Induction method and induction culture medium for inducing Helleborus thibetanus petiole to regenerate adventitious buds

Abstract

The invention provides an induction method and an induction culture medium for inducing Helleborus thibetanus petioles to regenerate adventitious buds, and solves the technical problem of low efficiency of inducing sprouted adventitious buds of Helleborus thibetanus rhizomes or overground stems in the prior art. A method for inducing regenerated adventitious buds of helleborus thibetanus petioles comprises the steps that petiole explants are disinfected and sterilized and transplanted to a regenerated adventitious bud induction culture medium, and the regenerated adventitious bud induction culture medium comprises 6-benayl aminopurine, forchlorfenuron, zeatin, 6-furfuryl aminopurine, indolebutyric acid, multivitamin tablets, a coagulator, cane sugar and a nutrient culture medium. According to the method, the helleborus thibetanus petioles are used as explants, adventitious buds can be efficiently induced, a helleborus thibetanus plant regeneration technical system is established, rapid propagation, high regeneration efficiency and rich explant sources are realized, meanwhile, a mature, stable and efficient helleborus thibetanus regeneration system is established, and the method has important significance on transgenic breeding of helleborus thibetanus.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to an induction method and an induction culture medium for inducing the regeneration of adventitious buds from the petioles of Helleborus truncatus. Background Art

[0002] Helleborus × hybridus is a perennial herbaceous perennial plant of the genus Helleborus in the family Ranunculaceae, also known as the "Christmas Rose". There are about 22 species in the world, most of which are distributed in Europe, and there is only one species in my country. Helleborus × hybridus has peculiar leaf shape, petal-shaped sepals, rich colors and diverse flower shapes. It blooms in late winter and early spring under natural conditions and has extremely high ornamental value.

[0003] The propagation of Helleborus can be done by sowing and ramet propagation. Seed sowing propagation has variation and separation in the offspring, and cannot maintain the same traits as the mother plant; although ramet propagation can maintain the traits of the mother plant, the propagation speed is slow and the production efficiency is low. With the in-depth development of Helleborus hybrid breeding, a large number of hybrid offspring with special and high ornamental value will be bred every year. The ramet propagation seriously restricts its mass production and commercial circulation. The existing tissue culture technology of Helleborus uses the sprouts of the plant rhizomes or aerial stems as explant materials, which is destructive and has little material. It is very harmful to the mother plant and often causes the death of the material. It is not suitable for very small or single mother materials. Summary of the invention

[0004] The invention provides an induction method and an induction culture medium for inducing the regeneration of adventitious buds from the petioles of Helleborus truncatus, aiming to solve the technical problem of low efficiency in inducing the germination and regeneration of adventitious buds from the rhizomes or aerial stems of Helleborus truncatus in the prior art.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a method for inducing adventitious bud regeneration from petioles of Helleborus truncatus, comprising:

[0007] S1. Clean the Helleborus plant, cut off the old petioles and bad roots, retain 8-10 cm of healthy roots, sterilize and disinfect the Helleborus roots, and then transplant them into the planting medium;

[0008] S2. After transplanting the Helleborus truncatus plants, wait for the rhizome buds of the Helleborus truncatus to germinate, soften and sterilize the petioles of the Helleborus truncatus, and obtain sterile Helleborus truncatus petiole explants for regenerating adventitious buds.

[0009] S3, inoculating the sterilized Helleborus petiole explants on a regenerated adventitious bud induction medium, and placing them under the conditions of 15-25° C., a light intensity of 1500-2000 LUX, a pH of 5.6-5.8, and a photoperiod of 8-10 hours, for induction culture for 30-40 days until adventitious buds are regenerated on the Helleborus petiole;

[0010] The regenerated adventitious bud induction medium comprises 2-4 parts by mass of 6-benzylaminopurine, 2-4 parts by mass of chlorfenapyr, 0.5-2 parts by mass of zeatin, 0.5-1.5 parts by mass of 6-furfurylaminopurine, 0.1-0.3 parts by mass of indolebutyric acid, 400-900 parts by mass of multivitamin tablets, 6000-7000 parts by mass of a coagulant, 18000-22000 parts by mass of sucrose and 950-1100 parts by volume of a nutrient medium;

[0011] Here, 1 part by mass: 1 part by volume = 1 mg: 1 mL.

[0012] A preferred solution, in step S3, the regenerated adventitious bud induction medium comprises 2.5 parts by mass of 6-benzylaminopurine, 3.8 parts by mass of chlorfenapyr, 1.8 parts by mass of zeatin, 1.2 parts by mass of 6-furfurylaminopurine, 0.3 parts by mass of indolebutyric acid and 490 parts by mass of multivitamin tablets, 6500 parts by mass of a coagulant, 21000 parts by mass of sucrose and 980 parts by volume of a nutrient medium;

[0013] Here, 1 part by mass: 1 part by volume = 1 mg: 1 ml.

[0014] A preferred solution, in step S3, the regenerated adventitious bud induction medium comprises 3.0 parts by mass of 6-benzylaminopurine, 3.0 parts by mass of chlorfenapyr, 1.5 parts by mass of zeatin, 0.8 parts by mass of 6-furfurylaminopurine, 0.2 parts by mass of indolebutyric acid and 560 parts by mass of multivitamin tablets, 6500 parts by mass of a coagulant, 20000 parts by mass of sucrose and 1000 parts by volume of a nutrient medium;

[0015] Here, 1 part by mass: 1 part by volume = 1 mg: 1 ml.

[0016] Based on the above scheme, experiments have shown that the combination of 3.0 mg / L 6-benzylaminopurine, 3.0 mg / L chlorfenapyr, 1.5 mg / L zeatin, 0.8 mg / L 6-furfurylaminopurine, 0.2 mg / L indolebutyric acid and 0.56 g / L multivitamin tablets, coagulant, sucrose and nutrient medium, these plant growth regulators, has a better effect in inducing the formation of adventitious buds.

[0017] A preferred solution is that in step S3, the regenerated adventitious bud induction medium comprises 4.0 parts by mass of 6-benzylaminopurine, 2.5 parts by mass of chlorfenapyr, 0.8 parts by mass of zeatin, 0.6 parts by mass of 6-furfurylaminopurine, 0.12 parts by mass of indolebutyric acid, 850 parts by mass of multivitamin tablets, 7000 parts by mass of coagulant, 22000 parts by mass of sucrose and 1100 parts by volume of nutrient medium;

[0018] Here, 1 part by mass: 1 part by volume = 1 mg: 1 ml.

[0019] A preferred solution is that in step S1, the Helleborus truncatus plant is cleaned until there is no attachment to the root system, the old leaves and petioles of the Helleborus truncatus are cut off, the bad roots are cleaned, the plant is soaked and sterilized with a fungicide, and the plant is transplanted into a planting medium with 1-2 cm of the root system exposed.

[0020] Based on the above plan, cleaning the iron chopstick plants can remove the dirt and other attachments on the roots, reduce the potential threat of pathogens and pests, and provide a more hygienic growth environment for the plants; cutting off old leaves and petioles and cleaning dead roots, diseased roots and redundant capillary roots can help reduce the burden on the plants, prevent ineffective consumption of nutrients, and promote the development of healthy roots; soaking the plants in fungicides can eliminate possible pathogens, reduce the risk of plant disease infection, and increase survival rate; exposing the roots 1-2 cm can help induce the growth of new roots, because new roots are usually easier to germinate close to the surface, which helps the plants better absorb water and nutrients.

[0021] In a preferred embodiment, the fungicide is 500-800 times of carbendazim or 1000-1500 times of potassium permanganate solution.

[0022] Based on the above scheme, carbendazim can interfere with the mitosis of pathogens, affect cell division, play a bactericidal role, and effectively prevent and treat a variety of crop diseases caused by fungi. Potassium permanganate can effectively kill various bacterial propagules, fungi, and tuberculosis bacteria, and has a strong bactericidal and disinfecting effect. Reasonable dilutions of these two fungicides can be used for the sterilization and disinfection of iron chopstick plants, reduce the occurrence of diseases, and improve the growth quality and health of iron chopstick plants.

[0023] Preferably, in step S1, the planting medium includes at least one of peat soil, perlite, and river sand.

[0024] Based on the above scheme, the porous structure of peat and perlite is conducive to the breathing of the roots of Helleborus plants, while preventing water accumulation at the roots and reducing diseases. The water retention and fertilizer retention of peat enable it to effectively retain water and nutrients to meet the growth needs of Helleborus. Perlite can absorb 2-3 times its own weight in water to keep the soil moist, and its chemical properties are stable. It will not react with the nutrient solution. It is sterile and does not contain organic matter, which avoids the growth of mold. It can also regulate soil temperature and protect the root system. River sand is suitable for seedling cultivation, cuttings and planting of Helleborus due to its loose, breathable and water-permeable characteristics. It is an economical and widely available planting medium, suitable for plantings of all sizes. Therefore, the above three planting media are all good choices for planting Helleborus.

[0025] The preferred solution is that in step S2, when the rhizome buds of the Helleborus chinensis germinate and the leaves of the Helleborus chinensis grow to 8-12 cm, the petioles of the Helleborus chinensis are covered with a black film, then covered with a light-proof container, and placed in a dark room for softening treatment, the softened petioles of the Helleborus chinensis are wiped with 70-75% alcohol, and then soaked in 1000 times mercuric chloride solution for 5-7 minutes, washed with sterile water for 3-5 times, and dried with sterile filter paper to obtain sterile Helleborus chinensis petiole explants with regenerated adventitious buds.

[0026] Based on the above scheme, by using black film covering and dark room treatment to simulate the growth environment under shade conditions in nature, the petiole of Helleborus can be softened, making the petiole tender, which is conducive to the absorption of hormones, thereby facilitating the formation of adventitious buds. This treatment method induces the production of adventitious buds. Subsequently, the steps of wiping with 70-75% alcohol and soaking in 1000 times mercuric chloride solution can effectively kill pathogenic microorganisms that may exist on the surface of the petiole of Helleborus and reduce the risk of pollution. Among them, alcohol and mercuric chloride are commonly used disinfectants, which can effectively kill bacteria and fungi, thereby improving the reproduction efficiency of Helleborus.

[0027] In a preferred embodiment, in step S3, the regenerated adventitious bud induction medium further comprises the nutrient medium being WPM medium, and the coagulant being carrageenan.

[0028] Based on the above scheme, WPM (Woody Plant Medium) medium is a medium specially designed for woody plant tissue culture. It improves the MS medium, replaces potassium nitrate with potassium sulfate, and reduces the content of ammonium nitrate, so that nitrogen salts are mainly supplied in the form of calcium nitrate. This low-salt concentration medium is suitable for the induction and growth of adventitious buds of woody plants such as Helleborus odoratus. It can provide trace elements, macroelements and vitamins required for plant growth. At the same time, sucrose provides energy and helps the growth and development of plant cells. Carrageenan helps the fixation and growth of Helleborus odoratus explants, and at the same time provides a uniform and nutritious environment, which is conducive to the induction and regeneration of adventitious buds of Helleborus odoratus petioles.

[0029] In a second aspect, the present invention provides an induction culture medium for inducing regeneration of adventitious buds from petioles of Helleborus truncatus, comprising 2-4 parts by mass of 6-benzylaminopurine, 2-4 parts by mass of chlorfenapyr, 0.5-2 parts by mass of zeatin, 0.5-1.5 parts by mass of 6-furfurylaminopurine, 0.1-0.3 parts by mass of indolebutyric acid, 400-900 parts by mass of multivitamin tablets, 6000-7000 parts by mass of a coagulant, 18000-22000 parts by mass of sucrose, and 950-1100 parts by volume of a nutrient culture medium;

[0030] Here, 1 part by mass: 1 part by volume = 1 mg: 1 mL.

[0031] The beneficial effects of the present invention are:

[0032] 1. The present invention provides an induction culture medium and an induction method for inducing the regeneration of adventitious buds from the petioles of Helleborus truncatus. For the first time, the petioles of Helleborus truncatus are used as explants for reproduction. This method causes minimal damage to the mother plant and is protective. At the same time, since the survival rate of the material is high and its source is wide, and it can better maintain the excellent traits of the mother plant, it is helpful to maintain the genetic stability of Helleborus truncatus and can achieve continuous rapid reproduction all year round. Therefore, the present invention has important practical value for the seedling breeding of Helleborus truncatus and is therefore particularly suitable for the breeding needs of Helleborus truncatus.

[0033] 2. The regenerated adventitious bud induction medium provided by the present invention includes: 3.0 parts by mass of 6-benzylaminopurine, 3.0 parts by mass of chlorfenapyr, 1.5 parts by mass of zeatin, 0.8 parts by mass of 6-furfurylaminopurine, 0.2 parts by mass of indolebutyric acid, 560 parts by mass of multivitamin tablets, 6500 parts by mass of coagulant, 20000 parts by mass of sucrose, and 1000 parts by volume of nutrient medium; the present invention adds zeatin and 6-furfurylaminopurine to the culture medium to promote cell division, which is conducive to inducing adventitious buds. At the same time, multivitamin tablets are also added to provide the required multivitamins for the petiole during the induction process, which significantly improves the induction success rate. The combination of these hormones is indispensable. They are combined with the induction method provided by the present invention, which can not only efficiently induce adventitious buds and achieve rapid reproduction, but also improve the reproduction efficiency and shorten the reproduction cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1This is a photo of the iron chopsticks of the present invention after removing leaves and trimming roots.

[0036] Figure 2 This is a photo of the iron chopsticks of the present invention after removing leaves and trimming roots and transplanting them into a planting medium.

[0037] Figure 3 It is a comparison photo of the softened petiole of the iron chopstick in the present invention and the normal (unsoftened) petiole (the left side is the softened petiole, and the right side is the normal (unsoftened) petiole).

[0038] Figure 4 This is a photograph of the experiment 2 of the present invention in which the regeneration adventitious bud induction medium provided by the present invention was not used and thus the induction was not successful.

[0039] Figure 5 This is a photograph of the experiment 3 of the present invention in which the regeneration adventitious bud induction medium provided by the present invention was not used and thus the induction was not successful.

[0040] Figure 6 This is a photograph of the induction of adventitious buds using the regenerated adventitious bud induction medium provided by the present invention in Experiment 7 of the present invention.

[0041] Figure 7 This is a photo of a plant that grew from adventitious buds. DETAILED DESCRIPTION

[0042] The technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiment of the present invention. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0043] Embodiment 1:

[0044] This embodiment provides a method for inducing adventitious bud regeneration from petioles of Helleborus truncatus, comprising:

[0045] S1. Clean the Helleborus plant, cut off the old petioles and bad roots, retain 8-10 cm of healthy roots, sterilize and disinfect the Helleborus roots, and then transplant them into the planting medium;

[0046] S2. After transplanting the Helleborus truncatus plants, wait for the rhizome buds of the Helleborus truncatus to germinate, soften and sterilize the petioles of the Helleborus truncatus, and obtain sterile Helleborus truncatus petiole explants for regenerating adventitious buds.

[0047] S3, inoculating the sterilized Helleborus petiole explants on a regenerated adventitious bud induction medium, and placing them under the conditions of 15-25° C., a light intensity of 1500-2000 LUX, a pH of 5.6-5.8, and a photoperiod of 8-10 hours, for induction culture for 30-40 days until adventitious buds are regenerated on the Helleborus petiole;

[0048] The regenerated adventitious bud induction medium comprises 2-4 parts by mass of 6-benzylaminopurine, 2-4 parts by mass of chlorfenapyr, 0.5-2 parts by mass of zeatin, 0.5-1.5 parts by mass of 6-furfurylaminopurine, 0.1-0.3 parts by mass of indolebutyric acid, 400-900 parts by mass of multivitamin tablets, 6000-7000 parts by mass of a coagulant, 18000-22000 parts by mass of sucrose and 950-1100 parts by volume of a nutrient medium;

[0049] Here, 1 part by mass: 1 part by volume = 1 mg: 1 mL.

[0050] The regenerated adventitious bud induction medium comprises 2.5 parts by mass of 6-benzylaminopurine, 3.8 parts by mass of chlorfenapyr, 1.8 parts by mass of zeatin, 1.2 parts by mass of 6-furfurylaminopurine, 0.3 parts by mass of indolebutyric acid, 490 parts by mass of multivitamin tablets, 6500 parts by mass of coagulant, 21000 parts by mass of sucrose and 980 parts by volume of nutrient medium;

[0051] Alternatively, in step S3, the regenerated adventitious bud induction medium comprises 3.0 parts by mass of 6-benzylaminopurine, 3.0 parts by mass of chlorfenapyr, 1.5 parts by mass of zeatin, 0.8 parts by mass of 6-furfurylaminopurine, 0.2 parts by mass of indolebutyric acid and 560 parts by mass of multivitamin tablets, 6500 parts by mass of a coagulant, 20000 parts by mass of sucrose and 1000 parts by volume of a nutrient medium;

[0052] Experiments have shown that the regenerated adventitious bud induction medium includes 4.0 parts by mass of 6-benzylaminopurine, 2.5 parts by mass of chlorfenapyr, 0.8 parts by mass of zeatin, 0.6 parts by mass of 6-furfurylaminopurine, 0.12 parts by mass of indolebutyric acid and 850 parts by mass of multivitamin tablets, 7000 parts by mass of coagulant, 22000 parts by mass of sucrose and 1100 parts by volume of nutrient medium. The combination of these plant growth regulators has a better effect in inducing the formation of adventitious buds.

[0053] Here, 1 part by mass: 1 part by volume = 1 mg: 1 mL.

[0054] In addition, in step S1, the Helleborus truncatus plant is cleaned until the root system is free of attachments, the old leaves and petioles of the Helleborus truncatus are cut off, the bad roots are cleaned, the bactericide is used for soaking and sterilization, and the plant is transplanted into a planting medium with 1-2 cm of the root system exposed, and the bactericide is 500-800 times of carbendazim or 1000-1500 times of potassium permanganate solution. The planting medium includes at least one of peat soil, perlite, and river sand.

[0055] Specifically, clean the iron chopstick plants, remove the growth medium supporting the iron chopstick plants, rinse with clean water until there is no attachment to the roots, cut off the old leaves and petioles of the iron chopsticks, leaving 0.5-1 cm, clean up dead roots, diseased roots, redundant capillary roots and other bad roots, trim the roots that are too long, leaving a length of 8-10 cm, soak and sterilize them in a fungicide for 5-10 minutes, dry the perennial roots in the shade until there is no liquid residue, and then transplant them to clean peat soil, perlite, river sand or their mixed media, with 1-2 cm of roots exposed.

[0056] In step S2, when the rhizome buds of the iron chopstick sprout and the leaves of the iron chopstick grow to 8-12 cm, the petioles of the iron chopstick are covered with a black film, then covered with an opaque container, and placed in a dark room for softening. The softened petioles of the iron chopstick are wiped with 70-75% alcohol, and then soaked in 1000 times mercuric chloride solution for 5-7 minutes, washed with sterile water for 3-5 times, and dried with sterile filter paper to obtain sterile iron chopstick petiole explants with regenerated adventitious buds.

[0057] Specifically, when the buds of the rhizomes or underground stems of the Helleborus plant begin to sprout, the softening treatment includes placing the petiole explant in a dark growth environment. The specific measures are to cover it with a black film and cover it with an opaque container, and then place it in a dark room. Then, use a 70-75% alcohol cotton ball to wipe the petiole explant material all over 1-2 times, then soak it in 1000 times mercuric chloride solution for 5-7 minutes, then wash it with sterile water 3-5 times, and finally use sterile filter paper to absorb the moisture, thereby obtaining a sterile Helleborus petiole explant with regenerated adventitious buds.

[0058] In step S3, the regenerated adventitious bud induction medium also includes the nutrient medium being WPM medium, and the coagulant being carrageenan.

[0059] Embodiment 2:

[0060] The present embodiment provides an induction culture medium for regenerating adventitious buds from petioles of Helleborus truncatus, comprising 2-4 parts by mass of 6-benzylaminopurine, 2-4 parts by mass of chlorfenapyr, 0.5-2 parts by mass of zeatin, 0.5-1.5 parts by mass of 6-furfurylaminopurine, 0.1-0.3 parts by mass of indolebutyric acid, 400-900 parts by mass of multivitamin tablets, 6000-7000 parts by mass of a coagulant, 18000-22000 parts by mass of sucrose, and 950-1100 parts by volume of a nutrient culture medium;

[0061] Here, 1 part by mass: 1 part by volume = 1 mg: 1 mL.

[0062] The present invention is further explained below in conjunction with experiments:

[0063] 1. Material preparation

[0064] Helleborus plants, peat soil, fungicide (70-75% alcohol and 1000 times mercuric chloride solution), sterile water, sterile filter paper, 6-benzylaminopurine, chlorfenapyr, zeatin, 6-furfurylaminopurine, indolebutyric acid, multivitamin tablets, carrageenan, sucrose, and WPM culture medium.

[0065] 2. Experimental Design

[0066] See also Figure 1-Figure 3 , select healthy and pest-free Helleborus plants, clean, remove leaves and roots, cut off bad roots, retain 8-10cm healthy roots, use fungicides to disinfect the roots of the plants, and transplant the disinfected plants into the planting medium. After the rhizomes and buds of the plants germinate and the leaves of the Helleborus grow to 8-12cm, cover the petioles of the Helleborus with black film and then cover them with opaque containers. Place them in a dark room for softening and then cut the petiole part. Use 70-75% alcohol cotton balls to wipe the petiole explant material 1-2 times. Soak the petiole explant in 1000 times mercuric chloride solution for 5-7 minutes. Wash with sterile water 3-5 times, absorb the water with sterile filter paper, and obtain sterile Helleborus petiole explants. The sterile petiole explants were inoculated on the regenerated adventitious bud induction medium. In this experiment, multiple groups of regenerated adventitious bud induction medium containing different formulas were set up to obtain the regenerated adventitious bud induction medium that can induce successful regeneration, and each group of regenerated adventitious bud induction medium was cultured at 22°C, light intensity 1800LUX, pH 5.7 and photoperiod of 9 hours. The induced culture was carried out for 35 days to observe the regeneration of adventitious buds on the petioles of Helleborus chinensis.

[0067] Control group: only WPM basal medium, no hormones

[0068] The experimental groups are as follows, to explore the effects of different hormones and their concentrations on the induction of adventitious bud regeneration from the petioles of Helleborus, in order to determine the best hormone combination and concentration, thereby optimizing the adventitious bud induction method of the petioles of Helleborus. In addition, the plant hormones explored in this experiment include 6-benzylaminopurine (6-BA), chlorpyrifos urone (CPPU), zeatin (ZT), 6-furfurylaminopurine (KT) and indolebutyric acid (IBA), as well as multivitamin tablets (Centrum).

[0069] Experimental group 1-all contained 1L WPM culture medium.

[0070]

[0071] 3. Conclusion

[0072] According to the test results, the following conclusions can be drawn: Figure 4 and Figure 5 The simple WPM basal medium failed to induce adventitious buds from the petioles of Helleborus chinensis, indicating that the addition of hormones is necessary for the induction of adventitious buds.

[0073] In the experiment on inducing adventitious buds from the petioles of Helleborus oleifera, test groups 1-5 failed to induce adventitious buds, indicating that these hormone combinations were not sufficient to support the formation of adventitious buds. However, starting from test groups 6-11, with the addition of specific hormones and the supplementation of multivitamin tablets, adventitious bud induction was successful. In particular, test group 11, whose formula: WPM+3.0mg / L6-BA+3.0mg / LCPPU+1.5mg / LZT+0.8mg / L KT+0.2mg / L IBA+0.56g / L had a suitable concentration ratio, successfully induced a large number of buds, such as Figure 6 As shown, Figure 7 Shown are plants grown from adventitious buds.

[0074] These results emphasize that a specific hormone combination and its appropriate concentration control are crucial for the induction of adventitious buds on the petioles of Helleborus. In particular, the addition of KT (6-furfurylaminopurine), ZT (zeatin) and multivitamin tablets have a positive effect on providing essential trace elements and nutrients and promoting plant growth. The addition of multivitamin tablets not only promotes plant growth, but also significantly improves the induction success rate. Therefore, this specific hormone combination combined with the induction method provided by the present invention has important practical value for the seedling breeding of Helleborus, helps to maintain the genetic stability of the plant, and can achieve continuous and rapid breeding all year round.

[0075] The present invention is not limited to the above-mentioned optional implementation modes. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that fall within the scope defined by the claims of the present invention fall within the protection scope of the present invention.

Claims

1. A method for inducing adventitious bud regeneration from petioles of Helleborus chinensis, characterized in that: The method comprises: S1. Clean the Helleborus plant, cut off the old petioles and bad roots, retain 8-10 cm of healthy roots, sterilize and disinfect the Helleborus roots, and then transplant them into the planting medium; S2, after transplanting the Helleborus truncatus plant, wait for the root and stem buds of the Helleborus truncatus to germinate, soften and sterilize the petioles of the Helleborus truncatus to obtain sterile Helleborus truncatus petiole explants for regenerating adventitious buds; S3, inoculating the sterilized Helleborus petiole explants on a regenerated adventitious bud induction medium, and placing them under the conditions of 15-25° C., a light intensity of 1500-2000 LUX, a pH of 5.6-5.8, and a photoperiod of 8-10 hours, for induction culture for 30-40 days until adventitious buds are regenerated on the Helleborus petiole; The regenerated adventitious bud induction medium comprises 2-4 parts by mass of 6-benzylaminopurine, 2-4 parts by mass of chlorfenapyr, 0.5-2 parts by mass of zeatin, 0.5-1.5 parts by mass of 6-furfurylaminopurine, 0.1-0.3 parts by mass of indolebutyric acid, 400-900 parts by mass of multivitamin tablets, 6000-7000 parts by mass of a coagulant, 18000-22000 parts by mass of sucrose and 950-1100 parts by volume of a nutrient medium; Here, 1 part by mass: 1 part by volume = 1 mg: 1 mL.

2. The method for inducing adventitious bud regeneration from petioles of Helleborus chinensis according to claim 1, characterized in that: In step S3, the regenerated adventitious bud induction medium includes 2.5 parts by mass of 6-benzylaminopurine, 3.8 parts by mass of chlorfenapyr, 1.8 parts by mass of zeatin, 1.2 parts by mass of 6-furfurylaminopurine, 0.3 parts by mass of indolebutyric acid, 490 parts by mass of multivitamin tablets, 6500 parts by mass of coagulant, 21000 parts by mass of sucrose and 980 parts by volume of nutrient medium; Here, 1 part by mass: 1 part by volume = 1 mg: 1 mL.

3. The method for inducing adventitious bud regeneration from petioles of Helleborus chinensis according to claim 1, characterized in that: In step S3, the regenerated adventitious bud induction medium includes 3.0 parts by mass of 6-benzylaminopurine, 3.0 parts by mass of chlorfenapyr, 1.5 parts by mass of zeatin, 0.8 parts by mass of 6-furfurylaminopurine, 0.2 parts by mass of indolebutyric acid, 560 parts by mass of multivitamin tablets, 6500 parts by mass of coagulant, 20000 parts by mass of sucrose and 1000 parts by volume of nutrient medium; Here, 1 part by mass: 1 part by volume = 1 mg: 1 mL.

4. The method for inducing adventitious bud regeneration from petioles of Helleborus chinensis according to claim 1, characterized in that: In step S3, the regenerated adventitious bud induction medium includes 4.0 parts by mass of 6-benzylaminopurine, 2.5 parts by mass of chlorfenapyr, 0.8 parts by mass of zeatin, 0.6 parts by mass of 6-furfurylaminopurine, 0.12 parts by mass of indolebutyric acid and 850 parts by mass of multivitamin tablets, 7000 parts by mass of a coagulant, 22000 parts by mass of sucrose and 1100 parts by volume of a nutrient medium; Here, 1 part by mass: 1 part by volume = 1 mg: 1 mL.

5. The method for inducing adventitious bud regeneration from petioles of Helleborus chinensis according to claim 1, characterized in that: In step S1, the Helleborus truncatus plant is cleaned until there is no attachment to the root system, the old leaves and petioles of the Helleborus truncatus are cut off, the bad roots are cleaned, the plant is soaked and sterilized with a fungicide, and the plant is transplanted into a planting medium with 1-2 cm of the root system exposed.

6. The method for inducing adventitious bud regeneration from petioles of Helleborus chinensis according to claim 5, characterized in that: The bactericide is 500-800 times of carbendazim or 1000-1500 times of potassium permanganate solution.

7. The method for inducing adventitious bud regeneration from petioles of Helleborus chinensis according to claim 1, characterized in that: In step S1, the planting medium includes at least one of peat soil, perlite, and river sand.

8. The method for inducing adventitious bud regeneration from petioles of Helleborus chinensis according to claim 1, characterized in that: In step S2, when the rhizome buds of the iron chopstick sprout and the leaves of the iron chopstick grow to 8-12 cm, the petioles of the iron chopstick are covered with a black film, then covered with a light-proof container, and placed in a dark room for softening. The softened petioles of the iron chopstick are wiped with 70-75% alcohol, and then soaked in 1000 times mercuric chloride solution for 5-7 minutes, washed with sterile water for 3-5 times, and dried with sterile filter paper to obtain sterile iron chopstick petiole explants with regenerated adventitious buds.

9. The method for inducing adventitious bud regeneration from petioles of Helleborus chinensis according to claim 1, characterized in that: In step S3, the regenerated adventitious bud induction medium also includes the nutrient medium being WPM medium, and the coagulant being carrageenan.

10. An induction culture medium for inducing the regeneration of adventitious buds from the petioles of Helleborus truncatus, characterized in that: The invention comprises 2-4 parts by weight of 6-benzylaminopurine, 2-4 parts by weight of chlorfenapyr, 0.5-2 parts by weight of zeatin, 0.5-1.5 parts by weight of 6-furfurylaminopurine, 0.1-0.3 parts by weight of indolebutyric acid, 400-900 parts by weight of multivitamin tablets, 6000-7000 parts by weight of a coagulant, 1800-2200 parts by weight of sucrose and 1000 parts by volume of a nutrient medium; Here, 1 part by mass: 1 part by volume = 1 mg: 1 ml.

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