CN105123519B - Method for Tissue Culture of Silver Birch - Google Patents

Abstract

The invention discloses a kind of by the method for silvery birch tissue cultures.The method is that the explant after sterilization is seeded in clump bud inducement cultivation base to induce clump bud；Clump bud is inoculated into subculture multiplication medium again to continue to cultivate；Then it is seeded in root media and is taken root, hardening and field planting management is carried out after taking root.The inventive method acquisition ground is high by the yield of silvery birch clone nursery stock, and the time is fast, and the nursery stock for being obtained is healthy and strong, and these nursery stocks are lacked with solving the problems, such as planting industry by silvery birch.

Description

Method for Tissue Culture of Silver Birch

technical field

The invention relates to a method for plant tissue culture, in particular to a method for tissue culture of silver birch.

Background technique

Silver birch (Grevillea) is a plant of the Proteaceae family (Proteaceae) that can bloom large inflorescences, and is native to Australia, New Guinea, New Caledonia, and Sulawesi in central Indonesia. Silver birch is the third largest tree species among Australian native plants next to acacia and eucalyptus. It has a wide distribution range, from tropical rainforest with annual rainfall of 3000mm to desert area with annual rainfall of 200mm, from alpine to coastal and arid inland. . There are about 360 kinds of genotypes, and the varieties vary from ground cover silver birch less than 0.5m to shrubs of 1-5m and trees up to 35m. Silver birch is easy to achieve interspecific hybridization and natural variation, so hundreds of cultivars and hybrids have been produced naturally or artificially.

The ground cover silver birch is a well-known drought-tolerant tree species, mostly distributed in the central and western deserts of Australia, where there is drought and little rain all year round, so many species of ground cover silver birch have strong drought resistance. There are many fine and dense fibrous roots in the root structure of ground cover silver birch, which is also the evolutionary result of long-term survival in barren soil. Such a structure is conducive to absorbing nutrients in poor soil, so ground cover silver birch is also a barren-tolerant tree species. The ground cover silver birch only needs to apply low-efficiency slow-release fertilizers, or no fertilizers. Therefore, the ground cover silver birch has the characteristics of low maintenance cost.

The ground cover silver birch is a raceme, and its flower shape is peculiar, most of which are brush-like or spider-like. Flowering throughout the year, the ground cover silver birch leaves are rich in leaf shape, ranging from needle-shaped to shallow and deeply lobed. The leaves are mostly dark green and gray green, and have high ornamental value in horticulture. The ground cover silver birch is suitable for intensive pruning, and can shape various horticultural tree shapes. Ground cover silver birch grafted to the rootstocks of shrub silver birch and tree silver birch has a high survival rate, and new weeping silver birch can be cultivated. Therefore, ground cover silver birch has been widely used in garden greening and landscape engineering in Australia. The ground cover silver birch is rich in nectar, which can provide food for humans, birds, and bees. Therefore, the ground cover silver birch is a famous tree species for attracting and raising birds.

At present, the technology of large-scale production of ground cover silver birch seedlings in my country is still immature, and it is difficult to achieve intensive operation and high production efficiency, and it is also unable to meet the large demand of the market for ground cover silver birch seedlings. The establishment of the ground cover silver birch tissue culture method with simple method, high rooting rate and high survival rate will play an important role in the popularization and application of ground cover silver birch.

Contents of the invention

In order to solve the above-mentioned existing problems, the present invention carried out tissue culture research of ground cover silver birch under the support of the introduction project "introduction of new varieties of ground cover silver birch and breeding technology", and established a mature ground cover silver birch tissue culture technology platform and The technological process of industrialized seedling cultivation has successfully propagated 30,000 ground cover silver birch tissue culture seedlings.

The object of the present invention is to provide a method for tissue culture of silver birch.

The technical scheme that the present invention takes is:

A method for ground cover silver birch tissue culture, comprising the following steps:

1) collection of explants;

2) disinfection of explants;

3) Cluster bud induction culture: Inoculate the explants sterilized in the previous step into the cluster bud induction medium, and induce culture for 3 to 5 months. During this period, they need to be transferred to the new cluster bud induction medium 3 to 5 times. The implant can be induced to produce multiple cluster buds;

4) Subculture proliferation culture: Inoculate the ground cover silver birch tissue culture seedlings that have induced cluster buds in the previous step into the subculture medium, and transfer the ground cover silver birch proliferation seedlings to new subcultures every 28 to 32 days. Continue to culture in the generation proliferation medium;

5) rooting induction culture: cut off the single buds with a height of more than 2 cm in the proliferation culture and insert them into the rooting medium, and cultivate them for 10 to 15 days;

6) seedling hardening: when at least 60% of the proliferating seedlings begin to take root, they enter the hardening stage, and the obtained ground cover silver birch rooted tissue culture seedlings are hardened for 15 to 20 hours under the conditions of light intensity 3000～6000Lx and temperature 26～30°C. sky;

7) Transplantation and management: Transplant the hardened seedlings into the transplanting medium for field cultivation and management.

Further, the explant collection time in step 1) is from June to September, and the 25-30 cm part of the top tip of the newly sprouted shoot is collected, and the leaves are cut off.

Further, the specific method of explant disinfection treatment in step 2) is: clean the collected branches, cut them into 3-4cm long stem segments with 1-2 leaf axils, and use 0.08-0.12 %v/v HgCl ₂ for 3 to 7 minutes, shake constantly to ensure that the disinfectant is in full contact with the branches, then clean with sterile water, and finally cut off 0.4 to 0.6 cm at both ends of the stem, leaving the middle containing Leaf axil stem segments were used as sterile explants.

Further, the formula of bud induction medium in step 3) is: 412-413mg/L NH ₄ NO ₃ , 1898-1902mg/L KNO ₃ , 84.5-85.5mg/L KH ₂ PO ₄ , 369-371mg/L MgSO ₄ 7HO ₂ , 131～133mg/LCaCl ₂ 2HO ₂ , 22～22.5mg/L MnSO ₄ 4HO ₂ , 8.4～8.8mg/L ZnSO ₄ 7HO ₂ , 6～6.4mg/LH ₃ BO ₃ , 0.22～0.27mg/L Na ₂ MoO ₄ ·2HO ₂ , 27.5～28mg/L FeSO ₄ ·7HO ₂ , 37～37.5mg/L Na ₂ ·EDTA·2HO ₂ , 0.8～0.85mg/L KI, 0.023～0.027 mg/L CuSO ₄ ·5HO ₂ , 0.023～0.027mg/L CoCl ₂ ·6HO ₂ , 1.8～2.2mg/L glycine, 0.08～0.12mg/L thiamine hydrochloride, 0.48～0.52mg/L pyridoxine hydrochloride , 0.48～0.52mg/L Niacin, 99～101mg/L Inositol, 0.8～1.2g/L PVP, 0.48～0.52mg/L 6-BA, 0.048～0.052mg/L IBA, 27～32g/L Sucrose , 7~8g/L carrageenan, pH5.75~5.85.

Further, the formulation of the subculture proliferation medium in step 4) is: 412-413mg/L NH ₄ NO ₃ , 1898-1902mg/L KNO ₃ , 84.5-85.5mg/L KH ₂ PO ₄ , 369-371mg/L MgSO ₄ 7HO ₂ , 131～133mg/LCaCl ₂ 2HO ₂ , 22～22.5mg/L MnSO ₄ 4HO ₂ , 8.4～8.8mg/L ZnSO ₄ 7HO ₂ , 6～6.4mg/LH ₃ BO ₃ , 0.22～0.27mg/L Na ₂ MoO ₄ ·2HO ₂ , 27.5～28mg/L FeSO ₄ ·7HO ₂ , 37～37.5mg/L Na ₂ ·EDTA·2HO ₂ , 0.8～0.85mg/L KI, 0.023～0.027 mg/L CuSO ₄ ·5HO ₂ , 0.023～0.027mg/L CoCl ₂ ·6HO ₂ , 1.8～2.2mg/L glycine, 0.08～0.12mg/L thiamine hydrochloride, 0.48～0.52mg/L pyridoxine hydrochloride , 0.48～0.52mg/L Niacin, 99～101mg/L Inositol, 0.8～1.2g/L PVP, 0.2～0.3mg/L 6-BA, 0.048～0.052mg/L IBA, 27～32g/L Sucrose , 7~8g/L carrageenan, pH5.75~5.85.

Further, the formulation of the rooting induction medium in step 5) is: 412-413mg/L NH ₄ NO ₃ , 473-477mg/L KNO ₃ , 42-43mg/L KH ₂ PO ₄ , 92-93mg/L MgSO ₄ · 7HO ₂ , 108～112mg/L CaCl ₂ 2HO ₂ , 11～11.5mg/L MnSO ₄ 4HO ₂ , 4～4.5mg/L ZnSO ₄ 7HO ₂ , 2.8～3.4mg/LH ₃ BO ₃ , 0.12～ 0.13mg/LNa ₂ MoO ₄ ·2HO ₂ , 13.5～14.5mg/L FeSO ₄ ·7HO ₂ , 18.3～18.9mg/L Na ₂ ·EDTA·2HO ₂ , 0.41～0.42mg/L KI, 0.012～0.013mg/ L CuSO ₄ 5HO ₂ , 0.012～0.013mg/L CoCl ₂ 6HO ₂ , 1.8～2.2mg/L glycine, 0.08～0.12mg/L thiamine hydrochloride, 0.45～0.55mg/L pyridoxine hydrochloride, 0.45 ~0.55mg/L niacin, 99~101mg/L inositol, 0.65~0.75mg/L NAA, 18~22g/L sucrose, 7~8g/L carrageenan, pH5.75~5.85.

Further, the culture conditions for cluster bud induction and subculture culture in step 3) and step 4) are: temperature 23-27°C, light 9-11h·d ^-1 , light intensity 2200-2700Lx; in step 5) The culture conditions for rooting induction culture are: temperature 24-26°C, light 8-10h·d ^-1 , and light intensity 2200-2700Lx.

Further, the transplanting medium in step 7) contains 45-55% yellow mud and 45-55% peat soil matrix.

Further, the specific method of field cultivation management described in step 7) is as follows: after transplanting, first cultivate 7～95% of shading rate, relative humidity of 85%～90%, and temperature of 25～33°C. 8 days; then the seedlings are placed in a shading rate of 75% to 80%, the humidity is 75% to 80%, and the temperature is 25 to 33°C and cultivated for 8 to 20 days; afterward, carry out shading and watering management according to conventional seedling cultivation .

Further, the time for transplantation in step 7) is March to June or September to November.

The beneficial effects of the present invention are:

The present invention establishes a tissue culture method capable of obtaining a large amount of silver birch clonal seedlings, wherein, the present invention improves the cluster bud induction medium, proliferation medium and rooting medium of silver birch through research, especially embodies The selection of the types and concentrations of the hormones in these three mediums, and the improvement of the concentration of trace and macroelements ensure the ideal results of high yield, fast growth and robust seedlings of ground cover silver birch clone seedlings.

The yield of silver birch clonal seedlings obtained by the method of the invention is high, the time is fast, and the obtained seedlings are robust, and these seedlings solve the problem of lack of ground cover silver birch seedlings in the planting industry.

The method of the invention is to obtain a large amount of high-quality and healthy ground cover silver birch seedlings through the asexual reproduction method of tissue culture, and the asexual reproduction method can ensure that the seedlings can completely inherit and maintain all the good qualities of the original mother plant.

Description of drawings

Fig. 1 is the influence that the rooting culture medium that contains the macroelement of different concentrations has rooting on ground cover silver birch tissue culture seedling;

Fig. 2 is the influence that the rooting culture medium that contains trace element of different concentration is rooted to ground cover silver birch tissue culture seedling;

Fig. 3 is the effect of different NAA concentrations (mg/L) on the rooting of ground cover silver birch tissue culture seedlings.

detailed description

A method for ground cover silver birch tissue culture, comprising the following steps:

1) collection of explants;

2) disinfection of explants;

3) Cluster bud induction culture: Inoculate the explants sterilized in the previous step into the cluster bud induction medium, and induce culture for 3 to 5 months. During this period, they need to be transferred to the new cluster bud induction medium 3 to 5 times. The implant can be induced to produce multiple cluster buds;

4) Subculture proliferation culture: Inoculate the ground cover silver birch tissue culture seedlings that have induced cluster buds in the previous step into the subculture medium, and transfer the ground cover silver birch proliferation seedlings to new subcultures every 28 to 32 days. Continue to culture in the generation proliferation medium;

5) rooting induction culture: cut off the single buds with a height of more than 2 cm in the proliferation culture and insert them into the rooting medium, and cultivate them for 10 to 15 days;

6) seedling hardening: when at least 60% of the proliferating seedlings begin to take root, they enter the hardening stage, and the obtained ground cover silver birch rooted tissue culture seedlings are hardened for 15 to 20 hours under the conditions of light intensity 3000～6000Lx and temperature 26～30°C. sky;

7) Transplantation and management: Transplant the hardened seedlings into the transplanting medium for field cultivation and management.

Preferably, the explant collection time in step 1) is from June to September, and the 25-30 cm part of the top tip of the newly sprouted shoot is collected, and the leaves are cut off.

More preferably, the explant collection time in step 1) is 9:00 to 10:00 in the morning after the weather continues to be sunny for 4 to 6 days in June to September.

Preferably, the specific method of explant disinfection treatment in step 2) is: clean the collected branches, cut them into 3-4cm long stem segments with 1-2 leaf axils, and use 0.08-0.12 %v/v HgCl ₂ for 3 to 7 minutes, shake constantly to ensure that the disinfectant is in full contact with the branches, then clean with sterile water, and finally cut off 0.4 to 0.6 cm at both ends of the stem, leaving the middle containing Leaf axil stem segments were used as sterile explants.

Preferably, the formula of bud induction medium in step 3) is: 412-413 mg/L NH ₄ NO ₃ , 1898-1902 mg/L KNO ₃ , 84.5-85.5 mg/L KH ₂ PO ₄ , 369-371 mg/L MgSO ₄ 7HO ₂ , 131～133mg/L CaCl ₂ 2HO ₂ , 22～22.5mg/L MnSO ₄ 4HO ₂ , 8.4～8.8mg/L ZnSO ₄ 7HO ₂ , 6～6.4mg/LH ₃ BO ₃ , 0.22～0.27mg/L Na ₂ MoO ₄ ·2HO ₂ , 27.5～28mg/L FeSO ₄ ·7HO ₂ , 37～37.5mg/L Na ₂ ·EDTA·2HO ₂ , 0.8～0.85mg/L KI, 0.023～0.027 mg/L CuSO ₄ ·5HO ₂ , 0.023～0.027mg/L CoCl ₂ ·6HO ₂ , 1.8～2.2mg/L glycine, 0.08～0.12mg/L thiamine hydrochloride, 0.48～0.52mg/L pyridoxine hydrochloride , 0.48～0.52mg/L Niacin, 99～101mg/L Inositol, 0.8～1.2g/L PVP, 0.48～0.52mg/L 6-BA, 0.048～0.052mg/L IBA, 27～32g/L Sucrose , 7~8g/L carrageenan, pH5.75~5.85.

Preferably, the culture conditions for bud induction in step 3) are: temperature 23-27°C, light 9-11h·d ^-1 , and light intensity 2200-2700Lx.

Preferably, the formulation of the subculture proliferation medium in step 4) is: 412-413 mg/L NH ₄ NO ₃ , 1898-1902 mg/L KNO ₃ , 84.5-85.5 mg/L KH ₂ PO ₄ , 369-371 mg/L MgSO ₄ 7HO ₂ , 131～133mg/L CaCl ₂ 2HO ₂ , 22～22.5mg/L MnSO ₄ 4HO ₂ , 8.4～8.8mg/L ZnSO ₄ 7HO ₂ , 6～6.4mg/LH ₃ BO ₃ , 0.22～0.27mg/L Na ₂ MoO ₄ ·2HO ₂ , 27.5～28mg/L FeSO ₄ ·7HO ₂ , 37～37.5mg/L Na ₂ ·EDTA·2HO ₂ , 0.8～0.85mg/L KI, 0.023～0.027 mg/L CuSO ₄ 5HO ₂ , 0.023～0.027mg/L CoCl ₂ 6HO ₂ , 1.8～2.2mg/L glycine, 0.08～0.12mg/Lmg/L thiamine hydrochloride, 0.48～0.52mg/L pyrimidine hydrochloride Pyridoxine, 0.48～0.52mg/L Niacin, 99～101mg/L Inositol, 0.8～1.2g/L PVP, 0.2～0.3mg/L 6-BA, 0.048～0.052mg/L IBA, 27～32g/L L sucrose, 7~8g/L carrageenan, pH5.75~5.85.

Preferably, the culture conditions for the subculture culture in step 4) are: temperature 23-27°C, light 9-11h·d ^-1 , and light intensity 2200-2700Lx.

Preferably, the formulation of the rooting induction medium in step 5) is: 412-413 mg/L NH ₄ NO ₃ , 473-477 mg/L KNO ₃ , 42-43 mg/L KH ₂ PO ₄ , 92-93 mg/L MgSO ₄ · 7HO ₂ , 108～112mg/L CaCl ₂ 2HO ₂ , 11～11.5mg/L MnSO ₄ 4HO ₂ , 4～4.5mg/L ZnSO ₄ 7HO ₂ , 2.8～3.4mg/LH ₃ BO ₃ , 0.12～ 0.13mg/LNa ₂ MoO ₄ ·2HO ₂ , 13.5～14.5mg/L FeSO ₄ ·7HO ₂ , 18.3～18.9mg/L Na ₂ ·EDTA·2HO ₂ , 0.41～0.42mg/L KI, 0.012～0.013mg/ L CuSO ₄ 5HO ₂ , 0.012～0.013mg/L CoCl ₂ 6HO ₂ , 1.8～2.2mg/L glycine, 0.08～0.12mg/L thiamine hydrochloride, 0.45～0.55mg/L pyridoxine hydrochloride, 0.45 ~0.55mg/L niacin, 99~101mg/L inositol, 0.65~0.75mg/L NAA, 18~22g/L sucrose, 7~8g/L carrageenan, pH5.75~5.85.

Preferably, the culture conditions for rooting induction culture in step 5) are: temperature 24-26°C, light 8-10h·d ^-1 , light intensity 2200-2700Lx.

Preferably, the transplant medium in step 7) contains 45-55% yellow mud and 45-55% peat soil matrix.

Preferably, the specific method of field cultivation and management described in step 7) is: after transplanting, firstly cultivate 7～95% sun-shading rate under the conditions of 85%～90% relative humidity and 25～33°C after transplanting. 8 days; then the seedlings are placed in a shading rate of 75% to 80%, the humidity is 75% to 80%, and the temperature is 25 to 33°C and cultivated for 8 to 20 days; afterward, carry out shading and watering management according to conventional seedling cultivation .

Preferably, the transplantation time in step 7) is 3-6 months or 9-11 months.

More preferably, the transplantation time in step 7) is 3-4 months.

Preferably, before transplantation, the transplant medium is sterilized with 1/1000 potassium permanganate solution, and sprayed with 1/800 chlorothalonil and carbendazim every week after transplantation to prevent the growth of miscellaneous bacteria, 15 days after transplantation Start fertilizing, and use 1/1000 compound fertilizer solution to spray once a week.

The present invention will be further described below in conjunction with specific examples, but is not limited thereto.

Embodiment 1 ground cover silver birch tissue culture method

The specific steps of ground cover silver birch tissue culture are as follows:

(1) Collection of explants

From June to September, when the rainfall is less, and the weather continues to be sunny for 5 days, from 9:00 to 10:00 in the morning, collect 25-30 cm of the top tip of the newly sprouted shoots, cut off the leaves and put them in a clean fresh-keeping bag, and bring them back to the experiment in time. The room is disinfected.

(2) Disinfection of explants

Soak the branches with washing powder solution for 30 minutes, and then rinse them under running water. Cut the branches into 3-4cm long stems with 1-2 leaf axils, soak and disinfect them with 0.1% HgCl ₂ for 3-7 minutes on the ultra-clean workbench, shake constantly to ensure that the disinfectant is in full contact with the branches, and then use Wash 4 times with sterile water, each time for 5 minutes, and finally cut off 0.5 cm at both ends of the stem segment, leaving the stem segment containing leaf axils in the middle as a sterile explant, and inoculating it on the cluster bud induction medium.

(3) Cluster bud induction culture

After the explants are collected and sterilized, the cluster buds are first induced to form cluster buds on the cluster bud induction medium, and the induction time is 3 to 5 months. The induction rate is about 15%. Experimental studies have found that a higher concentration of 6-BA is needed during the induction of ground cover silver birch buds. The culture conditions for cluster bud induction are: temperature 23-27°C, light 9-11h·d ^-1 , and light intensity 2200-2700Lx.

The formula of the above budding induction medium is: 412.5mg/LNH ₄ NO ₃ , 1900mg/L KNO ₃ , 85mg/L KH ₂ PO ₄ , 370mg/L MgSO ₄ 7HO ₂ , 132mg/L CaCl ₂ 2HO ₂ , 22.3mg/L MnSO ₄ ·4HO ₂ , 8.6mg/L ZnSO ₄ ·7HO ₂ , 6.2mg/LH ₃ BO ₃ , 0.25mg/LNa ₂ MoO ₄ ·2HO ₂ , 27.8mg/L FeSO ₄ ·7HO ₂ , 37.3 mg/L Na ₂ EDTA 2HO ₂ , 0.83mg/L KI, 0.025mg/L CuSO ₄ 5HO ₂ , 0.025mg/L CoCl ₂ 6HO ₂ , 2mg/L glycine, 0.1mg/L thiamine hydrochloride , 0.5mg/L pyridoxine hydrochloride, 0.5mg/L niacin, 100mg/L inositol, 1g/L PVP, 0.5mg/L6-BA, 0.05mg/L IBA, 30g/L sucrose and 7-8g/L L carrageenan, pH 5.8 (see Table 2).

(4) Subculture proliferation culture

The ground cover silver birch tissue culture seedlings induced to produce cluster buds in the previous step were transferred to the subculture proliferation medium for proliferation and expansion. The culture conditions for subculture are: temperature 25±2°C, light 10h/d, light intensity 2500Lx, transfer once every 30 days, and culture for 3-5 months. In order to configure the subculture proliferation medium suitable for the growth of silver birch tissue culture seedlings, this embodiment conducted in-depth research on relevant nutrient elements.

1. Macronutrients

Through the comparative test of macroelements, NH ₄ NO ₃ , KH ₂ PO ₄ , and CaCl ₂ in macroelements are the key factors affecting the state of proliferating seedlings. The medium contains 0.25MS NH ₄ NO ₃ +0.5MS KH ₂ PO ₄ + 0.3MS CaCl ₂ ·2HO ₂ +1MS KNO ₃ +1MSMgSO ₄ ·7HO ₂ can keep the tissue-cultured proliferation seedlings in good condition, otherwise the proliferation seedlings will have top wilting.

2. Hormone

Through the study, it was found that the concentration of 6-BA was the decisive factor in determining the multiplier, height and degree of vitrification of tissue cultured seedlings.

In order to study the effect of hormone concentration on the proliferation of silver birch seedlings, four concentration gradients of 0.2mg/L, 0.3mg/L, 0.4mg/L, and 0.5mg/L were designed for the hormone 6-BA concentration in the proliferation medium , 10 bottles of each concentration were treated each time, and 4 proliferating buds were inoculated in each bottle. After 30 days of cultivation, the proliferation results were counted.

Table 1 Effect of Hormone Concentration on Ground Cover Silver Birch Proliferation Seedlings

Studies have shown that the concentration of 6-BA has a significant effect on the multiplication factor, the height of the multiplication seedlings, and the degree of vitrification of the multiplication seedlings (see Table 1). Although when the concentration of 6-BA is 0.5 mg/L, the multiplication factor can reach 2 times, but the height of the multiplication seedlings is too low, which is not conducive to the rooting induction in the later stage, and the ratio of vitrification of the multiplication seedlings is also high, which will lead to the growth of healthy seedlings. proportion decreased. Considering various factors comprehensively, the concentration of 6-BA is 0.2～0.3mg/L, which is most suitable for the proliferation and cultivation of silver birch tissue culture seedlings.

In summary, the concentration of 6-BA should be appropriately reduced during subculture culture (Table 1), and the formula to obtain the best subculture medium is: 0.2-0.3mg/L 6-BA and 0.05mg/L Modified MS medium of LIBA;

The specific formula of subculture proliferation medium is: 412.5mg/LNH ₄ NO ₃ , 1900mg/L KNO ₃ , 85mg/L KH ₂ PO ₄ , 370mg/L MgSO ₄ 7HO ₂ , 132mg/L CaCl ₂ 2HO ₂ , 22.3 mg/L MnSO ₄ ·4HO ₂ , 8.6mg/L ZnSO ₄ ·7HO ₂ , 6.2mg/LH ₃ BO ₃ , 0.25mg/LNa ₂ MoO ₄ ·2HO ₂ , 27.8mg/L FeSO ₄ ·7HO ₂ , 37.3mg /L Na ₂ EDTA 2HO ₂ , 0.83mg/L KI, 0.025mg/L CuSO ₄ 5HO ₂ , 0.025mg/L CoCl ₂ 6HO ₂ , 2mg/L glycine, 0.1mg/L thiamine hydrochloride, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L niacin, 100mg/L inositol, 1g/L PVP, 0.2～0.3mg/L 6-BA, 0.05mg/L IBA, 30g/L sucrose, 7～ 8g/L carrageenan, pH5.8 (see Table 2).

(5) Rooting induction

When the proliferating seedlings are transferred each time, the single buds of the ground cover silver birch tissue culture with a height of more than 2 cm in the proliferating culture are cut off and inserted into the rooting induction medium, and cultivated for 10 to 15 days at a culture temperature of 24 to 26 ° C under light 8～10h·d ^-1 , light intensity 2500lx. In order to configure a rooting induction medium suitable for the rooting of the silver birch tissue culture seedlings, this example carried out in-depth research on the relevant nutrient elements.

By studying the content of macroelements, microelements and hormones in the rooting medium, it was found that when the content of macroelements was 0.25MS, the content of microelements was 0.5MS, and the concentration of NAA was 0.7mg/L, it was most suitable for the induction of silver birch. The tissue culture seedlings take root (see Fig. 1, Fig. 2, Fig. 3).

According to the experimental results in Figures 1 to 3, the rooting medium formula most suitable for inducing the rooting of silver birch tissue culture seedlings is: 412.5mg/L NH ₄ NO ₃ , 475mg/L KNO ₃ , 42.5mg/L KH ₂ PO _{4 . _ _ _ _ _ _ _ _ _} LNa ₂ MoO ₄ ·2HO ₂ , 13.9mg/L FeSO ₄ ·7HO ₂ , 18.65mg/L Na ₂ ·EDTA·2HO ₂ , 0.415mg/L KI, 0.0125mg/L CuSO ₄ ·5HO ₂ , 0.0125mg/L CoCl ₂ ·6HO ₂ , 2mg/L glycine, 0.1mg/L thiamine hydrochloride, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L niacin, 100mg/L inositol, 0.7mg/LNAA, 20g/L Sucrose, 7-8g/L carrageenan, pH 5.8 (see Table 2).

The medium formula of table 2 ground cover silver birch tissue culture rapid propagation

Note: The amount of carrageenan used can be adjusted according to the products of different manufacturers.

(6) Seedling hardening

When at least 60% of the proliferating seedlings begin to take root, they can turn to the seedling hardening stage. The suitable hardening conditions for silver birch rooted tissue culture seedlings are as follows: the light intensity is 6000-8000lx, the temperature is 26-30°C, and the suitable hardening time is 15-20 days. After hardening, the height of rooted seedlings can reach 4-5cm, the root system is developed, the seedlings are strong, and the leaves are emerald green. They can be directly transplanted into the nutrition bags in the nursery.

(7) Transplantation and management

Carefully take out the rooted seedlings that have completed hardening from the culture bottle, rinse the medium at the base of the seedlings with clean water, and transplant them into a nutrition bag containing 50% yellow mud and 50% peat soil matrix by mass percentage on the same day. The specification is 6×11cm.

In the initial stage of transplanting the ground cover silver birch tissue culture seedlings, the shading rate is 90-95%, the relative humidity is 85%-90%, and the temperature is 25-33°C and cultivated for 7-8 days; then the seedlings are placed in the shading rate 75% to 80%, humidity 75% to 80%, and temperature 25 to 33°C for 8 to 20 days; afterward, conventional seedling cultivation is carried out with sunshade and watering management, and the survival rate of seedling transplantation is greater than 85%.

The specific operation is as follows:

1) The 1st to 7th day of seedling transplantation: the seedbed is covered with a film, and a layer of shading net with a shading rate of 95% is added on the film; spray 5 to 6 times a day on sunny days, 4 to 5 times a day on cloudy days, and 4 to 5 times a day on rainy days. Spray 2 to 3 times a day; when the temperature is high, pour cooling water on the shading net to control the temperature of the seedbed to 25-33°C.

2) On the 8th to 30th day of seedling transplantation: a ventilating opening is arranged every 1 meter on the film at both ends and sides of the seedbed, and a sunshade net with a sunshade rate of 85% is covered during the day. On sunny days, spray water 4 to 5 times a day; on cloudy days, spray water 3 to 4 times a day; on rainy days, only cover the film throughout the day, and spray water 0 to 1 time a day.

3) Seedlings transplanted for 31-60 days: there is no need to cover the film again, but 50% of the sunshade net should be covered in the daytime on sunny days, and watered 2 to 3 times a day; on cloudy days, no sunshade net should be covered, and water should be poured 1 to 2 times a day. Second; in light rainy days, there is no need to cover the sunshade net, nor do you need to pour water; in heavy rain, you need to cover the film, but you need to set vents at both ends and sides of the seedbed, and you don't need to cover the sunshade net, and you don't need to pour water.

Before the seedlings are transplanted, the transplanting medium is disinfected with 1/1000 potassium permanganate solution, and sprayed with 1/800 chlorothalonil and carbendazim every week after transplantation to prevent the growth of miscellaneous bacteria. Fertilization was started 15 days after transplantation, and a 1/1000 compound fertilizer solution was used to spray once a week.

In Guangdong, the tissue-cultured seedlings of silver birch can be transplanted from March to June or from September to November. Among them, March to April is the best transplanting season for the tissue-cultured seedlings of silver birch, and the temperature and humidity are suitable for the growth of seedlings. Further cultivate to meet the height and requirements of afforestation. From July to August, the temperature is too high and the survival rate of transplantation is low. Through intensive fertilization, the seedlings can reach the height of afforestation. The survival rate of transplanting from October to November is also relatively high, but the growing season of seedlings has passed, and the seedlings cannot reach the height of afforestation in the spring of the following year.

Because most of the ground cover silver birches native to Australia with application value are artificially cultivated or natural hybrids, most of them are self-sterile, therefore, it is difficult to obtain the seeds of ground cover silver birch; on the other hand, it is also difficult to maintain hybridization in seed breeding For the excellent characteristics of new varieties, after introducing a small amount of mother plants, these new varieties can only be popularized and applied by breeding seedlings through the asexual reproduction technology of tissue culture. The yield of silver birch clone seedlings obtained by the method of the invention is high, the time is fast, and the obtained seedlings are robust, and these seedlings solve the problem of lack of silver birch seedlings in planting industry.

Embodiment 2 A kind of method of silver birch tissue culture

1) Collection of explants: From 9:00 to 10:00 in the morning after the weather continues to be sunny for 6 days from June to September every year, collect the 25-30cm part of the top tip of the new shoot, and cut off the leaves;

2) Disinfection of explants: clean the collected branches, cut them into 3-4cm long stem segments with _1-2 leaf axils, and soak them in 0.08-0.12% v/v HgCl in a sterile environment Sterilize for 3 to 7 minutes, shake constantly to ensure that the disinfectant is in full contact with the branches, then clean it with sterile water, and finally cut off 0.4 to 0.6 cm at both ends of the stem, leaving the stem with the leaf axil in the middle as a sterile explant body.

3) Cluster bud induction culture: inoculate the explants sterilized in the previous step into the cluster bud induction medium, and induce culture for 4 months. With one culture medium, the explant can be induced to produce multiple cluster buds; the culture conditions are: temperature 25°C, light 10h·d ^-1 , light intensity 2500Lx;

The formula of the above-mentioned cluster bud induction medium is: 412～413mg/L NH ₄ NO ₃ , 1898～1902mg/L KNO ₃ , 84.5～85.5mg/L KH ₂ PO ₄ , 369～371mg/L MgSO ₄ 7HO ₂ , 131～133mg/L CaCl ₂ 2HO ₂ , 22～22.5mg/L MnSO ₄ 4HO ₂ , 8.4～8.8mg/L ZnSO ₄ 7HO ₂ , 6～6.4mg/LH ₃ BO ₃ , 0.22～0.27mg/ LNa ₂ MoO ₄ 2HO ₂ , 27.5～28mg/L FeSO ₄ 7HO ₂ , 37～37.5mg/L Na ₂ EDTA 2HO ₂ , 0.8～0.85mg/L KI, 0.023～0.027mg/L CuSO ₄ 5HO ₂ , 0.023～0.027mg/L CoCl ₂ 6HO ₂ , 1.8～2.2mg/L glycine, 0.08～0.12mg/L thiamine hydrochloride, 0.48～0.52mg/L pyridoxine hydrochloride, 0.48～0.52mg/L L Niacin, 99～101mg/L Inositol, 0.8～1.2g/L PVP, 0.48～0.52mg/L 6-BA, 0.048～0.052mg/LIBA, 27～32g/L Sucrose, 7～8g/L Kara Glue, pH5.75～5.85.

4) Subculture proliferation culture: Inoculate the ground cover silver birch tissue culture seedlings that have induced cluster buds in the previous step into the subculture medium, and transfer the ground cover silver birch proliferation seedlings to new subcultures every 28 to 32 days. Continue to culture in the generation proliferation medium; the culture conditions are: temperature 25°C, light 10h·d ^-1 , light intensity 2500Lx;

The formula of the above subculture proliferation medium is: 412～413mg/L NH ₄ NO ₃ , 1898～1902mg/L KNO ₃ , 84.5～85.5mg/L KH ₂ PO ₄ , 369～371mg/L MgSO ₄ 7HO ₂ , 131～133mg/L CaCl ₂ 2HO ₂ , 22～22.5mg/L MnSO ₄ 4HO ₂ , 8.4～8.8mg/L ZnSO ₄ 7HO ₂ , 6～6.4mg/LH ₃ BO ₃ , 0.22～0.27mg/ LNa ₂ MoO ₄ 2HO ₂ , 27.5～28mg/L FeSO ₄ 7HO ₂ , 37～37.5mg/L Na ₂ EDTA 2HO ₂ , 0.8～0.85mg/L KI, 0.023～0.027mg/L CuSO ₄ 5HO ₂ , 0.023～0.027mg/L CoCl ₂ 6HO ₂ , 1.8～2.2mg/L glycine, 0.08～0.12mg/L thiamine hydrochloride, 0.48～0.52mg/L pyridoxine hydrochloride, 0.48～0.52mg/L L Niacin, 99～101mg/L Inositol, 0.8～1.2g/L PVP, 0.2～0.3mg/L 6-BA, 0.048～0.052mg/L IBA, 27～32g/L Sucrose, 7～8g/L Carrageenan, pH5.75～5.85.

5) Rooting induction culture: Cut out the single buds with a height of more than 2cm in the proliferation culture and insert them into the rooting medium, and cultivate them for 10-15 days; the culture conditions are: temperature 245°C, light 9h·d ^-1 , light intensity 2700Lx .

The formulation of the above rooting induction medium is: 412～413mg/LNH ₄ NO ₃ , 473～477mg/L KNO ₃ , 42～43mg/L KH ₂ PO ₄ , 92～93mg/L MgSO ₄ 7HO ₂ , 108～112mg /L CaCl ₂ 2HO ₂ , 11～11.5mg/LMnSO ₄ 4HO ₂ , 4～4.5mg/L ZnSO ₄ 7HO ₂ , 2.8～3.4mg/LH ₃ BO ₃ , 0.12～0.13mg/L Na ₂ MoO ₄ 2HO ₂ , 13.5～14.5mg/L FeSO ₄ 7HO ₂ , 18.3～18.9mg/L Na ₂ EDTA 2HO ₂ , 0.41～0.42mg/LKI, 0.012～0.013mg/L CuSO ₄ 5HO ₂ , 0.012～0.013mg/L CoCl ₂ ·6HO ₂ , 1.8～2.2mg/L Glycine, 0.08～0.12mg/L Thiamine Hydrochloride, 0.45～0.55mg/L Pyridoxine Hydrochloride, 0.45～0.55mg/L Niacin , 99～101mg/L inositol, 0.65～0.75mg/L NAA, 18～22g/L sucrose, 7～8g/L carrageenan, pH5.75～5.85.

6) seedling hardening: when at least 60% of the proliferating seedlings begin to take root, they enter the hardening stage, and the obtained ground cover silver birch rooted tissue culture seedlings are hardened for 15 days under the conditions of light intensity 3000～6000Lx and temperature 30°C;

7) Transplantation and management: Transplant the hardened seedlings into the transplanting medium containing 45-55% yellow mud and 45-55% peat soil matrix, and carry out field cultivation and management. The time of transplantation is 3-4 months. Before transplantation, the transplant medium was sterilized with 1/1000 potassium permanganate solution. After transplantation, it was sprayed alternately with 1/800 chlorothalonil and carbendazim every week to prevent the growth of miscellaneous bacteria. Fertilization began 15 days after transplantation. Spray once a week with 1/1000 compound fertilizer solution.

The specific method of field cultivation management is as follows: after transplanting, cultivate for 7 to 8 days under the conditions of shading rate of 90-95%, relative humidity of 85%-90%, and temperature of 25-33°C; The rate is 75% to 80%, the humidity is 75% to 80%, and the temperature is 25 to 33°C and cultivated for 8 to 20 days; afterward, sunshade and watering management will be carried out according to conventional seedling cultivation.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (7)

1. a method for ground cover silver birch tissue culture, is characterized in that: comprise the following steps: 1) collection of explants; the explants are stem segments containing axillary buds; 2) Disinfection of explants; 3) Cluster bud induction culture: Inoculate the explants sterilized in the previous step into the cluster bud induction medium, and induce culture for 3 to 5 months. During this period, they need to be transferred to the new cluster bud induction medium 3 to 5 times. The implant can be induced to produce multiple cluster buds; 4) Subculture proliferation culture: Inoculate the ground cover silver birch tissue culture seedlings that produced cluster buds induced in the previous step into the subculture medium, and transfer the ground cover silver birch proliferation seedlings to new subcultures every 28 to 32 days. Continue to culture in the generation proliferation medium; 5) Rooting induction culture: Cut out the single buds with a height of more than 2cm in the subculture culture and insert them into the rooting medium, and cultivate them for 10-15 days; 6) Seedling hardening: When at least 60% of the proliferating seedlings start to take root, they enter the hardening stage, and the obtained ground cover silver birch rooted tissue culture seedlings are hardened under the conditions of light intensity 3000-6000 Lx and temperature 26-30°C for 15-15 ～ 20 days; 7) Transplantation and management: Transplant the hardened seedlings into the transplanting medium for field cultivation and management; Step 3) The formula of medium bud induction medium is: 412~413 mg/L NH ₄ NO ₃ , 1898~1902 mg/L KNO ₃ , 84.5~85.5mg/L KH ₂ PO ₄ , 369~371mg/L MgSO ₄ 7H ₂ O, 131~133 mg/L CaCl ₂ 2H ₂ O, 22~22.5mg/L MnSO ₄ 4H ₂ O, 8.4~8.8 mg/L ZnSO ₄ 7H ₂ O, 6~6.4 mg/L LH ₃ BO ₃ , 0.22～0.27 mg/LNa ₂ MoO ₄ 2H ₂ O, 27.5～28 mg/L FeSO ₄ 7H ₂ O, 37～37.5 mg/L Na ₂ EDTA 2H ₂ O, 0.8～0.85 mg/L KI, 0.023～0.027 mg/L CuSO ₄ 5H ₂ O, 0.023～0.027 mg/L CoCl ₂ 6H ₂ O, 1.8～2.2 mg/L glycine, 0.08～0.12 mg/L thiamine hydrochloride, 0.48～0.52 mg/L pyridoxine hydrochloride, 0.48～0.52 mg/L niacin, 99～101 mg/L inositol, 0.8～1.2 g/L PVP, 0.48～0.52 mg/L 6-BA, 0.048～0.052 mg/L IBA, 27~32g/L sucrose, 7~8 g/L carrageenan, pH5.75~5.85; Step 4) The formula of subculture proliferation medium is: 412~413 mg/L NH ₄ NO ₃ , 1898~1902 mg/L KNO ₃ , 84.5~85.5mg/L KH ₂ PO ₄ , 369~371mg/L MgSO ₄ 7H ₂ O, 131~133 mg/L CaCl ₂ 2H ₂ O, 22~22.5mg/L MnSO ₄ 4H ₂ O, 8.4~8.8 mg/L ZnSO ₄ 7H ₂ O, 6~6.4 mg/L LH ₃ BO ₃ , 0.22～0.27 mg/LNa ₂ MoO ₄ 2H ₂ O, 27.5～28 mg/L FeSO ₄ 7H ₂ O, 37～37.5 mg/L Na ₂ EDTA 2H ₂ O, 0.8～0.85 mg/L KI, 0.023～0.027 mg/L CuSO ₄ 5H ₂ O, 0.023～0.027 mg/L CoCl ₂ 6H ₂ O, 1.8～2.2 mg/L glycine, 0.08～0.12 mg/L thiamine hydrochloride, 0.48～0.52 mg/L pyridoxine hydrochloride, 0.48～0.52 mg/L niacin, 99～101 mg/L inositol, 0.8～1.2 g/L PVP, 0.2～0.3 mg/L 6-BA, 0.048～0.052 mg/L IBA, 27~32g/L sucrose, 7~8 g/L carrageenan, pH5.75~5.85; The formulation of the rooting medium in step 5) is: 412-413 mg/L NH ₄ NO ₃ , 473-477 mg/L KNO ₃ , 42-43 mg/L KH ₂ PO ₄ , 92-93 mg/L MgSO ₄ · 7H ₂ O, 108～112 mg/L CaCl ₂ 2H ₂ O, 11～11.5 mg/LMnSO ₄ 4H ₂ O, 4～4.5 mg/L ZnSO ₄ 7H ₂ O, 2.8～3.4mg/LH ₃ BO _3. 0.12～0.13 mg/L Na ₂ MoO ₄ ·2H ₂ O, 13.5～14.5 mg/L FeSO ₄ ·7H ₂ O, 18.3～18.9mg/L Na ₂ ·EDTA·2H ₂ O, 0.41～0.42 mg/L LKI, 0.012～0.013 mg/L CuSO ₄ 5H ₂ O, 0.012～0.013 mg/L CoCl ₂ 6H ₂ O, 1.8～2.2 mg/L glycine, 0.08～0.12 mg/L thiamine hydrochloride, 0.45～0.55 mg/L pyridoxine hydrochloride, 0.45~0.55 mg/L niacin, 99~101mg/L inositol, 0.65~0.75 mg/L NAA, 18~22g/L sucrose, 7~8g/L carrageenan, pH5. 75~5.85. 2. The method according to claim 1, characterized in that: in step 1), the explants are collected from June to September, and the 25-30 cm portion of the top tip of the newly sprouted shoots is collected, and the leaves are cut off. 3. The method according to claim 1, characterized in that: the specific method of explant disinfection treatment in step 2) is: clean the collected branches, cut them into 3-4cm long, with 1-2 leaf axils For stems, sterilize them by soaking them in 0.08-0.12% v/v HgCl ₂ for 3-7 minutes in a sterile environment. During this period, shake them continuously to ensure that the disinfectant is in full contact with the branches, then clean them with sterile water, and finally clean both ends of the stems Cut off 0.4-0.6 cm each, leaving the stem segment containing leaf axils in the middle as sterile explants. 4. The method according to claim 1, characterized in that the culture conditions for bud induction and subculture culture in step 3) and step 4) are: temperature 23-27°C, light 9-11h·d ^{- 1.} The light intensity is 2200-2700Lx; the culture conditions for rooting induction culture in step 5) are: temperature 24-26°C, light 8-10h·d ^-1 , and light intensity 2200-2700Lx. 5. The method according to claim 1, characterized in that: the transplanting medium in step 7) contains 45-55% yellow mud and 45-55% peat soil matrix. 6. The method according to claim 1, characterized in that: the specific method of field cultivation management described in step 7) is: after transplanting, the shading rate is 90-95%, the relative humidity is 85%-90%, Cultivate for 7 to 8 days at a temperature of 25 to 33°C; then place the seedlings at a shading rate of 75% to 80%, humidity of 75% to 80%, and a temperature of 25 to 33°C for 8 to 20 days. Days; in the future, sunshade and watering management will be carried out according to conventional seedling cultivation. 7. The method according to claim 1, characterized in that: the time of transplantation in step 7) is 3-6 months or 9-11 months.