A tissue culture and rapid propagation method for Eucalyptus grandis

Abstract

The present invention discloses a tissue culture and rapid propagation method for Eucalyptus grandis, which comprises selecting an excellent individual plant of Eucalyptus grandis without pests and diseases for ring cutting to promote germination. When the budding strip grows to 30-35 cm, a lignified stem segment is cut to obtain an explant and the explant is disinfected. Induction culture, subculture, seedling culture and rooting culture are carried out in sequence. Finally, the rooted bottle seedlings with complete rooting and a seedling height of 4-6 cm are washed with a culture medium and transplanted into a nutrient cup containing a matrix. The present invention establishes a tissue culture system for excellent individual plants of Eucalyptus grandis, promotes the development of factory-based production of Eucalyptus grandis tissue culture seedlings, is conducive to promoting the planting of Eucalyptus grandis improved varieties, and has good economic and ecological benefits.

Description

Technical Field

[0001] The invention belongs to the field of plant cultivation, relates to a method for asexual propagation and seedling raising of Eucalyptus urophylla, and in particular to a method for rapid tissue culture propagation of Eucalyptus urophylla. Background Art

[0002] Eucalyptus grandis ( Eucalyptus urophyla × E. grandis ) is a fast-growing hybrid of Eucalyptus urophylla and Eucalyptus grandis. It combines the adaptability and disease resistance of Eucalyptus urophylla with the rapid growth and high pulp yield of Eucalyptus grandis, making it an ideal species for papermaking and wood-based panels. Eucalyptus urophylla grows quickly and produces high yields, typically requiring harvesting within 5-7 years of planting, resulting in significant economic benefits. In recent years, the development of artificial eucalyptus forests for industrial raw materials in my country has been rapid, with plantings expanding. This has led to an unprecedented surge in enthusiasm for establishing Eucalyptus urophylla forests and a sharp increase in demand for Eucalyptus urophylla seedlings.

[0003] While there are numerous tissue culture systems for eucalyptus hybrids, these systems vary significantly between strains. Consequently, tissue culture systems for individual plants, especially those for high-quality ones, essentially need to be re-established. Consequently, due to limitations in rapid tissue culture propagation technology, Eucalyptus urophylla varieties have been unable to achieve large-scale production and have not been widely promoted for cultivation. Summary of the Invention

[0004] In order to overcome the above-mentioned deficiencies in the prior art, the present invention provides a tissue culture rapid propagation method for Eucalyptus urophylla, which is targeted at the superior germplasm of Eucalyptus urophylla and can improve the tissue culture rooting rate and transplant survival rate.

[0005] The technical solution adopted in the present invention is as follows:

[0006] A tissue culture rapid propagation method for Eucalyptus urophylla comprises selecting high-quality individual Eucalyptus urophylla plants that are free of pests and diseases, performing girdling to promote budding, cutting lignified stem segments when budding shoots grow to 30 to 35 cm, and sterilizing the explants. Induction culture, subculture, seedling cultivation, and rooting culture are sequentially performed, and finally, the rooted seedlings that have fully rooted and are 4 to 6 cm tall are washed with culture medium and transplanted into nutrient cups containing a substrate.

[0007] The main steps include:

[0008] (1) Circumcision to promote germination

[0009] Select high-quality individual Eucalyptus grandis trees that are free of pests and diseases, remove surrounding shrubs, and spray with 1% potassium permanganate for disinfection the day before girdling. Girdle the tree 20 cm above the ground, 3 / 4 of the circumference, with a width of 3 cm. Scrape the cambium down to the phloem, and spray the phloem with a nutrient solution that promotes sprouting.

[0010] (2) Explant disinfection

[0011] When the sprouts grow to 30-35 cm, cut the semi-lignified stem segments, wash them with a detergent solution for 10 minutes, and then rinse them with tap water for 5 minutes. Bring them to a clean bench, cut them into small segments of 2-3 cm with 1-2 latent buds, place them in a sterile bottle, soak them in 75% alcohol for 30 seconds, rinse them with sterile water three times, soak them in 2% sodium hypochlorite for 3 minutes, and rinse them with sterile water three times.

[0012] (3) Induction culture

[0013] Use sterile filter paper to absorb the surface moisture of the small segment treated in step (2), cut off the ends of the small segment, inoculate it into the induction culture medium, and culture it at a temperature of 25±2℃, a light intensity of 12h / d, and a light intensity of 1500-2000lux for 20-25 days;

[0014] (4) Subculture

[0015] The sterile buds obtained in step (3) were cut and transferred to subculture medium. After culturing in the dark at 25±2°C for 5 days, they were cultured under the conditions of 12 h / d of light and 1500-2000 lux for 20-25 days.

[0016] (5) Cultivation of strong seedlings

[0017] The buds obtained in step (4) were cut into seedling medium and cultured for 15-20 days at a temperature of 25±2°C, a light intensity of 12h / d, and a light intensity of 2000-2500lux;

[0018] (6) Rooting culture

[0019] When the seedlings obtained by the seedling culture in step (5) grow to 3-4 cm, cut the seedlings and transfer them to the rooting medium. After culturing in the dark at a temperature of 25±2°C for 5 days, culture them under the conditions of 12 h / d of light and 1500-2000 lux for 20-25 days.

[0020] (7) Transplantation

[0021] Wash the culture medium of the rooted seedlings that have fully rooted and are 4-6 cm tall and transplant them into a nutrient cup containing a substrate. Drain the substrate thoroughly, cover with a film to keep it moist and cover with a shade net. Spray with 500-600 times diluted carbendazim every 5-7 days to prevent and control root rot; after 10 days, open the shade net and film, and spray the leaves with nutrient solution every 10 days.

[0022] Preferably, the raw material components of the germination-promoting nutrient solution in step (1) above are 1L solution containing 152mgKNO3, 132mgNH4NO3, 13.6mgKH2PO4, 26.56mgCaCl2, 29.6mgMgSO4, 0.1mgN 6 Benzyl adenine (6-BA), 0.2 mg naphthaleneacetic acid (NAA).

[0023] Preferably, the raw material components of the induction medium described in step (3) above are as follows: 1L induction medium contains 1520mgKNO3, 1320mgNH4NO3, 136mgKH2PO4, 265.6mgCaCl2, 296mgMgSO4, 6.2mgH3BO3, 22.6mgMnSO4, 8.6mgZnSO4, 0.25 mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 1000mgPVP, 0.24mgbiotin(VH), 100mgL-cysteine, 2.4mgD-calcium pantothenate(VB5), 2mgglycine, 5mgnic acid(VB3), 2mgthiamine(VB1), 0.5mgpyridoxine(VB6), 37.3mgNa2-EDTA, 27.8mgFeSO4, 100mginositol, 0.3-0.5mgN 6 Benzyl adenine (6-BA), 0.1-0.2 mg naphthaleneacetic acid (NAA), 30 g sucrose, 5 g agar powder, pH 5.8~6.

[0024] Preferably, the raw material components of the subculture medium in step (4) above are as follows: 1L of subculture medium contains 1520mgKNO3, 1320mgNH4NO3, 136mgKH2PO4, 265.6mgCaCl2, 296mgMgSO4, 6.2mgH3BO3, 22.6mgMnSO4, 8.6mgZnSO4, 0.25 mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 0.24mgbiotin(VH), 2mgglycine, 5mgnic acid(VB3), 2mgthiamine(VB1), 0.5mgpyridoxine(VB6), 37.3mgNa2-EDTA, 27.8mgFeSO4, 100mginositol, 0.4-0.6mgN 6 Benzyl adenine (6-BA), 0.2-0.3 mg naphthaleneacetic acid (NAA), 30 g sucrose, 5 g agar powder, pH 5.8~6.

[0025] Preferably, the raw material components of the seedling medium in step (5) above are as follows: 1L seedling medium contains 1520mgKNO3, 1320mgNH4NO3, 136mgKH2PO4, 265.6mgCaCl2, 296mgMgSO4, 6.2mgH3BO3, 22.6mgMnSO4, 8.6mgZnSO4, 0.25 mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 0.24mgbiotin(VH), 2mgglycine, 5mgnic acid(VB3), 2mgthiamine(VB1), 0.5mgpyridoxine(VB6), 37.3mgNa2-EDTA, 27.8mgFeSO4, 100mginositol, 0.1-0.3mgN 6 Benzyl adenine (6-BA), 0.3-0.5 mg naphthaleneacetic acid (NAA), 30 g sucrose, 5 g agar powder, pH 5.8~6.

[0026] Preferably, the raw material components of the rooting medium in step (6) above are as follows: 1L rooting medium contains 246mgKNO3, 335mgKH2PO4, 250mgCa(NO3)2, 76mgCaCl2, 258mgMgSO4, 3.1mgH3BO3, 11.15mgMnSO4, 4.3mgZnSO4, 0.125mgNa2MoO4, 0.0125mgCuSO4, 0.0125mgCoCl2, 0.415mgKI, 0.24mg biotin (VH), 100mgL-cysteine, 2.4mgD-pantothenate calcium (VB5), 2mgglycine, 5mgnic acid (VB3), 2mgthiamine (VB1), 0.5mgpyridoxine (VB6), 37.3mgNa2-EDTA, 27.8mg FeSO4, 100mg inositol, 0.3-0.5mg indolebutyric acid (IBA), 0.2-0.3mg naphthaleneacetic acid (NAA), 0.3-0.5mg paclobutrazol (PP333), 30g sucrose, 5g agar powder, 5g activated carbon, pH value 5.8~6.

[0027] Preferably, the matrix in step (7) above is a mixture of coconut husk, carbonized rice husk, peat soil and loess soil in a weight ratio of 5:2.5:2:0.5, and calcium magnesium phosphate fertilizer 4 kg / m 3 Mix well, cover with film and compost for one month to make a light substrate; the raw material components of the foliar nutrient solution are: 1L foliar nutrient solution contains 152mgKNO3, 132mgNH4NO3, 13.6mgKH2PO4, 26.56mgCaCl2, and 29.6mgMgSO4.

[0028] Preferably, the superior individual plant described in step (1) above is the Eucalyptus grandis GE19-2 variety. The parent of the Eucalyptus grandis GE19-2 variety is an F1 generation hybrid clone cultivated by artificial hybridization of Eucalyptus urophylla and Eucalyptus grandis. It is a forest tree variety approved by the Guangxi Zhuang Autonomous Region Forest Variety Approval Committee. It combines the excellent characteristics of the female parent Eucalyptus urophylla and the male parent Eucalyptus grandis. It has excellent characteristics such as fast growth, high yield, straight trunk shape, and strong resistance to insects (such as the eucalyptus leaf roller). The average breast diameter of a 5.5-year-old stand is 15.4 cm, the average tree height is 20.9 m, and the average annual cumulative growth is 47.0 m³ / hm. 2 Its wood is mainly used for plywood, fiberboard, pulp, sawn timber and construction.

[0029] The advantages and beneficial effects of the present invention are as follows:

[0030] 1. The present invention adopts the ring cutting method to obtain materials, which reduces the damage to the raw materials, can effectively retain the excellent germplasm of Eucalyptus urophylla and realize the efficient development of Eucalyptus urophylla asexual lines.

[0031] 2. Based on induction cultivation and subculture, the rooting culture medium of the present invention reduces the use of macroelements and some trace elements. By matching different elements, especially using paclobutrazol (PP333) and activated carbon, the rooting efficiency of Eucalyptus grandis is significantly improved, and the final rooting rate in the bottle can reach more than 95%.

[0032] 3. The present invention selects a specific matrix and foliar nutrient solution during transplantation, ensuring that the nutrients required for the growth and survival of Eucalyptus urophylla are comprehensive and sufficient, effectively improving the transplant survival rate of Eucalyptus urophylla tissue culture seedlings, which can reach more than 95%, reducing material loss, and promoting the large-scale production of Eucalyptus urophylla.

[0033] 4. There are many tissue culture systems for eucalyptus hybrid strains, but the differences in tissue culture systems between different eucalyptus strains are still relatively large. The tissue culture systems of different individual plants, especially excellent individual plants, basically need to be re-established. The present invention establishes a tissue culture system for excellent individual plants of Eucalyptus urophylla, promotes the development of factory production of Eucalyptus urophylla tissue culture seedlings, is conducive to the promotion and planting of Eucalyptus urophylla varieties, and has good economic and ecological benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a physical picture of the subcultured seedlings of Eucalyptus urophylla after subculture culture according to the present invention.

[0035] Figure 2 This is a real picture of the rooted seedlings of Eucalyptus grandis after the rooting culture of the present invention.

[0036] Figure 3 This is a real scene of Eucalyptus grandis seedlings that survived after transplantation by the present invention Figure I .

[0037] Figure 4 This is a real scene of Eucalyptus grandis seedlings that survived after transplantation by the present invention Figure II . DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the embodiments. Example

[0039] A tissue culture rapid propagation method for Eucalyptus grandis, the main steps comprising:

[0040] (1) Circumcision to promote germination

[0041] Select a single Eucalyptus grandis GE19-2 plant that is free of pests and diseases, remove surrounding shrubs, and spray with 1% potassium permanganate for disinfection the day before girdling. Girdle the tree 20 cm above the ground, 3 / 4 of the circumference, with a width of 3 cm. Scrape the cambium to the phloem, and spray the phloem with a bud-promoting nutrient solution.

[0042] The raw material components of the sprout-promoting nutrient solution are as follows: 1L solution contains 152mgKNO3, 132mgNH4NO3, 13.6mgKH2PO4, 26.56mgCaCl2, 29.6mgMgSO4, 0.1mgN 6 benzyl adenine (6-BA), 0.2 mg naphthaleneacetic acid (NAA);

[0043] (2) Explant disinfection

[0044] When the sprouts grow to 30-35 cm, cut the semi-lignified stem segments, wash them with a detergent solution for 10 minutes, and then rinse them with tap water for 5 minutes. Bring them to a clean bench, cut them into small segments of 2-3 cm with 1-2 latent buds, place them in a sterile bottle, soak them in 75% alcohol for 30 seconds, rinse them with sterile water three times, soak them in 2% sodium hypochlorite for 3 minutes, and rinse them with sterile water three times.

[0045] (3) Induction culture

[0046] Use sterile filter paper to absorb the surface moisture of the small segment treated in step (2), cut off the ends of the small segment, inoculate it into the induction culture medium, and culture it at a temperature of 25±2℃, a light intensity of 12h / d, and a light intensity of 1500-2000lux for 20-25 days;

[0047] The raw material components of the induction medium are as follows: 1L induction medium contains 1520mgKNO3, 1320mgNH4NO3, 136mgKH2PO4, 265.6mgCaCl2, 296mgMgSO4, 6.2mgH3BO3, 22.6mgMnSO4, 8.6mgZnSO4, 0.25mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 1000mgPVP, 0.24mgbiotin(VH), 100mgL-cysteine, 2.4mgD-pantothenatecalcium(VB5), 2mgglycine, 5mgnic acid(VB3), 2mgthiamine(VB1), 0.5mgpyridoxine(VB6), 37.3mgNa2-EDTA, 27.8mgFeSO4, 100mginositol, 0.4mgN 6 Benzyl adenine (6-BA), 0.1 mg naphthaleneacetic acid (NAA), 30 g sucrose, 5 g agar powder, pH 5.8-6;

[0048] (4) Subculture

[0049] The sterile buds obtained in step (3) were cut and transferred to subculture medium. After culturing in the dark at 25±2°C for 5 days, they were cultured under the conditions of 12 h / d of light and 1500-2000 lux for 20-25 days.

[0050] The raw material components of the subculture medium are as follows: 1L subculture medium contains 1520mgKNO3, 1320mgNH4NO3, 136mgKH2PO4, 265.6mgCaCl2, 296mgMgSO4, 6.2mgH3BO3, 22.6mgMnSO4, 8.6mgZnSO4, 0.25mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 0.24mgbiotin(VH), 2mgglycine, 5mgnic acid(VB3), 2mgthiamine(VB1), 0.5mgpyridoxine(VB6), 37.3mgNa2-EDTA, 27.8mgFeSO4, 100mginositol, 0.5mgN 6 Benzyl adenine (6-BA), 0.2 mg naphthaleneacetic acid (NAA), 30 g sucrose, 5 g agar powder, pH 5.8-6;

[0051] (5) Cultivation of strong seedlings

[0052] The buds obtained in step (4) were cut into seedling medium and cultured for 15-20 days at a temperature of 25±2°C, a light intensity of 12h / d, and a light intensity of 2000-2500lux;

[0053] The raw material components of the seedling culture medium are as follows: 1L seedling culture medium contains 1520mgKNO3, 1320mgNH4NO3, 136mgKH2PO4, 265.6mgCaCl2, 296mgMgSO4, 6.2mgH3BO3, 22.6mgMnSO4, 8.6mgZnSO4, 0.25mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 0.24mgbiotin(VH), 2mgglycine, 5mgnic acid(VB3), 2mgthiamine(VB1), 0.5mgpyridoxine(VB6), 37.3mgNa2-EDTA, 27.8mgFeSO4, 100mginositol, 0.2mgN 6 Benzyl adenine (6-BA), 0.4 mg naphthaleneacetic acid (NAA), 30 g sucrose, 5 g agar powder, pH 5.8-6;

[0054] (6) Rooting culture

[0055] When the seedlings obtained by the seedling culture in step (5) grow to 3-4 cm, cut the seedlings and transfer them to the rooting medium. After culturing in the dark at a temperature of 25±2°C for 5 days, culture them under the conditions of 12 h / d of light and 1500-2000 lux for 20-25 days.

[0056] The raw material components of the rooting medium are as follows: 1L of the rooting medium contains 246mgKNO3, 335mgKH2PO4, 250mgCa(NO3)2, 76mgCaCl2, 258mgMgSO4, 3.1mgH3BO3, 11.15mgMnSO4, 4.3mgZnSO4, 0.125mgNa2MoO4, 0.0125mgCuSO4, 0.0125mgCoCl2, 0.415mgKI, 0.24mg biotin (VH), 100mgL-cysteine, 2.4mgD-pantothenic acid calcium (VB5), 2mgglycine, 5mgnic acid (VB3), 2mgthiamine (VB1), 0.5mgpyridoxine (VB6), 37.3mgNa2-EDTA, 27.8mg FeSO4, 100 mg inositol, 0.4 mg indolebutyric acid (IBA), 0.2 mg naphthaleneacetic acid (NAA), 0.4 mg paclobutrazol (PP333), 30 g sucrose, 5 g agar powder, 5 g activated carbon, pH 5.8-6;

[0057] (7) Transplantation

[0058] Wash the culture medium of the rooted seedlings that have fully rooted and are 4-6 cm tall and transplant them into a nutrient cup filled with substrate. Drench the substrate thoroughly, cover with a film to keep it moist, and cover with a shade net. Spray with 500-600 times diluted carbendazim every 5-7 days to prevent root rot. After 10 days, open the shade net and film and spray the leaves with nutrient solution every 10 days.

[0059] The matrix is ​​a mixture of coconut husk, carbonized rice husk, peat soil and loess soil in a weight ratio of 5:2.5:2:0.5, and calcium magnesium phosphate fertilizer 4 kg / m 3 Mix well, cover with film and compost for one month to make a light substrate; the raw material components of the foliar nutrient solution are: 1L foliar nutrient solution contains 152mgKNO3, 132mgNH4NO3, 13.6mgKH2PO4, 26.56mgCaCl2, and 29.6mgMgSO4. Example

[0060] A tissue culture rapid propagation method for Eucalyptus grandis, the main steps comprising:

[0061] (1) Circumcision to promote germination

[0062] Select a single Eucalyptus grandis GE19-2 plant that is free of pests and diseases, remove surrounding shrubs, and spray with 1% potassium permanganate for disinfection the day before girdling. Girdle the tree 20 cm above the ground, 3 / 4 of the circumference, with a width of 3 cm. Scrape the cambium to the phloem, and spray the phloem with a bud-promoting nutrient solution.

[0063] The raw material components of the sprout-promoting nutrient solution are as follows: 1L solution contains 152mgKNO3, 132mgNH4NO3, 13.6mgKH2PO4, 26.56mgCaCl2, 29.6mgMgSO4, 0.1mgN 6 benzyl adenine (6-BA), 0.2 mg naphthaleneacetic acid (NAA);

[0064] (2) Explant disinfection

[0065] When the sprouts grow to 30-35 cm, cut the semi-lignified stem segments, wash them with a detergent solution for 10 minutes, and then rinse them with tap water for 5 minutes. Bring them to a clean bench, cut them into small segments of 2-3 cm with 1-2 latent buds, place them in a sterile bottle, soak them in 75% alcohol for 30 seconds, rinse them with sterile water three times, soak them in 2% sodium hypochlorite for 3 minutes, and rinse them with sterile water three times.

[0066] (3) Induction culture

[0067] Use sterile filter paper to absorb the surface moisture of the small segment treated in step (2), cut off the ends of the small segment, inoculate it into the induction culture medium, and culture it at a temperature of 25±2℃, a light intensity of 12h / d, and a light intensity of 1500-2000lux for 20-25 days;

[0068] The raw material components of the induction medium are as follows: 1L induction medium contains 1520mgKNO3, 1320mgNH4NO3, 136mgKH2PO4, 265.6mgCaCl2, 296mgMgSO4, 6.2mgH3BO3, 22.6mgMnSO4, 8.6mgZnSO4, 0.25mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 1000mgPVP, 0.24mgbiotin(VH), 100mgL-cysteine, 2.4mgD-pantothenate calcium(VB5), 2mgglycine, 5mgnic acid(VB3), 2mgthiamine(VB1), 0.5mgpyridoxine(VB6), 37.3mgNa2-EDTA, 27.8mgFeSO4, 100mginositol, 0.3mgN 6 Benzyl adenine (6-BA), 0.2 mg naphthaleneacetic acid (NAA), 30 g sucrose, 5 g agar powder, pH 5.8-6;

[0069] (4) Subculture

[0070] The sterile buds obtained in step (3) were cut and transferred to subculture medium. After culturing in the dark at 25±2°C for 5 days, they were cultured under the conditions of 12 h / d of light and 1500-2000 lux for 20-25 days.

[0071] The raw material components of the subculture medium are as follows: 1L subculture medium contains 1520mgKNO3, 1320mgNH4NO3, 136mgKH2PO4, 265.6mgCaCl2, 296mgMgSO4, 6.2mgH3BO3, 22.6mgMnSO4, 8.6mgZnSO4, 0.25mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 0.24mgbiotin(VH), 2mgglycine, 5mgnic acid(VB3), 2mgthiamine(VB1), 0.5mgpyridoxine(VB6), 37.3mgNa2-EDTA, 27.8mgFeSO4, 100mginositol, 0.4mgN 6 Benzyl adenine (6-BA), 0.3 mg naphthaleneacetic acid (NAA), 30 g sucrose, 5 g agar powder, pH 5.8-6;

[0072] (5) Cultivation of strong seedlings

[0073] The buds obtained in step (4) were cut into seedling medium and cultured for 15-20 days at a temperature of 25±2°C, a light intensity of 12h / d, and a light intensity of 2000-2500lux;

[0074] The raw material components of the seedling culture medium are as follows: 1L seedling culture medium contains 1520mgKNO3, 1320mgNH4NO3, 136mgKH2PO4, 265.6mgCaCl2, 296mgMgSO4, 6.2mgH3BO3, 22.6mgMnSO4, 8.6mgZnSO4, 0.25mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 0.24mgbiotin(VH), 2mgglycine, 5mgnic acid(VB3), 2mgthiamine(VB1), 0.5mgpyridoxine(VB6), 37.3mgNa2-EDTA, 27.8mgFeSO4, 100mginositol, 0.1mgN 6 Benzyl adenine (6-BA), 0.5 mg naphthaleneacetic acid (NAA), 30 g sucrose, 5 g agar powder, pH 5.8-6;

[0075] (6) Rooting culture

[0076] When the seedlings obtained by the seedling culture in step (5) grow to 3-4 cm, cut the seedlings and transfer them to the rooting medium. After culturing in the dark at a temperature of 25±2°C for 5 days, culture them under the conditions of 12 h / d of light and 1500-2000 lux for 20-25 days.

[0077] The raw material components of the rooting medium are as follows: 1L of the rooting medium contains 246mgKNO3, 335mgKH2PO4, 250mgCa(NO3)2, 76mgCaCl2, 258mgMgSO4, 3.1mgH3BO3, 11.15mgMnSO4, 4.3mgZnSO4, 0.125mgNa2MoO4, 0.0125mgCuSO4, 0.0125mgCoCl2, 0.415mgKI, 0.24mg biotin (VH), 100mgL-cysteine, 2.4mgD-pantothenic acid calcium (VB5), 2mgglycine, 5mgnic acid (VB3), 2mgthiamine (VB1), 0.5mgpyridoxine (VB6), 37.3mgNa2-EDTA, 27.8mg FeSO4, 100 mg inositol, 0.3 mg indolebutyric acid (IBA), 0.3 mg naphthaleneacetic acid (NAA), 0.3 mg paclobutrazol (PP333), 30 g sucrose, 5 g agar powder, 5 g activated carbon, pH 5.8-6;

[0078] (7) Transplantation

[0079] Wash the culture medium of the rooted seedlings that have fully rooted and are 4-6 cm tall and transplant them into a nutrient cup filled with substrate. Drench the substrate thoroughly, cover with a film to keep it moist, and cover with a shade net. Spray with 500-600 times diluted carbendazim every 5-7 days to prevent root rot. After 10 days, open the shade net and film and spray the leaves with nutrient solution every 10 days.

[0080] The matrix is ​​a mixture of coconut husk, carbonized rice husk, peat soil and loess soil in a weight ratio of 5:2.5:2:0.5, and calcium magnesium phosphate fertilizer 4 kg / m 3 Mix well, cover with film and compost for one month to make a light substrate; the raw material components of the foliar nutrient solution are: 1L foliar nutrient solution contains 152mgKNO3, 132mgNH4NO3, 13.6mgKH2PO4, 26.56mgCaCl2, and 29.6mgMgSO4. Example

[0081] A tissue culture rapid propagation method for Eucalyptus grandis, the main steps comprising:

[0082] (1) Circumcision to promote germination

[0083] Select a single Eucalyptus grandis GE19-2 plant that is free of pests and diseases, remove surrounding shrubs, and spray with 1% potassium permanganate for disinfection the day before girdling. Girdle the tree 20 cm above the ground, 3 / 4 of the circumference, with a width of 3 cm. Scrape the cambium to the phloem, and spray the phloem with a bud-promoting nutrient solution.

[0084] The raw material components of the sprout-promoting nutrient solution are as follows: 1L solution contains 152mgKNO3, 132mgNH4NO3, 13.6mgKH2PO4, 26.56mgCaCl2, 29.6mgMgSO4, 0.1mgN 6 benzyl adenine (6-BA), 0.2 mg naphthaleneacetic acid (NAA);

[0085] (2) Explant disinfection

[0086] When the sprouts grow to 30-35 cm, cut the semi-lignified stem segments, wash them with a detergent solution for 10 minutes, and then rinse them with tap water for 5 minutes. Bring them to a clean bench, cut them into small segments of 2-3 cm with 1-2 latent buds, place them in a sterile bottle, soak them in 75% alcohol for 30 seconds, rinse them with sterile water three times, soak them in 2% sodium hypochlorite for 3 minutes, and rinse them with sterile water three times.

[0087] (3) Induction culture

[0088] Use sterile filter paper to absorb the surface moisture of the small segment treated in step (2), cut off the ends of the small segment, inoculate it into the induction culture medium, and culture it at a temperature of 25±2℃, a light intensity of 12h / d, and a light intensity of 1500-2000lux for 20-25 days;

[0089] The raw material components of the induction medium are as follows: 1L induction medium contains 1520mgKNO3, 1320mgNH4NO3, 136mgKH2PO4, 265.6mgCaCl2, 296mgMgSO4, 6.2mgH3BO3, 22.6mgMnSO4, 8.6mgZnSO4, 0.25mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 1000mgPVP, 0.24mgbiotin(VH), 100mgL-cysteine, 2.4mgD-pantothenate calcium(VB5), 2mgglycine, 5mgnic acid(VB3), 2mgthiamine(VB1), 0.5mgpyridoxine(VB6), 37.3mgNa2-EDTA, 27.8mgFeSO4, 100mginositol, 0.5mgN 6 Benzyl adenine (6-BA), 0.1 mg naphthaleneacetic acid (NAA), 30 g sucrose, 5 g agar powder, pH 5.8-6;

[0090] (4) Subculture

[0091] The sterile buds obtained in step (3) were cut and transferred to subculture medium. After culturing in the dark at 25±2°C for 5 days, they were cultured under the conditions of 12 h / d of light and 1500-2000 lux for 20-25 days.

[0092] The raw material components of the subculture medium are as follows: 1L subculture medium contains 1520mgKNO3, 1320mgNH4NO3, 136mgKH2PO4, 265.6mgCaCl2, 296mgMgSO4, 6.2mgH3BO3, 22.6mgMnSO4, 8.6mgZnSO4, 0.25mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 0.24mgbiotin(VH), 2mgglycine, 5mgnic acid(VB3), 2mgthiamine(VB1), 0.5mgpyridoxine(VB6), 37.3mgNa2-EDTA, 27.8mgFeSO4, 100mginositol, 0.6mgN 6 Benzyl adenine (6-BA), 0.2 mg naphthaleneacetic acid (NAA), 30 g sucrose, 5 g agar powder, pH 5.8-6;

[0093] (5) Cultivation of strong seedlings

[0094] The buds obtained in step (4) were cut into seedling medium and cultured for 15-20 days at a temperature of 25±2°C, a light intensity of 12h / d, and a light intensity of 2000-2500lux;

[0095] The raw material components of the seedling culture medium are as follows: 1L seedling culture medium contains 1520mgKNO3, 1320mgNH4NO3, 136mgKH2PO4, 265.6mgCaCl2, 296mgMgSO4, 6.2mgH3BO3, 22.6mgMnSO4, 8.6mgZnSO4, 0.25mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 0.24mgbiotin(VH), 2mgglycine, 5mgnic acid(VB3), 2mgthiamine(VB1), 0.5mgpyridoxine(VB6), 37.3mgNa2-EDTA, 27.8mgFeSO4, 100mginositol, 0.3mgN 6 Benzyl adenine (6-BA), 0.3 mg naphthaleneacetic acid (NAA), 30 g sucrose, 5 g agar powder, pH 5.8-6;

[0096] (6) Rooting culture

[0097] When the seedlings obtained by the seedling culture in step (5) grow to 3-4 cm, cut the seedlings and transfer them to the rooting medium. After culturing in the dark at a temperature of 25±2°C for 5 days, culture them under the conditions of 12 h / d of light and 1500-2000 lux for 20-25 days.

[0098] The raw material components of the rooting medium are as follows: 1L of the rooting medium contains 246mgKNO3, 335mgKH2PO4, 250mgCa(NO3)2, 76mgCaCl2, 258mgMgSO4, 3.1mgH3BO3, 11.15mgMnSO4, 4.3mgZnSO4, 0.125mgNa2MoO4, 0.0125mgCuSO4, 0.0125mgCoCl2, 0.415mgKI, 0.24mg biotin (VH), 100mgL-cysteine, 2.4mgD-pantothenic acid calcium (VB5), 2mgglycine, 5mgnic acid (VB3), 2mgthiamine (VB1), 0.5mgpyridoxine (VB6), 37.3mgNa2-EDTA, 27.8mg FeSO4, 100 mg inositol, 0.5 mg indolebutyric acid (IBA), 0.2 mg naphthaleneacetic acid (NAA), 0.5 mg paclobutrazol (PP333), 30 g sucrose, 5 g agar powder, 5 g activated carbon, pH 5.8-6;

[0099] (7) Transplantation

[0100] Wash the culture medium of the rooted seedlings that have fully rooted and are 4-6 cm tall and transplant them into a nutrient cup filled with substrate. Drench the substrate thoroughly, cover with a film to keep it moist, and cover with a shade net. Spray with 500-600 times diluted carbendazim every 5-7 days to prevent root rot. After 10 days, open the shade net and film and spray the leaves with nutrient solution every 10 days.

[0101] The matrix is ​​a mixture of coconut husk, carbonized rice husk, peat soil and loess soil in a weight ratio of 5:2.5:2:0.5, and calcium magnesium phosphate fertilizer 4 kg / m 3 Mix well, cover with film and compost for one month to make a light substrate; the raw material components of the foliar nutrient solution are: 1L foliar nutrient solution contains 152mgKNO3, 132mgNH4NO3, 13.6mgKH2PO4, 26.56mgCaCl2, and 29.6mgMgSO4.

[0102] Comparative Example 1:

[0103] The only difference from Example 3 is that the sprout-promoting nutrient solution used in step (1) is different. Specifically, a single plant of Eucalyptus grandis GE19-2 variety without diseases and insect pests is selected, the surrounding shrubs are removed, and 1% potassium permanganate is sprayed for disinfection one day before ring cutting. The tree is ring cut at 20 cm above the ground, 3 / 4 of the circumference, with a width of 3 cm, the cambium is scraped to the phloem, and the sprout-promoting nutrient solution is sprayed to the phloem;

[0104] The germination-promoting nutrient solution used in this comparative example is: 200-300 mg / L gibberellin.

[0105] Comparative Example 2:

[0106] The only difference from Example 3 is that there is no seedling strengthening step, that is, after the subculture in step (4), the obtained clustered buds are directly subjected to rooting culture, and the rooting culture and transplanting steps after rooting are exactly the same as those in Example 3.

[0107] Comparative Example 3:

[0108] The only difference from Example 3 is that the formula of the rooting medium used in step (6) is different, and there is no paclobutrazol component in the rooting medium raw materials. Specifically, the rooting medium raw material components used in this comparative example are as follows: 1L rooting medium contains 246mgKNO3, 335mgKH2PO4, 250mgCa(NO3)2, 76mgCaCl2, 258mgMgSO4, 3.1mgH3BO3, 11.15mgMnSO4, 4.3mgZnSO4, 0.125mgNa2MoO4, 0.0125mgCuSO4, 0.0125mgCoCl2, 0.415mgKI, 0.24mg biotin (VH), 100mgL-cysteine, 2.4mgD-pantothenic acid calcium (VB5), 2mg glycine, 5mg nicotinic acid (VB3), 2mg thiamine (VB1), 0.5mg pyridoxine (VB6), 37.3mg Na2-EDTA, 27.8mg FeSO4, 100mg inositol, 0.5mg indolebutyric acid (IBA), 0.2mg naphthaleneacetic acid (NAA), 30g sucrose, 5g agar powder, 5g activated carbon, pH value 5.8~6.

[0109] Comparative Example 4:

[0110] The only difference from Example 3 is that the formula of the rooting culture medium used in step (6) is different, and there is no activated carbon component in the rooting culture medium raw material. Specifically, the rooting culture medium raw material components used in this comparative example are as follows: 1L rooting culture medium contains 246mgKNO3, 335mgKH2PO4, 250mgCa(NO3)2, 76mgCaCl2, 258mgMgSO4, 3.1mgH3BO3, 11.15mgMnSO4, 4.3mgZnSO4, 0.125mgNa2MoO4, 0.0125mgCuSO4, 0.0125mgCoCl2, 0.415mgKI, 0.24mg biotin (VH), 100mgL-cysteine, 2.4mgD-pantothenic acid calcium (VB5), 2mg glycine, 5mg nicotinic acid (VB3), 2mg thiamine (VB1), 0.5mg pyridoxine (VB6), 37.3mg Na2-EDTA, 27.8mg FeSO4, 100mg inositol, 0.5mg indolebutyric acid (IBA), 0.2mg naphthaleneacetic acid (NAA), 0.5mg paclobutrazol (PP333), 30g sucrose, 5g agar powder, pH value 5.8~6.

[0111] Comparative Example 5:

[0112] The only difference from Example 3 is that the formula of the rooting medium used in step (6) is different, and the rooting medium raw materials do not contain paclobutrazol and activated carbon materials. Specifically, the rooting medium raw material components used in this comparative example are as follows: 1L rooting medium contains 246mgKNO3, 335mgKH2PO4, 250mgCa(NO3)2, 76mgCaCl2, 258mgMgSO4, 3.1mgH3BO3, 11.15mgMnSO4, 4.3mgZnSO4, 0.125mgNa2MoO4, 0.0125mgCuSO4, 0.0125mgCoCl2, 0.415mgKI, 0.24mg biotin (VH), 100mgL-cysteine, 2.4mgD-pantothenic acid calcium (VB5), 2mg glycine, 5mg nicotinic acid (VB3), 2mg thiamine (VB1), 0.5mg pyridoxine (VB6), 37.3mg Na2-EDTA, 27.8mg FeSO4, 100mg inositol, 0.5mg indolebutyric acid (IBA), 0.2mg naphthaleneacetic acid (NAA), 30g sucrose, 5g agar powder, pH value 5.8~6.

[0113] Comparative Example 6:

[0114] The only difference from Example 3 is that the raw material components of the rooting culture medium described in step (6) are different, and the usage of elements such as K, N, P, Ca, Mg, B, Zn, Cu, and S is increased. Specifically, the raw material components of the rooting culture medium used in this comparative example are: 1L of rooting culture medium contains 1520mgKNO3, 335mgKH2PO4, 250mgCa(NO3)2, 265.6mgCaCl2, 296mgMgSO4, 6.1mgH3BO 3. 22.6mgMnSO4, 8.6mgZnSO4, 0.25mgNa2MoO4, 0.25mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 0.24mgbiotin(VH), 100mgL-cysteine, 2.4mgD-pantothenatecalcium(VB5), 2mgglycine, 5mgnic acid(VB3), 2mgthiamine(VB1), 0.5mgpyridoxine(VB6), 37.3mgNa2-EDTA, 27.8mgFeSO4, 100mginositol, 0.5mgindolebutyric acid(IBA), 0.2mgnaphthaleneacetic acid(NAA), 0.5mgpaclobutrazol(PP333), 30gsucrose, 5gagar powder, 5gactivated carbon, pH value 5.8~6.

[0115] Comparative Example 7:

[0116] The only difference from Example 3 is that the foliar nutrient solution in this comparative example is a nitrogen, phosphorus and potassium mixed solution (1 L of the mixed solution contains 20-30 g superphosphate + 3-5 g urea + 10-15 g potassium sulfate).

[0117] Test example:

[0118] The rooting rate and average root length of the Eucalyptus urophylla tissue culture seedlings in the bottle of Examples 1-3 and Comparative Examples 1-7, as well as the transplant survival rate three months after transplanting, were calculated. The results are shown in the following table:

[0119] Group Rooting rate in bottle Average root length of bottle seedlings Transplant survival rate Example 1 97.5% 3.1cm 99.5% Example 2 96.9% 3.0cm 98.2% Example 3 95.8% 3.1cm 96.0% Comparative Example 1 68.9% 2.7cm 70.2% Comparative Example 2 72.3% 2.7cm 75.3% Comparative Example 3 80.6% 2.3cm 76.2% Comparative Example 4 77.4% 2.4cm 80.1% Comparative Example 5 70.8% 2.4cm 72.6% Comparative Example 6 71.2% 2.6cm 70.5% Comparative Example 7 96.2% 3.0cm 82.1%

[0120] It can be seen from the above table that the technology of the present application is a complete technical solution, especially after the complete steps of ring cutting to promote germination, rooting culture and spraying of nutrient solution after transplanting of the present application, the rooting rate of Eucalyptus grandis tissue culture seedlings in the bottle can be increased to more than 95%, and the transplant survival rate can be increased to more than 95%.

Claims

1. A method for rapid propagation of Eucalyptus urophylla through tissue culture, characterized in that: Select high-quality individual Eucalyptus grandis plants that are free of pests and diseases and perform girdling to promote budding. When the buds grow to 30-35 cm, cut the lignified stem segments to obtain explants and disinfect the explants. Then, carry out induction culture, subculture, seedling culture and rooting culture in sequence. Finally, the rooted seedlings with complete rooting and a height of 4-6 cm are washed with the culture medium and transplanted into nutrient cups filled with substrate. The main steps include: (1) Circumcision to promote germination Select high-quality individual Eucalyptus grandis trees that are free of pests and diseases, remove surrounding shrubs, and spray with 1% potassium permanganate for disinfection the day before girdling. Girdle the tree 20 cm above the ground, 3 / 4 of the circumference, with a width of 3 cm. Scrape the cambium down to the phloem, and spray the phloem with a nutrient solution that promotes sprouting. (2) Explant disinfection When the sprouts grow to 30-35 cm, cut the semi-lignified stem segments, wash them with a detergent solution for 10 minutes, and then rinse them with tap water for 5 minutes. Bring them to a clean bench, cut them into small segments of 2-3 cm with 1-2 latent buds, place them in a sterile bottle, soak them in 75% alcohol for 30 seconds, rinse them with sterile water three times, soak them in 2% sodium hypochlorite for 3 minutes, and rinse them with sterile water three times. (3) Induction culture Use sterile filter paper to absorb the surface moisture of the small segment treated in step (2), cut off the ends of the small segment, inoculate it into the induction culture medium, and culture it at a temperature of 25±2℃, a light intensity of 12h / d, and a light intensity of 1500-2000lux for 20-25 days; (4) Subculture The sterile buds obtained in step (3) were cut and transferred to subculture medium. After culturing in the dark at 25±2°C for 5 days, they were cultured under the conditions of 12 h / d of light and 1500-2000 lux for 20-25 days. (5) Cultivation of strong seedlings The buds obtained in step (4) were cut into seedling medium and cultured for 15-20 days at a temperature of 25±2°C, a light intensity of 12h / d, and a light intensity of 2000-2500lux; (6) Rooting culture When the seedlings obtained by the seedling culture in step (5) grow to 3-4 cm, cut the seedlings and transfer them to the rooting medium. After culturing in the dark at a temperature of 25±2°C for 5 days, culture them under the conditions of 12 h / d of light and 1500-2000 lux for 20-25 days. (7) Transplantation Wash the culture medium of the rooted seedlings that have fully rooted and are 4-6 cm tall and transplant them into a nutrient cup filled with substrate. Drench the substrate thoroughly, cover with a film to keep it moist, and cover with a shade net. Spray with 500-600 times diluted carbendazim every 5-7 days to prevent root rot. After 10 days, open the shade net and film and spray the leaves with nutrient solution every 10 days. The raw material components of the germination-promoting nutrient solution in step (1) are as follows: 1L solution contains 152mgKNO3, 132mgNH4NO3, 13.6mgKH2PO4, 26.56mgCaCl2, 29.6mgMgSO4, 0.1mgN 6 benzyl adenine, 0.2 mg naphthaleneacetic acid; The raw material components of the induction medium described in step (3) are as follows: 1L induction medium contains 1520mgKNO3, 1320mgNH4NO3, 136mgKH2PO4, 265.6mgCaCl2, 296mgMgSO4, 6.2mgH3BO3, 22.6mgMnSO4, 8.6mgZnSO4, 0.25mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 1000mgPVP, 0.24mgbiotin, 100mgL-cysteine, 2.4mgD-calcium pantothenate, 2mgglycine, 5mgnic acid, 2mgthiamine, 0.5mgpyridoxine, 37.3mgNa2-EDTA, 27.8mgFeSO4, 100mginositol, 0.3-0.5mgN 6 Benzyl adenine (6-BA), 0.1-0.2 mg naphthaleneacetic acid, 30 g sucrose, 5 g agar powder, pH 5.8-6; The raw material components of the subculture medium in step (4) are as follows: 1L of subculture medium contains 1520mgKNO3, 1320mgNH4NO3, 136mgKH2PO4, 265.6mgCaCl2, 296mgMgSO4, 6.2mgH3BO3, 22.6mgMnSO4, 8.6mgZnSO4, 0.25 mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 0.24mgbiotin, 2mgglycine, 5mgnic acid, 2mgthiamine, 0.5mgpyridoxine, 37.3mgNa2-EDTA, 27.8mgFeSO4, 100mginositol, 0.4-0.6mgN 6 Benzyl adenine, 0.2-0.3 mg naphthaleneacetic acid, 30 g sucrose, 5 g agar powder, pH 5.8-6; The raw material components of the seedling culture medium in step (5) are as follows: 1L seedling culture medium contains 1520mgKNO3, 1320mgNH4NO3, 136mgKH2PO4, 265.6mgCaCl2, 296mgMgSO4, 6.2mgH3BO3, 22.6mgMnSO4, 8.6mgZnSO4, 0.25mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 0.24mgbiotin, 2mgglycine, 5mgnic acid, 2mgthiamine, 0.5mgpyridoxine, 37.3mgNa2-EDTA, 27.8mgFeSO4, 100mginositol, 0.1-0.3mgN 6 Benzyl adenine, 0.3-0.5 mg naphthaleneacetic acid, 30 g sucrose, 5 g agar powder, pH 5.8-6; The raw material components of the rooting medium in step (6) are as follows: 1L rooting medium contains 246mgKNO3, 335mgKH2PO4, 250mgCa(NO3)2, 76mgCaCl2, 258mgMgSO4, 3.1mgH3BO3, 11.15mgMnSO4, 4.3mgZnSO4, 0.125mgNa2MoO4, 0.0125mgCuSO4, 0.0125mgCoCl2, 0.415mgKI, 0.24mgbiotin, 100mgL-cysteine, 2.4mgD-pantothenate calcium, 2mgglycine, 5mgnic acid, 2mgthiamine, 0.5mgpyridoxine, 37.3mgNa2-EDTA, 27.8mg FeSO4, 100mg inositol, 0.3-0.5mg indolebutyric acid, 0.2-0.3mg naphthaleneacetic acid, 0.3-0.5mg paclobutrazol, 30g sucrose, 5g agar powder, 5g activated carbon, pH 5.8-6; The matrix in step (7) is a mixture of coconut husk, carbonized rice husk, peat soil and loess soil in a weight ratio of 5:2.5:2:0.5, and calcium magnesium phosphate fertilizer 4 kg / m 3 Mix well, cover with film and compost for one month to make a light substrate; the raw material components of the foliar nutrient solution are: 1L foliar nutrient solution contains 152mgKNO3, 132mgNH4NO3, 13.6mgKH2PO4, 26.56mgCaCl2, and 29.6mgMgSO4.

2. The method for rapid propagation of Eucalyptus grandis according to claim 1, wherein: The excellent individual plant described in step (1) is the Eucalyptus grandis GE19-2 variety.

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