Rapid seedling raising method of needle cushion flower tissue culture and its application

Abstract

The invention relates to the technical field of plant tissue culture, and specifically to a method and application of rapid seedling raising of pincushion flower tissue culture, wherein the end apical bud or axillary bud is used as an explant, which is placed on a clean bench for disinfection after cleaning, the explant is cut into single buds and inoculated in a primary generation cluster bud induction medium for dark culture, and then cultured in light and dark alternately, the induced cluster buds are cut into single buds and inoculated in a secondary proliferation medium for dark culture, and then cultured in light and dark alternately, and the proliferated cluster buds are cut into single buds and inoculated in a rooting medium for culture. The invention is a seedling breeding method using modern biotechnology, which grows in a completely artificially controlled environment, is not affected by factors such as climate and season, can be produced all over the country and all year round, and has a short reproduction cycle (single generation 40 to 50 days), a high reproduction coefficient (single generation proliferation coefficient is above 3.0), and can produce a large number of seedlings that can be used for planting in a short period of time.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for rapid seedling raising through tissue culture of pincushion flower and its application. Background Art

[0002] Pincushion ( Leucospermum nutans R.Br.) is an evergreen creeping shrub of the genus Pincushion in the family Proteaceae. The plant has many branches; the leaves are whorled, needle-shaped, heart-shaped, lanceolate, etc., with serrated edges and leaf tips, sessile, clustered, entire and leathery; the inflorescence is solitary or a few clusters, the corolla is small, needle-shaped, the flower color is red, yellow, orange, etc., the nuts are white, The flowering period is summer and the fruiting period is autumn. Because its flowers look like pins inserted into a spherical pin cushion when in full bloom, it is named "pin cushion flower". Pin cushion flower is native to South Africa and has been introduced and cultivated in China in recent years. It likes a warm, slightly dry and sunny environment. It is not cold-resistant. It should be cool in summer and avoid waterlogging. The suitable growth temperature is 15-27℃, and the winter temperature is not lower than 10℃. The soil should be fertile, loose and well-drained acidic sandy loam. Pin cushion flower is classified as Near Threatened (NT) in the "Red List of Endangered Species of the World Conservation Union".

[0003] Pincushion is suitable for small gardens in tropical areas of southern China. It is very elegant and fresh to decorate a few plants. In the north, it can be potted for viewing and placed in the lobby and guest room. It is very unique and novel. It is an ancient and primitive plant with rich varieties. When the flowers are in full bloom, they look like pins inserted in the spherical pincushion. It is peculiar and beautiful. It can be potted or used as fresh cut flowers. The flower language of pincushion is joy, luck and infinite blessings. It is a very popular variety in flower shops.

[0004] At present, the domestic propagation method of needle cushion flowers is cuttings, and its cutting survival rate is low (generally less than 20%), the cutting seedling production cycle is relatively long (generally requires 6 to 8 months of cuttings), and the cutting seedlings are very prone to root rot, so even if the cuttings are successful, their transplant survival rate is still low. Summary of the invention

[0005] The purpose of the invention is to provide a method for rapid seedling raising through tissue culture of Pincushion Flower and its application in view of the problems existing in the prior art, so as to realize its rapid propagation.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for rapid seedling raising of needle cushion flower tissue culture, comprising the following steps:

[0007] (1) Use the terminal bud or axillary bud as the explant, clean it and place it in a clean bench for disinfection;

[0008] (2) The sterilized explants were cut into single buds, inoculated into the primary induction medium, and cultured in the dark at 25±2°C for 7 to 10 days. They were then cultured in a light-dark alternation environment at 25±2°C, 8 hours of light per day, and a light intensity of 2000 to 3000 lx for 60 to 80 days.

[0009] The formula of the primary induction medium is: WPM medium + NAA 0.1-0.15 mg / L + 2-ip 0.05-0.1 mg / L + TDZ 3.0-4.0 mg / L;

[0010] (3) The induced clustered buds were cut into single buds, inoculated into subculture proliferation medium, and cultured in the dark at 25±2℃ for 4-7 days. Then, they were cultured in alternating light and dark conditions at 25±2℃, 10 hours of light per day, and a light intensity of 2000-3000 lx for 40-50 days.

[0011] The formula of the subculture proliferation medium is: WPM medium + NAA 0.1-0.15 mg / L + TDZ 1.0-2.0 mg / L + ZT 0.1-0.2 mg / L;

[0012] (4) The proliferated clustered buds are cut into single buds, inoculated into rooting medium, and cultured for 40 to 50 days at a temperature of 25 ± 2 °C, with 14 hours of light per day and a light intensity of 2000 to 3000 lx;

[0013] The formula of the rooting medium is: improved WPM medium + NAA 0.2-0.3 mg / L + IBA 0.5-0.6 mg / L + activated carbon 0.4-0.6 g / L;

[0014] The improved WPM culture medium is as follows: the macroelements are reduced by half, the concentrations of iron salts, organic substances and trace elements are 1.5 times of the original concentrations, and the others remain unchanged.

[0015] Further: the disinfection is: disinfection with 75% alcohol for 20-30s, rinsing with sterile water 3-4 times, disinfection with saturated bleach solution for 30-40min, rinsing with clean water 5-6 times, disinfection with a mixed solution of 20% alcohol + 2 drops of Tween-80 + 0.1% mercuric chloride for 5-6min, and rinsing with clean water 7-8 times.

[0016] Further: the formula of the primary induction medium is: WPM medium + NAA 0.15 mg / L + 2-ip 0.07 mg / L + TDZ 3.5 mg / L.

[0017] Furthermore: the formula of the subculture proliferation medium is: WPM medium + NAA 0.1 mg / L + TDZ 1.5 mg / L + ZT 0.15 mg / L.

[0018] Furthermore: the formula of the rooting medium is: improved WPM medium + NAA 0.2 mg / L + IBA 0.55 mg / L + activated carbon 0.5 g / L.

[0019] The beneficial technical effects of the present invention are:

[0020] 1. The present invention utilizes modern biotechnology to carry out seedling breeding. The cultivated seedlings carry almost no fungi and bacteria, and can basically prevent the situation where the seedlings carry pathogens, thereby increasing the survival rate of the seedlings after transplanting.

[0021] 2. The present invention utilizes modern biotechnology to carry out seedling breeding, grows in a completely artificially controlled environment, is not affected by factors such as climate and season, can be produced all over the country, all year round, and has a short breeding cycle (40 to 50 days for a single generation) and a high breeding coefficient (a single-generation proliferation coefficient is above 3.0), and can produce a large number of seedlings that can be used for planting in a short period of time.

[0022] 3. The disinfection success rate of the present invention can reach more than 40%, the disinfection mortality rate can be controlled within 12%, the primary induction rate can be as high as more than 74%, the secondary proliferation coefficient can be more than 3.0, and the rooting rate can reach more than 94%. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] Figure 1 This is the primary induction culture result of Example 1 of the present invention.

[0025] Figure 2 This is the result of the subculture proliferation culture of Example 1 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 seedling raising of pincushion flower tissue culture, comprising the following steps:

[0029] (1) Use the terminal bud or axillary bud as the explant, clean it and place it in a clean bench. Disinfect it with 75% alcohol for 25 seconds, rinse it with sterile water 4 times, disinfect it with saturated bleach solution for 35 minutes, rinse it with dry water 6 times, and disinfect it with a mixed solution of 20% alcohol + 2 drops of Tween-80 + 0.1% mercuric chloride for 6 minutes, rinse it with dry water 7 times.

[0030] (2) The sterilized explants were cut into single buds and inoculated into primary induction medium (formula: WPM medium + NAA 0.15 mg / L + 2-ip 0.07 mg / L + TDZ 3.5 mg / L), cultured in the dark at 25±2°C for 8 days, and then cultured in alternating light and dark conditions at 25±2°C, 8 h of light per day, and 2500 lx of light intensity for 70 days.

[0031] (3) The induced clustered buds were cut into single buds and inoculated into the subculture proliferation medium (formula: WPM medium + NAA 0.1 mg / L + TDZ 1.5 mg / L + ZT 0.15 mg / L), cultured in the dark at 25 ± 2 °C for 6 days, and then cultured in a light-dark alternating environment at 25 ± 2 °C, 10 h of light per day, and 2500 lx for 45 days. The culture results are as follows: Figure 1 shown.

[0032] (4) The proliferated clustered buds were cut into single buds and inoculated into rooting medium (formula: modified WPM medium + NAA 0.2 mg / L + IBA 0.55 mg / L + activated carbon 0.5 g / L), and cultured at 25 ± 2 °C, 14 h of light per day, and 2500 lx of light intensity for 45 days under alternating light and dark conditions. The culture results are as follows: Figure 2 shown.

[0033] Among them, the improved WPM culture medium is: the macroelements are halved, the iron salts, organic matter, and trace elements are 1.5 times the original concentration, and the others remain unchanged.

[0034] Example 2

[0035] A method for rapid seedling raising of pincushion flower tissue culture, comprising the following steps:

[0036] (1) Use the terminal bud or axillary bud as the explant, clean it and place it in a clean bench, and disinfect it with 75% alcohol for 20 seconds, rinse it with sterile water three times, disinfect it with saturated bleach solution for 30 minutes, rinse it with dry water five times, and disinfect it with a mixed solution of 20% alcohol + 2 drops of Tween-80 + 0.1% mercuric chloride for 5 minutes, and rinse it with dry water seven times.

[0037] (2) The sterilized explants were cut into single buds and inoculated into primary induction medium (formula: WPM medium + NAA 0.1 mg / L + 2-ip 0.05 mg / L + TDZ 3.0 mg / L), cultured in the dark at 25±2°C for 7 days, and then cultured in alternating light and dark conditions at 25±2°C, 8 h of light per day, and 2000 lx of light intensity for 80 days.

[0038] (3) The induced clustered buds were cut into single buds, inoculated into subculture proliferation medium (formula: WPM medium + NAA 0.1 mg / L + TDZ 1.0 mg / L + ZT 0.1 mg / L), cultured in the dark at 25±2℃ for 4 days, and then cultured in alternating light and dark conditions at 25±2℃, 10 h of light per day, and 2000 lx of light intensity for 50 days.

[0039] (4) The proliferated clustered buds were cut into single buds and inoculated into rooting medium (formula: modified WPM medium + NAA 0.2 mg / L + IBA 0.5 mg / L + activated carbon 0.4 g / L), and cultured at 25 ± 2 °C, with 14 h of light per day and a light intensity of 2000 lx under alternating light and dark conditions for 50 days;

[0040] Among them, the improved WPM culture medium is: the macroelements are halved, the iron salts, organic matter, and trace elements are 1.5 times the original concentration, and the others remain unchanged.

[0041] Example 3

[0042] A method for rapid seedling raising of pincushion flower tissue culture, comprising the following steps:

[0043] (1) Use the terminal bud or axillary bud as the explant, clean it and place it in a clean bench, and disinfect it with 75% alcohol for 30 seconds, rinse it with sterile water 4 times, disinfect it with saturated bleach solution for 40 minutes, rinse it with dry water 6 times, and disinfect it with a mixed solution of 20% alcohol + 2 drops of Tween-80 + 0.1% mercuric chloride for 6 minutes, and rinse it with dry water 8 times.

[0044] (2) The sterilized explants were cut into single buds and inoculated into primary induction medium (formula: WPM medium + NAA 0.15 mg / L + 2-ip 0.1 mg / L + TDZ 4.0 mg / L), cultured in the dark at 25±2°C for 10 days, and then cultured in alternating light and dark conditions at 25±2°C, 8 h of light per day, and 3000 lx of light intensity for 60 days.

[0045] (3) The induced clustered buds were cut into single buds and inoculated into subculture proliferation medium (formula: WPM medium + NAA 0.15 mg / L + TDZ 2.0 mg / L + ZT 0.2 mg / L), cultured in the dark at 25±2℃ for 7 days, and then cultured in alternating light and dark conditions at 25±2℃, 10 h of light per day, and 3000 lx of light intensity for 40 days.

[0046] (4) The proliferating clustered buds were cut into single buds and inoculated into rooting medium (formula: improved WPM medium + NAA 0.3 mg / L + IBA 0.6 mg / L + activated carbon 0.6 g / L), and cultured at 25 ± 2 °C, with 14 h of light per day and a light intensity of 3000 lx under alternating light and dark conditions for 40 days.

[0047] Among them, the improved WPM culture medium is: the macroelements are halved, the iron salts, organic matter, and trace elements are 1.5 times the original concentration, and the others remain unchanged.

[0048] Comparative Example 1

[0049] Use the end branches of the needle cushion flower for cutting seedling propagation and adopt conventional cutting management methods for management.

[0050] The disinfection contamination rate, disinfection mortality rate, cluster bud induction rate, proliferation coefficient and rooting rate of Examples 1-3 and Comparative Example 1 were statistically compared. The results are shown in Table 1.

[0051] Table 1 Statistical comparison results

[0052] Disinfection contamination rate Disinfection mortality rate Primary induction rate Subculture proliferation coefficient Rooting rate Example 1 51.3% 10.6% 74.6% 3.6 97.3% Example 2 50.8% 11.2% 78.2% 3.4 96.2% Example 3 52.7% 11.0% 75.3% 3.4 95.1% Comparative Example 1 - - - - 16.5%

[0053] 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 seedling raising of pincushion flower tissue culture, characterized in that the steps include: (1) Use the terminal bud or axillary bud as the explant, clean it and place it in a clean bench for disinfection; (2) The sterilized explants were cut into single buds, inoculated into the primary induction medium, and cultured in the dark at 25±2°C for 7 to 10 days. They were then cultured in a light-dark alternation environment at 25±2°C, 8 hours of light per day, and a light intensity of 2000 to 3000 lx for 60 to 80 days. The formula of the primary induction medium is: WPM medium + NAA 0.1-0.15 mg / L + 2-ip 0.05-0.1 mg / L + TDZ 3.0-4.0 mg / L; (3) The induced clustered buds were cut into single buds, inoculated into subculture proliferation medium, and cultured in the dark at 25±2℃ for 4-7 days. Then, they were cultured in alternating light and dark conditions at 25±2℃, 10 hours of light per day, and a light intensity of 2000-3000 lx for 40-50 days. The formula of the subculture proliferation medium is: WPM medium + NAA 0.1-0.15 mg / L + TDZ 1.0-2.0 mg / L + ZT 0.1-0.2 mg / L; (4) The proliferated clustered buds are cut into single buds, inoculated into rooting medium, and cultured for 40 to 50 days at a temperature of 25 ± 2 °C, with 14 hours of light per day and a light intensity of 2000 to 3000 lx; The formula of the rooting medium is: improved WPM medium + NAA 0.2-0.3 mg / L + IBA 0.5-0.6 mg / L + activated carbon 0.4-0.6 g / L; The improved WPM culture medium is as follows: the macroelements are reduced by half, the concentrations of iron salts, organic substances and trace elements are 1.5 times of the original concentrations, and the others remain unchanged.

2. The method according to claim 1, characterized in that: The disinfection is as follows: disinfect with 75% alcohol for 20 to 30 seconds, rinse with sterile water for 3 to 4 times, disinfect with saturated bleach solution for 30 to 40 minutes, rinse with sterile water for 5 to 6 times, disinfect with a mixed solution of 20% alcohol + 2 drops of Tween-80 + 0.1% mercuric chloride for 5 to 6 minutes, rinse with sterile water for 7 to 8 times.

3. The method according to claim 1, characterized in that: The formula of the primary induction medium is: WPM medium + NAA 0.15 mg / L + 2-ip 0.07 mg / L + TDZ 3.5 mg / L.

4. The method according to claim 1, characterized in that: The formula of the subculture proliferation medium is: WPM medium+NAA 0.1 mg / L+TDZ 1.5 mg / L+ZT 0.15 mg / L.

5. The method according to claim 1, characterized in that: The formula of the rooting medium is: improved WPM medium+NAA 0.2 mg / L+IBA 0.55 mg / L+activated carbon 0.5 g / L.

6. Application of the method according to any one of claims 1 to 5 in rapid seedling raising through tissue culture of Pincushion Flower.

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