A method for inducing callus and seedling from leaves of yunnan konjak and application thereof

Abstract

This invention provides a method for inducing callus and seedling formation using *Amorphophallus konjac* leaves as explants, and its application. The method for inducing callus using *Amorphophallus konjac* leaves as explants includes: inoculating *Amorphophallus konjac* leaves into a callus induction medium and culturing to obtain callus; the callus induction medium includes: 1 / 2 MS basal medium, naphthaleneacetic acid, 6-benzylaminopurine, thidiazuron, zeatin, sucrose, and agar. This invention uses *Amorphophallus konjac* leaves as explants for callus induction, which is convenient to obtain, and *Amorphophallus konjac* leaves are abundant, resulting in rapid propagation and high efficiency, greatly improving the quality and induction rate of callus.

Description

Technical Field

[0001] This invention belongs to the field of plant culture technology, and in particular relates to a method for inducing callus and seedlings using Amorphophallus konjac leaves as explants, and its application. Background Technology

[0002] The genus Amorphophallus is a large genus under the family Araceae, with approximately 300 species. Amorphophallus yunnanensis is a species within the genus Amorphophallus, mainly distributed in the Yunnan-Guizhou Plateau and adjacent areas of my country, with smaller distributions in surrounding areas such as Laos, Thailand, and northern Vietnam.

[0003] Konjac has wide applications in food, medicine, and chemical industries due to the high glucomannan content in its tubers, making it extremely valuable for development and utilization. With the development of the konjac processing industry, the demand for konjac raw materials is constantly increasing, driving the rapid development of konjac cultivation. Currently, in large-scale cultivation, asexual reproduction using tubers is the primary method of production. This not only requires a large amount of seed, has a low propagation coefficient, and high production costs, but also takes a long time, is difficult to store, and is susceptible to disease and rot after planting. Furthermore, long-term asexual reproduction also leads to quality degradation, seriously hindering production development and the promotion of new varieties.

[0004] Plant tissue culture, broadly speaking, refers to the technique of culturing isolated plant organs, tissues, cells, protoplasts, and even whole plants under sterile conditions on a specific culture medium. Its main characteristics are: (1) it is carried out in a culture container; (2) the sterile culture environment eliminates the invasion of microorganisms such as fungi, bacteria, and pests; (3) various environmental factors such as nutrients, hormones, and physical factors such as light and temperature are under artificial control and can reach optimal conditions; (4) it usually disrupts the normal plant development process and pattern.

[0005] CN102144562A discloses a method for efficient and rapid tissue culture propagation of konjac. The method involves inducing callus, adventitious buds and adventitious roots by in vitro culture of konjac corm cuttings, thereby forming a large number of konjac tissue culture seedlings in a short period of time. The tissue culture seedlings are then transplanted into a seedbed, supplemented with necessary agents and fertilizers, and the original konjac seed is obtained after normal seedling death.

[0006] CN116602213A discloses a tissue culture method for one-step seedling production from leaf corms of Amorphophallus bulbifera. By using the entire leaf corm of Amorphophallus bulbifera as explant, adding GA3 to the culture medium to break the dormancy of the leaf corm, promoting the germination of surface buds, and inducing bud differentiation with KT, seedlings can be produced in one step. The culture process does not require transfer bottles, which simplifies the steps of existing technologies, shortens the production cycle, and reduces the contamination rate and browning rate.

[0007] In existing technologies, explant corms are often used to induce callus. However, konjac corms grow underground, making them difficult to obtain. In addition, a single konjac plant usually has only one or a few corms, which greatly limits the number of explants that can be obtained. Furthermore, the formation and development of corms requires a long growth stage before they can be used for tissue culture. Therefore, using konjac corms as explants greatly limits the speed of konjac tissue culture.

[0008] In contrast, konjac leaves, which grow on the above-ground parts, are easy to observe and obtain. They can be continuously produced during the vigorous growth period of konjac, and the large number of leaves can provide a rich source of explants. However, because konjac leaves are exposed to the external environment for a long time, a large number of microorganisms will adhere to their surface, making disinfection difficult. Moreover, their active metabolic activity greatly affects the induction rate of callus tissue and the seedling rate when using leaves as explants.

[0009] Therefore, how to improve the efficiency of inducing callus and seedlings using konjac leaves as explants has become an urgent technical problem to be solved. Summary of the Invention

[0010] To address the shortcomings of existing technologies, the present invention aims to provide a method for inducing callus and seedling formation using *Amorphophallus konjac* leaves as explants, and its application. The method for inducing callus using *Amorphophallus konjac* leaves as explants provided by this invention can improve the quality and induction rate of callus, significantly enhancing propagation efficiency and quality.

[0011] To achieve this objective, the present invention employs the following technical solution:

[0012] In a first aspect, the present invention provides a method for inducing callus tissue using Amorphophallus konjac leaves as explants, the method comprising: inoculating Amorphophallus konjac leaves in a callus induction culture medium and culturing them to obtain callus tissue.

[0013] The callus induction medium includes: 1 / 2MS basal medium, naphthaleneacetic acid (NAA), 6-benzylaminopurine (6-BA), thidiazuron (TDZ), zeatin (ZT), sucrose, and agar.

[0014] 1 / 2MS basal medium provides essential nutritional support for callus induction from explants and a stable ionic environment for Amorphophallus konjac leaf cells, without causing abnormal explant growth due to nutrient overload. Sucrose is a carbon source in plant tissue culture, providing energy to cells and acting as an energy source and osmotic regulator. Agar, as a solidifying agent, maintains the morphological stability of the medium and increases its strength.

[0015] Meanwhile, naphthaleneacetic acid (NAA), 6-benzylaminopurine (6-BPA), thidiazuron, and zeatin were added to the culture medium to promote cell proliferation and differentiation, and improve the quality of callus tissue. NAA, as an auxin-like plant growth regulator, can stimulate the division and elongation of Amorphophallus konjac leaf cells; 6-BA, as a cytokinin, works in conjunction with auxins to regulate the balance between cell differentiation and proliferation. In the early stages of callus induction, 6-BA can promote the division of Amorphophallus konjac leaf cells, rapidly increasing the number of cells while maintaining callus viability; TDZ, as a plant growth regulator, has strong cytokinin activity and can also affect the sensitivity of cells to other hormones, thereby optimizing the induction and growth process of callus tissue; ZT, as a cytokinin, promotes cell division and growth, and improves the quality of callus tissue, resulting in a more compact cell structure and stronger cell viability.

[0016] In this invention, naphthaleneacetic acid, 6-benzylaminopurine, thiazolinone, and zeatin work together to enhance the formation speed and quality of callus tissue, providing a strong guarantee for improving the quality of Amorphophallus konjac seedlings.

[0017] Preferably, the callus induction culture medium, by mass-volume concentration, comprises: 1 / 2 MS basal medium, naphthaleneacetic acid 0.1-0.2 mg / L, 6-benzylaminopurine 2-3 mg / L, thiazidone 0.2-0.3 mg / L, zeatin 0.2-0.3 mg / L, sucrose 8-12 g / L, and agar 5-9 g / L.

[0018] Specific values ​​for the 0.1–0.2 mg / L range can be selected from 0.1 mg / L, 0.12 mg / L, 0.14 mg / L, 0.16 mg / L, 0.18 mg / L, and 0.2 mg / L, etc. Specific values ​​for the 2–3 mg / L range can be selected from 2 mg / L, 2.2 mg / L, 2.4 mg / L, 2.6 mg / L, 2.8 mg / L, and 3 mg / L, etc. Specific values ​​for the 0.2–0.3 mg / L range... Specific values ​​for the concentration can be selected from 0.2 mg / L, 0.22 mg / L, 0.24 mg / L, 0.26 mg / L, 0.28 mg / L, 0.3 mg / L, etc.; specific values ​​for the concentration range of 8–12 g / L can be selected from 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, etc.; and specific values ​​for the concentration range of 5–9 g / L can be selected from 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, etc.

[0019] Based on the potential interactions of the four hormones in improving the rate and quality of callus formation, when the components in the culture medium meet the above-mentioned specific concentration relationships, the synergistic relationship between naphthaleneacetic acid, 6-benzylaminopurine, thidiazuron, and zeatin can be better utilized to improve the quality of callus formation.

[0020] Preferably, the pH value of the callus induction culture medium is 5.5 to 6.5, for example, it can be 5.5, 5.7, 5.9, 6.1, 6.3, 6.5, etc.

[0021] Preferably, the culture temperature is 20–30°C and the humidity is 60–80%.

[0022] Among them, the specific point values ​​in the 20-30℃ range can be 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, etc., and the specific point values ​​in the 60-80% range can be 60%, 65%, 70%, 75%, 80%, etc.

[0023] Preferably, the step of inoculating the Yunnan konjac leaves into the callus induction medium further includes disinfecting the Yunnan konjac leaves.

[0024] Preferably, the disinfection process includes the following steps:

[0025] The explant Amorphophallus konjac leaves were sequentially washed with water, soaked in ethanol aqueous solution, filtered, soaked in sodium hypochlorite aqueous solution, filtered, soaked in dibromohydantoin aqueous solution, filtered, soaked in water, filtered, soaked in water a second time, and filtered.

[0026] Ethanol can quickly penetrate into microbial cells, denature proteins, and thus kill most bacteria and fungi. Sodium hypochlorite is a strong oxidant that can destroy the cell walls, cell membranes, and intracellular enzyme systems of microorganisms, thereby effectively killing various bacteria, fungi, and viruses. Dibromohydantoin can release bromide ions with bactericidal effects in water. These bromide ions can bind to the proteins and nucleic acids of microorganisms, interfere with the metabolic processes of microorganisms, and play a role in sterilization and disinfection.

[0027] In this invention, the leaves are soaked in an aqueous solution of ethanol, an aqueous solution of sodium hypochlorite, and an aqueous solution of dibromohydantoin in sequence, which greatly reduces the number of viruses and other microorganisms attached to the leaf surface and reduces the impact on the callus induction process.

[0028] Meanwhile, in this invention, after disinfectant treatment, soaking in water and a second soaking in water are performed respectively, which can better clean away the disinfectant residue on the surface and inside of the leaves, thereby reducing the impact of disinfectant on the callus induction process and improving the quality of callus induction.

[0029] Secondly, the present invention provides a callus tissue induced by the method of inducing callus tissue using Amorphophallus konjac leaves as explants as described in the first aspect.

[0030] Thirdly, the present invention provides a method for cultivating *Amorphophallus konjac* seedlings, the method comprising the following steps:

[0031] (1) Differentiation culture: The callus tissue described in the second aspect is inoculated into the differentiation culture medium for differentiation culture.

[0032] (2) Rooting culture: When the seedlings that have grown from the differentiation culture are 2-3 cm in length, they are cut and inoculated into the rooting culture medium to obtain seedlings.

[0033] (3) Transplanting and cultivation: When the roots are 1-2cm long, take out the seedlings and cultivate them in nutrient soil to obtain Yunnan konjac seedlings.

[0034] Among them, the specific point values ​​in the 2-3cm range can be 2cm, 2.2cm, 2.4cm, 2.6cm, 2.8cm, 3cm, etc., and the specific point values ​​in the 1-2cm range can be 1cm, 1.2cm, 1.4cm, 1.6cm, 1.8cm, 2cm, etc.

[0035] Preferably, the differentiation medium in step (1) comprises: MS basal medium, naphthaleneacetic acid (NAA), 6-benzylaminopurine (6-BA), sucrose and agar.

[0036] In this invention, adding naphthaleneacetic acid and 6-benzylaminopurine to MS basal medium can better regulate the balance of organ differentiation and improve the quality of differentiation culture.

[0037] Preferably, the differentiation medium in step (1) comprises, by mass-volume concentration: MS basal medium, naphthaleneacetic acid 0.3-0.5 mg / L, 6-benzylaminopurine 1-1.5 mg / L, sucrose 13-17 g / L and agar 4-8 g / L.

[0038] Specific values ​​for the 0.3–0.5 mg / L range can be 0.3 mg / L, 0.35 mg / L, 0.4 mg / L, 0.45 mg / L, 0.5 mg / L, etc.; specific values ​​for the 1–1.5 mg / L range can be 1 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, 1.5 mg / L, etc.; specific values ​​for the 13–17 g / L range can be 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, etc.; and specific values ​​for the 4–8 g / L range can be 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, etc.

[0039] Preferably, the pH value of the differentiation medium in step (1) is 5.5 to 6.5, for example, it can be 5.5, 5.7, 5.9, 6.1, 6.3, 6.5, etc.

[0040] Preferably, before inoculating the callus tissue described in the second aspect into the differentiation culture medium in step (1), the callus tissue is further cut into small pieces with a diameter of 0.3 to 0.5 cm.

[0041] Among them, the specific point values ​​in the range of 0.3 to 0.5 cm can be selected from 0.3 cm, 0.35 cm, 0.4 cm, 0.45 cm, 0.5 cm, etc.

[0042] Preferably, the rooting medium in step (2) includes: MS basal medium, naphthaleneacetic acid (NAA), indoleacetic acid (IAA), kinetin (KT), sucrose and agar.

[0043] MS medium provides energy support for root growth. NAA plays a key role in inducing root growth in the rooting medium, stimulating cell division and differentiation at the base of Amorphophallus konjac seedlings to form root primordia. IAA can induce cell differentiation and elongation, and participate in the physiological regulation of root growth. KT, as a cytokinin, can stimulate the division of root cells in Amorphophallus konjac seedlings, increase the number of root cells, and improve the efficiency of root cells in nutrient absorption and utilization.

[0044] In this invention, three hormones—naphthaleneacetic acid, indoleacetic acid, and kinetin—are added to MS basal medium. These three hormones work synergistically during the rooting culture process, improving the rooting rate of Amorphophallus yunnanensis and enhancing the quality of the seedling roots. This allows the seedlings to better absorb sufficient nutrients from the soil, thereby increasing the seedling survival rate.

[0045] Preferably, the rooting medium in step (2) comprises, by mass-volume concentration: MS basal medium, naphthaleneacetic acid 0.2-0.3 mg / L, indoleacetic acid 0.1-0.2 mg / L, kinetin 0.3-0.5 mg / L, sucrose 13-17 g / L and agar 4-8 g / L.

[0046] Specific values ​​within the 0.2–0.3 mg / L range can be 0.2 mg / L, 0.22 mg / L, 0.24 mg / L, 0.26 mg / L, 0.28 mg / L, 0.3 mg / L, etc. Specific values ​​within the 0.1–0.2 mg / L range can be 0.1 mg / L, 0.12 mg / L, 0.14 mg / L, 0.16 mg / L, 0.18 mg / L, 0.2 mg / L, etc. Specific values ​​for the 3–0.5 mg / L range can be 0.3 mg / L, 0.35 mg / L, 0.4 mg / L, 0.45 mg / L, 0.5 mg / L, etc.; specific values ​​for the 13–17 g / L range can be 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, etc.; and specific values ​​for the 4–8 g / L range can be 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, etc.

[0047] Based on the potential interaction of the three hormones in improving rooting rate, when the components in the culture medium meet the above-mentioned specific concentration relationships, the synergistic relationship between naphthaleneacetic acid, indoleacetic acid, and kinetin can be better utilized to improve rooting rate and root quality.

[0048] Preferably, the pH value of the rooting medium in step (2) is 5.5 to 6.5, for example, it can be 5.5, 5.7, 5.9, 6.1, 6.3, 6.5, etc.

[0049] Fourthly, the present invention provides an application of the method for inducing callus tissue using Amorphophallus konjac leaves as explants as described in the first aspect or the method for seedling formation of Amorphophallus konjac as described in the second aspect in the tissue culture of Amorphophallus konjac.

[0050] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] This invention uses Amorphophallus konjac leaves as explants to induce callus tissue. The material is readily available, and Amorphophallus konjac leaves are abundant, resulting in rapid propagation and high efficiency.

[0053] The callus induction medium provided by this invention contains four hormones—naphthaleneacetic acid, 6-benzylaminopurine, thiamethoxam, and zeatin—that work synergistically to improve the formation rate and quality of callus tissue, thus providing a sufficient guarantee for improving the quality of Amorphophallus konjac seedlings. When these hormones are added to 1 / 2 MS basal medium, the elemental concentrations in the medium are appropriate, allowing for better interaction with the four hormones, thereby improving the callus induction rate and the overall quality of the callus tissue.

[0054] Furthermore, when the concentrations of each hormone in the callus induction medium are controlled within the ranges of 0.1–0.2 mg / L for naphthaleneacetic acid, 2–3 mg / L for 6-benzylaminopurine, 0.2–0.3 mg / L for thiazidone, and 0.2–0.3 mg / L for zeatin, the synergistic relationship among the four hormones can be better utilized, thereby improving the callus induction rate and the overall quality of the callus.

[0055] Furthermore, disinfecting the leaves of *Amorphophallus konjac* sequentially with ethanol aqueous solution, sodium hypochlorite aqueous solution, and dibromohydantoin aqueous solution before inoculating them into the callus induction medium can significantly reduce the number of viruses and other microorganisms attached to the leaf surface, thus reducing their impact on the callus induction process. In addition, soaking in water and then soaking twice in water after disinfectant treatment can better wash away the disinfectant residues on the leaf surface and inside, thereby reducing the impact of disinfectant on the callus induction process.

[0056] Furthermore, during the seedling culture of callus tissue, three hormones—naphthaleneacetic acid, indoleacetic acid, and kinetin—were added to MS basal medium as a rooting medium. The three hormones worked together synergistically during the rooting culture process, which could further improve the rooting rate and root quality of Amorphophallus yunnanensis. Detailed Implementation

[0057] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0058] Example 1

[0059] This embodiment provides a method for inducing callus tissue using Amorphophallus konjac leaves as explants, including the following steps:

[0060] (1) Disinfection treatment of explant leaves: Fifty small leaves of Amorphophallus konjac were rinsed with tap water for 30 minutes and ultrapure water for 2 minutes in sequence; the leaves were trimmed to a length of 2.5 cm and a width of 0.8 cm, soaked in 75% ethanol aqueous solution for 2 minutes, filtered dry, soaked in 2% sodium hypochlorite aqueous solution for 15 minutes, filtered dry, soaked in 1 g / L dibromohydantoin aqueous solution for 5 minutes, and filtered dry; the leaves were then soaked in distilled water twice, each time for 2 minutes, and filtered dry after each soaking; the leaves were trimmed to a length of 1.2 cm and a width of 0.4 cm, and then cut and soaked in distilled water twice, for a total of 2 soakings, each time for 2 minutes, and filtered dry after each soaking, thus completing the disinfection treatment of explant leaves.

[0061] (2) Disinfected Amorphophallus konjac leaves were inoculated into callus induction medium to obtain callus tissue. The formula of the callus induction medium was as follows: 1 / 2 MS basal medium, 0.1 mg / L naphthaleneacetic acid, 2.0 mg / L 6-benzylaminopurine, 0.25 mg / L thidiazuron, 0.25 mg / L zeatin, 10 g / L sucrose, and 5 g / L agar, with a pH of 6; the culture temperature was 25℃ and the humidity was 70%.

[0062] Example 2

[0063] This embodiment provides a method for inducing callus tissue using Amorphophallus konjac leaves as explants, including the following steps:

[0064] (1) Disinfection treatment of explant leaves: Fifty small leaves of Amorphophallus konjac were rinsed with tap water for 25 minutes and ultrapure water for 4 minutes in sequence; the leaves were trimmed to 2 cm long and 1 cm wide, soaked in 75% ethanol aqueous solution for 3 minutes, filtered dry, soaked in 1% sodium hypochlorite aqueous solution for 17 minutes, filtered dry, soaked in 1.5 g / L dibromohydantoin aqueous solution for 3 minutes, and filtered dry; the leaves were then soaked in distilled water 3 times, 2 minutes each time, and filtered dry after each soaking; the leaves were trimmed to 1 cm long and 0.5 cm wide, and then cut and soaked in distilled water twice, for a total of 2 soakings, 3 minutes each time, and filtered dry after each soaking, thus completing the disinfection treatment of explant leaves.

[0065] (2) Disinfected Amorphophallus konjac leaves were inoculated into callus induction medium to obtain callus tissue. The formula of the callus induction medium was as follows: 1 / 2 MS basal medium, 0.1 mg / L naphthaleneacetic acid, 3 mg / L 6-benzylaminopurine, 0.2 mg / L thidiazuron, 0.2 mg / L zeatin, 12 g / L sucrose and 5 g / L agar, with a pH of 5.5; the culture temperature was 20℃ and the humidity was 80%.

[0066] Example 3

[0067] This embodiment provides a method for inducing callus tissue using Amorphophallus konjac leaves as explants, including the following steps:

[0068] (1) Disinfection treatment of explant leaves: Fifty small leaves of Amorphophallus konjac were rinsed with tap water for 35 min and ultrapure water for 5 min in sequence; the leaves were trimmed to 3 cm long and 0.5 cm wide, soaked in 75% ethanol aqueous solution for 3 min, filtered dry, soaked in 1% sodium hypochlorite aqueous solution for 20 min, filtered dry, soaked in 0.8 g / L dibromohydantoin aqueous solution for 4 min, and filtered dry; the leaves were then soaked in distilled water twice, each time for 3 min, and filtered dry after each soak; the leaves were trimmed to 1.5 cm long and 0.3 cm wide, and then cut and soaked in distilled water twice, for a total of 3 soaks, each time for 2 min, and filtered dry after each soak, thus completing the disinfection treatment of explant leaves.

[0069] (2) Disinfected Amorphophallus konjac leaves were inoculated into callus induction medium to obtain callus tissue. The formula of the callus induction medium was as follows: 1 / 2 MS basal medium, 0.2 mg / L naphthaleneacetic acid, 2 mg / L 6-benzylaminopurine, 0.3 mg / L thidiazuron, 0.3 mg / L zeatin, 8 g / L sucrose and 6 g / L agar, with a pH of 6.5; the culture temperature was 30℃ and the humidity was 70%.

[0070] Example 4

[0071] This embodiment provides a method for inducing callus tissue using Amorphophallus konjac leaves as explants. The only difference between this method and Example 1 is that the ethanol aqueous solution soaking and filtration are not performed in step (1), the soaking time of the leaves in sodium hypochlorite aqueous solution is adjusted to 16.5 min, and the soaking time of the leaves in dibromohydantoin aqueous solution is adjusted to 5.5 min. The remaining steps are the same as in Example 1.

[0072] Example 5

[0073] This embodiment provides a method for inducing callus tissue using Amorphophallus konjac leaves as explants. The only difference between this method and Example 1 is that the sodium hypochlorite aqueous solution soaking and filtration are not performed in step (1), the soaking time in the ethanol aqueous solution is adjusted to 6 min, and the soaking time in the dibromohydantoin aqueous solution is adjusted to 16 min. The remaining steps are consistent with Example 1.

[0074] Example 6

[0075] This embodiment provides a method for inducing callus tissue using Amorphophallus konjac leaves as explants. The only difference between this method and Example 1 is that in step (1), the dibromohydantoin aqueous solution is not used for soaking and filtration. The soaking time in the ethanol aqueous solution is adjusted to 3 min, and the soaking time in the sodium hypochlorite aqueous solution is adjusted to 19 min. The remaining steps are consistent with Example 1.

[0076] Example 7

[0077] This embodiment provides a method for inducing callus tissue using Amorphophallus konjac leaves as explants. The only difference between this embodiment and Embodiment 1 is that the disinfection treatment in step (1) is not performed. After the Amorphophallus konjac leaves are trimmed to a length of 1.2 cm and a width of 0.4 cm, callus tissue culture is performed directly. The callus tissue culture steps in step (2) are consistent with those in Embodiment 1.

[0078] Example 8

[0079] This embodiment provides a method for inducing callus tissue using Amorphophallus konjac leaves as explants. The only difference between this method and Example 1 is that the second soaking in distilled water is not performed in step (1). That is, the disinfection treatment of the explant leaves is completed after trimming the leaves to a length of 1.2 cm and a width of 0.4 cm. The remaining steps are consistent with Example 1.

[0080] Example 9

[0081] This embodiment provides a method for inducing callus tissue using Amorphophallus konjac leaves as explants. The only difference between this method and Example 1 is that the formula of the callus induction medium in step (2) is: 1 / 2 MS basal medium, naphthaleneacetic acid 0.25 mg / L, 6-benzylaminopurine 3.2 mg / L, thidiazuron 0.15 mg / L, zeatin 0.1 mg / L, sucrose 10 g / L and agar 5 g / L. The remaining steps are consistent with Example 1.

[0082] Example 10

[0083] This embodiment provides a method for inducing callus tissue using Amorphophallus konjac leaves as explants. The only difference between this method and Example 1 is that the formula of the callus induction medium in step (2) is: 1 / 2 MS basal medium, naphthaleneacetic acid 0.08 mg / L, 6-benzylaminopurine 1.8 mg / L, thidiazuron 0.32 mg / L, zeatin 0.4 mg / L, sucrose 10 g / L and agar 5 g / L. The remaining steps are consistent with Example 1.

[0084] Comparative Example 1

[0085] This comparative example provides a method for inducing callus using Amorphophallus konjac leaves as explants. The only difference between this method and Example 1 is that the callus induction medium in step (2) does not include naphthaleneacetic acid. The medium formula is: 1 / 2 MS basal medium, 6-benzylaminopurine 2.08 mg / L, thidiazuron 0.26 mg / L, zeatin 0.26 mg / L, sucrose 10 g / L and agar 5 g / L. The remaining steps are consistent with those in Example 1.

[0086] Comparative Example 2

[0087] This comparative example provides a method for inducing callus using Amorphophallus konjac leaves as explants. The only difference between this method and Example 1 is that the callus induction medium in step (2) does not include 6-benzylaminopurine. That is, the medium formula is: 1 / 2 MS basal medium, naphthaleneacetic acid 0.43 mg / L, thidiazuron 1.08 mg / L, zeatin 1.08 mg / L, sucrose 10 g / L and agar 5 g / L. The remaining steps are consistent with Example 1.

[0088] Comparative Example 3

[0089] This comparative example provides a method for inducing callus using Amorphophallus konjac leaves as explants. The only difference between this method and Example 1 is that the callus induction medium in step (2) does not include thiabendazole. The medium formula is: 1 / 2 MS basal medium, 0.11 mg / L naphthaleneacetic acid, 2.21 mg / L 6-benzylaminopurine, 0.28 mg / L zeatin, 10 g / L sucrose and 5 g / L agar. The remaining steps are the same as in Example 1.

[0090] Comparative Example 4

[0091] This comparative example provides a method for inducing callus using Amorphophallus konjac leaves as explants. The only difference between this method and Example 1 is that the callus induction medium in step (2) does not include zeatin. The medium formula is: 1 / 2 MS basal medium, naphthaleneacetic acid 0.11 mg / L, 6-benzylaminopurine 2.21 mg / L, thidiazuron 0.28 mg / L, sucrose 10 g / L and agar 5 g / L.

[0092] Comparative Example 5

[0093] This comparative example provides a method for inducing callus using Amorphophallus konjac leaves as explants. The only difference between this method and Example 1 is that in step (2), 1 / 2 MS basal medium is replaced with MS basal medium in the callus induction medium. The formula of the medium is: MS basal medium, naphthaleneacetic acid 0.1 mg / L, 6-benzylaminopurine 2.0 mg / L, thidiazuron 0.25 mg / L, zeatin 0.25 mg / L, sucrose 10 g / L and agar 5 g / L. The remaining steps are the same as in Example 1.

[0094] Comparative Example 6

[0095] This comparative example provides a method for inducing callus tissue using Amorphophallus konjac corms as explants. The only difference between this method and Example 1 is that the explant leaves are replaced with explant corms, while the remaining steps are the same as in Example 1.

[0096] Application Example 1

[0097] This application example provides a method for cultivating Yunnan konjac seedlings, including the following steps:

[0098] (1) Differentiation Culture: The callus tissue obtained by the method of inducing callus tissue in Example 1 was cut into small pieces with a side length of 0.4 cm and inoculated into differentiation medium for differentiation culture. The formulation of the differentiation medium was: MS basal medium, 0.4 mg / L naphthaleneacetic acid, 1.2 mg / L 6-benzylaminopurine, 15 g / L sucrose and 6 g / L agar, with a pH of 6; the differentiation culture temperature was 25℃ and the humidity was 70%.

[0099] (2) Rooting culture: When the seedlings that have grown from the differentiation culture reach a length of 2.5 cm, they are cut and inoculated into rooting medium to obtain seedlings. The formula of the rooting medium is as follows: MS basal medium, 0.25 mg / L naphthaleneacetic acid, 0.15 mg / L indoleacetic acid, 0.4 mg / L kinetin, 15 g / L sucrose and 6 g / L agar, with a pH of 6; the temperature for rooting culture is 26℃ and the humidity is 75%.

[0100] (3) Transplanting and cultivation: When the roots are 1.5cm long, take out the seedlings, wash off the culture medium on the seedlings, and cultivate them in nutrient soil. The temperature for transplanting and cultivation is 22℃ and the humidity is 60%. Weed regularly and apply compound fertilizer in time. After 1 month, Yunnan konjac seedlings will be obtained.

[0101] Application Example 2

[0102] This application example provides a method for cultivating Yunnan konjac seedlings, including the following steps:

[0103] (1) Differentiation Culture: The callus tissue obtained by the method in Example 2 was cut into small pieces with a side length of 0.3 cm and inoculated into differentiation medium for differentiation culture. The differentiation medium formula was: MS basal medium, 0.3 mg / L naphthaleneacetic acid, 1.5 mg / L 6-benzylaminopurine, 13 g / L sucrose and 8 g / L agar, with a pH of 5.5; the differentiation culture temperature was 26℃ and the humidity was 75%.

[0104] (2) Rooting culture: When the seedlings grown from the differentiation culture reach a length of 2 cm, they are cut and inoculated into rooting medium to obtain seedlings. The formula of the rooting medium is as follows: MS basal medium, naphthaleneacetic acid 0.2 mg / L, indoleacetic acid 0.2 mg / L, kinetin 0.3 mg / L, sucrose 13 g / L and agar 8 g / L, with a pH of 5.5; the rooting culture temperature is 26℃ and the humidity is 80%.

[0105] (3) Transplanting and cultivation: When the roots are 1cm long, take out the seedlings, wash off the culture medium on the seedlings, and cultivate them in nutrient soil. The temperature for transplanting and cultivation is 23℃ and the humidity is 65%. Weed regularly and apply compound fertilizer in time to obtain Yunnan konjac seedlings.

[0106] Application Example 3

[0107] This application example provides a method for cultivating Yunnan konjac seedlings, including the following steps:

[0108] (1) Differentiation Culture: The callus tissue obtained by the method in Example 3 was cut into small pieces with a side length of 0.5 cm and inoculated into differentiation medium for differentiation culture. The differentiation medium formula was: MS basal medium, 0.5 mg / L naphthaleneacetic acid, 1 mg / L 6-benzylaminopurine, 17 g / L sucrose and 4 g / L agar, with a pH of 6.5; the differentiation culture temperature was 27℃ and the humidity was 78%.

[0109] (2) Rooting culture: When the seedlings grown from the differentiation culture reach a length of 3 cm, they are cut and inoculated into rooting medium to obtain seedlings. The formula of the rooting medium is as follows: MS basal medium, naphthaleneacetic acid 0.3 mg / L, indoleacetic acid 0.1 mg / L, kinetin 0.5 mg / L, sucrose 17 g / L and agar 4 g / L, with a pH of 6.5; the rooting culture temperature is 26℃ and the humidity is 80%.

[0110] (3) Transplanting and cultivation: When the roots are 2cm long, take out the seedlings, wash off the culture medium on the seedlings, and cultivate them in nutrient soil. The temperature for transplanting and cultivation is 24℃ and the humidity is 80%. Weed regularly and spray foliar fertilizer to obtain Yunnan konjac seedlings.

[0111] Application Example 4-10

[0112] Application Examples 4-10 each provide a method for cultivating *Amorphophallus yunnanensis* seedlings. The only difference between them and Application Example 1 is that in step (1), the callus tissue obtained by the callus induction method of Example 1 is replaced with the callus tissue obtained by the callus induction method of Examples 4-10. The remaining steps are consistent with Application Example 1.

[0113] Application Example 11

[0114] This application example provides a method for cultivating Yunnan konjac seedlings. The only difference between this method and application example 1 is that the rooting medium in step (2) does not contain naphthaleneacetic acid. That is, the medium formula is: MS basal medium, indoleacetic acid 0.22 mg / L, kinetin 0.58 mg / L, sucrose 15 g / L and agar 6 g / L, with a pH of 6.

[0115] Application Example 12

[0116] This application example provides a method for cultivating *Amorphophallus yunnanensis* seedlings. The only difference between this method and Application Example 1 is that the rooting medium in step (2) does not contain indoleacetic acid. That is, the medium formula is: MS basal medium, naphthaleneacetic acid 0.31 mg / L, kinetin 0.49 mg / L, sucrose 15 g / L and agar 6 g / L, with a pH of 6.

[0117] Application Example 13

[0118] This application example provides a method for cultivating *Amorphophallus yunnanensis* seedlings. The only difference between this method and application example 1 is that the rooting medium in step (2) does not contain kinetin. The medium formula is: MS basal medium, naphthaleneacetic acid 0.5 mg / L, indoleacetic acid 0.3 mg / L, sucrose 15 g / L and agar 6 g / L, with a pH of 6.

[0119] Comparative Application Examples 1-6

[0120] Comparative Application Examples 1-6 each provide a method for cultivating *Amorphophallus yunnanensis* seedlings. The only difference between them and Application Example 1 is that the callus obtained by the callus induction method of Example 1 in step (1) is replaced by the callus obtained by the callus induction method of Comparative Examples 1-6. The remaining steps are consistent with Application Example 1.

[0121] Test Example 1

[0122] This test case examines the callus induction rate of each method used in the various examples / comparative examples, as well as the rooting rate and seedling rate in each application example / comparative application example. The formulas for calculating the callus induction rate, rooting rate, and seedling rate are as follows:

[0123] Callus induction rate = (Number of explants with callus formation / Number of inoculated explants) × 100%;

[0124] Rooting rate = Number of rooted seedlings / Number of seedlings inoculated in rooting medium for rooting culture × 100%;

[0125] Seedling survival rate = Number of seedlings that survive after transplanting / Total number of transplanted seedlings × 100%.

[0126] The results of callus induction rate, rooting rate and seedling rate for each implementation method are shown in the table below:

[0127]

[0128]

[0129] Data from Example 1 / Application Example 1 and Comparative Examples 1-4 show that when any one of naphthaleneacetic acid, 6-benzylaminopurine, thiazolinone, or zeatin is missing from the callus induction medium, the callus induction rate is significantly reduced. This indicates that the four hormones naphthaleneacetic acid, 6-benzylaminopurine, thiazolinone, and zeatin have a significant synergistic effect in improving the callus induction rate using Amorphophallus konjac leaves as explants. They can also improve the quality of callus tissue, thereby increasing the rooting rate and seedling survival rate of seedlings.

[0130] Data from Example 1 / Application Example 1 and Comparative Example 5 show that, compared to MS basal medium, 1 / 2MS basal medium has suitable concentrations of macro- and micro-elements, which can better complement naphthaleneacetic acid, 6-benzylaminopurine, thidiazuron, and zeatin, thereby improving the callus induction rate and the overall quality of callus.

[0131] Data from Example 1 / Application Example 1 and Comparative Example 6 show that, under the action of a callus induction medium containing 1 / 2 MS basal medium, naphthaleneacetic acid, 6-benzylaminopurine, thidiazuron, zeatin, sucrose, and agar, the callus induction rate was significantly higher when using Amorphophallus konjac leaves as explants compared to using Amorphophallus konjac corms as explants. Furthermore, the rooting rate and seedling rate were higher.

[0132] Data from Examples / Applications 1 and 4-7 show that disinfecting Amorphophallus konjac leaves before callus culture can reduce the impact of viruses and other microorganisms on the callus culture process, thereby increasing the callus induction rate. For the explant leaf disinfection process, under the condition that the total soaking time in the disinfectant is consistent, soaking the explant leaves sequentially in an ethanol aqueous solution, a sodium hypochlorite aqueous solution, and a dibromohydantoin aqueous solution can further improve the disinfection effect, reduce the number of microorganisms, and increase the callus induction rate.

[0133] As can be seen from the data in Examples / Applications 1 and 8, for the disinfection process of explant leaves, soaking the leaves twice in distilled water after trimming can better wash away the disinfectant residues on the leaf surface and inside, thereby reducing the impact of disinfectant on the callus induction process and improving the quality of callus induction.

[0134] Data from Examples / Applications 1 and 9-10 show that when the concentrations of each hormone in the callus induction medium are controlled within the ranges of 0.1-0.2 mg / L for naphthaleneacetic acid, 2-3 mg / L for 6-benzylaminopurine, 0.2-0.3 mg / L for thidiazuron, and 0.2-0.3 mg / L for zeatin, the synergistic relationship between the four hormones can be better utilized, thereby improving the callus induction rate and the overall quality of the callus.

[0135] Data from Application Examples 1 and 11-13 show that when any one of naphthaleneacetic acid, indoleacetic acid, or kinetin is missing from the rooting medium, the seedling rooting rate decreases significantly. This indicates that naphthaleneacetic acid, indoleacetic acid, and kinetin have a significant synergistic effect on the rooting process of Amorphophallus konjac seedlings, which can greatly improve the rooting rate. At the same time, the root quality of seedlings rooted under specific culture media is better, and they can better absorb sufficient nutrients from the soil during the transplanting stage, thereby improving the seedling survival rate.

[0136] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0137] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0138] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for inducing callus tissue using Amorphophallus konjac leaves as explants, characterized in that, The method includes: inoculating Amorphophallus konjac leaves into a callus induction medium and culturing them to obtain callus tissue; The callus induction medium, by mass-volume concentration, consists of 1 / 2 MS basal medium, naphthaleneacetic acid 0.1-0.2 mg / L, 6-benzylaminopurine 2-3 mg / L, thiazidone 0.2-0.3 mg / L, zeatin 0.2-0.3 mg / L, sucrose 8-12 g / L, and agar 5-9 g / L; The process of inoculating the Yunnan konjac leaves into the callus induction medium also includes disinfecting the Yunnan konjac leaves. The disinfection process includes the following steps: the explant Amorphophallus konjac leaves are sequentially washed with water, soaked in an ethanol aqueous solution, filtered, soaked in a sodium hypochlorite aqueous solution, filtered, soaked in a dibromohydantoin aqueous solution, filtered, soaked in water, filtered, soaked a second time in water, and filtered.

2. The method for inducing callus tissue using Amorphophallus konjac leaves as explants according to claim 1, characterized in that, The pH value of the callus induction culture medium is 5.5~6.

5.

3. The method for inducing callus tissue using Amorphophallus konjac leaves as explants according to claim 1, characterized in that, The culture temperature is 20~30℃ and the humidity is 60~80%.

4. A method for cultivating *Amorphophallus konjac* seedlings, characterized in that, The method for cultivating Yunnan konjac seedlings includes the following steps: (1) Differentiation culture: The callus obtained by the method according to any one of claims 1-3 is inoculated in differentiation culture medium for differentiation culture; (2) Rooting culture: When the seedlings that have grown from the differentiation culture are 2-3 cm in length, they are cut and inoculated into the rooting culture medium to obtain seedlings; (3) Transplanting and cultivation: When the roots are 1-2 cm long, take out the seedlings and cultivate them in nutrient soil to obtain Yunnan konjac seedlings; The differentiation medium in step (1) is MS basal medium, naphthaleneacetic acid 0.3~0.5 mg / L, 6-benzylaminopurine 1~1.5 mg / L, sucrose 13~17 g / L and agar 4~8 g / L, based on mass-volume concentration. The rooting medium in step (2) is MS basal medium, naphthaleneacetic acid 0.2~0.3 mg / L, indoleacetic acid 0.1~0.2 mg / L, kinetin 0.3~0.5 mg / L, sucrose 13~17 g / L and agar 4~8 g / L.

5. The method for cultivating *Amorphophallus konjac* seedlings according to claim 4, characterized in that, The pH value of the differentiation medium in step (1) is 5.5~6.

5.

6. The method for cultivating *Amorphophallus konjac* seedlings according to claim 4, characterized in that, Before inoculating the callus tissue into the differentiation culture medium in step (1), the callus tissue is cut into small pieces with a diameter of 0.3~0.5cm.

7. The method for cultivating *Amorphophallus konjac* seedlings according to claim 4, characterized in that, The pH value of the rooting medium in step (2) is 5.5~6.

5.

8. The application of a method for inducing callus using Amorphophallus konjac leaves as explants as described in any one of claims 1 to 3, or the method for seedling formation of Amorphophallus konjac as described in any one of claims 4 to 7, in the tissue culture of Amorphophallus konjac.

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