CN105265317B - A kind of For-carrying green onions rapid propagation method - Google Patents
A kind of For-carrying green onions rapid propagation method Download PDFInfo
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Abstract
The invention discloses a kind of For-carrying green onions rapid propagation method, by the cleaning of For-carrying green onions centre, sterilization.After explant sterilizes, in centre far from crosscutting at 3-5mm of bottom and remove top scale, along main growing point vertical tangent lines direction, it is longitudinally split into 4 parts, remove the plateau where main growing point and main growing point, induce Multiple Buds, and cultivate seedling, For-carrying green onion plateau rapid propagation systems are established by the optimization of above-mentioned incubation, substantial amounts of For-carrying green onions test tube seedling are obtained, the system generation time is short, growth coefficient is big, genetic stability is high, and the fast breeding of For-carrying green onions can be realized by the system, the plantation of For-carrying green onion industries is realized.
Description
Technical field
The invention belongs to agricultural technology field, and in particular to a kind of For-carrying green onions rapid propagation method.
Background technology
Plant tissue culture technique is the emerging technology grown up based on plant physiology, is referred to sterile
Under the conditions of, by vitro plant organ, tissue, cell, protoplast on the culture medium manually prepared, give suitable condition
Cultivated, induction produces callus or adventitious bud etc., or grows up to the process of intact plant.The technology is from 20 beginnings of the century
Since foundation, continued to develop in theoretical research and application technology, be widely used to the quick breeding of plant, breed improvement,
In terms of genetic engineering breeding, Germ-plasma resources protection, secondary metabolite production, generate huge economic benefit and society imitates
Benefit, profound influence is generated to modern agriculture and medicine and other fields.
For-carrying green onions, also known as cold green onion, are the plant of Liliaceae allium, perennial herb.In recent years, both at home and abroad many scholars from For-carrying
Many chemical compositions, predominantly sulfur-containing compound, flavone compound, Zi bodies compound and carbohydrate, and to this are isolated in green onion
Research is expanded in terms of the pharmacology of a little chemical compositions.Experiment proves that some of chemical compositions are to pre- preventing thrombosis, artery hard
Change, cerebral infarction is plugged with good action effect.Also, due to the verdant and thick thick flavors of For-carrying, slightly tasty and refreshing game, excellent taste.In addition its
Inclusion has significant pharmacological action, and dining table can be seen as delicious food, is the natural health care being free from side effects again.In Japan
With the in the market of South Korea, For-carrying green onions are one of extremely welcome vegetables.But, current China also rarely has on the introduction and acclimatization of For-carrying green onions
Report.Wild For-carrying green onions are grown on the area of 1,000 meter to 2,500 meters of height above sea level, are frequently grown in meadow, sylvan life, dark and damp hillside or ditch
Side.Due to the harvesting of the predation formulas of wild For-carrying green onions, causing this wild resource to be faced with exhaustion, in addition wild For-carrying green onions grow by
The limitation of the conditions such as weather, far can not meet and market needs.
The breeding of For-carrying green onions is more difficult, identical with most of alliums, its modes of reproduction can also be divided into generative propagation with it is asexual
Breeding.Research shows, plants better than with seminal propagation with bulb, because it is edible to need 3-5 to pluck with seminal propagation,
And the vitality reduction of seed is quickly, and germination is difficult, germination percentage is relatively low, and can improve bulb yield with bulb breeding；
Current foreign scholar proposes to be bred with the method for tissue cultures, and studies more deep, but because its is costly,
It can't be widely applied in production.Therefore, how rapid, high volume breeding For-carrying green onions turn into For-carrying green onion industrialization productions in the short time
It is crucial.
Tissue-culturing rapid propagation is carried out to For-carrying green onions using tissue culture technique, the fast breeding of For-carrying green onions in a short time can be realized,
But the height of proliferation rate is to determine that can For-carrying green onions carry out the bottleneck of factorial praluction, therefore invention one by tissue-culturing rapid propagation approach
It is from the effective way solved the above problems at all to plant For-carrying green onion rapid propagation methods.
The content of the invention
It is long the invention aims to solve For-carrying green onion growth cycles, and tissue-culturing rapid propagation propagation is inefficient, it is difficult to meet
The problem of large-scale production, and a kind of For-carrying green onions rapid propagation method is provided.
A kind of For-carrying green onions rapid propagation method, it includes：
1）The acquisition of For-carrying green onion plateaus
For-carrying green onion bulbs are taken, For-carrying green onion fibrous roots are removed, outer scale is divested, retained centered on Central growing point, it is a diameter of
The scale of 0.4-1cm plateau and its top parcel, obtains the centre of For-carrying green onions；
For-carrying green onions centre is rinsed, filter paper suck dry moisture is used in sterilization；
After explant sterilizes, in centre far from crosscutting at 3-5mm of bottom and remove top scale, along main growth
Point vertical tangent lines direction, it is longitudinally split into 4 parts, the plateau where main growing point and main growing point is removed, is cultivated respectively；
2）For-carrying green onions plateau is bred
The culture medium based on MS, addition 6-BA 0.5-2.0 mg/L, NAA 0.05-0.5 mg/L, PH is adjusted to 5.8,
Induce Multiple Buds；
3）Continue to be trained to plant and plant seedlings；
Described cuts into 4 parts, is, using main two vertical tangent lines of growing point as otch, to be cut, be cut into 4 parts, its
Middle 1/4 part of stripping and slicing of only one of which carries the plateau where main growing point, peels the squama where its main growing point and main growing point off
Stem disk；
The scale base portion on 1/4 part of described plateau top is cut again, high to remote main growing point end, nearly main growing point
The low inclined-plane in end.
Described 6-BA and NAA addition are respectively 1.0 mg/L and 0.1 mg/L；
Step 3）Described continuation culture seedling, is the culture medium based on MS, addition NAA 0.1-1 mg/L, PH regulations
To 5.8；Tufted seedling is inoculated in root media, seedling rooting of being grown thickly after 3 weeks；Tufted seedling after taking root is divided into individual plant,
It is inoculated in strong seedling culture base, obtains For-carrying green onion seedlings；Seedling is after domestication, plantation；
Described For-carrying green onions centre sterilization, after 75% alcohol-pickled sterilization 1min, is then soaked with 1%NaClO solution
15-20min of bubble sterilization.
The invention provides a kind of For-carrying green onions rapid propagation method, by the cleaning of For-carrying green onions centre, sterilization.Gone out by explant
After bacterium, in centre far from crosscutting at 3-5mm of bottom and remove top scale, along main growing point vertical tangent lines direction, longitudinal direction point
4 parts are cut into, the plateau where main growing point and main growing point is removed, Multiple Buds are induced, and cultivates seedling, passes through above-mentioned culture
The optimization of process establishes For-carrying green onion plateau rapid propagation systems, obtains substantial amounts of For-carrying green onions test tube seedling, the system generation time
Short, growth coefficient is big, and genetic stability is high, and the fast breeding of For-carrying green onions can be realized by the system, the kind of For-carrying green onion industries is realized
Plant.
Brief description of the drawings
The influence that Fig. 1 different hormone combinations are bred to tillered-onion stem apex（a：Tillered-onion stem apex；b：Stem apex 6-BA,
Proliferative conditions in NAA combinations；c：Proliferative conditions of the stem apex in 6-BA, IAA）；
The acquisition of Fig. 2 tillered-onion plateaus；
The micro- plateau cutting step flow of Fig. 3 tillered-onion test tubes；
The micro- plateau cutting step schematic flow sheet of Fig. 4 tillered-onion test tubes；
Fig. 5 tillered-onion test tube bulbs disk is bred（a：The micro- plateau of tillered-onion test tube；b：2nd week micro- Microscopic observation
The proliferative conditions arrived；c：3rd week adventitious buds proliferation, growing state；d：4th week adventitious buds proliferation, growing state）；
Fig. 6 tillered-onion the growth of plants cultures（a：Tillered-onion test tube breeds tufted seedling；b：Individual plant tillered-onion is tried
Guan Miao；c：Test tube seedling after strong seedling culture）；
The acquisition in Fig. 7 For-carrying green onions centre；
The acquisition of Fig. 8 For-carrying green onion plateaus；
Fig. 9 For-carrying green onions plateau breeds the acquisition of seedling；
Figure 10 For-carrying green onions breed seedling rooting；
The acquisition of Figure 11 garlic bulb disks；
Figure 12 garlic bulbs disk is inoculated with；
Figure 13 garlic bulbs disk is bred.
Embodiment
The tillered-onion stem apex of embodiment 1 is bred
Tillered-onion stem apex is inoculated in respectively in 6-BA, NAA combination of various concentrations, 6-BA, IAA combination culture medium
（Table 1）, two kinds of hormone combinations result in tillered-onion test tube propagation seedling, but the IAA and NAA of same concentrations are being bred
It is widely different during induction.The propagation seedling growing way wherein induced in 6-BA, NAA hormone combinations is preferable, and coefficient of differentiation is higher,
When cultivating 4 weeks, average coefficient of proliferation is up to 3.87；Although 6-BA, IAA hormone combinations can also realize that stem apex is bred, institute
The propagation seedling of acquisition is thin, weak, and coefficient of differentiation is low, and has Albino Seedling appearance（Such as Fig. 1）.It can be seen that, IAA action effect is not so good as
NAA.It is thus determined that MS+6-BA0.3mg/L+NAA0.1mg/L culture mediums, which are stem apex, breeds optimal medium.
Note：Culture medium based on MS.
The tillered-onion plateau of embodiment 2 is bred
One piece of tillered-onion is taken, the position of tillered-onion plateau suberification is removed, retained centered on Central growing point,
The bulb of a diameter of 0.4-1cm plateau and its top parcel, obtains tillered-onion centre.By tillered-onion central part
0.5 h is rinsed in position with flowing water, after 75% 1 min of alcohol-pickled sterilization, then with 1%NaClO solution soaking disinfections 15-20
Min, aseptic water washing 3-5 times is standby with filter paper suck dry moisture to remove remaining NaClO（Such as Fig. 2）.
Then top bulb is removed, is retained comprising the plateau including main growing point, plateau is cut into after 4 parts and removed
Main growing point and main growing point plateau, are inoculated in the culture medium based on MS respectively, add 6-BA（0.2-0.8 mg/L）、
NAA（0.1-2. 0mg/L）, PH adjusts into 5.8 increment culture medium culture, induces Multiple Buds.It is in 25 DEG C, light application time
Under the conditions of 16h, intensity of illumination are 2500lux, by the induction of 4 weeks, inducing effect is combined with 6-BA, NAA best；6-BA、IAA
Combination can also induce Multiple Buds, but growth coefficient is low, and regrowth has lopsided seedling.Wherein plateau is in addition 6-BA0.6mg/
In L, NAA0.1mg/L culture medium, after 4 weeks, differentiation number reaches maximum, the maximum growth coefficient of each plateau（Growth coefficient
=Bud Differentiation sum/inoculation number）Up to 20（It is shown in Table 2）.With the increase of induction time, plateau differentiation number increase, but inducing
Plateau differentiation number is not further added by after 4th week, and the differentiation rate of plateau reaches maximum（It is shown in Table 3）.
Note：Culture medium based on MS.
Note：Culture medium based on MS.
The tillered-onion detoxic seedling test tube balling of embodiment 3
Tillered-onion stem apex position is taken, flowing water rinses half an hour, after 75% alcohol-pickled sterilization 1min, then with 1%
NaClO solution 15-20min of soaking disinfection, aseptic water washing 3-5 times is standby with filter paper suck dry moisture to remove remaining NaClO
With stripping tillered-onion shoot tip meristem under microscope<0.3mm parts, in the culture medium based on MS, addition 6-BA, IAA's
Seedling is induced in primary culture medium.
After stem apex seedling, carbon source sets 30mg/L, 45mg/L, 60mg/L, 75mg/L, 90mg/L based on sucrose respectively
The sugared concentration of totally 5 carbon sources, inquires into influence of the different sugar concentration to tillered-onion test tube seedling balling, screens in optimal balling culture medium
Sugared concentration, is found by cultivating, increase bulb balling diameter, fresh weight increase with sugared concentration.When sugared concentration is higher than 75 mg/L
When, balling speed is accelerated but influences the growth of root, causes final bulb average diameter, fresh weight reduction（It is shown in Table 4）.
In detoxic seedling balling incubation, by the regulation and control to cultivating day and night temperature, the balling speed of test tube seedling is adjusted,
The preference temperature grown for tillered-onion sets 25 DEG C/25 DEG C, 25 DEG C/20 DEG C, 25 DEG C/15 DEG C totally 3 groups of diurnal temperatures respectively
Processing, screens optimum culturing temperature.When day and night temperature is 10 DEG C, bulb diameter and bulb fresh weight are better than other processing.Thus
It is determined that during tillered-onion detoxic seedling balling, optimum culturing temperature is 25 DEG C of daytime, 15 DEG C of night（It is shown in Table 5）.
In detoxic seedling balling incubation, by the intensity of illumination in incubation, the regulation and control of photoperiod, regulation examination
Guan Miao balling speed, sets 3 groups of intensities of illumination, photoperiod to handle respectively for tillered-onion growth conditions.As a result show, with
The extension of intensity of illumination and light application time, the average diameter and bulb fresh weight of test tube ball are stepped up, and are in intensity of illumination
Be conducive to tillered-onion detoxification test tube plantlet balling when 2500lux, 16h illumination, 8h dark conditions, its test tube ball bulb is averagely straight
Footpath, bulb fresh weight are above other condition of culture（Table 6）.
Efficient tillered-onion detoxic seedling test tube bulbs induction system is established by the optimization of balling condition, is tiller ocean
The fast breeding of green onion virus-elimination seedlingses provides favourable guarantee.
Note：Culture medium based on MS.
Note：Culture medium based on MS.
Note：Culture medium based on MS.
Embodiment 4 sets up tillered-onion virus-elimination seedlingses rapid propagation system using the micro- plateau multiplication technique of test tube
（1）The acquisition of the micro- plateau of test tube
Tillered-onion test tube seedling can be cut after balling culture 6 weeks or when tillered-onion test tube bulb diameter reaches more than 8mm
Take the micro- plateau of test tube.Using main two vertical tangent lines of growing point plateau as otch, progress is longitudinally cutting, is cut into 4 parts, wherein
1/4 part of stripping and slicing of only one of which carries main growing point plateau, peels the plateau where main growing point and main growing point off.Again by 1/
The scale base portion on 4 parts of plateau tops is cut again, high to remote main growing point end, the nearly low inclined-plane in main growing point end（As schemed
3rd, Fig. 4）.
The micro- plateau of test tube passes through the culture of 2 weeks in addition 6-BA0.6mg/L, NAA0.1mg/L culture medium, can be
The differentiation of Multiple Buds is observed under microscope, after 3 weeks, Multiple Buds plant height is maximum up to 1cm, and Multiple Buds plant height is up to 3cm after 4 weeks
（Such as Fig. 5）, and differentiation number reaches maximum, the maximum growth coefficient of each plateau（Growth coefficient=Bud Differentiation sum/inoculation
Number）Up to 34.With the increase of induction time, plateau differentiation number increase, but plateau breaks up number no longer after inducing the 4th week
Increase, the differentiation rate of plateau reaches maximum.
（2）The plateau that field is opened and influence of the micro- plateau of test tube to tillered-onion test tube seedling proliferation
The micro- bulb of plateau and test tube opened respectively using field carries out tillered-onion fast breeding, experiment as explant
As a result show, the propagation efficiency of the micro- bulb of test tube is 1.66 times of the plateau increment efficiency that field is opened（It is shown in Table 7）.Illustrate examination
Micro- plateau is managed with higher differentiation capability, the optimal explant of tillered-onion fast breeding is suitable as.
Note：Culture medium based on MS.
（3）Influence of the test tube ball development degree to test tube seedling proliferation
Detoxification bulb plateau using the offer of tillered-onion detoxification test tube plantlet is as explant, with the tiller of different growing stages
Onion test tube ball plateau is that explant carries out test tube seedling Multiplying culture, is as a result shown, with the increase of balling incubation time, examination
The differentiation capability of pipe ball plateau gradually strengthens, and balling culture proceeds to the 4th week, when bulb average diameter reaches 3.56mm, tool
There is differentiation capability, but the mean tillering number is not high；The test tube ball to after the 6th week is cultivated, differentiation capability is greatly improved, the mean tillering number
27.63 are reached, the mean tillering number reaches maximum 32.19 when cultivating the 10th week, and differentiation capability declines afterwards.Thus, we are true
Determine that balling incubation time is most short, differentiation capability higher time point-balling culture is used as tillered-onion test tube bulbs disk on the 6th week
It is optimal that the phase is provided（It is shown in Table 8）.
Note：Culture medium based on MS.
On this basis, the culture medium based on 1/2MS, by adjusting the sugared concentration in culture medium, NAA concentration, regulation
Tufted seedling growth, rooting rate.As a result show, in the culture medium that sugared concentration is 45mg/L, NAA0.1mg/L（PH=5.8）In, examination
Pipe seedling rooting most fast, growing way is optimal（It is shown in Table 9）.After 1 week, rooting rate is up to 100%, and test tube seedling color is dark green, sturdy, is obtained after one week
Obtain tiller Onion Seedling（See Fig. 6）.
Note：Culture medium based on MS.
Embodiment 5 sets up For-carrying green onion quick reproduction technique systems using For-carrying green onion plateau multiplication techniques
（1）The acquisition of For-carrying green onion plateaus
1）For-carrying green onion bulbs are taken, For-carrying green onion fibrous roots are removed, outer scale is divested, retained centered on Central growing point, it is a diameter of
The scale of 0.4-1cm plateau and its top parcel, obtains the centre of For-carrying green onions（Fig. 7）；
2）For-carrying green onions centre flowing water is rinsed into half an hour, after 75% alcohol-pickled sterilization 1min, then with 1%
NaClO solution 15-20min of soaking disinfection, aseptic water washing 3-5 times is standby with filter paper suck dry moisture to remove remaining NaClO
With；
3）After explant sterilizes, in centre far from crosscutting at 3-5mm of bottom and remove top scale, along main life
Long point vertical tangent lines direction, longitudinally split into 4 parts, the plateau where removing main growing point and main growing point is standby（Fig. 8）；
（2）For-carrying green onions plateau is bred
1）The culture medium based on MS, adds 6-BA（0.5-2.0 mg/L）、NAA（0.1-1.0 mg/L）, PH adjust to
5.8, prepare proliferated culture medium standby；
2）Plateau is averagely cut into 4 parts, is inoculated in proliferated culture medium（Fig. 9）, after the induction of 8 weeks, grow thickly
Seedling maximum growth coefficient is up to 20（It is shown in Table 10）.
Note：Culture medium based on MS.
（3）For-carrying green onions seedling is obtained
1）The culture medium based on MS, adds NAA（0.1-1.0 mg/L）, PH adjusted to 5.8, prepares root media standby
With；
2）Tufted seedling is inoculated in root media, seedling rooting of being grown thickly after 3 weeks（Table 11）.
3）Tufted seedling after taking root is divided into individual plant, is inoculated in strong seedling culture base, For-carrying green onion seedlings can be obtained after 4 months
（Such as Figure 10）.Seedling can be colonized in field after domestication to be used to produce.
Note：Culture medium based on MS.
Embodiment 6 sets up garlic quick reproduction technique system using the micro- plateau multiplication technique of garlic
（1）Garlic propagation seedling is obtained by explant of Allium fistulosum stem tips
Allium fistulosum stem tips are inoculated in respectively in 6-BA, NAA combination of various concentrations, 6-BA, IAA combination culture medium, two kinds
Hormone combinations result in garlic propagation seedling, but same concentrations IAA and NAA carry out proliferation-inducing when, it is widely different.Its
In the propagation seedling growing way that is induced in 6-BA, NAA hormone combinations preferably, and coefficient of differentiation is higher, when cultivating 4 weeks, average increasing
Coefficient is grown up to 7.75；Although 6-BA, IAA hormone combinations can also realize that stem apex is bred, the propagation seedling obtained is thin, weak,
Coefficient of differentiation is low.It can be seen that, IAA action effect is not so good as NAA.It is thus determined that MS+6-BA2.0mg/L+NAA 0.5mg/L are cultivated
Base is that stem apex breeds optimal medium（It is shown in Table 12）.
Note：Culture medium based on MS.
（2）Regrowth is obtained by explant of garlic bulb disk
Independent garlic clove is taken, crust and fibrous root is peelled off, 0.5 h is rinsed with flowing water, after 75% 1 min of alcohol-pickled sterilization,
1%NaClO solution soaking disinfection 15-20 min, the 3-5 NaClO to remove remnants of aseptic water washing are then used, is inhaled with filter paper
Solid carbon dioxide point is standby（See Figure 11）.2）
The white portion of outer wrap is removed, it is crosscutting at away from 3-5mm of bottom and remove top scale, along main growing point
Vertical tangent lines direction, it is longitudinally split into 4 parts, the plateau where main growing point and main growing point is removed, retains 2-3mm2Squama
Stem disk（See Figure 12）, the culture medium based on MS is inoculated in respectively, adds 6-BA（1.0-3.0mg/L）、NAA（0.5-3.0 mg/
L）, PH adjusts into 5.8 proliferated culture medium culture, induces Multiple Buds.It is that 16h, intensity of illumination are in 25 DEG C, light application time
Under the conditions of 2500lux, cultivated.
It is best with 6-BA, NAA combination inducing effect by the induction of 4 weeks；6-BA, IAA combination, which can also be induced, grows thickly
Bud, but growth coefficient is low, and there is lopsided seedling in regrowth.Wherein culture of the plateau in addition 6-BA2.0mg/L, NAA0.5mg/L
In base, the maximum growth coefficient of each plateau after 4 weeks（Growth coefficient=Bud Differentiation sum/inoculation number）Up to 40（It is shown in Table 13）.With
The increase of induction time, plateau differentiation number increase, but plateau differentiation number is not further added by after inducing the 4th week, plateau
Differentiation rate reach maximum（See Figure 13）.
Note：Culture medium based on MS.
Claims (5)
1. a kind of For-carrying green onions rapid propagation method, it includes：
1）The acquisition of For-carrying green onion plateaus
For-carrying green onion bulbs are taken, For-carrying green onion fibrous roots are removed, outer scale is divested, retained centered on Central growing point, a diameter of 0.4-1cm
Plateau and its top parcel scale, obtain For-carrying green onions centre；
For-carrying green onions centre is rinsed, filter paper suck dry moisture is used in sterilization；
After explant sterilizes, in centre far from crosscutting at 3-5mm of bottom and remove top scale, hung down along main growing point
Straight tangent line direction, it is longitudinally split into 4 parts, the plateau where main growing point and main growing point is removed, is cultivated respectively；
2）For-carrying green onions plateau is bred
The culture medium based on MS, addition 6-BA 0.5-2.0 mg/L, NAA 0.05-0.5 mg/L, pH is adjusted to 5.8, induction
Multiple Buds；
3）Continue to be trained to plant and plant seedlings；
Described plateau is the micro- plateau of laboratory test tube；
Described is divided into 4 parts, is, using main two vertical tangent lines of growing point as otch, to be cut, be cut into 4 parts, wherein only
There is 1/4 part of stripping and slicing to carry the plateau where main growing point, peel the plateau where its main growing point and main growing point off；
The scale base portion on 1/4 part of described plateau top is cut again, high to remote main growing point end, and nearly main growing point end is low
Inclined-plane.
2. a kind of For-carrying green onions rapid propagation method according to claim 1, it is characterised in that：Described 6-BA's and NAA adds
Dosage is respectively 1.0 mg/L and 0.1 mg/L.
3. a kind of For-carrying green onions rapid propagation method according to claim 2, it is characterised in that：Step 3）Described continuation culture
Planted seedlings into kind, be the culture medium based on MS, add NAA 0.1-1 mg/L, pH is adjusted to 5.8；Tufted seedling is inoculated in and taken root
In culture medium, seedling rooting of being grown thickly after 3 weeks；Tufted seedling after taking root is divided into individual plant, is inoculated in strong seedling culture base, For-carrying is obtained
Green onion seedling；Seedling is after domestication, plantation.
4. a kind of For-carrying green onions rapid propagation method according to claim 3, it is characterised in that：The micro- plateau of described test tube is
The micro- plateau of test tube cultivated by stem apex position.
5. a kind of For-carrying green onions rapid propagation method according to claim 1,2,3 or 4, it is characterised in that：Described sterilization is
With 75% alcohol-pickled sterilization 1min, then with 15-20min of 1%NaClO solution soaking disinfections.
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