CN106489734B - The breeding method of the nontoxic seedling of fortune paulownia - Google Patents
The breeding method of the nontoxic seedling of fortune paulownia Download PDFInfo
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- CN106489734B CN106489734B CN201610937949.3A CN201610937949A CN106489734B CN 106489734 B CN106489734 B CN 106489734B CN 201610937949 A CN201610937949 A CN 201610937949A CN 106489734 B CN106489734 B CN 106489734B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a kind of breeding method of the nontoxic seedling of fortune paulownia, comprise the following steps：1) using stem section of the fortune paulownia with axillary bud as explant, primary seedling is formed through Initial culture after sterilization；2) gained seedling is subjected to virus-free culture, obtains detoxification material；3) gained detoxification material is alternately accessed into illumination cultivation in high concentration plant hormone subculture medium and low-consistency plant hormone subculture medium, obtains tufted seedling；4) culture of rootage will be carried out after gained tufted seedling plant division, obtains rooted seedling.The method of the invention is simple to operate, detoxification, breeds and efficiency high of taking root, the rooted seedling well developed root system and fine hair of gained it is short (<1mm), seedling saving of work and time is washed, transplanting survival rate is high.
Description
Technical field
The present invention relates to the tissue culture technique of plant, and in particular to the breeding method of the nontoxic seedling of fortune paulownia.
Background technology
Fortune paulownia Paulownia fortunei (Seem.) Hemsl be Scrophulariaceae, the high megaphanerophyte of Paulownia, main product in
Southern china, high up to 30 meters, trunk is straight, and for the diameter of a cross-section of a tree trunk 1.3 meters above the ground up to 2 meters, being that a kind of purposes is wide, economic value is high, fast-growing is high-quality uses material
Deciduous species.The characteristics of fortune paulownia choiceness is：(1) good stress resistance, strong adaptability, can plant on the south the Yangtze river basin
Plant；(2) speed of growth soon, 5-6 can fell, mean DBH increment 42cm, per mu yield 33m3；(3) material is light, good toughness,
Density is low, intensity and bending strength are big, and economic value is higher.
To ensure that select tree character can stablize heredity, its sapling multiplication must be carried out by vegetative manner.Existing paulownia
Vegetative manner generally have grafting, divide the various ways such as root and tissue culture.Wherein, propagation by grafiting needs more manpower, thing
Power and financial resources, it is unsuitable for the large-scale production of paulownia seedling.And paulownia divide root nursery to be mainly to choose the raw paulownia kind roots of 1-2, one
As in late Febuary to early March bury root nursery, although its method simple and fast, emergence rate is high, and cost is low, needs relatively more
Vegetable material, speed and limits throughput；And requirement to soil is higher, it is necessary to which soil property is good, no disease and pests harm, dries without product
The edaphic conditions such as water, fertility abundance；The necessary no disease and pests harm of selected kind root, damage are lighter, if selection is improper, it is easy to cause
Emergence rate is low and triggers paulownia witches broom.Compared to grafting and root division, tissue culture detoxifying fast breeding more quickness and high efficiency, and not by point
The limitation of time of root nursery, material and soil property, a large amount of nontoxic high quality seedlings can be obtained in a short time, are steeped after mitigating transplanting
The generation and harm of paulownia withes broom.
Although the seedling detoxifying fast breeding technique of fortune paulownia is currently reported, different fortune paulownia fine individual plants,
Its optimal tissue culture detoxification technology and method difference (Li Fangdong, Sino-South African Forestry University of Science and Technology's journal, the phase of volume 2010,30 8,22-28
Page).The applicant has found in the prior art still in fortune paulownia choiceness seedling tissue-culturing rapid propagation industrialization process is studied
In the presence of the subject matter of three influence Industrial Efficiencies：First, the group training material detoxification time is grown, effect is not ideal enough, and detoxification efficiency is not
It is high；Second, on the single culture medium of high hormone concentration after multiple squamous subculture, the easy vitrifying of material, usual 5th generation
Glass rate is about 20%, and has elevated trend with the increase glass rate of subculture number, significantly influences seedling quality, numerous
Grow rate, rooting rate and survival rate；Third, tissue-cultured seedling, when being taken root on the culture medium that agar is supporter, root system surface is grown largely
The fine hair for being about 5mm, adsorb substantial amounts of culture medium, it is time-consuming to wash seedling, easily seedling is caused to damage washing seedling, influenceed
Wash seedling efficiency and transplanting survival rate.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of breeding method of the nontoxic seedling of fortune paulownia.This method operates
Simply, detoxification, breed and efficiency high of taking root, the rooted seedling robust plant of gained, well developed root system, the fine hair on root system it is short (<
1mm), seedling saving of work and time is washed, transplanting survival rate is high.
The breeding method of the nontoxic seedling of fortune paulownia of the present invention, comprises the following steps：
1) using stem section of the fortune paulownia with axillary bud as explant, it is inoculated in after sterilization in Initial culture base, illumination cultivation, directly
Axillary bud development on to explant grows to form primary seedling；
2) gained seedling is cut into the stem section with more than 1 or 2 axillary bud, be transferred in virus-free culture base, in 36-39 DEG C of knot
Virus-free culture is carried out under conditions of conjunction blue light illumination, obtains detoxification material；
3) gained detoxification material is cut into the stem section with more than 1 or 2 axillary bud, alternately access high concentration plant hormone after
For illumination cultivation in culture medium and low-consistency plant hormone subculture medium, tufted seedling is obtained；Wherein：
Described high concentration plant hormone squamous subculture based formulas is：MS+6-BA 3.5-4.5mg·L-1+IBA 0.35-
0.45mg·L-1+ sucrose 25-35gL-1+ agar 3.0-4.0gL-1, pH 5.6-6.0；
Described low-consistency plant hormone squamous subculture based formulas is：MS+6-BA 0.2-0.6mg·L-1+IBA 0.02-
0.06mg·L-1+ sucrose 25-35gL-1+ agar 3.0-4.0gL-1, pH 5.6-6.0；
4) by gained tufted seedling plant division, it is transferred in root media, illumination cultivation, obtains rooted seedling；Described training of taking root
Foster based formulas is：1/2MS+NAA 0.1-0.4mg·L-1+ sucrose 15-25gL-1+ carragheen 3.2-3.6gL-1, pH 5.6-
6.0。
It is same as the prior art to disinfecting for explant in the step 1) of above-mentioned breeding method, at specific sterilization
Reason can be carried out as follows：Take explant to be placed in the liquid detergent aqueous solution that mass concentration is 1-3% and soak 3-5min, take out
Rinsed well afterwards with flowing water, then be placed in the ethanol for being 70-75% with volumetric concentration on superclean bench and soak 50-70s, after taking-up
The HgCl that mass concentration is 0.1-0.2% is placed in again2Middle immersion 5-8min, take out, sterile water wash 3-5 times.
In the step 1) of above-mentioned breeding method, described Initial culture based formulas is preferably：MS+6-BA1.0-4.0mg·
L-1+IBA 0.1-0.4mg·L-1+ sucrose 25-35gL-1+ agar 3.0-4.0gL-1, pH5.6-6.0；More preferably：MS+
6-BA 2.0mg·L-1+IBA 0.2mg·L-1+ sucrose 30gL-1+ agar 3.5gL-1, pH 5.8.It is described in the step
Illumination cultivation condition is：Temperature is 27 ± 5 DEG C, light application time 10-15h/d, and intensity of illumination is 40 ± 5 μm of olm-2·s-1；
More preferably：Temperature is 28 ± 2 DEG C, light application time 10-12h/d, and intensity of illumination is 40 μm of olm-2·s-1.The step
In rapid, typically think to form primary sprout when the axillary bud development on explant grows to 2-3cm.
In the step 2) of above-mentioned breeding method, described virus-free culture based formulas is preferably：MS+6-BA3.5-4.5mg·
L-1+IBA 0.35-0.45mg·L-1+ sucrose 25-35gL-1+ agar 3.0-4.0gL-1, pH5.6-6.0；More preferably：MS
+6-BA 4.0mg·L-1+IBA 0.4mg·L-1+ sucrose 30gL-1+ agar 3.5gL-1, pH 5.8.It is described in the step
Blue light wavelength used is 465 ± 10nm during blue light illumination, and its intensity of illumination is 40 ± 5 μm of olm-2·s-1, light application time
10-15h/d；Further preferred blue light intensity of illumination is 40 ± 5 μm of olm-2·s-1, light application time 10-12h/d.
Applicants have discovered that in the step 3) of above-mentioned breeding method, by using high concentration plant hormone squamous subculture
The subculture method of base and low-consistency plant hormone subculture medium alternate culture, the same of material propagation efficiency can ensured
When, moreover it is possible to glass rate is reduced to less than 5%.The high concentration plant hormone subculture medium and low-consistency plant hormone after
For the subculture method of culture medium alternate culture, refer to detoxification material first accessing the training of high concentration plant hormone subculture medium
A subculture cycle is supported, material is then taken out and accesses low-consistency plant hormone subculture medium one subculture week of culture again
Phase；……；So move in circles alternate culture, until obtaining enough materials carries out culture of rootage.In the process, no matter material
Material is grown in high concentration plant hormone subculture medium, or is longer than in low-consistency plant hormone subculture medium, as long as not
There is vitrifying, plant height in more than 4cm sprout, be used equally for culture of rootage；Other materials for not reaching requirement of taking root
(as thinner and more delicate or short and small plant), can enter next squamous subculture cycle as subculture material, be that next round is taken root training
Support and carry out material stock.In the step, described high concentration plant hormone squamous subculture based formulas is preferably：MS+6-
BA4.0mg·L-1+IBA0.4mg·L-1+ sucrose 30gL-1+ agar 3.5gL-1, pH 5.8；Described low-consistency plant swashs
Plain squamous subculture based formulas is preferably：MS+6-BA0.5mg·L-1+IBA0.05mg·L-1+ sucrose 30gL-1+ agar 3.5g
L-1, pH 5.8.In the step, the illumination cultivation and in low-consistency plant hormone subculture in high concentration plant hormone subculture medium
Illumination cultivation condition is identical in culture medium, is：Temperature be 27 ± 5 DEG C, light application time 10-15h/d, intensity of illumination be 40 ±
5μmol·m-2·s-1；More preferably：Temperature is 28 ± 2 DEG C, light application time 10-12h/d, and intensity of illumination is 40 μ
mol·m-2·s-1。
In the step 4) of above-mentioned breeding method, described prescription of rooting medium is preferably：1/2MS+NAA0.2mg·L-1+
Sucrose 20gL-1+ carragheen 3.4gL-1, pH 5.8.Applicant has found in specific experimentation, when in culture of rootage
During using solid support of the carragheen as root media, not only rootage duration is short, rooting rate is high, well developed root system is pure white,
And the growth of fine hair on gained rooted seedling root system during using agar as solid support can be suppressed, control the length of fine hair
Degree is less than 1mm, so that rooted seedling is easier to wash, substantially increases and washes seedling efficiency and transplanting survival rate.It is described in the step
Illumination cultivation condition be：Temperature is 27 ± 5 DEG C, light application time 10-15h/d, and intensity of illumination is 40 ± 5 μm of olm-2·s-1；More preferably：Temperature is 28 ± 2 DEG C, light application time 10-12h/d, and intensity of illumination is 40 μm of olm-2·s-1.Should
In step, when by tufted seedling plant division, more than plant height 4.0cm healthy and strong tufted seedling is preferably selected to carry out plant division.
Under the condition of culture that appeal limits, explant is cultivated in Initial culture base obtains primary seedling (2-3cm) institute
Take time usually 25-35d；The time usual 10-15d of virus-free culture；Stem section with axillary bud is in high concentration plant hormone subculture
A subculture cycle in culture medium is identical with a subculture cycle in low-consistency plant hormone subculture medium, is usually
25-30d；It is usually 10-15d, now every plant long 3- that tufted seedling, which is cultivated in root media the time required to obtaining rooted seedling,
10, root long 3-5cm, and the root system of gained rooted seedling is white, the fine hair on root system it is short (<1mm), it is easy to wash, meets
Acclimatization and transplantses requirement.
Compared with prior art, the method have the characteristics that：
1st, before Multiple Buds largely expand, trained using the high temperature of 465 ± 10nm of wavelength 36-39 DEG C of blue light illumination combination
10-15d is supported, detoxification treatment is carried out to sprout, detoxification efficiency is improved, virus elimination rate is reached 100%；
2nd, using high concentration plant hormone subculture medium and low-consistency plant hormone subculture medium alternate culture after
For cultural method, the propagation efficiency of material was both ensured, the glass rate of material is reduced to less than 5% by and can；
3rd, in the culture of rootage stage, replace agar to be used as culture medium solid support using carragheen, and combine specific sharp
Plain concentration, not only makes that rootage duration is short, rooting rate is high, well developed root system is pure white, and inhibits fine hair on rooted seedling root system
Grow (villus length<1mm), make rooted seedling be easier to wash, so as to reduce the damage when washing seedling to rooted seedling, carry significantly
It is high to wash seedling efficiency and transplanting survival rate；
4th, the method for the invention is simple to operation, detoxification, breeds and efficiency high of taking root, and it is raw that industrialization batch can be achieved
Production.
Brief description of the drawings
Fig. 1 is the picture that the embodiment of the present invention 1 cultivates obtained primary seedling；
Fig. 2 is the picture that the embodiment of the present invention 1 cultivates obtained tufted seedling；
Fig. 3 is the picture that the embodiment of the present invention 1 cultivates obtained rooted seedling, wherein root system is pure white, fine hair is short (<1mm)；
Fig. 4 is that comparative example 3 cultivates the obtained picture of rooted seedling, and wherein root system is pale yellow, around overgrow with shallow white it is vaporific,
It is about 5mm fine hair；
Fig. 5 is the life that the embodiment of the present invention 1 and comparative example 3 cultivate picture, the wherein left side after obtained rooted seedling washing
Offspring (embodiment 1 cultivate obtain) root system fine hair it is short (<1mm), adhere to substantially without culture medium after washing；The rooted seedling on the right
(comparative example 3 is cultivated and obtained) root system fine hair is longer (about 5mm), and more culture medium is still adhered to after washing, and is difficult to clean；
Fig. 6 is the picture that the embodiment of the present invention 1 cultivates obtained transplanted seedling.
Embodiment
With reference to specific embodiment, the present invention is described in further detail, to more fully understand present disclosure, but
The present invention is not limited to following examples.
Embodiment 1
1) with fortune paulownia, (fortune paulownia is by existing in fortune paulownia out of osmanthus backlands area Guangxi Plant Inst.
Conventional screening assays are screened obtained fortune paulownia choiceness) stem section with axillary bud as explant, is placed in quality
Concentration is to soak 10min in 1% liquid detergent solution, is rinsed well under flowing tap water after taking-up, then be placed in ultra-clean work
Make to soak 60s with the ethanol that volumetric concentration is 70% on platform, be placed in the HgCl that mass concentration is 0.1% after taking-up again2Middle immersion
7min, take out, with sterile water wash 5 times；
2) explant after sterilization is inoculated in Initial culture base, illumination cultivation 30d, the growth of explant axillary bud development,
Form the high sprouts (as shown in Figure 1) of 2-3cm, as primary seedling；Wherein, described Initial culture based formulas is：MS+6-BA
2.0mg·L-1+IBA 0.2mg·L-1+ sucrose 30gL-1+ agar 3.5gL-1, pH 5.8；Described illumination cultivation condition
For：Temperature is 28 ± 3 DEG C, light application time 10h/d, and intensity of illumination is 40 μm of olm-2·s-1；Gained primary in aforementioned manners
Seedling is healthy and strong, blade emerald green, well-grown, inductivity 70%；
3) the seedling sub-cut of gained is accessed in virus-free culture base, in 37-39 DEG C of knot into the stem section with 1 or 2 axillary bud
Close under the conditions of blue light (465 ± 10nm of wavelength) irradiates and carry out virus-free culture 15d, detoxification treatment is carried out to sprout, obtains detoxification material
Material；Wherein, described virus-free culture based formulas is：MS+6-BA4.0mg·L-1+IBA0.4mg·L-1+ sucrose 30gL-1+ fine jade
Fat 3.5gL-1, pH 5.8；The intensity of illumination of blue light is 40 ± 5 μm of olm during virus-free culture-2·s-1, light application time 10h/
d；Detoxification efficiency is up to 100% in aforementioned manners；
4) gained detoxification material sub-cut is first accessed into the training of high concentration plant hormone subculture into the stem section with 1 or 2 axillary bud
Support in base, one subculture cycle (25d) of illumination cultivation, breeding coefficient 6.0/25d；Material sub-cut is then taken out into 1 or 2
The stem section of individual axillary bud, then access in low-consistency plant hormone subculture medium, one subculture cycle (25d) of illumination cultivation, breed
Coefficient 5.0/25d；……；So move in circles alternate culture, until obtaining enough materials is used for culture of rootage；Wherein, institute
The high concentration plant hormone squamous subculture based formulas stated is：MS+6-BA4.0mg·L-1+IBA 0.4mg·L-1+ sucrose 30gL-1+ agar 3.5gL-1, pH 5.8；Low-consistency plant hormone squamous subculture based formulas is：MS+6-BA 0.5mg·L-1+IBA
0.05mg·L-1+ sucrose 30gL-1+ agar 3.5gL-1, pH 5.8；Illumination cultivation condition in two kinds of culture mediums is identical,
It is：Temperature is 28 ± 3 DEG C, light application time 10h/d, and intensity of illumination is 40 μm of olm-2·s-1；Pass through above-mentioned alternate culture
The tufted seedling quantity of gained is more, average more than breeding coefficient 5.0/25d, and plant is relatively strengthened, grown fine, and glass rate is less than 5%,
The tufted seedling cultivated obtained by a cycle (25d) is highly 3-6cm (as shown in Figure 2)；
5) choose height to be transferred in root media after more than 4cm healthy and strong tufted seedling plant division, illumination cultivation 12d, obtain
To fortune paulownia rooted seedling；Wherein, described prescription of rooting medium is：1/2MS+NAA 0.2mg·L-1+ sucrose 20gL-1
+ carragheen 3.4gL-1, pH 5.8；The condition of culture of the illumination cultivation is：Temperature is 28 ± 3 DEG C, light application time 10h/
D, intensity of illumination are 40 μm of olm-2·s-1；In aforementioned manners gained rooted seedling plant strain growth it is good, well developed root system, it is pure white,
Fine hair it is short (<1mm), it is easy to wash, every plant long 5-10 bar, root long 3-5cm, rooting rate 100% is (left in such as Fig. 3 and Fig. 5
Shown in the rooted seedling on side)；
6) gained rooted seedling is subjected to hardening according to a conventional method, that is, obtains the nontoxic seedling of fortune paulownia (as shown in Figure 6).
Comparative example 1
Embodiment 1 is repeated, unlike：
Initial culture based formulas in step 2) is changed to：MS+6-BA 0.5mg·L-1+IBA 0.05mg·L-1+ sucrose
30g·L-1+ agar 3.5gL-1, pH 5.8；Primary sprout induced velocity is slow, and inductivity is low, and only 25%；
Poison-removing method in step 3) is changed to：Using white light, (intensity of illumination is 40 ± 5 μm of olm-2·s-1, illumination when
Between 10h/d) combine 33-35 DEG C of temperature culture 25d, detoxification efficiency 15%；
High concentration plant hormone culture medium prescription in step 4) is changed to：MS+6-BA 2.0mg·L-1+IBA
0.02mg·L-1+ sucrose 30gL-1+ agar 3.5gL-1(pH 5.8), Multiple Buds growth is general, and part sprouting pushes up after growing
It is withered to die；Other 10% material can not induce the direct withered death of Multiple Buds and part, dysoemia；Average line of breeding
Number 4.0/25d；Low-consistency plant hormone culture-medium formula is changed to：MS+6-BA 0.1mg·L-1+IBA 0.01mg·L-1+ sugarcane
Sugared 30gL-1+ agar 3.5gL-1(pH 5.8), tufted seedling induction is slow, inductivity is low, and invalid explant is more, breeding coefficient
2.0；Alternately the average coefficient of proliferation of subculture is 3.0/30d；
Prescription of rooting medium in step 5) is changed to：1/2MS+NAA 0.05mg·L-1+ sucrose 20gL-1+ OK a karaoke club
Glue 3.4gL-1, pH 5.8；Seedling well-grown, but rooting rate is low, only 53%, and root is thinner.
Comparative example 2
Embodiment 1 is repeated, unlike：
Initial culture based formulas in step 2) is changed to：MS+6-BA 5.0mg·L-1+IBA 0.5mg·L-1+ sucrose
30g·L-1+ agar 3.5gL-1, pH 5.8；There are a large amount of callus to produce, primary sprout can not be formed, and more than half explant is bad
Extremely；
Poison-removing method in step 3) is changed to：Using white light, (intensity of illumination is 40 ± 5 μm of olm-2·s-1, illumination when
Between 10h/d) combine 40-42 DEG C of temperature culture, because temperature is too high, material is most of dead after a few days for culture, does not reach and educates
Seedling effect；
High concentration plant hormone culture medium prescription in step 4) is changed to：MS+6-BA 6.0mg·L-1+IBA
0.6mg·L-1+ sucrose 30gL-1+ agar 3.5gL-1(pH 5.8)；Tufted seedling bottom produces a large amount of callus, most of
Tufted seedling growth is slow, and the later stage is slowly withered, breeding coefficient 1.6；Low-consistency plant hormone culture-medium formula is changed to：MS+6-BA
0.1mg·L-1+IBA 0.01mg·L-1+ sucrose 30gL-1+ agar 3.5gL-1(pH 5.8), material can induce sprouting,
Sprout part growth is normal, plant is sturdy, and a part of apical dieback is dead, breeding coefficient 2.6/30d；The alternately average proliferation of subculture
Coefficient is 2.1/30d；
Prescription of rooting medium in step 5) is revised as：1/2MS+NAA 0.8mg·L-1+ sucrose 20gL-1+ card
Draw glue 3.4gL-1, pH 5.8；Seedling rooting of growing thickly is few, and rootage duration is uneven, rooting rate 67%.
Comparative example 3
Embodiment 1 is repeated, unlike：
In step 5), the solid support of root media is changed to agar, i.e. prescription of rooting medium by carragheen
It is revised as：1/2MS+NAA 0.2mg·L-1+ sucrose 20gL-1+ agar 3.5gL-1, pH5.8；The growth of gained rooted seedling is good
Good, every plant long 3-7 bar, root long 3-6cm, rooting rate 100%, root color is pale yellow, but is covered with root system and be largely about the thin of 5mm
Fine hair (as shown in the rooted seedling on the right in Fig. 4 and Fig. 5), adhering to substantial amounts of culture medium, is not easy to clean up, wash seedling and take a lot of work expense
When.
Comparative example 4
Embodiment 1 is repeated, unlike：
Subculture method in step 4) is changed to：Only circulated with single low-consistency plant hormone subculture medium
Subculture, wherein, subculture medium is MS+6-BA 0.5mgL-1+IBA 0.05mg·L-1+ sucrose 30gL-1+ agar
3.5g·L-1(pH 5.8).As a result find, after more than 5 times continuous subcultures, only 70% culture materials, which can induce, grows thickly
Bud, and Multiple Buds quantity is on the low side, the plant of formation is sturdy tall and big, and leaf width is big, bud green；30% material, which can not induce, grows thickly
Bud, the direct withered death in part；Although without vitrified material, average breeding coefficient is too low, only 2.5/25d.
Comparative example 5
Embodiment 1 is repeated, unlike：
Subculture method in step 4) is changed to：Only with single intermediate concentration plant hormone culture medium circulated after
Generation, wherein, subculture medium is MS+6-BA 2.0mgL-1+IBA 0.2mg·L-1+ sucrose 30gL-1+ agar 3.5gL-1
(pH 5.8).As a result find, after more than 5 times continuous subcultures, 75% material can induce Multiple Buds, Multiple Buds growth one
As, Ye Lv, of medium size, it is dead that part sprouting grows rear apical dieback；25% material can not induce Multiple Buds and part is direct
Withered death；Average breeding coefficient 3.2/25d, glass rate are more than 10%.
Comparative example 6
Embodiment 1 is repeated, unlike：
Subculture method in step 4) is changed to：Only circulated with single high concentration plant hormone subculture medium
Subculture, wherein, subculture medium MS+6-BA4.0mgL-1+IBA 0.4mg·L-1+ sucrose 30gL-1+ agar 3.5g
L-1(pH 5.8).As a result find, the tufted seedling well-grown obtained by preceding 5 generation, leaf is bud green, of medium size, breeding coefficient 6.0/
25d；But to the 5th instead of after, start vitrified material occur, vitrifying yield be more than 20%, breeding coefficient drops to 3.0/
25d。
Embodiment 2
1) with fortune paulownia, (fortune paulownia is by existing in fortune paulownia out of osmanthus backlands area Guangxi Plant Inst.
Conventional screening assays are screened obtained fortune paulownia choiceness) stem section with axillary bud as explant, is placed in quality
Concentration is to soak 10min in 1% liquid detergent solution, is rinsed well under flowing tap water after taking-up, then be placed in ultra-clean work
Make to soak 50s with the ethanol that volumetric concentration is 70% on platform, be placed in the HgCl that mass concentration is 0.1% after taking-up again2Middle immersion
6min, take out, with sterile water wash 4 times；
2) explant after sterilization is inoculated in Initial culture base, illumination cultivation 25d, the growth of explant axillary bud development,
The high sprouts of 2-3cm, as primary seedling are formed, gained seedling is healthy and strong, blade emerald green, well-grown, inductivity 65%；Its
In, described Initial culture based formulas is：MS+6-BA 1.0mg·L-1+IBA 0.4mg·L-1+ sucrose 25gL-1+ agar
3.0g·L-1, pH 5.8；Described illumination cultivation condition is：Temperature is 28 ± 3 DEG C, light application time 12h/d, and intensity of illumination is
45μmol·m-2·s-1；
3) the seedling sub-cut of gained is accessed in virus-free culture base, in 37-39 DEG C of knot into the stem section with 1 or 2 axillary bud
Close under the conditions of blue light (465 ± 10nm of wavelength) irradiates and carry out virus-free culture 10d, detoxification treatment, detoxification efficiency are carried out to sprout
100%, obtain detoxification material；Wherein, described virus-free culture based formulas is：MS+6-BA 4.0mg·L-1+IBA 0.4mg·
L-1+ sucrose 30gL-1+ agar 3.5gL-1, pH 5.8；The intensity of illumination of blue light is 35 μm of olm during virus-free culture-2·s-1, light application time 12h/d；
4) gained detoxification material sub-cut is first accessed into the training of high concentration plant hormone subculture into the stem section with 1 or 2 axillary bud
Support in base culture, one subculture cycle (25d) of illumination cultivation, breeding coefficient 5.0/25d；Then take out material sub-cut into
The stem section of 1 or 2 axillary bud, then one subculture cycle (25d) of illumination cultivation in low-consistency plant hormone subculture medium is accessed, it is numerous
Grow coefficient 4.0/25d；……；So move in circles alternate culture, until obtaining enough materials is used for culture of rootage；Wherein,
Described high concentration plant hormone squamous subculture based formulas is：MS+6-BA 3.5mg·L-1+IBA 0.45mg·L-1+ sucrose
25g·L-1+ agar 3.0gL-1, pH 5.8；Low-consistency plant hormone squamous subculture based formulas is：MS+6-BA0.6mg·L-1+
IBA 0.02mg·L-1+ sucrose 25gL-1+ agar 3.0gL-1, pH 5.8；Illumination cultivation condition in two kinds of culture mediums
It is identical, be：Temperature is 28 ± 3 DEG C, light application time 12h/d, and intensity of illumination is 40 μm of olm-2·s-1；Pass through above-mentioned friendship
For more, the average breeding coefficient 4.5/25d of tufted seedling quantity of culture gained, plant grows fine, and leaf is bud green, of medium size,
Glass rate is less than 5%, and the tufted seedling obtained by culture a cycle (25d) is highly 2-6cm；
5) choose healthy and strong tufted seedling plant division of the height in more than 4cm to be transferred in root media, illumination cultivation 12d, obtain
Fortune paulownia rooted seedling；Wherein, described prescription of rooting medium is：1/2MS+NAA0.15mg·L-1+ sucrose 15gL-1+
Carragheen 3.3gL-1, pH 5.8；The condition of culture of the illumination cultivation is：Temperature is 28 ± 3 DEG C, light application time 10h/d,
Intensity of illumination is 40 μm of olm-2·s-1；The rooted seedling plant strain growth of gained is good in aforementioned manners, well developed root system, pure white, thin
Fine hair it is short (<1mm), it is easy to wash, every plant long 4-7 bar, root long 3-5cm, rooting rate 95%；
6) gained rooted seedling is subjected to hardening according to a conventional method, that is, obtains the nontoxic seedling of fortune paulownia.
Embodiment 3
1) explant is used as using the stem section of fortune paulownia (fortune paulownia is fortune paulownia choiceness C001) with axillary bud
Body, it is placed in the liquid detergent solution that mass concentration is 1% and soaks 10min, rinsed well under flowing tap water after taking-up, then
It is placed on superclean bench and soaks 70s with the ethanol that volumetric concentration is 75%, it is 0.2% to be placed in mass concentration after taking-up again
HgCl2Middle immersion 8min, take out, with sterile water wash 5 times；
2) explant after sterilization is inoculated in Initial culture base, illumination cultivation 25d, the growth of explant axillary bud development,
The high sprouts of 2-3cm, as primary seedling are formed, gained seedling is healthy and strong, blade emerald green, well-grown, inductivity 60%；Its
In, described Initial culture based formulas is：MS+6-BA 4.0mg·L-1+IBA 0.1mg·L-1+ sucrose 35gL-1+ agar
4.0g·L-1, pH 5.8；Described illumination cultivation condition is：Temperature is 27 ± 5 DEG C, light application time 15h/d, and intensity of illumination is
35μmol·m-2·s-1；
3) the seedling sub-cut of gained is accessed in virus-free culture base, in 36-38 DEG C of knot into the stem section with 1 or 2 axillary bud
Close under the conditions of blue light (465 ± 10nm of wavelength) irradiates and carry out virus-free culture 13d, detoxification treatment, detoxification efficiency are carried out to sprout
100%, obtain detoxification material；Wherein, described virus-free culture based formulas is：MS+6-BA 4.0mg·L-1+IBA 0.4mg·
L-1+ sucrose 35gL-1+ agar 4.0gL-1, pH 5.8；The intensity of illumination of blue light is 45 μm of olm during virus-free culture-2·s-1, light application time 10h/d；
4) gained detoxification material sub-cut is first accessed into the training of high concentration plant hormone subculture into the stem section with 1 or 2 axillary bud
Support in base culture, one subculture cycle (25d) of illumination cultivation, breeding coefficient 5.6/25d；Then take out material sub-cut into
The stem section of 1 or 2 axillary bud, then one subculture cycle (25d) of illumination cultivation in low-consistency plant hormone subculture medium is accessed, it is numerous
Grow coefficient 4.0/25d；……；So move in circles alternate culture, until obtaining enough materials is used for culture of rootage；Wherein,
Described high concentration plant hormone squamous subculture based formulas is：MS+6-BA 4.5mg·L-1+IBA 0.35mg·L-1+ sucrose
35g·L-1+ agar 4.0gL-1, pH 5.8；Low-consistency plant hormone squamous subculture based formulas is：MS+6-BA0.2mg·L-1+
IBA0.06mg·L-1+ sucrose 35gL-1+ agar 4.0gL-1, pH 5.8；Illumination cultivation condition phase in two kinds of culture mediums
Together, it is：Temperature is 27 ± 3 DEG C, light application time 15h/d, and intensity of illumination is 40 μm of olm-2·s-1；Pass through above-mentioned alternating
More, the average breeding coefficient 4.8/25d of tufted seedling quantity of gained is cultivated, plant grows fine, and leaf is bud green, of medium size, glass
Glass rate is less than 5%, and the tufted seedling obtained by culture a cycle (25d) is highly 2-6cm；
5) choose healthy and strong tufted seedling plant division of the height in more than 4cm to be transferred in root media, illumination cultivation 12d, obtain
Fortune paulownia rooted seedling；Wherein, described prescription of rooting medium is：1/2MS+NAA0.35mg·L-1+ sucrose 25gL-1+
Carragheen 3.5gL-1, pH 5.8；The condition of culture of the illumination cultivation is：Temperature is 28 ± 3 DEG C, light application time 15h/d,
Intensity of illumination is 35 μm of olm-2·s-1；The rooted seedling plant strain growth of gained is good in aforementioned manners, well developed root system, pure white, thin
Fine hair it is short (<1mm), it is easy to wash, every plant long 4-8 bar, root long 3-7cm, rooting rate 98%；
6) gained rooted seedling is subjected to hardening according to a conventional method, that is, obtains the nontoxic seedling of fortune paulownia.
Claims (7)
1. the breeding method of the nontoxic seedling of fortune paulownia, comprises the following steps：
1) using stem section of the fortune paulownia with axillary bud as explant, it is inoculated in after sterilization in Initial culture base, illumination cultivation, until outer
Axillary bud development in implant grows to form primary seedling；Described Initial culture based formulas is：MS+6-BA 1.0-4.0mg·L-1
+IBA 0.1-0.4mg·L-1+ sucrose 25-35gL-1+ agar 3.0-4.0gL-1, pH 5.6-6.0；
2) gained seedling is cut into the stem section with more than 1 or 2 axillary bud, be transferred in virus-free culture base, combined at 36-39 DEG C blue
Virus-free culture is carried out under conditions of light irradiation, obtains detoxification material；Described virus-free culture based formulas is：MS+6-BA 3.5-
4.5mg·L-1+IBA 0.35-0.45mg·L-1+ sucrose 25-35gL-1+ agar 3.0-4.0gL-1, pH 5.6-6.0；
3) gained detoxification material is cut into the stem section with more than 1 or 2 axillary bud, alternately accesses the training of high concentration plant hormone subculture
Illumination cultivation in base and low-consistency plant hormone subculture medium is supported, obtains tufted seedling；Wherein：
Described high concentration plant hormone squamous subculture based formulas is：MS+6-BA 3.5-4.5mg·L-1+IBA 0.35-
0.45mg·L-1+ sucrose 25-35gL-1+ agar 3.0-4.0gL-1, pH 5.6-6.0；
Described low-consistency plant hormone squamous subculture based formulas is：MS+6-BA 0.2-0.6mg·L-1+IBA 0.02-
0.06mg·L-1+ sucrose 25-35gL-1+ agar 3.0-4.0gL-1, pH 5.6-6.0；
4) by gained tufted seedling plant division, it is transferred in root media, illumination cultivation, obtains rooted seedling；Described root media
It is formulated and is：1/2MS+NAA 0.1-0.4mg·L-1+ sucrose 15-25gL-1+ carragheen 3.2-3.6gL-1, pH 5.6-6.0.
2. breeding method according to claim 1, it is characterised in that：In step 1), described Initial culture based formulas is：
MS+6-BA 2.0mg·L-1+IBA 0.2mg·L-1+ sucrose 30gL-1+ agar 3.5gL-1, pH 5.8.
3. breeding method according to claim 1, it is characterised in that：In step 2), described virus-free culture based formulas is：
MS+6-BA 4.0mg·L-1+IBA 0.4mg·L-1+ sucrose 30gL-1+ agar 3.5gL-1, pH 5.8.
4. breeding method according to claim 1, it is characterised in that：In step 3), described high concentration plant hormone after
It is for culture medium prescription：MS+6-BA 4.0mg·L-1+IBA 0.4mg·L-1+ sucrose 30gL-1+ agar 3.5gL-1, pH
5.8；
Described low-consistency plant hormone squamous subculture based formulas is：MS+6-BA 0.5mg·L-1+IBA0.05mg·L-1+ sucrose
30g·L-1+ agar 3.5gL-1, pH 5.8.
5. breeding method according to claim 1, it is characterised in that：In step 4), described prescription of rooting medium is：
1/2MS+NAA 0.2mg·L-1+ sucrose 20gL-1+ carragheen 3.4gL-1, pH 5.8.
6. according to the breeding method any one of claim 1-5, it is characterised in that：In step 2), during the irradiation of blue light
Between be 10-15h/d, intensity of illumination is 40 ± 5 μm of olm-2·s-1。
7. according to the breeding method any one of claim 1-5, it is characterised in that：Step 1), step 3) and step 4)
In, the condition of culture of the illumination cultivation is：Temperature is 27 ± 5 DEG C, light application time 10-15h/d, and intensity of illumination is 40 ± 5 μ
mol·m-2·s-1。
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| CN111406647B (en) * | 2020-04-17 | 2021-05-07 | 山东省林业科学研究院 | Efficient starting culture medium for directly inducing tetraploid paulownia petioles to regenerate adventitious buds and application |
| CN111374056B (en) * | 2020-04-17 | 2021-05-18 | 山东省林业科学研究院 | Proliferation medium formula for culturing stout tetraploid paulownia seedlings and application |
| CN112690211A (en) * | 2021-01-04 | 2021-04-23 | 海南品优种苗科技有限公司 | Method for virus-free tissue culture and domestication cultivation of paulownia saplings |
| CN113349055A (en) * | 2021-07-07 | 2021-09-07 | 广东粤恬生物科技有限公司 | Tissue culture method of paulownia |
| CN116135010B (en) * | 2023-04-10 | 2024-05-28 | 江苏省农业科学院宿迁农科所(宿迁市农业科学研究院) | Tissue culture and rapid propagation method for paulownia virus-free seedlings |
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