A tissue culture and rapid propagation method for Eucalyptus dunnii

Abstract

The invention discloses a tissue culture rapid propagation method for Eucalyptus dunnii, selects the excellent individual plants of Eucalyptus dunnii without disease and insect pests to carry out girdling and promote sprouting, when the budding bar grows to 1.5~2.0m, cuts the lignified branches and carries out grafting, when the grafted stem segment budding bar grows to 20~25cm, cuts the semi-lignified stem segment and obtains explant and carries out explant disinfection, carries out induction culture, subculture and rooting culture in sequence, finally will take root completely, the rooting bottle seedling of seedling height 4-6cm cleans culture medium and transplants into the nutrient cup equipped with matrix. After grafting, the present invention takes explant again, grafted seedling is in nursery ground, conveniently collects and easily controls disease and insect pests and pollution problems, ensures explant material growth vitality; The rooting culture medium used reduces the use of macroelements and some trace elements, by the collocation between different elements, significantly improves Eucalyptus dunnii rooting efficiency, and the rooting rate in the final bottle can reach more than 95%.

Description

Technical Field

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

[0002] Dunn's eucalyptus, commonly known as Dunn's white Gun in Australia, is a species of Eucalyptus in the Myrtaceae family, and its scientific name is Eucalyptus dunnii. Dunn's eucalyptus is naturally distributed in the tropical rainforests of the northeastern coast and coastal areas of Australia and southeastern Queensland. It is a tall tree with a narrow natural distribution area, and the tree height can generally reach 40~50m. Dunn's eucalyptus is a rare summer rain type, fast-growing and high-yielding tree species with strong cold resistance. This tree species grows fast, has high yield, good material, strong stress resistance, and especially strong cold resistance. According to measurements, the basic density of 10-year-old artificially cultivated Dunn's eucalyptus wood is 500kg / m 3 The basic density of naturally grown large-diameter Dunn eucalyptus wood is about 800kg / m 3 The wood is straight in texture and has a pale white heartwood. Its wood is widely used as a raw material for pulp fiber, as well as for peeled boards and sawn timber.

[0003] However, because E. dunnii is a cross-pollinated species that reproduces by seed and has a high degree of individual differentiation, rapid growth can lead to poor yields in high-yield forest establishment. Furthermore, E. dunnii's poor fruiting, low seed yield, and high seed prices further limit its promotion and application. Tissue culture is the primary method for exploring rapid asexual propagation of E. dunnii. While ongoing research on tissue culture of E. dunnii has been ongoing both domestically and internationally, it has generally failed to overcome the difficulty of rooting in tissue culture. Guan Chaoxu et al. (2012) achieved an 84.7% rooting rate using a low-salt DCR basal medium supplemented with the root promoter ABT1. Lin Yan (2004) used various traditional rooting methods, including one-step, two-step, and liquid methods containing only IBA, but achieved rooting rates of only 54.3% to 62.3%. This difficulty in rooting has severely hampered the development of industrial-scale production of E. dunnii tissue culture seedlings. 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 dunnii, which can effectively retain the excellent germplasm of Eucalyptus dunnii and 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 dunnii comprises selecting high-quality individual plants of Eucalyptus dunnii that are free of pests and diseases, performing girdling to promote sprouting, cutting lignified branches when the sprouts grow to 1.5-2.0 m, cutting semi-lignified stem segments to obtain explants when the sprouts of the grafted stem segments grow to 20-25 cm, sterilizing the explants, sequentially performing induction culture, subculture, and rooting culture, and finally, washing the culture medium of the rooted seedlings that have completely rooted and are 4-6 cm tall, and transplanting them into a nutrient cup containing a substrate.

[0007] The main steps include:

[0008] (1) Circumcision to promote germination

[0009] Select high-quality individual Eucalyptus dunnii plants 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 3 / 4 of its circumference at 20 cm above the ground, with a width of 3 cm. Scrape the cambium down to the phloem, and spray the phloem with a bud-promoting nutrient solution. Spray the trunk with 75% alcohol for disinfection on the fifth and tenth days after girdling.

[0010] (2) Bud grafting

[0011] When the buds grow to 1.5-2.0 m, the lignified branches are cut for grafting. Before grafting, the branches are soaked in 100 ppm naphthaleneacetic acid (NAA) for 1 hour.

[0012] (3) Explant disinfection

[0013] When the buds of the grafted stem segments grow to 20-25 cm, cut the semi-lignified stem segments, wash them with a detergent solution for 5 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 20 seconds, rinse them with sterile water three times, soak them in 2% sodium hypochlorite for 1 minute, and rinse them with sterile water three times.

[0014] (4) Induction culture

[0015] Use sterile filter paper to absorb the surface moisture of the small segment treated in step (3), cut off the ends of the small segment, inoculate it into the induction culture medium, and culture it for 20-25 days at a temperature of 25±2℃, a light intensity of 12h / d and a light intensity of 1500-2000lux;

[0016] (5) Subculture

[0017] The sterile buds obtained in step (4) were cut and transferred to subculture medium, and cultured for 20-25 days at a temperature of 25±2°C, a light intensity of 12 h / d, and a light intensity of 1500-2000 lux;

[0018] (6) Rooting culture

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

[0020] (7) Transplantation

[0021] Wash the culture medium for the rooted seedlings that have fully rooted and are 4-6 cm tall and transplant them into a nutrient cup containing a substrate. Pour the substrate thoroughly, cover with a film to keep it moist and cover with a shade net. You can use 800-1,000 times of thiophanate-methyl or 500-600 times of carbendazim alternately, spraying them once 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 once every 10 days.

[0022] Preferably, the grafting method described in step (2) above is:

[0023] Grafting time: spring, sunny days;

[0024] Rootstock selection: 25-35cm high, 2.5-5mm stem diameter, Eucalyptus dunnii, grafted at 10cm above the ground;

[0025] Selection of cuttings: 20-22 cm long, 2.5-5 mm in diameter, with 4-8 axillary buds, lignified Dunn's eucalyptus branches;

[0026] Before grafting, wrap the scion with medical sealing film. When grafting, cut the scion into a "V" shape on both sides, cut a 2-3 cm long incision in the middle of the grafting position of the rootstock, insert the scion into the rootstock, and wrap it with sealing film.

[0027] Move the grafted seedlings into the greenhouse and maintain the humidity at 80% and the temperature at 26±2℃. When the new leaves are half the size of mature leaves, move them under a shade net with a light transmittance of 50% for cultivation. When the new leaves are the size of mature leaves, cultivate them under full light.

[0028] Preferably, the raw material components of the sprout-promoting nutrient solution in step (1) above are as follows: 1L sprout-promoting nutrient solution contains 180mgKNO3, 36mgNH4NO3, 27mgKH2PO4, 20mgCaCl2, 36mgMgSO4, 0.1-0.2mgN 6 Benzyl adenine (6-BA), 0.1-0.2 mg naphthaleneacetic acid (NAA).

[0029] Preferably, the raw material components of the induction culture medium in step (4) above are as follows: 1L induction culture medium contains 1800mgKNO3, 360mgNH4NO3, 270mgKH2PO4, 200mgCaCl2, 360mgMgSO4, 10mgH3BO3, 25mgMnSO4, 10mgZnSO4, 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, 0.3-0.5mgN 6 Benzyl adenine (6-BA), 0.1-0.2mg naphthaleneacetic acid (NAA), 0.2-0.3mg kinetin (KT), 30g sucrose, 5g agar powder, pH 5.8-6. Adding kinetin to the induction medium can effectively promote the germination of Eucalyptus dunnii buds.

[0030] Preferably, the raw material components of the subculture medium in step (5) above are as follows: 1L of subculture medium contains 1800mgKNO3, 360mgNH4NO3, 270mgKH2PO4, 200mgCaCl2, 360mgMgSO4, 10mgH3BO3, 25mgMnSO4, 10mgZnSO4, 0.25mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 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, 0.4-0.6mgN 6 Benzyl adenine (6-BA), 0.2-0.3 mg naphthaleneacetic acid (NAA), 0.1-0.2 mg kinetin (KT), 30 g sucrose, 5 g agar powder, pH 5.8-6.

[0031] Preferably, the raw material components of the rooting medium in step (6) above are as follows: 1L rooting medium contains 600mgKNO3, 120mgNH4NO3, 90mgKH2PO4, 66mgCaCl2, 120mgMgSO4, 6.2mgH3BO3, 25mgMnSO4, 10mgZnSO4, 0.25mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 0.24mgbiotin(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, 0.4-0.6mg indolebutyric acid (IBA), 0.1-0.2mg naphthaleneacetic acid (NAA), 0.5-0.8mg paclobutrazol (PP333), 30g sucrose, 5g agar powder, 5g activated carbon, pH value 5.8~6.

[0032] Preferably, the matrix in step (7) above is loess soil: 95% loess soil + 5% superphosphate are mixed evenly or coconut husk: carbonized rice husk: peat soil: loess soil are mixed 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 let it ferment for one month to make a light substrate;

[0033] The raw material formula of the foliar nutrient solution is: 1L of foliar nutrient solution contains 180mgKNO3, 36mgNH4NO3, 27mgKH2PO4, 20mgCaCl2, and 36mgMgSO4.

[0034] Preferably, the excellent single plant of Eucalyptus dunnensis in step (1) above is Eucalyptus dunnensis GED9502 variety. Eucalyptus dunnensis GED9502 variety is a forest tree variety approved by the Guangxi Zhuang Autonomous Region Forestry Variety Approval Committee. It has excellent characteristics such as strong adaptability, fast growth, straight trunk, high yield, and strong cold resistance. At 5.5 years old, the average tree height can reach 19.5m, the average diameter at breast height can reach 17.1cm, and the average annual cumulative growth can reach 26.4m 3 .hm -2 .

[0035] The advantages and beneficial effects of the present invention are as follows:

[0036] 1. The invention takes explants after grafting, and the grafted seedlings are in a nursery, which is convenient for collection and easy to control pests and diseases and pollution problems, thereby ensuring the growth vitality of the explant material; the grafted seedlings can continuously provide explants, and the number of induction times and time are not limited; the invention avoids the problems of long transportation to a tissue culture base, high induction difficulty, low induction rate, and difficulty in successful single induction when budding strips are directly girdled as explant materials, thereby laying a foundation for the preservation of fine germplasm resources of Eucalyptus dunnii and the cultivation of later clones; and simultaneously, the branches are soaked in 100 ppm of naphthaleneacetic acid (NAA) for 1 hour before grafting, thereby overcoming the influence of the high tannin content of Eucalyptus dunnii and the easy formation of a tannin oxidation isolation layer on the cut surface, thereby contributing to the rapid healing of the incision.

[0037] 2. Based on induction cultivation and subculture, the rooting 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 dunnii is significantly improved, and the final rooting rate in the bottle can reach more than 95%.

[0038] 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 dunnii are comprehensive and sufficient, effectively improving the transplant survival rate of Eucalyptus dunnii tissue culture seedlings, which can reach more than 95%, reducing material loss, and promoting large-scale production of Eucalyptus dunnii.

[0039] 4. 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 dunnii and realize the efficient development of Eucalyptus dunnii clones. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a real picture of the grafted Eucalyptus dunnii seedlings after grafting according to the present invention.

[0041] Figure 2 This is a physical picture of the subcultured seedlings of Eucalyptus dunnii after subculture culture according to the present invention.

[0042] Figure 3 This is a real picture of the rooted seedlings of Eucalyptus dunnii after the rooting culture of the present invention.

[0043] Figure 4 This is a real picture of the eucalyptus dunnii seedlings that survived after being transplanted by the present invention. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to the accompanying drawings. Example

[0045] A tissue culture and rapid propagation method for Eucalyptus dunnii, comprising the following main steps:

[0046] (1) Circumcision to promote germination

[0047] Select a pest-free individual tree of Eucalyptus dunnii GED9502, remove surrounding shrubs, and spray with 1% potassium permanganate for disinfection the day before girdling. Girdle the tree 3 / 4 of its circumference at 20 cm above the ground, with a width of 3 cm. Scrape the cambium down to the phloem, and spray the phloem with a bud-promoting nutrient solution. Spray the trunk with 75% alcohol for disinfection on the fifth and tenth days after girdling.

[0048] The raw material components of the sprout-promoting nutrient solution are as follows: 1L of the sprout-promoting nutrient solution contains 180mgKNO3, 36mgNH4NO3, 27mgKH2PO4, 20mgCaCl2, 36mgMgSO4, 0.1mgN 6 benzyl adenine (6-BA), 0.2 mg naphthaleneacetic acid (NAA);

[0049] (2) Bud grafting

[0050] When the buds grow to 1.5-2.0 m, the lignified branches are cut for grafting. Before grafting, the branches are soaked in 100 ppm naphthaleneacetic acid (NAA) for 1 hour.

[0051] The method of described grafting is:

[0052] Grafting time: spring, sunny days;

[0053] Rootstock selection: 25-35cm high, 2.5-5mm stem diameter, Eucalyptus dunnii, grafted at 10cm above the ground;

[0054] Selection of cuttings: 20-22 cm long, 2.5-5 mm in diameter, with 4-8 axillary buds, lignified Dunn's eucalyptus branches;

[0055] Before grafting, wrap the scion with medical sealing film. When grafting, cut the scion into a "V" shape on both sides, cut a 2-3 cm long incision in the middle of the grafting position of the rootstock, insert the scion into the rootstock, and wrap it with sealing film.

[0056] Move the grafted seedlings into the greenhouse and maintain the humidity at 80% and the temperature at 26±2℃. When the new leaves are half the size of mature leaves, move them under a shade net with a light transmittance of 50% for cultivation. When the new leaves are the size of mature leaves, cultivate them under full light.

[0057] (3) Explant disinfection

[0058] When the buds of the grafted stem segments grow to 20-25 cm, cut the semi-lignified stem segments, wash them with a detergent solution for 5 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 20 seconds, rinse them with sterile water three times, soak them in 2% sodium hypochlorite for 1 minute, and rinse them with sterile water three times.

[0059] (4) Induction culture

[0060] Use sterile filter paper to absorb the surface moisture of the small segment treated in step (3), cut off the ends of the small segment, inoculate it into the induction culture medium, and culture it for 20-25 days at a temperature of 25±2℃, a light intensity of 12h / d and a light intensity of 1500-2000lux;

[0061] The raw material components of the induction culture medium are as follows: 1L of the induction culture medium contains 1800mgKNO3, 360mgNH4NO3, 270mgKH2PO4, 200mgCaCl2, 360mgMgSO4, 10mgH3BO3, 25mgMnSO4, 10mgZnSO4, 0.25 mgNa2MoO4, 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, 0.3mgN 6 Benzyl adenine (6-BA), 0.2mg naphthaleneacetic acid (NAA), 0.2mg kinetin (KT), 30g sucrose, 5g agar powder, pH 5.8-6; adding kinetin to the induction medium can effectively promote the germination of Eucalyptus dunnii buds;

[0062] (5) Subculture

[0063] The sterile buds obtained in step (4) were cut and transferred to subculture medium, and cultured for 20-25 days at a temperature of 25±2°C, a light intensity of 12 h / d, and a light intensity of 1500-2000 lux;

[0064] The raw material components of the subculture medium are as follows: 1L of subculture medium contains 1800mgKNO3, 360mgNH4NO3, 270mgKH2PO4, 200mgCaCl2, 360mgMgSO4, 10mgH3BO3, 25mgMnSO4, 10mgZnSO4, 0.25 mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 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, 0.4mgN 6Benzyl adenine (6-BA), 0.3 mg naphthaleneacetic acid (NAA), 0.1 mg kinetin (KT), 30 g sucrose, 5 g agar powder, pH 5.8-6;

[0065] (6) Rooting culture

[0066] When the seedlings obtained by step (5) grow to 3-4 cm, cut the seedlings and transfer them to rooting medium, and culture them for 20-25 days at a temperature of 25±2°C, a light intensity of 12h / d, and a light intensity of 1500-2000lux;

[0067] The raw material components of the rooting medium are as follows: 1L of the rooting medium contains 600mgKNO3, 120mgNH4NO3, 90mgKH2PO4, 66mgCaCl2, 120mgMgSO4, 6.2mgH3BO3, 25mgMnSO4, 10mgZnSO4, 0.25 mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 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, 100 mg inositol, 0.4 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;

[0068] (7) Transplantation

[0069] Wash the culture medium of the rooted seedlings with complete rooting and a height of 4-6 cm and transplant them into the nutrient cups filled with substrate. Drench the substrate thoroughly, cover with film to keep it moist and cover with shade net. You can use 800-1000 times dilution of thiophanate-methyl or 500-600 times dilution of carbendazim alternately, spraying them 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;

[0070] The matrix is ​​made 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 let it ferment for one month to make a light substrate;

[0071] The raw material formula of the foliar nutrient solution is: 1L of foliar nutrient solution contains 180mgKNO3, 36mgNH4NO3, 27mgKH2PO4, 20mgCaCl2, and 36mgMgSO4. Example

[0072] A tissue culture and rapid propagation method for Eucalyptus dunnii, comprising the following main steps:

[0073] (1) Circumcision to promote germination

[0074] Select a pest-free individual tree of Eucalyptus dunnii GED9502, remove surrounding shrubs, and spray with 1% potassium permanganate for disinfection the day before girdling. Girdle the tree 3 / 4 of its circumference at 20 cm above the ground, with a width of 3 cm. Scrape the cambium down to the phloem, and spray the phloem with a bud-promoting nutrient solution. Spray the trunk with 75% alcohol for disinfection on the fifth and tenth days after girdling.

[0075] The raw material components of the sprout-promoting nutrient solution are as follows: 1L of the sprout-promoting nutrient solution contains 180mgKNO3, 36mgNH4NO3, 27mgKH2PO4, 20mgCaCl2, 36mgMgSO4, 0.2mgN 6 benzyl adenine (6-BA), 0.2 mg naphthaleneacetic acid (NAA);

[0076] (2) Bud grafting

[0077] When the buds grow to 1.5-2.0 m, the lignified branches are cut for grafting. Before grafting, the branches are soaked in 100 ppm naphthaleneacetic acid (NAA) for 1 hour.

[0078] The method of described grafting is:

[0079] Grafting time: spring, sunny days;

[0080] Rootstock selection: 25-35cm high, 2.5-5mm stem diameter, Eucalyptus dunnii, grafted at 10cm above the ground;

[0081] Selection of cuttings: 20-22 cm long, 2.5-5 mm in diameter, with 4-8 axillary buds, lignified Dunn's eucalyptus branches;

[0082] Before grafting, wrap the scion with medical sealing film. When grafting, cut the scion into a "V" shape on both sides, cut a 2-3 cm long incision in the middle of the grafting position of the rootstock, insert the scion into the rootstock, and wrap it with sealing film.

[0083] Move the grafted seedlings into the greenhouse and maintain the humidity at 80% and the temperature at 26±2℃. When the new leaves are half the size of mature leaves, move them under a shade net with a light transmittance of 50% for cultivation. When the new leaves are the size of mature leaves, cultivate them under full light.

[0084] (3) Explant disinfection

[0085] When the buds of the grafted stem segments grow to 20-25 cm, cut the semi-lignified stem segments, wash them with a detergent solution for 5 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 20 seconds, rinse them with sterile water three times, soak them in 2% sodium hypochlorite for 1 minute, and rinse them with sterile water three times.

[0086] (4) Induction culture

[0087] Use sterile filter paper to absorb the surface moisture of the small segment treated in step (3), cut off the ends of the small segment, inoculate it into the induction culture medium, and culture it for 20-25 days at a temperature of 25±2℃, a light intensity of 12h / d and a light intensity of 1500-2000lux;

[0088] The raw material components of the induction culture medium are as follows: 1L of the induction culture medium contains 1800mgKNO3, 360mgNH4NO3, 270mgKH2PO4, 200mgCaCl2, 360mgMgSO4, 10mgH3BO3, 25mgMnSO4, 10mgZnSO4, 0.25 mgNa2MoO4, 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), 0.2mg naphthaleneacetic acid (NAA), 0.2mg kinetin (KT), 30g sucrose, 5g agar powder, pH 5.8-6; adding kinetin to the induction medium can effectively promote the germination of Eucalyptus dunnii buds;

[0089] (5) Subculture

[0090] The sterile buds obtained in step (4) were cut and transferred to subculture medium, and cultured for 20-25 days at a temperature of 25±2°C, a light intensity of 12 h / d, and a light intensity of 1500-2000 lux;

[0091] The raw material components of the subculture medium are as follows: 1L of subculture medium contains 1800mgKNO3, 360mgNH4NO3, 270mgKH2PO4, 200mgCaCl2, 360mgMgSO4, 10mgH3BO3, 25mgMnSO4, 10mgZnSO4, 0.25 mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 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), 0.2 mg naphthaleneacetic acid (NAA), 0.2 mg kinetin (KT), 30 g sucrose, 5 g agar powder, pH 5.8-6;

[0092] (6) Rooting culture

[0093] When the seedlings obtained by step (5) grow to 3-4 cm, cut the seedlings and transfer them to rooting medium, and culture them for 20-25 days at a temperature of 25±2°C, a light intensity of 12h / d, and a light intensity of 1500-2000lux;

[0094] The raw material components of the rooting medium are as follows: 1L of the rooting medium contains 600mgKNO3, 120mgNH4NO3, 90mgKH2PO4, 66mgCaCl2, 120mgMgSO4, 6.2mgH3BO3, 25mgMnSO4, 10mgZnSO4, 0.25 mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 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, 0.5mg indolebutyric acid (IBA), 0.1mg naphthaleneacetic acid (NAA), 0.6mg paclobutrazol (PP333), 30g sucrose, 5g agar powder, 5g activated carbon, pH 5.8-6;

[0095] (7) Transplantation

[0096] Wash the culture medium of the rooted seedlings with complete rooting and a height of 4-6 cm and transplant them into the nutrient cups filled with substrate. Drench the substrate thoroughly, cover with film to keep it moist and cover with shade net. You can use 800-1000 times dilution of thiophanate-methyl or 500-600 times dilution of carbendazim alternately, spraying them 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;

[0097] The matrix is ​​made 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 let it ferment for one month to make a light substrate;

[0098] The raw material formula of the foliar nutrient solution is: 1L of foliar nutrient solution contains 180mgKNO3, 36mgNH4NO3, 27mgKH2PO4, 20mgCaCl2, and 36mgMgSO4. Example

[0099] A tissue culture and rapid propagation method for Eucalyptus dunnii, comprising the following main steps:

[0100] (1) Circumcision to promote germination

[0101] Select a pest-free individual tree of Eucalyptus dunnii GED9502, remove surrounding shrubs, and spray with 1% potassium permanganate for disinfection the day before girdling. Girdle the tree 3 / 4 of its circumference at 20 cm above the ground, with a width of 3 cm. Scrape the cambium down to the phloem, and spray the phloem with a bud-promoting nutrient solution. Spray the trunk with 75% alcohol for disinfection on the fifth and tenth days after girdling.

[0102] The raw material components of the sprout-promoting nutrient solution are as follows: 1L of the sprout-promoting nutrient solution contains 180mgKNO3, 36mgNH4NO3, 27mgKH2PO4, 20mgCaCl2, 36mgMgSO4, 0.2mgN 6 benzyl adenine (6-BA), 0.1 mg naphthaleneacetic acid (NAA);

[0103] (2) Bud grafting

[0104] When the buds grow to 1.5-2.0 m, the lignified branches are cut for grafting. Before grafting, the branches are soaked in 100 ppm naphthaleneacetic acid (NAA) for 1 hour.

[0105] The method of described grafting is:

[0106] Grafting time: spring, sunny days;

[0107] Rootstock selection: 25-35cm high, 2.5-5mm stem diameter, Eucalyptus dunnii, grafted at 10cm above the ground;

[0108] Selection of cuttings: 20-22 cm long, 2.5-5 mm in diameter, with 4-8 axillary buds, lignified Dunn's eucalyptus branches;

[0109] Before grafting, wrap the scion with medical sealing film. When grafting, cut the scion into a "V" shape on both sides, cut a 2-3 cm long incision in the middle of the grafting position of the rootstock, insert the scion into the rootstock, and wrap it with sealing film.

[0110] Move the grafted seedlings into the greenhouse and maintain the humidity at 80% and the temperature at 26±2℃. When the new leaves are half the size of mature leaves, move them under a shade net with a light transmittance of 50% for cultivation. When the new leaves are the size of mature leaves, cultivate them under full light.

[0111] (3) Explant disinfection

[0112] When the buds of the grafted stem segments grow to 20-25 cm, cut the semi-lignified stem segments, wash them with a detergent solution for 5 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 20 seconds, rinse them with sterile water three times, soak them in 2% sodium hypochlorite for 1 minute, and rinse them with sterile water three times.

[0113] (4) Induction culture

[0114] Use sterile filter paper to absorb the surface moisture of the small segment treated in step (3), cut off the ends of the small segment, inoculate it into the induction culture medium, and culture it for 20-25 days at a temperature of 25±2℃, a light intensity of 12h / d and a light intensity of 1500-2000lux;

[0115] The raw material components of the induction culture medium are as follows: 1L of the induction culture medium contains 1800mgKNO3, 360mgNH4NO3, 270mgKH2PO4, 200mgCaCl2, 360mgMgSO4, 10mgH3BO3, 25mgMnSO4, 10mgZnSO4, 0.25 mgNa2MoO4, 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, 0.5mgN 6Benzyl adenine (6-BA), 0.1mg naphthaleneacetic acid (NAA), 0.3mg kinetin (KT), 30g sucrose, 5g agar powder, pH 5.8-6; adding kinetin to the induction medium can effectively promote the germination of Eucalyptus dunnii buds;

[0116] (5) Subculture

[0117] The sterile buds obtained in step (4) were cut and transferred to subculture medium, and cultured for 20-25 days at a temperature of 25±2°C, a light intensity of 12 h / d, and a light intensity of 1500-2000 lux;

[0118] The raw material components of the subculture medium are as follows: 1L of subculture medium contains 1800mgKNO3, 360mgNH4NO3, 270mgKH2PO4, 200mgCaCl2, 360mgMgSO4, 10mgH3BO3, 25mgMnSO4, 10mgZnSO4, 0.25 mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 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, 0.6mgN 6 Benzyl adenine (6-BA), 0.2 mg naphthaleneacetic acid (NAA), 0.2 mg kinetin (KT), 30 g sucrose, 5 g agar powder, pH 5.8-6;

[0119] (6) Rooting culture

[0120] When the seedlings obtained by step (5) grow to 3-4 cm, cut the seedlings and transfer them to rooting medium, and culture them for 20-25 days at a temperature of 25±2°C, a light intensity of 12h / d, and a light intensity of 1500-2000lux;

[0121] The raw material components of the rooting medium are as follows: 1L of the rooting medium contains 600mgKNO3, 120mgNH4NO3, 90mgKH2PO4, 66mgCaCl2, 120mgMgSO4, 6.2mgH3BO3, 25mgMnSO4, 10mgZnSO4, 0.25 mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 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, 0.6mg indolebutyric acid (IBA), 0.1mg naphthaleneacetic acid (NAA), 0.8mg paclobutrazol (PP333), 30g sucrose, 5g agar powder, 5g activated carbon, pH 5.8-6;

[0122] (7) Transplantation

[0123] Wash the culture medium of the rooted seedlings with complete rooting and a height of 4-6 cm and transplant them into the nutrient cups filled with substrate. Drench the substrate thoroughly, cover with film to keep it moist and cover with shade net. You can use 800-1000 times dilution of thiophanate-methyl or 500-600 times dilution of carbendazim alternately, spraying them 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;

[0124] The matrix is ​​loess soil: 95% loess soil + 5% superphosphate are mixed evenly;

[0125] The raw material formula of the foliar nutrient solution is: 1L of foliar nutrient solution contains 180mgKNO3, 36mgNH4NO3, 27mgKH2PO4, 20mgCaCl2, and 36mgMgSO4.

[0126] Comparative Example 1:

[0127] The only difference from Example 3 is that there is no grafting step, that is, after step (1) ring cutting to promote germination, when the buds grow to 1.5-2.0 m, the lignified branches are cut and cut into stem segments as explants, washed with a detergent solution for 5 min, and then rinsed with tap water for 5 min; brought to a clean bench, cut into small segments of 2-3 cm, placed in a sterile bottle, soaked in 75% alcohol for 30 s, washed with sterile water 3 times, soaked in 2% sodium hypochlorite by volume for 2 min, and washed with sterile water 3 times; the treated explants are cultured and transplanted according to steps (4) to (7) of Example 2.

[0128] Comparative Example 2:

[0129] The only difference from Example 3 is that there is no need to soak the buds generated by ring cutting to promote germination with 100 ppm naphthaleneacetic acid (NAA) before grafting, and the other steps are the same as in Example 3.

[0130] Comparative Example 3:

[0131] The only difference from Example 3 is that the raw material components of the rooting culture medium described in step (6) are different, and paclobutrazol and activated carbon materials are not included. Specifically, the raw material components of the rooting culture medium used in this comparative example are: 1L rooting culture medium contains 600mgKNO3, 120mgNH4NO3, 90mgKH2PO4, 66mgCaCl2, 120mgMgSO4, 6.2mgH3BO3, 25mgMnSO4, 10mgZnSO4, 0.25 mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 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, 0.6 mg indolebutyric acid (IBA), 0.1 mg naphthaleneacetic acid (NAA), 30 g sucrose, 5 g agar powder, pH 5.8~6.

[0132] Comparative Example 4:

[0133] 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 amounts of elements such as K, N, P, Ca, Mg, and B are increased. Specifically, the raw material components of the rooting culture medium used in this comparative example are: 1800 mg KNO3, 360 mg NH4NO3, 270 mg KH2PO4, 200 mg CaCl2, 360 mg MgSO4, 10 mg H3BO3, 0.25 mg Na2MoO4, 0.025 mg CuSO4, 0.025 mg CoCl2, 0.83 mg KI, 0.24 mg biotin (VH), 100 mg L-cysteine, 2.4 mg D-pantothenate calcium (VB5), 2 mg glycine, 5 mg nicotinic acid (VB3), 2 mg thiamine (VB1), 0.5 mg pyridoxine (VB6), 37.3 mg Na2-EDTA, 27.8 mg FeSO4, 100 mg inositol, 0.6 mg indolebutyric acid (IBA), 0.1 mg naphthaleneacetic acid (NAA), 0.8 mg paclobutrazol (PP333), 30 g sucrose, 5 g agar powder, 5 g activated carbon, pH 5.8~6.

[0134] Comparative Example 5:

[0135] 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).

[0136] Test example:

[0137] The rooting rate and average root length of the Eucalyptus dunnii tissue culture seedlings in bottles of Examples 1-3 and Comparative Examples 1-5, as well as the transplant survival rate three months after transplanting, were calculated. The results are shown in the following table:

[0138] Group Rooting rate in bottle Average root length of bottle seedlings Transplant survival rate Example 1 98.1% 3.1cm 97.3% Example 2 99.3% 2.9cm 98.8% Example 3 96.9% 3.0cm 96.9% Comparative Example 1 80.5% 2.5cm 81.4% Comparative Example 2 78.3% 2.6cm 72.1% Comparative Example 3 85.7% 2.1cm 82.6% Comparative Example 4 68.7% 2.2cm 75.8% Comparative Example 5 97.2% 3.0cm 80.8%

[0139] As can be seen from the above table, especially after the complete steps of girdling to promote germination, grafting, rooting culture and spraying nutrient solution after transplanting in this application, the rooting rate of Eucalyptus Dunn's tissue culture seedlings in the bottle can be increased to more than 95%, and the transplanting survival rate can be increased to more than 95%.

Claims

1. A tissue culture and rapid propagation method for Eucalyptus dunnii, characterized by: Select high-quality individual plants of Eucalyptus dunnii that are free of pests and diseases and perform girdling to promote budding. When the budding branches grow to 1.5-2.0m, cut the lignified branches for grafting. When the budding branches of the grafted stem segments grow to 20-25cm, cut the semi-lignified stem segments to obtain explants and disinfect the explants. Then, carry out induction culture, subculture and rooting culture in sequence. Finally, the rooting bottle seedlings that have fully rooted and are 4-6cm tall 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 dunnii plants 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 3 / 4 of its circumference at 20 cm above the ground, with a width of 3 cm. Scrape the cambium down to the phloem, and spray the phloem with a bud-promoting nutrient solution. Spray the trunk with 75% alcohol for disinfection on the fifth and tenth days after girdling. (2) Bud grafting When the buds grow to 1.5-2.0 m, the lignified branches are cut for grafting. Before grafting, the branches are soaked in 100 ppm naphthaleneacetic acid (NAA) for 1 hour. (3) Explant disinfection When the buds of the grafted stem segments grow to 20-25 cm, cut the semi-lignified stem segments, wash them with a detergent solution for 5 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 20 seconds, rinse them with sterile water three times, soak them in 2% sodium hypochlorite for 1 minute, and rinse them with sterile water three times. (4) Induction culture Use sterile filter paper to absorb the surface moisture of the small segment treated in step (3), cut off the ends of the small segment, inoculate it into the induction culture medium, and culture it for 20-25 days at a temperature of 25±2℃, a light intensity of 12h / d and a light intensity of 1500-2000lux; (5) Subculture The sterile buds obtained in step (4) were cut and transferred to subculture medium, and cultured for 20-25 days at a temperature of 25±2°C, a light intensity of 12 h / d, and a light intensity of 1500-2000 lux; (6) Rooting culture When the seedlings obtained by step (5) grow to 3-4 cm, cut the seedlings and transfer them to rooting medium, and culture them for 20-25 days at a temperature of 25±2°C, a light intensity of 12h / d, and a light intensity of 1500-2000lux; (7) Transplantation Wash the culture medium of the rooted seedlings with complete rooting and a height of 4-6 cm and transplant them into the nutrient cups filled with substrate. Drench the substrate thoroughly, cover with film to keep it moist and cover with shade net. You can use 800-1000 times dilution of thiophanate-methyl or 500-600 times dilution of carbendazim alternately, spraying them 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; The grafting method in step (2) is: Grafting time: spring, sunny days; Rootstock selection: 25-35cm high, 2.5-5mm stem diameter, Eucalyptus dunnii, grafted at 10cm above the ground; Selection of cuttings: 20-22 cm long, 2.5-5 mm in diameter, with 4-8 axillary buds, lignified Dunn's eucalyptus branches; Before grafting, wrap the scion with medical sealing film. When grafting, cut the scion into a "V" shape on both sides, cut a 2-3 cm long incision in the middle of the grafting position of the rootstock, insert the scion into the rootstock, and wrap it with sealing film. Move the grafted seedlings into the greenhouse and maintain the humidity at 80% and the temperature at 26±2℃. When the new leaves are half the size of mature leaves, move them to a shade net with a light transmittance of 50%. When the new leaves are the size of mature leaves, move them to full light cultivation. The raw material components of the germination-promoting nutrient solution in step (1) are as follows: 1L of germination-promoting nutrient solution contains 180mgKNO3, 36mgNH4NO3, 27mgKH2PO4, 20mgCaCl2, 36mgMgSO4, 0.1-0.2mgN 6 Benzyl adenine, 0.1-0.2 mg naphthaleneacetic acid; The raw material components of the induction culture medium in step (4) are as follows: 1L induction culture medium contains 1800mgKNO3, 360mgNH4NO3, 270mgKH2PO4, 200mgCaCl2, 360mgMgSO4, 10mgH3BO3, 25mgMnSO4, 10mgZnSO4, 0.25 mgNa2MoO4, 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, 0.1-0.2 mg naphthaleneacetic acid, 0.2-0.3 mg kinetin, 30 g sucrose, 5 g agar powder, pH 5.8-6; The raw material components of the subculture medium in step (5) are as follows: 1L of subculture medium contains 1800mgKNO3, 360mgNH4NO3, 270mgKH2PO4, 200mgCaCl2, 360mgMgSO4, 10mgH3BO3, 25mgMnSO4, 10mgZnSO4, 0.25 mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 0.24mgbiotin, 100mgL-cysteine, 2.4mgD-calcium pantothenate, 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, 0.1-0.2 mg kinetin, 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 600mgKNO3, 120mgNH4NO3, 90mgKH2PO4, 66mgCaCl2, 120mgMgSO4, 6.2mgH3BO3, 25mgMnSO4, 10mgZnSO4, 0.25 mgNa2MoO4, 0.025mgCuSO4, 0.025mgCoCl2, 0.83mgKI, 0.24mgbiotin, 100mgL-cysteine, 2.4mgD-calcium pantothenate, 2mgglycine, 5mgnic acid, 2mgthiamine, 0.5mgpyridoxine, 37.3mgNa2-EDTA, 27.8mg FeSO4, 100mg inositol, 0.4-0.6mg indolebutyric acid, 0.1-0.2mg naphthaleneacetic acid, 0.5-0.8mg paclobutrazol, 30g sucrose, 5g agar powder, 5g activated carbon, pH 5.8-6; The matrix in step (7) is loess soil: 95% loess soil + 5% superphosphate are mixed evenly or coconut husk: carbonized rice husk: peat soil: loess soil are mixed 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 let it ferment for one month to make a light substrate; The raw material formula of the foliar nutrient solution is: 1L of foliar nutrient solution contains 180mgKNO3, 36mgNH4NO3, 27mgKH2PO4, 20mgCaCl2, and 36mgMgSO4.

2. The method for rapid propagation of Eucalyptus dunnii according to claim 1, wherein: The excellent single plant of Eucalyptus dunnii in step (1) is the Eucalyptus dunnii GED9502 variety.

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