Hainan catalpa test tube seedlings and culture method thereof, and method for cultivating Hainan catalpa seedlings to be transplanted

Abstract

The present invention relates to the field of plant cultivation technology, and in particular to a Hainan catalpa test tube seedling, a cultivation method thereof, and a method for cultivating Hainan catalpa seedlings to be transplanted. The present invention includes a Hainan catalpa rooting culture medium, which is based on WPM culture medium and further includes the following components at the following concentrations: 0.1-0.5 mg / L indoleacetic acid, 0.1-0.5 mg / L naphthaleneacetic acid, 0.01-0.05 mg / L 2,4-dichlorophenoxyacetic acid, 15-25 g / L sucrose, and 6.0-8.0 g / L agar; the pH of the Hainan catalpa rooting culture medium is 5.7-5.9. Hainan catalpa test tube seedlings with 5-9 roots can be obtained using the rooting culture medium. The test tube seedlings are transplanted into the seedling culture medium of the present invention, and the transplant survival rate is as high as over 90%. This provides a new approach for the cultivation of Hainan catalpa and the selection of superior varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of plant cultivation, in particular to a Hainan catalpa test tube seedling and a cultivation method thereof, and a method for cultivating Hainan catalpa seedlings to be transplanted. Background Art

[0002] Hainan Catalpa, also known as Gmelina hainanensis Oliv, is a tree of the Verbenaceae family and the genus Gmelina, reaching heights of up to 15 meters. It is listed as a national Class II protected wild plant. Its tough texture rarely cracks after drying, remains stable, and is corrosion-resistant. Its cut surface is smooth and glossy, with beautiful ridges. It is often used in construction, shipbuilding, and high-quality furniture.

[0003] At present, the cultivation of Hainan catalpa mainly relies on seed propagation and cutting propagation, and no plan has been found to use tissue culture methods to cultivate Hainan catalpa with a high survival rate.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] The invention aims to provide a Hainan catalpa test tube seedling and a culture method thereof, and a method for cultivating Hainan catalpa seedlings to be transplanted, thereby providing a new approach for the rapid propagation of Hainan catalpa fine varieties.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a Hainan catalpa rooting culture medium, which is based on WPM culture medium and further includes the following components in the following concentrations:

[0008] 0.1-0.5 mg / L indoleacetic acid, 0.1-0.5 mg / L naphthaleneacetic acid, 0.01-0.05 mg / L 2,4-dichlorophenoxyacetic acid, 15-25 g / L sucrose, 6.0-8.0 g / L agar;

[0009] The pH of the Hainan catalpa rooting culture medium is 5.7-5.9.

[0010] The present invention also provides a method for culturing test tube seedlings of Hainan catalpa, comprising the following steps:

[0011] (1) Disinfecting the Hainan catalpa explant to obtain a sterile explant;

[0012] (2) inoculating the sterile explant into a differentiation medium and culturing for 28 to 32 days to obtain clustered shoots of Catalpa hainanensis;

[0013] (3) Take a single bud of the Hainan catalpa bud and transfer it to the subculture medium for 21-25 days to obtain the Hainan catalpa bud proliferation seedlings;

[0014] (4) Transferring the Hainan Ligusticum truncatum proliferating seedlings to Hainan Ligusticum truncatum rooting medium and culturing them for 40 to 50 days to obtain Hainan Ligusticum truncatum test tube seedlings;

[0015] The Hainan catalpa rooting culture medium in step (4) is the Hainan catalpa rooting culture medium.

[0016] Preferably, the explant of Catalpa hainanensis is a bud or stem of Catalpa hainanensis;

[0017] The method for disinfecting the Hainan catalpa explant is as follows: first, soaking the Hainan catalpa explant in alcohol for 30 to 40 seconds, and then soaking it in a mercuric chloride solution for 1 to 2 minutes;

[0018] The initial concentration of the alcohol is 70-80vt%;

[0019] The mercuric chloride solution is an aqueous solution of mercuric chloride;

[0020] The initial concentration of the mercuric chloride solution is 0.09-0.11 wt %.

[0021] Preferably, the differentiation medium is based on 1 / 2 MS medium and further comprises the following components at the following concentrations:

[0022] 0.3-1.5 mg / L 6-benzylaminopurine, 0.03-0.15 mg / L naphthaleneacetic acid, 20-40 g / L sucrose, 6.0-8.0 g / L agar;

[0023] The pH of the differentiation medium is 5.7-5.9.

[0024] Preferably, the secondary culture medium is based on MS medium and further comprises the following components at the following concentrations:

[0025] 0.6-2.0 mg / L 6-benzylaminopurine, 0.07-0.25 mg / L naphthaleneacetic acid, 0.1-0.4 mg / L kinetin, 20-40 g / L sucrose, 6.0-8.0 g / L agar;

[0026] The pH of the subculture medium is 5.7-5.9.

[0027] Preferably, the culture temperature in steps (2) to (4) is independently 23 to 25°C;

[0028] The humidity of the culture is independently 50-65%;

[0029] The light intensity of the culture is independently 2700-2900 Lux;

[0030] The light exposure time of the culture is independently 14 to 16 h / d.

[0031] The present invention also provides Hainan Ligusticum aviculare test tube seedlings cultured by the culture method, wherein the Hainan Ligusticum aviculare test tube seedlings have a plant height of 7 to 10 cm.

[0032] The present invention also provides a seedling-growing matrix, which comprises the following components in a mass ratio:

[0033] Vermiculite: river sand: perlite: peat soil is 1.5-2.5:1.5-2.5:0.5-1.5:4.5-5.5;

[0034] The seedling culture matrix is ​​used to cultivate the Hainan catalpa test tube seedlings into seedlings to be transplanted.

[0035] The present invention also provides a method for cultivating Hainan catalpa seedlings to be transplanted, comprising the following steps:

[0036] (1) Hardening the test tube seedlings of Ligusticum hainanensis for 4 to 7 days to obtain seedlings to be cultivated;

[0037] (2) transplanting the seedlings to be cultivated into a seedling culture medium and cultivating them for 15 to 20 days to obtain Hainan catalpa seedlings to be transplanted;

[0038] The hainan lily seedlings in the step (1) are the hainan lily seedlings in the test tube;

[0039] The seedling raising matrix in step (2) is the seedling raising matrix.

[0040] Preferably, the light intensity of the seedling hardening is 3800-4400 Lux;

[0041] The humidity of the seedling hardening is 60-70%;

[0042] The temperature of the seedling hardening is 25-27°C.

[0043] The present invention provides a Hainan catalpa test tube seedling and a culture method thereof, and a method for cultivating Hainan catalpa seedlings to be transplanted. The method of the present invention has the following advantages over the prior art methods:

[0044] The differentiation culture medium of the present invention can be used to differentiate 3-5 Hainan catalpa bud clusters. The clusters of buds can be cut into individual pieces and transferred to a subculture culture medium to obtain proliferated seedlings with stem segments. Transferring the proliferated seedlings to a rooting culture medium can produce Hainan catalpa test tube seedlings with 5-9 roots, a plant height of 7-10 cm, and 4-6 leaves. The entire process takes only 89-107 days. After hardening the test tube seedlings, they are transplanted, with a transplant survival rate of 90-95%. This provides a new approach for the cultivation of Hainan catalpa and the selection of superior varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the explant of Lithops hainanensis;

[0046] Figure 2 It is the clustered buds of Hainan catalpa;

[0047] Figure 3 It is the Hainan Catalpa hainanensis seedlings;

[0048] Figure 4 The rooting situation of Hainan catalpa;

[0049] Figure 5 This is a Hainan stone catalpa that survived after transplantation. DETAILED DESCRIPTION

[0050] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] In the present invention, the WPM culture medium includes components in the following concentrations: potassium sulfate 0.99 g / L, magnesium sulfate heptahydrate 0.37 g / L, potassium dihydrogen phosphate 0.17 g / L, ammonium nitrate 0.4 g / L, manganese sulfate tetrahydrate 0.0225 g / L, zinc sulfate heptahydrate 0.0086 g / L, boric acid 0.0062 g / L, anhydrous copper sulfate 0.00025 g / L, sodium molybdate dihydrate 0.00025 g / L, calcium nitrate tetrahydrate 0.556 g / L, ferrous sulfate heptahydrate 0.0278 g / L, disodium edetate 0.0373 g / L, inositol 0.1 g / L, glycine 0.002 g / L, vitamin B1 0.001 g / L, vitamin B6 0.0005 g / L, and vitamin B5 0.0005 g / L.

[0052] In the present invention, the MS culture medium includes the following components in the following concentrations: calcium chloride 0.44 g / L, ammonium sulfate 1.32 g / L, potassium nitrate 2.0 g / L, boric acid 0.0065 g / L, magnesium sulfate heptahydrate 0.36 g / L, copper sulfate 0.000025 g / L, potassium dihydrogen phosphate 0.17 g / L, cobalt chloride 0.000025 g / L, manganese sulfate 0.0168 g / L, zinc sulfate heptahydrate 0.0085 g / L, potassium iodide 0.00083 g / L, sodium molybdate 0.00025 g / L, disodium edetate 0.0373 g / L, glycine 0.0020 g / L, thiamine hydrochloride 0.0004 g / L, pyridoxine hydrochloride 0.0005 g / L, inositol 0.010 g / L, and ferric sulfate heptahydrate 0.0278 g / L.

[0053] Experimental Example 1

[0054] Select the young shoots of Hainan catalpa as explants ( Figure 1The explants were cleaned with sterile water, immersed in 75vt% alcohol for 30s, taken out, rinsed 3 times with sterile water, immersed in 0.1wt% mercuric chloride aqueous solution for 2min, and rinsed 3 times with sterile water to obtain sterile explants.

[0055] The sterile explants were inoculated into differentiation medium and cultured for 30 days under the conditions of 2800 Lux, 25° C., 60% humidity and 15 h / d of light intensity to obtain clustered buds of Catalpa hainanensis ( Figure 2 ), the number of clustered buds is 3 to 5. The clustered buds of Hainan catalpa were cut into individual buds, transferred to the subculture medium, and cultured for 23 days under the conditions of culture intensity of 2800 Lux, culture temperature of 25 ° C, humidity of 60%, and light time of 15 h / d to obtain the proliferated seedlings containing stem segments ( Figure 3 The mature buds in the proliferated seedlings were transferred to rooting medium with different hormone concentrations and cultured for 45 days to obtain test tube plantlets. The rooting conditions of Hainan catalpa tissue culture seedlings in different rooting medium were studied. The results are shown in Table 1.

[0056] The differentiation medium is based on 1 / 2 MS medium and also includes 1.0 mg / L 6-benzylaminopurine, 0.1 mg / L naphthaleneacetic acid, 30 g / L sucrose, and 7.0 g / L agar; the pH is 5.8.

[0057] The secondary culture medium is based on MS medium and also includes 1.0 mg / L 6-benzylaminopurine, 0.25 mg / L naphthaleneacetic acid + 0.4 mg / L kinetin, 30 g / L sucrose, and 7.0 g / L agar; the pH is 5.8.

[0058] The rooting medium was based on WPM medium, including different concentrations of hormones, 20 g / L sucrose, and 7.0 g / L agar; the pH was 5.8.

[0059] Table 1 Rooting conditions of different rooting media

[0060]

[0061]

[0062] IAA represents indoleacetic acid, NAA represents naphthylacetic acid, and 2,4-D represents 2,4-dichlorophenoxyacetic acid.

[0063] As shown in Table 1, the number of roots of Hainan Catalpa hainanensis in the rooting medium with different hormone concentrations is 5 to 7, the number of leaves is 4 to 6, the plant height is 7 to 10 cm, and the growth condition is good.

[0064] The rooting conditions in the rooting medium composed of 0.2mg / LIAA+0.5mg / LNAA+0.02mg / L2,4-D hormone are as follows Figure 4 (Left) shows the rooting situation in the rooting medium composed of 0.1mg / LIAA+0.5mg / LNAA+0.05mg / L2,4-D hormone. Figure 4 (middle) shows the rooting situation in the rooting medium composed of 0.5mg / L IAAA + 0.1mg / L NAA + 0.01mg / L 2,4-D hormone. Figure 4 (right) shown.

[0065] Experimental Example 2

[0066] Test tube seedlings obtained by culturing in a rooting medium containing 0.5 mg / L 1A A + 0.1 mg / L NAA + 0.01 mg / L 2,4-D from Experimental Example 1 were placed under diffuse light at a light intensity of 4000 Lux, a humidity of 60%, and a temperature of 25°C for 7 days to obtain seedlings to be cultivated. The culture medium in the seedlings to be cultivated was washed and transplanted into seedling cultivation media with different compositions and cultivated for 18 days to obtain seedlings to be transplanted. Transplant survival rate was calculated: Transplant survival rate = number of surviving seedlings / number of transplanted test tube seedlings × 100%. The results are shown in Table 2.

[0067] Table 2 Effects of different seedling culture media on the survival rate of Hainan catalpa test tube seedlings

[0068]

[0069]

[0070] Table 2 shows that the survival rate of Hainan catalpa seedlings in the seedling medium composed of 1 to 3 is the highest, at 90 to 95%. Figure 5 shown.

[0071] Example 1

[0072] Select the young shoots of Hainan catalpa as explants, clean them with sterile water, soak them in 75vt% alcohol for 30s, take them out, rinse them 3 times with sterile water, soak them in 0.1wt% mercuric chloride aqueous solution for 2min, and rinse them 3 times with sterile water to obtain sterile explants.

[0073] Sterile explants were inoculated into differentiation medium and cultured for 28 days under conditions of a light intensity of 2800 Lux, a culture temperature of 25°C, a humidity of 60%, and a photoperiod of 15 hours per day to obtain clustered buds of the Hainan catalpa. Clustered buds of the Hainan catalpa were cut into individual buds, transferred to a subculture medium, and cultured for 25 days under conditions of a culture intensity of 2800 Lux, a culture temperature of 25°C, a humidity of 60%, and a photoperiod of 15 hours per day to obtain proliferated seedlings containing stem segments. Mature buds from the proliferated seedlings were transferred to a rooting medium and cultured for 40 days to obtain Hainan catalpa test tube plantlets.

[0074] The differentiation medium is based on 1 / 2 MS medium and also includes 0.3 mg / L 6-benzylaminopurine, 0.15 mg / L naphthaleneacetic acid, 30 g / L sucrose, and 7.0 g / L agar; the pH is 5.8. Under this differentiation medium, clustered shoots of Hainan catalpa containing an average of four buds can be obtained.

[0075] The subculture medium is based on MS medium and also includes 2.0 mg / L 6-benzylaminopurine, 0.07 mg / L naphthaleneacetic acid + 0.2 mg / L kinetin, 30 g / L sucrose, and 7.0 g / L agar; the pH is 5.8.

[0076] The rooting medium is based on WPM medium and also includes 0.1 mg / L indoleacetic acid, 0.4 mg / L naphthaleneacetic acid, 0.03 mg / L 2,4-dichlorophenoxyacetic acid, 20 g / L sucrose, and 7.0 g / L agar; the pH is 5.8.

[0077] Hainan catalpa seedlings were placed in a diffuse light environment at 3800 lux, 65% humidity, and 27°C for 5 days to obtain seedlings. The culture medium was washed and transplanted into a seedling medium consisting of vermiculite, river sand, perlite, and peat at a mass ratio of 2:1.5:1.5:5. The seedlings were then incubated for 20 days to obtain seedlings ready for transplanting. The transplant survival rate was 95%.

[0078] Example 2

[0079] Select the young shoots of Hainan catalpa as explants, clean them with sterile water, soak them in 75vt% alcohol for 30s, take them out, rinse them 3 times with sterile water, soak them in 0.1wt% mercuric chloride aqueous solution for 2min, and rinse them 3 times with sterile water to obtain sterile explants.

[0080] Sterile explants were inoculated into differentiation medium and cultured for 32 days under conditions of a light intensity of 2900 Lux, a culture temperature of 23°C, a humidity of 65%, and a light duration of 16 h / d to obtain clustered buds of the Hainan catalpa. Clustered buds of the Hainan catalpa were cut into individual buds, transferred to a subculture medium, and cultured for 22 days under conditions of a culture intensity of 2900 Lux, a culture temperature of 23°C, a humidity of 65%, and a light duration of 16 h / d to obtain proliferated seedlings containing stem segments. Mature buds from the proliferated seedlings were transferred to a rooting medium and cultured for 45 days to obtain Hainan catalpa test tube plantlets.

[0081] The differentiation medium is based on 1 / 2 MS medium and also includes 1.2 mg / L 6-benzylaminopurine, 0.03 mg / L naphthaleneacetic acid, 30 g / L sucrose, and 7.0 g / L agar; the pH is 5.8. Under this differentiation medium, clustered shoots of Hainan catalpa containing an average of four buds can be obtained.

[0082] The secondary culture medium is based on MS medium and also includes 0.6 mg / L 6-benzylaminopurine, 0.15 mg / L naphthaleneacetic acid + 0.4 mg / L kinetin, 30 g / L sucrose, and 7.0 g / L agar; the pH is 5.8.

[0083] The rooting medium is based on WPM medium and also includes 0.5 mg / L indoleacetic acid, 0.2 mg / L naphthaleneacetic acid, 0.01 mg / L 2,4-dichlorophenoxyacetic acid, 20 g / L sucrose, and 7.0 g / L agar; the pH is 5.8.

[0084] Hainan catalpa seedlings were placed in a diffuse light environment at 4400 lux, 60% humidity, and 27°C for 5 days to obtain seedlings. The culture medium was washed and transplanted into a seedling medium consisting of vermiculite, river sand, perlite, and peat at a mass ratio of 1.8:1.8:1.2:5.5. The seedlings were then incubated for 15 days to obtain transplantable seedlings. The transplant survival rate was 92%.

[0085] As can be seen from the above embodiments, the present invention provides a Hainan catalpa test tube seedling and a culture method thereof and a method for cultivating Hainan catalpa seedlings to be transplanted. Using the differentiation culture medium of the present invention, clustered buds containing 3 to 5 Hainan catalpa buds can be differentiated, and the clustered buds are cut into individual pieces and transferred to a subculture culture medium to obtain a proliferation seedling with a stem segment. Transferring the proliferation seedling to a rooting culture medium can obtain Hainan catalpa test tube seedlings with 5 to 9 roots, a plant height of 7 to 10 cm, and 4 to 6 leaves. The entire process only takes 89 to 107 days. The test tube seedlings are transplanted after hardening, and the transplant survival rate is 90 to 95%. This provides a new approach for the cultivation of Hainan catalpa and the selection of superior varieties.

[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for cultivating test tube seedlings of Ligusticum hainanensis, characterized in that: The steps include: (1) Disinfect the Hainan catalpa explants to obtain sterile explants; (2) inoculating the sterile explants into differentiation medium and culturing for 28 to 32 days to obtain clustered shoots of Catalpa hainanensis; (3) Take a single clustered bud of Catalpa hainanensis and transfer it to the subculture medium for 21-25 days to obtain the proliferated seedlings of Catalpa hainanensis; (4) Transfer the propagated seedlings of Ligusticum hainanensis to the rooting medium of Ligusticum hainanensis and culture them for 40-50 days to obtain the test tube seedlings of Ligusticum hainanensis; The Hainan catalpa rooting medium in step (4) is based on the WPM medium and further contains the following components at the following concentrations: 0.1-0.5 mg / L indoleacetic acid, 0.1-0.5 mg / L naphthaleneacetic acid, 0.01-0.05 mg / L 2,4-dichlorophenoxyacetic acid, 15-25 g / L sucrose, 6.0-8.0 g / L agar; The pH of the Hainan catalpa rooting culture medium is 5.7-5.9; The Hainan catalpa explant is a bud or stem of Hainan catalpa; The differentiation medium is based on 1 / 2 MS medium, and the following components are added: 0.3-1.5 mg / L 6-benzylaminopurine, 0.03-0.15 mg / L naphthaleneacetic acid, 20-40 g / L sucrose, 6.0-8.0 g / L agar; The pH of the differentiation medium is 5.7-5.9; The secondary culture medium is based on MS medium, and the following components are added: 0.6-2.0 mg / L 6-benzylaminopurine, 0.07-0.25 mg / L naphthaleneacetic acid, 0.1-0.4 mg / L kinetin, 20-40 g / L sucrose, 6.0-8.0 g / L agar; The pH of the subculture medium is 5.7-5.

9.

2. The culture method according to claim 1, wherein The method for disinfecting the Hainan catalpa explant is as follows: first, soaking the Hainan catalpa explant in alcohol for 30 to 40 seconds, and then soaking it in a mercuric chloride solution for 1 to 2 minutes; The initial concentration of the alcohol is 70-80% vt%; The mercuric chloride solution is an aqueous solution of mercuric chloride; The initial concentration of the mercuric chloride solution is 0.09-0.11 wt %.

3. The culture method according to claim 1, wherein The culture temperature in steps (2) to (4) is independently 23 to 25°C; The humidity of the culture is independently 50-65%; The light intensity of the culture is independently 2700~2900 Lux; The light exposure time of the culture is independently 14-16 h / d.

4. The hainanensis seedlings obtained by the culture method according to any one of claims 1 to 3, characterized in that: The plant height of the test tube seedlings of the hainan lily is 7 to 10 cm.

5. A method for cultivating Hainan catalpa seedlings to be transplanted, characterized in that: The steps include: (1) Harden the test tube seedlings of Ligusticum hainanensis for 4-7 days to obtain seedlings to be cultivated; (2) transplanting the seedlings to be cultivated into a seedling culture medium and cultivating them for 15 to 20 days to obtain Hainan catalpa seedlings to be transplanted; The Hainan lily seedlings in the test tube are the Hainan lily seedlings in the test tube according to claim 4; The following components in a mass ratio are also added to the seedling culture medium in step (2): The ratio of vermiculite: river sand: perlite: peat soil is 1.5~2.5:1.5~2.5:0.5~1.5:4.5~5.

5.

6. The cultivation method according to claim 5, characterized in that The light intensity of the seedling hardening is 3800~4400Lux; The humidity of the seedling hardening is 60-70%; The temperature of the hardening is 25-27°C.

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