CN105265317A - Rapid propagation method of allium victorialis - Google Patents
Rapid propagation method of allium victorialis Download PDFInfo
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- CN105265317A CN105265317A CN201510730285.9A CN201510730285A CN105265317A CN 105265317 A CN105265317 A CN 105265317A CN 201510730285 A CN201510730285 A CN 201510730285A CN 105265317 A CN105265317 A CN 105265317A
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Abstract
The invention discloses a rapid propagation method of allium victorialis. The rapid propagation method of the allium victorialis comprises the following steps: washing and disinfecting the central part of the allium victorialis; carrying out explant sterilization, then transversely cutting from a position with a distance of 3-5mm from the bottom end in the central part and removing upper scales, longitudinally cutting into four parts along a vertical tangent direction of a main growth point, removing the main growth point and a bulb pan where the main growth point is located, inducing clustered shoots and culturing the clustered shoots into seedlings. A rapid propagation system of the bulb pan of the allium victorialis is built by optimizing the abovementioned culture process, and a large number of allium victorialis test tube seedlings are obtained. The system is short in proliferation time, high in proliferation coefficient and high in hereditary stability; through the system, the rapid propagation of the allium victorialis can be achieved, and the planting of the allium victorialis industry can be achieved.
Description
Technical field
The invention belongs to agricultural technology field, be specifically related to a kind of For-carrying green onion method for quickly breeding.
Background technology
Plant tissue culture technique is the emerging technology grown up based on plant physiology, refer to aseptically, by in vitro plant organ, tissue, cell, protoplast on the medium of artificial preparation, the condition be suitable for is cultivated, induction produces callus or indefinite bud etc., or grows up to the process of whole plant.This technology is since 20 beginnings of the century set up, at the technical development of theoretical investigation and application, the aspects such as the Fast-propagation of plant, breed improvement, genetic engineering breeding, Germ-plasma resources protection, secondary metabolite production are widely used in, create huge economic benefit and social benefit, profound influence is created to modern agriculture and medicine and other fields.
For-carrying green onion, has another name called cold green onion, is the plant of Liliaceae allium, perennial herb.In recent years, domestic and international many scholars isolate many chemical compositions from For-carrying green onion, are mainly sulfur-containing compound, flavonoids, Zi body compound and carbohydrate, and expand research to the pharmacology aspect of these chemical compositions.Through verification experimental verification, some of them chemical composition is plugged with good action effect to pre-preventing thrombosis, arteriosclerosis, cerebral infarction.Further, due to the verdant and thick thick flavor of For-carrying, slightly tasty and refreshing game, excellent taste.In addition its inclusion has significant pharmacological action, can see dining table as delicious food, is again the natural health care of being free from side effects.Japan and Korea S market on, For-carrying green onion is one of extremely welcome vegetables.But current China also rarely has report about the introduction and acclimatization of For-carrying green onion.Wild For-carrying green onion is grown on height above sea level 1, the area of 000 meter to 2,500 meters, often grow on meadow, sylvan life, dark and damp hillside or limes marginis.Owing to plucking the predation formula of wild For-carrying green onion, cause this wild resource to be faced with exhaustion, wild For-carrying green onion grows the restriction of the conditions such as climate in addition, can not meet far away and market demand.
The breeding of For-carrying green onion is more difficult, identical with most of allium, and its modes of reproduction also can be divided into sexual propagation and vegetative propagation.Research shows, with bulb plantation than good with seminal propagation, because it is edible to need 3-5 to pluck with seminal propagation, and the vitality of seed reduces very fast, and difficulty of germinateing, germination rate are lower, and can improve bulb yield with bulb breeding; Current foreign scholar proposes and breeds by the method for tissue cultures, and studies comparatively deep, but because of its costly, can't production on widely apply.Therefore, how rapid, high volume breeding For-carrying green onion becomes the key that the industrialization of For-carrying green onion produces in the short time.
Tissue culture technique is utilized to carry out tissue-culturing rapid propagation to For-carrying green onion, For-carrying green onion fast breeding at short notice can be realized, but the height of the rate of increase determines that can For-carrying green onion carry out the bottleneck of factorial praluction by tissue-culturing rapid propagation approach, a kind of method of therefore inventing For-carrying green onion Fast-propagation is the effective way from solving the problem at all.
Summary of the invention
The object of the invention is to solve For-carrying green onion growth cycle long, and tissue-culturing rapid propagation proliferate efficiency is not high, is difficult to the problem meeting large-scale production, and provides a kind of For-carrying green onion method for quickly breeding.
A kind of For-carrying green onion method for quickly breeding, it comprises:
1) acquisition of For-carrying green onion plateau
Get For-carrying green onion bulb, remove For-carrying green onion fibrous root, divest outer scale, retain centered by Central growing point, diameter is the plateau of 0.4-1cm and the scale of top parcel thereof, obtains the centre of For-carrying green onion;
Rinsed in For-carrying green onion centre, sterilization, uses filter paper suck dry moisture;
After explant sterilization, remove top scale in centre far from bottom 3-5mm place crosscut, along main growing point vertical tangent lines direction, be longitudinally divided into 4 parts, remove the plateau at main growing point and main growing point place, cultivate respectively;
2) For-carrying green onion plateau propagation
Medium based on MS, add 6-BA0.5-2.0mg/L, NAA0.05-0.5mg/L, PH is adjusted to 5.8, and induced bundle is sprouted;
3) continue to be trained kind to plant seedlings;
Described cuts into 4 parts, be with the vertical tangent line of main growing point two for otch, cut, be cut into 4 parts, wherein only have 1/4 part of stripping and slicing with the plateau at main growing point place, peel the plateau at its main growing point and main growing point place off;
The scale base portion on 1/4 part of described plateau top cuts again, high to main growing point end far away, the inclined-plane that nearly main growing point end is low.
The addition of described 6-BA and NAA is respectively 1.0mg/L and 0.1mg/L;
Seedling is cultivated in continuation described in step 3), is medium based on MS, adds NAA0.1-1mg/L, PH and is adjusted to 5.8; Tufted seedling is inoculated in root media, seedling rooting of growing thickly after 3 weeks; Tufted seedling after taking root is divided into individual plant, is inoculated in strong seedling culture base, obtain For-carrying green onion seedling; Seedling, after domestication, is planted;
Described For-carrying green onion centre sterilization, after 75% alcohol-pickled sterilization 1min, uses 1%NaClO solution soaking disinfection 15-20min subsequently.
The invention provides a kind of For-carrying green onion method for quickly breeding, For-carrying green onion centre is cleaned, sterilize.After explant sterilization, top scale is removed far from bottom 3-5mm place crosscut in centre, along main growing point vertical tangent lines direction, longitudinally be divided into 4 parts, remove the plateau at main growing point and main growing point place, induced bundle is sprouted, and cultivate seedling, establish For-carrying green onion plateau rapid propagation system by the optimization of above-mentioned incubation, obtain a large amount of For-carrying green onion test-tube plantlets, this system generation time is short, growth coefficient is large, genetic stability is high, can be realized the fast breeding of For-carrying green onion by this system, realizes the plantation of For-carrying green onion industry.
Accompanying drawing explanation
Impact (a: tillered-onion stem apex that Fig. 1 different hormone combinations is bred tillered-onion stem apex; B: the proliferative conditions of stem apex in 6-BA, NAA combination; C: the proliferative conditions of stem apex in 6-BA, IAA);
The acquisition of Fig. 2 tillered-onion plateau;
Fig. 3 tillered-onion test tube micro-plateau cutting step flow process;
Fig. 4 tillered-onion test tube micro-plateau cutting step schematic flow sheet;
Fig. 5. tillered-onion test tube bulbs dish propagation (a: the micro-plateau of tillered-onion test tube; The proliferative conditions that basis of microscopic observation arrived in b: the 2 week; C: the 3 week adventitious buds proliferation, growing state; D: the 4 week adventitious buds proliferation, growing state);
Fig. 6. tillered-onion the growth of plants cultivation (a: tillered-onion test tube propagation tufted seedling; B: individual plant tillered-onion test-tube plantlet; C: the test-tube plantlet after strong seedling culture);
The acquisition in Fig. 7 For-carrying green onion centre;
The acquisition of Fig. 8 For-carrying green onion plateau;
The acquisition of Fig. 9 For-carrying green onion plateau propagation seedling;
Figure 10 For-carrying green onion propagation seedling rooting;
The acquisition of Figure 11 garlic bulb dish;
Figure 12 garlic bulb dish is inoculated;
Figure 13 garlic bulb dish is bred.
Embodiment
embodiment 1 tillered-onion stem apex is bred
Tillered-onion stem apex is inoculated in respectively 6-BA, NAA combination of variable concentrations, 6-BA, IAA combine in medium (table 1), two kinds of hormone combinations all can obtain tillered-onion test tube propagation seedling, but IAA and NAA of same concentrations is when carrying out proliferation-inducing, widely different.The propagation seedling growing way of wherein inducing in 6-BA, NAA hormone combinations is better, and coefficient of differentiation is higher, and when cultivation 4 weeks, average proliferation coefficient can reach 3.87; Although 6-BA, IAA hormone combinations also can realize stem apex propagation, the propagation seedling obtained is thin, weak, and coefficient of differentiation is low, and has Albino Seedling to occur (as Fig. 1).Visible, the action effect of IAA is not as NAA.Therefore determine that MS+6-BA0.3mg/L+NAA0.1mg/L medium is stem apex propagation optimal medium.
Note: medium based on MS.
embodiment 2 tillered-onion plateau is bred
Get tillered-onion one piece, remove the position of tillered-onion plateau suberification, retain centered by Central growing point, diameter is the plateau of 0.4-1cm and the bulb of top parcel thereof, obtains tillered-onion centre.By tillered-onion centre running water 0.5h, after 75% alcohol-pickled sterilization 1min, use 1%NaClO solution soaking disinfection 15-20min subsequently, aseptic water washing 3-5 time to remove remaining NaClO, with filter paper suck dry moisture (as Fig. 2) for subsequent use.
Then top bulb is removed, retain the plateau comprising main growing point, main growing point and main growing point plateau is removed after plateau being cut into 4 parts, be inoculated in medium based on MS respectively, add 6-BA(0.2-0.8mg/L), NAA(0.1-2.0mg/L), PH is adjusted in the increment medium of 5.8 and cultivates, and induced bundle is sprouted.25 DEG C, under light application time is 16h, intensity of illumination is 2500lux condition, through the induction of 4 weeks, combine inducing effect with 6-BA, NAA best; 6-BA, IAA combination also can induce Multiple Buds, but growth coefficient is low, and regrowth exists lopsided seedling.Wherein plateau is in the medium adding 6-BA0.6mg/L, NAA0.1mg/L, and after 4 weeks, differentiation number reaches maximum, and the maximum growth coefficient of each plateau (growth coefficient=Bud Differentiation sum/inoculation number) can reach 20(in table 2).Along with the increase of induction time, plateau differentiation number increases, but no longer increases at induction the 4th week rear plateau differentiation number, and the differentiation rate of plateau reaches maximum (see table 3).
Note: medium based on MS.
Note: medium based on MS.
embodiment 3 tillered-onion detoxic seedling test tube balling
Get tillered-onion stem apex position, running water half an hour, after 75% alcohol-pickled sterilization 1min, use 1%NaClO solution soaking disinfection 15-20min subsequently, aseptic water washing 3-5 times is to remove remaining NaClO, for subsequent use with filter paper suck dry moisture, peels off tillered-onion shoot tip meristem <0.3mm part under microscope, at medium based on MS, add in the Primary culture base of 6-BA, IAA and induce seedling.
After stem apex seedling, based on sucrose, carbon source arranges 30mg/L, 45mg/L, 60mg/L, 75mg/L, 90mg/L totally 5 carbon source sugar concentration respectively, inquire into different sugar concentration to the impact of tillered-onion test-tube plantlet balling, screen sugared concentration in best balling medium, find by cultivating, along with increase bulb balling diameter, the fresh weight increase of sugared concentration.When sugared concentration is higher than 75mg/L, balling speed is accelerated but is affected the growth of root, causes final bulb average diameter, fresh weight to reduce (see table 4).
In detoxic seedling balling incubation, by the regulation and control to cultivation day and night temperature, regulate the balling speed of test-tube plantlet, the preference temperature for tillered-onion growth arranges 25 DEG C/25 DEG C, 25 DEG C/20 DEG C, 25 DEG C/15 DEG C totally 3 groups of diurnal temperature process respectively, screening optimum culturing temperature.When day and night temperature is 10 DEG C, bulb diameter and bulb fresh weight are better than other process.Determine that, in tillered-onion detoxic seedling balling process, optimum culturing temperature is daytime 25 DEG C thus, night 15 DEG C (see table 5).
In detoxic seedling balling incubation, by the intensity of illumination in incubation, photoperiodic regulation and control, regulate the balling speed of test-tube plantlet, 3 groups of intensities of illumination, photoperiod process are set respectively for tillered-onion growth conditions.Result shows, with the prolongation of intensity of illumination and light application time, average diameter and the bulb fresh weight of test tube ball progressively increase, be 2500lux in intensity of illumination, be conducive to tillered-onion detoxification test tube plantlet balling when 16h illumination, 8h dark condition, its test tube ball bulb average diameter, bulb fresh weight are all higher than other condition of culture (table 6).
Establish efficient tillered-onion detoxic seedling test tube bulbs induction system by the optimization of balling condition, the fast breeding for tillered-onion virus-elimination seedlings provides favourable guarantee.
Note: medium based on MS.
Note: medium based on MS.
Note: medium based on MS.
embodiment 4 utilizes the micro-plateau multiplication technique of test tube to set up tillered-onion virus-elimination seedlings rapid propagation system
(1) acquisition of the micro-plateau of test tube
Tillered-onion test-tube plantlet, after balling cultivates 6 weeks or when tillered-onion test tube bulb diameter reaches more than 8mm, can cut the micro-plateau of test tube.With the vertical tangent line of main growing point plateau two for otch, carry out longitudinal cutting, be cut into 4 parts, wherein only have 1/4 part of stripping and slicing with main growing point plateau, peel the plateau at main growing point and main growing point place off.Again the scale base portion on 1/4 part of plateau top is cut again, high to main growing point end far away, the inclined-plane (as Fig. 3, Fig. 4) that nearly main growing point end is low.
The micro-plateau of test tube in the medium adding 6-BA0.6mg/L, NAA0.1mg/L through the cultivation of 2 weeks, the differentiation of Multiple Buds can be examined under a microscope, after 3 weeks, Multiple Buds plant height is maximum reaches 1cm, after 4 weeks, Multiple Buds plant height can reach 3cm(as Fig. 5), and differentiation number reaches maximum, the maximum growth coefficient (growth coefficient=Bud Differentiation sum/inoculation number) of each plateau can reach 34.Along with the increase of induction time, plateau differentiation number increases, but no longer increases at induction the 4th week rear plateau differentiation number, and the differentiation rate of plateau reaches maximum.
(2) plateau that field is open and the impact that the micro-plateau of test tube is bred tillered-onion test-tube plantlet
Plateau open using field respectively and the micro-bulb of test tube carry out tillered-onion fast breeding as explant, and experimental result shows, the proliferate efficiency of the micro-bulb of test tube is 1.66 times (see table 7) of the plateau increment efficiency that field opens.Illustrate that the micro-plateau of test tube has higher differentiation capability, be suitable as the best explant of tillered-onion fast breeding.
Note: medium based on MS.
(3) test tube ball development degree impact that test-tube plantlet is bred
The detoxification kind ball plateau provided using tillered-onion detoxification test tube plantlet is as explant, with the tillered-onion test tube ball plateau of different growing stages for explant carries out test-tube plantlet Multiplying culture, result shows, along with the increase of balling incubation time, the differentiation capability of test tube ball plateau strengthens gradually, and balling is cultivated and proceeded to the 4th week, when bulb average diameter reaches 3.56mm, there is differentiation capability, but the mean tillering number is not high; Cultivate the test tube ball after the 6th week, differentiation capability significantly improves, and the mean tillering number reaches 27.63, and when cultivation the 10th week, the mean tillering number reached maximum 32.19, differentiation capability decline afterwards.Thus, we determine that balling incubation time is the shortest, and the time point that differentiation capability is higher-balling is cultivated the 6th week the best as tillered-onion test tube bulbs dish and provided the phase (see table 8).
Note: medium based on MS.
On this basis, medium based on 1/2MS, by regulating sugared concentration, the NAA concentration in medium, regulates tufted seedling growth, rooting rate.Result shows, is in the medium (PH=5.8) of 45mg/L, NAA0.1mg/L in sugared concentration, and rooting of vitro seedling is the fastest, growing way best (see table 9).After 1 week, rooting rate reaches 100%, and test-tube plantlet color is dark green, sturdy, obtains tillered-onion seedling (see figure 6) after one week.
Note: medium based on MS.
embodiment 5 utilizes For-carrying green onion plateau multiplication technique to set up For-carrying green onion quick reproduction technique system
(1) acquisition of For-carrying green onion plateau
1) get For-carrying green onion bulb, remove For-carrying green onion fibrous root, divest outer scale, retain centered by Central growing point, diameter is the plateau of 0.4-1cm and the scale of top parcel thereof, obtains the centre (Fig. 7) of For-carrying green onion;
2) by For-carrying green onion centre running water half an hour, after 75% alcohol-pickled sterilization 1min, use 1%NaClO solution soaking disinfection 15-20min subsequently, aseptic water washing 3-5 times is to remove remaining NaClO, for subsequent use with filter paper suck dry moisture;
3) after explant sterilization, remove top scale in centre far from bottom 3-5mm place crosscut, along main growing point vertical tangent lines direction, be longitudinally divided into 4 parts, remove the plateau (Fig. 8) for subsequent use at main growing point and main growing point place;
(2) For-carrying green onion plateau propagation
1) medium based on MS, add 6-BA(0.5-2.0mg/L), NAA(0.1-1.0mg/L), PH is adjusted to 5.8, preparation proliferated culture medium for subsequent use;
2) plateau is on average cut into 4 parts, be inoculated in (Fig. 9) in proliferated culture medium, after the induction of 8 weeks, the maximum growth coefficient of tufted seedling can reach 20(in table 10).
Note: medium based on MS.
(3) For-carrying green onion seedling obtains
1) medium based on MS, adds NAA(0.1-1.0mg/L), PH is adjusted to 5.8, and preparation root media is for subsequent use;
2) tufted seedling is inoculated in root media, seedling rooting (table 11) of growing thickly after 3 weeks.
3) tufted seedling after taking root is divided into individual plant, is inoculated in strong seedling culture base, For-carrying green onion seedling (as Figure 10) after 4 months, can be obtained.Seedling through domestication after can be colonizated in field for the production of.
Note: medium based on MS.
embodiment 6 utilizes the micro-plateau multiplication technique of garlic to set up garlic quick reproduction technique system
(1) be that explant obtains garlic propagation seedling with Allium fistulosum stem tips
Allium fistulosum stem tips is inoculated in respectively 6-BA, NAA combination of variable concentrations, 6-BA, IAA combine in medium, and two kinds of hormone combinations all can obtain garlic propagation seedling, but IAA and NAA of same concentrations is when carrying out proliferation-inducing, widely different.The propagation seedling growing way of wherein inducing in 6-BA, NAA hormone combinations is better, and coefficient of differentiation is higher, and when cultivation 4 weeks, average proliferation coefficient can reach 7.75; Although 6-BA, IAA hormone combinations also can realize stem apex propagation, the propagation seedling obtained is thin, weak, and coefficient of differentiation is low.Visible, the action effect of IAA is not as NAA.Therefore determine that MS+6-BA2.0mg/L+NAA0.5mg/L medium is stem apex propagation optimal medium (see table 12).
Note: medium based on MS.
(2) with garlic bulb dish for explant obtains regrowth
Get independent garlic clove, peel off crust and fibrous root, use running water 0.5h, after 75% alcohol-pickled sterilization 1min, use 1%NaClO solution soaking disinfection 15-20min subsequently, aseptic water washing 3-5 time to remove remaining NaClO, with filter paper suck dry moisture (see Figure 11) for subsequent use.2）
Remove the white portion of outer wrap, removing top scale apart from bottom 3-5mm place crosscut, along main growing point vertical tangent lines direction, being longitudinally divided into 4 parts, removing the plateau at main growing point and main growing point place, reservation 2-3mm
2plateau (see Figure 12), be inoculated in medium based on MS respectively, add 6-BA(1.0-3.0mg/L), NAA(0.5-3.0mg/L), PH is adjusted in the proliferated culture medium of 5.8 and cultivates, and induced bundle is sprouted.25 DEG C, under light application time is 16h, intensity of illumination is 2500lux condition, cultivate.
Through the induction of 4 weeks, combine inducing effect with 6-BA, NAA best; 6-BA, IAA combination also can induce Multiple Buds, but growth coefficient is low, and regrowth exists lopsided seedling.Wherein plateau is in the medium adding 6-BA2.0mg/L, NAA0.5mg/L, and after 4 weeks, the maximum growth coefficient of each plateau (growth coefficient=Bud Differentiation sum/inoculation number) can reach 40(in table 13).Along with the increase of induction time, plateau differentiation number increases, but no longer increases at induction the 4th week rear plateau differentiation number, and the differentiation rate of plateau reaches maximum (see Figure 13).
Note: medium based on MS.
Claims (6)
1. a For-carrying green onion method for quickly breeding, it comprises:
1) acquisition of For-carrying green onion plateau
Get For-carrying green onion bulb, remove For-carrying green onion fibrous root, divest outer scale, retain centered by Central growing point, diameter is the plateau of 0.4-1cm and the scale of top parcel thereof, obtains the centre of For-carrying green onion;
Rinsed in For-carrying green onion centre, sterilization, uses filter paper suck dry moisture;
After explant sterilization, remove top scale in centre far from bottom 3-5mm place crosscut, along main growing point vertical tangent lines direction, be longitudinally divided into 4 parts, remove the plateau at main growing point and main growing point place, cultivate respectively;
2) For-carrying green onion plateau propagation
Medium based on MS, add 6-BA0.5-2.0mg/L, NAA0.05-0.5mg/L, PH is adjusted to 5.8, and induced bundle is sprouted;
3) continue to be trained kind to plant seedlings.
2. a kind of For-carrying green onion method for quickly breeding according to claim 1, is characterized in that: the addition of described 6-BA and NAA is respectively 1.0mg/L and 0.1mg/L.
3. a kind of For-carrying green onion method for quickly breeding according to claim 1 and 2, it is characterized in that: described cuts into 4 parts, for otch with the vertical tangent line of main growing point two, cut, be cut into 4 parts, wherein only have 1/4 part of stripping and slicing with the plateau at main growing point place, peel the plateau at its main growing point and main growing point place off.
4. a kind of For-carrying green onion method for quickly breeding according to claim 3, is characterized in that: the scale base portion on 1/4 part of described plateau top cuts again, high to main growing point end far away, the inclined-plane that nearly main growing point end is low.
5. a kind of For-carrying green onion method for quickly breeding according to claim 4, is characterized in that: seedling is cultivated in the continuation described in step 3), is medium based on MS, adds NAA0.1-1mg/L, PH and is adjusted to 5.8; Tufted seedling is inoculated in root media, seedling rooting of growing thickly after 3 weeks; Tufted seedling after taking root is divided into individual plant, is inoculated in strong seedling culture base, obtain For-carrying green onion seedling; Seedling, after domestication, is planted.
6. a kind of For-carrying green onion method for quickly breeding according to claim 1 or 5, is characterized in that: described For-carrying green onion centre sterilization, after 75% alcohol-pickled sterilization 1min, uses 1%NaClO solution soaking disinfection 15-20min subsequently.
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| CN111264390A (en) * | 2020-03-16 | 2020-06-12 | 北华大学 | Method for regenerating plant by somatic cells of allium victorialis |
| CN111492981A (en) * | 2020-06-11 | 2020-08-07 | 包头市农牧业科学研究院 | Tissue culture rapid propagation method of red onions |
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