An induction method and an induction medium for inducing iron ladle petiole to regenerate adventitious buds

Abstract

The application provides an induction method and an induction medium for inducing adventitious buds of leaf petioles of Asparagus cochinchinensis, and solves the technical problem of low efficiency of inducing germination of adventitious buds of rhizomes or aerial stems in the prior art. The induction method of the adventitious buds of the leaf petioles of the Asparagus cochinchinensis comprises the following steps: sterilizing and disinfecting leaf petiole explants and transplanting and transferring the leaf petiole explants to an induction medium of the adventitious buds, wherein the induction medium of the adventitious buds comprises 6-benzylaminopurine, forchlorfenuron, zeatin, 6-furfurylaminopurine, indole-3-butyric acid, a multi-vitamin tablet, a coagulant, sucrose and a nutrient medium. The method uses the leaf petiole of the Asparagus cochinchinensis as the explant, can efficiently induce the adventitious buds, establishes a plant regeneration technical system of the Asparagus cochinchinensis, realizes rapid propagation, has high regeneration efficiency, and has abundant sources of the explants. Meanwhile, the method establishes a mature, stable and efficient regeneration system of the Asparagus cochinchinensis, and has important significance for transgenic breeding of the Asparagus cochinchinensis.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to an induction method and induction culture medium for inducing the regeneration of adventitious buds from the petioles of Helleborus thunbergii. Background Technology

[0002] Iron chopsticks Helleborus ×hybridus It belongs to the genus *Heliotropium* of the family Ranunculaceae. Helleborus The Christmas rose is a perennial herbaceous plant, with about 22 species worldwide, mostly distributed in Europe, and only one species in my country. It has uniquely shaped leaves, petal-like sepals, and a rich variety of flower colors and shapes. Under natural conditions, it blooms in late winter and early spring, making it highly ornamental.

[0003] Helleborine can be propagated through sowing and division. Seed propagation results in offspring exhibiting variations and segregation, failing to maintain the same traits as the parent plant. While division can preserve parent plant traits, it is slow and inefficient. With the advancement of hybridization breeding of Helleborine, a large number of unique and highly ornamental hybrids are selected annually, severely hindering their mass production and commercial distribution through division. Current tissue culture techniques for Helleborine use rhizomes or shoots from above-ground stems as explants, which is destructive, yields limited material, and is highly damaging to the parent plant, often causing plant death. This method is unsuitable for very small numbers or single plants of parent material. Summary of the Invention

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

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a method for inducing adventitious bud regeneration from the petiole of *Hemlock*, comprising:

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

[0008] S2. After transplanting the Helleborus plants, wait for the rhizomes to sprout, then soften and sterilize the petioles of the Helleborus plants to obtain sterile Helleborus petiole explants with regenerated adventitious buds.

[0009] S3. The sterile Helleborus petiole explants are inoculated onto the regeneration adventitious bud induction medium and placed at 15-25℃, light intensity of 1500-2000 LUX, pH of 5.6-5.8 and photoperiod of 8-10 hours for 30-40 days until adventitious buds regenerate on the Helleborus petiole.

[0010] The regenerated adventitious shoot induction medium consists of 2-4 parts by weight of 6-benzylaminopurine, 2-4 parts by weight of chlorpyrifos, 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 coagulant, 18000-22000 parts by weight of sucrose, and 950-1100 parts by volume of nutrient medium.

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

[0012] In a preferred embodiment, in step S3, the regenerated adventitious bud induction medium consists of 2.5 parts by weight of 6-benzylaminopurine, 3.8 parts by weight of chlorpyrifos, 1.8 parts by weight of zeatin, 1.2 parts by weight of 6-furfurylaminopurine, 0.3 parts by weight of indolebutyric acid, 490 parts by weight of multivitamin tablets, 6500 parts by weight of coagulant, 21000 parts by weight of sucrose, and 980 parts by volume of nutrient medium.

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

[0014] In a preferred embodiment, in step S3, the regenerated adventitious shoot induction medium consists of 3.0 parts by weight of 6-benzylaminopurine, 3.0 parts by weight of chlorpyrifos, 1.5 parts by weight of zeatin, 0.8 parts by weight of 6-furfurylaminopurine, 0.2 parts by weight of indolebutyric acid, 560 parts by weight of multivitamin tablets, 6500 parts by weight of coagulant, 20000 parts by weight of sucrose, and 1000 parts by volume of nutrient medium.

[0015] Wherein, 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 plant growth regulators, namely 3.0 mg / L of 6-benzylaminopurine, 3.0 mg / L of chlorpyrifos, 1.5 mg / L of zeatin, 0.8 mg / L of 6-furfurylaminopurine, 0.2 mg / L of indolebutyric acid, 0.56 g / L of multivitamin tablets, coagulant, sucrose, and nutrient culture medium, has a better effect on inducing the formation of adventitious buds.

[0017] In a preferred embodiment, in step S3, the regenerated adventitious shoot induction medium consists of 4.0 parts by weight of 6-benzylaminopurine, 2.5 parts by weight of chlorpyrifos, 0.8 parts by weight of zeatin, 0.6 parts by weight of 6-furfurylaminopurine, 0.12 parts by weight of indolebutyric acid, 850 parts by weight of multivitamin tablets, 7000 parts by weight of coagulant, 22000 parts by weight of sucrose, and 1100 parts by volume of nutrient medium.

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

[0019] In the preferred embodiment, in step S1, the hellebore plant is cleaned until there are no attached substances on the roots, old leaves and petioles are removed, bad roots are cleaned, the plant is soaked in a fungicide for sterilization, and then transplanted into the planting medium with the roots exposed for 1-2 cm.

[0020] Based on the above methods, cleaning the Helleborus plant can remove soil and other attachments from the roots, reducing the potential threat of pathogens and pests and providing a more hygienic growing environment for the plant; pruning old leaves and petioles, as well as cleaning dead roots, diseased roots, and redundant fine roots, helps reduce the plant's burden, prevents ineffective nutrient consumption, and promotes the development of healthy roots; soaking the plant in a fungicide can eliminate any pathogens that may be present, reducing the risk of disease infection and improving the survival rate; exposing the roots to 1-2 cm helps induce the growth of new roots, as new roots usually sprout more easily near the ground, which helps the plant better absorb water and nutrients.

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

[0022] Based on the above methods, carbendazim can interfere with the mitosis of pathogens, affecting cell division and exerting a bactericidal effect, effectively preventing and controlling various crop diseases caused by fungi. Potassium permanganate can effectively kill various vegetative bacteria, fungi, and tubercle bacilli, exhibiting strong bactericidal and disinfectant effects. Appropriate dilutions of both fungicides can be used for the sterilization and disinfection of Helleborus plants, reducing disease occurrence and improving the growth quality and health of Helleborus plants.

[0023] In a preferred embodiment, in step S1, the planting medium includes at least one of peat moss, perlite, and river sand.

[0024] Based on the above scheme, the porous structure of peat moss and perlite is beneficial for the respiration of the roots of Helleborus plants, while preventing waterlogging and reducing disease. Peat moss's water and fertilizer retention properties effectively hold moisture and nutrients, meeting the growth needs of Helleborus. Perlite can absorb 2-3 times its own weight in water, keeping the soil moist, and its chemical stability prevents reaction with nutrient solutions. It is sterile, contains no organic matter, avoids mold growth, and regulates soil temperature, protecting the root system. River sand, due to its loose, breathable, and permeable properties, is suitable for Helleborus seedling cultivation, cuttings, and planting. It is an economical and widely available planting medium suitable for planting at all scales. Therefore, all three planting media are good choices for planting Helleborus.

[0025] In the preferred embodiment, in step S2, when the rhizomes of *Heliotropium indicum* sprout and the leaves grow to 8-12 cm, the petioles of *Heliotropium indicum* are covered with a black film, then covered with an opaque container and placed in a dark room for softening treatment. The softened petioles are wiped with 70-75% alcohol, then soaked in a 1000-fold diluted mercuric chloride solution for 5-7 minutes, rinsed 3-5 times with sterile water, and the moisture is absorbed with sterile filter paper to obtain sterile *Heliotropium indicum* petiole explants with regenerated adventitious buds.

[0026] Based on the above scheme, by using black film covering and dark chamber treatment to simulate the natural shaded growth environment, the petioles of *Heliotropium indicum* can be softened, making them more tender and facilitating hormone absorption, thus promoting the formation of adventitious buds. This treatment method induces the production of adventitious buds. Subsequently, wiping with 70-75% alcohol and soaking in a 1000-fold diluted mercuric chloride solution effectively kills any pathogenic microorganisms that may be present on the surface of the petioles, reducing the risk of contamination. Alcohol and mercuric chloride, as commonly used disinfectants, can effectively kill bacteria and fungi, thereby improving the reproductive efficiency of *Heliotropium indicum*.

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

[0028] Based on the above scheme, WPM (Woody Plant Medium) is a culture medium specifically designed for the tissue culture of woody plants. It modifies MS medium by replacing potassium nitrate with potassium sulfate and reducing the ammonium nitrate content, ensuring that nitrogen salts are primarily supplied in the form of calcium nitrate. This low-salt-concentration medium is suitable for the induction and growth of adventitious buds in woody plants such as Helleborus, providing the micronutrients, macronutrients, and vitamins necessary for plant growth. Sucrose provides energy, which is beneficial for plant cell growth and development. Carrageenan helps with the fixation and growth of Helleborus explants, while also providing a uniform and nutrient-rich environment, which is conducive to the induction and regeneration of adventitious buds on Helleborus petioles.

[0029] Secondly, the present invention provides an induction culture medium for inducing the regeneration of adventitious buds from the petioles of Helleborus thunbergii, comprising 2-4 parts by weight of 6-benzylaminopurine, 2-4 parts by weight of chlorpyrifos, 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 coagulant, 18000-22000 parts by weight of sucrose, and 950-1100 parts by volume of nutrient culture medium;

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

[0031] The beneficial effects of this invention are as follows:

[0032] 1. This invention provides an induction culture medium and method for inducing adventitious bud regeneration from Helleborus petioles. For the first time, Helleborus petioles are used as explants for propagation. This method causes minimal damage to the mother plant and is protective. Furthermore, due to the high survival rate and wide availability of the material, it effectively maintains the superior traits of the parent plant, contributing to the genetic stability of Helleborus and enabling continuous, rapid propagation year-round. Therefore, this invention has significant practical value for Helleborus seedling propagation and is particularly suitable for the propagation needs of Helleborus.

[0033] 2. The regeneration adventitious bud induction culture medium provided by this invention consists of: 3.0 parts by weight of 6-benzylaminopurine, 3.0 parts by weight of chlorpyrifos, 1.5 parts by weight of zeatin, 0.8 parts by weight of 6-furfurylaminopurine, 0.2 parts by weight of indolebutyric acid, 560 parts by weight of multivitamin tablets, 6500 parts by weight of solidifying agent, 20000 parts by weight of sucrose, and 1000 parts by volume of nutrient medium. The addition of zeatin and 6-furfurylaminopurine to the culture medium promotes cell division, which is beneficial for inducing adventitious buds. Simultaneously, the addition of multivitamin tablets provides the petioles with the necessary vitamins during the induction process, significantly improving the induction success rate. This combination of hormones is indispensable; their combination with the induction method provided by this invention not only efficiently induces adventitious buds and achieves rapid propagation, but also improves propagation efficiency and shortens the propagation cycle. Attached Figure Description

[0034] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This is a photo of the iron chopsticks after removing the leaves and trimming the roots, as described in this invention.

[0036] Figure 2 This is a photo of the *Heliotropium indicum* transplanted into the planting medium after the leaves and roots have been removed in this invention.

[0037] Figure 3 These are comparison photos of the petioles of the iron chopsticks in this invention after softening and normal (unsoftened) petioles (the left side is the softened petiole, and the right side is the normal (unsoftened) petiole).

[0038] Figure 4 This is a photograph of Experiment 2 in this invention, which failed to induce adventitious shoots because the regenerated adventitious shoot induction medium provided by this invention was not used.

[0039] Figure 5 This is a photograph of Experiment 3 in this invention, which failed to induce adventitious shoots because the regenerated adventitious shoot induction medium provided by this invention was not used.

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

[0041] Figure 7 This is a photo of a plant that has grown from adventitious buds. Detailed Implementation

[0042] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the protection scope of the present invention.

[0043] Example 1:

[0044] This embodiment provides a method for inducing adventitious bud regeneration from the petioles of *Hemlock*, including:

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

[0046] S2. After transplanting the Helleborus plants, wait for the rhizomes to sprout, then soften and sterilize the petioles of the Helleborus plants to obtain sterile Helleborus petiole explants with regenerated adventitious buds.

[0047] S3. The sterile Helleborus petiole explants are inoculated onto the regeneration adventitious bud induction medium and placed at 15-25℃, light intensity of 1500-2000 LUX, pH of 5.6-5.8 and photoperiod of 8-10 hours for 30-40 days until adventitious buds regenerate on the Helleborus petiole.

[0048] The regenerated adventitious shoot induction medium consists of 2-4 parts by weight of 6-benzylaminopurine, 2-4 parts by weight of chlorpyrifos, 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 coagulant, 18000-22000 parts by weight of sucrose, and 950-1100 parts by volume of nutrient medium.

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

[0050] The regenerated adventitious shoot induction medium consists of 2.5 parts by weight of 6-benzylaminopurine, 3.8 parts by weight of chlorpyrifos, 1.8 parts by weight of zeatin, 1.2 parts by weight of 6-furfurylaminopurine, 0.3 parts by weight of indolebutyric acid, 490 parts by weight of multivitamin tablets, 6500 parts by weight of coagulant, 21000 parts by weight of sucrose, and 980 parts by volume of nutrient medium.

[0051] Alternatively, in step S3, the regenerated adventitious shoot induction medium consists of 3.0 parts by weight of 6-benzylaminopurine, 3.0 parts by weight of chlorpyrifos, 1.5 parts by weight of zeatin, 0.8 parts by weight of 6-furfurylaminopurine, 0.2 parts by weight of indolebutyric acid, 560 parts by weight of multivitamin tablets, 6500 parts by weight of coagulant, 20000 parts by weight of sucrose, and 1000 parts by volume of nutrient medium.

[0052] Experiments have shown that the regenerated adventitious bud induction medium, composed of 4.0 parts by weight of 6-benzylaminopurine, 2.5 parts by weight of chlorpyrifos, 0.8 parts by weight of zeatin, 0.6 parts by weight of 6-furfurylaminopurine, 0.12 parts by weight of indolebutyric acid, 850 parts by weight of multivitamin tablets, 7000 parts by weight of coagulant, 22000 parts by weight of sucrose, and 1100 parts by volume of nutrient medium, has a better effect on inducing the formation of adventitious buds.

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

[0054] In addition, in step S1, the hellebore plant is cleaned until the roots are free of any attached substances, old leaves and petioles are removed, and unhealthy roots are cleaned. It is then soaked in a fungicide for sterilization and transplanted into the planting medium, with the roots exposed for 1-2 cm. The fungicide is a 500-800 times dilution of carbendazim or a 1000-1500 times dilution of potassium permanganate solution. The planting medium includes at least one of peat moss, perlite, and river sand.

[0055] Specifically, clean the helleborus plant, remove the growing medium supporting the plant, rinse with clean water until the roots are free of any attached substances, cut off old leaves and petioles, leaving 0.5-1 cm, remove dead roots, diseased roots, and redundant fine roots, trim excessively long roots to 8-10 cm, soak in a fungicide for 5-10 minutes to sterilize, and then transplant the rootstock into a clean peat moss, perlite, river sand or a mixture thereof, exposing 1-2 cm of the roots.

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

[0057] Specifically, after the buds of the rhizomes or underground stems of the Helleborus plant begin to sprout, the softening treatment involves placing the petiole explant in a dark growing environment. The specific measures are to cover it with a black film and cover it with an opaque container, then place it in a dark room. Next, wipe the petiole explant material with cotton balls soaked in 70-75% alcohol 1-2 times, then soak it in a 1000-fold diluted mercuric chloride solution for 5-7 minutes, then rinse it with sterile water 3-5 times, and finally blot dry with sterile filter paper, thus obtaining sterile Helleborus petiole explants with regenerated adventitious buds.

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

[0059] Example 2:

[0060] This embodiment provides an induction culture medium for regenerating adventitious buds from the petioles of *Helianthus angustifolius*, which consists of 2-4 parts by weight of 6-benzylaminopurine, 2-4 parts by weight of chlorpyrifos, 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 coagulant, 18000-22000 parts by weight of sucrose, and 950-1100 parts by volume of nutrient culture medium.

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

[0062] The invention will be further explained and illustrated below with reference to experiments:

[0063] 1. Material preparation

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

[0065] 2. Experimental Design

[0066] See Figures 1-3 Select healthy, pest-free *Helleborus* plants, clean them, remove leaves and trim roots, cutting away any unhealthy roots and retaining 8-10cm of healthy roots. Disinfect the plant roots with a fungicide, then transplant the disinfected plants into the planting medium. Once the plant's rootstock and stem buds have sprouted and the leaves have grown to 8-12cm, cover the petioles with black film and then cover them with an opaque container. Place them in a dark room to soften them before cutting off the petioles. Wipe the petiole explant material with cotton balls soaked in 70-75% alcohol 1-2 times. Soak the petiole explant in a 1000-fold diluted mercuric chloride solution for 5-7 minutes. Rinse 3-5 times with sterile water, then blot dry with sterile filter paper to obtain sterile *Helleborus* petiole explants. Sterile petiole explants were inoculated onto a regeneration adventitious bud induction medium. In this experiment, multiple groups of regeneration adventitious bud induction media with different formulations were set up to obtain a regeneration adventitious bud induction medium that could successfully induce regeneration adventitious buds. Each group of regeneration adventitious bud induction media was cultured at 22℃, light intensity of 1800LUX, pH of 5.7 and photoperiod of 9 hours for 35 days, and the regeneration of adventitious buds on the petioles of Helleborus thunbergii was observed.

[0067] Control group: containing only WPM basal culture medium, without hormones.

[0068] The experimental group is as follows: This study investigated the effects of different hormones and their concentrations on the induction of adventitious buds from Helleborus petioles, aiming to determine the optimal hormone combination and concentration, thereby optimizing the method for inducing adventitious buds from Helleborus petioles. Additionally, the plant hormones investigated in this experiment included 6-benzylaminopurine (6-BA), chlorpyrifos (CPPU), zeatin (ZT), 6-furfurylaminopurine (KT), and indolebutyric acid (IBA), as well as multivitamins (Centrum).

[0069] Experimental groups 1-11 all contained 1 L WPM culture medium.

[0070] Culture medium formulation growth status 1 WPM+3.0mg6-BA+3.0mgCPPU+0.2mg IBA No buds, induction failed. 2 WPM+2.8mg6-BA+2.4mg CPPU+0.2mgIBA +0.5mg ZT No buds, induction failed. 3 WPM+2.6mg6-BA+3.2mg CPPU0.2mg IBA+0.7mg KT+ No buds, induction failed. 4 WPM+2.2mg6-BA+2.6mg CPPU+0.3mgIBA+0.5mg ZT+0.7mg KT No buds, induction failed. 5 WPM+2.8mg6-BA+2.4mg CPPU+0.2mgIBA+0.5mg ZT+0.7mg KT No buds, induction failed. 6 WPM + 3.9mg 6-BA + 2.5mg CPPU + 0.12mg IBA + 0.8mg ZT + 0.6mg KT + 0.4g Multivitamin Tablets A small number of buds were successfully induced. 7 WPM + 2.5mg 6-BA + 3.8mg CPPU + 0.3mg IBA + 1.8mg ZT + 1.2mg KT + 0.56g Multivitamin Tablets A small number of buds were successfully induced. 8 WPM + 3.5mg 6-BA + 2.9mg CPPU + 0.2mg IBA + 0.7mg ZT + 1.3mg KT + 0.48g Multivitamin Tablets A small number of buds were successfully induced. 9 WPM + 2.9mg 6-BA + 3.1mg CPPU + 0.3mg IBA + 2.4mg ZT + 1.4mg KT + 0.57g Multivitamin Tablets A small number of buds were successfully induced. 10 WPM + 3.2mg 6-BA + 2.2mg CPPU + 0.1mg IBA + 1.3mg ZT + 1.0mg KT++ + 0.6g Multivitamin Tablets A small number of buds were successfully induced. 11 WPM + 3.0 mg / L 6-BA + 3.0 mg / L PPU + 0.2 mg / L IBA + 1.5 mg / L ZT + 0.8 mg / L KT + 0.56 g / L Multivitamin Tablets The number of buds was high, indicating successful induction.

[0071] 3. Conclusion

[0072] Based on the experimental results, the following conclusions can be drawn: (See also) Figure 4 and Figure 5 The WPM basal medium alone failed to induce adventitious buds in Helleborus petioles, indicating that the addition of hormones is necessary for the induction of adventitious buds.

[0073] In the experiment on the induction of adventitious buds on the petioles of *Heliotropium indicum*, groups 1-5 failed to induce adventitious buds, indicating that the hormone combinations were insufficient to support adventitious bud formation. However, starting from groups 6-11, with the addition of specific hormones and the supplementation of multivitamins, adventitious bud induction was successful. In particular, group 11, with its appropriate formulation of WPM + 3.0 mg / L 6-BA + 3.0 mg / L PPU + 1.5 mg / L ZT + 0.8 mg / L KT + 0.2 mg / L IBA + 0.56 g / L, successfully induced a large number of buds, such as... Figure 6 As shown, Figure 7 The plant was shown as a result of adventitious buds.

[0074] These results highlight the crucial role of specific hormone combinations and appropriate concentration control in inducing adventitious buds on Helleborus petioles. In particular, the addition of KT (6-furfurylaminopurine), ZT (zeatin), and multivitamins plays a positive role in providing essential micronutrients and promoting plant growth. The addition of multivitamins not only promotes plant growth but also significantly improves the induction success rate. Therefore, this specific hormone combination, coupled with the induction method provided by this invention, has significant practical value for the propagation of Helleborus seedlings, helping to maintain the genetic stability of the plant and enabling continuous, rapid propagation throughout the year.

[0075] This invention is not limited to the above-described optional embodiments. Anyone can derive other various forms of products under the guidance of this invention. However, regardless of any changes made in their shape or structure, any technical solution that falls within the scope of the claims of this invention shall be protected by this invention.

Claims

1. A method for inducing adventitious bud regeneration from the petioles of *Hematoxylin and elegans*, characterized in that, The method includes: S1. Clean the Helleborus plant, cut off old petioles and bad roots, retain 8-10cm of healthy roots, sterilize the Helleborus roots and then transplant them into the planting medium. S2. After transplanting the Helleborus plants, wait for the rhizomes of Helleborus to sprout, soften and sterilize the petioles of Helleborus to obtain sterile Helleborus petiole explants with regenerated adventitious buds. S3. The sterile Helleborus petiole explants are inoculated onto the regeneration adventitious bud induction medium and placed at 15-25℃, light intensity of 1500-2000 LUX, pH of 5.6-5.8 and photoperiod of 8-10 hours for 30-40 days until adventitious buds regenerate on the Helleborus petiole. The regenerated adventitious shoot induction medium consists of 2-4 parts by weight of 6-benzylaminopurine, 2-4 parts by weight of chlorpyrifos, 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 coagulant, 18000-22000 parts by weight of sucrose, and 950-1100 parts by volume of nutrient medium. Wherein, 1 part by mass : 1 part by volume = 1 mg : 1 mL.

2. The method for inducing adventitious bud regeneration from the petiole of *Hematoxylin and elegans* according to claim 1, characterized in that: In step S3, the regenerated adventitious shoot induction medium consists of 2.5 parts by weight of 6-benzylaminopurine, 3.8 parts by weight of chlorpyrifos, 1.8 parts by weight of zeatin, 1.2 parts by weight of 6-furfurylaminopurine, 0.3 parts by weight of indolebutyric acid, 490 parts by weight of multivitamin tablets, 6500 parts by weight of coagulant, 21000 parts by weight of sucrose, and 980 parts by volume of nutrient medium. Wherein, 1 part by mass : 1 part by volume = 1 mg : 1 mL.

3. The method for inducing adventitious bud regeneration from the petiole of *Hematoxylin and elegans* according to claim 1, characterized in that: In step S3, the regenerated adventitious shoot induction medium consists of 3.0 parts by weight of 6-benzylaminopurine, 3.0 parts by weight of chlorpyrifos, 1.5 parts by weight of zeatin, 0.8 parts by weight of 6-furfurylaminopurine, 0.2 parts by weight of indolebutyric acid, 560 parts by weight of multivitamin tablets, 6500 parts by weight of coagulant, 20000 parts by weight of sucrose, and 1000 parts by volume of nutrient medium. Wherein, 1 part by mass : 1 part by volume = 1 mg : 1 mL.

4. The method for inducing adventitious bud regeneration from the petiole of *Hemlock* according to claim 1, characterized in that: In step S3, the regenerated adventitious shoot induction medium consists of 4.0 parts by weight of 6-benzylaminopurine, 2.5 parts by weight of chlorpyrifos, 0.8 parts by weight of zeatin, 0.6 parts by weight of 6-furfurylaminopurine, 0.12 parts by weight of indolebutyric acid, 850 parts by weight of multivitamin tablets, 7000 parts by weight of coagulant, 22000 parts by weight of sucrose, and 1100 parts by volume of nutrient medium. Wherein, 1 part by mass : 1 part by volume = 1 mg : 1 mL.

5. The method for inducing adventitious bud regeneration from the petiole of *Hematoxylin and elegans* according to claim 1, characterized in that, In step S1, clean the hellebore plant until there are no attached substances on the roots, cut off the old leaves and petioles, clean the bad roots, soak it in a fungicide to sterilize it, and transplant it into the planting medium, exposing the roots 1-2 cm.

6. The method for inducing adventitious bud regeneration from the petiole of *Hematoxylin and elegans* according to claim 5, characterized in that, The bactericide is a 500-800 times dilution of carbendazim or a 1000-1500 times dilution of potassium permanganate solution.

7. The method for inducing adventitious bud regeneration from the petiole of *Hemlock* according to claim 1, characterized in that, In step S1, the planting medium includes at least one of peat moss, perlite, and river sand.

8. The method for inducing adventitious bud regeneration from the petiole of *Hematoxylin and elegans* according to claim 1, characterized in that, In step S2, when the rhizomes of *Heliotropium indicum* sprout and the leaves grow to 8-12 cm, the petioles of the *Heliotropium indicum* are covered with a black film, then covered with an opaque container and placed in a dark room for softening. The softened petioles are wiped with 70-75% alcohol, then soaked in a 1000-fold diluted mercuric chloride solution for 5-7 minutes, rinsed with sterile water 3-5 times, and dried with sterile filter paper to obtain sterile *Heliotropium indicum* petiole explants with regenerated adventitious buds.

9. The method for inducing adventitious bud regeneration from the petiole of *Hematoxylin and elegans* according to claim 1, characterized in that, In step S3, the regenerated adventitious bud induction medium further includes WPM medium as the nutrient medium and carrageenan as the coagulant.

10. An induction culture medium for inducing the regeneration of adventitious buds from the petioles of *Hemlock*, characterized in that, It consists of 2-4 parts by weight of 6-benzylaminopurine, 2-4 parts by weight of chlorpyrifos, 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 coagulant, 1800-2200 parts by weight of sucrose, and 1000 parts by volume of nutrient culture medium; Wherein, 1 part by mass : 1 part by volume = 1 mg : 1 ml.

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