Tissue culture detoxification method for bergamot Chinese yam
Through the detoxification method of Foshou yam tissue culture, including alcohol disinfection, disinfectant treatment, culture medium culture and heat treatment, the problems of yield decline and variety degradation caused by virus accumulation of Foshou yam were solved, and the effect of improving the sterility and budding rate was achieved, ensuring the stability and quality of the detoxification seedlings.
Patent Information
- Application Number
- CN202510076754.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-17
- Publication Date
- 2025-02-28
AI Technical Summary
After years of cultivation of Foshou Yam, viruses accumulate on its taro seeds, leaves shrink, underground tubers deformed, and black spots appearing in the fur. The yield decreases year by year, seriously affecting the market value. The existing yam detoxification methods lack sterile culture of plant ex vivo tissues and high-temperature detoxification, and cannot obtain detoxification seedlings stably and effectively.
The detoxification method of Foshou yam tissue culture is adopted. The specific steps include preparing experimental materials, disinfecting alcohol, disinfecting disinfectant, putting in culture medium for dark culture and light culture, heat treatment of sterile seedlings and culturing detoxification seedlings. Through these steps, it is possible to effectively reduce toxins inside and outside the plant, improve the sterility and budding rate, and obtain stable detoxification seedlings.
Through the tissue culture detoxification method, the problem of easy browning of tissue culture of Foshou yam is solved, the sterile tissue culture system is improved, the survival rate, sterile rate and germination rate are improved, and the stability and quality of the detoxification seedlings are ensured.
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Figure CN119522830A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of yam detoxification, in particular to a bergamot yam tissue culture detoxification method. Background Art
[0002] Yam is a general term for the underground tubers of plants in the Dioscoreaceae family and its closely related species. It is an annual or perennial monocotyledonous twining vine. Yams are widely grown in China, and are grown in Henan, Anhui, Shandong, Hubei, Jiangxi, Hunan, Guizhou, northern Yunnan, Sichuan, eastern Gansu and southern Shaanxi. Buddha's hand yam is one of the typical local varieties. Buddha's hand yam is an underground tuber crop, which is mainly propagated asexually by tubers to keep seeds. Because yam is planted all year round in the main production areas of Buddha's hand yam, the varieties are mixed, the strains are impure, and the vegetative propagation is single, resulting in the accumulation of diseases, reduced yields, degeneration of varieties, and poor commerciality in the main production areas of Buddha's hand yam, which seriously restricts the development of the Buddha's hand yam industry.
[0003] After years of cultivation, the virus on the seed tuber of Buddha's hand yam accumulates all year round, causing the yam vines to be short, the leaves to curl, the underground tubers to be deformed, the fur to have black spots, and the yield to decrease year by year, which seriously affects the market value of Buddha's hand yam. However, the existing yam detoxification method lacks aseptic culture and high temperature detoxification of plant in vitro tissues, so it is impossible to stably and effectively obtain Buddha's hand yam detoxification seedlings.
[0004] Therefore, we proposed a method for detoxification of yam tissue culture in order to solve the above problems. Summary of the invention
[0005] The purpose of the present invention is to provide a method for detoxification of bergamot yam tissue culture, so as to solve the problem that the bergamot yam currently on the market has been planted for many years, and the virus on its seed tuber has accumulated all year round, resulting in the yam vines being short, the leaves curling, the underground tubers being deformed, the fur being prone to black spots, and the yield decreasing year by year, which seriously affects the market value of bergamot yam. However, the existing yam detoxification method cannot stably and effectively obtain bergamot yam detoxification seedlings due to the lack of aseptic culture and high temperature detoxification of plant in vitro tissues.
[0006] To achieve the above object, the present invention provides the following technical solution: a method for detoxification of yam tissue culture, the specific steps of the method are as follows: Step 1: Prepare experimental materials The stems and vines of yam grown in the field were used as experimental materials. A young stem segment of about 10 cm long was taken from the top of the yam and then cut into stem segments with nodes of about 3 cm to obtain the initial sterile seedling material. Step 2: Alcohol disinfection The cut stem segments with nodes are first rinsed with tap water, then soaked in 75% pure alcohol for 30-60 seconds, and then sterile water is taken out and the soaked stem segments with nodes are placed on the clean bench for a rinse; Step 3: Disinfection with Disinfectant The noded stem segments were treated with a disinfectant containing 0.1% mercuric chloride and 3% sodium hypochlorite solution. The disinfection time was 8-15 minutes. The noded stem segments were then taken out and rinsed five times with sterile water. Step 4: Place in culture medium for dark and light culture After rinsing, the stem segments with nodes were placed on a clean workbench, and the stem segments were cut into small segments of about 1 cm with only one node. The cut stem segments were placed in a culture medium of MS+6-BA (0.4-0.5 mg / L)+NAA (0.01-0.10 mg / L), pH 5.8 for culture; the culture temperature was 25°C, and the cells were first cultured in the dark for 1 week, and then transferred to a light condition of 1000-1500 lx, 16 h / d for 2 weeks; the disinfection effects of different disinfectants and treatments for different time periods on the young stem segments of yam were compared; Step 5: Heat treatment of sterile seedlings The rooted sterile seedlings were cultured under gradually increasing temperature conditions (25→38°C) for 2 weeks, then cultured under high temperature conditions (39-40°C) for 3 weeks, and then cultured under gradually decreasing temperature conditions (39→25°C) for 1-2 weeks; Step 6: Shoot tip culture virus-free seedlings The stem tips of the sterile seedlings after heat treatment were cut and transferred to a culture medium of MS+6-BA (0.1-0.5 mg / L) + NAA (0.01-0.05 mg / L) + 30 g / L sucrose + 5 g / L agar, pH 5.8, to obtain virus-free tissue culture seedlings of yam.
[0007] Due to the detoxification of the tissue culture of Buddha's hand yam plant tissue, the problem of easy browning of Buddha's hand yam tissue culture has been solved, the sterile tissue culture system of Buddha's hand yam has been improved, and there is sufficient experimental data for the next experiment or comparison, which can effectively reduce the toxins inside and outside the Buddha's hand yam, so that it can better avoid withering or other unexpected situations during the growth process, and increase the survival rate, sterility rate and germination rate of Buddha's hand yam.
[0008] 2. The method for detoxification of yam tissue culture according to claim 1, characterized in that the formula for the sterility rate and germination rate of the young stem segments of yam is: Sterility rate = (number of sterile materials / total number of explants) × 100% Survival rate = (survival material rate / total number of explants) × 100%.
[0009] The adoption of the above technical solution enables the user to determine and calculate the survival rate and sterility rate of the yam stem segments through a formula, thereby increasing the accuracy of the experimental data.
[0010] As a preferred technical scheme of the present invention, the longer the disinfection time of the yam stem segments in the disinfectant of 0.1% mercuric chloride and 3% sodium hypochlorite solution in step 3 is, the more sterile materials will be produced. However, if the disinfection time of 3% sodium hypochlorite is too long, the number of surviving materials will decrease. Therefore, if the disinfection time is too long, the toxins produced by the stem segments are not conducive to the growth of the explants. The above technical scheme can increase the sterile materials of the yam stem segments after extending the disinfection time, and the toxins produced by the stem segments can be controlled by controlling the disinfection time.
[0011] As the preferred technical solution of the present invention, in step 3, the sterile material obtained by treating the stem segments of yam with 0.1% mercuric chloride is significantly more than that obtained by treating the stem segments with 3% sodium hypochlorite at the same treatment time, and the survival rate is also relatively high, indicating that the disinfection effect of 0.1% mercuric chloride is better.
[0012] As a preferred technical solution of the present invention, if 0.1% mercuric chloride is used for disinfection of the yam stem segments in step 3 for 15 minutes, the optimized disinfection effect can be achieved. In the process of debugging the disinfectant, it can be adjusted according to different dosages.
[0013] As a preferred technical solution of the present invention, in step 4, the sterilized stem segments with nodes cut into pieces of about 1 cm are placed on a culture medium with different components at a pH of 5.8 to 6.2 for culture; after about 15 days, white callus will appear at the axillary buds of the stem segments. As the callus grows, the culture medium gradually turns brown, and the callus also turns brown.
[0014] As the preferred technical solution of the present invention, in step 4, when 1 / 2MS and 1 / 5MS are not added with any hormones, both 1 / 2MS and 1 / 5MS are conducive to the production of root systems, and the roots produced by 1 / 2MS are the earliest, and the root system is relatively robust compared with 1 / 5MS, which indicates that 1 / 2MS is more conducive to the rooting of Buddha's hand yam; from the perspective of the adventitious bud differentiation rate of disinfected noded stem segments, the bud differentiation rates of MS+6-BA0.4 mg / L +NAA0.01 mg / L and MS+6-BA 0.4 mg / L +NAA 0.04 mg / L are relatively good in this group of experiments.
[0015] As a preferred technical solution of the present invention, in step 4, the disinfected stem segments with nodes are heated to 40°C in the presence of MS+6-BA 0.4 mg·L -1 +NAA0.01mg·L -1In the culture medium under the conditions of MS+6-BA0.4 mg / L +NAA 0.04 mg / L, most of the sterile materials directly differentiated into buds from stem nodes; while under the conditions of MS+6-BA0.4 mg / L +NAA 0.04 mg / L, not only was the callus rate high, the amount of callus was also large, and the stem segments first had callus tissue and then produced adventitious buds; therefore, MS+6-BA 0.4 mg / L +NAA 0.04 mg / L was more conducive to the formation of adventitious buds of yam.
[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. Due to the detoxification of the plant tissue of bergamot yam, the problem of easy browning of bergamot yam tissue culture is solved, the aseptic tissue culture system of bergamot yam is improved, and sufficient experimental data are available for the next step of experiment or comparison, so that the toxins inside and outside the bergamot yam can be effectively reduced, so that it can better avoid withering or other unexpected situations during the growth process, increase the survival rate, sterility rate and germination rate of bergamot yam, and obtain stable bergamot yam tissue culture detoxification seedlings through heat treatment of sterile seedlings; 2. By soaking the stem segments of bergamot yam for different time periods, we can find out which one has the best detoxification effect and increase the sterility rate of bergamot yam, making it easier for users to conduct experimental comparisons. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 It is a schematic diagram of the detoxification technology route of the present invention; Figure 2 It is a schematic diagram of the sterility rate and survival rate of stems and roots of the present invention; Figure 3 This is a graph showing the effects of different culture media of the present invention on the formation of adventitious buds from virus-free stems and roots. DETAILED DESCRIPTION
[0018] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Example
[0019] See also Figure 1-Figure 3 The present invention provides a technical solution: a method for detoxification of yam tissue culture, characterized in that the method comprises the following specific steps: Step 1: Prepare experimental materials The stems and vines of yam grown in the field were used as experimental materials. A young stem segment of about 10 cm long was taken from the top of the yam and then cut into stem segments with nodes of about 3 cm to obtain the initial sterile seedling material. Step 2: Alcohol disinfection The cut stem segments with nodes are first rinsed with tap water, then soaked in 75% pure alcohol for 30-60 seconds, and then sterile water is taken out and the soaked stem segments with nodes are placed on the clean bench for a rinse; Step 3: Disinfection with disinfectant The stem segments with nodes were treated with a disinfectant, which consisted of 0.1% mercuric chloride and 3% sodium hypochlorite solution. The disinfection time was 8 minutes, and then the stem segments with nodes were taken out and rinsed five times with sterile water. When disinfected with 0.1% mercuric chloride disinfectant, the number of inoculated materials with node stem segments was 43, the number of sterile materials was 19, the number of surviving materials was 17, the sterility rate was 44.18%, and the survival rate was 39.53%; When disinfected with 3% sodium hypochlorite solution, the number of inoculated materials with noded stem segments was 44, the number of sterile materials was 3, the number of surviving materials was 1, the sterility rate was 6.81%, and the survival rate was 2.77%; Step 4: Place in culture medium for dark and light culture After rinsing, the stem segments with nodes were placed on a clean workbench, and the stem segments were cut into small segments of about 1 cm with only one node. The cut materials were placed in a culture medium of MS+6-BA (0.4-0.5 mg / L)+NAA (0.01-0.10 mg / L)+sucrose 30 g / L+agar 5 g / L, pH 5.8 for culture; the culture temperature was 25°C, and the cells were cultured in the dark for 1 week, and then transferred to a light condition of 1000-1500 lx, 16 h / d for 2 weeks; the disinfection effects of different disinfectants and treatments for different time periods on the young stem segments of yam were compared; The formulas for the sterility rate and germination rate of the young stem segments of yam are: Sterility rate = (number of sterile materials / total number of explants) × 100% Survival rate = (survival material rate / total number of explants) × 100%.
[0020] Step 5: Heat treatment of sterile seedlings The rooted sterile seedlings were cultured under gradually increasing temperature conditions (25→38°C) for 2 weeks, then cultured under high temperature conditions (39-40°C) for 3 weeks, and then cultured under gradually decreasing temperature conditions (39→25°C) for 1-2 weeks; Step 6: Shoot tip culture virus-free seedlings The stem tips of the sterile seedlings after heat treatment were cut and transferred to a culture medium of MS+6-BA (0.1-0.5 mg / L) + NAA (0.01-0.05 mg / L) + 30 g / L sucrose + 5 g / L agar, pH 5.8, to obtain virus-free tissue culture seedlings of yam.
[0021] In step 3, the longer the disinfection time of the stem segments of the yam of the bergamot hand in the disinfectant of 0.1% mercuric chloride and 3% sodium hypochlorite solution is, the more sterile materials will be. However, if the disinfection time of 3% sodium hypochlorite is too long, the survival number of the materials will decrease. Therefore, if the disinfection time is too long, the toxins produced by the stem segments are not conducive to the growth of the explants. In step 3, the amount of sterile material obtained by 0.1% mercuric chloride was significantly more than that by 3% sodium hypochlorite at the same treatment time, and the survival rate was relatively high, indicating that 0.1% mercuric chloride had a better disinfection effect; In step 3, if 0.1% mercuric chloride is used for 15 minutes to disinfect the stem segments of yam, the optimized disinfection effect can be achieved; in step 4, the noded stem segments that have been disinfected and cut into about 1 cm are placed on a culture medium with different components of pH 5.8-6.2 for culture; after about 15 days, white callus will appear at the axillary buds of the stem segments, and as the callus grows, the culture medium gradually turns brown, and the callus also turns brown; In step 4, when no hormones were added to 1 / 2MS and 1 / 5MS, both 1 / 2MS and 1 / 5MS were beneficial to the production of root system, and the root system produced by 1 / 2MS was the earliest and stronger than that of 1 / 5MS, which indicated that 1 / 2MS was more conducive to the rooting of yam. From the perspective of adventitious bud differentiation rate of stem segments, MS+6-BA0.4mg·L -1 +NAA0.01mg·L -1 and MS+6-BA0.4mg·L -1 +NAA 0.04mg·L -1 The bud differentiation rate was relatively good in this set of experiments; In step 4, under the conditions of MS+6-BA0.4 mg / L +NAA0.01 mg / L, most of the sterile materials directly differentiated into buds from the nodes of the noded stem segments; while under the conditions of MS+6-BA 0.4 mg / L +NAA 0.04 mg / L, not only was the callus rate high, but the amount of callus was also large, and the stem segments first had callus tissue and then produced adventitious buds; therefore, MS+6-BA 0.4 mg / L +NAA 0.04mg / L was more conducive to the formation of adventitious buds of yam. Example
[0022] The difference between this embodiment and embodiment 1 is: a method for detoxification of yam tissue culture, characterized in that the method has the following specific steps: Step 1: Prepare experimental materials The stems and vines of yam grown in the field were used as experimental materials. A young stem segment of about 10 cm long was taken from the top of the yam and then cut into stem segments with nodes of about 3 cm to obtain the initial sterile seedling material. Step 2: Alcohol disinfection The cut stem segments with nodes are first rinsed with tap water, then soaked in 75% pure alcohol for 30-60 seconds, and then sterile water is taken out and the soaked stem segments with nodes are placed on the clean bench for a rinse; Step 3: Disinfection with disinfectant The noded stem segments were treated with a disinfectant containing 0.1% mercuric chloride and 3% sodium hypochlorite solution for 12 minutes. The noded stem segments were then removed and rinsed five times with sterile water. When disinfected with 0.1% mercuric chloride disinfectant, the number of inoculated materials with node stem segments was 43, the number of sterile materials was 24, the number of surviving materials was 24, the sterility rate was 55.81%, and the survival rate was 55.81%; When disinfected with 3% sodium hypochlorite solution, the number of inoculated materials with noded stem segments was 48, the number of sterile materials was 6, the number of surviving materials was 4, the sterility rate was 12.50%, and the survival rate was 8.33%; Step 4: Place in culture medium for dark and light culture After rinsing, the stem segments with nodes were placed on a clean workbench, and the stem segments were cut into small segments of about 1 cm with only one node. The cut materials were placed in a culture medium of MS+6-BA (0.4-0.5 mg / L)+NAA (0.01-0.10 mg / L)+sucrose 30 g / L+agar 5 g / L, pH 5.8 for culture; the culture temperature was 25°C, and the cells were cultured in the dark for 1 week, and then transferred to a light condition of 1000-1500 lx, 16 h / d for 2 weeks; the disinfection effects of different disinfectants and treatments for different time periods on the young stem segments of yam were compared; The formulas for the sterility rate and germination rate of the young stem segments of yam are: Sterility rate = (number of sterile materials / total number of explants) × 100% Survival rate = (survival material rate / total number of explants) × 100% Step 5: Heat treatment of sterile seedlings The rooted sterile seedlings were cultured under gradually increasing temperature conditions (25→38℃) for 2 weeks, then cultured under high temperature conditions (39-40℃) for 3 weeks, and then cultured under gradually decreasing temperature conditions (39→25℃) for 1-2 weeks.
[0023] Step 6: Shoot tip culture virus-free seedlings The stem tips of the sterile seedlings after heat treatment were cut and transferred to a culture medium of MS+6-BA (0.1-0.5 mg / L) + NAA (0.01-0.05 mg / L) + 30 g / L sucrose + 5 g / L agar, pH 5.8, to obtain virus-free tissue culture seedlings of yam.
[0024] In step 3, the longer the disinfection time of the stem segments of the yam of the bergamot hand in the disinfectant of 0.1% mercuric chloride and 3% sodium hypochlorite solution is, the more sterile materials will be. However, if the disinfection time of 3% sodium hypochlorite is too long, the survival number of the materials will decrease. Therefore, if the disinfection time is too long, the toxins produced by the stem segments are not conducive to the growth of the explants. In step 3, the amount of sterile material obtained by 0.1% mercuric chloride was significantly more than that by 3% sodium hypochlorite at the same treatment time, and the survival rate was relatively high, indicating that 0.1% mercuric chloride had a better disinfection effect; In step 3, if 0.1% mercuric chloride is used for 15 minutes to disinfect the stem segments of yam, the optimized disinfection effect can be achieved; in step 4, the noded stem segments that have been disinfected and cut into about 1 cm are placed on a culture medium with different components of pH 5.8-6.2 for culture; after about 15 days, white callus will appear at the axillary buds of the stem segments, and as the callus grows, the culture medium gradually turns brown, and the callus also turns brown; In step 4, when no hormones were added to 1 / 2MS and 1 / 5MS, both 1 / 2MS and 1 / 5MS were beneficial to the production of root system, and the root system produced by 1 / 2MS was the earliest and more robust than that of 1 / 5MS, which indicated that 1 / 2MS was more conducive to the rooting of yam. From the perspective of adventitious bud differentiation rate of stem segments, the bud differentiation rates of MS+6-BA 0.4 mg / L +NAA 0.01 mg / L and MS+6-BA0.4 mg / L +NAA 0.04 mg / L were relatively good in this group of experiments. In step 4, under the conditions of MS+6-BA0.4 mg / L +NAA0.01 mg / L, most of the sterile materials of the noded stem segments directly differentiated into buds from the stem nodes; while under the conditions of MS+6-BA 0.4 mg / L +NAA 0.04 mg / L, not only was the callus rate high, the amount of callus was also large, and the stem segments first had callus tissue and then produced adventitious buds; therefore, MS+6-BA 0.4 mg / L +NAA 0.04mg / L was more conducive to the formation of adventitious buds of yam. Example
[0025] The present embodiment is different from the embodiment 1 and the embodiment 2 in that: a method for detoxification of yam tissue culture, characterized in that the method comprises the following specific steps: Step 1: Prepare experimental materials The stems and vines of yam grown in the field were used as experimental materials. A young stem segment of about 10 cm long was taken from the top of the yam and then cut into stem segments with nodes of about 3 cm to obtain the initial sterile seedling material. Step 2: Alcohol disinfection The cut stem segments with nodes are first rinsed with tap water, then soaked in 75% pure alcohol for 30-60 seconds, and then sterile water is taken out and the soaked stem segments with nodes are placed on the clean bench for a rinse; Step 3: Disinfection with disinfectant The noded stem segments were treated with a disinfectant containing 0.1% mercuric chloride and 3% sodium hypochlorite solution for 15 minutes. The noded stem segments were then removed and rinsed five times with sterile water. When disinfected with 0.1% mercuric chloride disinfectant, the number of inoculated materials with node stem segments was 46, the number of sterile materials was 32, the number of surviving materials was 27, the sterility rate was 69.56%, and the survival rate was 58.69%; When disinfected with 3% sodium hypochlorite solution, the number of inoculated materials with noded stem segments was 41, the number of sterile materials was 23, the number of surviving materials was 0, the sterility rate was 56.09%, and the survival rate was 0%; Step 4: Place in culture medium for dark and light culture After rinsing, the stem segments with nodes were placed on a clean workbench, and the stem segments were cut into small segments of about 1 cm with only one node. The cut materials were placed in a culture medium of MS+6-BA (0.4-0.5 mg / L)+NAA (0.01-0.10 mg / L)+sucrose 30 g / L+agar 5 g / L, pH 5.8 for culture; the culture temperature was 25°C, and the cells were cultured in the dark for 1 week, and then transferred to a light condition of 1000-1500 lx, 16 h / d for 2 weeks; the disinfection effects of different disinfectants and treatments for different time periods on the young stem segments of yam were compared; The formulas for the sterility rate and germination rate of the young stem segments of yam are: Sterility rate = (number of sterile materials / total number of explants) × 100% Survival rate = (survival material rate / total number of explants) × 100%.
[0026] Step 5: Heat treatment of sterile seedlings The rooted sterile seedlings were cultured under gradually increasing temperature conditions (25→38℃) for 2 weeks, then cultured under high temperature conditions (39-40℃) for 3 weeks, and then cultured under gradually decreasing temperature conditions (39→25℃) for 1-2 weeks.
[0027] Step 6: Shoot tip culture virus-free seedlings The stem tips of the sterile seedlings after heat treatment were cut and transferred to a culture medium of MS+6-BA (0.1-0.5 mg / L) + NAA (0.01-0.05 mg / L) + 30 g / L sucrose + 5 g / L agar, pH 5.8, to obtain virus-free tissue culture seedlings of yam.
[0028] In step 3, the longer the disinfection time of the stem segments of the yam of the bergamot hand in the disinfectant of 0.1% mercuric chloride and 3% sodium hypochlorite solution is, the more sterile materials will be. However, if the disinfection time of 3% sodium hypochlorite is too long, the survival number of the materials will decrease. Therefore, if the disinfection time is too long, the toxins produced by the stem segments are not conducive to the growth of the explants. In step 3, the amount of sterile material obtained by 0.1% mercuric chloride was significantly more than that by 3% sodium hypochlorite at the same treatment time, and the survival rate was relatively high, indicating that 0.1% mercuric chloride had a better disinfection effect; In step 3, if 0.1% mercuric chloride is used for 15 minutes to disinfect the stem segments of yam, the optimized disinfection effect can be achieved; in step 4, the noded stem segments that have been disinfected and cut into pieces of about 1 cm are placed on a culture medium with different components at a pH of 5.8 to 6.2 for culture; after about 15 days, white callus tissue will appear at the axillary buds of the stem segments. As the callus tissue grows, the culture medium gradually turns brown, and the callus tissue also turns brown.
[0029] In step 4, when no hormones were added to 1 / 2MS and 1 / 5MS, both 1 / 2MS and 1 / 5MS were beneficial to the production of root system, and the root system produced by 1 / 2MS was the earliest and more robust than that of 1 / 5MS, which indicated that 1 / 2MS was more conducive to the rooting of yam. From the perspective of adventitious bud differentiation rate of stem segments, the bud differentiation rates of MS+6-BA 0.4 mg / L +NAA 0.01 mg / L and MS+6-BA0.4 mg / L +NAA 0.04 mg / L were relatively good in this group of experiments. In step 4, under the conditions of MS+6-BA0.4 mg / L +NAA0.01 mg / L, most of the sterile materials directly differentiated into buds from the nodes of the noded stem segments; while under the conditions of MS+6-BA 0.4 mg / L +NAA 0.04 mg / L, not only was the callus rate high, but the amount of callus was also large, and the stem segments first had callus tissue and then produced adventitious buds; therefore, MS+6-BA 0.4 mg / L +NAA 0.04mg / L was more conducive to the formation of adventitious buds of yam.
[0030] Thereby completing a series of tasks, the contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.
[0031] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.
Claims
1. A method for detoxification of yam tissue culture, characterized in that The specific steps of the method are as follows: Step 1: Prepare experimental materials The stems and vines of yam grown in the field were used as experimental materials. A young stem segment of about 10 cm long was taken from the top of the yam and then cut into stem segments with nodes of about 3 cm to obtain the initial sterile seedling material. Step 2: Alcohol disinfection First, rinse the surface of the cut noded stem segments with tap water, then soak them in 75% alcohol for 30-60 seconds, and then quickly rinse them with sterile water. Step 3: Disinfection with Disinfectant The noded stem segments were treated with disinfectants, which were 0.1% mercuric chloride and 3% sodium hypochlorite solution. The disinfection time was 8-15 minutes. The noded stem segments were then taken out and rinsed five times with sterile water. Step 4: Place in culture medium for dark and light culture After rinsing, the stem segments with nodes were placed on a clean workbench, and the stem segments were cut into small segments of about 1 cm with only one node. The cut materials were placed in a culture medium of MS+6-BA (0.4-0.5 mg / L)+NAA (0.01-0.10 mg / L)+sucrose 30 g / L+agar 5 g / L, pH 5.8 for culture; the culture temperature was 25°C, and the cells were cultured in the dark for 1 week, and then transferred to a light condition of 1000-1500 lx, 16 h / d for 2 weeks; the disinfection effects of different disinfectants and treatments for different time periods on the young stem segments of yam were compared; Step 5: Heat treatment of sterile seedlings The rooted sterile seedlings were cultured under gradually increasing temperature conditions (25→38°C) for 2 weeks, then cultured under high temperature conditions (39-40°C) for 3 weeks, and then cultured under gradually decreasing temperature conditions (39→25°C) for 1-2 weeks; Step 6: Shoot tip culture virus-free seedlings The stem tips of the sterile seedlings after heat treatment were cut and transferred to a culture medium of MS+6-BA (0.1-0.5 mg / L) + NAA (0.01-0.10 mg / L) + 30 g / L sucrose + 5 g / L agar, pH 5.8, to obtain virus-free tissue culture seedlings of yam.
2. The method for detoxification of yam tissue culture according to claim 1, characterized in that: The formulas for the sterility rate and germination rate of the young stem segments of yam are as follows: Sterility rate = (number of sterile materials / total number of explants) × 100% Survival rate = (survival material rate / total number of explants) × 100%.
3. The method for detoxification of yam tissue culture according to claim 1, characterized in that: The longer the disinfection time of the yam stem segments in the disinfectant of 0.1% mercuric chloride and 3% sodium hypochlorite solution in step 3 is, the more sterile materials will be. However, if the disinfection time of 3% sodium hypochlorite is too long, the number of surviving materials will decrease. Therefore, if the disinfection time is too long, the toxins produced by the stem segments are not conducive to the growth of the explants.
4. The method for detoxification of yam tissue culture according to claim 1, characterized in that: In step 3, the amount of sterile material obtained by treating the yam stem segments with 0.1% mercuric chloride is significantly more than that obtained by treating the yam stem segments with 3% sodium hypochlorite at the same treatment time, and the survival rate is also relatively high, indicating that the disinfection effect of 0.1% mercuric chloride is better.
5. The method for detoxification of yam tissue culture according to claim 1, characterized in that: In step 3, if 0.1% mercuric chloride is used for disinfection for 15 minutes, the optimized disinfection effect can be achieved.
6. The method for detoxification of yam tissue culture according to claim 1, characterized in that: In step 4, the sterilized and cut stem segments with nodes of about 1 cm are placed on a culture medium with different components and a pH of 5.8-6.2 for culture; after about 15 days, white callus will appear at the axillary buds of the stem segments. As the callus grows, the culture medium gradually turns brown, and the callus also turns brown.
7. The method for detoxification of yam tissue culture according to claim 1, characterized in that: In step 4, when 1 / 2MS and 1 / 5MS were not added with any hormones, both 1 / 2MS and 1 / 5MS were beneficial to the production of root systems, and the roots produced by 1 / 2MS were the earliest and the root systems were relatively robust compared with those of 1 / 5MS, which indicated that 1 / 2MS was more conducive to the rooting of yam. From the perspective of the adventitious bud differentiation rate of the disinfected noded stem segments, the bud differentiation rates of MS+6-BA 0.4 mg / L +NAA 0.01 mg / L and MS+6-BA 0.4 mg / L +NAA0.04 mg / L were relatively good in this group of experiments.
8. The method for detoxification of yam tissue culture according to claim 1, characterized in that: In step 4, under the conditions of MS+6-BA0.4 mg / L +NAA0.01 mg / L, most of the sterile materials of the noded stem segments directly differentiate into buds from the stem nodes; while under the conditions of MS+6-BA 0.4 mg / L +NAA 0.04 mg / L, not only is the callus rate high, the amount of callus is also large, and the stem segments first have callus tissue and then produce adventitious buds; therefore, MS+6-BA 0.4 mg / L +NAA 0.04 mg / L is more conducive to the formation of adventitious buds of yam.