Tissue culture and rapid propagation method of Delphinium grandiflorum and its application

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a method for tissue culture and rapid propagation of delphinium grandiflorum and its application. The method comprises the following steps: selecting young stems of delphinium grandiflorum as explants, cleaning them with soap solution, and disinfecting them with potassium permanganate solution, alcohol and mercuric chloride solution in sequence; inoculating the disinfected explants in a primary induction medium and cultivating them for 50 to 60 days; cutting the induced clustered buds into single buds and inoculating them in a secondary proliferation medium and cultivating them for 40 to 45 days; cutting the clustered buds of secondary proliferation into single buds and inoculating them in a rooting medium and cultivating them for 30 to 40 days; and controlling the disinfection contamination rate within 40%, the disinfection mortality rate within 10%, achieving a clustered bud induction rate of more than 40%, and stabilizing the proliferation coefficient at about 4.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a tissue culture and rapid propagation method of delphinium grandiflorum and its application. Background Art

[0002] Delphinium granaiflorum, also known as "delphinium flower" and "big delphinium", is a perennial grass plant of the genus Delphinium in the family Ranunculaceae. Because the flower shape resembles blue delphiniums falling all over the branches, it is called "delphinium". There are many varieties of delphiniums. Relatively short varieties with a plant height of 35-60cm are widely used in gardens and have high ornamental value. Delphiniums are drought-tolerant and like the sun. They can grow well in semi-shady environments. They are widely distributed in my country and are widely planted in both the north and the south. At present, they are mainly used for mountain greening and landscaping of gardens. The demand for some greening projects is also very large.

[0003] There are many ways to propagate Delphinium grandiflorum. It can be sown, cut, and divided in spring and autumn. When the seedlings grow to a certain height, they can be transplanted and planted. Fertilization before flowering can make the plants grow rapidly and produce a large number of flowers. The publication number is CN117481024A, and the name is: A short-cycle high-yield soilless cultivation method for cut Delphinium grandiflorum and its application. The method adopts a soilless cultivation matrix and adopts integrated precise management of fertilizer and water circulation, combined with precise control of light, temperature, and humidity, to shorten the growth period of Delphinium grandiflorum from 90 to 120 days to 50 to 60 days, greatly shortening the growth cycle. The publication number is CN102598994A, and the name is: Artificial cultivation method of Delphinium grandiflorum. The method mainly includes the steps of seed collection, seedbed preparation, seed treatment, site selection and land preparation, transplanting, and post-transplant management. The publication number is CN117682914A, and the name is: a vase culture solution for delphinium cut flowers and its application. Delphinium is harvested when 6 to 8 flowers are in bloom in winter and when 30% of the flowers are in bloom in spring; the harvested fresh cut flowers are immersed in an STS solution for 15 to 20 hours and stored at 2 to 5° C. The leaves at the lower part of the stems of the delphinium cut flowers are removed and inserted into the vase culture solution for vase culture, thereby extending the vase life of the delphinium cut flowers to 25 days.

[0004] The above disclosed methods are traditional ways of cultivating flowers, all of which are propagated by seeds or cuttings. However, most of the current domestic methods of propagation are by cuttings, which have the problems of serious accumulation of diseases and pests, and degradation of multiple generations of cutting varieties. Summary of the invention

[0005] The purpose of the invention is to provide a tissue culture and rapid propagation method of delphinium grandiflorum and its application in view of the problems existing in the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for rapid propagation of Delphinium grandiflorum through tissue culture, comprising the following steps:

[0007] (1) Selection of explants: Select young stems of Delphinium grandiflorum as explants;

[0008] (2) Cleaning and disinfection of explants: After the explants are cleaned with soap solution, they are disinfected with potassium permanganate solution, alcohol and mercuric chloride solution in sequence;

[0009] (3) Primary induction culture: Cut off the wounds at both ends of the sterilized explants, inoculate them into the primary induction culture medium, and culture them for 50 to 60 days at a temperature of 25 ± 2 °C, with light exposure for 12 h per day and a light intensity of 2000 to 3000 lx;

[0010] The formula of the primary induction medium is: MS+NAA 0.02-0.04 mg / L+2-iP 0.5-0.8 mg / L+KT8.0-9.0 mg / L+GA3 0.6-0.8 mg / L;

[0011] (4) Subculture proliferation culture: The induced clustered buds are cut into single buds, inoculated into subculture proliferation medium, and cultured for 40 to 45 days at a temperature of 25 ± 2 °C, with 12 hours of light per day and a light intensity of 2000 to 3000 lx;

[0012] The formula of the secondary proliferation medium is: MS+NAA 0.05-0.08 mg / L+KT 5.0-6.0 mg / L+TDZ 0.3-0.5 mg / L;

[0013] (5) Rooting culture: Cut the subcultured clustered buds into single buds, inoculate them into rooting medium, and culture them for 30 to 40 days at a temperature of 25 ± 2 °C, with 12 hours of light per day and a light intensity of 2000 to 3000 lx;

[0014] The formula of the rooting culture medium is: MS+NAA 0.2-0.3 mg / L+IBA 0.5-0.7 mg / L.

[0015] Furthermore, the explant young stem is a newly grown, smooth stem that is not hollow and is located on the soil surface at the base of the plant.

[0016] Furthermore, the specific steps of disinfection are: use 0.02% potassium permanganate to disinfect by standing for 60 to 80 minutes, rinse with sterile water 6 to 7 times, use 75% alcohol to shake for 40 to 50 seconds, rinse with sterile water 3 to 4 times, use 0.15% mercuric chloride solution to shake for 2 to 3 minutes, rinse with sterile water 7 to 8 times.

[0017] Furthermore, the formula of the primary induction medium is: MS+NAA 0.03mg / L+2-iP 0.6mg / L+KT8.5mg / L+GA3 0.7mg / L.

[0018] Furthermore, the formula of the secondary proliferation medium is: MS+NAA 0.06mg / L+KT 5.5mg / L+TDZ0.4mg / L.

[0019] Furthermore, the formula of the rooting medium is: MS+NAA 0.2mg / L+IBA 0.6mg / L.

[0020] The beneficial technical effects of the present invention are:

[0021] 1. There are axillary buds on the stems of Delphinium grandiflorum. From the perspective of tissue culture theory, its stems should be the best explant material, but in actual operation, the stems of Delphinium grandiflorum mostly have villi, and the stems are hollow. No matter which disinfectant is used for treatment, its contamination rate reaches more than 90%. When high-dose and long-term disinfection methods are adopted, pollution cannot be effectively controlled, and excessive disinfection can also cause explant death. At the same time, the growth of axillary buds is often accompanied by the differentiation of flower buds, so that cluster bud induction is more difficult in the tissue culture process. The present invention creatively adopts the newly-sprouted stems at the base of the plant, and simultaneously adopts potassium permanganate, alcohol and high-concentration mercuric chloride for short-term disinfection, which can control the disinfection contamination rate within 40% and the disinfection mortality rate within 10%.

[0022] 2. In the primary induction process, the present invention creatively adds GA3 at an appropriate concentration, which can effectively inhibit the flower bud differentiation of Delphinium grandiflorum during the cluster bud induction process. At the same time, combined with appropriate concentrations of NAA, 2-iP and high concentrations of KT, the cluster bud induction rate can reach more than 40%.

[0023] 3. During the induction of the primary generation of clustered buds of the giant petal, it was found that a certain amount of vitrified seedlings occurred. If the vitrified seedlings were not inhibited, a large area of ​​stunted seedlings or even dead seedlings would easily appear during further propagation. During the subculture proliferation culture, the present invention appropriately increases the auxin concentration and greatly reduces the KT concentration. At the same time, TDZ of a suitable concentration is used instead of 2-iP, which can promote the reversal of vitrified seedlings. When the subculture proliferation is carried out for many times, the vitrification phenomenon will not occur. When the proliferation generation is within 10 generations, the proliferation coefficient can also be stabilized at about 4. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0025] Figure 1 This is the proliferation culture result of Example 3 of the present invention. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1

[0028] A method for rapid propagation of Delphinium grandiflorum tissue culture, comprising the following steps:

[0029] (1) Selection of explants: Select young stems of Delphinium grandiflorum as explants, which should be newly grown, smooth stems at the base of the plant and not hollow.

[0030] (2) Cleaning and disinfection of explants: After the explants are cleaned with soap solution, they are disinfected with 0.02% potassium permanganate for 60 minutes, rinsed with sterile water for 6 times, disinfected with 75% alcohol by shaking for 40 seconds, rinsed with sterile water for 3 times, and disinfected with 0.15% mercuric chloride solution by shaking for 2 minutes, rinsed with sterile water for 7 times.

[0031] (3) Primary induction culture: Cut off the wounds at both ends of the sterilized explants and inoculate them into primary induction culture medium (formula: MS + NAA 0.02mg / L + 2-iP 0.5mg / L + KT 8.0mg / L + GA3 0.6mg / L), place them at a temperature of 25±2℃, light for 12h per day, and culture them under alternating light and dark conditions with a light intensity of 2000lx for 50 days.

[0032] (4) Subculture: The induced clustered buds were cut into single buds and inoculated into subculture proliferation medium (formula: MS + NAA 0.05 mg / L + KT 5.0 mg / L + TDZ 0.3 mg / L), and cultured at 25 ± 2 °C, with 12 h of light per day and a light intensity of 2000 lx for 40 days under alternating light and dark conditions.

[0033] (5) Rooting culture: Cut the subcultured clustered buds into single buds, inoculate them into rooting medium (formula: MS + NAA 0.3 mg / L + IBA 0.7 mg / L), place them at a temperature of 25 ± 2 °C, light exposure for 12 h per day, and culture them under alternating light and dark conditions with a light intensity of 2000 lx for 30 days.

[0034] Example 2

[0035] A method for rapid propagation of Delphinium grandiflorum tissue culture, comprising the following steps:

[0036] (1) Selection of explants: Select young stems of Delphinium grandiflorum as explants, which should be newly grown, smooth stems at the base of the plant and not hollow.

[0037] (2) Cleaning and disinfection of explants: After the explants are cleaned with soap solution, they are disinfected with 0.02% potassium permanganate for 80 minutes, rinsed with sterile water 7 times, disinfected with 75% alcohol by shaking for 50 seconds, rinsed with sterile water 4 times, and disinfected with 0.15% mercuric chloride solution by shaking for 3 minutes, rinsed with sterile water 8 times.

[0038] (3) Primary induction culture: Cut off the wounds at both ends of the sterilized explants and inoculate them into primary induction culture medium (formula: MS + NAA 0.04mg / L + 2-iP 0.8mg / L + KT 9.0mg / L + GA3 0.8mg / L), place them at a temperature of 25±2℃, light for 12h per day, and culture them under alternating light and dark conditions with a light intensity of 3000lx for 60 days.

[0039] (4) Subculture: The induced clustered buds were cut into single buds and inoculated into subculture proliferation medium (formula: MS + NAA 0.08 mg / L + KT 6.0 mg / L + TDZ 0.5 mg / L), and cultured at 25 ± 2 °C, with 12 h of light per day and a light intensity of 3000 lx for 45 days under alternating light and dark conditions.

[0040] (5) Rooting culture: Cut the subcultured clustered buds into single buds, inoculate them into rooting medium (formula: MS + NAA 0.3 mg / L + IBA 0.7 mg / L), place them at a temperature of 25 ± 2 °C, light exposure for 12 h per day, and culture them under alternating light and dark conditions with a light intensity of 3000 lx for 40 days.

[0041] Example 3

[0042] A method for rapid propagation of Delphinium grandiflorum tissue culture, comprising the following steps:

[0043] (1) Selection of explants: Select young stems of Delphinium grandiflorum as explants, which should be newly grown, smooth stems at the base of the plant and not hollow.

[0044] (2) Cleaning and disinfection of explants: After the explants are cleaned with soap solution, they are disinfected with 0.02% potassium permanganate for 70 minutes, rinsed with sterile water 7 times, disinfected with 75% alcohol by shaking for 45 seconds, rinsed with sterile water 4 times, and disinfected with 0.15% mercuric chloride solution by shaking for 2.5 minutes, rinsed with sterile water 8 times.

[0045] (3) Primary induction culture: Cut off the wounds at both ends of the sterilized explants and inoculate them into primary induction culture medium (formula: MS + NAA 0.03mg / L + 2-iP 0.6mg / L + KT 8.5mg / L + GA3 0.7mg / L), place them at a temperature of 25±2℃, light for 12h per day, and culture them under alternating light and dark conditions with a light intensity of 2500lx for 55 days.

[0046] (4) Subculture proliferation culture: The induced clustered buds were cut into single buds and inoculated into subculture proliferation medium (formula: MS + NAA 0.06 mg / L + KT 5.5 mg / L + TDZ 0.4 mg / L), and cultured at 25 ± 2 °C, with 12 h of light per day and a light intensity of 2500 lx for 43 days under alternating light and dark conditions. Figure 1 shown.

[0047] (5) Rooting culture: Cut the subcultured clustered buds into single buds, inoculate them into rooting medium (formula: MS + NAA 0.2 mg / L + IBA 0.6 mg / L), place them at a temperature of 25 ± 2 °C, light exposure for 12 h per day, and culture them under alternating light and dark conditions with a light intensity of 2500 lx for 35 days.

[0048] The above Examples 1-3 were used to perform statistics on disinfection contamination rate, disinfection mortality rate, primary generation clustered bud induction rate, proliferation coefficient and rooting rate. The results are shown in Table 1.

[0049] Table 1 Statistical results

[0050] Disinfection contamination rate Disinfection mortality rate Primary generation bud induction rate Proliferation coefficient Rooting rate Example 1 36.7% 8.7% 54.3% 3.9 98.6% Example 2 37.2% 9.5% 51.9% 4.1 99.2% Example 3 33.5% 8.1% 55.8% 4.5 98.3%

[0051] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for rapid propagation of Delphinium grandiflorum through tissue culture, characterized in that the steps include: (1) Selection of explants: Select young stems of Delphinium grandiflorum as explants; (2) Cleaning and disinfection of explants: After the explants are cleaned with soap solution, they are disinfected with potassium permanganate solution, alcohol and mercuric chloride solution in sequence; (3) Primary induction culture: Cut off the wounds at both ends of the sterilized explants, inoculate them in the primary induction culture medium, place them at a temperature of 25±2℃, light for 12 hours per day, and culture them under alternating light and dark conditions with a light intensity of 2000-3000lx for 50-60 days; The formula of the primary induction medium is: MS+NAA 0.02-0.04 mg / L+2-iP 0.5-0.8 mg / L+KT 8.0-9.0 mg / L+GA3 0.6-0.8 mg / L; (4) Subculture proliferation culture: The induced clustered buds are cut into single buds, inoculated into subculture proliferation medium, and cultured for 40 to 45 days at a temperature of 25 ± 2 °C, with 12 hours of light per day and a light intensity of 2000 to 3000 lx; The formula of the subculture proliferation medium is: MS+NAA 0.05-0.08 mg / L+KT 5.0-6.0 mg / L+TDZ 0.3-0.5 mg / L; (5) Rooting culture: The subcultured clustered buds are cut into single buds, inoculated into rooting medium, and cultured for 30 to 40 days at a temperature of 25 ± 2 °C, with 12 h of light per day and a light intensity of 2000 to 3000 lx; The formula of the rooting culture medium is: MS+NAA 0.2-0.3 mg / L+IBA 0.5-0.7 mg / L.

2. The method for rapid propagation of Delphinium grandiflorum by tissue culture according to claim 1, characterized in that: The explant young stem is a newly grown smooth stem located on the soil surface at the base of the plant.

3. The method for rapid propagation of Delphinium grandiflorum by tissue culture according to claim 1, characterized in that: The specific steps for disinfection are: use 0.02% potassium permanganate to disinfect by standing for 60 to 80 minutes, rinse with sterile water 6 to 7 times, use 75% alcohol to shake for 40 to 50 seconds, rinse with sterile water 3 to 4 times, use 0.15% mercuric chloride solution to shake for 2 to 3 minutes, rinse with sterile water 7 to 8 times.

4. The method for rapid propagation of Delphinium grandiflorum by tissue culture according to claim 1, characterized in that: The formula of the primary induction medium is: MS+NAA 0.03mg / L+2-iP 0.6mg / L+KT 8.5mg / L+GA3 0.7mg / L.

5. The method for rapid propagation of Delphinium grandiflorum by tissue culture according to claim 1, characterized in that: The formula of the secondary proliferation medium is: MS+NAA 0.06mg / L+KT 5.5mg / L+TDZ 0.4mg / L.

6. The method for rapid propagation of Delphinium grandiflorum by tissue culture according to claim 1, characterized in that: The formula of the rooting medium is: MS+NAA 0.2mg / L+IBA 0.6mg / L.

7. Application of the method according to claim 1 in tissue culture and rapid propagation of Delphinium grandiflorum.

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