A kind of allium plateau rapid propagation method
Technical field
The invention belongs to agricultural technology field, and in particular to a kind of allium plateau rapid propagation method.
Background technology
Allium more Cai in production are bred Yong the raw cloves of Tiller, belong to vegetative propagation.Carry out for a long time asexual numerous
Grow, cause a kind of virus virosis that individually infection or several viral compound infections are triggered extremely serious.It is raw after plant is susceptible
Growing way is weak, and the short and small , Fen Tiller of plant are reduced, and plant causes yield and quality to be greatly lowered because degeneration diminishes, to productive consumption and
Foreign exchange earning brings serious obstacle.Allium detoxification technology uses Shoot Tip Culture mostly, achieves good effect, but
Breeding potential is not high, is difficult to mass produce.
Tillered-onion belongs to Liliaceae allium, there is the laudatory title of health food, is current known uniquely to contain prostate
Plain A plant.This material is a kind of stronger vasodilator, energy vasodilator, reduces the viscosity of blood, is increased coronal dynamic
Arteries and veins CBF, also there is reduction and antithrombotic, and the boosting of internal catecholamine can be resisted, so as to steady
Determine blood pressure.But the few bolting of tillered-onion blossoms and bears fruit, can only be bred in production with the raw clove of tiller, line of breeding
Number is relatively low, and its bulb belongs to shortening stem, and the foundation of fast breeding system has certain difficulty.At present on tillered-onion
Fast numerous relevant report is actually rare, and Xu Qijiang strips 0.3-0.5mm stem apexs and induced, then activated culture, culture of rootage
Tillered-onion stem apex detoxic seedling can be obtained.Jiang Yudong thinks that paclobutrazol has the function that very strong promotion test tube bulbs formation.
Old allusion quotation and Xu Qijiang obtain regeneration plant using tillered-onion stem apex as explant through different development ways, wherein, with tillered-onion
Stem apex be explant after initial culture culture, be transferred in root media, obtain test tube seedling.Wang Yong, Liu Zhaokun, Xiang Yuanping
Attempt to obtain tillered-onion regeneration plant by callus induction and again differentiation pathway Deng, but differentiation rate is not high, and pass through
Callus tissue culture is easy to produce variation, it is difficult to realizes the factorial praluction of tillered-onion.
For-carrying green onions, also known as cold green onion, are Liliaceae allium, perennial herb.In recent years, both at home and abroad many scholars from For-carrying green onions
In isolate many chemical compositions, experiment proves that some of chemical compositions are plugged with to pre- preventing thrombosis, artery sclerosis, cerebral infarction
Good action effect.Also, due to the verdant and thick thick flavors of For-carrying, slightly tasty and refreshing game, excellent taste.But at present China on
The introduction and acclimatization of For-carrying green onions is also rarely reported.The breeding of For-carrying green onions is more difficult, identical with most of alliums, and its modes of reproduction can also divide
For generative propagation and vegetative propagation.Research shows that it is better than with seminal propagation to be planted with bulb, because needing 3- with seminal propagation
5 years could be ripe, and the vitality of seed reduces quickly, and germination is difficult, germination percentage is relatively low, and can be carried with bulb breeding
High bulb yield.Foreign scholar proposes at present is bred with the method for tissue cultures, and studies more deep, but because
Its is costly, can't be widely applied in production.Therefore, how rapid, high volume breeding For-carrying green onions turn into the production of For-carrying green onions in the short time
The key of industry metaplasia production.
Garlic is Liliaceae allium herbaceos perennial, and the garlic of field planting is asexually propagated crop, can only be passed through
Valve or the method for formation aerosol index is divided to be bred.It is long-term to carry out vegetative propagation, the garlic of virus infection can be made constantly to accumulate
Virus and pass to offspring, the quality and yield of garlic is declined, it is difficult to meet foreign trader to export garlic quality and specification want
Ask.Simultaneously because of kind of a garlic virus infection, the general underproduction 30% -60% of garlic, the income of peasant greatly affected.Therefore, it is necessary to
Detoxification rejuvenation periodically is carried out to garlic and just can guarantee that yield.But garlic is using dividing valve method to be increased in Production of Large Fields
Grow, this results in the garlic after the detoxification of laboratory can only breed several times or more than ten times in 1 year in big Tanaka, breeding coefficient
It is relatively low, and Garlic Virus is easily infected once again in breeding.At present, to how quickly to obtain substantial amounts of detoxification test tube plantlet,
Increase detoxification garlic breeding coefficient, shorten the production cycle, reduce detoxification garlic seeling industry cost etc., carry out specifically
Research, be the key for promoting garlic detoxification production industrialization.
Plant tissue culture technique is the emerging technology to be 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,
Genetic engineering breeding, Germ-plasma resources protection, secondary metabolite production etc., generate huge economic benefit and society imitates
Benefit, profound influence is generated to modern agriculture and medicine and other fields.The tissue-culturing rapid propagation of allium can realize that allium exists
Fast breeding in short time, but the height of proliferation rate is always to determine that can allium be carried out by tissue-culturing rapid propagation approach
The bottleneck of factorial praluction, therefore it is the effective way to solve the above problems to invent a kind of allium rapid propagation method.
The content of the invention
It is not high the invention aims to solve allium tissue-culturing rapid propagation proliferation rate, it is difficult to meet large-scale production
Problem, and a kind of allium plateau rapid propagation method is provided.
Allium plateau rapid propagation method, it includes:
1)Allium bulb is taken, allium fibrous root is removed, divests outer scale, retain using Central growing point in
The scale of the heart, a diameter of 0.4-1cm plateau and its top parcel, obtain the centre of allium;Cleaning, sterilization;
2)In centre far from crosscutting at 3-5mm of bottom and remove top scale, along main growing point vertical tangent lines longitudinally point
4 parts are cut into, removes the plateau where main growing point and main growing point, is inoculated in PH 5.8, MS+6-BA 0.1-2.0mg/L,
Cultivated in NAA 0.05-1.0mg/L culture mediums, induce Multiple Buds;
3)Continue to cultivate seedling;
Described, MS+6-BA 0.1-1mg/L, NAA 0.05-0.10mg/L;
Described cleaning, sterilization rinse 0.5 h for flowing water, after 75% 1 min of alcohol-pickled sterilization, then use 1%NaClO
The min of solution soaking disinfection 15-20,3-5 NaClO with removing remnants of aseptic water washing, with filter paper suck dry moisture;
Described allium plateau is the micro- plateau of laboratory test tube;
The Fiber differentiation time of described test tube seedling balling is 6 weeks;
Described cuts into 4 parts, is using main two vertical tangent lines of growing point as otch, is longitudinally cut, be cut into 4
Part, 1/4 part of stripping and slicing of only one of which carries main growing point plateau, peels the bulb where main growing point and main growing point off
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
Hold low inclined-plane.
The invention provides a kind of allium plateau rapid propagation method, by the cleaning of allium centre, disappears
Poison, it is longitudinally split into 4 along main growing point vertical tangent lines in centre far from crosscutting rear removal top scale at 3-5mm of bottom
Part, the plateau where main growing point and main growing point is removed, induces Multiple Buds, and cultivate seedling.Pass through above-mentioned incubation
Optimization establish allium plateau rapid propagation system, obtain substantial amounts of detoxification test tube plantlet, the system generation time
Short, growth coefficient is big, and genetic stability is high, and the fast breeding of allium can be realized by the system.Can be that allium is planted
The factorial praluction of thing virus-elimination seedlingses provides low price cost, the powerful guarantee of low time cost, promotes allium industry
Development.
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 tries
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
6-BA, NAA that tillered-onion stem apex is inoculated in various concentrations respectively are combined, in 6-BA, IAA combination culture medium
(Table 1), two kinds of hormone combinations can obtain 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, retains 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 uses 1%NaClO solution soaking disinfections 15-20
Min, aseptic water washing 3-5 times are standby with filter paper suck dry moisture to remove remaining NaClO(Such as Fig. 2).
Then top bulb is removed, retains comprising the plateau including main growing point, is removed after plateau is cut into 4 parts
Main growing point and main growing point plateau, the culture medium based on MS is inoculated in respectively, add 6-BA(0.2-0.8 mg/L)、
NAA(0.1-2. 0mg/L), PH adjusts into 5.8 increment culture medium and cultivates, and induces Multiple Buds.It is in 25 DEG C, light application time
It is best with 6-BA, NAA combination inducing effect by the induction of 4 weeks under the conditions of 16h, intensity of illumination are 2500lux;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/
L, in NAA0.1mg/L culture medium, after 4 weeks, differentiation number reaches maximum, each plateau maximum growth coefficient(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 are 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, add 6-BA, IAA
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
Totally 5 carbon source sugar concentration, influence of the different sugar concentration to tillered-onion test tube seedling balling is inquired into, is screened in optimal balling culture medium
Sugared concentration, found by cultivating, with the increase bulb balling diameter of sugared concentration, fresh weight increase.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 to reduce(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,
For the preference temperature of tillered-onion growth, 25 DEG C/25 DEG C, 25 DEG C/20 DEG C, 25 DEG C/15 DEG C totally 3 groups of diurnal temperatures are set respectively
Processing, screen 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, 3 groups of intensities of illumination, photoperiod are set 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 advantageous 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 establishes 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
Only 1/4 part of stripping and slicing 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(Such as figure
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 break up number and reach 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 reach maximum.
(2)The plateau that field opens and influence of the micro- plateau of test tube to tillered-onion test tube seedling proliferation
The plateau and the micro- bulb of 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 opens(It is shown in Table 7).Illustrate examination
Managing micro- plateau has higher differentiation capability, is suitable as the optimal explant of tillered-onion fast breeding.
Note:Culture medium based on MS.
(3)Influence of the test tube ball development degree to test tube seedling proliferation
The detoxification bulb plateau provided using 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, the results showed that, 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 greatly improves, the mean tillering number
Reach 27.63, 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 the 6th week is as tillered-onion test tube bulbs disk
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 is most fast, growing way is optimal(It is shown in Table 9).After 1 week, for rooting rate up to 100%, 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 establishes 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 is removed, divests outer scale, retain centered on Central growing point, it is a diameter of
The scale of 0.4-1cm plateau and its top parcel, obtain 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 are 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, add 6-BA(0.5-2.0 mg/L)、NAA(0.1-1.0 mg/L), PH adjust to
5.8, it is standby to prepare proliferated culture medium;
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 obtains
1)The culture medium based on MS, add NAA(0.1-1.0 mg/L), PH adjusted to 5.8, and it is standby to prepare root media
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 and be used to produce.
Note:Culture medium based on MS.
Embodiment 6 establishes garlic quick reproduction technique system using the micro- plateau multiplication technique of garlic
(1)Garlic is obtained as explant breed seedling using Allium fistulosum stem tips
6-BA, NAA that Allium fistulosum stem tips are inoculated in various concentrations respectively are combined, in 6-BA, IAA combination culture medium, two kinds
Hormone combinations can obtain garlic propagation seedling, but the IAA and NAA of same concentrations are when carrying out proliferation-inducing, widely different.Its
In the propagation seedling growing way that is induced in 6-BA, NAA hormone combinations it is preferable, and coefficient of differentiation is higher, average to increase when cultivating 4 weeks
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, peels off crust and fibrous root, 0.5 h is rinsed with flowing water, after 75% 1 min of alcohol-pickled sterilization,
1%NaClO solution soaking disinfection 15-20 min are then used, the 3-5 NaClO with removing remnants of aseptic water washing, are 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 and cultivates, and induces Multiple Buds.It is in 25 DEG C, light application time 16h, intensity of illumination
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 induce, grows thickly
Bud, but growth coefficient is low, and lopsided seedling be present in regrowth.Wherein culture of the plateau in addition 6-BA2.0mg/L, NAA0.5mg/L
In base, each plateau maximum growth coefficient 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.