Method for inducing calluses and seedlings by taking konjac yunnanensis leaves as explants and application of method for inducing calluses and seedlings by taking konjac yunnanensis leaves as explants

Abstract

The invention provides a method for inducing calluses and seedlings by taking konjac yunnanensis leaves as explants and application of the method. The method for inducing the calluses by taking the konjac yunnanensis leaves as the explants comprises the following steps: inoculating the konjac yunnanensis leaves into a callus induction culture medium for culturing to obtain the calluses; the callus induction culture medium comprises a 1 / 2 MS basal culture medium, naphthylacetic acid, 6-benzylamino adenine, thidiazuron, zeatin, cane sugar and agar. According to the method, the konjac yunnanensis leaves are used as explants to induce the callus, materials are convenient to obtain, the number of the konjac yunnanensis leaves is large, the propagation speed is high, the efficiency is high, and the quality of the callus and the induction rate of the callus are greatly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant cultivation, and particularly relates to a method for inducing callus and seedling formation by taking Konjac konjac leaves as explants and application thereof. Background Art

[0002] Arum is a large genus under the Araceae family, with about 300 species. Yunnan konjac is a species under the genus Arum. It is mainly distributed in my country's Yunnan-Guizhou Plateau and neighboring areas, and in small quantities in neighboring Laos, Thailand, northern Vietnam and other regions.

[0003] Konjac has a wide range of applications in food, medicine and chemical industry because it contains a large amount of glucomannan in its tuber, and development and utilization value is extremely high. Along with the development of konjac processing industry, the demand for konjac raw material continues to increase, has driven the rapid development of konjac planting industry. In the current large-scale promotion planting, mainly use tuber to carry out asexual reproduction in production, not only the amount of seeds is large, the reproduction coefficient is low, the production cost is high, and the time is long, the storage difficulty of seed taro, the susceptible disease rot after sowing, and long-term asexual reproduction also causes quality degradation, which seriously hinders the development of production and the promotion of new varieties.

[0004] Plant tissue culture refers to the technology of culturing isolated plant organs, tissues, cells, protoplasts and even complete plants on a specific culture medium under sterile conditions. Its main characteristics are: (1) It is carried out in a culture container; (2) The sterile culture environment excludes the invasion of microorganisms such as fungi, bacteria and pests; (3) Various environmental factors such as nutritional factors, hormone factors and physical factors such as light and temperature are under artificial control and can reach the optimal conditions; (4) The normal plant development process and pattern are usually disrupted.

[0005] CN102144562A discloses a method for the efficient and rapid tissue culture propagation of konjac, which comprises the steps of in vitro culture of konjac corm cut materials to induce the production of callus, adventitious buds and adventitious roots, thereby forming a large number of konjac tissue culture seedlings in a relatively short period of time, transplanting the tissue culture seedlings into a seedbed, supplemented with necessary drugs and fertilizers, and obtaining konjac stock after normal seedling settling.

[0006] CN116602213A discloses a tissue culture method for one-step seedling formation of leaf corms of pearly konjac. The method comprises the following steps: selecting the entire leaf corm of pearly konjac as an explant, adding GA3 to a culture medium to break the dormancy of the leaf corm, promoting the buds on the surface to sprout, and inducing bud differentiation with KT to achieve one-step seedling formation. The culture process does not require bottle transfer, thus simplifying the steps of the prior art, shortening the production cycle, and reducing the contamination rate and browning rate.

[0007] In the prior art, explant corms are often used to induce callus tissue. Then, konjac corms grow underground and are difficult to obtain. At the same time, a konjac plant usually has only one or a few corms, which greatly limits the number of explants that can be obtained. In addition, the formation and development of the corms require a long growth stage before they can be used for tissue culture. Therefore, using konjac corms as explants greatly limits the tissue culture speed of konjac.

[0008] On the contrary, konjac leaves grow above ground, are easy to observe and obtain, can be continuously produced during the vigorous growth period of konjac, and have a large number of leaves, which can provide a rich source of explants. However, due to long-term exposure to the external environment, a large number of microorganisms will adhere to the surface of konjac leaves, making it difficult to disinfect, and the metabolic activity is active, which greatly affects the induction rate and seedling rate of callus induction using leaves as explants.

[0009] Therefore, how to improve the efficiency of inducing callus and seedling formation using konjac leaves as explants has become a technical problem that needs to be solved urgently. Summary of the invention

[0010] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for inducing callus and seedlings using Konjac leaves as explants and its application. The method for inducing callus using Konjac leaves as explants provided by the present invention can improve the quality of callus and callus induction rate, and greatly improve the reproduction efficiency and reproduction quality.

[0011] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0012] In a first aspect, the present invention provides a method for inducing callus using Konjac konjac leaves as explants, the method comprising: inoculating Konjac konjac leaves in a callus induction medium for cultivation to obtain callus.

[0013] The callus induction medium comprises: 1 / 2MS basic medium, naphthylacetic acid (NAA), 6-benzylaminoadenine (6-BA), thidiazuron (TDZ), zeatin (ZT), sucrose and agar.

[0014] 1 / 2MS basal medium can provide basic nutritional support for explant callus induction and a stable ion environment for Konjac leaf cells without causing abnormal growth of explants due to excess nutrition. Sucrose is a carbon source in plant tissue culture, providing energy for cells and acting as an energy source and osmotic regulator. Agar, as a coagulant, can maintain the morphological stability of the culture medium and increase the strength of the culture medium.

[0015] At the same time, naphthylacetic acid, 6-benzylaminoadenine, thiadiazole and zeatin were added to the culture medium to promote cell proliferation and differentiation and improve the quality of callus. Among them, NAA, as an auxin plant growth regulator, can stimulate the division and elongation of Konjac leaf cells; 6-BA, as a cytokinin, can work together with auxin to regulate the balance of cell differentiation and proliferation. In the early stage of callus induction, 6-BA can promote the division of Konjac leaf cells, increase the number of cells rapidly, and maintain the vitality of callus; TDZ, as a plant growth regulator, has strong cytokinin activity and can affect the sensitivity of cells to other hormones, thereby optimizing the induction and growth process of callus; ZT, as a cytokinin, has the effect of promoting cell division and growth, and can improve the quality of callus, making the cell structure of callus more compact and the cell vitality stronger.

[0016] In the present invention, naphthylacetic acid, 6-benzylaminoadenine, thidiazuron and zeatin cooperate with each other to synergistically enhance the efficiency, thereby jointly improving the formation speed and quality of callus tissue, and providing sufficient guarantee for improving the quality of Yunnan konjac seedlings.

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

[0018] Among them, the specific point values ​​in 0.1-0.2mg / L can be selected from 0.1mg / L, 0.12mg / L, 0.14mg / L, 0.16mg / L, 0.18mg / L, 0.2mg / L, etc., the specific point values ​​in 2-3mg / L can be selected from 2mg / L, 2.2mg / L, 2.4mg / L, 2.6mg / L, 2.8mg / L, 3mg / L, etc., 0.2-0.3mg / L The specific point values ​​in the range of 8 to 12 g / L can be selected from 8g / L, 9g / L, 10g / L, 11g / L, 12g / L, etc., and the specific point values ​​in the range of 5 to 9 g / L can be selected from 5g / L, 6g / L, 7g / L, 8g / L, 9g / L, etc.

[0019] Based on the potential interaction of the four hormones in improving the formation speed and quality of callus tissue, when the components in the culture medium meet the above-mentioned specific concentration relationship, the synergistic relationship between naphthylacetic acid, 6-benzylaminoadenine, thidiazuron and zeatin can be better exerted to improve the formation quality of callus tissue.

[0020] Preferably, the pH value of the callus induction medium is 5.5-6.5, 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 range of 20 to 30°C can be selected from 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, etc., and the specific point values ​​in the range of 60 to 80% can be selected from 60%, 65%, 70%, 75%, 80%, etc.

[0023] Preferably, the method further comprises disinfecting the Konjac leaves before inoculating the Konjac leaves into the callus induction medium.

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

[0025] The explant konjac leaves are sequentially washed with water, soaked in an ethanol aqueous solution, drained, soaked in a sodium hypochlorite aqueous solution, drained, soaked in a dibromohydantoin aqueous solution, drained, soaked in water, drained, soaked in water twice, and drained.

[0026] Ethanol can quickly penetrate into microbial cells and denature proteins, thereby killing most bacterial and fungal nutrients; sodium hypochlorite is a strong oxidant that can destroy microbial cell walls, cell membranes, and intracellular enzyme systems, thereby effectively killing various bacteria, fungi, and viruses; dibromocyanate can release bromide ions with bactericidal effects in water. These bromide ions can combine with microbial proteins and nucleic acids, interfere with the metabolic process of microorganisms, and play a role in sterilization and disinfection.

[0027] In the present invention, ethanol aqueous solution, sodium hypochlorite aqueous solution and dibromohydantoin aqueous solution are used sequentially for soaking, which greatly reduces the number of microorganisms such as viruses attached to the leaf surface and reduces the influence on the callus induction process.

[0028] At the same time, in the present invention, after the disinfectant treatment, water soaking and water secondary soaking are respectively performed, which can better clean the disinfectant remaining on the surface and inside of the leaves, thereby reducing the influence of the disinfectant water on the callus induction process, and further improving the callus induction quality.

[0029] In a second aspect, the present invention provides a callus tissue induced by the method for inducing callus tissue using Konjac konjac leaves as explants as described in the first aspect.

[0030] In a third aspect, the present invention provides a method for growing seedlings of Konjac yunnanensis, the method comprising the following steps:

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

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

[0033] (3) Transplanting and cultivation: When the root length is 1 to 2 cm, the seedlings are taken out and cultivated in nutrient soil to obtain Yunnan konjac seedlings.

[0034] Among them, the specific point values ​​in 2 to 3 cm can be selected from 2cm, 2.2cm, 2.4cm, 2.6cm, 2.8cm, 3cm, etc., and the specific point values ​​in 1 to 2cm can be selected from 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-benzylaminoadenine (6-BA), sucrose and agar.

[0036] In the present invention, naphthylacetic acid and 6-benzylaminoadenine are added to the MS basic culture medium, which can better regulate the balance of organ differentiation and improve the quality of differentiation culture.

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

[0038] Among them, the specific point values ​​in 0.3-0.5mg / L can be selected from 0.3mg / L, 0.35mg / L, 0.4mg / L, 0.45mg / L, 0.5mg / L, etc., the specific point values ​​in 1-1.5mg / L can be selected from 1mg / L, 1.1mg / L, 1.2mg / L, 1.3mg / L, 1.4mg / L, 1.5mg / L, etc., the specific point values ​​in 13-17g / L can be selected from 13g / L, 14g / L, 15g / L, 16g / L, 17g / L, etc., and the specific point values ​​in 4-8g / L can be selected from 4g / L, 5g / L, 6g / L, 7g / L, 8g / L, etc.

[0039] Preferably, the pH value of the differentiation medium in step (1) is 5.5-6.5, 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 medium in step (1), the step further includes cutting the callus tissue into small pieces with a diameter of 0.3 to 0.5 cm.

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

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

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

[0044] In the present invention, three hormones, namely, naphthylacetic acid, indoleacetic acid and kinetin, are added to the MS basic culture medium. The three hormones cooperate with each other in the rooting culture process to synergistically enhance the efficiency, thereby improving the rooting rate of the Yunnan konjac and improving the quality of the seedling root system, so that sufficient nutrients can be better absorbed from the soil, thereby improving the seedling rate.

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

[0046] Among them, the specific point values ​​in 0.2-0.3mg / L can be selected from 0.2mg / L, 0.22mg / L, 0.24mg / L, 0.26mg / L, 0.28mg / L, 0.3mg / L, etc., the specific point values ​​in 0.1-0.2mg / L can be selected from 0.1mg / L, 0.12mg / L, 0.14mg / L, 0.16mg / L, 0.18mg / L, 0.2mg / L, etc., 0. The specific point values ​​in the range of 3-0.5mg / L can be selected from 0.3mg / L, 0.35mg / L, 0.4mg / L, 0.45mg / L, 0.5mg / L, etc.; the specific point values ​​in the range of 13-17g / L can be selected from 13g / L, 14g / L, 15g / L, 16g / L, 17g / L, etc.; the specific point values ​​in the range of 4-8g / L can be selected from 4g / L, 5g / L, 6g / L, 7g / L, 8g / L, etc.

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

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

[0049] In a fourth aspect, the present invention provides an application of the method for inducing callus using Konjac leaves as explants as described in the first aspect or the method for forming seedlings of Konjac as described in the second aspect in tissue culture of Konjac.

[0050] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

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

[0052] The invention uses the leaves of Konjac yunnanensis as explants to induce callus tissue, and the material is convenient to obtain, and the number of the leaves of Konjac yunnanensis is large, the propagation speed is fast, and the efficiency is high.

[0053] In the callus induction culture medium provided by the present invention, four hormones, namely, naphthylacetic acid, 6-benzylaminoadenine, thidiazuron and zeatin, cooperate with each other, synergize and enhance the efficiency, and jointly improve the formation speed and quality of callus, thereby providing sufficient guarantee for improving the quality of Yunnan konjac seedlings; the above hormones are added to a 1 / 2MS basic culture medium, and the element concentrations in the culture medium are appropriate, so that the four hormones can be better cooperated with each other, thereby improving the callus induction rate and the overall quality of the callus.

[0054] Furthermore, when the concentration of each hormone in the callus induction medium is controlled within the range of 0.1-0.2 mg / L of naphthylacetic acid, 2-3 mg / L of 6-benzylaminopurine, 0.2-0.3 mg / L of thidiazuron, and 0.2-0.3 mg / L of zeatin, the synergistic relationship between the four hormones can be better exerted, thereby improving the induction rate of callus and the overall quality of callus.

[0055] Furthermore, before the Yunnan konjac leaves are inoculated into the callus induction medium, they are disinfected in sequence using an ethanol aqueous solution, a sodium hypochlorite aqueous solution, and a dibromohydantoin aqueous solution, which can greatly reduce the number of microorganisms such as viruses attached to the leaf surface and reduce the impact on the callus induction process; and after the disinfectant treatment, soaking in water and soaking in water twice are respectively performed, which can better clean the disinfectant remaining on the surface and inside of the leaves, thereby reducing the impact of the disinfectant on the callus induction process.

[0056] Furthermore, in the process of seedling cultivation of callus tissue, three hormones, naphthylacetic acid, indoleacetic acid and kinetin, are added to the MS basal culture medium as a rooting medium. The three hormones cooperate with each other and synergistically enhance their effects during the rooting culture process, which can further improve the rooting rate and root quality of Konjac. DETAILED DESCRIPTION

[0057] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0058] Example 1

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

[0060] (1) Disinfection of explant leaves: 50 small leaves of Yunnan konjac were rinsed with tap water for 30 minutes and ultrapure water for 2 minutes in turn; leaves were trimmed to 2.5 cm in length and 0.8 cm in width, immersed in 75% ethanol aqueous solution for 2 minutes, filtered, immersed in 2% sodium hypochlorite aqueous solution for 15 minutes, filtered, immersed in 1 g / L dibromohydantoin aqueous solution for 5 minutes, filtered; then the leaves were soaked in distilled water for 2 minutes each time and filtered after each soaking; leaves were trimmed to 1.2 cm in length and 0.4 cm in width, cut and immersed in distilled water for a second time, immersed twice in total, immersed for 2 minutes each time, filtered after each soaking, and the disinfection of explant leaves was completed.

[0061] (2) Inoculating the disinfected Konjac leaves in a callus induction medium to obtain callus tissue, wherein the formula of the callus induction medium is: 1 / 2MS basal medium, 0.1 mg / L naphthaleneacetic acid, 2.0 mg / L 6-benzylaminoadenine, 0.25 mg / L thidiazuron, 0.25 mg / L zeatin, 10 g / L sucrose and 5 g / L agar, pH value is 6; the culture temperature is 25°C, and the humidity is 70%.

[0062] Example 2

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

[0064] (1) Disinfection of explant leaves: 50 small leaves of Amorphophallus yunnanensis were rinsed with tap water for 25 minutes and ultrapure water for 4 minutes; the leaves were trimmed to 2 cm in length and 1 cm in width, immersed in 75% ethanol aqueous solution for 3 minutes, filtered, immersed in 1% sodium hypochlorite aqueous solution for 17 minutes, filtered, immersed in 1.5 g / L dibromohydantoin aqueous solution for 3 minutes, filtered; the leaves were then immersed in distilled water for 3 times, each time for 2 minutes, and filtered after each immersion; the leaves were trimmed to 1 cm in length and 0.5 cm in width, cut and immersed in distilled water for a second time, immersed twice in total, each time for 3 minutes, and filtered after each immersion, to complete the disinfection of the explant leaves.

[0065] (2) Inoculate the disinfected Konjac leaves in a callus induction medium to obtain callus tissue, wherein the formula of the callus induction medium is: 1 / 2MS basal medium, 0.1 mg / L naphthaleneacetic acid, 3 mg / L 6-benzylaminoadenine, 0.2 mg / L thidiazuron, 0.2 mg / L zeatin, 12 g / L sucrose and 5 g / L agar, pH value is 5.5; the culture temperature is 20°C and the humidity is 80%.

[0066] Example 3

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

[0068] (1) Disinfection of explant leaves: 50 small leaves of Amorphophallus yunnanensis were rinsed with tap water for 35 minutes and ultrapure water for 5 minutes in turn; leaves were trimmed to 3 cm in length and 0.5 cm in width, soaked in 75% ethanol aqueous solution for 3 minutes, filtered, soaked in 1% sodium hypochlorite aqueous solution for 20 minutes, filtered, soaked in 0.8 g / L dibromohydantoin aqueous solution for 4 minutes, filtered; then the leaves were soaked in distilled water for 2 times, each time for 3 minutes, and filtered after each soaking; leaves were trimmed to 1.5 cm in length and 0.3 cm in width, cut and soaked in distilled water for a second time, and soaked for a total of 3 times, each time for 2 minutes, and filtered after each soaking, completing the disinfection of explant leaves.

[0069] (2) Inoculating the disinfected Konjac leaves in a callus induction medium to obtain callus tissue, wherein the formula of the callus induction medium is: 1 / 2MS basal medium, 0.2 mg / L naphthaleneacetic acid, 2 mg / L 6-benzylaminoadenine, 0.3 mg / L thidiazuron, 0.3 mg / L zeatin, 8 g / L sucrose and 6 g / L agar, pH value is 6.5; the culture temperature is 30°C and the humidity is 70%.

[0070] Example 4

[0071] This embodiment provides a method for inducing callus tissue using Konjac konjac leaves as explants, which differs from Embodiment 1 only in that in step (1), the immersion in ethanol aqueous solution and the filtration are not performed, the immersion time of the leaves in the sodium hypochlorite aqueous solution is adjusted to 16.5 min, and the immersion time of the leaves in the dibromohydantoin aqueous solution is adjusted to 5.5 min. The remaining steps are consistent with Embodiment 1.

[0072] Example 5

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

[0074] Example 6

[0075] This embodiment provides a method for inducing callus tissue using Konjac konjac leaves as explants, which differs from Embodiment 1 only in that in step (1), the dibromohydantoin aqueous solution is not used for soaking and filtering, the soaking time in the ethanol aqueous solution is adjusted to 3 minutes, and the soaking time in the sodium hypochlorite aqueous solution is adjusted to 19 minutes. The remaining steps are consistent with Embodiment 1.

[0076] Example 7

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

[0078] Example 8

[0079] This embodiment provides a method for inducing callus tissue using Konjac leaves as explants. The only difference between this method and Embodiment 1 is that in step (1), no secondary immersion in distilled water is performed, that is, the disinfection of the explant leaves is completed after the leaves are trimmed to 1.2 cm in length and 0.4 cm in width. The remaining steps are consistent with Embodiment 1.

[0080] Example 9

[0081] This embodiment provides a method for inducing callus tissue using Konjac 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 / 2MS basal medium, 0.25 mg / L naphthaleneacetic acid, 3.2 mg / L 6-benzylaminoadenine, 0.15 mg / L thidiazuron, 0.1 mg / L zeatin, 10 g / L sucrose and 5 g / L agar. The remaining steps are consistent with Example 1.

[0082] Example 10

[0083] This embodiment provides a method for inducing callus tissue using Konjac 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 / 2MS basal medium, 0.08 mg / L naphthaleneacetic acid, 1.8 mg / L 6-benzylaminoadenine, 0.32 mg / L thidiazuron, 0.4 mg / L zeatin, 10 g / L sucrose and 5 g / L agar. The remaining steps are consistent with Example 1.

[0084] Comparative Example 1

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

[0086] Comparative Example 2

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

[0088] Comparative Example 3

[0089] The comparative example provides a method for inducing callus using Konjac konjac leaves as explants, which differs from Example 1 only in that in step (2), the callus induction medium does not include thidiazuron, that is, the medium formula is: 1 / 2MS basal medium, 0.11 mg / L naphthaleneacetic acid, 2.21 mg / L 6-benzylaminoadenine, 0.28 mg / L zeatin, 10 g / L sucrose and 5 g / L agar, and the remaining steps are consistent with Example 1.

[0090] Comparative Example 4

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

[0092] Comparative Example 5

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

[0094] Comparative Example 6

[0095] This comparative example provides a method for inducing callus tissue using Konjac corms as explants, which differs from Example 1 only in that the explant leaves are replaced by explant corms, and the remaining steps are consistent with Example 1.

[0096] Application Example 1

[0097] This application example provides a method for growing seedlings of Konjac yunnanensis, comprising the following steps:

[0098] (1) Differentiation culture: The callus obtained by the method of inducing callus in Example 1 was cut into small pieces with a side length of 0.4 cm, inoculated into a differentiation medium, and subjected to differentiation culture. The differentiation medium was formulated as follows: MS basal medium, 0.4 mg / L naphthaleneacetic acid, 1.2 mg / L 6-benzylaminoadenine, 15 g / L sucrose, and 6 g / L agar, with a pH value of 6; the temperature of the differentiation culture was 25° C., and the humidity was 70%.

[0099] (2) Rooting culture: When the length of the seedlings grown from the differentiation culture is 2.5 cm, they are cut and inoculated into a rooting medium to obtain seedlings. The formula of the rooting medium is: 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 value of 6; the temperature for rooting culture is 26°C and the humidity is 75%.

[0100] (3) Transplanting and cultivation: When the root length is 1.5 cm, the seedlings are taken out, the culture medium on the seedlings is rinsed clean, and the seedlings are cultivated in nutrient soil. The temperature for transplanting and cultivation is 22° C. and the humidity is 60%. Weeding is done on time and compound fertilizer is applied in time. After 1 month, the Yunnan konjac seedlings are obtained.

[0101] Application Example 2

[0102] This application example provides a method for growing seedlings of Konjac yunnanensis, comprising the following steps:

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

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

[0105] (3) Transplanting and cultivation: When the root length is 1 cm, the seedlings are taken out, the culture medium on the seedlings is rinsed clean, and the seedlings are cultivated in nutrient soil. The temperature for transplanting and cultivation is 23° C. and the humidity is 65%. Weeding is carried out on time and compound fertilizer is applied in time to obtain Yunnan konjac seedlings.

[0106] Application Example 3

[0107] This application example provides a method for growing seedlings of Konjac yunnanensis, comprising the following steps:

[0108] (1) Differentiation culture: The callus obtained by the method of inducing callus in Example 3 was cut into small pieces with a side length of 0.5 cm, inoculated into a differentiation medium, and then cultured. The differentiation medium was formulated as follows: MS basal medium, 0.5 mg / L naphthaleneacetic acid, 1 mg / L 6-benzylaminoadenine, 17 g / L sucrose, and 4 g / L agar, with a pH value of 6.5; the temperature of the differentiation culture was 27° C., and the humidity was 78%.

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

[0110] (3) Transplanting and cultivation: When the root length is 2 cm, the seedlings are taken out, the culture medium on the seedlings is rinsed clean, and the seedlings are cultivated in nutrient soil. The temperature for transplanting and cultivation is 24° C. and the humidity is 80%. Weeding is carried out on time and foliar fertilizer is sprayed to obtain Yunnan konjac seedlings.

[0111] Application Example 4-10

[0112] Application Examples 4-10 each provide a method for growing seedlings of Konjac konjac, and the difference between them and Application Example 1 is that, in step (1), the callus obtained by the method for inducing callus tissue in Example 1 is replaced by the callus obtained by the method for inducing callus tissue in Examples 4-10, and the remaining steps are consistent with Application Example 1.

[0113] Application Example 11

[0114] This application example provides a method for growing seedlings of Amorphophallus yunnanensis, which differs from Application Example 1 only in that the rooting medium in step (2) does not include naphthaleneacetic acid, that is, the medium formula is: MS basal medium, 0.22 mg / L indoleacetic acid, 0.58 mg / L kinetin, 15 g / L sucrose and 6 g / L agar, and the pH value is 6.

[0115] Application Example 12

[0116] This application example provides a method for growing seedlings of Amorphophallus yunnanensis, which differs from application example 1 only in that the rooting medium in step (2) does not include indoleacetic acid, that is, the medium formula is: MS basal medium, 0.31 mg / L naphthaleneacetic acid, 0.49 mg / L kinetin, 15 g / L sucrose and 6 g / L agar, and the pH value is 6.

[0117] Application Example 13

[0118] This application example provides a method for growing seedlings of Amorphophallus yunnanensis, which differs from application example 1 only in that the rooting medium in step (2) does not include kinetin, that is, the medium formula is: MS basal medium, 0.5 mg / L naphthaleneacetic acid, 0.3 mg / L indoleacetic acid, 15 g / L sucrose and 6 g / L agar, and the pH value is 6.

[0119] Comparative Application Examples 1-6

[0120] Comparative Application Examples 1-6 each provide a method for growing seedlings of Konjac konjac, which differ from Application Example 1 in that the callus obtained by the method for inducing callus in Example 1 in step (1) is replaced by the callus obtained by the method for inducing callus in Comparative Examples 1-6, respectively, and the remaining steps remain the same as in Application Example 1.

[0121] Test Example 1

[0122] This test example tests the callus induction rate of each callus induction method of each embodiment / comparative example, and the rooting rate and seedling rate in each application example / comparative application example seedling method. The calculation formulas for callus induction rate, rooting rate and seedling rate are as follows:

[0123] Callus induction rate = number of explants with callus / 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 rate = number of surviving seedlings after transplanting / total number of transplanted seedlings × 100%.

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

[0127]

[0128]

[0129] It can be seen from the data in Example 1 / Application Example 1 and Comparative Examples / Comparative Application Examples 1-4 that when the callus induction medium lacks any one of naphthylacetic acid, 6-benzylaminoadenine, thidiazuron or zeatin, the callus induction rate is greatly reduced, indicating that the four hormones of naphthylacetic acid, 6-benzylaminoadenine, thidiazuron and zeatin have a significant synergistic effect in improving the callus induction rate in the callus induction process using Konjac konjac leaves as explants, and can also improve the quality of callus, thereby improving the rooting rate and seedling rate of seedlings.

[0130] It can be seen from the data in Example 1 / Application Example 1 and Comparative Example / Comparative Application Example 5 that, compared with the MS basal medium, the concentrations of macroelements and trace elements in the 1 / 2MS basal medium are appropriate, which can better cooperate with naphthylacetic acid, 6-benzylaminoadenine, thidiazuron and zeatin, thereby improving the callus induction rate and the overall quality of the callus.

[0131] It can be seen from the data in Example 1 / Application Example 1 and Comparative Example / Comparative Application Example 6 that under the action of a callus induction medium comprising 1 / 2MS basal medium, naphthylacetic acid, 6-benzylaminopurine, thidiazuron, zeatin, sucrose and agar, the callus induction rate of inducing callus with Konjac leaves as explants is significantly improved compared to that with Konjac corms as explants, and further, the rooting rate is high and the seedling rate is high.

[0132] It can be seen from the data in Example / Application Example 1 and Example / Application Examples 4-7 that disinfecting the leaves of Konjac konjac before callus culture can reduce the influence of microorganisms such as viruses on the callus culture process, thereby improving the induction rate of callus. For the disinfection process of explant leaves, when the total time of disinfectant immersion is consistent, the explant leaves are sequentially immersed in ethanol aqueous solution, sodium hypochlorite aqueous solution, and dibromohydantoin aqueous solution, which can further improve the disinfection ability, reduce the number of microorganisms, and improve the callus induction rate.

[0133] It can be seen from the data in Example / Application Example 1 and Example / Application Example 8 that for the explant leaf disinfection process, trimming the leaves and then soaking them in distilled water for a second time can better clean the disinfectant remaining on the surface and inside of the leaves, thereby reducing the impact of the disinfectant on the callus induction process and improving the callus induction quality.

[0134] It can be seen from the data of Example / Application Example 1 and Example / Application Examples 9-10 that when the concentration of each hormone in the callus induction medium is controlled within the range of 0.1-0.2 mg / L of naphthylacetic acid, 2-3 mg / L of 6-benzylaminopurine, 0.2-0.3 mg / L of thidiazuron, and 0.2-0.3 mg / L of zeatin, the synergistic relationship between the four hormones can be better exerted, thereby improving the induction rate of callus and the overall quality of callus.

[0135] It can be seen from the data in Application Examples 1 and 11-13 that when any one of naphthylacetic acid, indoleacetic acid or kinetin is lacking in the rooting culture medium, the rooting rate of the seedlings is greatly reduced, indicating that naphthylacetic acid, indoleacetic acid and kinetin have a significant synergistic effect in the rooting process of Konjac seedlings, which can greatly improve the rooting rate. At the same time, the root system quality of the seedlings rooted in the specific culture medium is better, and they can better absorb sufficient nutrients from the soil during the transplanting stage, thereby improving the seedling rate.

[0136] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0137] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to 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 further describe various possible combinations.

Claims

1. A method for inducing callus using Konjac konjac leaves as explants, characterized in that: The method comprises: inoculating Konjac konjac leaves into a callus induction medium for culture to obtain callus; The callus induction medium comprises: 1 / 2MS basic medium, naphthylacetic acid, 6-benzylaminoadenine, thidiazuron, zeatin, sucrose and agar.

2. The method of inducing callus using Konjac konjac leaves as explants according to claim 1, wherein: The callus induction medium comprises, in terms of mass volume concentration, 1 / 2MS basic medium, 0.1-0.2 mg / L of naphthaleneacetic acid, 2-3 mg / L of 6-benzylaminoadenine, 0.2-0.3 mg / L of thidiazuron, 0.2-0.3 mg / L of zeatin, 8-12 g / L of sucrose and 5-9 g / L of agar; Preferably, the pH value of the callus induction medium is 5.5-6.5; Preferably, the culture temperature is 20-30° C. and the humidity is 60-80%.

3. The method for inducing callus using Konjac konjac leaves as explants according to claim 1 or 2, wherein: The method further comprises disinfecting the Konjac leaves before inoculating the Konjac leaves into the callus induction medium; Preferably, the disinfection process comprises the following steps: The explant konjac leaves are sequentially washed with water, soaked in an ethanol aqueous solution, drained, soaked in a sodium hypochlorite aqueous solution, drained, soaked in a dibromohydantoin aqueous solution, drained, soaked in water, drained, soaked in water twice, and drained.

4. A callus obtained by the method for inducing callus using Konjac konjac leaves as explants as claimed in any one of claims 1 to 3.

5. A method for growing seedlings of Konjac yunnanensis, characterized in that: The seedling growing method of the described konjac comprises the steps: (1) Differentiation culture: inoculating the callus tissue described in claim 4 into a differentiation medium for differentiation culture; (2) Rooting culture: When the length of the seedlings grown from the differentiation culture is 2 to 3 cm, they are cut and inoculated into a rooting medium to obtain seedlings; (3) Transplanting and cultivation: When the root length is 1 to 2 cm, the seedlings are taken out and cultivated in nutrient soil to obtain Yunnan konjac seedlings.

6. The seedling method of Konjac konjac according to claim 5, wherein: The differentiation medium in step (1) comprises: MS basal medium, naphthylacetic acid, 6-benzylaminoadenine, sucrose and agar; Preferably, in terms of mass volume concentration, the differentiation medium in step (1) comprises: MS basal medium, 0.3-0.5 mg / L naphthaleneacetic acid, 1-1.5 mg / L 6-benzylaminoadenine, 13-17 g / L sucrose and 4-8 g / L agar.

7. The seedling growing method of the Yunnan konjac according to claim 5 or 6, characterized in that, The pH value of the differentiation medium in step (1) is 5.5 to 6.5; Preferably, before inoculating the callus tissue of claim 4 into the differentiation medium in step (1), the step further comprises cutting the callus tissue into small pieces with a diameter of 0.3 to 0.5 cm.

8. The method for growing seedlings of Konjac according to any one of claims 5 to 7, characterized in that: The rooting medium in step (2) comprises: MS basal medium, naphthylacetic acid, indoleacetic acid, kinetin, sucrose and agar; Preferably, in terms of mass volume concentration, the rooting medium in step (2) comprises: MS basal medium, 0.2-0.3 mg / L naphthaleneacetic acid, 0.1-0.2 mg / L indoleacetic acid, 0.3-0.5 mg / L kinetin, 13-17 g / L sucrose and 4-8 g / L agar.

9. The method for growing seedlings of Konjac according to any one of claims 5 to 8, characterized in that: The pH value of the rooting medium in step (2) is 5.5-6.

5.

10. Use of the method for inducing callus using Konjac leaves as explants as claimed in any one of claims 1 to 3 or the method for forming seedlings of Konjac as claimed in any one of claims 5 to 9 in Konjac tissue culture.

Images