Tissue culture medium for rapid seedling raising of apple rootstock and rapid seedling raising method

Abstract

The invention discloses a tissue culture medium for rapid seedling raising of apple rootstocks and a rapid seedling raising method, and belongs to the technical field of plant tissue culture. The method specifically comprises the following steps: (1) obtaining a sterile material; (2) induction culture: inoculating the treated explants into an induction culture medium, and culturing at room temperature for 15-20 days to obtain adventitious buds; (3) multiplication culture: inoculating the adventitious buds into a multiplication culture medium, and carrying out light-dark culture at room temperature for 25-30 days to obtain cluster buds; (4) rooting culture: inoculating the cluster buds into a rooting culture medium, and carrying out light-dark culture at room temperature until the root length is 2-4cm; (5) transplanting. Through cooperation of the specific induction culture medium, the proliferation culture medium and the rooting culture medium, the growth and development rate and the survival rate of the apple rootstock can be effectively increased.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a tissue culture medium and a rapid seedling raising method for apple rootstock. Background Technology

[0002] Apples are a staple fruit and one of the most widely cultivated fruit trees in the world. The vitamin C in apples is a protector of the cardiovascular system and a vital element for heart disease patients. Apples are sweet, sour, and slightly salty in taste, and are non-toxic. They have the effects of quenching thirst, strengthening the spleen and stopping diarrhea, and harmonizing the stomach and relieving nausea. People who eat more apples are far less likely to catch a cold than those who eat few or no apples. The pectin and trace element chromium in apples help stabilize blood sugar, making apples not only a healthy snack but also an essential fruit for anyone wanting to control their blood sugar. They can also effectively lower cholesterol. Apples can also prevent cancer and lead poisoning. Traditional Chinese medicine believes that apples have the effects of quenching thirst, moistening the lungs and relieving irritability, strengthening the spleen and stomach, nourishing the heart and qi, moistening the intestines, stopping diarrhea, relieving summer heat, and sobering up. Therefore, their cultivation techniques are of great significance for improving fruit quality and yield.

[0003] During apple tree growth, the root system easily produces adventitious buds and root suckers, which not only affect the growth of the above-ground parts of the tree and ventilation and light penetration under the canopy, but also bring inconvenience to daily tree management. However, directly cutting off the root suckers from the base will cause even more root suckers to sprout around the trunk, wasting the tree's organic nutrients. Using natural root sucker seedlings for propagation is not only beneficial to improving apple quality, but also greatly improves propagation efficiency. Older apple trees, especially some aging ones, can produce some root suckers around them every year. Using this method for propagation is simple, easy, and low-cost. However, it also has many disadvantages: First, the number of seedlings is small, with only a few to a dozen seedlings being propagated from a single mother tree over the years; second, the seedlings are uneven, because the root suckers of the current year mainly rely on the mother tree for nutrients, and due to the different locations of the root suckers and the different strengths of their root systems, the seedlings show significant differences in size and thickness after emergence; third, the number of self-grown roots is not large, because the growth of the root suckers of the current year mainly relies on the mother tree, so very few self-grown roots are produced, the root system is poorly developed, and the survival rate after transplanting is low.

[0004] In traditional apple tree cultivation, apple seedlings are mainly cultivated using single-trunk or tall spindle-shaped tree systems. These techniques allow for relatively loose spacing between plants and rows, resulting in a smaller number of seedlings planted per unit area, which affects land utilization and early orchard yields. Furthermore, traditional cultivation techniques have limitations in terms of seedling resilience, growth rate, and fruit quality. Summary of the Invention

[0005] In view of the above-mentioned shortcomings in the prior art, the present invention provides a tissue culture medium and a rapid seedling raising method for apple rootstock, which can effectively improve the survival rate of seedlings.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows:

[0007] A rapid seedling cultivation method for apple rootstock includes the following steps:

[0008] (1) Obtaining sterile materials

[0009] In spring, cut one-year-old apple branches, wash them with running water, cut them diagonally into stem segments with 1-2 buds, soak them in pure water for 20-30 minutes, clean and disinfect them, and then pat them dry.

[0010] (2) Induction culture

[0011] The treated explants were inoculated into an induction medium and cultured at room temperature for 15-20 days to obtain adventitious shoots. The induction medium included MS medium and 1.5-2.5 mg / L 6-BA, 1-1.5 mg / L 2,4-D, 5-8 g / L agar, and 25-30 g / L sucrose.

[0012] (3) Proliferation culture

[0013] Adventitious buds were inoculated into a proliferation medium and cultured in light and dark at room temperature for 25-30 days to obtain clustered buds. The proliferation medium included MS medium and 0.05-0.5 mg / L aminoethyl ester, 0.5-1.5 mg / L zeatin, 25-30 mg / L sodium thiosulfate, 5-8 g / L agar, and 25-30 g / L sucrose.

[0014] (4) Rooting culture

[0015] The bud clusters were inoculated into the rooting medium and cultured in the dark at room temperature until the roots were 2-4 cm long. The rooting medium included MS medium and 1-1.5 mg / L IAA, 0.5-1.0 mg / L melatonin, 5-8 g / L agar, and 25-30 g / L sucrose.

[0016] (5) Transplanting

[0017] When the roots are 2-4cm long, they are shaded to harden off. After disinfecting the rooted seedlings, they are transplanted into nutrient pots and cultured under light for 5-7 days. Then they are transplanted to outdoor soil and given regular water and fertilizer management.

[0018] Furthermore, the specific process of cleaning and disinfection in step (1) is as follows:

[0019] After rinsing with running water, soak in 75-85% ethanol for 30-60 seconds, then rinse with sterile water 3-5 times, then soak in 0.1% mercuric chloride solution for 5-10 minutes, and then rinse with sterile water 3-5 times.

[0020] Furthermore, the induction medium in step (2) includes MS medium and 1.5~2.0 mg / L 6-BA, 1~1.2 mg / L 2,4-D, 5~8 g / L agar, and 25~30 g / L sucrose.

[0021] Furthermore, the light-dark culture process in step (3) is as follows:

[0022] After one week of dark treatment, the light was applied for 12-16 hours per day at a light intensity of 2000-3500 lx.

[0023] Furthermore, the proliferation medium in step (3) includes MS medium and 0.05~0.1 mg / L amino acid ester, 0.5~1.0 mg / L zeatin, 25 mg / L sodium thiosulfate, 5~8 g / L agar, and 25~30 g / L sucrose.

[0024] Furthermore, the rooting medium in step (4) includes MS medium and 1~1.2 mg / L IAA, 0.5~0.8 mg / L melatonin, 5~8 g / L agar, and 25~30 g / L sucrose.

[0025] Furthermore, the light-dark culture process in step (4) is as follows:

[0026] After dark culture for 3-7 days, the light is provided for 12-16 hours per day at a light intensity of 2000-2500 lx.

[0027] Furthermore, the substrate in the nutrient pot is a mixture of peat moss, perlite, vermiculite, and well-rotted animal manure in a mass ratio of 3:1:1:2.

[0028] Furthermore, in step (5), a 5% potassium permanganate solution is used for disinfection.

[0029] A tissue culture medium for rapid seedling cultivation of apple rootstock includes an induction medium, a proliferation medium, and a rooting medium.

[0030] The induction medium includes MS medium and 1.5~2.5 mg / L 6-BA, 1~1.5 mg / L 2,4-D, 5~8 g / L agar, and 25~30 g / L sucrose;

[0031] The proliferation medium includes MS medium and 0.05~0.5 mg / L aminoethyl ester, 0.5~1.5 mg / L zeatin, 25~30 mg / L sodium thiosulfate, 5~8 g / L agar, and 25~30 g / L sucrose;

[0032] The rooting medium includes MS medium and 1-1.5 mg / L IAA, 0.5-1.0 mg / L melatonin, 5-8 g / L agar, and 25-30 g / L sucrose.

[0033] Furthermore, the pH values ​​of the induction medium, proliferation medium, and rooting medium were all 5.5–6.3.

[0034] The beneficial effects of this invention are:

[0035] This invention, through the combination of specific induction culture medium, proliferation culture medium and rooting culture medium, can effectively improve the growth and development speed and survival rate of apple rootstocks.

[0036] This invention uses apple rootstock, which is difficult to induce callus, as explant material. Aminopropyl esters are added to the culture medium, which enhances cell activity, accelerates the dedifferentiation of isolated tissue into callus, and makes the callus more robust. Furthermore, in combination with zeatin, it can improve bud differentiation rate, prevent seedling etiolation, promote thicker stems and leaves in rootstock seedlings, enhance photosynthesis, and reduce the occurrence of "vitrification" (transparent leaves, easy lodging) in tissue culture seedlings. Aminopropyl esters can also activate antioxidant systems such as superoxide dismutase (SOD) and catalase (CAT) in plants, reducing the damage of zeatin by reactive oxygen species, thereby blocking the enzymatic degradation process and slowing down the degradation of hormones in the culture medium and plant. The inventors found in their research that the addition of sodium thiosulfate can further enhance the efficacy of aminopropyl esters.

[0037] This invention combines a proliferation medium and a rooting medium to reduce the risk of high concentrations of auxin inhibiting the root system, while promoting an increase in the number of roots (more lateral roots and fibrous roots) and enhancing root vitality (stronger roots with stronger absorption capacity), thus laying the foundation for subsequent transplant survival. Detailed Implementation

[0038] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0039] Example 1:

[0040] A rapid seedling cultivation method for apple rootstock includes the following steps:

[0041] (1) Obtaining sterile materials

[0042] In spring, cut one-year-old apple branches, wash them with running water, cut them diagonally into stem segments with 1-2 buds, soak them in pure water for 20 minutes, rinse them with running water, soak them in 75% ethanol for 30 seconds, then rinse them with sterile water 3-5 times, then soak them in a 0.1% mercuric chloride solution for 5 minutes, and then rinse them with sterile water 3-5 times.

[0043] (2) Induction culture

[0044] The treated explants were inoculated into induction medium and cultured at room temperature for 16 days to obtain adventitious shoots. The induction medium included MS medium and 1.5 mg / L 6-BA, 1 mg / L 2,4-D, 5 g / L agar, and 25 g / L sucrose.

[0045] (3) Proliferation culture

[0046] Adventitious buds were inoculated into proliferation medium and treated in the dark at room temperature for one week. Then, they were exposed to light for 16 hours a day at a light intensity of 2000 lx. After 25-30 days, clustered buds were obtained. The proliferation medium included MS medium and 0.1 mg / L aminoethyl ester, 0.5 mg / L zeatin, 30 mg / L sodium thiosulfate, 5 g / L agar, and 25 g / L sucrose.

[0047] (4) Rooting culture

[0048] The shoot clusters were inoculated into rooting medium and cultured in the dark at room temperature for 7 days, then exposed to light for 16 hours a day at a light intensity of 2500 lx until the roots reached a length of 2-4 cm. The rooting medium included MS medium and 1.5 mg / L IAA, 0.5 mg / L melatonin, 5 g / L agar, and 25 g / L sucrose.

[0049] (5) Transplanting

[0050] When the roots are 2-4cm long, move them outdoors to the shade for 10-12 days to harden them off. Then, after disinfecting the rooted seedlings, transplant them into nutrient pots and cultivate them under light for 5-7 days. After that, take them out and soak them in a 0.1% carbendazim solution for 5-10 minutes. Then, wash the roots and transplant them into outdoor soil and carry out regular water and fertilizer management.

[0051] Example 2:

[0052] A rapid seedling cultivation method for apple rootstock includes the following steps:

[0053] (1) Obtaining sterile materials

[0054] In spring, cut one-year-old apple branches, wash them with running water, cut them diagonally into stem segments with 1-2 buds, soak them in pure water for 25 minutes, rinse them with running water, soak them in 75% ethanol for 35 seconds, then rinse them with sterile water 3-5 times, then soak them in a 0.1% mercuric chloride solution for 5 minutes, and then rinse them with sterile water 3-5 times.

[0055] (2) Induction culture

[0056] The treated explants were inoculated into induction medium and cultured at room temperature for 16 days to obtain adventitious shoots. The induction medium included MS medium and 2.5 mg / L 6-BA, 1.5 mg / L 2,4-D, 8 g / L agar, and 30 g / L sucrose.

[0057] (3) Proliferation culture

[0058] Adventitious buds were inoculated into proliferation medium and treated in the dark at room temperature for one week. Then, they were exposed to light for 16 hours a day at a light intensity of 2000 lx. After 25-30 days, clustered buds were obtained. The proliferation medium included MS medium and 0.5 mg / L aminoethyl ester, 1.5 mg / L zeatin, 30 mg / L sodium thiosulfate, 8 g / L agar, and 30 g / L sucrose.

[0059] (4) Rooting culture

[0060] The shoots were inoculated into rooting medium and cultured in the dark at room temperature for 7 days, with 16 hours of light per day at a light intensity of 2500 lx, until the roots were 2-4 cm long. The rooting medium included MS medium and 1 mg / L IAA, 0.5 mg / L melatonin, 8 g / L agar, and 30 g / L sucrose.

[0061] (5) Transplanting

[0062] When the roots are 2-4cm long, move them outdoors to the shade for 10-15 days to harden them off. Then, after disinfecting the rooted seedlings, transplant them into nutrient pots and cultivate them under light for 5-7 days. After that, take them out and soak them in a 0.1% carbendazim solution for 5 minutes. Then wash the roots and transplant them into outdoor soil and carry out regular water and fertilizer management.

[0063] Example 3:

[0064] A rapid seedling cultivation method for apple rootstock includes the following steps:

[0065] (1) Obtaining sterile materials

[0066] In spring, cut one-year-old apple branches, wash them with running water, cut them diagonally into stem segments with 1-2 buds, soak them in pure water for 30 minutes, rinse them with running water, soak them in 75% ethanol for 40 seconds, then rinse them with sterile water 3-5 times, then soak them in a 0.1% mercuric chloride solution for 5 minutes, and then rinse them with sterile water 3-5 times.

[0067] (2) Induction culture

[0068] The treated explants were inoculated into induction medium and cultured at room temperature for 16 days to obtain adventitious shoots. The induction medium included MS medium and 2.0 mg / L 6-BA, 1.2 mg / L 2,4-D, 8 g / L agar, and 30 g / L sucrose.

[0069] (3) Proliferation culture

[0070] Adventitious shoots were inoculated into a proliferation medium and treated in the dark at room temperature for one week. Then, they were exposed to light for 16 hours a day at a light intensity of 2000 lx. After 25-30 days, clustered shoots were obtained. The proliferation medium included MS medium and 0.1 mg / L aminoethyl ester, 1.0 mg / L zeatin, 28 mg / L sodium thiosulfate, 8 g / L agar, and 30 g / L sucrose.

[0071] (4) Rooting culture

[0072] The shoots were inoculated into rooting medium and cultured in the dark at room temperature for 7 days, then exposed to light for 16 hours a day at a light intensity of 2500 lx until the roots were 2-4 cm long. The rooting medium included MS medium and 1.2 mg / L IAA, 1.0 mg / L melatonin, 8 g / L agar, and 30 g / L sucrose.

[0073] (5) Transplanting

[0074] When the roots are 2-4cm long, move them outdoors to the shade for 10-15 days to harden them off. Then, after disinfecting the rooted seedlings, transplant them into nutrient pots and cultivate them under light for 5-7 days. After that, take them out and soak them in a 0.1% carbendazim solution for 5 minutes. Then wash the roots and transplant them into outdoor soil and carry out regular water and fertilizer management.

[0075] Comparative Example 1:

[0076] Compared with Example 1, the difference is that amino acid ester was added to the induction medium, while the rest of the process is the same as in Example 1.

[0077] Comparative Example 2:

[0078] Compared with Example 1, the difference is that amino acid ester was added to the induction medium and GA3 was used to replace amino acid ester in the proliferation medium, while the rest of the process is the same as in Example 1.

[0079] Comparative Example 3:

[0080] The difference from Example 1 is that the amount of amino acid ester used in the proliferation medium is 5 mg / L, while the rest of the process is the same as in Example 1.

[0081] Comparative Example 4:

[0082] The difference from Example 1 is that sodium thiosulfate is not added to the proliferation medium, while the rest of the process is the same as in Example 1.

[0083] Comparative Example 5:

[0084] Compared with Example 1, the difference is that dimethyl sulfoxide is used instead of sodium thiosulfate in the proliferation medium, while the rest of the process is the same as in Example 1.

[0085] Comparative Example 6:

[0086] The difference from Example 1 is that IBA was used instead of IAA in the rooting medium, while the rest of the process remained the same as in Example 1.

[0087] The treatment was carried out in accordance with the methods described in Examples 1-3 and Comparative Examples 1-6, with 30 replicates for each treatment. The germination rate, rooting rate of tissue culture seedlings and transplant survival rate during the induction culture process were calculated. The results are shown in Table 1.

[0088] Table 1. Effects of different treatments on bud break, rooting, and survival of apple rootstocks.

[0089] Germination rate (%) Rooting rate (%) Survival rate (%) Example 1 100 100 100 Example 2 100 100 96.7 Example 3 100 100 100 Comparative Example 1 83.3 73.3 70 Comparative Example 2 80 70 50 Comparative Example 3 100 50 40 Comparative Example 4 100 80 76 Comparative Example 5 100 66.7 60 Comparative Example 6 100 100 83.3

[0090] According to the test results in Table 1, the scheme constructed in this application can significantly improve the rooting rate and survival rate of apple rootstocks. Compared with the comparative examples, when the components in the induction medium, proliferation medium and rooting medium are replaced or added or subtracted, the overall germination, rooting and survival rates are significantly affected, such as in comparative examples 1 to 5. It can be seen that only by using the technical solution defined in this application can apple rootstock seedlings with high germination rate, rooting rate and survival rate be obtained.

[0091] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for rapid seedling raising of an apple rootstock, characterized by, The method comprises the following steps: (1) obtaining sterile materials In spring, apple one-year branches are cut, washed with running water, and cut into stem segments with 1-2 buds. After being soaked in pure water for 20-30 minutes, the stem segments are cleaned and disinfected, and then the surface water is absorbed; (2) induction culture The treated explants are inoculated into an induction medium, and cultured at room temperature for 15-20 days to obtain adventitious buds; the induction medium comprises MS medium and 1.5-2.5 mg / L 6-BA, 1-1.5 mg / L 2,4-D, 5-8 g / L agar and 25-30 g / L sucrose; (3) proliferation culture The adventitious buds are inoculated into a proliferation medium, and light and dark culture is carried out at room temperature for 25-30 days to obtain multiple shoots; the proliferation medium comprises MS medium and 0.05-0.5 mg / L alminium, 0.5-1.5 mg / L zeatin, 25-30 mg / L sodium thiosulfate, 5-8 g / L agar and 25-30 g / L sucrose; (4) rooting culture The multiple shoots are inoculated into a rooting medium, and light and dark culture is carried out at room temperature until the roots grow to 2-4 cm; the rooting medium comprises MS medium and 1-1.5 mg / L IAA, 0.5-1.0 mg / L melatonin, 5-8 g / L agar and 25-30 g / L sucrose; (5) transplanting When the roots grow to 2-4 cm, the seedlings are shaded and acclimated, then the rooted seedlings are disinfected and transplanted into pots, and after 5-7 days of light culture, the seedlings are transplanted into outdoor soil and subjected to conventional water and fertilizer management.

2. The method of rapid seedling raising of the apple rootstock according to claim 1, characterized in that, In step (1), the specific process of cleaning and disinfecting is as follows: After being washed with running water, the explants are soaked in 75-85% ethanol for 30-60 seconds, then washed with sterile water for 3-5 times, then soaked in 0.1% mercuric chloride solution for 5-10 minutes, then washed with sterile water for 3-5 times.

3. The method of rapid seedling raising of the apple rootstock according to claim 1, characterized in that, In step (2), the induction medium comprises MS medium and 1.5-2.0 mg / L 6-BA, 1-1.2 mg / L 2,4-D, 5-8 g / L agar and 25-30 g / L sucrose.

4. The method of rapid seedling raising of the apple rootstock according to claim 1, characterized by, In step (3), the process of light and dark culture is as follows: After dark treatment for one week, daily light exposure is 12-16 hours, and the light intensity is 2000-3500 lx.

5. The method of rapid seedling raising of the apple rootstock according to claim 1, characterized in that, In step (3), the proliferation medium comprises MS medium and 0.05-0.1 mg / L alminium, 0.5-1.0 mg / L zeatin, 25 mg / L sodium thiosulfate, 5-8 g / L agar and 25-30 g / L sucrose.

6. The method of rapid seedling raising of the apple rootstock according to claim 1, characterized in that, In step (4), the rooting medium comprises MS medium and 1-1.2 mg / L IAA, 0.5-0.8 mg / L melatonin, 5-8 g / L agar and 25-30 g / L sucrose.

7. The method of rapid seedling raising of the apple rootstock according to claim 1, characterized in that, In step (4), the process of light and dark culture is as follows: After dark culture for 3-7 days, daily light exposure is 12-16 hours, and the light intensity is 2000-2500 lx.

8. The method of rapid seedling raising of the apple rootstock according to claim 1, characterized in that, In step (5), the seedlings are disinfected with 5% potassium permanganate solution.

9. A tissue culture medium for rapid seedling raising of apple rootstock, characterized by, The tissue culture medium comprises the induction medium, the proliferation medium and the rooting medium. The induction medium comprises MS medium and 1.5-2.5 mg / L 6-BA, 1-1.5 mg / L 2,4-D, 5-8 g / L agar, and 25-30 g / L sucrose; The proliferation medium comprises MS medium and 0.05-0.5 mg / L prohexadione, 0.5-1.5 mg / L zeatin, 25-30 mg / L sodium thiosulfate, 5-8 g / L agar, and 25-30 g / L sucrose; The rooting medium comprises MS medium and 1-1.5 mg / L IAA, 0.5-1.0 mg / L melatonin, 5-8 g / L agar, and 25-30 g / L sucrose.