CN104145821A - In-vitro culture plant regeneration method of grevillea orange marmalade - Google Patents
In-vitro culture plant regeneration method of grevillea orange marmalade Download PDFInfo
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Abstract
The invention discloses an in-vitro culture plant regeneration method of grevillea orange marmalade. The method includes following steps: collecting semi-lignified branches on the top of a crown of the grevillea orange marmalade, wherein the branches are strong and robust in growth and a free of diseases and insect pests; washing the branches; cutting the branches to obtain a branch segment which is 2-3 cm in length and has 1-2 axillary buds; disinfecting the branch segment and washing the branch segment through sterilized water; inducing the axillary buds to germinate under artificial control conditions; performing a cutting operation to obtain germinated sterilized buds; inoculating the germinated sterilized buds onto a proliferation culture medium; inducing cluster buds to grow; enlarging a reproduction quantity; cutting the culture medium into single buds; inducing the single buds to root to obtain complete tissue cultured rooted bottle seedlings; moving the rooted tissue cultured bottle seedlings to a substrate for culturing; and moving the rooted tissue cultured bottle seedlings to a flowerpot for culturing large seedlings after growth of new buds and new roots. By means of the method, problems of high contamination rate, serious browning, low inductivity and low transplanting survival rate in a tissue culture propagation technology of the grevillea orange marmalade. Quick propagation and cultivation of the grevillea orange marmalade are achieved. The invention has an important significance on wide popularization of proteaceae grevillea plants in Chinese landscaping.
Description
Technical field
The present invention relates to a kind of cultured in vitro regeneration plant method, specifically a kind of cultured in vitro regeneration plant method of yellow fruit silvery birch, belongs to biological in vitro tissue and cultivates seedling growing process field.
Background technology
Silvery birch, another name thin,tough silk cypress, silk tree, silver-colored Oak Tree, originates in Oceania, evergreen, trunk is straight, tree-like attractive in appearance, especially during the blossom season joint, in Wan Lvcong, lining, with orange-yellow flower, is landscape tree and street tree, and all introduce a fine variety in Chinese Guangdong, Guangxi, each cities and towns, Yunnan, is one of main green tree species in city.This kind of trunk is perfectly straight, and tall and big tall and sturdy, tree crown is neat, should do street tree, shade tree; Also be applicable to rural area " four is other " greening, should build quick-forming of landscape woods, timber forest in low mountain.What also have as decorative indoor plant cultivation compared with cryogenic region.Timber is coarse and hard, and look light red, existing beautiful speckle on section, and medullary ray is arranged very close, and elastic force and resistance to rotten power are strong, and construction easily, can supply furniture, engraving, the purposes such as decoration and vehicle manufacture.
Yellow fruit silvery birch (Grevillea orange marmalade) is the evergreen medium-sized screen type shrub of Proteaceae Grevillea, high 1 ~ 4 meter, circular tree crown, and branches and leaves are luxuriant; Blade celadon, long lanceolar; Spend orangely or golden yellow, tripod flower type, is rich in honey, and the florescence is the winter in spring and autumn.Originate in Australia, full sun plant, is adapted to good, the fertile acid sandy soil of draining, the resistance to frost of moderate, drought-enduring, high temperature resistant, antipollution.Yellow fruit silvery birch is the seeds with Australia landscape, Mediterranean landscape, beach landscape and Tropic Affair.Australia is widely used in roadside streetscape, flower garden greening, hedgerow and potted plant, is to have to keep out the wind, and attracts the important plant of the sweet animal of food.
The conventional method that is usually used in cultivating silvery birch nursery stock is seed propagation, and efficiency is lower, and plant percent is also lower, cannot meet the demand of supply in enormous quantities.Existing as follows about cultivation and the quick propagating technology of silvery birch in prior art: CN 102301909A discloses the method for the efficient Fast-propagation of a kind of orange silvery birch, it is by selecting suitable cutting, cutting is carried out to suitable processing, in suitable cottage propagation period, cuttage is in being equipped with the nutrition cup of Nutrition Soil, be positioned over and in cuttage cool canopy, breed child care management, then through suitable cultivation management technology, and obtain the orange silvery birch seedling of high-quality; The method of this type of cuttage often exists the cuttage seeding root system of acquisition poor, can not form main root, the lower deficiency of survival rate after less, the cuttage of taking root.CN 200910214053.2 discloses a kind of method of fast breeding Grevillea banksii R. Br, mainly comprises selection and sterilization, the cultivation of callus, the induction of the propagation of callus, indefinite bud of material and expands the steps such as culture of rootage, transplanting and hardening numerous, indefinite bud.
Existing Proteaceae Grevillea plant tissue culture technique exists that pollution rate is high, brownization is serious, inductivity is low, transplanting success is low, production cost is high, is difficult to adapt to the development of a large amount of nursery stock demands.Have no the report of a kind of cultured in vitro regeneration plant method of yellow fruit silvery birch.
Summary of the invention
A kind of cultured in vitro regeneration plant method that the object of this invention is to provide yellow fruit silvery birch, the method can overcome the problem that pollution rate is high, brownization is serious, inductivity is low, transplanting success is low existing in yellow fruit silvery birch tissue culture propagation technology, realizing the Fast-propagation of yellow fruit silvery birch cultivates, extensive popularization important in inhibiting to Proteaceae Grevillea plant in classical Chinese garden greening, to the Plant Tissue Breeding of research Proteaceae Grevillea, overcome and there is extremely valuable research value.
Technical scheme of the present invention is as follows: a kind of cultured in vitro regeneration plant method of yellow fruit silvery birch, comprises the following steps:
(1) gather explant and material processed thereof: gather robust growth, the branches without the semi-lignified on 5 years raw yellow fruit silvery birch tree crown tops of damage by disease and insect, cut off the blade on branch, retain petiole, pack into and in sealed bag, take back laboratory; Described material processed is first branch to be used to liquid detergent solution foam washing 15 ~ 20 minutes, in foam washing process, constantly shake bottle, with flowing water, rinse well, more alcohol-pickled 10 seconds that are 95% by concentration, then with flowing water, rinse 3 times, after cleaning, be cut into the stem section with 1 ~ 2 axillalry bud of 2 ~ 3 centimetres;
(2) set up sterile propagation body: the stem section that step (1) is obtained in aseptic experiment chamber with 75% alcohol sterilizing 1 ~ 2 minute, use again 0.1% mercuric chloride solution sterilizing 10 ~ 15 minutes, then use sterile water wash 3 ~ 4 times, one bottle of stem section is inoculated on MS1 medium, in culturing room, cultivate induction axillary bud sprouting; Described condition of culture is: 20 ~ 30 ℃ of room temperatures, and illumination every day 8 ~ 12 hours, intensity of illumination 800 ~ 2000LX, cultivates 30 ~ 32 days; When sprout green bud from axil, through cutting apart, transfer and can making the continuous growth and breeding of green bud, complete the foundation of sterile propagation body;
(3) propagation is cultivated: the sterile propagation body that step (2) is obtained is inoculated in the proliferated culture medium of MS2, being placed on manual control room temperature is 30 ~ 32 ℃, illumination every day 8 ~ 12 hours, cultivates under the condition of intensity of illumination 800 ~ 2000LX 30 ~ 32 days, from axil, sprouts aseptic green bud, the green bud of clip is inoculated on proliferated culture medium MS2, bud clump propagation, cultivated after 30 days, 5 ~ 6 times of bud clump propagation multiples, under aseptic condition, cut apart again switching, through subculture repeatedly, cultivated expanding propagation;
(4) culture of rootage: in the propagation incubation of step (3), Multiple Buds is high 20 millimeters when above, and Multiple Buds is cut from base portion, is divided into individual plant and is inoculated in MS3 root media, be placed on 20 ~ 30 ℃ of left and right of room temperature, illumination every day 8 ~ 12 hours, intensity of illumination is cultivated 20 ~ 25 days under the condition of 800 ~ 2000LX, and seedling starts root, be placed into again under 75% shading condition in greenhouse hardening 7 ~ 14 days, bottle seedling leaf of taking root launches, and elongation is healthy and strong, forms whole plant;
(5) transplant: the good bottle seedling of root growth that cultivation is obtained moves into greenhouse domestication, bottle seedling is from manual control condition of culture and give nutritive element to reform of nature environmental condition, and after the domestication process of oneself's absorption nutrient, be transplanted in the seedling-raising cup that grow seedling nutritional matrix is housed; Trasplanting method is: the seedling in bottle is carefully taken out, clean and stick the medium on seedling, transplant seedlings in the seedling-raising cup of filling up nutrient matrix, each cup plantation one young plant, transplant thickness of earth-fill cover and just cover root system, be generally no more than 1 centimetre, compress matrix, make shoot root and matrix close contact, the final singling water of drenching after having transplanted;
(6) seedling culture: the seedling after transplanting will be strengthened water and fertilizer management and sick insect protected control in month, and suitably shade, guarantee that seedling survives, when growth of seedling is stablized, grow after sprouting and Xin Gen, progressively increase intensity of illumination, until carry out full exposure Routine Management, when seedling culture to 15 is centimetre high, can move bigger flowerpot cultivating large seedling.
Described MS1 medium is first culture base, its preparation is that carragheen 7000mg/L is added to 500ml distilled water, being heated to carragheen dissolves completely, then add the nutritive element composition in the culture medium prescription being dissolved in the water in advance, add water to 1000ml, stir, pH is adjusted to 5.8, bottling, sterilizing, standby; Nutritive element composition and quality proportioning thereof in described culture medium prescription are as follows: 6 ~ benzyladenine, 0.05 ~ 5.0mg/L, methyl α-naphthyl acetate 0.01 ~ 0.5mg/L, NH
4nO
3990 ~ 1650mg/L, KNO
31140 ~ 1900mg/L, CaCl
22H
2o 240 ~ 400mg/L, MgSO
47H
2o 74 ~ 370mg/L, KH
2pO
4102 ~ 170mg/L, KI 0.5 ~ 0.83mg/L, H
3bO
33.72 ~ 6.2mg/L, MnSO
44H
2o 13.38 ~ 22.3mg/L, ZnSO
47H
2o 5.16 ~ 8.6mg/L, NaMoO
22H
2o 0.15 ~ 0.25mg/L, CuSO
45H
2o 0.015 ~ 0.025mg/L, CoCl
26H
2o 0.015 ~ 0.025mg/L, Na
2eDTA 22.38 ~ 37.3mg/L, FeSO
47H
2o 16.68 ~ 27.8mg/L, inositol 60 ~ 100mg/L, nicotinic acid 0.3 ~ 0.5mg/L, puridoxine hydrochloride 0.3 ~ 0.5mg/L, nicotinic acid thiamine 0.06 ~ 0.1mg/L, glycine 1.2 ~ 2.0mg/L, sucrose 30000mg/L.
Described MS2 medium is proliferated culture medium, its preparation is that carragheen 7000mg/L is added to 500ml distilled water, being heated to carragheen dissolves completely, then add the nutritive element composition in the culture medium prescription being dissolved in the water in advance, add water to 1000ml, stir, pH is adjusted to 5.8, bottling, sterilizing, standby; Nutritive element composition and quality proportioning thereof in described culture medium prescription are as follows: 6 ~ benzyladenine, 0.05 ~ 0.2mg/L, methyl α-naphthyl acetate 0.1 ~ 0.3mg/L, active carbon 1.0 ~ 3.0g/L, NH
4nO
3990 ~ 1650mg/L, KNO
31140 ~ 1900mg/L, CaCl
22H
2o 240 ~ 400mg/L, MgSO
47H
2o 222 ~ 370mg/L, KH
2pO
4102 ~ 170mg/L, KI 0.5 ~ 0.83mg/L, H
3bO
33.72 ~ 6.2mg/L, MnSO
44H
2o 13.38 ~ 22.3mg/L, ZnSO
47H
2o 5.16 ~ 8.6mg/L, NaMoO
22H
2o 0.15 ~ 0.25mg/L, CuSO
45H
2o 0.015 ~ 0.025mg/L, CoCl
26H
2o 0.015 ~ 0.025mg/L, Na
2eDTA 22.38 ~ 37.3mg/L, FeSO
47H
2o 16.68 ~ 27.8mg/L, inositol 60 ~ 100mg/L, nicotinic acid 0.3 ~ 0.5mg/L, puridoxine hydrochloride 0.3 ~ 0.5mg/L, nicotinic acid thiamine 0.06 ~ 0.1mg/L, glycine 1.2 ~ 2.0mg/L, sucrose 30000mg/L.
Described MS3 medium is root induction medium, its preparation is that carragheen 7000mg/L is added to 500ml distilled water, being heated to carragheen dissolves completely, then add the nutritive element composition in the culture medium prescription being dissolved in the water in advance, add water to 1000ml, stir, pH is adjusted to 5.8, bottling, sterilizing, standby; Nutritive element composition and quality proportioning thereof in described formula are as follows: methyl α-naphthyl acetate 2.0 ~ 4.0mg/L, active carbon 1.0 ~ 3.0g/L, NH
4nO
3200 ~ 800mg/L, KNO
3250 ~ 900mg/L, CaCl
22H
2o 50 ~ 200mg/L, MgSO
47H
2o 222 ~ 370mg/L, Ca (NO
3)
2200 ~ 400mg/L, KH
2pO
4102 ~ 170mg/L, KI 0.5 ~ 0.83mg/L, H
3bO
33.72 ~ 6.2mg/L, MnSO
44H
2o 13.38 ~ 22.3mg/L, ZnSO
47H
2o 516 ~ 8.6mg/L, NaMoO
22H
2o 0.15 ~ 0.25mg/L, CuSO
45H
2o 0.015 ~ 0.025mg/L, CoCl
26H
2o 0.015 ~ 0.025mg/L, Na
2eDTA 22.38 ~ 37.3mg/L, FeSO
47H
2o 16.68 ~ 27.8mg/L, inositol 50 ~ 100mg/L, nicotinic acid 0.25 ~ 0.5mg/L, puridoxine hydrochloride 0.5 ~ 1.0mg/L, nicotinic acid thiamine 0.1 ~ 0.25mg/L, glycine 1.0 ~ 2.0mg/L, sucrose 15000 ~ 30000mg/L.
Preferably, the nutritive element composition in MS1, MS2, MS3 culture medium prescription and quality proportioning thereof are as follows respectively:
(1) MS1 medium: 6 ~ benzyladenine 2.5mg/L, methyl α-naphthyl acetate 0.25mg/L, NH
4nO
31650mg/L, KNO
31900mg/L, CaCl
22H
2o 400mg/L, MgSO
47H
2o 370mg/L, KH
2pO
4170mg/L, KI 0.83mg/L, H
3bO
36.2mg/L, MnSO
44H
2o 22.3mg/L, ZnSO
47H
2o 8.6mg/L, NaMoO
22H
2o 0.25mg/L, CuSO
45H
2o 0.025mg/L, CoCl
26H
2o 0.025mg/L, Na
2eDTA 37.3mg/L, FeSO
47H
2o 27.8mg/L, inositol 100mg/L, nicotinic acid 0.5mg/L, puridoxine hydrochloride 0.5mg/L, nicotinic acid thiamine 0.1mg/L, glycine 2.0mg/L, sucrose 30000mg/L.
(2) MS2 medium: 6 ~ benzyladenine 0.1mg/L, methyl α-naphthyl acetate 0.2mg/L, active carbon 2.0 g/L, NH
4nO
31650mg/L, KNO
31900mg/L, CaCl
22H
2o 400mg/L, MgSO
47H
2o 370mg/L, KH
2pO
4170mg/L, KI 0.83mg/L, H
3bO
36.2mg/L, MnSO
44H
2o 22.3mg/L, ZnSO
47H
2o 8.6mg/L, NaMoO
22H
2o 0.25mg/L, CuSO
45H
2o 0.025mg/L, CoCl
26H
2o 0.025mg/L, Na
2eDTA 37.3mg/L, FeSO
47H
2o 27.8mg/L, inositol 100mg/L, nicotinic acid 0.5mg/L, puridoxine hydrochloride 0.5mg/L, nicotinic acid thiamine 0.1mg/L, glycine 2.0mg/L, sucrose 30000mg/L.
(3) MS3 medium: methyl α-naphthyl acetate 3.0mg/L, active carbon 2.0g/L, NH
4nO
3800mg/L, KNO
3900mg/L, CaCl
22H
2o 200mg/L, MgSO
47H
2o 370mg/L, Ca (NO
3)
2400mg/L, KH
2pO
4170mg/L, KI 0.83mg/L, H
3bO
36.2mg/L, MnSO
44H
2o 22.3mg/L, ZnSO
47H
2o 8.6mg/L, NaMoO
22H
2o 0.25mg/L, CuSO
45H
2o 0.025mg/L, CoCl
26H
2o 0.025mg/L, Na
2eDTA 37.3mg/L, FeSO
47H
2o 27.8mg/L, inositol 100mg/L, nicotinic acid 0.5mg/L, puridoxine hydrochloride 1mg/L, nicotinic acid thiamine 0.25mg/L, glycine 2.0mg/L, sucrose 30000mg/L.
The described grow seedling nutritional matrix of step (5) preferably adopts peat soil, yellow soil and perlite to be fully mixed to get in the ratio of 2:3:1.
The water and fertilizer management method of the described seedling of step (6) after transplanting is preferably as follows: keep matrix moistening, relative air humidity, more than 95%, prevents plant dehydration, 25 ~ 28 ℃ of cultivation temperature, 75% shading net shading for cool canopy; Transplant and progressively control water supply in media after 15 days, relative moisture is controlled at 60 ~ 80%, promotes new root sprouting to occur.
Sick insect protected the prevent and treat method of the described seedling of step (6) after transplanting is preferably as follows: within after sprigging the 3rd day, adopt 0.1% tpn spraying disinfection sterilizing, afterwards every spraying in 10 ~ 15 days once; When there being sprouting to sprout, when new root grows, spray 1200 ~ 1800 times of nutrient solutions, according to seedling growth, every two weeks, spray once.
Described nutrient solution is formed by the compounding medicine of following quality proportioning: NH
4nO
3700 ~ 900mg/L, KNO
3800 ~ 1000mg/L, CaCl
22H
2o 150 ~ 250mg/L, MgSO
47H
2o 280 ~ 400mg/L, Ca (NO
3)
2300 ~ 500mg/L, KH
2pO
4120 ~ 220mg/L, KI 0.6 ~ 1.0mg/L, H
3bO
35 ~ 8mg/L, MnSO
44H
2o 20 ~ 25mg/L, ZnSO
47H
2o 6.5 ~ 10mg/L, NaMoO
22H
2o 0.1 ~ 0.5mg/L, CuSO
45H
2o 0.01 ~ 0.05mg/L, CoCl
26H
2o 0.01 ~ 0.05mg/L.
Described nutrient solution is preferably formed by the compounding medicine of following quality proportioning: NH
4nO
3800mg/L, KNO
3900mg/L, CaCl
22H
2o 200mg/L, MgSO
47H
2o 370mg/L, Ca (NO
3)
2400mg/L, KH
2pO
4170mg/L, KI 0.83mg/L, H
3bO
36.2mg/L, MnSO
44H
2o 22.3mg/L, ZnSO
47H
2o 8.6mg/L, NaMoO
22H
2o 0.25mg/L, CuSO
45H
2o 0.025mg/L, CoCl
26H
2o 0.025mg/L.
The present invention is as follows with respect to the beneficial effect of prior art: the present invention provides a kind of method of Fast-propagation for originating in Australian yellow fruit silvery birch, Proteaceae Grevillea plant is extensively promoted in classical Chinese garden greening to important in inhibiting; To the Plant Tissue Breeding of research Proteaceae Grevillea, overcome the tissue culture propagation technology such as pollution rate is high, brownization is serious, inductivity is low, transplanting success is low and there is extremely valuable research value.
Embodiment
Below by embodiment, the present invention is described in further details, these embodiment are only used for illustrating the present invention, do not limit the scope of the invention.
Embodiment 1 adopts following steps to realize the present invention.
1, gather explant and material processed: in Ai Jia farm, Sanshui District, Fushan City, Guangdong Province, gather robust growth, 5 years raw yellow really branches of the semi-lignified on silvery birch tree crown top without damage by disease and insect, cut off the blade on branch, retain petiole, pack into and in sealed bag, take back laboratory; First branch is used to liquid detergent solution foam washing 15 minutes, in foam washing process, constantly shaken bottle, with flowing water, rinse well, more alcohol-pickled 10 seconds that are 95% by concentration, then with flowing water, rinse 3 times, after cleaning, be cut into the stem section with 1 ~ 2 axillalry bud of 2 ~ 3 centimetres.
2, set up sterile propagation body:
(1) the just preparation of culture base: MS1 medium is first culture base, its preparation is that carragheen 7000mg/L is added to 500ml distilled water, being heated to carragheen dissolves completely, then add the nutritive element composition in the culture medium prescription being dissolved in the water in advance, add water to 1000ml, stir, pH is adjusted to 5.8, bottling, sterilizing, standby; Nutritive element composition and quality proportioning thereof in described formula are as follows: 6 ~ benzyladenine 2.5mg/L, methyl α-naphthyl acetate 0.25mg/L, NH
4nO
31650mg/L, KNO
31900mg/L, CaCl
22H
2o 400mg/L, SO
47H
2o 370mg/L, KH
2pO
4170mg/L, KI 0.83mg/L, H
3bO
36.2mg/L, MnSO
44H
2o 22.3mg/L, ZnSO
47H
2o 8.6mg/L, NaMoO
22H
2o 0.25mg/L, CuSO
45H
2o 0.025mg/L, CoCl
26H
2o 0.025mg/L, Na
2eDTA 37.3mg/L, FeSO
47H
2o 27.8mg/L, inositol 100mg/L, nicotinic acid 0.5mg/L, puridoxine hydrochloride 0.5mg/L, nicotinic acid thiamine 0.1mg/L, glycine 2.0mg/L, sucrose 30000mg/L;
(2) by the stem section after cleaning in aseptic experiment chamber with 75% alcohol sterilizing 1 minute, use again 0.1% mercuric chloride solution sterilizing 12 minutes, then use sterile water wash 3 ~ 4 times, one bottle of stem section is inoculated on MS1 medium, in culturing room, cultivate, induction axillary bud sprouting, when sprout green bud from axil, through cutting apart, transfer and can making the continuous growth and breeding of green bud, complete the foundation of sterile propagation body; Condition of culture is: 20 ~ 30 ℃ of room temperatures, and illumination every day 10 hours, intensity of illumination 1000 ~ 1500LX, cultivates 30 days.
3, propagation is cultivated:
(1) preparation of proliferated culture medium: MS2 medium is proliferated culture medium, its preparation is that carragheen 7000mg/L is added to 500ml distilled water, being heated to carragheen dissolves completely, then add the nutritive element composition in the culture medium prescription being dissolved in the water in advance, add water to 1000ml, stir, pH is adjusted to 5.8, bottling, sterilizing, standby; Nutritive element composition and quality proportioning thereof in described formula are as follows: 6 ~ benzyladenine 0.1mg/L, methyl α-naphthyl acetate 0.2mg/L, active carbon 2.0 g/L, NH
4nO
31650mg/L, KNO
31900mg/L, CaCl
22H
2o 400mg/L, MgSO
47H
2o 370mg/L, KH
2pO
4170mg/L, KI 0.83mg/L, H
3bO
36.2mg/L, MnSO
44H
2o 22.3mg/L, ZnSO
47H
2o 8.6mg/L, NaMoO
22H
2o 0.25mg/L, CuSO
45H
2o 0.025mg/L, CoCl
26H
2o 0.025mg/L, Na
2eDTA 37.3mg/L, FeSO
47H
2o 27.8mg/L, inositol 100mg/L, nicotinic acid 0.5mg/L, puridoxine hydrochloride 0.5mg/L, nicotinic acid thiamine 0.1mg/L, glycine 2.0mg/L, sucrose 30000mg/L;
(2) the sterile propagation body of acquisition is inoculated in the proliferated culture medium of the MS2 preparing, being placed on manual control room temperature is 30 ℃, illumination every day 10 hours, cultivates under the condition of intensity of illumination 1600LX 32 days, from axil, sprouts aseptic green bud, the green bud of clip is inoculated on proliferated culture medium MS2, bud clump propagation, cultivated after 30 days, 6 times of bud clump propagation multiples, under aseptic condition, cut apart again switching, through subculture repeatedly, cultivated expanding propagation.
4, culture of rootage:
(1) preparation of root media: MS3 medium is root induction medium, its preparation is that carragheen 7000mg/L is added to 500ml distilled water, being heated to carragheen dissolves completely, then add the nutritive element composition in the culture medium prescription being dissolved in the water in advance, add water to 1000ml, stir, pH is adjusted to 5.8, bottling, sterilizing, standby; Nutritive element composition and quality proportioning thereof in described formula are as follows: methyl α-naphthyl acetate 3.0mg/L, active carbon 2.0g/L, NH
4nO
3800mg/L, KNO
3900mg/L, CaCl
22H
2o 200mg/L, MgSO
47H
2o 370mg/L, Ca (NO
3)
2400mg/L, KH
2pO
4170mg/L, KI 0.83mg/L, H
3bO
36.2mg/L, MnSO
44H
2o 22.3mg/L, ZnSO
47H
2o 8.6mg/L, NaMoO
22H
2o 0.25mg/L, CuSO
45H
2o 0.025mg/L, CoCl
26H
2o 0.025mg/L, Na
2eDTA 37.3mg/L, FeSO
47H
2o 27.8mg/L, inositol 100mg/L, nicotinic acid 0.5mg/L, puridoxine hydrochloride 1mg/L, nicotinic acid thiamine 0.25mg/L, glycine 2.0mg/L, sucrose 30000mg/L;
(2) in propagation incubation, Multiple Buds is high 22 millimeters when above, and Multiple Buds is cut from base portion, is divided into individual plant and is inoculated in MS3 root media, be placed on 20 ~ 30 ℃ of left and right of room temperature, illumination every day 10 hours, intensity of illumination is cultivated 22 days under the condition of 1800LX, and seedling starts root, be placed into again under 75% shading condition in greenhouse hardening 10 days, bottle seedling leaf of taking root launches, and elongation is healthy and strong, forms whole plant.
5, transplant:
(1) domestication: the good bottle seedling of root growth that cultivation is obtained moves into greenhouse domestication, make bottle seedling from manual control condition of culture and give nutritive element to reform of nature environmental condition, and oneself draws nutrient;
(2) transplant: with peat soil, yellow soil and perlite, in the ratio of 2:3:1, fully mix and make nutrient matrix, be filled up in seedling-raising cup, seedling in bottle is carefully taken out, clean and stick the medium on seedling, transplant seedlings to the seedling-raising cup of filling up nutrient matrix, each cup plantation one young plant, transplant thickness of earth-fill cover and just cover root system, be generally no more than 1 centimetre, compress matrix, make shoot root and matrix close contact, the final singling water of drenching after having transplanted.
6, seedling culture: the seedling after transplanting will be strengthened water and fertilizer management and sick insect protected control in month, and suitably shade, guarantee that seedling survives, when growth of seedling is stablized, grow after sprouting and Xin Gen, progressively increase intensity of illumination, until carry out full exposure Routine Management, when seedling culture to 15 is centimetre high, can move bigger flowerpot cultivating large seedling.
The method of water and fertilizer management: keep matrix moistening, relative air humidity, more than 95%, prevents plant dehydration, 25 ~ 28 ℃ of cultivation temperature, 75% shading net shading for cool canopy; Transplant and progressively control water supply in media afterwards in 15 days, relative moisture is controlled at 75 ~ 80%, promotes new root sprouting to occur.
Diseases and pests controlling method: adopt 0.1% tpn spraying disinfection sterilizing for after sprigging the 3rd day, afterwards every spraying in 12 days once; When there being sprouting to sprout, when new root grows, spray 1500 times of nutrient solutions, according to seedling growth, every two weeks, spray once; The nutrient solution spraying is that the compounding medicine by following proportioning obtains: NH
4nO
3800mg/L, KNO
3900mg/L, CaCl
22H
2o 200mg/L, MgSO
47H
2o 370mg/L, Ca (NO
3)
2400mg/L, KH
2pO
4170mg/L, KI 0.83mg/L, H
3bO
36.2mg/L, MnSO
44H
2o 22.3mg/L, ZnSO
47H
2o 8.6mg/L, NaMoO
22H
2o 0.25mg/L, CuSO
45H
2o 0.025mg/L, CoCl
26H
2o 0.025mg/L.
Embodiment 2 adopts following steps to realize the present invention.
1, gather explant and material processed: in Ai Jia farm, Sanshui District, Fushan City, Guangdong Province, gather robust growth, 5 years raw yellow really branches of the semi-lignified on silvery birch tree crown top without damage by disease and insect, cut off the blade on branch, retain petiole, pack into and in sealed bag, take back laboratory; First branch is used to liquid detergent solution foam washing 18 minutes, in foam washing process, constantly shaken bottle, with flowing water, rinse well, more alcohol-pickled 10 seconds that are 95% by concentration, then with flowing water, rinse 3 times, after cleaning, be cut into the stem section with 1 ~ 2 axillalry bud of 2 ~ 3 centimetres.
2, set up sterile propagation body:
(1) the just preparation of culture base: MS1 medium is first culture base, its preparation is that carragheen 7000mg/L is added to 500ml distilled water, being heated to carragheen dissolves completely, then add the nutritive element composition in the culture medium prescription being dissolved in the water in advance, add water to 1000ml, stir, pH is adjusted to 5.8, bottling, sterilizing, standby; Nutritive element composition and quality proportioning thereof in described formula are as follows: 6 ~ benzyladenine 3.0mg/L, methyl α-naphthyl acetate 0.2mg/L, NH
4nO
3990mg/L, KNO
31140mg/L, CaCl
22H
2o 240mg/L, MgSO
47H
2o 222mg/L, KH
2pO
4102mg/L, KI 0.5mg/L, H
3bO
33.7mg/L, MnSO
44H
2o 13.38mg/L, ZnSO
47H
2o 5.2mg/L, NaMoO
22H
2o 0.15mg/L, CuSO
45H
2o 0.015mg/L, CoCl
26H
2o 0.015mg/L, Na
2eDTA 22.38mg/L, FeSO
47H
2o 16.68mg/L, inositol 60mg/L, nicotinic acid 0.3mg/L, puridoxine hydrochloride 0.3mg/L, nicotinic acid thiamine 0.06mg/L, glycine 1.2mg/L, sucrose 30000mg/L;
(2) by the stem section after cleaning in aseptic experiment chamber with 75% alcohol sterilizing 1 minute, use again 0.1% mercuric chloride solution sterilizing 10 minutes, then use sterile water wash 3 ~ 4 times, one bottle of stem section is inoculated on MS1 medium, in culturing room, cultivate, induction axillary bud sprouting, when sprout green bud from axil, through cutting apart, transfer and can making the continuous growth and breeding of green bud, complete the foundation of sterile propagation body; Condition of culture is: 20 ~ 30 ℃ of room temperatures, and illumination every day 8 hours, intensity of illumination 1800 ~ 2000LX, cultivates 31 days.
3, propagation is cultivated:
(1) preparation of proliferated culture medium: MS2 medium is proliferated culture medium, its preparation is that carragheen 7000mg/L is added to 500ml distilled water, being heated to carragheen dissolves completely, then add the nutritive element composition in the culture medium prescription being dissolved in the water in advance, add water to 1000ml, stir, pH is adjusted to 5.8, bottling, sterilizing, standby; Nutritive element composition and quality proportioning thereof in described formula are as follows: 6 ~ benzyladenine 0.05mg/L, methyl α-naphthyl acetate 0.3mg/L, active carbon 1.0g/L, NH
4nO
3990mg/L, KNO
31140mg/L, CaCl
22H
2o 240mg/L, MgSO
47H
2o 222mg/L, KH
2pO
4102mg/L, KI 0.5mg/L, H
3bO
33.7mg/L, MnSO
44H
2o 13.38mg/L, ZnSO
47H
2o 5.2mg/L, NaMoO
22H
2o 0.15mg/L, CuSO
45H
2o 0.015mg/L, CoCl
26H
2o 0.015mg/L, Na
2eDTA 22.38mg/L, FeSO
47H
2o 16.68mg/L, inositol 60mg/L, nicotinic acid 0.3mg/L, puridoxine hydrochloride 0.3mg/L, nicotinic acid thiamine 0.06mg/L, glycine 1.2mg/L, sucrose 30000mg/L;
(2) the sterile propagation body of acquisition is inoculated in the proliferated culture medium of the MS2 preparing, being placed on manual control room temperature is 31 ℃, illumination every day 12 hours, cultivates under the condition of intensity of illumination 1200LX 31 days, from axil, sprouts aseptic green bud, the green bud of clip is inoculated on proliferated culture medium MS2, bud clump propagation, cultivated after 30 days, 5 times of bud clump propagation multiples, under aseptic condition, cut apart again switching, through subculture repeatedly, cultivated expanding propagation.
4, culture of rootage:
(1) preparation of root media: MS3 medium is root induction medium, its preparation is that carragheen 7000mg/L is added to 500ml distilled water, being heated to carragheen dissolves completely, then add the nutritive element composition in the culture medium prescription being dissolved in the water in advance, add water to 1000ml, stir, pH is adjusted to 5.8, bottling, sterilizing, standby; Nutritive element composition and quality proportioning thereof in described formula are as follows: 2.0mg/L, active carbon 1.0g/L, NH
4nO
3600mg/L, KNO
3800mg/L, CaCl
22H
2o 150mg/L, MgSO
47H
2o 222mg/L, Ca (NO
3)
2300mg/L, KH
2pO
4102mg/L, KI 0.5mg/L, H
3bO
33.7mg/L, MnSO
44H
2o 13.38mg/L, ZnSO
47H
2o 5.2mg/L, NaMoO
22H
2o 0.15mg/L, CuSO
45H
2o 0.015mg/L, CoCl
26H
2o 0.015mg/L, Na
2eDTA 22.38mg/L, FeSO
47H
2o 16.68mg/L, inositol 80mg/L, nicotinic acid 0.5mg/L, puridoxine hydrochloride 1mg/L, nicotinic acid thiamine 0.25mg/L, glycine 2.0mg/L, sucrose 28000mg/L;
(2) in propagation incubation, Multiple Buds is high 25 millimeters when above, and Multiple Buds is cut from base portion, is divided into individual plant and is inoculated in MS3 root media, be placed on 20 ~ 30 ℃ of left and right of room temperature, illumination every day 11 hours, intensity of illumination is cultivated 25 days under the condition of 1400LX, and seedling starts root, be placed into again under 75% shading condition in greenhouse hardening 7 days, bottle seedling leaf of taking root launches, and elongation is healthy and strong, forms whole plant.
5, transplant:
(1) domestication: the good bottle seedling of root growth that cultivation is obtained moves into greenhouse domestication, make bottle seedling from manual control condition of culture and give nutritive element to reform of nature environmental condition, and oneself draws nutrient;
(2) transplant: with peat soil, yellow soil and perlite, in the ratio of 2:3:1, fully mix and make nutrient matrix, be filled up in seedling-raising cup, seedling in bottle is carefully taken out, clean and stick the medium on seedling, transplant seedlings to the seedling-raising cup of filling up nutrient matrix, each cup plantation one young plant, transplant thickness of earth-fill cover and just cover root system, be generally no more than 1 centimetre, compress matrix, make shoot root and matrix close contact, the final singling water of drenching after having transplanted.
6, seedling culture: the seedling after transplanting will be strengthened water and fertilizer management and sick insect protected control in month, and suitably shade, guarantee that seedling survives, when growth of seedling is stablized, grow after sprouting and Xin Gen, progressively increase intensity of illumination, until carry out full exposure Routine Management, when seedling culture to 15 is centimetre high, can move bigger flowerpot cultivating large seedling.
The method of water and fertilizer management: keep matrix moistening, relative air humidity, more than 95%, prevents plant dehydration, 25 ~ 28 ℃ of cultivation temperature, 75% shading net shading for cool canopy; Transplant and progressively control water supply in media afterwards in 15 days, relative moisture is controlled at 60 ~ 65%, promotes new root sprouting to occur.
Diseases and pests controlling method: adopt 0.1% tpn spraying disinfection sterilizing for after sprigging the 3rd day, afterwards every spraying in 15 days once; When there being sprouting to sprout, when new root grows, spray 1200 times of nutrient solutions, according to seedling growth, every two weeks, spray once; The nutrient solution spraying is that the compounding medicine by following proportioning obtains: NH
4nO
3900mg/L, KNO
3800mg/L, CaCl
22H
2o 150mg/L, MgSO
47H
2o 280mg/L, Ca (NO
3)
2300mg/L, KH
2pO
4220mg/L, KI 1.0mg/L, H
3bO
38mg/L, MnSO
44H
2o 20mg/L, ZnSO
47H
2o 6.5mg/L, NaMoO
22H
2o 0.1mg/L, CuSO
45H
2o 0.01mg/L, CoCl
26H
2o 0.01mg/L.
Embodiment 3 adopts following steps to realize the present invention.
1, gather explant and material processed: in Ai Jia farm, Sanshui District, Fushan City, Guangdong Province, gather robust growth, 5 years raw yellow really branches of the semi-lignified on silvery birch tree crown top without damage by disease and insect, cut off the blade on branch, retain petiole, pack into and in sealed bag, take back laboratory; First branch is used to liquid detergent solution foam washing 20 minutes, in foam washing process, constantly shaken bottle, with flowing water, rinse well, more alcohol-pickled 10 seconds that are 95% by concentration, then with flowing water, rinse 3 times, after cleaning, be cut into the stem section with 1 ~ 2 axillalry bud of 2 ~ 3 centimetres.
2, set up sterile propagation body:
(1) the just preparation of culture base: MS1 medium is first culture base, its preparation is that carragheen 7000mg/L is added to 500ml distilled water, being heated to carragheen dissolves completely, then add the nutritive element composition in the culture medium prescription being dissolved in the water in advance, add water to 1000ml, stir, pH is adjusted to 5.8, bottling, sterilizing, standby; Nutritive element composition and quality proportioning thereof in described formula are as follows: 6 ~ benzyladenine 5.0mg/L, methyl α-naphthyl acetate 0.5mg/L, NH
4nO
31250mg/L, KNO
31450mg/L, CaCl
22H
2o 320mg/L, MgSO
47H
2o 290mg/L, KH
2pO
4140mg/L, KI 0.65mg/L, H
3bO
34.82mg/L, MnSO
44H
2o 18.55mg/L, ZnSO
47H
2o 6.52mg/L, NaMoO
22H
2o 0.2mg/L, CuSO
45H
2o 0.02mg/L, CoCl
26H
2o 0.02mg/L, Na
2eDTA 28.5mg/L, FeSO
47H
2o 21.5mg/L, inositol 80mg/L, nicotinic acid 0.4mg/L, puridoxine hydrochloride 0.4mg/L, nicotinic acid thiamine 0.08mg/L, glycine 1.6mg/L, sucrose 30000mg/L;
(2) by the stem section after cleaning in aseptic experiment chamber with 75% alcohol sterilizing 2 minutes, use again 0.1% mercuric chloride solution sterilizing 15 minutes, then use sterile water wash 3 ~ 4 times, one bottle of stem section is inoculated on MS1 medium, in culturing room, cultivate, induction axillary bud sprouting, when sprout green bud from axil, through cutting apart, transfer and can making the continuous growth and breeding of green bud, complete the foundation of sterile propagation body; Condition of culture is: 20 ~ 30 ℃ of room temperatures, and illumination every day 12 hours, intensity of illumination 800LX, cultivates 32 days.
3, propagation is cultivated:
(1) preparation of proliferated culture medium: MS2 medium is proliferated culture medium, its preparation is that carragheen 7000mg/L is added to 500ml distilled water, being heated to carragheen dissolves completely, then add the nutritive element composition in the culture medium prescription being dissolved in the water in advance, add water to 1000ml, stir, pH is adjusted to 5.8, bottling, sterilizing, standby; Nutritive element composition and quality proportioning thereof in described formula are as follows: 6 ~ benzyladenine 0.2mg/L, methyl α-naphthyl acetate 0.1mg/L, active carbon 3.0 g/L, NH
4nO
31250mg/L, KNO
31450mg/L, CaCl
22H
2o 320mg/L, MgSO
47H
2o 290mg/L, KH
2pO
4140mg/L, KI 0.65mg/L, H
3bO
34.82mg/L, MnSO
44H
2o 18.55mg/L, ZnSO
47H
2o 6.52mg/L, NaMoO
22H
2o 0.2mg/L, CuSO
45H
2o 0.02mg/L, CoCl
26H
2o 0.02mg/L, Na
2eDTA 28.5mg/L, FeSO
47H
2o 21.5mg/L, inositol 80mg/L, nicotinic acid 0.4mg/L, puridoxine hydrochloride 0.4mg/L, nicotinic acid thiamine 0.08mg/L, glycine 1.6mg/L, sucrose 30000mg/L;
(2) the sterile propagation body of acquisition is inoculated in the proliferated culture medium of the MS2 preparing, being placed on manual control room temperature is 32 ℃, illumination every day 8 hours, cultivates under the condition of intensity of illumination 2000LX 30 days, from axil, sprouts aseptic green bud, the green bud of clip is inoculated on proliferated culture medium MS2, bud clump propagation, cultivated after 30 days, 5.5 times of bud clump propagation multiples, under aseptic condition, cut apart again switching, through subculture repeatedly, cultivated expanding propagation.
4, culture of rootage:
(1) preparation of root media: MS3 medium is root induction medium, its preparation is that carragheen 7000mg/L is added to 500ml distilled water, being heated to carragheen dissolves completely, then add the nutritive element composition in the culture medium prescription being dissolved in the water in advance, add water to 1000ml, stir, pH is adjusted to 5.8, bottling, sterilizing, standby; Nutritive element composition and quality proportioning thereof in described formula are as follows: 4.0mg/L, active carbon 3.0g/L, NH
4nO
3200mg/L, KNO
3250mg/L, CaCl
22H
2o 100mg/L, MgSO
47H
2o 320mg/L, Ca (NO
3)
2200mg/L, KH
2pO
4140mg/L, KI 0.65mg/L, H
3bO
34.82mg/L, MnSO
44H
2o 18.85mg/L, ZnSO
47H
2o 6.52mg/L, NaMoO
22H
2o 0.2mg/L, CuSO
45H
2o 0.02mg/L, CoCl
26H
2o 0.02mg/L, Na
2eDTA 28.5mg/L, FeSO
47H
2o 21.5mg/L, inositol 50mg/L, nicotinic acid 0.25mg/L, puridoxine hydrochloride 0.5mg/L, nicotinic acid thiamine 0.1mg/L, glycine 1.0mg/L, sucrose 15000mg/L;
(2) in propagation incubation, Multiple Buds is high 20 millimeters when above, and Multiple Buds is cut from base portion, is divided into individual plant and is inoculated in MS3 root media, be placed on 20 ~ 30 ℃ of left and right of room temperature, illumination every day 9 hours, intensity of illumination is cultivated 20 days under the condition of 1700LX, and seedling starts root, be placed into again under 75% shading condition in greenhouse hardening 14 days, bottle seedling leaf of taking root launches, and elongation is healthy and strong, forms whole plant.
5, transplant:
(1) domestication: the good bottle seedling of root growth that cultivation is obtained moves into greenhouse domestication, make bottle seedling from manual control condition of culture and give nutritive element to reform of nature environmental condition, and oneself draws nutrient;
(2) transplant: with peat soil, yellow soil and perlite, in the ratio of 2:3:1, fully mix and make nutrient matrix, be filled up in seedling-raising cup, seedling in bottle is carefully taken out, clean and stick the medium on seedling, transplant seedlings to the seedling-raising cup of filling up nutrient matrix, each cup plantation one young plant, transplant thickness of earth-fill cover and just cover root system, be generally no more than 1 centimetre, compress matrix, make shoot root and matrix close contact, the final singling water of drenching after having transplanted.
6, seedling culture: the seedling after transplanting will be strengthened water and fertilizer management and sick insect protected control in month, and suitably shade, guarantee that seedling survives, when growth of seedling is stablized, grow after sprouting and Xin Gen, progressively increase intensity of illumination, until carry out full exposure Routine Management, when seedling culture to 15 is centimetre high, can move bigger flowerpot cultivating large seedling.
The method of water and fertilizer management: keep matrix moistening, relative air humidity, more than 95%, prevents plant dehydration, 25 ~ 28 ℃ of cultivation temperature, 75% shading net shading for cool canopy; Transplant and progressively control water supply in media afterwards in 15 days, relative moisture is controlled at 65 ~ 75%, promotes new root sprouting to occur.
Diseases and pests controlling method: adopt 0.1% tpn spraying disinfection sterilizing for after sprigging the 3rd day, afterwards every spraying in 10 days once; When there being sprouting to sprout, when new root grows, spray 1800 times of nutrient solutions, according to seedling growth, every two weeks, spray once; The nutrient solution spraying is that the compounding medicine by following proportioning obtains: NH
4nO
3700mg/L, KNO
31000mg/L, CaCl
22H
2o 250mg/L, MgSO
47H
2o 400mg/L, Ca (NO
3)
2500mg/L, KH
2pO
4120mg/L, KI 0.6mg/L, H
3bO
35mg/L, MnSO
44H
2o 25mg/L, ZnSO
47H
2o 10mg/L, NaMoO
22H
2o 0.5mg/L, CuSO
45H
2o 0.05mg/L, CoCl
26H
2o 0.05mg/L.
Claims (8)
1. a cultured in vitro regeneration plant method for yellow fruit silvery birch, is characterized in that: comprise the following steps:
(1) gather explant and material processed thereof: gather robust growth, the branches without the semi-lignified on 5 years raw yellow fruit silvery birch tree crown tops of damage by disease and insect, cut off the blade on branch, retain petiole, pack into and in sealed bag, take back laboratory; Described material processed is first branch to be used to liquid detergent solution foam washing 15 ~ 20 minutes, in foam washing process, constantly shake bottle, with flowing water, rinse well, more alcohol-pickled 10 seconds that are 95% by concentration, then with flowing water, rinse 3 times, after cleaning, be cut into the stem section with 1 ~ 2 axillalry bud of 2 ~ 3 centimetres;
(2) set up sterile propagation body: the stem section that step (1) is obtained in aseptic experiment chamber with 75% alcohol sterilizing 1 ~ 2 minute, use again 0.1% mercuric chloride solution sterilizing 10 ~ 15 minutes, then use sterile water wash 3 ~ 4 times, one bottle of stem section is inoculated on MS1 medium, in culturing room, cultivate induction axillary bud sprouting; Described condition of culture is: 20 ~ 30 ℃ of room temperatures, and illumination every day 8 ~ 12 hours, intensity of illumination 800 ~ 2000LX, cultivates 30 ~ 32 days; When sprout green bud from axil, through cutting apart, transfer and can making the continuous growth and breeding of green bud, complete the foundation of sterile propagation body;
(3) propagation is cultivated: the sterile propagation body that step (2) is obtained is inoculated in the proliferated culture medium of MS2, being placed on manual control room temperature is 30 ~ 32 ℃, illumination every day 8 ~ 12 hours, cultivates under the condition of intensity of illumination 800 ~ 2000LX 30 ~ 32 days, from axil, sprouts aseptic green bud, the green bud of clip is inoculated on proliferated culture medium MS2, bud clump propagation, cultivated after 30 days, 5 ~ 6 times of bud clump propagation multiples, under aseptic condition, cut apart again switching, through subculture repeatedly, cultivated expanding propagation;
(4) culture of rootage: in the propagation incubation of step (3), Multiple Buds is high 20 millimeters when above, and Multiple Buds is cut from base portion, is divided into individual plant and is inoculated in MS3 root media, be placed on 20 ~ 30 ℃ of left and right of room temperature, illumination every day 8 ~ 12 hours, intensity of illumination is cultivated 20 ~ 25 days under the condition of 800 ~ 2000LX, and seedling starts root, be placed into again under 75% shading condition in greenhouse hardening 7 ~ 14 days, bottle seedling leaf of taking root launches, and elongation is healthy and strong, forms whole plant;
(5) transplant: the good bottle seedling of root growth that cultivation is obtained moves into greenhouse domestication, bottle seedling is from manual control condition of culture and give nutritive element to reform of nature environmental condition, and after the domestication process of oneself's absorption nutrient, be transplanted in the seedling-raising cup that grow seedling nutritional matrix is housed; Trasplanting method is: the seedling in bottle is carefully taken out, clean and stick the medium on seedling, transplant seedlings in the seedling-raising cup of filling up nutrient matrix, each cup plantation one young plant, transplant thickness of earth-fill cover and just cover root system, be generally no more than 1 centimetre, compress matrix, make shoot root and matrix close contact, the final singling water of drenching after having transplanted;
(6) seedling culture: the seedling after transplanting will be strengthened water and fertilizer management and sick insect protected control in month, and suitably shade, guarantee that seedling survives, when growth of seedling is stablized, grow after sprouting and Xin Gen, progressively increase intensity of illumination, until carry out full exposure Routine Management, when seedling culture to 15 is centimetre high, can move bigger flowerpot cultivating large seedling.
2. the cultured in vitro regeneration plant method of yellow fruit silvery birch according to claim 1, it is characterized in that: described MS1 medium is first culture base, its preparation is that carragheen 7000mg/L is added to 500ml distilled water, be heated to carragheen and dissolve completely, then add the nutritive element composition in the culture medium prescription being dissolved in the water in advance, add water to 1000ml, stir, pH is adjusted to 5.8, and bottling, sterilizing are standby; Nutritive element composition and quality proportioning thereof in described culture medium prescription are as follows: 6 ~ benzyladenine, 0.05 ~ 5.0mg/L, methyl α-naphthyl acetate 0.01 ~ 0.5mg/L, NH
4nO
3990 ~ 1650mg/L, KNO
31140 ~ 1900mg/L, CaCl
22H
2o 240 ~ 400mg/L, MgSO
47H
2o 74 ~ 370mg/L, KH
2pO
4102 ~ 170mg/L, KI 0.5 ~ 0.83mg/L, H
3bO
33.72 ~ 6.2mg/L, MnSO
44H
2o 13.38 ~ 22.3mg/L, ZnSO
47H
2o 5.16 ~ 8.6mg/L, NaMoO
22H
2o 0.15 ~ 0.25mg/L, CuSO
45H
2o 0.015 ~ 0.025mg/L, CoCl
26H
2o 0.015 ~ 0.025mg/L, Na
2eDTA 22.38 ~ 37.3mg/L, FeSO
47H
2o 16.68 ~ 27.8mg/L, inositol 60 ~ 100mg/L, nicotinic acid 0.3 ~ 0.5mg/L, puridoxine hydrochloride 0.3 ~ 0.5mg/L, nicotinic acid thiamine 0.06 ~ 0.1mg/L, glycine 1.2 ~ 2.0mg/L, sucrose 30000mg/L.
3. the cultured in vitro regeneration plant method of yellow fruit silvery birch according to claim 1, it is characterized in that: described MS2 medium is proliferated culture medium, its preparation is that carragheen 7000mg/L is added to 500ml distilled water, be heated to carragheen and dissolve completely, then add the nutritive element composition in the culture medium prescription being dissolved in the water in advance, add water to 1000ml, stir, pH is adjusted to 5.8, and bottling, sterilizing are standby; Nutritive element composition and quality proportioning thereof in described culture medium prescription are as follows: 6 ~ benzyladenine, 0.05 ~ 0.2mg/L, methyl α-naphthyl acetate 0.1 ~ 0.3mg/L, active carbon 1.0 ~ 3.0g/L, NH
4nO
3990 ~ 1650mg/L, KNO
31140 ~ 1900mg/L, CaCl
22H
2o 240 ~ 400mg/L, MgSO
47H
2o 222 ~ 370mg/L, KH
2pO
4102 ~ 170mg/L, KI 0.5 ~ 0.83mg/L, H
3bO
33.72 ~ 6.2mg/L, MnSO
44H
2o 13.38 ~ 22.3mg/L, ZnSO
47H
2o 5.16 ~ 8.6mg/L, NaMoO
22H
2o 0.15 ~ 0.25mg/L, CuSO
45H
2o 0.015 ~ 0.025mg/L, CoCl
26H
2o 0.015 ~ 0.025mg/L, Na
2eDTA 22.38 ~ 37.3mg/L, FeSO
47H
2o 16.68 ~ 27.8mg/L, inositol 60 ~ 100mg/L, nicotinic acid 0.3 ~ 0.5mg/L, puridoxine hydrochloride 0.3 ~ 0.5mg/L, nicotinic acid thiamine 0.06 ~ 0.1mg/L, glycine 1.2 ~ 2.0mg/L, sucrose 30000mg/L.
4. the cultured in vitro regeneration plant method of yellow fruit silvery birch according to claim 1, it is characterized in that: described MS3 medium is root induction medium, its preparation is that carragheen 7000mg/L is added to 500ml distilled water, be heated to carragheen and dissolve completely, then add the nutritive element composition in the culture medium prescription being dissolved in the water in advance, add water to 1000ml, stir, pH is adjusted to 5.8, and bottling, sterilizing are standby; Nutritive element composition and quality proportioning thereof in described formula are as follows: methyl α-naphthyl acetate 2.0 ~ 4.0mg/L, active carbon 1.0 ~ 3.0g/L, NH
4nO
3200 ~ 800mg/L, KNO
3250 ~ 900mg/L, CaCl
22H
2o 50 ~ 200mg/L, MgSO
47H
2o 222 ~ 370mg/L, Ca (NO
3)
2200 ~ 400mg/L, KH
2pO
4102 ~ 170mg/L, KI 0.5 ~ 0.83mg/L, H
3bO
33.72 ~ 6.2mg/L, MnSO
44H
2o 13.38 ~ 22.3mg/L, ZnSO
47H
2o 5.16 ~ 8.6mg/L, NaMoO
22H
2o 0.15 ~ 0.25mg/L, CuSO
45H
2o 0.015 ~ 0.025mg/L, CoCl
26H
2o 0.015 ~ 0.025mg/L, Na
2eDTA 22.38 ~ 37.3mg/L, FeSO
47H
2o 16.68 ~ 27.8mg/L, inositol 50 ~ 100mg/L, nicotinic acid 0.25 ~ 0.5mg/L, puridoxine hydrochloride 0.5 ~ 1.0mg/L, nicotinic acid thiamine 0.1 ~ 0.25mg/L, glycine 1.0 ~ 2.0mg/L, sucrose 15000 ~ 30000mg/L.
5. the cultured in vitro regeneration plant method of yellow fruit silvery birch according to claim 1, is characterized in that: the described grow seedling nutritional matrix of step (5) is that peat soil, yellow soil and perlite are fully mixed to get in the ratio of 2:3:1.
6. the cultured in vitro regeneration plant method of yellow fruit silvery birch according to claim 1, it is characterized in that: the water and fertilizer management method of the described seedling of step (6) after transplanting is: keep matrix moistening, relative air humidity is more than 95%, prevent plant dehydration, 25 ~ 28 ℃ of cultivation temperature, 75% shading net shading for cool canopy; Transplant and progressively control water supply in media after 15 days, relative moisture is controlled at 60 ~ 80%, promotes new root sprouting to occur.
7. the cultured in vitro regeneration plant method of yellow fruit silvery birch according to claim 1, it is characterized in that: sick insect protected the prevent and treat method of the described seedling of step (6) after transplanting is: within after sprigging the 3rd day, adopt 0.1% tpn spraying disinfection sterilizing, afterwards every spraying in 10 ~ 15 days once; When there being sprouting to sprout, when new root grows, spray 1200 ~ 1800 times of nutrient solutions, according to seedling growth, every two weeks, spray once.
8. the cultured in vitro regeneration plant method of yellow fruit silvery birch according to claim 7, is characterized in that: described nutrient solution is that the compounding medicine by following quality proportioning forms: NH
4nO
3700 ~ 900mg/L, KNO
3800 ~ 1000mg/L, CaCl
22H
2o 150 ~ 250mg/L, MgSO
47H
2o 280 ~ 400mg/L, Ca (NO
3)
2300 ~ 500mg/L, KH
2pO
4120 ~ 220mg/L, KI 0.6 ~ 1.0mg/L, H
3bO
35 ~ 8mg/L, MnSO
44H
2o 20 ~ 25mg/L, ZnSO
47H
2o 6.5 ~ 10mg/L, NaMoO
22H
2o 0.1 ~ 0.5mg/L, CuSO
45H
2o 0.01 ~ 0.05mg/L, CoCl
26H
2o 0.01 ~ 0.05mg/L.
Priority Applications (1)
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| CN105766586A (en) * | 2016-03-30 | 2016-07-20 | 孙德灿 | Cultivation method of scindapsus aureus |
| CN105941147A (en) * | 2016-05-06 | 2016-09-21 | 江苏东郁园林科技有限公司 | Betula papyrifera tissue culture seedling propagation method |
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| WO2007121518A1 (en) * | 2006-04-21 | 2007-11-01 | The State Of Western Australia Through Its Department Of Agriculture | Improved plant culture methods using a modified auxin treatment step |
| CN101743908A (en) * | 2009-12-22 | 2010-06-23 | 佛山市林业科学研究所 | Tissue culture, rapid propagation and cultivation method of grevillea banksii |
| CN103975832A (en) * | 2014-01-28 | 2014-08-13 | 华南农业大学 | Out-of-tube rooting method for tissue culture seedlings of silver birch |
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| WO2007121518A1 (en) * | 2006-04-21 | 2007-11-01 | The State Of Western Australia Through Its Department Of Agriculture | Improved plant culture methods using a modified auxin treatment step |
| CN101743908A (en) * | 2009-12-22 | 2010-06-23 | 佛山市林业科学研究所 | Tissue culture, rapid propagation and cultivation method of grevillea banksii |
| CN103975832A (en) * | 2014-01-28 | 2014-08-13 | 华南农业大学 | Out-of-tube rooting method for tissue culture seedlings of silver birch |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105766586A (en) * | 2016-03-30 | 2016-07-20 | 孙德灿 | Cultivation method of scindapsus aureus |
| CN105941147A (en) * | 2016-05-06 | 2016-09-21 | 江苏东郁园林科技有限公司 | Betula papyrifera tissue culture seedling propagation method |
| CN105941147B (en) * | 2016-05-06 | 2018-06-01 | 江苏东郁园林科技有限公司 | Paper birch tissue culture of sprout mating system |
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