CN102499071B - Chemical mutation breeding method for bougainvillea - Google Patents
Chemical mutation breeding method for bougainvillea Download PDFInfo
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- CN102499071B CN102499071B CN 201110420276 CN201110420276A CN102499071B CN 102499071 B CN102499071 B CN 102499071B CN 201110420276 CN201110420276 CN 201110420276 CN 201110420276 A CN201110420276 A CN 201110420276A CN 102499071 B CN102499071 B CN 102499071B
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Abstract
The invention discloses a chemical mutation breeding method for bougainvillea, and relates to the field of the vegetative propagation and chemical mutation breeding of xylophyta and vines. The method treats cottage pieces of bougainvillea with sodium azide (NaN3), adopts continuous cottage propagation to create a time and a space which are good for growth of leaf color mutants, and obtains new lines with stable heredity and ornamental value through generation selection, thereby achieving the purpose of chemical mutation breeding. By adopting the technical scheme in the invention, large quantities of female parent materials can be treated in one step; the base number of variants is relatively increases greatly; the mutation frequency is increased; the treatment cost is relatively low; and the breeding period can be shorten effectively. By utilizing the continuous cottage propagation manner, the growth of leaf color mutants is ensured to be competitive, which is helpful for overcoming the chimera formation, thereby obtaining stable and uniform variants. The chemical mutation breeding method can obtain high-frequency mutation and a wide spectrum of variation, and is helpful for the variety improvement of bougainvillea.
Description
Technical field
The present invention relates to gardening wood, liane breeding field, the chemical mutation field of Te other Shi le cuckoo.
Background technology
Le cuckoo (Bougainvillea spp.) is the precious towel platymiscium of Nyctaginaceae (Nyetaginaceae), have another name called precious towel, Bougainvillea spectabilis, paper flower, bougainvillea etc., originate in tropical America, 19th century were imported Europe into, imported Southeast Asian countries into by the U.S. and Japan.Beginning the 1950's to be imbued with sight a large amount of introduction of plant ， of south China le cuckoo with the changeful of its bract color, become one of principal item of greening in many big cities of southern china, is important ornamental plants in garden.
The mutation breeding technology has unique effect in the ornamental plants breed improvement, that a kind of useful of breeding technique deficiency replenished, it can suddenly change by modificator gene, produce newtype, new proterties, new gene that conventional method that natural world did not have originally or general can't obtain, can break gene linkage, improve recombination fraction, on dna level, mutation breeding tends to cause than homology and the larger transformation of heterologous transgene.
The mutation breeding technology mainly can be divided into irradiation mutagenesis and two kinds of forms of mutagenesis.Irradiation mutagenesis mainly is to select to utilize various rays (such as Co
60-gamma-rays) on the doses level, the biological living material is shone, cause the distortion of Chromosome level in the biological cell, form sudden change such as inversion, disappearance etc.Be characterized in larger to the chromosome injury, so lethal mutation is more, this is the main flow form of early stage induced-mutation technique.The mutagenesis then chemical mutagen (such as ethylmethane sulfonate, sodium azide etc.) by the finite concentration scope produces mutant to living materials is smeared, injected, the processing mode such as immersion causes DNA in the biological cell damage and errors repair etc.Possess easy to operately, dose rate is easy to control, and is little to the genome damage, and the mutation rate high is popular a kind of breeding mode widely now.
Domestic Jue Da Bu Fen le cuckoo kind all can not be gathered in the crops seed, thereby all is utilize to transplant a cutting to carry out cottage propagation (vegetative propagation), can not hybridize as sexual generation and creates new varieties, and the breeding work of Zhe Gei le cuckoo brings certain difficulty.The method of physical mutagenesis exists that processing cost is high, long-distance transport affects the weak point such as cuttage bar survival rate.
Enzyme is the class active substance with biocatalysis, exists multi-form isodynamic enzyme between same genus and species different cultivars.Isodynamic enzyme is as the direct product of gene expression, can directly reflect the gene difference between the kind, the different spectral bands that shows isodynamic enzyme by isozyme electrophoretic techique, can be used between the species, the field (Wang Zhongren such as the hereditary difference between the same species different population is identified, genetic diversity Journal of Sex Research and Origin of Species evolution, the plant allozyme is analyzed [M]. Beijing: Science Press, 1996), now be widely used in the variety identification research of ornamental plants.Peroxidase (POD) is a kind of isodynamic enzyme that can utilize the hydrogen peroxide oxidation hydrogen donor, demand to hydrogen peroxide is very single-minded, in higher plant, extensively and in a large number exist, have obvious kind, tissue and developmental stage specificity, reflect that to a certain extent the system of plant occurs.Because peroxidase enzymes is the product of gene expression, therefore can judge well existence and the expression rule thereof of gene to the analysis of its zymogram, can disclose from molecular level the hereditary basis of species.
Summary of the invention
For understanding the problems such as the breeding approach that exists in the le cuckoo conventional breeding work of determining is few, breeding efficiency is low, the invention provides a kind of simply and efficiently chemical mutation method.The clear superiorities such as the present invention has that processing cost is cheap, the easy easy operation of method and mutation frequency height.
The technical solution adopted for the present invention to solve the technical problems is:
The chemical mutation method of Yi Zhong le cuckoo is characterized in that, comprises the steps:
Draw materials: select the female parent of peony flower system, draw materials winter; Described drawing materials chosen annual De le cuckoo branch on the sunny side, is cut into the cuttage bar about 10～12cm, and each cuttage bar stays 2～4 buds.The biology top of described cuttage bar is sealed with solid wax, and it is stand-by that the lower end is whittled into wedge shape.
The material mutagenic treatment: ready material is all immersed in the mutagen treatment fluid, and sealing is placed on dark place, soaks certain hour, keeps certain temperature; Described mutagen are NaN
3Described NaN
3Be 1-10mmol/L.In the described material mutagenic treatment, immerse the material in the mutagen treatment fluid, soak time is 24h, and treatment temperature is 25 ℃.
The cuttage of mutant materials: the material after the immersion washes with flowing water, and immediately cuttage is in the seedling bed in greenhouse, and the control temp. and humidity keeps certain solar radiation; Material after soaking in the cuttage of described mutant materials washes 45min with flowing water, and the temperature after the cuttage is controlled at 25-30 ℃, and air humidity remains on more than 60%, the solar radiation of 2000Lux.
The selection plantation of mutant strain: after 50～60 days, by comparing with contrast, select the leaf look mutant character of survival, carry out continuous cottage propagation, until leaf look mutant character keeps stable;
Determine new lines: the mutant strain of determining leaf look mutant character inheritance stability is new lines.The mutant strain of described definite leaf look mutant character inheritance stability is that the concrete grammar of new lines is the Polyacrylamide Gel Electrophoresis that the comparatively stable variant of phenotype is carried out isodynamic enzyme, relatively contrast and the zymogram of variant isodynamic enzyme, get rid of potential chimera, the stability of definitive variation strain phenotypic character heredity filters out new lines.
Utilize chemical mutagen sodium azide (NaN
3) the cuttage bar of Dui le cuckoo carries out immersion treatment, produce variant by mutagenesis, then utilize continuous cuttage to separate the mode of purpose proterties, create the time and the space that are conducive to the growth of purpose proterties, namely select to obtain the strain of inheritance stability by the continuous generation, reach the purpose of chemical mutation Gai Liang le cuckoo kind.
Xuan is Dinged the maternal cuttage bar of De le cuckoo determine that the chemical mutagen of concentration processes, separate leaf look mutant character plant by continuous cuttage and generation selection, finally select and meet the breeding intermediate materials of viewing and admiring characteristic.
Its concrete steps are as follows:
(1) draw materials: select the maternal branch of annual De le cuckoo on the sunny side, be cut into the cuttage bar about 10～12cm, each cuttage bar stays 2～4 vegetative buds.The biology top of cuttage bar is sealed with solid wax, and it is stand-by that the lower end is whittled into wedge shape.
(2) preparation of buffer solution and mutagenesis liquid: 12g KH
2PO
4Be dissolved in the 1L distilled water, then use H
3PO
4Regulate pH value to 3.0 and be buffer solution; Get the NaN of the 1mol/L of different volumes number
3Stoste is dissolved in above-mentioned buffer solution, is mixed with the mutagenesis liquid of each concentration level of 1～10mmol/L.
(3) mutagenic treatment of material and cuttage: above-mentioned material is all immersed in the mutagenesis liquid of each concentration level, process 24h, 25 ℃ for the treatment of temperatures in the sealing dark; To place flowing water to wash 30～45min through the material of mutagenic treatment, and carry out immediately cuttage, cutting medium be peat soil: perlite=1: 1, and the control temperature is at 25～30 ℃, and air humidity remains on more than 60%, keeps the solar radiation of 2000Lux.
(4) the phenotype mutant strain is selected and continuous cottage propagation: after cuttage 50～60 days, and different on leaf look proterties of contrast (not by mutagenic treatment) and mutagenic fungi relatively, selected leaf look proterties of suddenling change.After choosing the purpose proterties, can begin continuous cottage propagation, its role is to separate leaf look mutant character.The embodiment of cottage propagation is continuously: the base portion (VM that selects the nascent branch of mutant strain
1Generation) carries out successive propagation (VM as the cuttage bar
2Generation), cultivation step is the same; Select VM
2Nascent branch (still the keeping the purpose proterties) base portion in generation carries out successive propagation (VM as the cuttage bar
3Generation), successive propagation like this is until leaf look mutant character keeps stable.
In the actual breeding work, take into account the variation that survives number and mutation rate, simultaneously according to the breeding objective of oneself, can determine the mutagen concentration of suitable proportion.After the selected purpose proterties, can separate the continuous cottage propagation of proterties.
The different maternal propagating materialss of Dui le cuckoo carry out mutagenesis process (according to method provided herein) can realize the genetic variation of leaf look (by in a large number, repeatedly test proved), in practical operation, can adjust treatment dosage to reach the promising result that obtains the good variation individual plant of target according to different maternal propagating materialss.
(5) VM
3Protein fingerprint pattern for rear variant detects: the Polyacrylamide Gel Electrophoresis that the comparatively stable variant of phenotype is carried out isodynamic enzyme, relatively contrast and the zymogram situation of variant isodynamic enzyme (such as peroxidase), get rid of potential chimera, the stability of definitive variation strain leaf look mutant character heredity filters out new lines.
The efficient chemical mutagen of mutagenesis utilization of the present invention is processed, and can obtain more leaf look sudden change, and processing cost is low, can process on the spot and cultivate, simple easy operation.
Adopt this programme can receive following remarkable result:
1, disposablely can process on the spot large quantities of maternal materials, relatively greatly increase the radix of variant.Processing cost is very cheap, and can effectively shorten breeding cycle.
2, by the vegetative propagation mode of continuous cuttage, guarantee that the growth of mutant has competitiveness, be conducive to overcome chimeric generation, obtain the variation plant of stable uniform.
3, adopt NaN
3The maternal cuttage bar of Dui le cuckoo carries out mutagenesis, can obtain the leaf look sudden change (mutation frequency of medial lethal dose reaches 70.37%) of upper frequency, and spectrum of variation is wider, is conducive to the sight of put forward Gao le cuckoo kind.
Description of drawings
Fig. 1 is the continuous cottage propagation method schematic diagram that separates the purpose proterties.
Fig. 2 is leaf variegation material (VM
3) protein fingerprint pattern in generation.CK is contrast, and BN1-9 is variant.
Fig. 3 A is contrast, and Fig. 3 B and Fig. 3 C are mutant strain BN6 and the BN7 plant leaf situations of inheritance stability.
Embodiment
The present invention adopts following embodiment, and following examples are intended to illustrate the present invention, rather than the present invention is limited.If no special instructions, be conventional method (with reference to Xu Guanren chief editor's " plant mutagenesis thremmatology ", 1996, front page, Chinese agriculture publishing house and distribution wait clearly chief editor's " biological heredity mark and application ", 2008, front page, Chemical Industry Press).The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
(1) draws materials: in the test nursery in Shenzhen Chinese Academy of Sciences Xian Hu botanical garden science popularization experiment garden, get the annual purple flower of common single-lobe le cuckoo (the Bougainvillea glabra choisy cv.) branch that faces south, be cut into the cuttage bar about 10～12cm, each cuttage bar stays 2～4 vegetative buds.The biology top of cuttage bar is sealed with solid wax, and it is stand-by that the lower end is whittled into wedge shape.
(2) preparation of buffer solution: get 12g KH
2PO
4Be dissolved in the 1L distilled water, use H
3PO
4Regulate pH value to 3.0.
(3) NaN
3The preparation for the treatment of fluid: get 1～10ml NaN
3Stoste (1mol/L) is dissolved in the above-mentioned buffer solution of 1L, is made into respectively 1.25,2.5,5.0, the 10.0mmol/L NaN of totally four concentration levels
3Treatment fluid.
(4) material mutagenic treatment: the NaN that ready material is all immersed above-mentioned varying level concentration
3In the treatment fluid, sealing is placed on dark place, soaks 24h.
(5) cuttage of mutant materials: the material after the immersion washes 45min with flowing water, and immediately cuttage is in the seedling bed in greenhouse, and temperature is controlled at 25 ℃ (25 ℃～30 ℃ all can), and humidity remains on more than 60%, keeps the solar radiation of 2000Lux.
(6) Phenotypic Selection of mutant strain and continuously cottage propagation: after 55 days (50～60 days all can), select the morph cuttage bar of (selected purpose proterties in this test example) of leaf look to transplant plantation, primary part observation leaf look has the plant of variation.Specifically survive strain number and mutation rate and see Table one (take leaf variegation as example), survival rate (%)=survival number/maternal cuttage number * 100%, mutation frequency (%)=phenotypic variation number/cuttage bar survives number * 100%.Table one is NaN
3Each concentration level survival rate of plant and leaf variegation number statistics, as can be known, raising along with mutagenesis liquid concentration (dosage), surviving number reduces, but the phenotypic variation rate has improved, and therefore in actual breeding work, takes into account the variation that survives number and aberration rate, according to the breeding objective of oneself, can determine the mutagen concentration of suitable proportion simultaneously.Find out from table one, the preferred mutagen concentration of the present invention is 5.0mmol/L.After the selected leaf variegation proterties, can separate the continuous cottage propagation of proterties.The mode of continuous cottage propagation: the base portion (VM that selects the nascent branch of mutant strain
1Generation) carries out successive propagation (VM as the cuttage bar
2Generation); Select M
2Nascent branch (still the keeping the purpose proterties) base portion in generation carries out successive propagation (VM as the cuttage bar
3Generation), successive propagation like this is until the purpose proterties keeps stable.The concrete operations flow process is seen Fig. 1.
(7) VM3 detects for the protein fingerprint pattern of rear variant: the Polyacrylamide Gel Electrophoresis that the comparatively stable variant of phenotype is carried out isodynamic enzyme.Concrete steps: adopt the Proteau II xi cell vertical slab electrophoresis instrument of BIO-RAD company, the gel slab preparation of peroxidase and esterase is with reference to clear etc. the method for distribution.Point sample (the preparation method of sample: get each 0.1g of young leaflet tablet that contrasts and supply the examination variant, Tris-Hcl (pH8.8) the buffer solution ice bath that adds 0.05mol/L grinds, and through the centrifugal 15min of 6000r/min, gets supernatant, adding is with 40% sucrose of volume, and-20 ℃ save backup.) complete rear plugged, initial voltage 100V adds to 200V behind the 0.5h.When bromophenol blue indicator moves to apart from separation gel bottom 1cm, the powered-down stop electrophoresis.The method that peroxidase stain waits clearly with reference to distribution, employing acetic acid-Differentiation (distribution is clear etc., biological heredity mark and application [M]. Beijing: Chemical Industry Press, 2008).
From VM
3Select 9 comparatively stable strain variants of leaf variegation to analyze in generation, contrast and variant peroxidase enzymes characteristic strip situation about occurring relatively, and add up, whether definitive variation strain purpose character inheritance is stable, gets rid of chimeric possibility.Result of the test is seen Fig. 2, and Fig. 2 shows, the selected 9 strain variants of this test example, and 7 strains (BN1-BN5, and BN8-BN9) are chimera, and 2 strains (BN6 and BN7) are the actual plant that morphs.The fish tail arrow is that BN6 is depicted as the disappearance characteristic strip among the figure, and another arrow is that BN7 is depicted as newly-increased characteristic strip, illustrates that variation has all occured for BN6 and BN7, is new lines, can further train improved variety.The picture information of the actual BN6 that morphs and BN7 is seen Fig. 3, and the performance of BN6 blade face is the minus green state comparatively uniformly, and tikka point is light yellow, and characteristics are not minus greens of main vein; The BN7 blade face then is irregular bulk macula lutea, and skewness, vein color and patch solid colour in the patch zone.
Table one
Claims (5)
1. the chemical mutation method of Yi Zhong le cuckoo is characterized in that, comprises the steps:
Draw materials: select the female parent of peony flower system, draw materials winter;
The material mutagenic treatment: ready material is all immersed in the mutagen treatment fluid, and sealing is placed on dark place, soaks certain hour, keeps certain temperature;
The cuttage of mutant materials: the material after the immersion washes with flowing water, and immediately cuttage is in the seedling bed in greenhouse, and the control temp. and humidity keeps certain solar radiation;
The selection plantation of mutant strain: after 50～60 days, by comparing with contrast, select the leaf look mutant character of survival, carry out continuous cottage propagation, until leaf look mutant character keeps stable;
Determine new lines: the mutant strain of determining leaf look mutant character inheritance stability is new lines;
Described mutagen are NaN
3
Described NaN
3Concentration is 1-10mmol/L;
In the described material mutagenic treatment, immerse the material in the mutagen treatment fluid, soak time is 24h, and treatment temperature is 25 ℃.
2. the chemical mutation method of claim 1 Suo Shu De le cuckoo is characterized in that, described drawing materials chosen annual De le cuckoo branch on the sunny side, is cut into the cuttage bar about 10～12cm, and each cuttage bar stays 2～4 buds.
3. the chemical mutation method of claim 2 Suo Shu De le cuckoo is characterized in that, the biology top of described cuttage bar is sealed with solid wax, and it is stand-by that the lower end is whittled into wedge shape.
4. the chemical mutation method of claim 1 Suo Shu De le cuckoo, it is characterized in that, the material after soaking in the cuttage of described mutant materials washes 45min with flowing water, and the temperature after the cuttage is controlled at 25-30 ℃, air humidity remains on more than 60%, the solar radiation of 2000Lux.
5. the chemical mutation method of claim 1 Suo Shu De le cuckoo, it is characterized in that, the stable mutant strain of described definite leaf variegation character inheritance is that the concrete grammar of new lines is the Polyacrylamide Gel Electrophoresis that the comparatively stable variant of phenotype is carried out isodynamic enzyme, relatively contrast and the zymogram of mutant strain isodynamic enzyme, get rid of potential chimera, the stability of definitive variation strain purpose character inheritance filters out new lines.
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| CN104719015B (en) * | 2015-03-13 | 2017-08-29 | 湖南省园艺研究所 | A kind of sodium azide induces the processing method of citrus bud mutation |
| CN107873511B (en) * | 2017-11-15 | 2019-12-10 | 湖北省农业科学院粮食作物研究所 | Sweet potato stem section 60 Co-gamma ray radiation mutation breeding method |
| CN111357556A (en) * | 2020-04-17 | 2020-07-03 | 山东胜伟盐碱地科技有限公司 | Method for breeding new variety of vitex rotundifolia on saline-alkali soil |
| CN113575158B (en) * | 2021-08-13 | 2022-07-15 | 泰安市泰山林业科学研究院 | Mutagenesis method of new variation variety of pyrus betulaefolia |
| CN115152626A (en) * | 2022-08-08 | 2022-10-11 | 中国热带农业科学院热带作物品种资源研究所 | Creation method and application of bougainvillea spectabilis polyploid germplasm |
| CN115281077A (en) * | 2022-08-08 | 2022-11-04 | 中国热带农业科学院热带作物品种资源研究所 | Bougainvillea spectabilis chromosome doubling method based on chemical seed soaking and application thereof |
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