CN103190344A - Tissue culture method of fargesii - Google Patents
Tissue culture method of fargesii Download PDFInfo
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- CN103190344A CN103190344A CN2013101099039A CN201310109903A CN103190344A CN 103190344 A CN103190344 A CN 103190344A CN 2013101099039 A CN2013101099039 A CN 2013101099039A CN 201310109903 A CN201310109903 A CN 201310109903A CN 103190344 A CN103190344 A CN 103190344A
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- 238000012136 culture method Methods 0.000 title 1
- 238000000034 method Methods 0.000 claims abstract description 34
- 241000723422 Catalpa Species 0.000 claims abstract description 11
- 230000006698 induction Effects 0.000 claims abstract description 8
- 230000035784 germination Effects 0.000 claims abstract description 6
- 230000004069 differentiation Effects 0.000 claims description 56
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 43
- 239000002609 medium Substances 0.000 claims description 37
- 230000035755 proliferation Effects 0.000 claims description 31
- 230000001939 inductive effect Effects 0.000 claims description 24
- 239000008272 agar Substances 0.000 claims description 20
- 239000005720 sucrose Substances 0.000 claims description 20
- 239000001963 growth medium Substances 0.000 claims description 12
- 229920001817 Agar Polymers 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 8
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 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
- 239000003415 peat Substances 0.000 claims description 3
- 235000019362 perlite Nutrition 0.000 claims description 3
- 239000010451 perlite Substances 0.000 claims description 3
- 239000012882 rooting medium Substances 0.000 claims description 3
- 239000002689 soil Substances 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims 4
- 229930006000 Sucrose Natural products 0.000 claims 4
- 239000003501 hydroponics Substances 0.000 claims 2
- 239000000758 substrate Substances 0.000 claims 2
- 239000005708 Sodium hypochlorite Substances 0.000 claims 1
- 238000002791 soaking Methods 0.000 claims 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims 1
- 239000008223 sterile water Substances 0.000 claims 1
- 239000008399 tap water Substances 0.000 claims 1
- 235000020679 tap water Nutrition 0.000 claims 1
- 230000004083 survival effect Effects 0.000 abstract description 10
- 239000000463 material Substances 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 238000012090 tissue culture technique Methods 0.000 abstract description 2
- 230000001133 acceleration Effects 0.000 abstract 1
- 238000009395 breeding Methods 0.000 abstract 1
- 240000004528 Catalpa ovata Species 0.000 description 45
- 235000010005 Catalpa ovata Nutrition 0.000 description 45
- 238000012546 transfer Methods 0.000 description 12
- 230000003203 everyday effect Effects 0.000 description 8
- 206010020649 Hyperkeratosis Diseases 0.000 description 7
- 239000002245 particle Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 6
- 238000005507 spraying Methods 0.000 description 6
- 238000005286 illumination Methods 0.000 description 3
- 230000008774 maternal effect Effects 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 241001090347 Bignoniaceae Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 241001236296 Catalpa fargesii Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000000280 densification Methods 0.000 description 1
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- 230000035876 healing Effects 0.000 description 1
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- 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
- 239000006870 ms-medium Substances 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000031068 symbiosis, encompassing mutualism through parasitism Effects 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Cultivation Of Plants (AREA)
Abstract
本发明涉及一种灰楸组织培养方法，包括：采集灰楸母树一年生萌生枝条依次进行枝条水培催芽、无菌化处理、芽的诱导和增殖、不定芽生根培养、炼苗与移栽，得到灰楸组培苗。本发明方法选择特定的健壮无病虫害灰楸母本，通过特定的组织培养技术和培养条件，能够在短时间内获得大批量优质灰楸组培苗，满足科研和生产所需；与其他繁殖方法相比，本发明需要的母本材料少，有1～2个茎段即可大批量繁殖，且更好地保持母本的优良性状，将幼苗移栽室外，成活率能达到95%以上；繁殖速度快，苗木质量好，出苗率高；无需培育砧木与接穗，极大地减少占地面积，并降低人力及管理成本。The invention relates to a method for tissue culture of ash catalpa, which comprises: collecting annual sprouted branches of ash catalpa tree, carrying out branch hydroponic germination acceleration, aseptic treatment, bud induction and multiplication, adventitious bud rooting culture, seedling hardening and transplanting in sequence, to obtain Ash catalpa tissue culture seedlings. The method of the present invention selects a specific strong and healthy female parent of Catalpa spp., and through specific tissue culture techniques and culture conditions, can obtain large quantities of high-quality plantlets in a short period of time to meet the needs of scientific research and production; and other breeding methods In comparison, the present invention requires less female parent material, and can reproduce in large quantities with 1 to 2 stem segments, and better maintain the excellent traits of the female parent. When the seedlings are transplanted outdoors, the survival rate can reach more than 95%; The propagation speed is fast, the quality of seedlings is good, and the emergence rate is high; there is no need to cultivate rootstocks and scions, which greatly reduces the occupied area, and reduces labor and management costs.
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 the tall and big deciduous tree of Bignoniaceae (Bignoniaceae) Catalpa (Catalpa), is that the high-quality preciousness of China's traditional cultivation is with material and ornamental plantation seeds.It has a very wide distribution, and mainly being distributed in ,Jing river, the ，Yi Weihe River, China central and west regions, Fenhe river basin and Qinling Mountain is 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, perfectly straight, the tough and tensile densification of material texture, be high-quality fast-growing commerical tree species.
Usually, grey Chinese catalpa breeds by grafting and cuttage.Propagation by grafiting need shift to an earlier date 1 year cultivates stock (Chinese catalpa), gathers the bud of maternal 1 year living branch or Miao Ganshang next year as scion; In the grafting process, because scion growth in thickness generally is greater than stock, cause interface healing poor, survival rate is generally in 70% left and right, the grafting wind resistance a little less than, and many rudiments of stock base portion, need continuous bud picking, increased management cost.Cottage propagation need gather current-year branch, seedling is dry or root is buried dry (root) vernalization, gather the semi-lignified spray and carry out cuttage, comparatively strict to germination bed and cuttage bed temperature, humidity and relative air humidity requirement, management cost is high, cuttage survival rate is lower, is generally 50% left and right.
It is a kind of efficient vegetative propagation mode that tissue is cultivated, and reproductive efficiency is high and cost is lower, is to solve the fast numerous effective means of grey Chinese catalpa choiceness, has not yet to see the report that relevant grey Chinese catalpa choiceness is organized the cultivation aspect.
Summary of the invention
In order to overcome the deficiency of the vegetative propagation techniques existence such as grafting and cuttage, 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 group training seedling of high-quality efficiently.
Grey Chinese catalpa method for tissue culture provided by the invention comprises: gather that the annual germinating branch of grey Chinese catalpa elite stand carries out that bough water planting vernalization, asepticize are processed successively, the inducing and propagation, adventitious bud rooting cultivation, hardening and transplanting of bud, obtain grey Chinese catalpa group training seedling.
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 the sprouting bud.Preferably bud length to be sprouted is about 10cm, most preferably wins during 8～12cm.
Described asepticize is processed and comprised: the sprouting bud after water planting vernalization is rinsed to 30min with running water, and the liquor natrii hypochloritis who is 10% by mass concentration afterwards soaks 6～7min, again through aseptic water washing 4～5 times, finally with aseptic filter paper, blots surface moisture afterwards.
Described bud induce and propagation comprises: the sprouting bud that asepticize was processed is cut into the stem section, then is inoculated on inducing culture, cultivates 35～50 days, obtains Bud Differentiation; And then this Bud Differentiation is cut into to the stem section is inoculated on proliferated culture medium, cultivate after 30～45 days and form indefinite bud.
Preferably, the stem section that the sprouting bud that asepticize was processed is cut into, and differentiation
bud is cut into 'shas an axillalry bud on the stem section at least.
More preferably, described bud induce and propagation comprises: it is about 1.5cm that the sprouting bud that asepticize was processed is cut into length, and most preferably the stem section of 1.4～1.6cm, then be inoculated on inducing culture, cultivates 35～50 days, obtains Bud Differentiation; And then this Bud Differentiation is cut into to length is about 1.0cm, most preferably the stem section of 0.8～1.2cm is inoculated on proliferated culture medium, cultivates after 30～45 days and forms indefinite bud.
Wherein, described bud induces Differentiation and proliferation to cultivate used medium to be the DKW minimal medium, also to comprise: 0.6～1.4mgL
-1the 6-BA(6-benzylaminopurine, benzylaminmopurine), 0.1～0.3mgL
-1the IBA(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 that induces the Differentiation and proliferation medium of described bud is 5.8.
Further, the inducing in atomization of bud, after cultivating 3 days, stem segment with axillary buds place bud starts to expand; Within 8～10 days, stem segment base section callus starts to expand; Coinduction is cultivated the bud of 35～50 days (preferably 40～48 days) Bud Differentiations and is grown up in 0.7cm.
Further, this Bud Differentiation is cut into to the stem section and is inoculated on proliferated culture medium, cultivate after 8～10 days and start Proliferation, Differentiation formation indefinite bud, more than after 30～45 days (preferably 34～40 days), indefinite bud can grow to 1.0cm.
Described adventitious bud rooting is cultivated and comprised: the indefinite bud that propagation is obtained is cut into the stem section, and root induction in the root media of transferring grew up to the seedling of taking root after 28～40 days.
Wherein, it is the 1/2MS minimal medium that described adventitious bud rooting is cultivated medium used, also comprises: 0.25～0.35mgL
-1iBA, 0.025～0.035mgL
-1the NAA(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 at least to the stem section with a blade.
More preferably, after 30～45 days, indefinite bud is cut into to the long stem section in 1.5cm left and right.
Wherein, indefinite bud is in the root induction process, after 5～6 days, stem segment base section starts to expand greening, and there is light green pinprick size particles to produce, after 8～10 days, start to take root, the seedling that obtains taking root after 28～40 days (preferably 30～40 days) (after 28～40 days (preferably 30～40 days), seedling can grow 1.0～2.5cm).
Described hardening and transplanting comprise: the seedling of taking root that adventitious bud rooting is turned out is transplanted after being placed in greenhouse hardening (16～20 days), after transplanting, with shade net, shelters from heat or light.
Wherein, the seedling of taking root is placed in greenhouse and conforms and within 6～8 days, unclamp envelope bottle rope, every other day the bottle film is unclamped, and within 10～12 days, transplants afterwards.
Wherein, the matrix of the transplantation of seedlings of taking root is peat soil: perlite (V/V)=3～5:1, preferably 4:1.
Further, the matrix of the transplantation of seedlings of taking root also can comprise carbendazim, and consumption is 200g/m
3.
Wherein, described bud induce and temperature that propagation, adventitious bud rooting are cultivated is 22～26 ℃, intensity of illumination is 1800～2200 luxs (lx), light application time is 12～15 hours/every day.Preferably: intensity of illumination is 2000 luxs (lx), and light application time is 14 hours/day.
With the method for tissue culture of existing Chinese catalpa, compare:
1. the difference of Chinese catalpa and grey Chinese catalpa: Chinese catalpa and grey Chinese catalpa are two different kinds that all belong to the Bignoniaceae Catalpa, and its area, growth, adaptability, wood property have very large difference;
2. the existing ripe method for tissue culture of Chinese catalpa, but ripe grey Chinese catalpa method for tissue culture is not also arranged at present, according to current had Chinese catalpa method for tissue culture, can't be normally, the tissue that efficiently carries out grey Chinese catalpa cultivates.
Therefore, the inventive method is selected specific healthy and strong without damage by disease and insect ash Chinese catalpa female parent, by specific tissue culture technique and condition of culture, can obtain at short notice high-quality ash Chinese catalpa group training seedling in enormous quantities, meets research and production required.With other propagation methods, compare, the maternal material that the present invention needs is few, has 1～2 stem section to breed in enormous quantities, and keeps better maternal merit, 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 cultivating stock and scion, greatly reduce floor space, and reduce manpower and management cost.
The accompanying drawing explanation
Fig. 1 is the sprouting bud that 1 water planting vernalization obtained after 30 days according to embodiment.
Fig. 2 is that the Bud Differentiation obtained after 15 days is induced in 1 asepticize material cultivation according to embodiment.
The Bud Differentiation of Fig. 3 for according to embodiment 1 asepticize material, inducing cultivation to obtain after 45 days.
The indefinite bud of Fig. 4 for according to embodiment 1 Bud Differentiation, transferring and induce propagation to obtain after 40 days.
The seedling of taking root that Fig. 5 obtains after 30 days for the root induction cultivation of transferring according to embodiment 1 indefinite bud.
Fig. 6 for the shoot root of taking root that 1 indefinite bud switching root induction cultivation obtained after 30 days according to embodiment is.
Embodiment
Following examples are used for the present invention is described, 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 modification that the inventive method, step or condition are done 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, i.e. the macroelement constituent standard recipe of MS medium (Murashing and Skoog medium, 1962) of (other elements do not reduce by half) that respectively reduces by half:
If do not specialize, the conventional means that in embodiment, technological means used 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, treat to win about the long 10cm of bud.
The number of days that the present invention is given is all calculated from starting cultivation.
Embodiment 1
The method for tissue culture of a kind of grey Chinese catalpa, get the annual germinating branch of grey Chinese catalpa elite stand, indoor, carries out water planting (running water) vernalization, after sprouting length and reaching more than 8cm, wins and sprout bud (as shown in Figure 1), next comprises the following steps:
(1) asepticize is processed
Win the sprouting bud after the vernalization of grey Chinese catalpa bough water planting, remove all blades, rinse 30min with running water, liquor natrii hypochloritis with 10% on superclean bench soaks 6～7min, blot surface moisture with aseptic filter paper after aseptic water washing 4～5 times, bud is cut into to the long stem section of 1.5cm (guaranteeing to have an axillalry bud at least on the stem section) 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) bud induces and breeds
After the stem section is inoculated in inducing culture 3d, stem segment with axillary buds place bud starts to expand, 9d left and right stem segment base section callus starts to expand, grow up and (induce cultivation totally 45 days) after 1.0cm until the bud of Bud Differentiation, get Bud Differentiation and be cut into the stem section of 1.0cm, transfer upper in proliferated culture medium (composition is identical with inducing culture), cultivate after 10d and start Proliferation, Differentiation 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 section starts to expand greening, and has light green pinprick size particles to produce, and after 10d, starts to take root, and after root symbiosis is cultivated 30d, seedling can long 1.2cm.
(4) hardening and transplanting
When the culture of rootage root system grows to 2.5cm (after culture of rootage 30～40d), bottle seedling of taking root is placed in greenhouse, and envelope bottle rope is unclamped in the 7d left and right that conforms, every other day the bottle film is unclamped, 10d transplants left and right, cleans the root medium during transplanting, transplants rear overlay film (spraying water every day 3 times) in time, shelter from heat or light, loose film after 7d, take off film after 10d, after 40d, transplant outdoor, 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 to use carbendazim sterilizing, consumption be 200g/m
3.Bud induce and temperature that propagation, adventitious bud rooting are cultivated is 25 ℃, intensity of illumination is 2000 luxs (lx), 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
The method for tissue culture of a kind of grey Chinese catalpa, after getting the annual germinating branch of grey Chinese catalpa elite stand and carrying out water planting vernalization, comprises the following steps:
(1) asepticize is processed
Win the sprouting bud after the vernalization of grey Chinese catalpa bough water planting, remove all blades, rinse 30min with running water, liquor natrii hypochloritis with 10% on superclean bench soaks 6～7min, blot surface moisture with aseptic filter paper after aseptic water washing 4～5 times, bud is cut into to the long stem section of 1.5cm (guaranteeing to have an axillalry bud at least on the stem section) 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) bud induces Differentiation and proliferation
After the stem section is inoculated in inducing culture 3d, stem segment with axillary buds place bud starts to expand, and 10d left and right stem segment base section callus starts to expand, and after cultivation 40d, Bud Differentiation bud number reaches 5, the long 2.86cm of bud; Get Bud Differentiation and be cut into the stem section of 1.0cm, transfer upper in proliferated culture medium (composition is identical with inducing culture), cultivate after 10d and start Proliferation, Differentiation and form indefinite bud, after 36d, the Proliferation, Differentiation bud is several 4.7, the long 2.97cm of Bud Differentiation, and 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 section starts to expand greening, and has light green pinprick size particles to produce, and after 10d, starts to take root, and 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, bottle seedling of taking root is placed in greenhouse, and envelope bottle rope is unclamped in the 7d left and right that conforms, every other day the bottle film is unclamped, 10d transplants left and right, cleans the root medium during transplanting, transplants rear overlay film (spraying water every day 3 times) in time, shelter from heat or light, loose film after 7d, take off film after 10d, after 40d, transplant outdoor, enter field management, transplanting survival rate is 98%.Other conditions are with embodiment 1.
Embodiment 3
The method for tissue culture of a kind of grey Chinese catalpa, after getting the annual germinating bar of grey Chinese catalpa elite stand and carrying out water planting vernalization, comprises the following steps:
(1) asepticize is processed
Win the sprouting bud after the vernalization of grey Chinese catalpa bough water planting, remove all blades, rinse 30min with running water, liquor natrii hypochloritis with 10% on superclean bench soaks 6～7min, blot surface moisture with aseptic filter paper after aseptic water washing 4～5 times, bud is cut into to the long stem section of 1.5cm (guaranteeing to have an axillalry bud at least on the stem section) 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) bud induces Differentiation and proliferation
After the stem section is inoculated in inducing culture 3d, stem segment with axillary buds place bud starts to expand, and 8d left and right stem segment base section callus starts to expand, and after cultivation 38d, the Bud Differentiation bud is several 3.43, the long 0.765cm of bud; Get Bud Differentiation and be cut into the stem section of 1.0cm, transfer upper in proliferated culture medium (composition is identical with inducing culture), cultivate after 10d and start Proliferation, Differentiation and form indefinite bud, after 34d, the Proliferation, Differentiation bud is several 3.567, the long 1.672cm of Bud Differentiation, and 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 section starts to expand greening, and has light green pinprick size particles to produce, and after 10d, starts to take root, and 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, bottle seedling of taking root is placed in greenhouse, and envelope bottle rope is unclamped in the 7d left and right that conforms, every other day the bottle film is unclamped, 10d transplants left and right, cleans the root medium during transplanting, transplants rear overlay film (spraying water every day 3 times) in time, shelter from heat or light, loose film after 7d, take off film after 10d, after 40d, transplant outdoor, enter field management, transplanting survival rate is 96%.Other conditions are with embodiment 1.
Embodiment 4
The method for tissue culture of a kind of grey Chinese catalpa, after getting the annual germinating bar of grey Chinese catalpa elite stand and carrying out water planting vernalization, comprises the following steps:
(1) asepticize is processed
Win the sprouting bud after the vernalization of grey Chinese catalpa bough water planting, remove all blades, rinse 30min with running water, liquor natrii hypochloritis with 10% on superclean bench soaks 6～7min, blot surface moisture with aseptic filter paper after aseptic water washing 4～5 times, bud is cut into to the long stem section of 1.5cm (guaranteeing to have an axillalry bud at least on the stem section) 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) bud induces Differentiation and proliferation
After the stem section is inoculated in inducing culture 3d, stem segment with axillary buds place bud starts to expand, and 10d left and right stem segment base section callus starts to expand, and after cultivation 48d, the Bud Differentiation bud is several 2, the long 0.506cm of bud; Get Bud Differentiation and be cut into the stem section of 1.0cm, transfer upper in proliferated culture medium (composition is identical with inducing culture), cultivate after 10d and start Proliferation, Differentiation and form indefinite bud, after 43d, the Proliferation, Differentiation bud is several 4.033, the long 1.893cm of Bud Differentiation, and 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 section starts to expand greening, and has light green pinprick size particles to produce, and after 8d, starts to take root, and 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, bottle seedling of taking root is placed in greenhouse, and envelope bottle rope is unclamped in the 7d left and right that conforms, every other day the bottle film is unclamped, 10d transplants left and right, cleans the root medium during transplanting, transplants rear overlay film (spraying water every day 3 times) in time, shelter from heat or light, loose film after 7d, take off film after 10d, after 40d, transplant outdoor, enter field management, transplanting survival rate is 99%.Other conditions are with embodiment 1.
Embodiment 5
The method for tissue culture of a kind of grey Chinese catalpa, after getting the annual germinating bar of grey Chinese catalpa elite stand and carrying out water planting vernalization, comprises the following steps:
(1) asepticize is processed
Win the sprouting bud after the vernalization of grey Chinese catalpa bough water planting, remove all blades, rinse 30min with running water, liquor natrii hypochloritis with 10% on superclean bench soaks 6～7min, blot surface moisture with aseptic filter paper after aseptic water washing 4～5 times, bud is cut into to the long stem section of 1.5cm (guaranteeing to have an axillalry bud at least on the stem section) 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) bud induces Differentiation and proliferation
After the stem section is inoculated in inducing culture 3d, stem segment with axillary buds place bud starts to expand, and 10d left and right stem segment base section callus starts to expand, and after cultivation 44d, the Bud Differentiation bud is several 4.333, the long 1.1cm of bud; Get Bud Differentiation and be cut into the stem section of 1.0cm, transfer upper in proliferated culture medium (composition is identical with inducing culture), cultivate after 10d and start Proliferation, Differentiation and form indefinite bud, after 45d, the Proliferation, Differentiation bud is several 7.037, the long 1.992cm of Bud Differentiation, and 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 section starts to expand greening, and has light green pinprick size particles to produce, and after 10d, starts to take root, and 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, bottle seedling of taking root is placed in greenhouse, and envelope bottle rope is unclamped in the 7d left and right that conforms, every other day the bottle film is unclamped, 10d transplants left and right, cleans the root medium during transplanting, transplants rear overlay film (spraying water every day 3 times) in time, shelter from heat or light, loose film after 7d, take off film after 10d, after 40d, transplant outdoor, enter field management, transplanting survival rate is 96%.Other conditions are with embodiment 1.
Embodiment 6
The method for tissue culture of a kind of grey Chinese catalpa, after getting the annual germinating bar of grey Chinese catalpa elite stand and carrying out water planting vernalization, comprises the following steps:
(1) asepticize is processed
Win the sprouting bud after the vernalization of grey Chinese catalpa bough water planting, remove all blades, rinse 30min with running water, liquor natrii hypochloritis with 10% on superclean bench soaks 6～7min, blot surface moisture with aseptic filter paper after aseptic water washing 4～5 times, bud is cut into to the long stem section of 1.5cm (guaranteeing to have an axillalry bud at least on the stem section) 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) bud induces Differentiation and proliferation
After the stem section is inoculated in inducing culture 3d, stem segment with axillary buds place bud starts to expand, and 8d left and right stem segment base section callus starts to expand, and after cultivation 45d, the Bud Differentiation bud is several 1.143, the long 0.131cm of bud; Get Bud Differentiation and be cut into the stem section of 1.0cm, transfer upper in proliferated culture medium (composition is identical with inducing culture), cultivate after 10d and start Proliferation, Differentiation and form indefinite bud, after 50d, the Proliferation, Differentiation bud is several 4.37, the long 2.437cm of Bud Differentiation, and 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 section starts to expand greening, and has light green pinprick size particles to produce, and after 9d, starts to take root, and 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, bottle seedling of taking root is placed in greenhouse, and envelope bottle rope is unclamped in the 7d left and right that conforms, every other day the bottle film is unclamped, 10d transplants left and right, cleans the root medium during transplanting, transplants rear overlay film (spraying water every day 3 times) in time, shelter from heat or light, loose film after 7d, take off film after 10d, after 40d, transplant outdoor, 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 that does not break away from the technology of the present invention principle; can also make the part improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
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| 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 |
| CN107889744A (en) * | 2017-11-14 | 2018-04-10 | 山东省林木种质资源中心 | The tissue culture and rapid propagation method that a kind of spun gold is seized |
| 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 |
| CN115812605A (en) * | 2023-02-17 | 2023-03-21 | 中国林业科学研究院 | Method for inducing adventitious buds of kalopanax septemlobus leaves and regenerating plants |
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| CN105724253A (en) * | 2016-03-21 | 2016-07-06 | 广西壮族自治区农业科学院花卉研究所 | Tissue culture method of Mansoa alliacea |
| CN106613961A (en) * | 2016-11-09 | 2017-05-10 | 湖北省林业科学研究院 | Cultivation method for micropropagation of catalpa bungei |
| 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 |
| CN107889744A (en) * | 2017-11-14 | 2018-04-10 | 山东省林木种质资源中心 | The tissue culture and rapid propagation method that a kind of spun gold is seized |
| 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 |
| CN115812605A (en) * | 2023-02-17 | 2023-03-21 | 中国林业科学研究院 | Method for inducing adventitious buds of kalopanax septemlobus leaves and regenerating plants |
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