Rapid Propagation Methods and Applications of Ruby Cluster Silver Leaf Helichrysum Tissue Culture

Abstract

The invention relates to the technical field of plant tissue culture, and specifically to a method and application of tissue culture rapid propagation of ruby ​​cluster silver leaf immortelle; the steps include: explant selection and cleaning, explant disinfection, primary generation cluster bud induction, secondary generation cluster bud proliferation and cluster bud rooting. The disinfection contamination rate of the method of the invention is 8%, the disinfection mortality rate is 24%, the primary generation cluster bud induction rate is as high as 100%, the comprehensive proliferation coefficient can reach more than 20, the rooting rate stably reaches 100%, and the vitrified seedlings in the secondary generation proliferation process can also be devitrified, and the seedlings are strong and green. The production cycle is greatly shortened, thereby greatly reducing the production cost and management cost.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a tissue culture rapid propagation method and application of ruby ​​cluster silverleaf immortelle. Background Art

[0002] Helichrysum arvense (scientific name: Jacobaea maritima ) is a plant of the genus Senecio of the Asteraceae family. It is a perennial herb with thick rhizomes and erect flower stems covered with dense grayish-white cotton hairs. The lower part is not bald. The head inflorescence is inverted cone or bell-shaped, with more than ten or dozens of them arranged in compound umbels at the ends of stems or branches; the peduncle is long and cottony.

[0003] The flower language of Helichrysum includes pure love, elegant temperament, perseverance and beautiful memories. Its flowers are as white as snow, symbolizing pure and flawless love; its elegant shape and waxy and crystal clear leaves imply elegant temperament; Helichrysum has strong vitality and can grow tenaciously in harsh environments, representing the spirit of perseverance; at the same time, its rich colors and various shapes can leave a deep impression on people. Giving Helichrysum as a gift can also express the wish to leave beautiful memories.

[0004] Silver leaf immortelle can be used to arrange flower beds and flower borders, or can be potted for viewing. It can also be used as dried flowers, cut flowers and flower arrangements. Silver leaf immortelle generally adopts two methods of seed propagation and cutting propagation. Among them, the seed propagation method is generally sown in a greenhouse in December, and the seedlings grow slowly. The cutting propagation method is generally to cut about 10cm of tender shoots from the end of February to March 15, the wound at the front of the knife can be stained with rooting ridges, remove the two leaves at the base, and then cut into well-drained sandy soil. However, cutting propagation has low reproduction rate, long reproduction cycle, high production cost, and cannot achieve large-scale mass production in a short time, so it is necessary to develop a method for rapid tissue culture of ruby ​​cluster silver leaf immortelle. Summary of the invention

[0005] The invention aims to provide a tissue culture rapid propagation method and application of ruby ​​cluster silverleaf immortelle, so as to solve the problems of slowness and long cycle of traditional propagation methods.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for rapid propagation of ruby ​​cluster silver leaf immortelle tissue culture, comprising the following steps:

[0007] (1) Explant selection and cleaning: Take the buds at the base of the Ruby Cluster Helichrysum and wash them;

[0008] (2) Explant disinfection: The explants were disinfected with a mixture of hydrogen peroxide, silver nitrate and Tween-80, 75% alcohol, and a mixture of mercuric chloride and Tween-80 in turn;

[0009] (3) Primary bud induction: The sterilized explants were inoculated into the primary bud induction medium at 25±2℃, with a light intensity of 2000-3000 lx and a light duration of 10 h / d for 40-50 days under alternating light and dark conditions;

[0010] The formula of the primary generation cluster bud induction medium is: MS medium + IBA 0.05-0.1 mg / L + TDZ 1.0-2.0 mg / L + CPPU 0.2-0.3 mg / L;

[0011] (4) Subculture of clustered buds: The induced clustered buds were cut into single buds, and the leaves were cut off and cut into small segments of 1 to 1.5 cm. The single buds were inoculated in clustered bud subculture proliferation medium A, and cultured for 30 to 35 days at a temperature of 25±2℃, a light intensity of 2000 to 3000 lx, and a light duration of 10 h / d under alternating light and dark conditions; the leaves were inoculated in clustered bud subculture proliferation medium B, and cultured for 10 to 15 days under a temperature of 25±2℃ and total darkness. After the wounds began to turn white, they were cultured for another 30 to 35 days at a temperature of 25±2℃, a light intensity of 2000 to 3000 lx, and a light duration of 10 h / d under alternating light and dark conditions;

[0012] The formula of the cluster bud subculture proliferation medium A is: MS medium + IBA 0.02-0.05 mg / L + TDZ 0.5-1.0 mg / L + BR 1.0-2.0 ug / L;

[0013] The formula of the cluster bud subculture proliferation medium B is: MS medium + 2,4-D 0.2-0.4 mg / L + 6-BA 2.0-3.0 mg / L + TDZ 3.0-4.0 mg / L;

[0014] (5) Rooting of clustered buds: Cut the clustered buds obtained by subculture into single buds and inoculate them into rooting medium at 25±2℃, light intensity of 2000-3000lx, and light duration of 10h / d for 20-25d of alternating light and dark.

[0015] The rooting culture medium formula is: 1 / 2WPM culture medium + IBA 0.5-0.7 mg / L + 2,4-D 0.05-0.1 mg / L + applesauce 5-10 mg / L.

[0016] Furthermore, in step (2), the disinfection is specifically as follows: a mixed solution of 1% hydrogen peroxide, 0.1% silver nitrate and 2 to 3 drops of Tween-80 is allowed to stand for disinfection for 12 to 24 hours, and then rinsed with sterile water 4 to 5 times; a mixed solution of 75% alcohol is shaken for disinfection for 30 to 40 seconds, and then rinsed with sterile water 2 to 3 times; a mixed solution of 0.05% mercuric chloride is added with 2 to 3 drops of Tween-80 and then shaken for disinfection for 5 to 6 minutes, and then rinsed with sterile water 6 to 7 times.

[0017] Furthermore, in step (3), the formula of the primary cluster bud induction medium is: MS medium + IBA 0.07 mg / L + TDZ 1.5 mg / L + CPPU 0.25 mg / L.

[0018] Furthermore, in step (4), the formula of the cluster bud subculture proliferation medium A is: MS medium + IBA 0.03 mg / L + TDZ 0.7 mg / L + BR 1.5 ug / L.

[0019] Furthermore, in step (4), the formula of the cluster bud subculture proliferation medium B is: MS medium + 2,4-D 0.3 mg / L + 6-BA 2.5 mg / L + TDZ 3.5 mg / L.

[0020] Furthermore, in step (5), the rooting medium formula is: 1 / 2WPM medium + IBA 0.6 mg / L + 2,4-D 0.08 mg / L + applesauce 7 mg / L.

[0021] The beneficial technical effects of the present invention are:

[0022] 1. The ruby ​​stems and leaves are all covered with a large amount of villi. When conventional 75% alcohol and 0.1% mercuric chloride solution are used for disinfection, no matter how the time is combined, either a large amount of pollution occurs, or a certain amount of pollution occurs but a large number of explants die. When the villi are scraped off with a knife, the contamination rate can be greatly reduced during long-term disinfection, but the explants can hardly withstand the disinfectant and cause death or damage and cannot sprout. The present invention adopts a mixed solution composed of hydrogen peroxide, silver nitrate and Tween-80 of appropriate concentration to stand for 12 to 24 hours in the initial stage of disinfection, which is conducive to the killing of surface microorganisms on the one hand, and on the other hand, the surface activity of the explants can also be increased, and the alcohol and low-concentration mercuric chloride solution are collocated, which is conducive to substantially killing the microorganisms of the explants, while minimizing the damage to the explants.

[0023] 2. The present invention adopts a combination of NAA, TDZ and CPPU during the primary induction culture, which can make the primary cluster bud induction rate as high as 100%, and the cluster bud tillering rate is high.

[0024] 3. The present invention utilizes both the tillering buds and the leaves of the buds during the subculture proliferation culture, and its comprehensive proliferation coefficient can reach more than 20. In the field of tissue culture, the increase in the proliferation coefficient can greatly shorten the production cycle and reduce the amount of mother bottles used, thereby greatly reducing production costs and management costs.

[0025] 4. The present invention adopts a combination of IBA and 2,4-D at a suitable concentration during rooting culture, and simultaneously adds applesauce at a suitable concentration, which can stabilize the rooting rate to 100%, and the vitrified seedlings in the process of subculture and proliferation can also be devitrified, and the seedlings are strong and green. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] Figure 1 This is a diagram of the primary generation of clustered bud induction results of Example 3 of the present invention.

[0028] Figure 2 This is a diagram showing the results of the proliferation of subcultured clustered buds in Example 3 of the present invention.

[0029] Figure 3 This is a diagram showing the rooting results of clustered buds in Example 3 of the present invention. DETAILED DESCRIPTION

[0030] 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.

[0031] Example 1

[0032] A method for rapid propagation of ruby ​​cluster Helichrysum cinnamomeum tissue culture, comprising the following steps:

[0033] (1) Explant selection and cleaning: Take the buds at the base of the ruby ​​cluster silverleaf helichrysum and wash them.

[0034] (2) Explant disinfection: The explants were disinfected with a mixed solution of 1% hydrogen peroxide, 0.1% silver nitrate and 2 drops of Tween-80 for 12 h, and then rinsed with sterile water for 4 times; disinfected with 75% alcohol by shaking for 30 s, and then rinsed with sterile water twice; disinfected with 0.05% mercuric chloride solution plus 2 drops of Tween-80 by shaking for 5 min, and then rinsed with sterile water for 6 times.

[0035] (3) Primary bud induction: The sterilized explants were inoculated into the primary bud induction medium at 25±2℃, with a light intensity of 2000 lx and a light duration of 10 h / d for 40 days under alternating light and dark conditions;

[0036] Among them, the formula of the primary cluster bud induction medium is: MS medium + IBA 0.05mg / L + TDZ 1.0mg / L + CPPU 0.2mg / L.

[0037] (4) Subculture of clustered buds: The induced clustered buds were cut into single buds, and the leaves were cut off and cut into 1 cm small segments. The single buds were inoculated in clustered bud subculture proliferation medium A, and cultured for 30 days at a temperature of 25±2℃, a light intensity of 2000lx, and a light duration of 10h / d with alternating light and dark conditions; the leaves were inoculated in clustered bud subculture proliferation medium B, and cultured for 10 to 15 days at a temperature of 25±2℃ and in complete darkness. After the wounds began to turn white, they were cultured for another 30 days at a temperature of 25±2℃, a light intensity of 2000lx, and a light duration of 10h / d with alternating light and dark conditions;

[0038] Among them, the formula of cluster bud subculture proliferation medium A is: MS medium + IBA 0.02mg / L + TDZ 0.5mg / L + BR1.0ug / L;

[0039] Among them, the formula of cluster bud subculture proliferation medium B is: MS medium + 2,4-D 0.2mg / L + 6-BA 2.0mg / L + TDZ 3.0mg / L.

[0040] (5) Rooting of clustered buds: The clustered buds obtained by subculture were cut into single buds and inoculated into rooting medium at 25±2℃, light intensity of 2000lx, and light duration of 10h / d with alternating light and dark culture for 20 days;

[0041] Among them, the rooting culture medium formula is: 1 / 2WPM culture medium + IBA 0.5~0.7mg / L + 2,4-D 0.05~0.1mg / L + apple paste 5~10mg / L.

[0042] Example 2

[0043] A method for rapid propagation of ruby ​​cluster Helichrysum cinnamomeum tissue culture, comprising the following steps:

[0044] (1) Explant selection and cleaning: Take the buds at the base of the Ruby Cluster Helichrysum and wash them;

[0045] (2) Explant disinfection: The explants were disinfected with a mixed solution of 1% hydrogen peroxide, 0.1% silver nitrate and 3 drops of Tween-80 for 24 h, and then rinsed with sterile water 5 times; disinfected with 75% alcohol by shaking for 40 s, and then rinsed with sterile water 3 times; disinfected with 0.05% mercuric chloride solution plus 3 drops of Tween-80 by shaking for 6 min, and then rinsed with sterile water 7 times.

[0046] (3) Primary bud induction: The sterilized explants were inoculated into the primary bud induction medium at 25±2℃, with a light intensity of 3000 lx and a light duration of 10 h / d for 50 days under alternating light and dark conditions;

[0047] Among them, the formula of the primary cluster bud induction medium is: MS medium + IBA 0.1mg / L + TDZ 2.0mg / L + CPPU 0.3mg / L.

[0048] (4) Subculture of cluster buds: The induced cluster buds were cut into single buds, and the leaves were cut off and cut into 1.5 cm small segments. The single buds were inoculated in cluster bud subculture proliferation medium A, and cultured for 35 days at a temperature of 25±2℃, a light intensity of 3000lx, and a light duration of 10h / d under alternating light and dark conditions; the leaves were inoculated in cluster bud subculture proliferation medium B, and cultured for 15 days at a temperature of 25±2℃ and in complete darkness. After the wounds began to turn white, they were cultured for another 35 days at a temperature of 25±2℃, a light intensity of 3000lx, and a light duration of 10h / d under alternating light and dark conditions;

[0049] Among them, the formula of cluster bud subculture proliferation medium A is: MS medium + IBA 0.05mg / L + TDZ 1.0mg / L + BR2.0ug / L;

[0050] Among them, the formula of cluster bud subculture proliferation medium B is: MS medium + 2,4-D 0.4mg / L + 6-BA 3.0mg / L + TDZ 4.0mg / L;

[0051] (5) Rooting of clustered buds: The clustered buds obtained by subculture were cut into single buds and inoculated into rooting medium at a temperature of 25±2℃, a light intensity of 3000lx, and a light duration of 10h / d with alternating light and dark for 25 days;

[0052] Among them, the rooting culture medium formula is: 1 / 2WPM culture medium + IBA 0.7 mg / L + 2,4-D 0.1 mg / L + applesauce 10 mg / L.

[0053] Example 3

[0054] A method for rapid propagation of ruby ​​cluster Helichrysum cinnamomeum tissue culture, comprising the following steps:

[0055] (1) Explant selection and cleaning: Take the buds at the base of the ruby ​​cluster silverleaf helichrysum and wash them.

[0056] (2) Explant disinfection: The explants were disinfected with a mixed solution of 1% hydrogen peroxide, 0.1% silver nitrate and 2 drops of Tween-80 for 18 h, and then rinsed with sterile water for 4 times; disinfected with 75% alcohol by shaking for 35 seconds, and then rinsed with sterile water for 3 times; disinfected with 0.05% mercuric chloride solution plus 2 drops of Tween-80 by shaking for 5 minutes, and then rinsed with sterile water for 6 times.

[0057] (3) Primary bud induction: The sterilized explants were inoculated into the primary bud induction medium, and the temperature was 25±2℃, the light intensity was 2500lx, and the light duration was 10h / d. The culture was carried out under light-dark alternation for 45 days. The culture results were as follows: Figure 1 As shown;

[0058] Among them, the formula of the primary cluster bud induction medium is: MS medium + IBA 0.07mg / L + TDZ 1.5mg / L + CPPU 0.25mg / L.

[0059] (4) Subculture of clustered buds: The induced clustered buds were cut into single buds, and the leaves were cut off and cut into 1 cm small segments. The single buds were inoculated in clustered bud subculture proliferation medium A, and cultured for 33 days at a temperature of 25±2℃, a light intensity of 2500lx, and a light duration of 10h / d under alternating light and dark conditions; the leaves were inoculated in clustered bud subculture proliferation medium B, and cultured for 13 days under a temperature of 25±2℃ and total darkness. After the wounds began to turn white, they were cultured for another 33 days at a temperature of 25±2℃, a light intensity of 2500lx, and a light duration of 10h / d under alternating light and dark conditions;

[0060] Among them, the formula of cluster bud subculture proliferation medium A is: MS medium + IBA 0.03mg / L + TDZ 0.7mg / L + BR1.5ug / L;

[0061] Among them, the formula of cluster bud subculture proliferation medium B is: MS medium + 2,4-D 0.3mg / L + 6-BA 2.5mg / L + TDZ 3.5mg / L.

[0062] (5) Rooting of clustered buds: The clustered buds obtained by subculture were cut into single buds and inoculated into rooting medium. The temperature was 25±2℃, the light intensity was 2500lx, and the light duration was 10h / d. The light was alternating with dark for 23 days. The culture results were as follows: Figure 3 As shown;

[0063] Among them, the rooting medium formula is: 1 / 2WPM medium + IBA 0.6mg / L + 2,4-D 0.08mg / L + applesauce 7mg / L.

[0064] Comparative Example 1

[0065] Take the buds at the base of the ruby ​​cluster Cineraria, wash them, and follow the procedure described in Gong Wei, Hu Tingxing, Gong Yuanbo, et al. Research on tissue culture propagation of Cineraria finely cracked leaves [J]. Journal of Tropical and Subtropical Botany, 2004, 12(5):471-472.DOI:10.3969 / j.issn.1005-3395.2004.5.016.

[0066] Comparative Example 2

[0067] Take the buds at the base of the ruby ​​cluster Cineraria, wash them, and follow the procedure described by Gong Wei, Hu Tingxing, Gong Yuanbo, et al. Tissue culture and rapid propagation of Cineraria slenderis[J]. Acta Physiologica Sinica, 2004, 040(002):195-195.

[0068] Comparative Example 3

[0069] Take the buds at the base of the ruby ​​cluster Helichrysum citrinum, wash them, and follow the procedure described by Zhao Hongbo, Chen Fadi, Fang Weimin. Tissue culture of Helichrysum citrinum[J]. Acta Physiologica Sinica, 2004, 40(005):578-578.

[0070] The disinfection contamination rate, disinfection mortality rate, primary generation cluster bud induction rate, secondary generation cluster bud proliferation coefficient, vitrification rate and rooting rate of the above Examples 1-3 and Comparative Examples 1-3 were statistically analyzed, and the results are shown in Table 1.

[0071] Table 1 Statistical results

[0072] Disinfection contamination rate Disinfection mortality rate Primary generation bud induction rate Subculture shoot proliferation coefficient Glass transition rate Rooting rate Example 1 7.9% 23.7% 98.6% 20 (no healing) 6.7% 100% Example 2 8.1% 23.8% 98.1% 21 (no healing) 7.1% 100% Example 3 7.6% 22.9% 100% 23 (no healing) 6.4% 100% Comparative Example 1 43.2% 54.1% 8.4% (about 13% induced callus) 3.7 (mostly healed) 19.3% 80.3% Comparative Example 2 45.3% 53.7% 32.6% 6.4 (partial healing) 10.9% 85.1% Comparative Example 3 18.8% 64.9% 18.4% (about 6% induced callus) 5.7 (partial healing) 9.6% 94.2%

[0073] 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 Ruby Cluster Helichrysum cinnamomeum tissue culture, characterized in that the steps include: (1) Explant selection and cleaning: Take the buds at the base of the Ruby Cluster Helichrysum and wash them; (2) Explant disinfection: The explants were disinfected with a mixture of hydrogen peroxide, silver nitrate and Tween-80, 75% alcohol, and a mixture of mercuric chloride and Tween-80 in turn; (3) Primary bud induction: The sterilized explants were inoculated into the primary bud induction medium at 25±2℃, with a light intensity of 2000-3000 lx and a light duration of 10 h / d for 40-50 days under alternating light and dark conditions; The formula of the primary generation cluster bud induction medium is: MS medium + IBA 0.05-0.1 mg / L + TDZ 1.0-2.0 mg / L + CPPU 0.2-0.3 mg / L; (4) Subculture of clustered buds: The induced clustered buds were cut into single buds, and the leaves were cut off and cut into small segments of 1 to 1.5 cm. The single buds were inoculated in clustered bud subculture proliferation medium A, and cultured for 30 to 35 days at a temperature of 25±2℃, a light intensity of 2000 to 3000 lx, and a light duration of 10 h / d under alternating light and dark conditions; the leaves were inoculated in clustered bud subculture proliferation medium B, and cultured for 10 to 15 days under a temperature of 25±2℃ and total darkness. After the wounds began to turn white, they were cultured for another 30 to 35 days at a temperature of 25±2℃, a light intensity of 2000 to 3000 lx, and a light duration of 10 h / d under alternating light and dark conditions; The formula of the cluster bud subculture proliferation medium A is: MS medium + IBA 0.02-0.05 mg / L + TDZ 0.5-1.0 mg / L + BR 1.0-2.0 ug / L; The formula of the cluster bud subculture proliferation medium B is: MS medium + 2,4-D 0.2-0.4 mg / L + 6-BA 2.0-3.0 mg / L + TDZ 3.0-4.0 mg / L; (5) Rooting of clustered buds: Cut the clustered buds obtained by subculture into single buds and inoculate them into rooting medium at 25±2℃, light intensity of 2000-3000lx, and light duration of 10h / d for 20-25d of alternating light and dark. The rooting culture medium formula is: 1 / 2WPM culture medium + IBA 0.5-0.7 mg / L + 2,4-D 0.05-0.1 mg / L + applesauce 5-10 mg / L.

2. The method according to claim 1, characterized in that: In step (2), the disinfection is specifically as follows: a mixed solution of 1% hydrogen peroxide, 0.1% silver nitrate and 2 to 3 drops of Tween-80 is allowed to stand for disinfection for 12 to 24 hours, and then rinsed with sterile water 4 to 5 times; a mixed solution of 75% alcohol is shaken for disinfection for 30 to 40 seconds, and then rinsed with sterile water 2 to 3 times; a mixed solution of 0.05% mercuric chloride is added with 2 to 3 drops of Tween-80 and then shaken for disinfection for 5 to 6 minutes, and then rinsed with sterile water 6 to 7 times.

3. The method according to claim 1, characterized in that: In step (3), the formula of the primary cluster bud induction medium is: MS medium + IBA 0.07 mg / L + TDZ 1.5 mg / L + CPPU 0.25 mg / L.

4. The method according to claim 1, characterized in that: In step (4), the formula of the cluster bud subculture proliferation medium A is: MS medium + IBA 0.03 mg / L + TDZ 0.7 mg / L + BR 1.5 ug / L.

5. The method according to claim 1, characterized in that: In step (4), the formula of the cluster bud subculture proliferation medium B is: MS medium + 2,4-D 0.3 mg / L + 6-BA 2.5 mg / L + TDZ 3.5 mg / L.

6. The method according to claim 1, characterized in that: In step (5), the rooting medium formula is: 1 / 2WPM medium + IBA 0.6 mg / L + 2,4-D 0.08 mg / L + applesauce 7 mg / L.

7. Use of the method according to any one of claims 1 to 6 in tissue culture and rapid propagation of Ruby Cluster Helichrysum cineraria.

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