CN103340152B - Chrysanthemum screening, detoxification and rapid propagation method - Google Patents
Chrysanthemum screening, detoxification and rapid propagation method Download PDFInfo
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Abstract
The invention relates to the field of flower cultivation, in particular to a chrysanthemum screening and detoxification rapid propagation method, which rapidly identifies bud mutation and ornament mutation through a tissue culture rapid propagation technology, and comprises the following steps: collecting aseptic and healthy female parent seedlings of chrysanthemum, cutting into stem segments with buds, and sterilizing; inoculating the stem with buds on a culture medium A for growth; after one week, new axillary buds germinateWhen the plant grows, the plant is transferred to a new culture medium for growth, wherein the culture medium is a culture medium A, and rooting sterile healthy female parent seedlings are obtained after two weeks of growth; cutting the sterile and robust female parent seedling obtained in the step S3
Description
Technical field
The present invention relates to field of flower culture, be specifically related to a kind of chrysanthemum screening detoxifying fast breeding method.
Background technology
Chrysanthemum is composite family Dendranthema, perennial root herbage flower.Chrysanthemum originates in the ground such as Henan China, Luoyang, Hangzhou, is one of large famous flower of China ten, is described as among " plum, orchid, bamboo, chrysanthemum " four gentleman.The cultivation history of more than 3000 year is had in China.Chrysanthemum imports Japan about the Tang Dynasty into through Korea greatly, and the 17th century passes European and American countries, has now become one of world-renowned flowers.Well-known chrysanthemum is lucuriant in design, various in style, and attitude is graceful, can withstand the frost in full bloom, always by people are liked and praise in the time of the year when autumn changes into winter.
The bud mutation of chrysanthemum is one of important means of seed selection chrysanthemum new varieties, i.e. the sudden change of the abiogenous inhereditary material of the meristematic cell of bud, and the branch of being grown by the bud suddenlyd change also breeding forms new kind.Bud mutation is that in plant soma, inhereditary material changes, and crosses kind of a change and often occurs in the meristematic cell of bud.When bud sprouts into new plant, namely performance makes new advances individual different in proterties with former type.So bud mutation is that breeding of new variety provides an important channel.The wide range of bud mutation, in many ornamental flowers, often occur the change of pattern, Hua Xing, the mode such as leaf, people utilize these to make a variation, and through being separated, directive breeding, create many new varieties.But also some is variety, be namely the temporary variation occurred due to the impact of environmental condition or cultivation condition etc., gene does not change, and recovers again the proterties of the first generation when the third generation is cultivated.
Plant Tissue Breeding refers to and is seeded on synthetic media by a part for plant, makes it grow into new plant according to predeterminated target.In recent years, flower tissue cultivation and fast numerous detoxification technology are applied in flower seed plantlet breeding production more and more.The application of tissue cultures in the industry of flowers and plants can fast, amount reproduction improved seeds, can breeding coefficient be increased, accelerate reproduction speed, the seedling consistent with female parent gene proterties height can be produced fast.
Summary of the invention
The present invention seeks to by group culturation rapid propagating technology, differentiate bud mutation and variety fast, realize the Fast-propagation of chrysanthemum, promote chrysanthemum standardized cultivation.
Technical scheme of the present invention screens detoxifying fast breeding method for providing a kind of chrysanthemum, and it comprises the following steps:
S1: gather the aseptic healthy and strong female parent seedling of chrysanthemum and be cut into stem with bud, sterilization;
S2: be inoculated in by stem with bud in culture medium A and grow, described culture medium A comprises B
5, 2.0mg/L the GA of TDZ, 0.1mg/L of NAA, 0.005mg/L of BA, 0.5mg/L
3;
After S3: one week, treat new axillary bud sprouting extremely
time, transfer grows on new culture medium, and described culture medium is culture medium A, obtains aseptic healthy and strong female parent seedling of taking root after growing two weeks;
S4: the aseptic healthy and strong female parent seedling obtained in step S3 is cut into
stem with bud, be inoculated on culture medium and carry out subculture and culture of rootage simultaneously, described culture medium is still culture medium A, and after one week, stem with bud grows up to whole plant.
Preferably, in above-mentioned chrysanthemum screening detoxifying fast breeding method, in described step S1, sterilizing operation is: alcohol sterilizing 20-40s, mercuric chloride sterilizing 4-6min, aseptic water washing 4-6 time.
Preferably, in above-mentioned chrysanthemum screening detoxifying fast breeding method, described step S2 is specially: be inoculated in by stem with bud on culture medium and grow, and described culture medium is culture medium A, at illumination 200LX, 16h, cultivates under the condition of culture that temperature is 24 degree.
Preferably, in above-mentioned chrysanthemum screening detoxifying fast breeding method, described step S3 is specially: after one week, treats new axillary bud sprouting extremely
time, transfer grows on new culture medium, and described culture medium is culture medium A, at illumination 2000LX, 16h, cultivates under the condition of culture that temperature is 24 degree, obtains aseptic healthy and strong female parent seedling of taking root after growing two weeks.
Preferably, in above-mentioned chrysanthemum screening detoxifying fast breeding method, described step S4 is specially: be cut into by the aseptic healthy and strong female parent seedling obtained in step S3
stem with bud, be inoculated on culture medium and carry out subculture and culture of rootage simultaneously, described culture medium is still culture medium A, and at illumination 2000LX, 16h, cultivate under the condition of culture that temperature is 24 degree, after one week, stem with bud grows up to whole plant.
Preferably, in above-mentioned chrysanthemum screening detoxifying fast breeding method, " the aseptic healthy and strong female parent seedling obtained in step S3 is cut in described step S4
stem with bud " concrete operation method be: by the cutting mode of aseptic healthy and strong female parent seedling by excision bud seedling terminal bud, cut into
stem with bud.
The present invention utilizes plant tissue culture technique, obtains the aseptic plantlet in vitro of chrysanthemum, low production cost, and the plantlet in vitro obtained heredity shape is consistent, and breeding coefficient is high, and the breeding cycle is short, is the effective way that chrysanthemum bud mutation is differentiated fast.Of the present invention group of training culture medium and strong sprout are same culture medium, reduce operation easier, are beneficial to raising culture efficiency.What the present invention shortened chrysanthemum bud mutation sorts the time, compares traditional use bud mutation plant and produces pin bud, reaches the method for seedlings plugging field planting after some, saves time more than 8 months.
Detailed description of the invention
By describing technology contents of the present invention, structural feature in detail, being realized object and effect, be explained in detail below in conjunction with embodiment.
Institute of the present invention medicament component illustrates:
B
5culture medium is the nineteen sixty-eight designs such as Gan Boge (Gamborg), and compared with other culture mediums, its main feature is containing lower ammonium, and this nutritional labeling of ammonium may have the effect of Developing restraint to some culture medium.To B in callus and suspension are cultivated
5when culture medium and MS culture medium carry out contrast culture experiment, find that the callus of some plant and cell culture grow well on MS culture medium, some is at B
5culture medium grow preferably.
NAA refers to methyl α-naphthyl acetate, and 6-BA refers to 6-benzyl aminopurine.
Phenyl thiadiazoles (N-Phenyl-N '-1,2,3 ,-thiadiazol-5-ylurea; Thidiazuron) namely TDZ is a kind of new plant growth regulator of Schering company of West Germany Prof. Du Yucang, and its active ingredient is phenylurea diazonium thiazole, and Mok thinks that TDZ has strong cell division activity.The existing a large amount of report of the application of TDZ in Vitro Plant is cultivated, as the concentration TDZ that is 1.0mg/L significantly improves the propagation number of shore plum adventitious bud, TDZ facilitates the Fast-propagation of Chinese lobelia.
GA
3i.e. gibberellin, be the elongation promoting stem to the effect that plant growth is the most outstanding, more obvious to hereditary dwarf-type effect especially, the application of gibberellin in cultured in vitro also has a large amount of reports, as GA
3promote the propagation of bud in Chinese toon cultured in vitro.
The present invention B used
5culture medium is as shown in table 1.
Table 1
| Composition | Working concentration (mg/L) | |
| A great number of elements | Potassium nitrate KNO 3 | 2500 |
| MgSO 4·7H 2O | 250 | |
| CaCl 2·2H 2O | 150 | |
| (NH 4)2SO 4 | 134 | |
| NaH 2PO 4.H 2O | 150 |
| Trace element | KI | 0.75 |
| H 3BO 4 | 3.0 | |
| MnSO 4·4H 2O | 10 | |
| ZnSO 4·7H 2O | 2.0 | |
| Na 2MoO 4·2H 2O | 0.25 | |
| CoCl 2·6H 2O | 0.025 | |
| Molysite | CuSO 4·5H 2O | 0.025 |
| Na 2-EDTA | 37.3 | |
| FeSO 4·7H 2O | 27.8 | |
| Organic principle | Inositol | 100 |
| Nicotinic acid | 1.0 | |
| Puridoxine hydrochloride | 1.0 | |
| Tyiamine Hd | 10 |
Embodiment 1
The present invention is after the doubtful bud mutation plant of discovery, and utilize the bud point tissue of plant location side shoot, the method for use group training, Fast-propagation goes out bottled seedling, and by the cutting mode of excision bud seedling terminal bud, subculture medium adds TDZ and GA
3the measures such as two kinds of hormone combinations, to improve shoot proliferation coefficient, finally realize the object of rapid batch breeding, are differentiated whether be bud mutation fast.
The invention provides a kind of chrysanthemum screening detoxifying fast breeding method, it comprises the following steps:
S1: gather the aseptic healthy and strong female parent seedling of chrysanthemum and be cut into stem with bud, alcohol sterilizing 20-40s, mercuric chloride sterilizing 4-6min, aseptic water washing 4-6 time;
S2: be inoculated in by stem with bud on culture medium and grow, described culture medium is culture medium A, and at illumination 200LX, 16h, cultivate under the condition of culture that temperature is 24 degree, described culture medium A comprises B
5, 2.0mg/L the GA of TDZ, 0.1mg/L of NAA, 0.005mg/L of BA, 0.5mg/L
3;
After S3: one week, treat new axillary bud sprouting extremely
time, transfer grows on new culture medium, and described culture medium is culture medium A, at illumination 2000LX, 16h, cultivates under the condition of culture that temperature is 24 degree, obtains aseptic healthy and strong female parent seedling of taking root after growing two weeks.
S4: by the cutting mode of aseptic healthy and strong female parent seedling by excision bud seedling terminal bud, cut into
stem with bud, be inoculated on culture medium and carry out subculture and culture of rootage simultaneously, described culture medium is still culture medium A, and at illumination 2000LX, 16h, cultivate under the condition of culture that temperature is 24 degree, after one week, stem with bud grows up to whole plant.
1.TDZ and GA
3application to improving the result of the test of the rate of increase
Shoot proliferation coefficient and subculture cycle are the important indicators of tissue-culturing rapid propagation efficiency, but result confirms that these two indexs always change along with the change of cytokinin concentration, and there is certain negative variable relation, namely along with the raising of shoot proliferation coefficient, its squamous subculture cycle to certainly will be extended to a certain extent.For reducing each culture medium shoot proliferation coefficient and subculture cycle statistical error, subculture medium is transferred to the single plant sprout of chrysanthemum new varieties band axillalry bud or bud seedling stem section, more than 80% grows thickly budlet height of seedling when reaching 2.0cm, as the survey time of subculture cycle and shoot proliferation coefficient.Above-mentioned chrysanthemum is used to screen detoxifying fast breeding method at B
5culture medium add basic element of cell division TDZ0.005,0.01, a 0.1mg/L3 concentration gradient and gibberellin GA
30.0, to form the result of the test that 9 kinds of culture mediums add up as shown in table 2 for 0.1mg/L2 concentration gradient.
In his-and-hers watches 2, the 1st, 2,3,4 tested number data analyses are visible, replace 6-BA with variable concentrations TDZ and also can reach same seedling and bud proliferation object, within the scope of finite concentration, chrysanthemum seedling and bud proliferation coefficient improves along with the raising of TDZ concentration, concentration is the effect that namely TDZ of 0.05mg/L is equivalent to the 6-BA of 2.0mg/L, at interpolation TDZ0.01 ~ 0.1mg/L, namely there is the generation of a large amount of vitreous shoot.Result of the test shows that hormone kind is different simultaneously, and the position of buds differentiation propagation is also different, and be separately that in the culture medium of mitogen, buds differentiation mainly comes from bud seedling basal part of stem with 6-BA, namely bud seedling bottom stem section forms a large amount of adventitious bud.In the combination of additional TDZ, bud seedling not only comes from bud seedling base portion, also induces generation adventitious bud from budlet seedling internode simultaneously as seen.Additional GA at the same time
3, BA, TDZ hormone combinations in, find the axillary bud sprouting of a large amount of budlet seedling, and the crowd shoots growth of differentiation is fast, greatly shortens subculture cycle.Add in the culture medium of different hormone combinations, although the position that its Multiple Buds produces is different, its Reproduction methods is all the direct adventitious organogenesis without callus phase.This ways for training, because the multiple of cell chromosome can keep stability, therefore, chrysanthemum new varieties realize numerous soon by this approach cultured in vitro, maternal merit (leaf look, color and luster etc.) can be kept well, the frequency morphed is extremely low, is also confirmed further in this research at plantlet in vitro application.
If table 2 is TDZ, 6-BA, NAA, GA
3hormone combinations is on the impact of chrysanthemum shoot proliferation coefficient, subculture cycle and buds differentiation mode.
Table 2
The middle aged rate of increase of table 2=［ growth coefficient × (1-10%-vitreous shoot %) ］
year breeds algebraically, (wherein 10% is each subculture pollution rate, year propagation algebraically=365 day ÷ subculture cycle), in his-and-hers watches 2, the 4th to the 9th tested number subculture cycle and Shoot propagation coefficient carry out variance analysis, as shown in table 3, TDZ and GA
3two factors have significant impact effect to subculture cycle, not remarkable on growth coefficient impact.Year, the rate of increase was the composite target of reflection tissue-culturing rapid propagation efficiency, and from year, the computing formula of the rate of increase can be analyzed, and subculture cycle is the material impact index of year rate of increase.According to table 2 middle aged rate of increase size sequence number (to being greater than the control group year rate of increase 3.87
9.6the year rate of increase sort), can infer that the 7th tested number should be optimal hormone combinations, its shoot proliferation rate can reach 8.3, and effective seedling and bud proliferation rate reaches 6.5, year the rate of increase be 5.65
13, be up to ten thousand times of control group (CK).Visible TDZ and GA
3the interpolation of two kinds of hormones facilitates the tissue-culturing rapid propagation efficiency of Dendranthema morifolium Varieties greatly.The culture medium that " 346 " obtain the highest micropropagation efficiency is B
5+ BA2.0mg/L+NAA0.5mg/L+TDZ0.005mg/L+GA
30.1mg/L.Test also finds along with gibberellin concentration increases, and the rate of increase declines, and when concentration is increased to 0.5mg/L, also occurs elongated tender stem and undergrown leaf, deformity bud seedling, visible GA
3use should control certain concentration well.
Table 3
In the subculture medium of additional TDZ hormone, the bud seedling of chrysanthemum new varieties propagation not only comes from gemmule seedling base portion, and the induction of bud seedling internode produces a large amount of adventitious bud.Additional GA at the same time
3culture medium in, also find the axillary bud sprouting of a large amount of budlet seedling, and the crowd shoots growth of differentiation is fast, greatly shortens subculture cycle.Visible TDZ and GA
3the interpolation of two kinds of hormones can promote chrysanthemum new varieties tissue-culturing rapid propagation efficiency.The culture medium obtaining the highest micropropagation efficiency is B
5+ BA2.0mg/L+NAA0.5mg/L+TDZ0.005mg/L+GA
30.1mg/L.
2. extract bud seedling terminal bud, promote propagation
With the subculture bud seedling containing 2 axillalry buds and 1 terminal bud for material, the bud seedling clipping terminal bud and non-truncation is turned same subculture multiplication medium, compares two kinds of bud seedling cutting modes to the impact of two kind rates of increase.
During chrysanthemum new varieties squamous subculture inoculation operation, after tender stem is cut terminal bud, then be transferred to (B on fresh culture
5+ BA2.0mg/L+NAA0.5mg/L+GA
30.1mg/L), observe the multiplying power of its every strain buds differentiation Multiple Buds, every bottle of switching 1 goes to push up simple bud, the growth coefficient of statistics 10 simple buds, contrast test is carried out with the bud seedling not excising terminal bud subculture of transferring, all high than the seedling and bud proliferation rate of not excising a bud from result of the test (the different cutting mode of table 4-subculture bud seedling is on the impact of growth coefficient) the average proliferation rate of visible Regenerated plant through excising terminal bud, excision terminal bud can promote sprouting of the growth of bud seedling middle and lower part axillalry bud and adventitious bud.
It is as shown in table 5 that his-and-hers watches 4 carry out two-way analysis of variance result, the variance analysis of the rate of increase between different cutting mode, individual plant, and analyzing visible cutting mode difference by table 5 has remarkable impact to the chrysanthemum rate of increase.
Table 4
Table 5
In tissue cultures, logical Plant Growth Regulator suppresses the apical dominance of plant to promote that bottom axillary bud sprouting is budlet seedling.Chrysanthemum new varieties have stronger apical dominance, in cultured in vitro, also have same performance, and the vigorous terminal bud that grows often suppresses the sprouting of lateral bud consumingly, and branch propagation is less, affects multiplication rate.Result of the test shows, in squamous subculture, except utilizing plant growth regulator to except promoting seedling and bud proliferation, takes excision terminal bud to affect the redistribution of plant hormone different parts in plant, the propagation promoting bud seedling also very effective method.After tender stem is cut terminal bud, then switching can promote the multiplying power of buds differentiation Multiple Buds.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize description of the present invention to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (3)
1. a chrysanthemum screening detoxifying fast breeding method, is characterized in that: it comprises the following steps:
S1: gather the aseptic healthy and strong female parent seedling of chrysanthemum and be cut into stem with bud, sterilization;
S2: be inoculated in by stem with bud on culture medium and grow, at illumination 200LX, 16h, cultivates under the condition of culture that temperature is 24 degrees Celsius, and described culture medium is culture medium A, and described culture medium A comprises B
5, 2.0mg/L the GA of TDZ, 0.1mg/L of NAA, 0.005mg/L of BA, 0.5mg/L
3;
After S3: one week, when new axillary bud sprouting to 2 ~ 3cm, transfer grows on new culture medium, and described culture medium is culture medium A, at illumination 2000LX, 16h, cultivates under the condition of culture that temperature is 24 degrees Celsius, obtains aseptic healthy and strong female parent seedling of taking root after growing two weeks;
S4: the stem with bud aseptic healthy and strong female parent seedling obtained in step S3 being cut into 2 ~ 3cm, be inoculated on culture medium and carry out subculture and culture of rootage simultaneously, described culture medium is still culture medium A, at illumination 2000LX, 16h, cultivate under the condition of culture that temperature is 24 degrees Celsius, after one week, stem with bud grows up to whole plant.
2. chrysanthemum screening detoxifying fast breeding method according to claim 1, it is characterized in that, in described step S1, sterilizing operation is: alcohol sterilizing 20-40s, mercuric chloride sterilizing 4-6min, aseptic water washing 4-6 time.
3. chrysanthemum screening detoxifying fast breeding method according to claim 1, it is characterized in that, the concrete operation method " the aseptic healthy and strong female parent seedling obtained in step S3 being cut into the stem with bud of 2 ~ 3cm " in described step S4 is: by the cutting mode of aseptic healthy and strong female parent seedling by excision bud seedling terminal bud, cut into the stem with bud of 2 ~ 3cm.
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| US5567599A (en) * | 1990-08-21 | 1996-10-22 | Florigene Europe B.V. | Method for producing transformed chrysanthemum plants |
| CN1631106A (en) * | 2004-12-27 | 2005-06-29 | 华南师范大学 | Breeding method of Wedelia trilobata |
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| CN1631106A (en) * | 2004-12-27 | 2005-06-29 | 华南师范大学 | Breeding method of Wedelia trilobata |
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