CN103190344B - Tissue culture method of fargesii - Google Patents
Tissue culture method of fargesii Download PDFInfo
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- CN103190344B CN103190344B CN201310109903.9A CN201310109903A CN103190344B CN 103190344 B CN103190344 B CN 103190344B CN 201310109903 A CN201310109903 A CN 201310109903A CN 103190344 B CN103190344 B CN 103190344B
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- 238000012136 culture method Methods 0.000 title abstract 5
- 238000000034 method Methods 0.000 claims abstract description 42
- 230000001939 inductive effect Effects 0.000 claims abstract description 30
- 230000004069 differentiation Effects 0.000 claims description 58
- 240000004528 Catalpa ovata Species 0.000 claims description 49
- 235000010005 Catalpa ovata Nutrition 0.000 claims description 49
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 40
- 239000002609 medium Substances 0.000 claims description 35
- 239000008272 agar Substances 0.000 claims description 20
- 239000005720 sucrose Substances 0.000 claims description 20
- 230000006698 induction Effects 0.000 claims description 19
- 230000035755 proliferation Effects 0.000 claims description 19
- 230000008569 process Effects 0.000 claims description 16
- 239000001963 growth medium Substances 0.000 claims description 13
- 230000003203 everyday effect Effects 0.000 claims description 9
- 239000008399 tap water Substances 0.000 claims description 8
- 235000020679 tap water Nutrition 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- 239000011159 matrix material Substances 0.000 claims description 6
- 238000002054 transplantation Methods 0.000 claims description 5
- 238000005286 illumination Methods 0.000 claims description 4
- 238000000338 in vitro Methods 0.000 claims description 4
- 239000003415 peat Substances 0.000 claims description 4
- 235000019362 perlite Nutrition 0.000 claims description 4
- 239000010451 perlite Substances 0.000 claims description 4
- 239000012882 rooting medium Substances 0.000 claims description 4
- 239000002689 soil Substances 0.000 claims description 4
- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 claims description 3
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 claims description 3
- 239000006013 carbendazim Substances 0.000 claims description 3
- 230000004083 survival effect Effects 0.000 abstract description 10
- 239000000463 material Substances 0.000 abstract description 6
- 238000009395 breeding Methods 0.000 abstract description 3
- 241000238631 Hexapoda Species 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 238000012090 tissue culture technique Methods 0.000 abstract description 2
- 238000012258 culturing Methods 0.000 abstract 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 7
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- 229920001817 Agar Polymers 0.000 description 6
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- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 description 1
- YGGXZTQSGNFKPJ-UHFFFAOYSA-N methyl 2-naphthalen-1-ylacetate Chemical compound C1=CC=C2C(CC(=O)OC)=CC=CC2=C1 YGGXZTQSGNFKPJ-UHFFFAOYSA-N 0.000 description 1
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Cultivation Of Plants (AREA)
Abstract
The invention relates to a tissue culture method of fargesii. The method comprises the following steps of: acquiring annual produced branches of a fargesii seed tree, and sequentially carrying out branches hydroponic sprouting, asepticizing, buds inducing and multiplying, adventitious bud rooting culturing, hardening-seedling, and transplanting to obtain fargesii tissue cultured seedling. After the tissue culture method is used, the specific, robust and plant diseases and insect pests-free fargesii female parent is selected, a large batch of high-quality fargesii tissue cultured seedling can be obtained within a short time through a specific tissue culture technique and a specific culture condition, so that the scientific research and production requirements can be met; compared with the other breeding methods, the tissue culture method needs less female parent materials, 1-2 stem(s) can be bred on a large scale, the good properties of the female parent can be preferably kept, the seedling is transplanted to outdoors, and the rate of survival can be more than 95%; the tissue culture method is quick in breeding speed, good in nursery stock quality, and high in emergence rate; and the stock and the cion do not need to be bred, so that the floor space can be greatly reduced, and the labor cost and the management cost can be reduced.
Description
Technical field
The present invention relates to the method for tissue culture of a kind of woody plant, especially relate to the method for tissue culture of a kind of grey Chinese catalpa.
Background technology
Ash Chinese catalpa (Catalpa fargesii) is Bignoniaceae (Bignoniaceae) Catalpa (Catalpa) tall and big deciduous tree, is high-quality preciousness material and the ornamental plantation seeds of China's traditional cultivation.It has a very wide distribution, and is mainly distributed in China central and west regions, with the Weihe River, Jing He, Fenhe river basin and Qinling Mountain for concentration zones, scattered in village periphery and mountain valley.Ash Chinese catalpa well developed root system, fast growth, wind resistance, solid native ability are strong, and cold-resistant drought-enduring, material texture leads to straight, tough and tensile densification, is high-quality fast-growing commerical tree species.
Usually, grey Chinese catalpa is bred by grafting and cuttage.Propagation by grafiting need shift to an earlier date 1 year and cultivate stock (Chinese catalpa), gathers the bud of maternal 1 year raw branch or Miao Ganshang next year as scion; In grafting procedures, because scion growth in thickness is generally greater than stock, cause interface to heal poor, survival rate is generally about 70%, and grafting wind resistance is more weak, and the many rudiments of stock base portion, need continuous bud picking, add management cost.Cottage propagation need gather current-year branch, seedling is done or root carries out burying dry (root) vernalization, gather semi-lignified spray and carry out cuttage, require comparatively strict to germination bed and cutting bed epidemic disaster and relative air humidity, management cost is high, cuttage survival rate is lower, is generally about 50%.
Tissue cultures is a kind of vegetative manner efficiently, and reproductive efficiency is high and cost is lower, is solve grey Chinese catalpa choiceness effective means numerous soon, has not yet to see the report about grey Chinese catalpa choiceness tissue cultures aspect.
Summary of the invention
In order to overcome the deficiency that the vegetative propagation technique such as grafting and cuttage exists, the object of the present invention is to provide the method for tissue culture of a kind of grey Chinese catalpa, the method can obtain the grey Chinese catalpa plantlet in vitro of high-quality efficiently.
Grey Chinese catalpa method for tissue culture provided by the invention, comprising: gather that the annual germinating branch of grey Chinese catalpa elite stand carries out that bough water planting vernalization, asepticize process, the induction of bud and propagation, adventitious bud rooting are cultivated successively, hardening and transplanting, obtains grey Chinese catalpa plantlet in vitro.
Described bough water planting vernalization comprises: gather the annual germinating branch of grey Chinese catalpa elite stand, after indoor water planting (running water) vernalization, win sprouting bud.Preferably wait that sprouting bud length is about 10cm, most preferably wins during 8 ~ 12cm.
Described asepticize process comprises: by the sprouting bud tap water 30min after water planting vernalization, soak 6 ~ 7min afterwards with the liquor natrii hypochloritis that mass concentration is 10%, afterwards again through aseptic water washing 4 ~ 5 times, finally blots surface moisture with aseptic filter paper.
Induction and the propagation of described bud comprise: the sprouting bud of asepticize process is cut into stem section, is then inoculated on inducing culture, cultivate 35 ~ 50 days, obtain Bud Differentiation; And then this Bud Differentiation is cut into stem section is inoculated on proliferated culture medium, cultivate and form indefinite bud after 30 ~ 45 days.
Preferably, the stem section that the sprouting bud of asepticize process is cut into, and differentiation
bud is cut into 'sstem section has an axillalry bud at least.
More preferably, induction and the propagation of described bud comprise: it is about 1.5cm that the sprouting bud of asepticize process is cut into length, and most preferably the stem section of 1.4 ~ 1.6cm, is then inoculated on inducing culture, cultivate 35 ~ 50 days, obtain Bud Differentiation; And then this Bud Differentiation is cut into length is about 1.0cm, most preferably the stem section of 0.8 ~ 1.2cm is inoculated on proliferated culture medium, cultivates and forms indefinite bud after 30 ~ 45 days.
Wherein, the differentiation-inducing and Multiplying culture used medium of described bud is DKW minimal medium, also comprises: 0.6 ~ 1.4mgL
-16-BA(6-benzylaminopurine, benzylaminmopurine), 0.1 ~ 0.3mgL
-1iBA(indolebutyric acid, Indole-3-Butytric acid), 25gL
-1sucrose and 4.5gL
-1agar.Be preferably: DKW minimal medium, 0.75 ~ 1.2mgL
-16-BA, 0.13 ~ 0.23mgL
-1iBA, 25gL
-1sucrose and 4.5gL
-1agar.
Further, the pH of the differentiation-inducing and proliferated culture medium of described bud is 5.8.
Further, in the differentiation-inducing process of bud, cultivate after 3 days, stem segment with axillary buds place bud starts to expand; Within 8 ~ 10 days, stem segment base portion callus starts to expand; The bud that coinduction cultivates 35 ~ 50 days (preferably 40 ~ 48 days) Bud Differentiations is grown up in 0.7cm.
Further, this Bud Differentiation is cut into stem section and is inoculated on proliferated culture medium, cultivate after 8 ~ 10 days and start Proliferation, Differentiation formation indefinite bud, 30 ~ 45 days (preferably 34 ~ 40 days) afterwards indefinite bud can grow to more than 1.0cm.
Described adventitious bud rooting is cultivated and is comprised: being cut into stem section, root induction in root media of transferring by breeding the indefinite bud obtained, growing up to seedling of taking root after 28 ~ 40 days.
Wherein, it is 1/2MS minimal medium that described adventitious bud rooting cultivates medium used, also comprises: 0.25 ~ 0.35mgL
-1iBA, 0.025 ~ 0.035mgL
-1nAA(methyl α-naphthyl acetate, 1-naphthlcetic acid), 10gL
-1sucrose and 5.0gL
-1agar.Be preferably: 1/2MS minimal medium, 0.28 ~ 0.32mgL
-1iBA, 0.029 ~ 0.032mgL
-1nAA, 10gL
-1sucrose and 5.0gL
-1agar.
Further, the pH of described adventitious bud rooting medium is 5.8.
Preferably, indefinite bud is cut into the stem section at least with a blade.
More preferably, after 30 ~ 45 days, indefinite bud is cut into the stem section that about 1.5cm is long.
Wherein, indefinite bud is in root induction process, after 5 ~ 6 days, stem segment base portion starts to expand greening, and have light green pinprick size particles to produce, start to take root after 8 ~ 10 days, after 28 ~ 40 days (preferably 30 ~ 40 days), namely obtain seedling (28 ~ 40 days (preferably 30 ~ 40 days) afterwards seedling can grow 1.0 ~ 2.5cm) of taking root.
Described hardening and transplanting comprise: the seedling of taking root of being turned out by adventitious bud rooting is placed in greenhouse hardening (16 ~ 20 days) and transplants afterwards, shelters from heat or light after transplanting with shade net.
Wherein, seedling of taking root is placed in greenhouse and conforms and within 6 ~ 8 days, unclamp envelope bottle rope, is unclamped by bottle film every other day, transplants after 10 ~ 12 days.
Wherein, the matrix of transplantation of seedlings of taking root is peat soil: perlite (V/V)=3 ~ 5:1, preferred 4:1.
Further, the matrix of transplantation of seedlings of taking root also can comprise carbendazim, and consumption is 200g/m
3.
Wherein, the temperature that the induction of described bud and propagation, adventitious bud rooting are cultivated is 22 ~ 26 DEG C, and intensity of illumination is 1800 ~ 2200 luxs (lx), and light application time is 12 ~ 15 hours/every day.Preferred: intensity of illumination is 2000 luxs (lx), and light application time is 14 hours/day.
Compare with the method for tissue culture of existing Chinese catalpa:
1. the difference of Chinese catalpa and grey Chinese catalpa: Chinese catalpa and grey Chinese catalpa all belong to the different kind of two of Bignoniaceae Catalpa, and its area, growth, adaptability, wood property have very large difference;
2. Chinese catalpa has ripe method for tissue culture, but does not also have ripe grey Chinese catalpa method for tissue culture at present, according to current had Chinese catalpa method for tissue culture, cannot normally, efficiently carry out the tissue cultures of grey Chinese catalpa.
Therefore, the inventive method is selected specific healthy and strong maternal without damage by disease and insect ash Chinese catalpa, by specific tissue culture technique and condition of culture, can obtain high-quality in enormous quantities ash Chinese catalpa plantlet in vitro at short notice, meet needed for research and production.Compared with other propagation methods, the female parent material that the present invention needs is few, has 1 ~ 2 stem section to breed in enormous quantities, and keeps maternal merit better, and seedling replanting is outdoor, and survival rate can reach more than 95%; Reproduction speed is fast, and seedling quality is good, and emergence rate is high; Without the need to cultivating stock and scion, greatly reduce floor space, and reduce manpower and management cost.
Accompanying drawing explanation
Fig. 1 is the sprouting bud obtained after 30 days according to the vernalization of embodiment 1 water planting.
Fig. 2 is the Bud Differentiation obtained after 15 days according to embodiment 1 asepticize material cultivation induction.
Fig. 3 is the Bud Differentiation obtained after 45 days according to embodiment 1 asepticize material Fiber differentiation.
Fig. 4 is the indefinite bud obtained after 40 days according to embodiment 1 Bud Differentiation switching proliferative induction.
Fig. 5 is the seedling of taking root obtained after cultivating 30 days according to embodiment 1 indefinite bud switching root induction.
Fig. 6 is the shoot root system of taking root obtained after cultivating 30 days according to embodiment 1 indefinite bud switching root induction.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention; Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to protection scope of the present invention.
The formula of minimal medium involved in the present invention is as follows:
The standard recipe of DKW minimal medium:
The standard recipe of MS minimal medium (Murashing and Skoog medium, 1962):
1/2 basic MS culture medium, namely macroelement constituent respectively reduces by half the standard recipe of MS medium (Murashing and Skoog medium, 1962) of (other elements do not reduce by half):
If do not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art.
The present invention gets the annual germinating bar of grey Chinese catalpa elite stand and carries out water planting vernalization, is specially: get the annual germinating branch of grey Chinese catalpa elite stand, in indoor water planting (running water) vernalization, treats that long about the 10cm of bud wins.
Number of days given by the present invention, all calculates from starting to cultivate.
Embodiment 1
A method for tissue culture for grey Chinese catalpa, gets the annual germinating branch of grey Chinese catalpa elite stand, carries out water planting (running water) vernalization in indoor, after sprouting length and reaching more than 8cm, wins and sprouts bud (as shown in Figure 1), next comprise the following steps:
(1) asepticize process
Win the sprouting bud after the vernalization of grey Chinese catalpa bough water planting, remove all blades, use tap water 30min, superclean bench soaks 6 ~ 7min with the liquor natrii hypochloritis of 10%, after aseptic water washing 4 ~ 5 times, blot surface moisture with aseptic filter paper, bud is cut into the long stem section of 1.5cm (ensureing that stem section has an axillalry bud at least) and is inoculated in inducing culture: DKW minimal medium+1.0mgL
-16-BA+0.2mgL
-1iBA+25gL
-1sucrose+4.5gL
-1on agar (pH5.8).
(2) induction of bud and propagation
After stem section is inoculated in inducing culture 3d, stem segment with axillary buds place bud starts to expand, about 9d stem segment base portion callus starts to expand, bud until Bud Differentiation grows up after 1.0cm (Fiber differentiation totally 45 days), get the stem section that Bud Differentiation is cut into 1.0cm, transfer on proliferated culture medium (composition is identical with inducing culture), start Proliferation, Differentiation after cultivating 10d and form indefinite bud, can grow to 2.2cm after 35d, growth coefficient is 8.8.
(3) culture of rootage of indefinite bud
Get the Proliferation, Differentiation bud that length is greater than 1.5cm, being cut into length is that 1.5cm is with a blade stem section, transfers into root media: 1/2MS minimal medium+0.3mgL
-1iBA+0.03mgL
-1nAA+10gL
-1sucrose+5.0gL
-1agar (pH5.8), after 5d, stem segment base portion starts to expand greening, and has light green pinprick size particles to produce, and starts to take root after 10d, and after root symbiosis cultivates 30d, seedling can long 1.2cm.
(4) hardening and transplanting
When culture of rootage root system grows to 2.5cm (after culture of rootage 30 ~ 40d), a bottle seedling of taking root is placed in greenhouse, and about the 7d that conforms unclamps envelope bottle rope, every other day bottle film is unclamped, about 10d transplants, and cleans root medium during transplanting, timely overlay film (every day sprays water 3 times) after transplanting, shelter from heat or light, after 7d, loose film, takes off film after 10d, transplants outdoor after 40d, enter field management, transplanting survival rate is 99%.The matrix of transplantation of seedlings of taking root is peat soil: perlite (V/V)=4:1, and uses carbendazim sterilizing, and consumption is 200g/m
3.The temperature that the induction of bud and propagation, adventitious bud rooting are cultivated is 25 DEG C, and intensity of illumination is 2000 luxs (lx), and light application time is 14 hours/every day.
In incubation, the picture in each stage is as shown in Fig. 2 ~ Fig. 6.
Embodiment 2
A method for tissue culture for grey Chinese catalpa, getting after the annual germinating branch of grey Chinese catalpa elite stand carries out water planting vernalization, comprising the following steps:
(1) asepticize process
Win the sprouting bud after the vernalization of grey Chinese catalpa bough water planting, remove all blades, use tap water 30min, superclean bench soaks 6 ~ 7min with the liquor natrii hypochloritis of 10%, after aseptic water washing 4 ~ 5 times, blot surface moisture with aseptic filter paper, bud is cut into the long stem section of 1.5cm (ensureing that stem section has an axillalry bud at least) and is inoculated in inducing culture: DKW minimal medium+0.8mgL
-16-BA+0.15mgL
-1iBA+25gL
-1sucrose+4.5gL
-1on agar (pH5.8).
(2) the differentiation-inducing and propagation of bud
After stem section is inoculated in inducing culture 3d, stem segment with axillary buds place bud starts to expand, and about 10d stem segment base portion callus starts to expand, and after cultivating 40d, Bud Differentiation bud number reaches 5, the long 2.86cm of bud; Get the stem section that Bud Differentiation is cut into 1.0cm, transfer on proliferated culture medium (composition is identical with inducing culture), start Proliferation, Differentiation after cultivating 10d and form indefinite bud, Proliferation, Differentiation bud number 4.7 after 36d, the long 2.97cm of Bud Differentiation, growth coefficient is 14.
(3) culture of rootage of indefinite bud
Get Proliferation, Differentiation bud (length is greater than 1.5cm), being cut into length is that 1.5cm is with a blade stem section, transfers into root media: 1/2MS minimal medium+0.31mgL
-1iBA+0.029mgL
-1nAA+10gL
-1sucrose+5.0gL
-1agar (pH5.8), after 5d, stem segment base portion starts to expand greening, and has light green pinprick size particles to produce, and start after 10d to take root, after 32d, seedling root of hair number is 1.13, the long 0.734cm of root, the long 1.197cm of seedling sprouting.
(4) hardening and transplanting
After culture of rootage 36d, a bottle seedling of taking root is placed in greenhouse, and about the 7d that conforms unclamps envelope bottle rope, every other day bottle film is unclamped, about 10d transplants, and cleans root medium during transplanting, timely overlay film (every day sprays water 3 times) after transplanting, shelter from heat or light, after 7d, loose film, takes off film after 10d, transplants outdoor after 40d, enter field management, transplanting survival rate is 98%.Other conditions are with embodiment 1.
Embodiment 3
A method for tissue culture for grey Chinese catalpa, getting after the annual germinating bar of grey Chinese catalpa elite stand carries out water planting vernalization, comprising the following steps:
(1) asepticize process
Win the sprouting bud after the vernalization of grey Chinese catalpa bough water planting, remove all blades, use tap water 30min, superclean bench soaks 6 ~ 7min with the liquor natrii hypochloritis of 10%, after aseptic water washing 4 ~ 5 times, blot surface moisture with aseptic filter paper, bud is cut into the long stem section of 1.5cm (ensureing that stem section has an axillalry bud at least) and is inoculated in inducing culture: DKW minimal medium+1.2mgL
-16-BA+0.16mgL
-1iBA+25gL
-1sucrose+4.5gL
-1on agar (pH5.8).
(2) the differentiation-inducing and propagation of bud
After stem section is inoculated in inducing culture 3d, stem segment with axillary buds place bud starts to expand, and about 8d stem segment base portion callus starts to expand, Bud Differentiation bud number 3.43 after cultivation 38d, the long 0.765cm of bud; Get the stem section that Bud Differentiation is cut into 1.0cm, transfer on proliferated culture medium (composition is identical with inducing culture), start Proliferation, Differentiation after cultivating 10d and form indefinite bud, Proliferation, Differentiation bud number 3.567 after 34d, the long 1.672cm of Bud Differentiation, growth coefficient is 6.0.
(3) culture of rootage of indefinite bud
Get the Proliferation, Differentiation bud that length is greater than 1.5cm, being cut into length is that 1.5cm is with a blade stem section, transfers into root media: 1/2MS minimal medium+0.29mgL
-1iBA+0.03mgL
-1nAA+10gL
-1sucrose+5.0gL
-1agar (pH5.8), after 5d, stem segment base portion starts to expand greening, and has light green pinprick size particles to produce, and start after 10d to take root, after 36d, seedling root of hair number is 3.3, the long 0.906cm of root, the long 0.736cm of seedling sprouting.
(4) hardening and transplanting
After culture of rootage 32d, a bottle seedling of taking root is placed in greenhouse, and about the 7d that conforms unclamps envelope bottle rope, every other day bottle film is unclamped, about 10d transplants, and cleans root medium during transplanting, timely overlay film (every day sprays water 3 times) after transplanting, shelter from heat or light, after 7d, loose film, takes off film after 10d, transplants outdoor after 40d, enter field management, transplanting survival rate is 96%.Other conditions are with embodiment 1.
Embodiment 4
A method for tissue culture for grey Chinese catalpa, getting after the annual germinating bar of grey Chinese catalpa elite stand carries out water planting vernalization, comprising the following steps:
(1) asepticize process
Win the sprouting bud after the vernalization of grey Chinese catalpa bough water planting, remove all blades, use tap water 30min, superclean bench soaks 6 ~ 7min with the liquor natrii hypochloritis of 10%, after aseptic water washing 4 ~ 5 times, blot surface moisture with aseptic filter paper, bud is cut into the long stem section of 1.5cm (ensureing that stem section has an axillalry bud at least) and is inoculated in inducing culture: DKW minimal medium+0.9mgL
-16-BA+0.2mgL
-1iBA+25gL
-1sucrose+4.5gL
-1on agar (pH5.8).
(2) the differentiation-inducing and propagation of bud
After stem section is inoculated in inducing culture 3d, stem segment with axillary buds place bud starts to expand, and about 10d stem segment base portion callus starts to expand, Bud Differentiation bud number 2 after cultivation 48d, the long 0.506cm of bud; Get the stem section that Bud Differentiation is cut into 1.0cm, transfer on proliferated culture medium (composition is identical with inducing culture), start Proliferation, Differentiation after cultivating 10d and form indefinite bud, Proliferation, Differentiation bud number 4.033 after 43d, the long 1.893cm of Bud Differentiation, growth coefficient is 7.6.
(3) culture of rootage of indefinite bud
Get Proliferation, Differentiation bud (length is greater than 1.5cm), being cut into length is that 1.5cm is with a blade stem section, transfers into root media: 1/2MS minimal medium+0.32mgL
-1iBA+0.031mgL
-1nAA+10gL
-1sucrose+5.0gL
-1agar (pH5.8), after 6d, stem segment base portion starts to expand greening, and has light green pinprick size particles to produce, and start after 8d to take root, after 38d, seedling root of hair number is 1.433, the long 1.249cm of root, the long 0.488cm of seedling sprouting.
(4) hardening and transplanting
After culture of rootage 30d, a bottle seedling of taking root is placed in greenhouse, and about the 7d that conforms unclamps envelope bottle rope, every other day bottle film is unclamped, about 10d transplants, and cleans root medium during transplanting, timely overlay film (every day sprays water 3 times) after transplanting, shelter from heat or light, after 7d, loose film, takes off film after 10d, transplants outdoor after 40d, enter field management, transplanting survival rate is 99%.Other conditions are with embodiment 1.
Embodiment 5
A method for tissue culture for grey Chinese catalpa, getting after the annual germinating bar of grey Chinese catalpa elite stand carries out water planting vernalization, comprising the following steps:
(1) asepticize process
Win the sprouting bud after the vernalization of grey Chinese catalpa bough water planting, remove all blades, use tap water 30min, superclean bench soaks 6 ~ 7min with the liquor natrii hypochloritis of 10%, after aseptic water washing 4 ~ 5 times, blot surface moisture with aseptic filter paper, bud is cut into the long stem section of 1.5cm (ensureing that stem section has an axillalry bud at least) and is inoculated in inducing culture: DKW minimal medium+0.75mgL
-16-BA+0.23mgL
-1iBA+25gL
-1sucrose+4.5gL
-1on agar (pH5.8).
(2) the differentiation-inducing and propagation of bud
After stem section is inoculated in inducing culture 3d, stem segment with axillary buds place bud starts to expand, and about 10d stem segment base portion callus starts to expand, Bud Differentiation bud number 4.333 after cultivation 44d, the long 1.1cm of bud; Get the stem section that Bud Differentiation is cut into 1.0cm, transfer on proliferated culture medium (composition is identical with inducing culture), start Proliferation, Differentiation after cultivating 10d and form indefinite bud, Proliferation, Differentiation bud number 7.037 after 45d, the long 1.992cm of Bud Differentiation, growth coefficient is 14.0.
(3) culture of rootage of indefinite bud
Get Proliferation, Differentiation bud (length is greater than 1.5cm), being cut into length is that 1.5cm is with a blade stem section, transfers into root media: 1/2MS minimal medium+0.3mgL
-1iBA+0.032mgL
-1nAA+10gL
-1sucrose+5.0gL
-1agar (pH5.8), after 5d, stem segment base portion starts to expand greening, and has light green pinprick size particles to produce, and start after 10d to take root, after 40d, seedling root of hair number is 2.8, the long 0.801cm of root, the long 0.764cm of seedling sprouting.
(4) hardening and transplanting
After culture of rootage 35d, a bottle seedling of taking root is placed in greenhouse, and about the 7d that conforms unclamps envelope bottle rope, every other day bottle film is unclamped, about 10d transplants, and cleans root medium during transplanting, timely overlay film (every day sprays water 3 times) after transplanting, shelter from heat or light, after 7d, loose film, takes off film after 10d, transplants outdoor after 40d, enter field management, transplanting survival rate is 96%.Other conditions are with embodiment 1.
Embodiment 6
A method for tissue culture for grey Chinese catalpa, getting after the annual germinating bar of grey Chinese catalpa elite stand carries out water planting vernalization, comprising the following steps:
(1) asepticize process
Win the sprouting bud after the vernalization of grey Chinese catalpa bough water planting, remove all blades, use tap water 30min, superclean bench soaks 6 ~ 7min with the liquor natrii hypochloritis of 10%, after aseptic water washing 4 ~ 5 times, blot surface moisture with aseptic filter paper, bud is cut into the long stem section of 1.5cm (ensureing that stem section has an axillalry bud at least) and is inoculated in inducing culture: DKW minimal medium+1.1mgL
-16-BA+0.13mgL
-1iBA+25gL
-1sucrose+4.5gL
-1on agar (pH5.8).
(2) the differentiation-inducing and propagation of bud
After stem section is inoculated in inducing culture 3d, stem segment with axillary buds place bud starts to expand, and about 8d stem segment base portion callus starts to expand, Bud Differentiation bud number 1.143 after cultivation 45d, the long 0.131cm of bud; Get the stem section that Bud Differentiation is cut into 1.0cm, transfer on proliferated culture medium (composition is identical with inducing culture), start Proliferation, Differentiation after cultivating 10d and form indefinite bud, Proliferation, Differentiation bud number 4.37 after 50d, the long 2.437cm of Bud Differentiation, growth coefficient is 10.7.
(3) culture of rootage of indefinite bud
Get Proliferation, Differentiation bud (length is greater than 1.5cm), being cut into length is that 1.5cm is with a blade stem section, transfers into root media: 1/2MS minimal medium+0.28mgL
-1iBA+0.029mgL
-1nAA+10gL
-1sucrose+5.0gL
-1agar (pH5.8), after 5d, stem segment base portion starts to expand greening, and has light green pinprick size particles to produce, and start after 9d to take root, after 30d, seedling root of hair number is 0.867, the long 0.679cm of root, the long 0.706cm of seedling sprouting.
(4) hardening and transplanting are
After culture of rootage 40d, a bottle seedling of taking root is placed in greenhouse, and about the 7d that conforms unclamps envelope bottle rope, every other day bottle film is unclamped, about 10d transplants, and cleans root medium during transplanting, timely overlay film (every day sprays water 3 times) after transplanting, shelter from heat or light, after 7d, loose film, takes off film after 10d, transplants outdoor after 40d, enter field management, transplanting survival rate is 95%.Other conditions are with embodiment 1.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the technology of the present invention principle; can also make part improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (4)
1. a grey Chinese catalpa method for tissue culture, comprising: gather that the annual germinating branch of grey Chinese catalpa elite stand carries out that bough water planting vernalization, asepticize process, the induction of bud and propagation, adventitious bud rooting are cultivated successively, hardening and transplanting, obtains grey Chinese catalpa plantlet in vitro;
Described bough water planting vernalization comprises: gather the annual germinating branch of grey Chinese catalpa elite stand, after indoor water planting vernalization, win sprouting bud;
Described asepticize process comprises: by the sprouting bud tap water 30min after water planting vernalization, soak 6 ~ 7min afterwards with the liquor natrii hypochloritis that mass concentration is 10%, afterwards again through aseptic water washing 4 ~ 5 times, finally blots surface moisture with aseptic filter paper;
Induction and the propagation of described bud comprise: the sprouting bud of asepticize process is cut into stem section, is then inoculated on inducing culture, cultivate 35 ~ 50 days, obtain Bud Differentiation; And then this Bud Differentiation is cut into stem section is inoculated on proliferated culture medium, cultivate and form indefinite bud after 30 ~ 45 days;
Induction and the Multiplying culture used medium of described bud are DKW minimal medium, also comprise: 0.6 ~ 1.4mgL
-16-BA, 0.1 ~ 0.3mgL
-1iBA, 25gL
-1sucrose and 4.5gL
-1agar; The induction of described bud and the pH of proliferated culture medium are 5.8;
Described adventitious bud rooting is cultivated and is comprised: the indefinite bud obtained by Proliferation, Differentiation is cut into stem section, and root induction in adventitious bud rooting medium of transferring, grew up to seedling of taking root after 28 ~ 40 days;
It is 1/2MS minimal medium that described adventitious bud rooting cultivates medium used, also comprises: 0.25 ~ 0.35mgL
-1iBA, 0.025 ~ 0.035mgL
-1nAA, 10gL
-1sucrose and 5.0gL
-1agar; The pH of described adventitious bud rooting medium is 5.8;
Described hardening and transplanting comprise: transplant after the seedling of taking root of being turned out by adventitious bud rooting is placed in greenhouse hardening, shelter from heat or light after transplanting; The matrix of transplantation of seedlings of taking root is peat soil: perlite=3 ~ 5:1; Described matrix also comprises carbendazim, and consumption is 200g/m
3; The temperature that the induction of described bud and propagation, adventitious bud rooting are cultivated is 22 ~ 26 DEG C, and intensity of illumination is 1800 ~ 2200 luxs, and light application time is 12 ~ 15 hours/every day.
2. method according to claim 1, is characterized in that, the induction of described bud and Multiplying culture DKW minimal medium used, also comprise: 0.75 ~ 1.2mgL
-16-BA, 0.13 ~ 0.23mgL
-1iBA, 25gL
-1sucrose and 4.5gL
-1agar.
3. method according to claim 1, is characterized in that, described adventitious bud rooting medium is 1/2MS minimal medium, also comprises: 0.28 ~ 0.32mgL
-1iBA, 0.029 ~ 0.032mgL
-1nAA, 10gL
-1sucrose and 5.0gL
-1agar.
4. method according to claim 1, is characterized in that, the matrix of transplantation of seedlings of taking root is peat soil: perlite=4:1.
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| CN104041418B (en) * | 2014-07-08 | 2016-06-01 | 江苏省中国科学院植物研究所 | A kind of method inducing Chinese catalpa somatic embryo to occur |
| CN105724253B (en) * | 2016-03-21 | 2018-01-12 | 广西壮族自治区农业科学院花卉研究所 | A kind of method for tissue culture of garlic perfume (or spice) rattan |
| CN106613961B (en) * | 2016-11-09 | 2018-11-02 | 湖北省林业科学研究院 | A kind of breeding method of Chinese catalpa micropropagation |
| CN106718883A (en) * | 2016-11-28 | 2017-05-31 | 华中农业大学 | A kind of Yunnan Chinese catalpa and the miniature engrafting method of test tube seedling of Chinese catalpa |
| CN107889744B (en) * | 2017-11-14 | 2019-08-13 | 山东省林木种质资源中心 | A kind of tissue culture and rapid propagation method of spun gold Chinese catalpa |
| CN111631137A (en) * | 2020-07-14 | 2020-09-08 | 郑州市农林科学研究所 | Culture medium and culture method for tissue culture of Sorbus sophorae |
| CN115812605B (en) * | 2023-02-17 | 2023-05-16 | 中国林业科学研究院 | Method for inducing adventitious buds of ash tree leaves and regenerating plants |
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