, 1-2, and 1-3.

[0044] Figure 3 Effects of adding different concentrations of potato extract on regeneration of adventitious buds from leaf discs. The numbers in the figure represent the concentration of potato extract (in g / L). Each treatment was repeated with 6 dishes. The average germination rate was calculated after 35 days of culture and representative dishes were selected for photographing and recording. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0046] The chrysanthemum used in this experiment was conventional chrysanthemum; the potatoes (TD), purple sweet potatoes (ZS), and red dates (HZ) used in this experiment were all purchased from ordinary vegetable markets.

[0047] Example 1: A method for inducing the regeneration of adventitious buds from chrysanthemum leaves using potato extract and obtaining chrysanthemum plants using the adventitious buds from chrysanthemum leaves, comprising the following steps:

[0048] 1) Explant disinfection and sterile seedling culture:

[0049] Select chrysanthemums that are in good growth (dark green leaves, strong plants, free of diseases and pests) and in the peak period of vegetative growth, cut stem segments with buds (with 1 to 2 buds) about 2 cm long as explant materials, rinse them under running water 1 to 2 times, and absorb the moisture with filter paper.

[0050] Then, the tube was disinfected with 75% (volume %) ethanol for 1 min, washed three times with sterile water, and then disinfected with 0.1% (mass %) mercuric chloride solution for 12 min (shaking occasionally during the period), and washed three times with sterile water.

[0051] The disinfected stem segments were cut off from the browned part and inoculated into 1 / 2MS medium. The culture conditions were illumination for 12 h / d and the light intensity was 30-40 μmol·m -2 ·s -1 The culture temperature is 24±1°C. The material obtained after 4-6 weeks of culture is sterile seedlings of chrysanthemum (usually with 5-10 leaves).

[0052] Every 4 to 6 weeks, individual seedlings can be cut into stem segments with 1 to 2 buds (the length of the stem segment is about 2 cm) for subculture.

[0053] 2) Leaf disc regeneration of adventitious buds:

[0054] The 3-4 leaves from the top of the sterile chrysanthemum seedlings obtained in step 1) were removed, and leaf discs with a diameter of 6 mm were taken from each leaf near the petiole using a hole puncher, and immediately inoculated onto adventitious bud induction medium (TD400) with the adaxial surface facing upwards, and cultured in a plant tissue culture room. The culture conditions were dark culture for the first week, followed by light culture for 12 h / d, and the light intensity was 30-40 μmol·m -2 ·s -1 , the culture temperature was 24±1°C. After about 5 weeks (i.e., 4 weeks of light culture), the production of adventitious buds was observed.

[0055] The adventitious bud induction medium (TD400) is an MS basic medium supplemented with 1.0 mg / L 6-BA, 0.5 mg / L NAA, 400 g / L potato extract, 30 g / L white sugar, 3.5 g / L Phytogel, and pH 5.8±0.2.

[0056] The preparation method of the adventitious bud induction medium (TD400) is as follows: based on MS basic medium (4.43g), 1mg 6-benzylaminoadenine (6-BA), 0.5mg naphthaleneacetic acid (NAA), potato extract, 30g white sugar are added, deionized water is added to make the volume to 1L and then evenly mixed, 1mol / L NaOH or 1mol / L HCl is used to adjust the pH to 5.8±0.2, 3.5g plant gel is added and the medium is sterilized at 121°C under high pressure for 20min.

[0057] The potato extract is prepared by washing and peeling potatoes, weighing 400 g, and cutting into 2×2 cm 2The potato extract was defined as the potato extract after the small pieces were put into 500 mL of deionized water, boiled for 20 min, filtered with a sieve after cooling, and the filtrate was added to the MS medium, and the amount of the extract was calculated based on the weight of the peeled potatoes before boiling.

[0058] The culture time of step 2) was 5 weeks and the germination rate was 78%.

[0059] Germination rate = number of leaf discs induced with adventitious buds / total number of leaf discs.

[0060] 3) Rooting culture:

[0061] Cut each adventitious bud with a length of ≥0.5 cm obtained in step 2) with a scalpel to obtain a single bud;

[0062] The single buds were transferred to 1 / 2MS medium and cultured in the plant tissue culture room under the conditions of 12h / d of light and 30-40μmol·m -2 ·s -1 , the culture temperature is 24±1℃.

[0063] The cultivation time of step 3) is 4 weeks, and the rooting rate is 100%. Generally, there are 2 to 5 roots, and the length of the roots is about 5 to 10 cm.

[0064] 4) Acclimation and transplantation of tissue culture seedlings

[0065] The rooted chrysanthemum seedlings obtained in step 3) are acclimated for 7 to 10 days under normal temperature and natural light conditions (temperature 20°C, light conditions of 1500lx), the root culture medium of the seedlings is washed, and the seedlings are planted in a plug tray. After planting, water is poured thoroughly, shade is provided, and the humidity is maintained at 75% to 90%, and the culture temperature is 24±1°C. The growth matrix is ​​peat, vermiculite, and perlite mixed in a volume ratio of 2:1:1.

[0066] 14 days after planting in the plug tray, the leaves of the chrysanthemum are dark green and the stems are upright normally, so it can be planted in the field.

[0067] Example 2: The culture time in step 2) of Example 1 was changed from 5 weeks to 3 weeks (ie, 2 weeks of light culture), and the germination rate was 25%.

[0068] Comparative Example 1-1,

[0069] The "adventitious bud induction medium (TD400)" in step 2) of Example 1 was replaced with "adventitious bud induction medium (CK)", that is, no potato extract was added. The rest was the same as step 1) to step 2) of Example 1.

[0070] After 5 weeks of culture, the leaf disk became thicker and yellow-green callus appeared on the edge, but no adventitious buds were produced and the budding rate was 0.

[0071] Comparative Examples 1-2

[0072] The "adventitious bud induction medium (TD400)" in step 2) of Example 1 is replaced with "adventitious bud induction medium (ZS400)", that is, the purple sweet potato extract obtained by referring to the preparation method of "potato extract" from 400g of peeled purple sweet potatoes is substituted for the "potato extract" in the "adventitious bud induction medium (TD400)"; the rest is equivalent to steps 1) to 2) of Example 1.

[0073] The purple sweet potato extract is prepared by washing and peeling the purple sweet potato, weighing 400 g, and cutting into 2×2 cm 2 The small pieces were put into 500 mL of deionized water, boiled for 20 min, cooled and filtered with a sieve, and the filtrate was defined as purple sweet potato extract.

[0074] After 5 weeks of culture, the leaf disks produced dense white-green calli with a budding rate of 11.1%.

[0075] Comparative Examples 1-3

[0076] The "adventitious bud induction medium (TD400)" in step 2) of Example 1 was replaced with "adventitious bud induction medium (HZ400)", that is, the red date extract obtained by referring to the preparation method of "potato extract" from 400g of red dates was substituted for the "potato extract" in the "adventitious bud induction medium (TD400)"; the rest was the same as steps 1) to 2) of Example 1.

[0077] The red date extract is prepared by washing red dates, weighing 400 g of the red dates, cutting them in half, and putting them into 500 mL of deionized water, boiling them for 20 minutes, and filtering them with a sieve after cooling. The obtained filtrate is defined as the red date extract.

[0078] After 5 weeks of cultivation, the leaf disks turned brown or white, half of them died, and the germination rate was 0.

[0079] By analyzing the experimental results of comparative examples 1-1, 1-2, and 1-3, we found that different supplementary additives have different regulatory effects on the growth state of the chrysanthemum leaf disc. Among them, the addition of potato extract can significantly promote the bud regeneration of the chrysanthemum leaf disc.

[0080] Comparative Example 2-1

[0081] The "adventitious bud induction medium (TD400)" in step 2) of Example 1 was changed to "adventitious bud induction medium (TD200)", that is, "potato extract corresponding to 400g of potatoes" was changed to "potato extract corresponding to 200g of potatoes". The rest was the same as step 1) to step 2) of Example 1.

[0082] The culture time of step 2) was 5 weeks and the germination rate was 43%.

[0083] Comparative Example 2-2

[0084] The "adventitious bud induction medium (TD400)" in step 2) of Example 1 was changed to "adventitious bud induction medium (TD600)", that is, "potato extract corresponding to 400g of potatoes" was changed to "potato extract corresponding to 600g of potatoes". The rest was the same as step 1) to step 2) of Example 1.

[0085] The culture time of step 2) was 5 weeks and the germination rate was 67%.

[0086] Comparative Examples 2-3

[0087] The "adventitious bud induction medium (TD400)" in step 2) of Example 1 was changed to "adventitious bud induction medium (TD800)", that is, "potato extract corresponding to 400g of potatoes" was changed to "potato extract corresponding to 800g of potatoes". The rest was the same as step 1) to step 2) of Example 1.

[0088] The culture time of this step 2) was 5 weeks; the leaf disc was yellow-green, and the germination rate was 33%.

[0089] By analyzing the experimental results of the above comparative examples, it was found that different concentrations of potato extract had different promoting effects on the regeneration of leaf disc buds of chrysanthemum - with the increase of potato extract concentration, the germination rate showed a trend of first increasing and then decreasing. Among them, when the concentration of potato extract was 400g / L, the leaf disc germination rate was the highest, reaching 78%.

[0090] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A method for inducing adventitious bud regeneration from chrysanthemum leaves using potato extract, characterized in that The following steps are included in sequence: 1) Explant disinfection and sterile seedling culture: Using the stem segments of chrysanthemum with buds as explant materials, cultivating chrysanthemum sterile seedlings with at least 4 leaves; 2) Leaf disc regeneration of adventitious buds: Remove 3 to 4 leaves from the top of the sterile chrysanthemum seedlings obtained in step 1), use a hole puncher to take a leaf disc with a diameter of 6±1 mm near the petiole of each leaf, and immediately inoculate it on the adventitious bud induction medium (adaxial side facing up), and culture it in a plant tissue culture room; The culture conditions were: dark culture for the first week, followed by light culture for 12 h / d, with a light intensity of 30-40 μmol·m -2 ·s -1 The culture temperature was 24±1℃; the light culture time was 2 to 4 weeks; and adventitious buds from the leaves of chrysanthemum were obtained.

2. The method for regenerating adventitious buds from chrysanthemum leaves according to claim 1, characterized in that: The adventitious bud induction medium is: based on 4.43g of MS basic medium, 0.9-1.1mg of 6-benzylaminoadenine, 0.4-0.6mg of naphthaleneacetic acid, 200-800g of potato extract corresponding to potatoes, 29-31g of white sugar, deionized water is added to make the volume to 1L and then evenly mixed, the pH is adjusted to 5.8±0.2, and 3.4-3.6g of plant gel is added; The preparation method of potato extract is as follows: wash and peel potatoes, weigh 200-800 g, cut into small pieces, put into 400-600 mL deionized water, boil for 18-22 minutes, filter with a sieve after cooling, and the obtained filtrate is potato extract.

3. The method for regenerating adventitious buds from chrysanthemum leaves according to claim 2, characterized in that: The adventitious bud induction medium is: based on 4.43g of MS basic medium, 1mg of 6-benzylaminoadenine, 0.5mg of naphthaleneacetic acid (NAA), a potato extract corresponding to 400g of potatoes, 30g of white sugar are added, deionized water is added to make the volume to 1L and then evenly mixed, the pH is adjusted to 5.8±0.2, and 3.5g of plant gel is added.

4. The method for regenerating adventitious buds from chrysanthemum leaves according to any one of claims 1 to 3, characterized in that The step 1) is: Select chrysanthemums with good growth, cut stem segments with 1 to 2 buds as explant materials, and then sterilize them; The disinfected stem segments were cut off from the browned part and inoculated into 1 / 2MS medium. The culture conditions were illumination culture for 12 h / d and the light intensity was 30-40 μmol·m -2 ·s -1 The culture temperature is 24±1℃; after culturing for 4 to 6 weeks, sterile seedlings of Hangzhou chrysanthemum are obtained.

5. The method for regenerating adventitious buds from chrysanthemum leaves according to claim 4, characterized in that: Using the sterile chrysanthemum seedlings obtained in step 1), stem segments with 1 to 2 buds are cut for subculture.

6. A method for obtaining chrysanthemum plants using adventitious buds from chrysanthemum leaves, characterized in that: Including rooting culture and tissue culture seedling acclimatization and transplanting; 1. Rooting and cultivation Cut each adventitious bud with a length of ≥0.5 cm obtained in step 2) to obtain a single bud; The single buds were transferred to 1 / 2MS medium and cultured in the plant tissue culture room under the conditions of 12h / d of light and 30-40μmol·m -2 ·s -1 , the culture temperature was 24±1℃; The culture time of this step 3) is 3 to 4 weeks; 2. Acclimation and transplantation of tissue culture seedlings The rooted chrysanthemum bottle seedlings obtained in step 1 are acclimated under normal temperature and natural light conditions for 7 to 10 days, the culture medium at the roots of the seedlings is cleaned, and they are planted in plug trays. After planting, they are watered thoroughly, shaded, and the humidity is maintained at 75% to 90%. After being planted in the plug trays for 2 weeks, subsequent field planting can be carried out.

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