CN104365474A - Method applied to chemical mutagenesis of adventitious buds of bananas - Google Patents
Method applied to chemical mutagenesis of adventitious buds of bananas Download PDFInfo
- Publication number
- CN104365474A CN104365474A CN201410657745.5A CN201410657745A CN104365474A CN 104365474 A CN104365474 A CN 104365474A CN 201410657745 A CN201410657745 A CN 201410657745A CN 104365474 A CN104365474 A CN 104365474A
- Authority
- CN
- China
- Prior art keywords
- indefinite bud
- mutagenesis
- seedling
- banana
- days
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Cultivation Of Plants (AREA)
Abstract
The invention discloses a method applied to chemical mutagenesis of adventitious buds of bananas. The method comprises the following steps: firstly, preparing a phosphate buffer solution with the pH value being 3.0, preparing a mutagenic agent sodium azide solution by the phosphate buffer solution with the pH value being 3.0, selecting the third and fourth generations of clustered adventitious buds of the adventitious buds of the bananas, cutting the clustered adventitious buds into single adventitious buds, putting the adventitious buds into the sodium azide solution for immersing and carrying out mutagenesis, transferring the adventitious buds subjected to mutagenesis to a proliferation medium, putting the proliferation medium in an axenic cultivation chamber for carrying out recovery culture for about 20 days; transferring the survival adventitious buds subjected to the recovery culture into a rooting medium, strengthening seedlings and rooting, and carrying out rooting culture for about 25 days; transferring rooting seedlings into a seedling culturing greenhouse and hardening the seedlings for 5 days, then strengthening seedlings and rooting in a temporary planting sand tank for 20 days, then planting the seedlings in a seedling culturing tray; and screening variant strains when ten leaves of the seedlings in test tubes grow. The method has the beneficial effects that the banana breeding has the characteristics of being high in mutagenesis frequency, wide in range, quick to separate and easy to stabilize.
Description
Technical field
The invention belongs to crops select index technical field, relate to a kind of mutagenesis method of banana indefinite bud.
Background technology
Banana (Musa spp.) is a kind of important fruit tree in many tropical and subtropical countries, has larger area under cultivation in provinces and regions such as China Guangdong, Guangxi, Fujian, Hainan, Yunnan and Taiwan.The main breed overwhelming majority of banana is triploid, is difficult to the improved Varieties being obtained high-quality, high yield, resistance by traditional crossbreeding mode.The mode utilizing the technology such as mutation breeding, gene engineering and multiple technologies to combine is carried out seed selection new variety of banana and is necessitated.
Mutagenesis have easy to operate, Induced dosage is easy to control, mutation rate high little to Genomic damage, becomes the mutation breeding technologies of extensive use in recent years.Since first Spence finds sodium azide (NaN in nineteen sixty-five
3) since mutagenesis to barley, many researchers both domestic and external are to NaN
3mutagenesis on crops conducts extensive research, and proves NaN
3it is a kind of effective chemical mutagen.But NaN
3report is rarely had to the mutagenesis of banana indefinite bud.This pilot system have studied NaN
3to mutagenesis and the Mutagenic Effect thereof of banana indefinite bud, for the select index work of banana provides foundation.
Summary of the invention
The object of the present invention is to provide a kind of mutagenesis method of banana indefinite bud, the invention has the beneficial effects as follows the breeding for banana, have that mutagenic frequency is high, scope is wide, be separated soon, easily stable feature.
The technical solution adopted in the present invention is carried out according to following steps:
Step 1: the phosphate buffer of configuration pH value 3.0;
First liquid: get SPA 4.2ml, the 1000ml that adds water dissolves and shakes up;
Second liquid: claim sodium hydrogen phosphate 17.9g, the 1000ml that adds water dissolves and shakes up;
Get first liquid 1000ml to mix with second liquid 1000ml, i.e. first liquid: second liquid=1:1 mixes, and can obtain the phosphate buffer of pH value 3.0.
Step 2: prepare mutagen sodium azide solution;
1, be the sodium azide solution of 0.15g/L, 0.20g/L, 0.25g/L with the phosphate buffer configuration concentration of pH value 3.0.
2, the sodium azide solution 110ml loading volume getting variable concentrations is respectively in the clean blake bottle of 220ml, builds bottle cap.
3, getting 120ml running water loading volume is in the clean blake bottle of 220ml, builds bottle cap, as the sterile water of cleaning.
4, the blake bottle that sodium azide mutagenesis liquid and sterile water are housed above is put into high-pressure sterilizing pot high-temperature sterilization, temperature is set as 105 DEG C, and the time is set as 30 minutes, after sterilizing take out put into desinfection chamber cooling for subsequent use.
Step 3: the mutagenic treatment of banana indefinite bud;
1, in Brazil that growth selection is basically identical, healthy and strong, No. 1, agriculture section, anti-withered No. 5 3rd ~ 4 generations, grow thickly indefinite bud, are cut into single indefinite bud in superclean bench.
2, the single indefinite bud after cutting is put into the sodium azide solution of variable concentrations, every bottle of leaching about 30 buds.The mutagenic treatment of Brazil is that 0.15g/L soaks 3h, or 0.20g/L soaks 2h; No. 1, agriculture section, the mutagenic treatment of anti-withered No. 5 are that 0.15g/L soaks 3h, or 0.25g/L soaks 2h.
3, every 15 minutes double swerve blake bottles once, indefinite bud is made to soak evenly.
4, after the time arrives, the indefinite bud after soaking is put into the cleaning of the sterile water after sterilization 3 times.
5, the indefinite bud after cleaning is transferred on proliferated culture medium (MS+6-BA 3mg/L+NAA 0.3mg/L+Ad 5mg/L+ sucrose 30g/L, PH5.8), every bottle graft kind 5 ~ 6 buds.
6, the indefinite bud after mutagenesis is put into Sterile culture room and carry out renewal cultivation, cultivation temperature 28 DEG C, intensity of illumination 1000lx, illumination every day 8h, about 20 days renewal cultivation time.
Step 4: the culture of rootage after the mutagenesis of banana indefinite bud;
The indefinite bud of surviving after renewal cultivation is proceeded to root media (1/2MS+IBA 1.0mg/L+NAA0.2mg/L+ sucrose 30g/L+ carbon dust 2g/L, PH5.8), put into Sterile culture room strengthening seedling and rooting, cultivation temperature 28 DEG C, intensity of illumination 2000lx, illumination every day 12h, about 25 days culture of rootage time.
Step 5: the transplanting of seedling of taking root and management;
1, seedling (bottle seedling) of taking root goes to seedling growth greenhouse hardening 5 days.
2, practice after seedling terminates, seedling of taking root takes out from bottle, is cleaned up by the medium that shoot root portion is adhered to clear water.
3, the transplantation of seedlings of taking root after cleaning is carried out heeling in strengthening seedling and rooting to husky groove.
4, heeling in seedling after about 20 days, to be colonizated in specification be in 12cm × 10cm seedling-raising cup.
5, rich water quality management and the extermination of disease and insect pest is strengthened.
Step 6: the screening of variant after mutagenesis
When test-tube plantlet grows to 10 leaves, carry out the screening of variant.The variants such as height is large-scale, dwarf-type, abnormal blade profile, flower leaf type, narrow leaf type, close nodal pattern, blue or green stalk type are screened from the test-tube plantlet colony after mutagenesis.Variant properties and characteristics main manifestations is as follows:
Normal strain: plant strain growth is normal, and blade is verticillate, and interval is evenly moderate, leaf green.
High large-scale: growth rate is very fast, and plant is higher, sturdy, and blade interval is larger.
Dwarf-type: plant is shorter, sturdy, and petiole is short, the close life of blade, blade is thicker, and leaf look more dark green.
Abnormal blade profile: occur some vanelets made a variation in the middle of blade, or leaf growth is abnormal.
Flower leaf type: yellow-white striped, minus green patch appear in blade, or uneven distribution decorative pattern.
Narrow leaf type: blade is narrower, blade is less and thin, and partial blade vein is anisopleual, and false stem is thinner.
Close nodal pattern: blade interval is very short, blade is intensive at plant top, and petiole is shorter.
Blue or green stalk type: false stem light green color or yellow green, blade is many and thin, and leaf look pale green, plant is more fragile, frangibility.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail.
One, materials and methods
(1) experiment material
With banana variety Brazil, No. 1, agriculture section, anti-withered No. 5 for material, the banana germplasm garden that material source is set up in Donggnan Banana and Vegetable Institute.
(2) experimental technique
From resource garden, take the banana that proterties is excellent, growing way is healthy and strong inhale bud seedling, after cutting growing point sterilization, set up explant.Explant is inoculated in the differentiation of inducing culture evoking adventive bud, the indefinite bud of differentiation transfers to proliferated culture medium, and to carry out expansion numerous, through 3 ~ 4 generations shoot proliferation obtain a large amount of indefinite bud after cultivating, select indefinite bud of the same size to carry out mutagenesis process.Be inoculated in proliferated culture medium renewal cultivation 1 generation with the Brazilian indefinite bud after the mutagenesis of suitable mutagenesis concentration, proceed to root media culture of rootage, be then transplanted to seedling-raising cup and carry out variation condition survey.
1, the foundation of explant
The excellent suction bud of band sword-like leave resource garden chosen takes back laboratory, to prune blade and the false stem of part with cutter, only stays the stem section that 8 ~ 10cm of band growing point is long, peels off outside leaf sheath, finally only stays 2 ~ 3cm of band growing point long as explant material.Then 1min is steeped, the sterile water wash after taking-up sterilizing 1 time with the alcohol of 75%, with 0.1% clorox process 20min, sterile water wash 3 times.Repaired further by material with scalpel in aseptic dish, brown stain periphery of pruning, make false stem portion retain about 0.5cm, bulb part retains about 1cm.Then explant is inoculated in inducing culture (MS+Ad 5mg/L+ sucrose 30g/L, PH5.8).Cultivation temperature is 26 DEG C, light culture.Within about 30 days, axillalry bud grows from leaf sheath base, namely obtains 1st generation indefinite bud.
2, Multiplying culture
Cut out by the 1st generation indefinite bud of acquisition, be inoculated in proliferated culture medium (MS+6-BA 3mg/L+NAA0.3mg/L+Ad 5mg/L+ sucrose 30g/L, PH5.8), about 15d subculture 1 time, so can make indefinite bud obtain continuous differentiation and proliferation.Cultivation temperature 28 DEG C, intensity of illumination 1000lx, illumination every day 8h.In subculture 3 ~ 4 generation, choose the relatively consistent indefinite bud of growing way as experiment material.
3, mutagenesis
Be dipped into respectively in the sodium azide solution of variable concentrations by the banana indefinite bud of subculture to 3 ~ 4 generations, soak time is respectively 3 hours and 2 hours, obtains the suitable mutagenesis concentration of banana indefinite bud mutagenesis by experiment.By suitable mutagenesis concentration, mutagenesis is carried out to banana indefinite bud again, then the banana indefinite bud after mutagenesis is inoculated in proliferated culture medium (the same) and carries out renewal cultivation.
4, culture of rootage
Banana indefinite bud after recovery is proceeded to root media (1/2MS+IBA 1.0mg/L+NAA 0.2mg/L+ sucrose 30g/L+ carbon dust 2g/L, PH5.8) and carries out culture of rootage.Cultivation temperature is 28 DEG C, and intensity of illumination is 2000lx, and illumination every day is 12h.
5, secondary seedling is cultivated
Seedling (bottle seedling) of taking root goes to seedling growth greenhouse hardening 5 days.Practice after seedling terminates, seedling of taking root takes out from bottle, cleans up with the medium of clear water by shoot root portion, is then transplanted to husky groove and carries out heeling in strengthening seedling and rooting, and heeling in seedling after about 30 days, to be colonizated in specification be in 12cm × 10cm seedling-raising cup.Rich water quality management and the same daily management of the extermination of disease and insect pest, when test-tube plantlet grows to 10 leaves, carry out observation and the investigation of variant.
(3) data processing and statistics
Inoculation strain number refers to the indefinite bud sum be inoculated on medium, dead strain number refers to be inoculated in indefinite bud sum dead on medium, differentiation strain number is inoculated in the indefinite bud sum that differentiation on medium has indefinite bud, effective bud number refers to effective indefinite bud sum on medium, the high average plant height referring to indefinite bud in bottle of bud, variant number refers to that colony's height after mutagenesis is large-scale, the summation of the dissimilar variant number such as dwarf-type, abnormal blade profile, flower leaf type, narrow leaf type, close nodal pattern, blue or green stalk type.Survey data uses SPSS to carry out adding up and variance analysis.Each index calculate formula is as follows.
Dead strain number/inoculation strain number × 100% of lethality rate %=
Differentiation rate %=breaks up strain number/inoculation strain number × 100%
Growth coefficient=effectively bud number/inoculation strain number
Aberration rate %=variant number/colony's strain number × 100%
Two, the phosphate buffer collocation method research of pH value 3.0
Most plants application NaN
3when carrying out mutagenesis, it is all phosphate buffer NaN3 being dissolved in pH value 3.0.Therefore, the collocation method of phosphate buffer of pH value 3.0 and most important on the impact of tissue culture seedlings of bananas is studied.
(1) pH value 3.0 phosphate buffered saline technique study
1, experimental technique
Search for " phosphate buffer " keyword by ***, retrieval obtains the phosphate buffered saline method that pH value is 2.0, as follows, does not find the phosphate buffered saline method of pH value 3.0.
Phosphate buffer (PH2.0)
First liquid: get phosphatase 11 6.6ml, add water to 1000ml, shake up.
Second liquid: get sodium hydrogen phosphate 71.6g, adds water and makes to be dissolved into 1000ml.
Get above-mentioned first liquid 72.5ml to mix with second liquid 27.5ml, shake up, obtain the phosphate buffer that pH value is 2.0.
With reference to PH2.0 phosphate buffered saline method in above data, get first liquid 60ml to mix with 30ml, 40ml, 50ml, 55ml, 60ml, 65ml, 70ml, 80ml second liquid respectively, determine the pH value of the mixed liquor of different volumes ratio with the portable PH measurement of IQ150 produced in USA.
2, result of the test
Table 1 first liquid and second liquid different volumes are than the pH value of mixed liquor
| Test number | First liquid | Second liquid | PH value |
| 1 | 60ml | 30ml | 1.95 |
| 2 | 60ml | 40ml | 2.22 |
| 3 | 60ml | 50ml | 2.56 |
| 4 | 60ml | 55ml | 2.73 |
| 4 | 60ml | 60ml | 2.96 |
| 5 | 60ml | 65ml | 3.25 |
| 4 | 60ml | 70ml | 3.68 |
| 6 | 60ml | 80ml | 4.27 |
As can be seen from Table 1, when first liquid amasss as 60ml, second liquid is long-pending when being also 60ml, and both ph value of mixture are 2.96, close to 3.0.Therefore, PH3.0 phosphate-buffered formula of liquid is obtained by experiment as follows:
First liquid: get phosphatase 11 6.6ml, the 1000ml that adds water dissolves and shakes up;
Second liquid: claim sodium hydrogen phosphate 71.6g, the 1000ml that adds water dissolves and shakes up;
Get first liquid 60ml to mix with second liquid 60ml, i.e. first liquid: second liquid=1:1, shakes up, and can obtain the phosphate buffer of pH value 3.0.
(2) variable concentrations phosphate buffer is on the impact of Banana Growth
1, experiment process
Can obtain the phosphate buffer of pH value 3.0 by above method, but whether the phosphate buffer of this concentration can impact to the growth of banana seedlings, needs to be probed into further.Therefore arranging four kinds of different phosphate buffering liquid concentrations to verify, take distilled water as contrast.
Process 1: measure phosphatase 11 6.6ml, add water to 1000ml, shake up; Take sodium hydrogen phosphate 71.6g, add water and make to be dissolved into 1000ml, shake up; Both are mixed.
Process 2: measure phosphoric acid 8.3ml, add water to 1000ml, shake up; Take sodium hydrogen phosphate 35.8g, add water and make to be dissolved into 1000ml, shake up; Both are mixed.
Process 3: measure phosphatase 24 .2ml, add water to 1000ml, shake up; Take sodium hydrogen phosphate 17.9g, add water and make to be dissolved into 1000ml, shake up; Both are mixed.
Process 4: measure phosphoric acid 2.1ml, add water to 1000ml, shake up; Take sodium hydrogen phosphate 9.0g, add water and make to be dissolved into 1000ml, shake up; Both are mixed.
Contrast: measure 1000ml distilled water and contrast.
2, experimentation
(1) by each 2 liters of the phosphate buffer of above process preparation variable concentrations.
(2) pH value of the phosphate buffer of variable concentrations is determined with the portable PH measurement of IQ150 produced in USA.
(3) the phosphate buffer 1 10ml loading volume getting variable concentrations is respectively in the clean blake bottle of 220ml, and build bottle cap, often process fills 6 bottles.
(4) 110ml distilled water loading volume is got in contrast is in the clean blake bottle of 220ml, builds bottle cap, fills 6 bottles.
(5) getting 120ml running water loading volume is in the clean blake bottle of 220ml, builds bottle cap, fills 100 bottles, as the sterile water of cleaning.
(6) blake bottle that phosphate buffer, distilled water and running water are housed above is put into high-pressure sterilizing pot high-temperature sterilization, temperature is set as 105 DEG C, and the time is set as 30 minutes, after sterilizing take out put into desinfection chamber cooling for subsequent use.
(7) in Brazil 3rd ~ 4 generation that growth selection is basically identical, healthy and strong, grows thickly indefinite bud as experiment material, is cut into single indefinite bud in superclean bench.
(8) the single Brazilian indefinite bud after cutting is put into the phosphate buffer after sterilizing and distilled water soaks 3 hours, every bottle of leaching about 30 buds, every 15 minutes double swerves once, make indefinite bud soak evenly.
(9), after the time arrives, the indefinite bud soaked is put into the cleaning of the sterile water after sterilizing 3 times.
(10) indefinite bud after cleaning is transferred on solid culture medium and carries out renewal cultivation.Often process repetition 3 times, every repeated inoculation 12 bottles, every bottle graft kind 4 buds.
(11) after 20 days, inoculation strain number, dead strain number is investigated at renewal cultivation, differentiation strain number, the indexs such as effective bud number, bud are high.
(12) after having investigated, film recording is carried out to the representational sample of each processing selecting.
3, experimental result
Table 2 variable concentrations phosphate buffer is on the impact of Brazilian indefinite bud
| Process | PH value | Lethality rate (%) | Differentiation rate (%) | Growth coefficient | Bud high (cm) |
| Process 1 | 2.87±0.03c | 14.6±2.10a | 72.9±2.07c | 2.00±0.06c | 4.89±0.22b |
| Process 2 | 3.00±0.06c | 7.9±2.31b | 83.6±2.55b | 2.55±0.05b | 5.16±0.05ab |
| Process 3 | 3.07±0.03c | 0.0±0.00c | 91.9±1.17a | 3.16±0.04a | 5.27±0.04a |
| Process 4 | 3.33±0.09b | 0.0±0.00c | 92.9±1.51a | 3.26±0.05a | 5.29±0.02a |
| Distilled water | 5.50±0.12a | 0.0±0.00c | 92.5±1.44a | 3.28±0.06a | 5.27±0.04a |
As can be seen from Table 2, the pH value 3.0.07 of process 3, very close with pH value 3.0, lethality rate, differentiation rate, growth coefficient, bud be high is respectively 0.0%, 91.9%, 3.16,5.27cm, not remarkable with contrast difference, illustrate that the normal growth of this concentration on banana indefinite bud does not affect.Therefore, the pH value 3.0 phosphate-buffered formula of liquid obtaining the mutagenesis of banana indefinite bud sodium azide by experiment suitable is as follows:
First liquid: get SPA 4.2ml, the 1000ml that adds water dissolves and shakes up;
Second liquid: claim sodium hydrogen phosphate 17.9g, the 1000ml that adds water dissolves and shakes up;
Get first liquid 1000ml to mix with second liquid 1000ml, i.e. first liquid: second liquid=1:1 mixes, and can obtain the phosphate buffer of pH value 3.0.
Three, different sodium azide concentration is to the mutagenesis of banana indefinite bud
(1) test process
Test arranges 3 factors, i.e. sodium azide concentration, mutation time and kind.Sodium azide concentration has 0.00g/L (contrast), 0.05g/L, 0.10g/L, 0.15g/L, 0.20g/L, 0.25g/L, 0.30g/L, 0.35g/L, 0.40g/L, 0.45g/L, 0.50g/L, 0.55g/L, 0.60g/L, 0.65g/L, 0.70g/L, 0.75g/L, 0.80g/L totally 17 levels, mutation time has 3 hours and 2 hours totally 2 levels, kind has Brazil, No. 1, agriculture section, anti-withered No. 5 totally 3 levels, amounts to 102 process.
(2) process of the test and method
1, configure the phosphate buffer 80 liters of pH value 3.0, i.e. each 40 liters of first liquid, second liquid, both mix by equal-volume.
2, by each 4 liters of the sodium azide solution of test processing configuration variable concentrations, solvent is the phosphate buffer of pH value 3.0.
3, the sodium azide solution 110ml loading volume getting variable concentrations is respectively in the clean blake bottle of 220ml, and build bottle cap, often process fills 6 bottles.
4, getting 120ml running water loading volume is in the clean blake bottle of 220ml, builds bottle cap, fills 1900 bottles, as the sterile water of cleaning.
5, the blake bottle that sodium azide mutagenesis liquid and sterile water are housed above is put into high-pressure sterilizing pot high-temperature sterilization, temperature is set as 105 DEG C, and the time is set as 30 minutes, after sterilizing take out put into desinfection chamber cooling for subsequent use.
6, in Brazil that growth selection is basically identical, healthy and strong, No. 1, agriculture section, anti-withered No. 5 3rd ~ 4 generations, grow thickly indefinite bud as experiment material, are cut into single indefinite bud in superclean bench.
7, the single indefinite bud after cutting is put into the sodium azide solution of variable concentrations, every bottle of leaching about 30 buds, soak time is respectively 3 hours and 2 hours, every 15 minutes double swerves once, indefinite bud is soaked even.
8, after the time arrives, the indefinite bud after soaking is put into the cleaning of the sterile water after sterilization 3 times.
9, the indefinite bud after cleaning is transferred on solid culture medium and carries out renewal cultivation, often process 3 times and repeat, every repeated inoculation 12 bottles, every bottle graft kind 4 buds.
10, after 20 days, inoculation strain number, dead strain number is investigated at renewal cultivation, differentiation strain number, the indexs such as effective bud number, bud are high.
11, according to the growing way situation of indefinite bud after mutagenesis, indefinite bud is divided into 6 grades, the grade of investigation different disposal indefinite bud.
1 grade: growing way is strong, without browning, differentiation rate is high
2 grades: growing way is comparatively strong, and a small amount of brownization, differentiation rate is higher
3 grades: growing way is medium, moderate brownization, differentiation rate is medium
4 grades: growing way is more weak, major part brownization is dead, breaks up lower
5 grades: growing way is extremely weak, serious brownization is dead, seldom breaks up
6 grades: do not have growing way, whole brownization is dead, not differentiation
12, after having investigated, film recording is carried out to the representational sample of each processing selecting.
(3) result of the test and analysis
The suitable mutagenesis concentration of table 3 banana indefinite bud mutagenesis
Experiment shows, Brazil, No. 1, agriculture section, anti-withered No. 5 NaN through variable concentrations
3mutagenic treatment, with the raising of NaN3 mutagenesis concentration, the lethality rate of Multi bud body significantly improves, and differentiation rate, growth coefficient and bud are high obviously to be reduced, and growing way dies down.Work as NaN
3mutagenesis concentration be 0.15g/L and mutation time is 3h time, Brazil, No. 1, agriculture section, the lethality rate of anti-withered No. 5 are respectively 48.5%, 46.7%, 45.9%, close to half lethal concentration; Value-added coefficient is respectively 1.51,1.47,1.59, does not increase to some extent before the mutagenesis of fixed number number ratio; Growing way is respectively 3.2,3.5,3.2, and indefinite bud growing way is medium, and moderate brownization, differentiation rate is medium.Work as NaN
3mutagenesis concentration be 0.20g/L and mutation time is 2h time, 0.25g/L and mutation time be when being 2h, Brazil, No. 1, agriculture section, the lethality rate of anti-withered No. 5 are respectively 43.6%, 45.4%, 47.6%, close to half lethal concentration; Value-added coefficient is respectively 1.71,1.45,1.48, does not increase to some extent before the mutagenesis of fixed number number ratio; Growing way is respectively 3.3,3.6,3.7, and indefinite bud growing way is medium, and moderate brownization, differentiation rate is medium.Therefore, 0.15g/L is the suitable mutagenesis concentration of Brazil, No. 1, agriculture section, anti-withered No. 5 mutagenic treatment 3h; 0.20g/L is the mutagenesis concentration that Brazilian mutagenic treatment 2h is suitable for, the mutagenesis concentration that 0.25g/L is No. 1, agriculture section, anti-withered No. 5 mutagenic treatment 2h are suitable for.
Four, sodium azide solution is to the Mutagenic Effect of banana indefinite bud
(1) test process
With Brazilian any of several broadleaf plants indefinite bud for test material, with suitable NaN
3mutagenesis concentration carries out mutagenesis, namely arranges 0.15g/L mutagenesis 3h and 0.20g/L mutagenesis 2h two process, respectively with 0.00g/L mutagenesis 3 hours and 2 hours for contrast.Be seeded to proliferated culture medium after mutagenesis and carry out renewal cultivation, be then seeded to root media and carry out culture of rootage, be finally colonizated in seedling-raising cup the investigation carrying out plant aberration rate after mutagenesis.
(2) process of the test and method
1, configure the phosphate buffer 8 liters of pH value 3.0, i.e. each 4 liters of first liquid, second liquid, both mix by equal-volume.
2, by test process configure respectively 0.00g/L, 0.15g/L, 0.20g/L sodium azide solution 4 liters, 2 liters, 2 liters, solvent is the phosphate buffer of pH value 3.0.
3, the sodium azide solution 110ml loading volume getting variable concentrations is respectively in the clean blake bottle of 220ml, and build bottle cap, often process fills 14 bottles.
4, getting 120ml running water loading volume is in the clean blake bottle of 220ml, builds bottle cap, fills 200 bottles, as the sterile water of cleaning.
5, the blake bottle that sodium azide treatment fluid and sterile water are housed above is put into high-pressure sterilizing pot high-temperature sterilization, temperature is set as 105 DEG C, and the time is set as 30 minutes, after sterilizing take out put into desinfection chamber cooling for subsequent use.
6, Brazil 3rd ~ 4 generation indefinite bud 1200 strain that growth selection is basically identical, healthy and strong, as mutagenesis inoculation material, is cut into single indefinite bud in superclean bench.
7, the single indefinite bud after cutting is put into the sodium azide solution of variable concentrations, every bottle is about soaked about 30 buds, and soak time is respectively 3 hours and 2 hours, rolled every 15 minutes dynamic once, indefinite bud is soaked even.
8, after the time arrives, the indefinite bud after soaking is put into the cleaning of the sterile water after sterilization 3 times.
9, be transferred on solid culture medium and carry out renewal cultivation by the indefinite bud after cleaning, every bottle graft kind 5 ~ 6 buds, incubation time is 20 days.
10, Regenerated plant is forwarded to root media and carries out culture of rootage, rootage duration is 25 days.
11, seedling of taking root goes to seedling growth greenhouse and practices seedling 5 days, is then colonizated in seedling-raising cup.
12, practice after seedling terminates, seedling of taking root takes out from bottle, is cleaned up by the medium that shoot root portion is adhered to clear water.
13, the transplantation of seedlings of taking root after cleaning is carried out heeling in strengthening seedling and rooting to husky groove.
14, heeling in seedling after about 20 days, to be colonizated in specification be in 12cm × 10cm seedling-raising cup.
15, when test-tube plantlet grows to 10 leaves, observation and the investigation of variant is carried out.
16, after having investigated, film recording is carried out to the representational sample of each processing selecting.
(3) result of the test and analysis
1, the observation of variant and classification after mutagenesis
By observing the Brazilian any of several broadleaf plants secondary seedling after mutagenesis, find that most of test-tube plantlet growth is normal, minority test-tube plantlet morphs, and variation type mainly contains high large-scale, dwarf-type, abnormal blade profile, flower leaf type, narrow leaf type, close nodal pattern, blue or green stalk type totally 7 classes, and its properties and characteristics main manifestations is as follows:
Normal strain: plant strain growth is normal, and blade is verticillate, and interval is evenly moderate, leaf green.
High large-scale: growth rate is very fast, and plant is higher, sturdy, and blade interval is larger.
Dwarf-type: plant is shorter, sturdy, and petiole is short, the close life of blade, blade is thicker, and leaf look more dark green.
Abnormal blade profile: occur some vanelets made a variation in the middle of blade, or leaf growth is abnormal.
Flower leaf type: yellow-white striped, minus green patch appear in blade, or uneven distribution decorative pattern.
Narrow leaf type: blade is narrower, blade is less and thin, and partial blade vein is anisopleual, and false stem is thinner.
Close nodal pattern: blade interval is very short, blade is intensive at plant top, and petiole is shorter.
Blue or green stalk type: false stem light green color or yellow green, blade is many and thin, and leaf look pale green, plant is more fragile, frangibility.
2, the investigation of secondary seedling variant number after mutagenesis
As can be seen from Table 4, Brazilian any of several broadleaf plants sodium azide is suitable for the process of mutagenesis concentration 0.15g/L mutagenesis 3h, and populational variation rate is 6.8%, is 2.0 times of contrast aberration rate 3.4%; Brazil any of several broadleaf plants is suitable for the process of mutagenesis concentration 0.20g/L mutagenesis 2h with sodium azide, populational variation rate is 6.6%, and be 2.1 times of contrast aberration rate 3.1%, visible, after being suitable for the mutagenesis of mutagenesis concentration with sodium azide, aberration rate significantly improves.In addition, the aberration rate being suitable for mutagenesis concentration 0.15g/L mutagenesis 3h and 0.20g/L mutagenesis 2h colony plant with sodium azide is respectively 6.8% and 6.6%, and both are more or less the same, and can combine as mutagenic treatment.
Secondary seedling variant number investigation statistics table after table 4 mutagenesis
Five, brief summary and discussion
1, the phosphate buffer of pH value 3.0
NaN
3be a kind of point mutation agent, the mode of action of itself and DNA is that base is replaced, and DNA single chain is ruptured.NaN
3when pH value 3.0, NaN
3mainly HN is produced in solution
3molecule, it is in neutral, and easy permeate through cell membranes enters in cell, affects the normal synthesis of DNA, thus cause the generation of point mutation with base substitute mode.Therefore, at application NaN
3need the sour environment providing pH value 3.0 when carrying out mutagenesis, Mutagenic Effect is best.
This pilot system have studied the collocation method of the phosphate buffer of pH value 3.0 and the impact on tissue culture seedlings of bananas thereof, screens the pH value 3.0 phosphate-buffered formula of liquid that the mutagenesis of banana indefinite bud sodium azide is suitable.
2, sodium azide concentration is to the mutagenesis of banana indefinite bud and effect thereof
Sodium azide have high to the efficiency of inducing mutation of crops, physiological damage is little, low toxin, bringing out very effective to chlorophyll disappearance and morphological mutation in an acidic solution, is one chemical mutagen safely and efficiently.
Determining that the mutagenesis concentration that mutagen are suitable for will consider the effect of mutagenesis and the extent of injury to explant, is generally reference with medial lethal dose.Bhagwat and Duncan then thinks and determines that the mutagenesis concentration be suitable for not is definitely suitable by medial lethal dose, also will consider the recovery of material after process, propagation, regeneration and variation situation.
This experimental study shows, 0.15g/L is the suitable mutagenesis concentration of Brazil, No. 1, agriculture section, anti-withered No. 5 mutagenic treatment 3h; 0.20g/L is the mutagenesis concentration that Brazilian mutagenic treatment 2h is suitable for, the mutagenesis concentration that 0.25g/L is No. 1, agriculture section, anti-withered No. 5 mutagenic treatment 2h are suitable for.Under the combination of these mutagenic treatment, lethality rate is close to semilethal rate, and the indefinite bud of survival can restoration ecosystem and increment, obtains regeneration plant easy, and after mutagenesis the aberration rate of secondary test-tube plantlet apparently higher than contrast.It is feasible that application sodium azide carries out mutagenesis to banana indefinite bud.
Claims (6)
1. a mutagenesis method for banana indefinite bud, is characterized in that, carries out according to following steps:
Step 1: the phosphate buffer of configuration pH value 3.0;
Step 2: prepare mutagen sodium azide solution;
Step 3: the mutagenic treatment of banana indefinite bud;
1) select Brazil, No. 1, agriculture section, resist 3rd ~ 4 generations of withered No. 5 three kinds to grow thickly indefinite bud, in superclean bench, be cut into single indefinite bud;
2) the single indefinite bud after cutting is put into sodium azide solution and carry out mutagenic treatment, every bottle of leaching 30 buds;
3) indefinite bud every 15 minutes double swerve blake bottles once, is made to soak evenly;
4), after the time arrives, the indefinite bud after soaking is put into the cleaning of the sterile water after sterilization 3 times;
5) indefinite bud after cleaning is transferred on proliferated culture medium, every bottle graft kind 5 ~ 6 buds;
6) indefinite bud after mutagenesis is put into Sterile culture room and carry out renewal cultivation, cultivation temperature 28 DEG C, intensity of illumination 1000lx, illumination every day 8h, about 20 days renewal cultivation time;
Step 4: the culture of rootage after the mutagenesis of banana indefinite bud;
The indefinite bud of surviving after renewal cultivation is proceeded to root media, puts into Sterile culture room strengthening seedling and rooting, cultivation temperature 28 DEG C, intensity of illumination 2000lx, illumination every day 12h, about 25 days culture of rootage time;
Step 5: the transplanting of seedling of taking root and management;
1) seedling of taking root goes to seedling growth greenhouse hardening 5 days;
2) practice after seedling terminates, seedling of taking root takes out from bottle, is cleaned up by the medium that shoot root portion is adhered to clear water;
3) transplantation of seedlings of taking root after cleaning is carried out heeling in strengthening seedling and rooting to husky groove;
4) heeling in seedling after 20 days, to be colonizated in specification be in 12cm × 10cm seedling-raising cup;
Step 6: the screening of variant after mutagenesis;
When test-tube plantlet grows to 10 leaves, carry out the screening of variant.
2. according to a kind of described in claim 1 mutagenesis method of banana indefinite bud, it is characterized in that: in described step 1, the phosphate buffer method of configuration pH value 3.0 is:
First liquid: get SPA 4.2ml, the 1000ml that adds water dissolves and shakes up;
Second liquid: claim sodium hydrogen phosphate 17.9g, the 1000ml that adds water dissolves and shakes up;
First liquid: second liquid=1:1 mixes, and can obtain the phosphate buffer of pH value 3.0.
3. according to a kind of described in claim 1 mutagenesis method of banana indefinite bud, it is characterized in that: in described step 2, mutagen sodium azide solution is the sodium azide solution of 0.15g/L, 0.20g/L, 0.25g/L by the concentration that the phosphate buffer of pH value 3.0 is configured to.
4. according to a kind of described in claim 1 mutagenesis method of banana indefinite bud, it is characterized in that: in described step 3, the mutagenic treatment of Brazil is that 0.15g/L soaks 3h, or 0.20g/L soaks 2h; No. 1, agriculture section, the mutagenic treatment of anti-withered No. 5 are that 0.15g/L soaks 3h, or 0.25g/L soaks 2h.
5. according to a kind of described in claim 1 mutagenesis method of banana indefinite bud, it is characterized in that: in described step 3, proliferated culture medium is MS+6-BA 3mg/L+NAA 0.3mg/L+Ad 5mg/L+ sucrose 30g/L, PH5.8.
6. according to a kind of described in claim 1 mutagenesis method of banana indefinite bud, it is characterized in that: in described step 4, root media is 1/2MS+IBA 1.0mg/L+NAA 0.2mg/L+ sucrose 30g/L+ carbon dust 2g/L, PH5.8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410657745.5A CN104365474A (en) | 2014-11-18 | 2014-11-18 | Method applied to chemical mutagenesis of adventitious buds of bananas |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410657745.5A CN104365474A (en) | 2014-11-18 | 2014-11-18 | Method applied to chemical mutagenesis of adventitious buds of bananas |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104365474A true CN104365474A (en) | 2015-02-25 |
Family
ID=52544872
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410657745.5A Pending CN104365474A (en) | 2014-11-18 | 2014-11-18 | Method applied to chemical mutagenesis of adventitious buds of bananas |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104365474A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112293248A (en) * | 2020-11-30 | 2021-02-02 | 中国热带农业科学院海口实验站 | Chemical mutagenesis method for inducing and increasing mutation range of banana |
| CN112369323A (en) * | 2020-11-30 | 2021-02-19 | 中国热带农业科学院海口实验站 | Mutagenesis method for inducing ideal mutation density of bananas |
| CN112753572A (en) * | 2021-01-04 | 2021-05-07 | 华南农业大学 | Mutation breeding method for resisting blight of bananas |
| CN115349407A (en) * | 2022-08-17 | 2022-11-18 | 广西壮族自治区农业科学院 | Method for simply judging banana dwarfing time node and dwarfing effect |
-
2014
- 2014-11-18 CN CN201410657745.5A patent/CN104365474A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112293248A (en) * | 2020-11-30 | 2021-02-02 | 中国热带农业科学院海口实验站 | Chemical mutagenesis method for inducing and increasing mutation range of banana |
| CN112369323A (en) * | 2020-11-30 | 2021-02-19 | 中国热带农业科学院海口实验站 | Mutagenesis method for inducing ideal mutation density of bananas |
| CN112293248B (en) * | 2020-11-30 | 2021-12-21 | 中国热带农业科学院海口实验站 | Chemical mutagenesis method for inducing and increasing mutation range of banana |
| WO2022110864A1 (en) * | 2020-11-30 | 2022-06-02 | 中国热带农业科学院海口实验站 | Chemical mutagenesis method for increasing mutation range of dwarf bananas by means of induction |
| CN112753572A (en) * | 2021-01-04 | 2021-05-07 | 华南农业大学 | Mutation breeding method for resisting blight of bananas |
| CN115349407A (en) * | 2022-08-17 | 2022-11-18 | 广西壮族自治区农业科学院 | Method for simply judging banana dwarfing time node and dwarfing effect |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105191805B (en) | A kind of micro-propagation method of tilia miqueliana | |
| CN109430063B (en) | A kind of directed screening method of floorboard with high oil content peanut | |
| CN101116424B (en) | Highly effective lily bulblet inducement culture method | |
| CN104054583A (en) | Rapid-reproduction method for apple rootstocks T337 | |
| CN111972288B (en) | Passion fruit in-vitro preservation and proliferation regeneration method | |
| CN104365474A (en) | Method applied to chemical mutagenesis of adventitious buds of bananas | |
| CN103461143A (en) | Method for tissue culture and rapid propagation of camellia oleifera | |
| CN101816286B (en) | Method for tissue culture and rapid propagation of narcissus pseudonarcissus by using ramentum | |
| Browne et al. | Resistance to Phytophthora and graft compatibility with Persian walnut among selections of Chinese wingnut | |
| CN109294930A (en) | A method of obtaining dendrobium candidum plantlet stage mycorrhizal fungi | |
| CN105409748B (en) | A kind of fast breeding method of extra large scirpus scirpus | |
| CN103109728B (en) | Rapid seedling culturing method of pinus sylvestris in tube | |
| CN102612962A (en) | Method for identifying resistance of sweet potatoes in seedling stage to black spot | |
| Hossain et al. | Mass propagation of Dendrocalamus asper by branch cutting | |
| CN114375640B (en) | Method for promoting growth of camellia oleifera seedlings by using dark-color endophytic fungi | |
| Majidian et al. | The effect of four levels of GA3 and BA on the quantitative and qualitative characteristics of Zantedeschia aethiopica cv. Childsiana pot plant | |
| CN103125391B (en) | Local one-year multi-generation breeding method of soybeans | |
| CN102318478B (en) | New high-quality seed reserving method of spring cabbage | |
| CN108934713A (en) | A kind of acer catalpifolium seed seedling-raising method | |
| CN103999680B (en) | Plasma treatment willow branch produces the method for bud mutation | |
| CN103734021A (en) | Collection and induction method for alnus nepalensis axillary bud explants | |
| Galli et al. | Training systems and rootstocks for high density planting (HDP) of the cultivar'Abbé Fétel': developmental trials in Italy | |
| KR102042899B1 (en) | The mass inoculation system for selecting resistant varieties of Lettuce Wilt and the selection method of resistant varieties using the same | |
| CN106605595B (en) | It is cultivated fine seed strains the method for fiery fiber crops by stem apex | |
| CN111373901A (en) | Rapid germination propagation method for sweet potato seeds |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150225 |