Tissue culture method of high-yield and high-quality tomatoes

Abstract

The invention relates to the technical field of tomato culture, and discloses a high-yield and high-quality tomato tissue culture method, which comprises the following steps: disinfecting a stem section on a tomato plant as a tomato explant, and inserting the stem section into a preset induction culture medium for induction culture so as to generate a callus; transferring into a preset proliferation culture medium, and performing adventitious bud differentiation induction; transferring the induced explant into a preset differential culture medium, and carrying out adversity differentiation induction; transferring the explant subjected to adversity differentiation induction into a rooting culture medium for rooting culture to obtain a complete regenerated plant; the regenerated plants are subjected to seedling hardening treatment, and the regenerated plants subjected to seedling hardening are transplanted. Plant hormones with different concentrations and PEG-8000 are added into a culture medium to cause osmotic stress, permeation-resistant mutant cells in tomato calluses are screened, callus cell regeneration is induced, and mutants are screened out through leaf phenotypes and growth vigor of regenerated plants and serve as resistant germplasm resources for tomato breeding.

Description

Technical Field

[0001] The invention relates to the technical field of tomato culture, in particular to a tissue culture method for high-yield and high-quality tomatoes. Background Art

[0002] Tomato (Solanum lycopersium), a plant of the Solanaceae family, has a unique fruit flavor and rich nutritional value. It is one of the most commonly cultivated fruits and vegetables in the world. Plants are affected by various adverse environmental factors during their growth and development, including biotic and abiotic stresses. Tomatoes have the highest demand for water during vegetative growth and fruit formation among Solanaceous vegetables. Most cultivated tomato varieties are sensitive to drought stress and moderately sensitive to salt stress. With the trend of increasingly serious environmental problems such as soil salinization, breeding tomato varieties with good stress resistance and high yield plays an important role in the development of the tomato industry. Summary of the invention

[0003] The present invention provides a tissue culture method for high-yield and high-quality tomatoes. The method comprises the following steps: adding different concentrations of plant hormones and PEG-8000 to a culture medium to cause osmotic stress, screening osmotic-resistant mutant cells in tomato callus, inducing callus cell regeneration, screening mutants through leaf phenotypes and growth potential of regenerated plants, and the mutant plants can be used as resistant germplasm resources for tomato breeding.

[0004] The present invention provides a tissue culture method for high-yield and high-quality tomatoes, comprising:

[0005] S1. Select high-yield and high-quality tomato varieties, and use healthy and pest-free stem segments with axillary buds on tomato plants as tomato explants;

[0006] S2, disinfecting the tomato explants with running water, ethanol and sodium hypochlorite solution;

[0007] S3, cutting the sterilized tomato explants and inserting them into a preset induction medium for induction culture, so that callus tissue appears on the tomato explants;

[0008] S4, transferring the tomato explant with callus tissue to a preset proliferation medium to induce adventitious bud differentiation of tomato callus tissue;

[0009] S5, transferring the tomato explant after adventitious bud differentiation induction to a preset differentiation medium to induce callus differentiation under stress;

[0010] S6, transferring the tomato explants after stress differentiation induction to a rooting medium for rooting culture to obtain complete regenerated plants;

[0011] S7, hardening the regenerated plants, and transplanting the hardened regenerated plants.

[0012] Furthermore, the step S2 specifically includes:

[0013] S201, washing the tomato explants with running water, and then soaking the tomato explants in 75% ethanol for 30-60 seconds to disinfect the surface;

[0014] S202, quickly taking out the tomato explants, and soaking them in a sodium hypochlorite solution containing 2.0% effective chlorine for 4-7 minutes for deep disinfection;

[0015] S203, washing the tomato explants with sterile water for 3-5 times, each time for 5 minutes, and drying the surface of the explants with sterile filter paper after washing.

[0016] Furthermore, the step S3 specifically includes:

[0017] S301, on a clean bench, using scissors or a scalpel to cut off 3 mm from each end of the sterilized tomato explant;

[0018] S302, using tweezers to inoculate the tomato explants onto an MS medium without plant hormones for primary culture, and after 20 days of primary culture, cutting off the germinated axillary buds from the base, transferring them onto an MS medium for subculture proliferation culture, and obtaining tomato tissue culture seedlings;

[0019] S303, selecting tomato tissue culture seedlings with normal terminal buds, strong growth and consistent growth, using petioles of leaves that have just expanded after clipping as explants, and inoculating them on an induction medium for induction culture; wherein the induction medium uses MS basic medium + plant hormones, and the plant hormones are 1.0 mg / L BA + 2.0 mg / L 2,4-D;

[0020] S304. About 30 mL of culture medium was added to a 100 mL culture bottle, and 6 explants were inoculated. After 30 days of induction culture, callus tissue appeared on the tomato explants.

[0021] Furthermore, the step S4 specifically includes:

[0022] S401, dividing the tomato callus induced in step S304 into small pieces of set size in a clean bench;

[0023] S402, transferring the divided small pieces to a preset proliferation medium; wherein the proliferation medium adopts MS basic medium + plant hormone, and the plant hormone is 1.0 mg / L BA;

[0024] S403. Six pieces of callus tissue were inoculated into a culture bottle, and callus tissue with differentiated adventitious buds was obtained after 30 days.

[0025] Furthermore, the step S5 specifically includes:

[0026] S501, transferring the callus of the differentiated adventitious buds obtained in step S403 to a differentiation medium supplemented with 5% by mass of PEG-8000 to induce callus differentiation under stress;

[0027] S502, observing the growth of adventitious buds, counting the number of adventitious buds differentiated from the callus after 30 days, and screening adventitious buds with strong stress resistance through the growth of the differentiated adventitious buds after 15 days of continuous cultivation.

[0028] Furthermore, the step S6 specifically includes:

[0029] S601. When the adventitious buds grow to 2-3 cm in height, cut them off from the differentiation medium and transfer them to a rooting medium; wherein the rooting medium is 1 / 2MS+NAA 0.5 mg / L;

[0030] S602, rooting culture was carried out under the conditions of temperature of 20±2℃, light intensity of 1000-1500lx, and light duration of 10-12 hours / day;

[0031] S603. After 7-14 days of cultivation, adventitious roots begin to grow from the base of the adventitious buds, forming complete regenerated plants.

[0032] Furthermore, the step S6 specifically includes:

[0033] S701, when the regenerated plant obtained in step S603 has 3-5 main roots and a set number of lateral roots, the culture bottle is taken out from the culture room and placed in a greenhouse or shed for seedling hardening;

[0034] S702. Select nutrient soil or substrate as the transplanting substrate, take out the regenerated plants from the culture bottle after hardening, wash the culture medium at the roots with clean water, transplant them into nutrient pots or seedling trays, water them with enough water to establish roots, place them in a cool and ventilated place, keep the air humidity at 80%, the temperature at 20-25°C, and carry out normal cultivation management after the plants grow new leaves.

[0035] The beneficial effects of the present invention are:

[0036] The present invention induces ideal callus and improves the differentiation effect of adventitious buds of callus by adding different concentrations of plant hormones to the culture medium; at the same time, by adding 5% of PEG-8000 by mass to the culture medium, the osmotic pressure of the culture medium is increased, and osmotic stress conditions are created for tomato callus regeneration, and regeneration is carried out under adverse conditions, so that cells with vigorous division or adaptation to adversity express embryonic properties to regenerate plants, and preliminary selection is carried out according to the growth performance of differentiated adventitious buds to obtain stress-resistant regenerated plants that can still grow normally under osmotic stress. Finally, stress-resistant regenerated plants are obtained by directional screening of tomato callus mutant cells, which can provide a reference for plant resistance breeding. DETAILED DESCRIPTION

[0037] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0038] The present invention provides a tissue culture method for high-yield and high-quality tomatoes, comprising:

[0039] S1. Select high-yield, high-quality tomato varieties, and use healthy and pest-free stem segments with axillary buds on tomato plants as tomato explants. Select stem segments with consistent growth and robustness, cut off the leaves and part of the petiole, and cut them into 2-3 cm stem segments with one axillary bud in a clean bench.

[0040] S2. Disinfecting the tomato explants with running water, ethanol and sodium hypochlorite solution.

[0041] S201, washing the tomato explants with running water to remove dust and impurities on the surface, and then soaking the tomato explants in 75% ethanol for 30-60 seconds to disinfect the surface;

[0042] S202, quickly taking out the tomato explants, and soaking them in a sodium hypochlorite solution containing 2.0% effective chlorine for 4-7 minutes for deep disinfection;

[0043] S203, washing the tomato explants with sterile water for 3-5 times, each time for 5 minutes, and after washing, blotting the surface of the explants with sterile filter paper to remove residual disinfectant.

[0044] S3, cutting the sterilized tomato explants and inserting them into a preset induction culture medium for induction culture, so that callus tissue appears on the tomato explants.

[0045] S301, on a clean bench, using scissors or a scalpel to cut off 3 mm from each end of the sterilized tomato explant;

[0046] S302, using tweezers to inoculate the tomato explants onto an MS medium without plant hormones for primary culture, observing the germination of axillary buds every day and removing contaminated seedlings in time, and after 20 days of primary culture, cutting the germinated axillary buds from the base and transferring them onto an MS medium for subculture proliferation culture to obtain a large number of tomato tissue culture seedlings;

[0047] S303, select tomato tissue culture seedlings with normal terminal buds, strong growth and consistent growth, use petioles of newly unfolded leaves as explants, cut into small sections of about 0.5 cm in length, and inoculate them on callus induction medium containing different hormones for induction culture. The induction medium uses MS as the basic medium, adds plant hormones of different concentrations, changes the multiple of MS inorganic salts, adds BA and 2,4-D of different concentrations (i.e., the medium is: MS+1.0mg / L BA+2.0mg / L2,4-D), the medium pH is 5.8, the carbon source is 30g / L sucrose, and 7g / L agar powder is solidified.

[0048] S304. Add about 30 mL of culture medium to a 100 mL culture flask, inoculate 6 explants, and observe the changes in the explants and the growth of callus tissue. After 30 days of induction culture, callus tissue appears in the tomato explants.

[0049] The inoculated culture bottle was placed in a tissue culture room at a culture temperature of (25±2)°C and a light intensity of 2000 lx for continuous light culture. The callus induction rate / % can be calculated as: the number of explants induced with callus / the number of inoculated explants×100.

[0050] S4. Transferring the tomato explants with callus tissue to a preset proliferation medium to induce adventitious bud differentiation of tomato callus tissue.

[0051] S401. Divide the tomato callus tissue induced in step S304 into small pieces of about 3 mm×3 mm in size in an ultra-clean workbench.

[0052] S402, transferring the divided small pieces to a tomato callus proliferation medium, wherein the proliferation medium uses MS as the basic medium, adds different concentrations of plant hormones (i.e., the medium is: MS+1.0 mg / L BA), adjusts the pH value to 5.8, uses 30 g / L sucrose as the carbon source, and solidifies with 7 g / L agar powder.

[0053] S403. Six pieces of callus tissue were inoculated into a culture bottle, and callus tissue with differentiated adventitious buds was obtained after 30 days.

[0054] The inoculated culture bottle is placed in a tissue culture room at a culture temperature of (25±2)°C and a light intensity of 2000 lx for continuous light culture. The adventitious bud differentiation rate / % can be calculated as follows: the number of calli differentiated from adventitious buds / the number of inoculated calli×100.

[0055] S5. Transferring the tomato explants after adventitious bud differentiation induction to a preset differentiation medium to perform callus differentiation induction under stress conditions.

[0056] S501. Determine the best differentiation medium for tomato callus: MS+1.0 mg / L BA through step S403, transfer the callus of the differentiated adventitious buds obtained in step S403 to the differentiation medium supplemented with 5% PEG-8000, and induce callus differentiation under adverse conditions.

[0057] S502, observing the growth of adventitious buds, counting the number of adventitious buds differentiated from the callus after 30 days, and screening adventitious buds with strong stress resistance by judging the growth of the differentiated adventitious buds after 15 days of continuous cultivation.

[0058] The inoculated culture bottle is placed in a tissue culture room at a culture temperature of (25±2)°C and a light intensity of 2000 lx for continuous light culture. Among them, the normal growth rate of adventitious buds can be calculated / %=the number of adventitious buds with normal growth potential / the number of adventitious buds differentiated from callus tissue×100.

[0059] The adventitious buds that differentiated under adverse conditions and grew normally were transferred to MS medium supplemented with 5% PEG-8000 for subculture. During the subculture process, the seedlings grew normally and there was no leaf curling under osmotic stress. It can be seen that the seedlings obtained by screening have a certain degree of stress resistance.

[0060] S6. Transferring the tomato explants after stress differentiation induction to a rooting medium for rooting culture to obtain complete regenerated plants.

[0061] S601. When the adventitious buds grow to 2-3 cm in height, they are cut off from the differentiation medium and transferred to the rooting medium, which is 1 / 2MS + NAA 0.5 mg / L.

[0062] S602, rooting culture was carried out under the conditions of temperature of 20±2℃, light intensity of 1000-1500lx, and light time of 10-12 hours / day.

[0063] S603. After 7-14 days of cultivation, adventitious roots begin to grow from the base of the adventitious buds, forming complete regenerated plants.

[0064] S7, hardening the regenerated plants, and transplanting the hardened regenerated plants.

[0065] S701. When the root system of the regenerated plant is well developed with 3-5 main roots and a set number of lateral roots, the culture bottle is taken out of the culture room and placed in a greenhouse or shed for seedling hardening. The seedling hardening time is generally 3-5 days to allow the regenerated plant to gradually adapt to the external environmental conditions.

[0066] S702. Choose loose, fertile, well-drained nutrient soil or substrate as the transplanting substrate, such as vermiculite, perlite, peat soil, etc. mixed in a certain proportion. Take the regenerated plants out of the culture bottle after hardening, wash the culture medium at the roots with clean water, and then transplant them into the nutrient pot or seedling tray, and water them enough for rooting. After transplanting, place the plants in a cool and ventilated place, keep the air humidity at about 80%, and the temperature at 20-25℃. When the plants grow new leaves, indicating that the transplant survives, normal cultivation management can be carried out.

[0067] The present invention induces ideal callus and improves the differentiation effect of adventitious buds of callus by adding different concentrations of plant hormones to the culture medium; at the same time, by adding 5% of PEG-8000 by mass to the culture medium, the osmotic pressure of the culture medium is increased, and osmotic stress conditions are created for tomato callus regeneration, and regeneration is carried out under adverse conditions, so that cells with vigorous division or adaptation to adversity express embryonic properties to regenerate plants, and preliminary selection is carried out according to the growth performance of differentiated adventitious buds to obtain stress-resistant regenerated plants that can still grow normally under osmotic stress. Finally, stress-resistant regenerated plants are obtained by directional screening of tomato callus mutant cells, which can provide a reference for plant resistance breeding.

[0068] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, device, article or method including a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, device, article or method. In the absence of further restrictions, an element defined by the sentence "includes a ..." does not exclude the presence of other identical elements in the process, device, article or method including the element.

[0069] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A tissue culture method for high-yield and high-quality tomatoes, characterized in that: include: S1. Select high-yield and high-quality tomato varieties, and use healthy and pest-free stem segments with axillary buds on tomato plants as tomato explants; S2, disinfecting the tomato explants with running water, ethanol and sodium hypochlorite solution; S3, cutting the sterilized tomato explants and inserting them into a preset induction medium for induction culture, so that callus tissue appears on the tomato explants; S4, transferring the tomato explant with callus tissue to a preset proliferation medium to induce adventitious bud differentiation of tomato callus tissue; S5, transferring the tomato explant after adventitious bud differentiation induction to a preset differentiation medium to induce callus differentiation under stress; S6, transferring the tomato explants after stress differentiation induction to a rooting medium for rooting culture to obtain complete regenerated plants; S7, hardening the regenerated plants, and transplanting the hardened regenerated plants.

2. The tissue culture method for high-yield and high-quality tomatoes according to claim 1, characterized in that: The step S2 specifically includes: S201, washing the tomato explants with running water, and then soaking the tomato explants in 75% ethanol for 30-60 seconds to disinfect the surface; S202, quickly taking out the tomato explants, and soaking them in a sodium hypochlorite solution containing 2.0% effective chlorine for 4-7 minutes for deep disinfection; S203, washing the tomato explants with sterile water for 3-5 times, each time for 5 minutes, and drying the surface of the explants with sterile filter paper after washing.

3. The tissue culture method for high-yield and high-quality tomatoes according to claim 2, characterized in that: The step S3 specifically includes: S301, on a clean bench, using scissors or a scalpel to cut off 3 mm from each end of the sterilized tomato explant; S302, using tweezers to inoculate the tomato explants onto an MS medium without plant hormones for primary culture, and after 20 days of primary culture, cutting off the germinated axillary buds from the base, transferring them onto an MS medium for subculture proliferation culture, and obtaining tomato tissue culture seedlings; S303, selecting tomato tissue culture seedlings with normal terminal buds, strong growth and consistent growth, using petioles of leaves that have just expanded after clipping as explants, and inoculating them on an induction medium for induction culture; wherein the induction medium uses MS basic medium + plant hormones, and the plant hormones are 1.0 mg / L BA + 2.0 mg / L 2,4-D; S304. About 30 mL of culture medium was added to a 100 mL culture bottle, and 6 explants were inoculated. After 30 days of induction culture, callus tissue appeared on the tomato explants.

4. The tissue culture method for high-yield and high-quality tomatoes according to claim 3, characterized in that: The step S4 specifically includes: S401, dividing the tomato callus induced in step S304 into small pieces of set size in a clean bench; S402, transferring the divided small pieces to a preset proliferation medium; wherein the proliferation medium adopts MS basic medium + plant hormone, and the plant hormone is 1.0 mg / L BA; S403. Six pieces of callus tissue were inoculated into a culture bottle, and callus tissue with differentiated adventitious buds was obtained after 30 days.

5. The tissue culture method for high-yield and high-quality tomatoes according to claim 4, characterized in that: The step S5 specifically includes: S501, transferring the callus of the differentiated adventitious buds obtained in step S403 to a differentiation medium supplemented with 5% by mass of PEG-8000 to induce callus differentiation under stress; S502, observing the growth of adventitious buds, counting the number of adventitious buds differentiated from the callus after 30 days, and screening adventitious buds with strong stress resistance through the growth of the differentiated adventitious buds after 15 days of continuous cultivation.

6. The tissue culture method for high-yield and high-quality tomatoes according to claim 5, characterized in that: The step S6 specifically includes: S601. When the adventitious buds grow to 2-3 cm in height, cut them off from the differentiation medium and transfer them to a rooting medium; wherein the rooting medium is 1 / 2MS+NAA 0.5 mg / L; S602, rooting culture was carried out under the conditions of temperature of 20±2℃, light intensity of 1000-1500lx, and light duration of 10-12 hours / day; S603. After 7-14 days of cultivation, adventitious roots begin to grow from the base of the adventitious buds, forming complete regenerated plants.

7. The tissue culture method for high-yield and high-quality tomatoes according to claim 6, characterized in that: The step S6 specifically includes: S701, when the regenerated plant obtained in step S603 has 3-5 main roots and a set number of lateral roots, the culture bottle is taken out from the culture room and placed in a greenhouse or shed for seedling hardening; S702. Select nutrient soil or substrate as the transplanting substrate, take out the regenerated plants from the culture bottle after hardening, wash the culture medium at the roots with clean water, transplant them into nutrient pots or seedling trays, water them with enough water to establish roots, place them in a cool and ventilated place, keep the air humidity at 80%, the temperature at 20-25°C, and carry out normal cultivation management after the plants grow new leaves.