CN106489734A - 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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- CN106489734A CN106489734A CN201610937949.3A CN201610937949A CN106489734A CN 106489734 A CN106489734 A CN 106489734A CN 201610937949 A CN201610937949 A CN 201610937949A CN 106489734 A CN106489734 A CN 106489734A
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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, comprises the following steps：1) with stem section of the fortune paulownia with axillary bud as explant, formed just for seedling through initial culture after sterilization；2) gained seedling is carried out virus-free culture, obtains detoxification material；3) gained detoxification material is alternately accessed illumination cultivation in high concentration phytohormone subculture medium and low-consistency plant hormone subculture medium, obtains tufted seedling；4) root culture will be carried out after gained tufted seedling plant division, obtains Seedling of taking root.The method of the invention is simple to operate, detoxification, efficiency high of breeding and take root, gained take root Seedling well developed root system and fine hair short (<1mm), Seedling saving of work and time is washed, and 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 the diameter of a cross-section of a tree trunk 1.3 meters above the ground is that a kind of purposes is wide, economic worth is high, fast-growing high-quality uses material up to 2 meters
Deciduous species.The characteristics of fortune paulownia superior clone is：(1) good stress resistance, strong adaptability, can plant on the south the Yangtze river basin
Plant；(2) fast growth, 5-6 can felling, 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 worth is higher.
For ensureing that select tree character can stablize heredity, its sapling multiplication must be carried out by vegetative manner.Existing Paulownia
Vegetative manner generally have grafting, point various ways such as root and tissue culture.Wherein, propagation by grafiting needs more manpower, thing
Power and financial resources, are unsuitable for Paulownia seedling large-scale production.And the Paulownia point root nursery mainly raw Paulownia kind roots of selection 1-2, one
As bury root nursery in late Febuary to early March, although its method simple and fast, emergence rate is high, and low cost needs relatively more
Vegetable material, speed and limits throughput；And the requirement to soil is higher, needs good soil property, no disease and pests harm, dry without product
The edaphic conditions such as water, fertility abundance；Selected kind of root must no disease and pests harm, damage relatively light, if select improper, it is easy to cause
Emergence rate is low and causes paulownia witches broom.Grafting and root division, tissue culture detoxifying fast breeding more quickness and high efficiency is compared, and is not divided
The restriction of the time of root nursery, material and soil property, can obtain nontoxic high quality seedling in a large number at short notice, mitigate
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 optimum tissue culture detoxification technology different with method (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 is had found in prior art still in research fortune paulownia superior clone seedling tissue-culturing rapid propagation industrialization process
There is the subject matter of three impact Industrial Efficiencies：One is that the group training material detoxification time is long, and effect is not ideal enough, and detoxification efficiency is not
High；Two is the easy vitrification of material in the single culture medium of high hormone concentration after multiple successive transfer culture, usual 5th generation
Glass rate is about 20%, and as the increase glass rate of subculture number has elevated trend, significantly affects seedling quality, numerous
Grow rate, rooting rate and survival rate；Three is tissue cultured seedling when taking root in culture medium of the agar for supporter, and root system surface grows in a large number
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 Seedling is washed, is affected
Wash Seedling efficiency and transplanting survival rate.
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.The method is operated
Simply, detoxification, efficiency high of breeding and take root, the fine hair that takes root on Seedling robust plant, well developed root system, root system of gained short (<
1mm), Seedling saving of work and time is washed, and transplanting survival rate is high.
The breeding method of the nontoxic seedling of fortune paulownia of the present invention, comprises the following steps：
1) with 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 to explant grows to be formed just for seedling；
2) gained seedling is cut into the stem section with more than 1 or 2 axillary bud, is proceeded in virus-free culture base, tied at 36-39 DEG C
Virus-free culture is carried out under conditions of closing blue light illumination, detoxification material is obtained；
3) gained detoxification material is cut into the stem section with more than 1 or 2 axillary bud, alternately access high concentration phytohormone after
Illumination cultivation in culture base and low-consistency plant hormone subculture medium, obtains tufted seedling；Wherein：
Described high concentration phytohormone successive transfer culture based formulas are：MS+6-BA 3.5-4.5mg·L-1+IBA 0.35-
0.45mg·L-1+ sucrose 25-35g L-1+ agar 3.0-4.0g L-1, pH 5.6-6.0；
Described low-consistency plant hormone successive transfer culture based formulas are：MS+6-BA 0.2-0.6mg·L-1+IBA 0.02-
0.06mg·L-1+ sucrose 25-35g L-1+ agar 3.0-4.0g L-1, pH 5.6-6.0；
4) by gained tufted seedling plant division, proceed in root media, illumination cultivation obtains Seedling of taking root；Described training of taking root
Foster based formulas are：1/2MS+NAA 0.1-0.4mg·L-1+ sucrose 15-25g L-1+ carrageenan 3.2-3.6g L-1, pH 5.6-
6.0.
The step of above-mentioned breeding method 1) in, same as the prior art to disinfecting for explant, at specific sterilization
Reason can be carried out as follows：Take explant and be placed in the liquid detergent aqueous solution that mass concentration is 1-3% and soak 3-5min, take out
Rinsed well with flowing water afterwards, then be placed on superclean bench with ethanol that volumetric concentration is 70-75% immersion 50-70s, after taking-up
HgCl that mass concentration be 0.1-0.2% is placed in again2Middle immersion 5-8min, takes out, sterile water wash 3-5 time.
The step of above-mentioned breeding method 1) in, described initial culture based formulas are preferably：MS+6-BA1.0-4.0mg·
L-1+IBA 0.1-0.4mg·L-1+ sucrose 25-35g L-1+ agar 3.0-4.0g L-1, pH5.6-6.0；More preferably：MS+
6-BA 2.0mg·L-1+IBA 0.2mg·L-1+ sucrose 30g L-1+ agar 3.5g L-1, pH 5.8.In the step, described
Illumination cultivation condition is：Temperature is 27 ± 5 DEG C, and light application time is 10-15h/d, and intensity of illumination is 40 ± 5 μm of ol m-2·s-1；
More preferably：Temperature is 28 ± 2 DEG C, and light application time is 10-12h/d, and intensity of illumination is 40 μm of ol m-2·s-1.The step
In rapid, typically think to be formed just when the axillary bud development on explant grows to 2-3cm for sprout.
The step of above-mentioned breeding method 2) in, described virus-free culture based formulas are preferably：MS+6-BA3.5-4.5mg·
L-1+IBA 0.35-0.45mg·L-1+ sucrose 25-35g L-1+ agar 3.0-4.0g L-1, pH5.6-6.0；More preferably：MS
+6-BA 4.0mg·L-1+IBA 0.4mg·L-1+ sucrose 30g L-1+ agar 3.5g L-1, pH 5.8.In the step, described
During blue light illumination, blue light wavelength used is 465 ± 10nm, and its intensity of illumination is 40 ± 5 μm of ol m-2·s-1, light application time
10-15h/d；Further preferably blue light intensity of illumination is 40 ± 5 μm of ol m-2·s-1, light application time 10-12h/d.
Applicants have discovered that, the step of above-mentioned breeding method 3) in, by adopt high concentration phytohormone successive transfer culture
Base and the subculture method of low-consistency plant hormone subculture medium alternate culture, can ensure that the same of material propagation efficiency
When, moreover it is possible to glass rate is reduced to less than 5%.The high concentration phytohormone subculture medium and low-consistency plant hormone after
The subculture method of culture base alternate culture, refers to and for detoxification material first to access the training of high concentration phytohormone subculture medium
A subculture cycle is supported, material is then taken out and is accessed 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 root culture.In the process, no matter material
Material is grown in high concentration phytohormone subculture medium, or is longer than in low-consistency plant hormone subculture medium, as long as not
There is vitrification, plant height in the sprout of more than 4cm, be used equally to root culture；Other do not reach the material of requirement of taking root
(such as the thin and delicate or short and small plant of comparison), can enter the next successive transfer culture cycle as subculture material, be that next round is taken root training
Supporting carries out material stock.In the step, described high concentration phytohormone successive transfer culture based formulas are preferably：MS+6-
BA4.0mg·L-1+IBA0.4mg·L-1+ sucrose 30g L-1+ agar 3.5g L-1, pH 5.8；Described low-consistency plant swashs
Plain successive transfer culture based formulas are preferably：MS+6-BA0.5mg·L-1+IBA0.05mg·L-1+ sucrose 30g L-1+ agar 3.5g
L-1, pH 5.8.In the step, illumination cultivation and in low-consistency plant hormone subculture in high concentration phytohormone subculture medium
In culture medium, illumination cultivation condition is identical, is：Temperature is 27 ± 5 DEG C, and light application time is 10-15h/d, intensity of illumination is 40 ±
5μmol·m-2·s-1；More preferably：Temperature is 28 ± 2 DEG C, and light application time is 10-12h/d, and intensity of illumination is 40 μ
mol·m-2·s-1.
The step of above-mentioned breeding method 4) in, described prescription of rooting medium is preferably：1/2MS+NAA0.2mg·L-1+
Sucrose 20g L-1+ carrageenan 3.4g L-1, pH 5.8.Applicant is had found in specific experimentation, when in root culture
Using carrageenan as root media solid support when, not only rootage duration is short, rooting rate is high, well developed root system is pure white,
And the growth of the fine hair on Seedling root system of can suppressing to take root using agar as gained during solid support, controls the length of fine hair
Degree so that Seedling of taking root is easier to wash, is substantially increased and washes Seedling efficiency and transplanting survival rate less than 1mm.In the step, described
Illumination cultivation condition be：Temperature is 27 ± 5 DEG C, and light application time is 10-15h/d, and intensity of illumination is 40 ± 5 μm of ol m-2·s-1；More preferably：Temperature is 28 ± 2 DEG C, and light application time is 10-12h/d, and intensity of illumination is 40 μm of ol m-2·s-1.Should
In step, when by tufted seedling plant division, the healthy and strong tufted seedling of more than plant height 4.0cm is preferably selected to carry out plant division.
Under the condition of culture that appeal is limited, explant is cultivated in Initial culture base and is obtained just for seedling (2-3cm) institute
Take time usually 25-35d；Time of virus-free culture usual 10-15d；Stem section with axillary bud is in high concentration phytohormone subculture
A subculture cycle in culture medium is identical with a subculture cycle in low-consistency plant hormone subculture medium, usually
25-30d；Tufted seedling is usually 10-15d the time required to cultivating the Seedling that obtains taking root in root media, now per a plant long 3-
10, the long 3-5cm of root, and gained take root Seedling root system for white, the fine hair on root system short (<1mm), it is easy to wash, meets
Acclimatization and transplantses are required.
Compared with prior art, the method have the characteristics that：
1st, before Multiple Buds are expanded in a large number, the high temperature for combining 36-39 DEG C using the blue light illumination of 465 ± 10nm of wavelength is trained
Foster 10-15d, carries out detoxification treatment to sprout, improves detoxification efficiency, makes virus elimination rate reach 100%；
2nd, using high concentration phytohormone subculture medium and low-consistency plant hormone subculture medium alternate culture after
Culture method, had both ensured the propagation efficiency of material, the glass rate of material can be reduced to less than 5% again；
3rd, in the root culture stage, agar is replaced as culture medium solid support using carrageenan, and is combined 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 Seedling root system of taking root
Growth (villus length<1mm), the Seedling that makes to take root is easier to wash, and so as to reduce the damage of the opposite root when Seedling is washed, carries significantly
High wash Seedling efficiency and transplanting survival rate；
4th, the method for the invention is simple to operation, detoxification, efficiency high of breeding and take root, and can achieve industrialization batch raw
Produce.
Description of the drawings
Fig. 1 is that the embodiment of the present invention 1 cultivates the first picture for seedling for obtaining；
Fig. 2 is the picture that the embodiment of the present invention 1 cultivates the tufted seedling for obtaining；
Fig. 3 is that the embodiment of the present invention 1 cultivates the picture of Seedling of taking root for obtaining, wherein root system is pure white, fine hair short (<1mm)；
Fig. 4 is that comparative example 3 cultivates the picture of Seedling of taking root for obtaining, and wherein root system is pale yellow, around overgrow with shallow white vaporific,
It is about the fine hair of 5mm；
Fig. 5 is that the embodiment of the present invention 1 and comparative example 3 cultivate the picture after the Seedling washing of taking root for obtaining, the wherein life on the left side
Root (embodiment 1 cultivate obtain) root system fine hair short (<1mm), adhere to without culture medium after washing substantially；The Seedling of taking root on the right
(comparative example 3 is cultivated and obtained) root system fine hair longer (about 5mm), still adheres to more culture medium after washing, and is difficult to clean；
Fig. 6 is the picture that the embodiment of the present invention 1 cultivates the transplanted seedling for obtaining.
Specific 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 from Guangxi Plant Inst. of osmanthus backlands area
Conventional screening assays carry out screening the fortune paulownia superior clone for obtaining) stem section with axillary bud, as explant, is placed in quality
Concentration be 1% liquid detergent solution in soak 10min, rinsed well after taking-up under flowing tap water, then be placed in ultra-clean work
Make on platform with the ethanol immersion 60s that volumetric concentration is 70%, after taking-up, be placed in the HgCl that mass concentration is 0.1% again2Middle immersion
7min, takes out, with sterile water wash 5 times；
2) by sterilization after explant be inoculated in Initial culture base, illumination cultivation 30d, explant axillary bud development grow,
The high sprouts (as shown in Figure 1) of 2-3cm are formed, as just for seedling；Wherein, described initial culture based formulas are：MS+6-BA
2.0mg·L-1+IBA 0.2mg·L-1+ sucrose 30g L-1+ agar 3.5g L-1, pH 5.8；Described illumination cultivation condition
For：Temperature is 28 ± 3 DEG C, and light application time is 10h/d, and intensity of illumination is 40 μm of ol m-2·s-1；Gained first generation in aforementioned manners
Seedling is healthy and strong, blade emerald green, well-grown, inductivity 70%；
3) by the seedling sub-cut of gained into the stem section with 1 or 2 axillary bud, access in virus-free culture base, tie at 37-39 DEG C
Virus-free culture 15d is carried out under the conditions of closing blue light (465 ± 10nm of wavelength) irradiation, detoxification treatment is carried out to sprout, detoxification material is obtained
Material；Wherein, described virus-free culture based formulas are：MS+6-BA4.0mg·L-1+IBA0.4mg·L-1+ sucrose 30g L-1+ fine jade
Fat 3.5g L-1, pH 5.8；During virus-free culture, the intensity of illumination of blue light is 40 ± 5 μm of ol m-2·s-1, light application time is 10h/
d；Detoxification efficiency is up to 100% in aforementioned manners；
4) sub-cut of gained detoxification material is first accessed the training of high concentration phytohormone subculture into the stem section with 1 or 2 axillary bud
In foster base, one subculture cycle (25d) of illumination cultivation, breeding coefficient is 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 for root culture；Wherein, institute
The high concentration phytohormone successive transfer culture based formulas that states are：MS+6-BA4.0mg·L-1+IBA 0.4mg·L-1+ sucrose 30g L-1+ agar 3.5g L-1, pH 5.8；Low-consistency plant hormone successive transfer culture based formulas are：MS+6-BA 0.5mg·L-1+IBA
0.05mg·L-1+ sucrose 30g L-1+ agar 3.5g L-1, pH 5.8；Illumination cultivation condition in two kinds of culture medium is identical,
It is：Temperature is 28 ± 3 DEG C, and light application time is 10h/d, and intensity of illumination is 40 μm of ol m-2·s-1；By 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, grows fine, and glass rate is less than 5%,
The tufted seedling of culture a cycle (25d) gained is highly 3-6cm (as shown in Figure 2)；
5) choose height to proceed in root media after the healthy and strong tufted seedling plant division of more than 4cm, illumination cultivation 12d is obtained
Take root Seedling to fortune paulownia；Wherein, described prescription of rooting medium is：1/2MS+NAA 0.2mg·L-1+ sucrose 20g L-1
+ carrageenan 3.4g L-1, pH 5.8；The condition of culture of the illumination cultivation is：Temperature is 28 ± 3 DEG C, and light application time is 10h/
D, intensity of illumination are 40 μm of ol m-2·s-1；The Seedling plant strain growth of taking root of gained is good in aforementioned manners, well developed root system, pure white,
Fine hair short (<1mm), it is easy to wash, per plant long 5-10 bar, the long 3-5cm of root, rooting rate 100% is (such as a left side in Fig. 3 and Fig. 5
Side is taken root shown in Seedling)；
6) gained Seedling of taking root is carried out seedling exercising according to a conventional method, that is, obtains the nontoxic seedling of fortune paulownia (as shown in Figure 6).
Comparative example 1
Repeat embodiment 1, except for the difference that：
By step 2) in initial culture based formulas be changed to：MS+6-BA 0.5mg·L-1+IBA 0.05mg·L-1+ sucrose
30g·L-1+ agar 3.5g L-1, pH 5.8；Just slow for sprout induced velocity, inductivity is low, and only 25%；
By step 3) in poison-removing method be changed to：(intensity of illumination is 40 ± 5 μm of ol m to adopt white light-2·s-1, illumination when
Between 10h/d) combine 33-35 DEG C of temperature culture 25d, detoxification efficiency 15%；
By step 4) in high concentration phytohormone culture medium prescription be changed to：MS+6-BA 2.0mg·L-1+IBA
0.02mg·L-1+ sucrose 30g L-1+ agar 3.5g L-1(pH 5.8), Multiple Buds growth are general, and part sprouting is pushed up after growing
Withered 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 30g L-1+ agar 3.5g L-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；
By step 5) in prescription of rooting medium be changed to：1/2MS+NAA 0.05mg·L-1+ sucrose 20g L-1+ OK a karaoke club
Glue 3.4g L-1, pH 5.8；Seedling well-grown, but rooting rate is low, only 53%, and root is thinner.
Comparative example 2
Repeat embodiment 1, except for the difference that：
By step 2) in initial culture based formulas be changed to：MS+6-BA 5.0mg·L-1+IBA 0.5mg·L-1+ sucrose
30g·L-1+ agar 3.5g L-1, pH 5.8；There are a large amount of calluss to produce, just cannot be formed for sprout, more than half explant is bad
Extremely；
By step 3) in poison-removing method be changed to：(intensity of illumination is 40 ± 5 μm of ol m to adopt white light-2·s-1, illumination when
Between 10h/d) combine 40-42 DEG C of temperature culture, as temperature is too high, material is most of dead after a few days for culture, does not reach and educates
Seedling effect；
By step 4) in high concentration phytohormone culture medium prescription be changed to：MS+6-BA 6.0mg·L-1+IBA
0.6mg·L-1+ sucrose 30g L-1+ agar 3.5g L-1(pH 5.8)；Tufted seedling bottom produces a large amount of calluss, most of
Tufted seedling growth is slow, later stage are 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 30g L-1+ agar 3.5g L-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；Replace the average proliferation of subculture
Coefficient is 2.1/30d；
By step 5) in prescription of rooting medium be revised as：1/2MS+NAA 0.8mg·L-1+ sucrose 20g L-1+ card
Draw glue 3.4g L-1, pH 5.8；Seedling rooting of growing thickly is few, and rootage duration is uneven, and rooting rate is 67%.
Comparative example 3
Repeat embodiment 1, except for the difference that：
In step 5) in, the solid support of root media is changed to agar, i.e. prescription of rooting medium by carrageenan
It is revised as：1/2MS+NAA 0.2mg·L-1+ sucrose 20g L-1+ agar 3.5g L-1, pH5.8；Gained take root Seedling growth good
Good, per plant long 3-7 bar, the long 3-6cm of root, rooting rate 100%, root color are pale yellow, but cover with root system and be about the thin of 5mm in a large number
Fine hair (in Fig. 4 and Fig. 5 the right taking root shown in Seedling), adhering to substantial amounts of culture medium, is difficult to clean up, and washes Seedling and takes a lot of work expense
When.
Comparative example 4
Repeat embodiment 1, except for the difference that：
By step 4) in subculture method be changed to：Only it is circulated with single low-consistency plant hormone subculture medium
Subculture, wherein, subculture medium is MS+6-BA 0.5mg L-1+IBA 0.05mg·L-1+ sucrose 30g L-1+ agar
3.5g·L-1(pH 5.8).As a result find, through more than 5 times continuous subcultures after, only 70% culture materials can be induced 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 can not be induced 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
Repeat embodiment 1, except for the difference that：
By step 4) in subculture method be changed to：Only with single intermediate concentration phytohormone culture medium be circulated after
In generation, wherein, subculture medium is MS+6-BA 2.0mg L-1+IBA 0.2mg·L-1+ sucrose 30g L-1+ agar 3.5g L-1
(pH 5.8).As a result find, through more than 5 times continuous subcultures after, 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
Repeat embodiment 1, except for the difference that：
By step 4) in subculture method be changed to：Only it is circulated with single high concentration phytohormone subculture medium
Subculture, wherein, subculture medium is MS+6-BA4.0mg L-1+IBA 0.4mg·L-1+ sucrose 30g L-1+ agar 3.5g
L-1(pH 5.8).As a result find, the tufted seedling well-grown of front 5 generation gained, leaf are bud green, of medium size, breeding coefficient 6.0/
25d；But to the 5th instead of after, start vitrified material occur, vitrification 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 from Guangxi Plant Inst. of osmanthus backlands area
Conventional screening assays carry out screening the fortune paulownia superior clone for obtaining) stem section with axillary bud, as explant, is placed in quality
Concentration be 1% liquid detergent solution in soak 10min, rinsed well after taking-up under flowing tap water, then be placed in ultra-clean work
Make on platform with the ethanol immersion 50s that volumetric concentration is 70%, after taking-up, be placed in the HgCl that mass concentration is 0.1% again2Middle immersion
6min, takes out, with sterile water wash 4 times；
2) by sterilization after explant be inoculated in Initial culture base, illumination cultivation 25d, explant axillary bud development grow,
The high sprouts of 2-3cm are formed, as just for seedling, gained seedling is healthy and strong, blade emerald green, well-grown, inductivity 65%；Its
In, described initial culture based formulas are：MS+6-BA 1.0mg·L-1+IBA 0.4mg·L-1+ sucrose 25g L-1+ agar
3.0g·L-1, pH 5.8；Described illumination cultivation condition is：Temperature is 28 ± 3 DEG C, and light application time is 12h/d, and intensity of illumination is
45μmol·m-2·s-1；
3) by the seedling sub-cut of gained into the stem section with 1 or 2 axillary bud, access in virus-free culture base, tie at 37-39 DEG C
Virus-free culture 10d is carried out under the conditions of closing blue light (465 ± 10nm of wavelength) irradiation, and detoxification treatment, detoxification efficiency is carried out to sprout
100%, obtain detoxification material；Wherein, described virus-free culture based formulas are：MS+6-BA 4.0mg·L-1+IBA 0.4mg·
L-1+ sucrose 30g L-1+ agar 3.5g L-1, pH 5.8；During virus-free culture, the intensity of illumination of blue light is 35 μm of ol m-2·s-1, light application time is 12h/d；
4) sub-cut of gained detoxification material is first accessed the training of high concentration phytohormone subculture into the stem section with 1 or 2 axillary bud
In foster base culture, one subculture cycle (25d) of illumination cultivation, breeding coefficient is 5.0/25d；Material sub-cut is then taken out into carrying
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, numerous
Grow coefficient 4.0/25d；……；So move in circles alternate culture, until obtaining enough materials for root culture；Wherein,
Described high concentration phytohormone successive transfer culture based formulas are：MS+6-BA 3.5mg·L-1+IBA 0.45mg·L-1+ sucrose
25g·L-1+ agar 3.0g L-1, pH 5.8；Low-consistency plant hormone successive transfer culture based formulas are：MS+6-BA0.6mg·L-1+
IBA 0.02mg·L-1+ sucrose 25g L-1+ agar 3.0g L-1, pH 5.8；Illumination cultivation condition in two kinds of culture medium
Identical, it is：Temperature is 28 ± 3 DEG C, and light application time is 12h/d, and intensity of illumination is 40 μm of ol m-2·s-1；By above-mentioned friendship
More, the average breeding coefficient 4.5/25d of the tufted seedling quantity for cultivating gained, plant is replaced to grow fine, leaf is bud green, of medium size,
Less than 5%, the tufted seedling for cultivating a cycle (25d) gained is highly 2-6cm to glass rate；
5) choose height to proceed in root media in the healthy and strong tufted seedling plant division of more than 4cm, illumination cultivation 12d is obtained
Fortune paulownia is taken root Seedling；Wherein, described prescription of rooting medium is：1/2MS+NAA0.15mg·L-1+ sucrose 15g L-1+
Carrageenan 3.3g L-1, pH 5.8；The condition of culture of the illumination cultivation is：Temperature is 28 ± 3 DEG C, and light application time is 10h/d,
Intensity of illumination is 40 μm of ol m-2·s-1；The Seedling plant strain growth of taking root of gained is good in aforementioned manners, well developed root system, pure white, thin
Fine hair short (<1mm), it is easy to wash, per plant long 4-7 bar, the long 3-5cm of root, rooting rate 95%；
6) gained Seedling of taking root is carried out seedling exercising according to a conventional method, that is, obtains the nontoxic seedling of fortune paulownia.
Embodiment 3
1) using the stem section of (fortune paulownia is fortune paulownia superior clone C001) with axillary bud fortune paulownia as explant
Body, is placed in immersion 10min in the liquid detergent solution that mass concentration is 1%, is rinsed well after taking-up under flowing tap water, then
Being placed on superclean bench and 70s being soaked 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, takes out, with sterile water wash 5 times；
2) by sterilization after explant be inoculated in Initial culture base, illumination cultivation 25d, explant axillary bud development grow,
The high sprouts of 2-3cm are formed, as just for seedling, gained seedling is healthy and strong, blade emerald green, well-grown, inductivity 60%；Its
In, described initial culture based formulas are：MS+6-BA 4.0mg·L-1+IBA 0.1mg·L-1+ sucrose 35g L-1+ agar
4.0g·L-1, pH 5.8；Described illumination cultivation condition is：Temperature is 27 ± 5 DEG C, and light application time is 15h/d, and intensity of illumination is
35μmol·m-2·s-1；
3) by the seedling sub-cut of gained into the stem section with 1 or 2 axillary bud, access in virus-free culture base, tie at 36-38 DEG C
Virus-free culture 13d is carried out under the conditions of closing blue light (465 ± 10nm of wavelength) irradiation, and detoxification treatment, detoxification efficiency is carried out to sprout
100%, obtain detoxification material；Wherein, described virus-free culture based formulas are：MS+6-BA 4.0mg·L-1+IBA 0.4mg·
L-1+ sucrose 35g L-1+ agar 4.0g L-1, pH 5.8；During virus-free culture, the intensity of illumination of blue light is 45 μm of ol m-2·s-1, light application time is 10h/d；
4) sub-cut of gained detoxification material is first accessed the training of high concentration phytohormone subculture into the stem section with 1 or 2 axillary bud
In foster base culture, one subculture cycle (25d) of illumination cultivation, breeding coefficient is 5.6/25d；Material sub-cut is then taken out into carrying
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, numerous
Grow coefficient 4.0/25d；……；So move in circles alternate culture, until obtaining enough materials for root culture；Wherein,
Described high concentration phytohormone successive transfer culture based formulas are：MS+6-BA 4.5mg·L-1+IBA 0.35mg·L-1+ sucrose
35g·L-1+ agar 4.0g L-1, pH 5.8；Low-consistency plant hormone successive transfer culture based formulas are：MS+6-BA0.2mg·L-1+
IBA0.06mg·L-1+ sucrose 35g L-1+ agar 4.0g L-1, pH 5.8；Illumination cultivation condition phase in two kinds of culture medium
Same, it is：Temperature is 27 ± 3 DEG C, and light application time is 15h/d, and intensity of illumination is 40 μm of ol m-2·s-1；By above-mentioned alternating
More, the average breeding coefficient 4.8/25d of tufted seedling quantity of culture gained, plant grow fine, and leaf is bud green, of medium size, glass
Less than 5%, the tufted seedling for cultivating a cycle (25d) gained is highly 2-6cm to glass rate；
5) choose height to proceed in root media in the healthy and strong tufted seedling plant division of more than 4cm, illumination cultivation 12d is obtained
Fortune paulownia is taken root Seedling；Wherein, described prescription of rooting medium is：1/2MS+NAA0.35mg·L-1+ sucrose 25g L-1+
Carrageenan 3.5g L-1, pH 5.8；The condition of culture of the illumination cultivation is：Temperature is 28 ± 3 DEG C, and light application time is 15h/d,
Intensity of illumination is 35 μm of ol m-2·s-1；The Seedling plant strain growth of taking root of gained is good in aforementioned manners, well developed root system, pure white, thin
Fine hair short (<1mm), it is easy to wash, per plant long 4-8 bar, the long 3-7cm of root, rooting rate 98%；
6) gained Seedling of taking root is carried out seedling exercising according to a conventional method, that is, obtains the nontoxic seedling of fortune paulownia.
Claims (9)
1. the breeding method of the nontoxic seedling of fortune paulownia, comprises the following steps：
1) with 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 be formed just for seedling；
2) gained seedling is cut into the stem section with more than 1 or 2 axillary bud, is proceeded in virus-free culture base, combined at 36-39 DEG C blue
Virus-free culture is carried out under conditions of light irradiation, detoxification material is obtained；
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 phytohormone subculture
Illumination cultivation in foster base and low-consistency plant hormone subculture medium, obtains tufted seedling；Wherein：
Described high concentration phytohormone successive transfer culture based formulas are：MS+6-BA 3.5-4.5mg·L-1+IBA 0.35-
0.45mg·L-1+ sucrose 25-35g L-1+ agar 3.0-4.0g L-1, pH5.6-6.0；
Described low-consistency plant hormone successive transfer culture based formulas are：MS+6-BA 0.2-0.6mg·L-1+IBA 0.02-
0.06mg·L-1+ sucrose 25-35g L-1+ agar 3.0-4.0g L-1, pH5.6-6.0；
4) by gained tufted seedling plant division, proceed in root media, illumination cultivation obtains Seedling of taking root；Described root media
Formula is：1/2MS+NAA 0.1-0.4mg·L-1+ sucrose 15-25g L-1+ carrageenan 3.2-3.6g L-1, pH 5.6-6.0.
2. breeding method according to claim 1, it is characterised in that：Step 1) in, described initial culture based formulas are：
MS+6-BA 1.0-4.0mg·L-1+IBA 0.1-0.4mg·L-1+ sucrose 25-35g L-1+ agar 3.0-4.0g L-1, pH
5.6-6.0.
3. breeding method according to claim 1, it is characterised in that：Step 1) in, described initial culture based formulas are：
MS+6-BA 2.0mg·L-1+IBA 0.2mg·L-1+ sucrose 30g L-1+ agar 3.5g L-1, pH 5.8.
4. breeding method according to claim 1, it is characterised in that：Step 2) in, described virus-free culture based formulas are：
MS+6-BA3.5-4.5mg·L-1+IBA0.35-0.45mg·L-1+ sucrose 25-35g L-1+ agar 3.0-4.0g L-1, pH
5.6-6.0.
5. breeding method according to claim 1, it is characterised in that：Step 2) in, described virus-free culture base is：MS+
6-BA4.0mg·L-1+IBA0.4mg·L-1+ sucrose 30g L-1+ agar 3.5g L-1, pH 5.8.
6. breeding method according to claim 1, it is characterised in that：Step 3) in, described high concentration phytohormone after
Culture based formulas are：MS+6-BA 4.0mg·L-1+IBA 0.4mg·L-1+ sucrose 30g L-1+ agar 3.5g L-1, pH
5.8；
Described low-consistency plant hormone successive transfer culture based formulas are：MS+6-BA 0.5mg·L-1+IBA0.05mg·L-1+ sucrose
30g·L-1+ agar 3.5g L-1, pH 5.8.
7. breeding method according to claim 1, it is characterised in that：Step 4) in, described prescription of rooting medium is：
1/2MS+NAA0.2mg·L-1+ sucrose 20g L-1+ carrageenan 3.4g L-1, pH 5.8.
8. the breeding method according to any one of claim 1-7, it is characterised in that：Step 2) in, during the irradiation of blue light
Between be 10-15h/d, intensity of illumination be 40 ± 5 μm of ol m-2·s-1.
9. the breeding method according to any one of claim 1-7, 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, and light application time is 10-15h/d, and intensity of illumination is 40 ± 5 μ
mol·m-2·s-1.
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| CN111374056A (en) * | 2020-04-17 | 2020-07-07 | 山东省林业科学研究院 | Proliferation medium formula for culturing stout tetraploid paulownia seedlings and application |
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| 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 |
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| 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 |
| CN116135010A (en) * | 2023-04-10 | 2023-05-19 | 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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