CN101591629B - Bacillus subtilis and application thereof in banana tissue culture - Google Patents
Bacillus subtilis and application thereof in banana tissue culture Download PDFInfo
- Publication number
- CN101591629B CN101591629B CN2009101117971A CN200910111797A CN101591629B CN 101591629 B CN101591629 B CN 101591629B CN 2009101117971 A CN2009101117971 A CN 2009101117971A CN 200910111797 A CN200910111797 A CN 200910111797A CN 101591629 B CN101591629 B CN 101591629B
- Authority
- CN
- China
- Prior art keywords
- banana
- bud
- substratum
- subtilis
- tissue culture
- 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.)
- Expired - Fee Related
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a bacillus subtilis and application thereof in banana tissue culture. The bacillus subtilis T122F adopted by the invention has the functions of promoting growth and resisting diseases. For using the functions of the bacillus subtilis such as promoting growth and resisting diseases, a culture solution of bacillus subtilis T122F and metabolites of the bacillus subtilis T122F are added in a banana tissue culture medium and then banana tissue culture material is put in the banana tissue culture medium to be cultured into a healthy banana seedlings improved in growth vigor, toxic tolerance, disease resistance and the like quickly. The application of the bacillus subtilis in the banana tissue culture has the advantages of simple and convenient operation, high rate of emergence of healthy banana seedlings, excellent economic benefits in terms of banana tissue culture seedling culture period and production cost conservation, and significant practical values in terms of expansion of the application range of the bacillus subtilis and development of a novel banana tissue culture medium for improving and creatively utilizing the resistance of banana germplasm resources.
Description
Technical field
The technology of the present invention field belongs to the Plant Protection and the plant cell engineering of agronomy class and learns; More specifically relate to a kind of subtilis and the application in the banana tissue culture thereof.
Background technology
A large amount of chemical pesticide control Plant diseasess of using have caused harm to environment and human health, utilize safety and eco-friendly biological or chemical inducible factor to come the defensive raction of activated plant be exactly a kind of efficient strategy.Subtilis (Bacillussubtilis) is that a class is had a liking for temperature, aerobic or amphimicrobian saprophytic bacteria, and widely distributed at occurring in nature, very easily separation and Culture is to the person poultry harmless, free from environmental pollution.Can produce the gemma that heat, ultraviolet ray, electromagnetic radiation is had very strong resistance, tolerate various bad environment conditions.Produce multiple antibiotic (as anti-fungus peptide, lipopeptide antibiotics, micromolecular polysaccharide material, macromolecular antibacterial protein) and enzyme (as cell wall degrading enzymes such as proteolytic enzyme, amylase, esterase, chitinase and acetylamino Polyglucosidases), have broad spectrum antibiotic activity and extremely strong anti-adversity ability.To the multiple pathogenic bacteria of plant (as banana blight bacteria, wheat powdery mildew, Pyricularia oryzae, cotton rhizoctonia solani, pathogen of soybean root rot, botrytis cinerea, pepper anthracnose bacterium etc.) produce and suppress, on the main diseases of farm crop such as cucumber, capsicum, banana, paddy rice, wheat, corn, cotton, show good prevention effect at present.
All plants have the effective protection mechanism of multiple potential; induction of resistance (induced resistance) is meant physics, chemistry and the biological method utilized; anticipate plant, inducing plant starts the defense mechanism of self, strengthens resistivity to external world.Induction of resistance also is a kind of important mechanisms of subtilis controlling plant diseases; the intravital multiple defense response of subtilis energy inducing plant; induce the content of the activity of some plant defense expression of gene, increase and the relevant protective enzyme of resistance and plant protecting chemical, xylogen, phenolic compound; produce many new disease-resistant fungus matter; thereby improve the system resistance of plant to multiple pathogenic bacteria, this inducible system resistance has broad spectrum, systematicness and non-specific usually.Subtilis not only contains plant hormone and similar metabolite such as phytokinin, zeatin, dormin, gibberic acid etc., and can also inducing plant endogenous growth hormone (indolylacetic acid, Plant hormones regulators,gibberellins, zeatin) and chlorophyll content increase, promote plant-growth, improve the plant adaptability and the resistance of environment to external world.The application of subtilis is very extensive, how to make microbial inoculum with this bacterium both at home and abroad in agriculture field, is used for agriculture production with the biological pesticide form, and Agro-ecology safety and minimizing environmental pollution are played important meaning.
Summary of the invention
The purpose of this invention is to provide a kind of subtilis and the application in the banana tissue culture thereof, the subtilis T122F that turns out is used for the banana tissue culture, economical convenient on material source, technical easy and simple to handle, has tissue culture seedlings of bananas growth safety, it is fast, neat to emerge, and seedling raise period is short, low cost and other advantages.The tissue culture seedlings of bananas robust growth that obtains, well developed root system, toxinicide, resistance against diseases are strong, all can reach a bottle seedling and heel in quality standard.Implement this invention to developing a kind of novel banana tissue culture medium (TCM), improvement of banana germ plasm resource resistance and creative utilization are had vital role.Also has important practice significance to expanding the new application and the banana breeding for disease resistance research new way of subtilis in the banana tissue culture.
Subtilis of the present invention (Bacillus subtilis) T122F bacterial strain, its 16s rDNA sequence is shown in SEQ IDNO.1 in the sequence table.
Subtilis of the present invention (Bacillus subtilis) T122F bacterial strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 19th, 2009, and it is called for short CGMCC, and deposit number is: CGMCCNo.3043.
Subtilis of the present invention (Bacillus subtilis) T122F strain morphology is learned and is identified: as shown in following table 1:
Table 1 cellular form and physiological and biochemical test result
The selection of subtilis of the present invention: described subtilis T122F bacterial strain separation screening in the healthy banana plant body in banana blight lesion obtains.
Subtilis T122F bacterial strain of the present invention is used for the banana tissue culture.
Remarkable advantage of the present invention is:
The present invention adopts a bacillus subtilis T122F of seed selection, add this finite concentration subtilis T122F nutrient solution to the banana tissue culture medium (TCM) equably and (contain proliferated culture medium, division culture medium, root media), insert corresponding banana cultivated material (callus then, the bud of growing thickly, the differentiation seedling), make it healthy growth in the banana tissue culture medium (TCM) that contains subtilis T122F meta-bolites, by comparing the growing state and the toxinicide of banana different growing stage cultivated material, after the disease resistance situation, find to use this technology can obviously improve banana adventitious buds proliferation and seedling differentiation capability, improve the grow thickly toxinicide of bud and tissue cultured seedling of banana, resistance against diseases makes banana plant just can obtain induction of resistance in the tissue cultured seedling phase.
The T122F bacterial strain that the present invention uses is that separation screening obtains in the healthy banana plant body in banana blight lesion a kind ofly has a banana endogenous spore bacillus short sick, the control effect, this bacterial strain by conventional cellular form, Physiology and biochemistry and 16SrRNA sequencing, identifies that the T122F bacterial strain is a subtilis through Institute of Microorganism, Academia Sinica.With compare from the subtilis in the intravital genus bacillus of banana and other source; this T122F bacterial strain has stronger anti-microbial activity and biocontrol effect to banana blight; can obviously promote banana grow thickly bud tissue and plant strain growth; improve the activity of banana seedlings protective enzyme; and in the banana body, grow surely rapidly and breed; strengthening the resistance of plant to disease, is a bacterial strain that is worth further investigation and has potentiality to be exploited.
Induce crop disease-resistant and short natural disposition about subtilis, mostly based on research and application on some crop seeds and the plant level, do not see at banana cells, organize the application of inducing on the level at present.Banana cells engineering tissue culture technique makes the banana seedling realize batch production production, plays a part very important to the development of banana industry.The present invention is through experimental study for many years, draw the culturing filtrate that adds subtilis T122F in the banana tissue culture procedures, can obviously promote on the one hand the grow thickly growth of bud tissue of banana, improve adventitious buds proliferation and seedling differentiation capability, can also improve anti-toxin, anti-sick ability that banana is grown thickly bud and broken up seedling on the other hand.The present invention shortens seedling raise period to the seedling rate of further raising tissue culture seedlings of bananas, joint medicine cost, and the resistance against diseases that improves tissue culture seedlings of bananas has important effect.
The present invention adopts this subtilis T122F to carry out the banana tissue culture, it is simple that the bacterial strain T122F that uses cultivates raw material, easy and simple to handle on the using method, banana is grown thickly, and bud, regrowth incubation time cycle shorten, robust growth, to promoting that the growth of banana seedlings safety, batch production are emerged fast, saving production cost has vital role and good economic benefit; The tissue culture seedlings of bananas robust growth that obtains, it is neat to emerge, well developed root system, toxinicide, resistance against diseases are strong, can finish and obtain the induction of resistance effect of function stem T122F in the tissue culture seedlings of bananas phase to banana plant, banana plant is possessed and improve, can reach a bottle seedling and heel in quality standard the anti-adversity ability under the conditions such as disease, toxin.Implement this invention to developing a kind of novel banana tissue culture medium (TCM), improvement of banana germ plasm resource resistance and creative utilization are had vital role.Also has important practice significance to expanding the new application and the banana breeding for disease resistance research new way of subtilis in the banana tissue culture.
Description of drawings
Fig. 1 is the substratum of the T122F of containing of the present invention, wherein a:T122F bacterial strain NB nutrient solution; B: banana proliferated culture medium; C: contain 10% T122F strain cultured solution banana proliferated culture medium; D: banana root media; E: contain 10%T122F strain cultured solution banana root media.
Fig. 2 is each stage growing way situation, wherein a of banana bud of the present invention: the bud growing way of (20 days) in containing 10%T122F strain cultured solution proliferated culture medium of growing thickly; B: the bud growing way of (20 days) in containing 10% BS08 strain cultured solution proliferated culture medium of growing thickly; C: the bud growing way of (20 days) in blank proliferated culture medium of growing thickly; D: the bud growing way of (20 days) in containing 10%T122F strain cultured solution division culture medium of growing thickly; E: the bud growing way of (20 days) in blank division culture medium of growing thickly.
Fig. 3 is that employing T122F bacterial strain of the present invention carries out the banana cultivation and do not adopt the T122F bacterial strain to carry out the contrast situation that banana is cultivated, a wherein: the symptom (show resistance to poison good, blade normal) of banana regrowth after 25% crude venom soaks 72h of inducing processing through the 10%T122F strain cultured solution; B: without the symptom (show resistance to poison poor, partly blade wilt, root blackening easily broken) of banana regrowth after 25% crude venom soaks 72h of any processing; C: the symptom of the banana regrowth of inducing processing through the 10%T122F strain cultured solution after 25% crude venom soaks 120h (show that resistance to poison is good, minority blade wither vertical, chlorosis); D: without symptom after 25% crude venom soaks 120h of the banana regrowth of any processing (show that resistance to poison is poor, all blades are wilted, chlorosis, the root blackening is easily broken); E: induce the symptom (show that tolerance to diseases is good, the plant leaf yellow is few, and bulb interior tissue browning area is few) behind the banana regrowth wilt spore inoculating 60d of processing through the 10%T122F strain cultured solution; F: without the symptom (show that tolerance to diseases is poor, the plant leaf yellow is many, and the blade that has is withered dies, and bulb interior tissue browning area is big) behind the banana regrowth wilt spore inoculating 60d of any processing.
Fig. 4 is the banana seedlings growing way comparison of passing through T122F strain culturing of the present invention and not passing through the T122F strain culturing, wherein a: the banana seedlings of inducing processing through the 10%T122F strain cultured solution is in warm canopy growing way (showing that the leaf look dark green is, tall and big); B: without any processing banana seedlings in warm canopy growing way (show growth normal).
Embodiment
The seed selection step of subtilis is:
Gather healthy banana plant from the banana blight lesion, after getting the about 4g of its false stem middle part tissue and drying with aqua sterilisa flushing, behind 70% (V/V) alcohol surface sterilization 30S, (getting mercuric chloride 1 gram adds 1000 ml distilled waters and can obtain 0.1% mercuric chloride solution to use 0.1% mercuric chloride solution again,) surface sterilization 3.0min, dry behind the aseptic water washing 3 times.Every sample adds the 10ml sterilized water and pulverizes, behind the static 15min, get 50ul and be applied in the KB culture medium flat plate, 28 ℃ of dark culturing 48～72h, according to picking list bacterium colonies such as the colonial morphology of subtilis, colors, carry out bacterial strain purifying and preservation with the NA substratum according to a conventional method.
Bacterial strain adopts dull and stereotyped face-off growth method to the biological assay of banana blight pathogenic bacteria, be that the dull and stereotyped central authorities of PDA of 9cm connect a little ring bacterial strain bacterium colony (under 28 ℃ with the inoculation circling point promptly at diameter, the last cultivation of NA 24h), inserting diameter at plate edge simultaneously is the pathogenic fungi bacterium piece of 8mm, 28 ℃ of following dark culturing 5～7d, further screening obtains the tangible banana endogenetic bacteria of germ growth-inhibiting effect T122F.
Wherein separatory KB culture medium prescription is: peptone 20.0g, MgSO
47H
2O 1.5g, K
2HPO
4H
2O 1.8g, glycerine 10.0ml, agar 16.0～18.0g, distilled water 1000.0ml, Ph7.2.
The NA solid culture based formulas of cultivating usefulness is NA: peptone 5.0g, beef extract 3.0g, glucose 2.5g, agar 16.0～18.0g, distilled water 1000.0ml, Ph7.0～7.2.
The NB liquid culture based formulas of cultivating usefulness is NB: peptone 5.0g, beef extract 3.0g, glucose 2.5g, distilled water 1000.0ml, Ph7.0～7.2.
The PDA culture medium prescription that usefulness is cultivated in face-off is: potato 200.0g, peptone 5.0g, glucose 20.0g, agar 16.0～18.0g, distilled water 1000.0ml, Ph7.0～7.2.
The applying step of subtilis T122F bacterial strain in the banana tissue culture comprises in regular turn:
(1) making of T122F strain cultured solution: with conventional bacterial liquid substratum shaking culture, nutrient solution filtration and sterilization back are standby under 28 ℃ of conditions for bacterial strain.
(2) making of banana tissue culture medium (TCM): on MS basic medium prescription, preparation banana adventitious buds proliferation substratum, grow thickly bud division culture medium and mitogenetic seedling rooting substratum.
(3) contain the banana tissue culture matrix manufacturing of T122F strain cultured solution: the T122F nutrient solution adds in the banana tissue culture medium (TCM) according to a certain volume equably.
(4) cultivation of banana group training material: in containing the banana tissue culture medium (TCM) of T122F culturing filtrate, move into and wait to cultivate banana group training material, and cultivate down in suitable condition.
(5) test-results relatively: banana group training material grow in corresponding substratum behind the suitable fate, is blank with untreated banana group training substratum, and relatively the banana group is trained material growing state and resistance situation.
(6) healthy tissue culture seedlings of bananas obtains: obtain fast through the T122F nutrient solution induce processing, robust growth, banana group training that resistance capacity the is strong seedling of taking root.
Implementation step:
The method of the embodiment of the invention 1 comprises in regular turn: cultivation, the test-results of the making of T122F strain cultured solution, the making of banana tissue culture medium (TCM), the banana tissue culture matrix manufacturing that contains the T122F strain cultured solution, banana group training material compares, the acquisition of healthy tissue culture seedlings of bananas.Wherein T122F strains separation, preservation, cultivation and banana tissue culture belong to conventional plant pathology organon and plant tissue culture organon.
(1) the T122F aseptic culture fluid is made: bacterial strain is used conventional bacterium NB liquid nutrient medium shaking culture 3 days under 28 ℃ of conditions, behind the double-deck filter paper filtering of nutrient solution, and moist heat sterilization 20min under 121 ℃ of conditions.(Fig. 1 a).
(2) making of banana tissue culture medium (TCM): on MS basic medium prescription, preparation banana adventitious buds proliferation substratum, grow thickly bud division culture medium and mitogenetic seedling rooting substratum.Wherein the adventitious buds proliferation substratum is MS 1000ml+6-benzylaminopurine (6-BA) 5mg+ naphthylacetic acid (NAA) 2mg (Fig. 1 b); The bud division culture medium of growing thickly is MS 1000ml+6-benzylaminopurine (6-BA) 3mg+ naphthylacetic acid (NAA) 1mg; Mitogenetic seedling rooting substratum is MS 500ml+6-furfuryl group aminopurine (KT) 1mg+ naphthylacetic acid (NAA) 1mg and activity charcoal powder+2g+ pure water 500ml (Fig. 1 d).Each is cultivated based on standby behind the moist heat sterilization 20min under 121 ℃ of conditions.
(3) contain the banana tissue culture matrix manufacturing of T122F nutrient solution: the T122F strain cultured solution is added into 50 ℃ banana adventitious buds proliferation substratum, grow thickly bud division culture medium and mitogenetic seedling rooting substratum respectively by 10% volume ratio, and mixing cooling back is standby; (Fig. 1 c, e).
(4) cultivation of banana group training material:
The step 1) bud shoot proliferation of growing thickly is cultivated: in containing the banana adventitious buds proliferation substratum of T122F strain cultured solution, every bottle (containing substratum 30ml) moves into banana variety platform any of several broadleaf plants 5 in the bud tissue (about 10 buds) of growing thickly for No. 2, in 28 ℃～30 ℃, cultivate under light intensity 2000Lx, 10h light/14h dark condition.Cultivate after 12～15 days, the bud of growing thickly can change propagation of future generation (Fig. 2 a, b, c),
Step 2) the bud differentiation culture of growing thickly: grow thickly bud in division culture medium in 28 ℃～30 ℃, every bottle (containing substratum 30ml), cultivate under light intensity 2000Lx, 12h light/12h dark condition after 20 days, the bud of growing thickly be divided into can move into behind the qualified seedling root culture in the root media (Fig. 2 d, e).
Step 3) differentiation seedling rooting cultivation: get step 2) the differentiation seedling moves into and contains in the banana root media of T122F strain cultured solution, every bottle is moved 8 strains of banana differentiation seedling, in 28 ℃～30 ℃, cultivate under light intensity 2000Lx, 12h light/12h dark condition, cultivate after 20 days, the differentiation seedling can grow good root, stem and leaf (Fig. 2 f, g).
(5) test-results relatively: after banana group training material is grown in corresponding substratum, with untreated banana group training substratum is blank, the bud fresh weight increased value of growing thickly that sample survey T122F strain cultured solution is handled, propagation number, qualified seedling differentiation rate, the seedling fresh weight of taking root, take root seedling plant height and the seedling toxinicide of taking root, disease-resistant bacterium ability, relatively the banana group is trained material growing state and resistance situation (Fig. 3 a, b, c, d, e, f).
(6) healthy banana seedlings obtains: obtain fast through the T122F nutrient solution induce processing, robust growth, resistance capacity strong, can reach bottle seedling and heel in banana seedlings quality standard, that become strain phase growing way health (Fig. 4 a, b).
Following examples further specify the present invention, but the present invention not only is limited to this.
Subtilis T122F nutrient solution is organized inductive effect to the banana bud of growing thickly
(simultaneous test)
One, materials and methods
1, for examination genus bacillus inoculum preparation
Respectively that subtilis T122F of the present invention and control strain subtilis is (commercially available, derive from soil) bacterial strain under 28 ℃ of conditions with conventional NB liquid nutrient medium shaking culture 3 days, behind the double-deck filter paper filtering of nutrient solution, moist heat sterilization 20min under 121 ℃ of conditions.
2, for examination banana blight bacteria and thick toxin preparation thereof
No. 4 physiological strains of banana blight bacteria (Fusarium oxysporum f.sp.cubense) separate the banana of falling ill from the field, and germ is (28 ℃) behind PSA culture medium culturing 7d, are made into 1 * 10 with sterilized water
6The spore suspension of individual spore/mL is used for inoculation.Germ in the Cha Shi substratum shaking culture 10d (28 ℃, 140rpm), filter centrifugal (10000rpm, 10min) after, in 70 ℃ of following water-bath 30min, the culturing filtrate of acquisition is the thick toxin solution of germ.
3, contain the banana tissue culture medium (TCM) preparation of T122F strain cultured solution
On MS basic medium prescription, preparation banana adventitious buds proliferation substratum, grow thickly bud division culture medium and mitogenetic seedling rooting substratum, and on these three kinds of substratum, add T122F and BS08 nutrient solution, making nutrient solution final volume ratio is 10%, and is contrast with the substratum that adds NB nutrient solution (5%～40%) or do not do any processing.Wherein the adventitious buds proliferation substratum is MS1000ml+6-benzylaminopurine (6-BA) 5mg+ naphthylacetic acid (NAA) 2mg; The bud division culture medium of growing thickly is MS 1000ml+6-benzylaminopurine (6-BA) 3mg+ naphthylacetic acid (NAA) 1mg; Mitogenetic seedling rooting substratum is MS 500ml+6-furfuryl group aminopurine (KT) 1mg+ naphthylacetic acid (NAA) 1mg and activity charcoal powder+2g+ pure water 500ml.Substratum is sub-packed in the banana group training vial, every bottled 30ml.
4, banana seedlings induces processing
Platform any of several broadleaf plants No. 2 (AAA) bud of growing thickly was bred 20 days in the proliferated culture medium that contains strain culturing filtrate, seedling differentiation is after 20 days in division culture medium, immigration contains in the root media of strain culturing filtrate took root 20 days, can obtain the regrowth that the genus bacillus nutrient solution is induced processing.Induce in the process observe banana grow thickly bud and break up seedling, the growing way of the seedling of taking root, investigate and growth parameter(s)s such as the strain weight of untreated banana adventitious buds proliferation rate, weightening finish, seedling differentiation rate (more than the height of seedling 2cm), the seedling of taking root, false stem height.Each is handled and investigates 50 bottles altogether, tests twice.The adventitious buds proliferation culture condition is 28 ℃～30 ℃, and light intensity 2000Lx, 10h light/14h are dark.The bud of growing thickly is 28 ℃～30 ℃ in differentiation and differentiation seedling rooting culture condition, and light intensity 2000Lx, 12h light/12h are dark.
5, banana regrowth toxin and germ are handled
The banana regrowth (hinder root and keep the long 2cm of root) that access method 4 obtains is immersed in 25% the toxin dilution, and the immersion liquid height is to false stem 2cm place, 120h " Invest, Then Investigate " plant wilting situation, calculating regrowth wilting index, every processing 10 young plants, 3 repetitions.60 days banana seedlings of seedling age (hinder root and keep the long 3cm of root) is moved in flowerpot, every basin 1 strain, and every strain banana is irritated spore suspension, and (concentration is 1 * 10
6/ ml) 100ml is in the banana seedlings root, earthing then, and each handles 10 young plants, repeats for 3 times.Connect bacterium 60d " Invest, Then Investigate " banana seedlings incidence, the test temperature condition is 28～35 ℃, and relative temperature is about 90%, test twice, and to induce the banana seedlings of processing to make blank without bacteria culture fluid.
Two, interpretation of result
1, strain culturing medium (NB substratum) is to the grow thickly influence of bud hyperblastosis of banana
As known from Table 1, compare with blank, proliferated culture medium contains NB substratum 5%～10% (V/V) does not have obvious influence to the banana adventitious buds proliferation, but with the increase of NB nutrient solution concentration, banana adventitious buds proliferation rate obviously descends.The strain culturing medium of considering high density has a significant effect to the banana adventitious buds proliferation, thereby it is comparatively suitable to select for use 10% (V/V) concentration of T122F strain cultured solution to carry out the banana inducing clumping bud.
Table 1 NB substratum different concns is handled the grow thickly influence (20 days) of bud hyperblastosis of banana
| Handle | Bud proliferation rate (%) |
| Blank | 111.00Aa |
| 5％NB | 108.50Aa |
| 10％NB | 115.47Aa |
| 20％NB | 76.92Bb |
| 30％NB | 27.25Cc |
| 40％NB | 0Dd |
2, the T122F nutrient solution is to the grow thickly influence of bud hyperblastosis and weight of banana
As known from Table 2,10% (V/V) T122F nutrient solution has promoter action to banana adventitious buds proliferation and weightening finish, with blank mutually specific energy obviously improve adventitious buds proliferation rate and weight increase.Compare with the processing of control strain BS08 nutrient solution, the bud that the T122F nutrient solution is handled is thicker.
Table 2 10%T122F nutrient solution to banana grow thickly the bud hyperblastosis and the weightening finish result
| Handle | The class frequency of sprouting (%) | Bud proliferation rate (%) | Weight increase (%) | The bud growing way |
| T122F | 97.50Aa | 172.00Aa | 711.88Aa | Bud is thick |
| Control strain (BS08) | 95.90Aa | 159.52Aa | 381.99Bb | Bud is thin |
| Blank | 91.67Aa | 111.00Bb | 410.78Bb | Normally |
3, the T122F nutrient solution is grown thickly to banana, and the bud tissue increases weight and the influence of differentiation
As known from Table 3, in division culture medium, 10% (V/V) T122F nutrient solution still has obvious promoter action to the banana adventitious buds proliferation, and the banana of its processing is grown thickly, and to be divided into standard seedling rate be 51.97% to bud, apparently higher than two kinds of contrasts.The differentiation seedling that the T122F nutrient solution is handled is strong, high, the leaf look dark green.
Table 3 10%T122F nutrient solution is to the banana bud differentiation influence of growing thickly
| Strains tested | Seedling and bud proliferation rate (%) | Differentiation seedling rate * (％) | The seedling growing way |
| T122F | 125.63Aa | 51.97Aa | Miao Zhuan, height, leaf look dark green |
| Control strain (BS08) | 104.17Bb | 40.48Bb | Seedling is on the weak side, the leaf look normal |
| Blank | 92.86Bc | 45.95ABc | Normally |
*Annotate: differentiation standard seedling refers to the high above bud of 2cm that is.
4, the T122F nutrient solution is induced the banana regrowth growing way and the resistance capacity of acquisition
As known from Table 4, the banana regrowth that the bud of growing thickly obtains after the T122F nutrient solution is induced propagation, breaks up and taken root is taken root normally, compare with blank, banana seedlings after inducing is strong, high, the leaf look dark green, stem is thick, root system is prosperous, standard seedling rate reaches 100%, and the anti-poison of standard seedling, to heel in the anti-sick ability of seedling all good than blank.
Banana regrowth growing way and resistance capacity were relatively after table 4 T122F nutrient solution was induced and handled
| Handle | Strain heavy (g/ strain) | False stem height (cm/ strain) | Standard seedling rate * (％) | Standard seedling wilting index (%) | Heel in seedling diseases feelings index (%) |
| T122F | 1.30Aa | 4.43Aa | 100.00Aa | 6.67Bb | 61.90Bb |
| Blank | 0.82Bb | 3.10Bb | 82.37Bb | 76.54Aa | 82.14Aa |
*Annotate: the standard jar seedling is that seedling is pollution-free, and the thick shape of root system has the white root more than 2; The high about 2.5-3cm of false stem, the about 0.3cm of diameter stem, false stem are green; Natural leaf more than 2 is arranged, and the leaf look dark green.
Three, evaluation of result
1, subtilis T122F has disease-resistant, the short function of giving birth to, and it is cultivated and preserves conveniently, adopts conventional NB bacteria culture medium and freezing, stored refrigerated all can.
2, the T122F nutrient solution adds in banana adventitious buds proliferation substratum, division culture medium and the root media with 10% volume ratio, helps banana the growing of bud of growing thickly to increase, break up, take root and regrowth growth, improves the standard seedling rate of banana seedlings.The bud of growing thickly is cultivated in the proliferated culture medium that contains the 10%T122F nutrient solution can change the propagation next generation in 12～15 days, grew thickly the bud differentiation culture after 20 days, and differentiation seedling ratio is handled and blank apparently higher than subtilis BS08 nutrient solution.
3, the T122F nutrient solution induces adventitious buds proliferation rate height, the bud of processing thick, and the differentiation seedling is strong, high, the leaf look dark green, and growing way all is better than subtilis BS08 nutrient solution and handles and blank.Induce the banana regrowth that handle to obtain to take root normally by the T122F nutrient solution, seedling is strong, high, the leaf look dark green, stem slightly, standard seedling rate reaches 100%, the anti-poison of standard seedling, to heel in the anti-sick ability of seedling all good than blank, banana seed growing way in warm canopy is all good.
4, adopt the present invention can shorten the tissue culture seedlings of bananas incubation time, save cost, improve tissue cultured seedling seedling rate and standard rate, optimize the tissue cultured seedling growing way, improve the resistance of tissue cultured seedling, have vital role promoting healthy seedling industrialized the emerging fast of banana.
The nucleotides sequence tabulation
＜110〉Inst. of Plant Protection, fujian Academy of Agricultural Science
＜120〉a kind of subtilis and the application in the banana tissue culture thereof
<160>1
<210>1
<211>
<212>DNA
＜213〉subtilis (Bacillus subtilis)
<220>
＜223〉this subtilis (Bacillus subtilis) T122F bacterial strain 16S rDNA length overall 1353.
<400>1
tgctccatga ttcagcggcg gacgggtgag taatgcctag gaatctgcct ggtagtgggg 60
gacaacgttt cgaaaggaac gctaataccg catacgtcct acgggagaaa gtgggggatc 120
ttcggacctc acgctatcag atgagcctag gtcggattag ctagttgttg aggtaaaggc 180
tcaccaaggc gacgatccgt aactggtctg ataggatgat cagtccacct ggaactgaga 240
cacggtccag actcctacgg gaggcagcag tggggaatat tggagaatgg gcgaaaggct 300
gatccagcca tgccgcgtgt gtgaagaagg tcttcggatt gtaaagcact ttaagttggg 360
aggaagggca gttagttaat accttgctgt tttgacgtta ccaacagaat aagcaccggc 420
taacttcgtg ccagcagccg cggtaatacg aagggtgcaa gcgttaatcg gaattactgg 480
gcgtaaagcg cgcgtaggtg gttcgttaag ttggatgtga aagccccggg ctcaacctgg 540
gaactgcatc caaaactggc gagctagagt atggcagagg gtggtggaat ttcctgtgta 600
gcggtgaaat gcgtagatat aggaaggaac accagtggcg aaggcgacca cctgggctaa 660
tactgacact gatgtgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca 720
cgccgtaaac gatgtcgact agccgttggg atccttgaga tcttagtggc gcagctaacg 780
cattaagtcg accgcctggg gagtacggcc gcaaggttaa aactcaaatg aattgacggg 840
ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 900
gccttgacat gcagagaact ttccagagat ggattggtgc cttcgggaac tctgacacag 960
gtgctgcatg gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgtaacgagc 1020
gcaacccttg ttcttagtta ccagcacgtt aaggtgggca ctctaaggag actgccggtg 1080
acaaaccgga ggaaggtggg gatgacgtca agtcatcatg gcccttacgg cctgggctac 1140
acacgtgcta caatggtcgg tacaaagggt tgccaagccg cgaggtggag ctaatcccat 1200
aaaaccgatc gtagtccgga tcgcagtctg caactcgact gcgtgaagtc ggaatcgcta 1260
gtaatcgtga atcagaatgt cacggtgaat acgttcccgg gccttgtaca caccgcccgt 1320
cacaccatgg gagtggtttg ctccagaagt agc 1353
Claims (4)
1. subtilis is characterized in that: described subtilis (Bacillus subtilis) T122F bacterial strain, and its 16srDNA sequence is shown in SEQ ID NO.1 in the sequence table; The preserving number of described subtilis T122F bacterial strain is CGMCC No.3043.
2. the purposes of a subtilis as claimed in claim 1, it is characterized in that: described subtilis T122F bacterial strain is used for the banana tissue culture.
3. the purposes of subtilis according to claim 2, it is characterized in that: the concrete steps of described subtilis T122F bacterial strain in the banana tissue culture are:
(1) the T122F aseptic culture fluid is made: described T122F bacterial strain is used conventional bacterium NB liquid nutrient medium shaking culture 3 days under 28 ℃ of conditions, and behind the double-deck filter paper filtering of nutrient solution, standby behind sterilization 20min under 121 ℃ of conditions;
(2) making of banana tissue culture medium (TCM): on MS basic medium prescription, preparation banana adventitious buds proliferation substratum, grow thickly bud division culture medium and mitogenetic seedling rooting substratum: described adventitious buds proliferation substratum is: MS substratum 1000ml+6-benzylaminopurine 6-BA 5mg+ naphthylacetic acid NAA2mg; The described bud division culture medium of growing thickly is: MS substratum 1000ml+6-benzylaminopurine 6-BA 3mg+ naphthylacetic acid NAA 1mg; Described mitogenetic seedling rooting substratum is: MS substratum 500ml+6-furfuryl group aminopurine KT 1mg+ naphthylacetic acid NAA 1mg+ activity charcoal powder 2g+ pure water 500ml; Described respectively the cultivation based on standby behind the sterilization 20min under 121 ℃ of conditions;
(3) contain the banana tissue culture matrix manufacturing of T122F nutrient solution: the T122F strain cultured solution of step (1) is added into 50 ℃ banana adventitious buds proliferation substratum, grow thickly bud division culture medium and mitogenetic seedling rooting substratum respectively by 10% volume ratio, and mixing cooling back is standby;
(4) cultivation of banana group training material:
The step 1) bud shoot proliferation of growing thickly is cultivated: in containing the banana adventitious buds proliferation substratum of T122F strain cultured solution, every bottle moves into grow thickly 5 in bud tissue of banana, in 28 ℃～30 ℃, cultivates under light intensity 2000Lx, 10h light/14h dark condition; Cultivate after 12～15 days, it is of future generation that the bud of growing thickly changes propagation;
Step 2) the bud differentiation culture of growing thickly: get the bud of growing thickly of step 1), in 28 ℃～30 ℃, cultivate under light intensity 2000Lx, 10h light/14h dark condition after 20 days in the bud division culture medium of growing thickly, the bud of growing thickly moves into root culture in the root media after being divided into qualified seedling;
Step 3) differentiation seedling rooting cultivation: get step 2) the differentiation seedling moves into and contains in the banana root media of T122F strain cultured solution, every bottle is moved 8 strains of banana differentiation seedling, in 28 ℃～30 ℃, cultivate under light intensity 2000Lx, 12h light/12h dark condition, cultivate after 20 days, the differentiation seedling grows good root, stem and leaf.
4. the purposes of subtilis according to claim 3, it is characterized in that: in the bud shoot proliferation culturing step of growing thickly in the described step (4), every bottle contains substratum 30ml, moves into grow thickly 10 of buds of banana; In the described bud differentiation culture step of growing thickly, every bottle contains substratum 30ml and moves into 10 of buds of growing thickly behind the shoot proliferation, and described differentiation seedling rooting culturing step contains substratum 30ml for every bottle.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101117971A CN101591629B (en) | 2009-05-19 | 2009-05-19 | Bacillus subtilis and application thereof in banana tissue culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101117971A CN101591629B (en) | 2009-05-19 | 2009-05-19 | Bacillus subtilis and application thereof in banana tissue culture |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101591629A CN101591629A (en) | 2009-12-02 |
| CN101591629B true CN101591629B (en) | 2011-08-24 |
Family
ID=41406510
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009101117971A Expired - Fee Related CN101591629B (en) | 2009-05-19 | 2009-05-19 | Bacillus subtilis and application thereof in banana tissue culture |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101591629B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2636649T3 (en) * | 2010-03-01 | 2017-10-06 | University Of Delaware | Compositions and methods to increase biomass and tolerance to pathogens in plants |
| CN102433269B (en) * | 2011-10-21 | 2012-10-17 | 西北农林科技大学 | Bacillus and application thereof |
| CN103627659B (en) * | 2013-11-27 | 2015-10-28 | 福建省农业科学院植物保护研究所 | A kind of subtilis and the application in rice green smut control thereof |
| CN107699530B (en) * | 2017-11-27 | 2021-05-04 | 周口师范学院 | Bacillus fermentation medium, fermentation method and application |
| CN108476981B (en) * | 2018-03-27 | 2024-05-03 | 中国热带农业科学院热带生物技术研究所 | Screening method of banana germplasm with resistance to fusarium wilt |
| CN112205230B (en) * | 2020-09-18 | 2022-05-06 | 广西壮族自治区农业科学院 | Support-free and anti-sunburn cultivation method for spring fruiting of citrus |
| CN112961810B (en) * | 2021-04-15 | 2022-11-08 | 福建省农业科学院植物保护研究所 | Bacterial strains and microbial inoculum for preventing and treating crop diseases, and preparation method and application thereof |
| CN113632730A (en) * | 2021-09-17 | 2021-11-12 | 内蒙古中加农业生物科技有限公司 | Formula of culture medium for potato virus-free seedlings |
| CN114940959A (en) * | 2022-06-09 | 2022-08-26 | 河北农业大学 | Application of bacillus subtilis in promoting growth of crops |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186887A (en) * | 2007-12-04 | 2008-05-28 | 华南农业大学 | Bacillus subtilis 203 for highly effective preventing and curing banana wilt and application thereof |
| CN101250495A (en) * | 2008-03-03 | 2008-08-27 | 中国热带农业科学院环境与植物保护研究所 | Plant pathogenic fungi antagonistic bacteria strain and its use in control of plant diseases |
-
2009
- 2009-05-19 CN CN2009101117971A patent/CN101591629B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186887A (en) * | 2007-12-04 | 2008-05-28 | 华南农业大学 | Bacillus subtilis 203 for highly effective preventing and curing banana wilt and application thereof |
| CN101250495A (en) * | 2008-03-03 | 2008-08-27 | 中国热带农业科学院环境与植物保护研究所 | Plant pathogenic fungi antagonistic bacteria strain and its use in control of plant diseases |
Non-Patent Citations (4)
| Title |
|---|
| JP昭59-212416A 1984.12.01 |
| 付业勤 等.内生拮抗细菌BEB2的分子鉴定及其对香蕉枯萎病的抑制作用.《热带作物学报》.2009,第30卷(第1期),80-85. * |
| 孙正祥 等.香蕉枯萎病拮抗细菌的分离筛选与鉴定.《中国生物防治》.2008,第24卷(第2期),143-147. * |
| 殷晓敏 等.香蕉内在拮抗细菌研究——菌株B215的分离及分子鉴定.《热带作物学报》.2008,第29卷(第5期),636-640. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101591629A (en) | 2009-12-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101591629B (en) | Bacillus subtilis and application thereof in banana tissue culture | |
| CN113151059B (en) | Multifunctional Siamese bacillus and application thereof | |
| CN104498386B (en) | The preparation method and application of raw Bacillus amyloliquefaciens strain SZ23 and zymotic fluid in wild jujube | |
| CN102433282B (en) | Bacillus subtilis NB12, as well as culture method and application thereof | |
| CN102086444B (en) | Paenibacillus elgii strain and application thereof | |
| CN101575574B (en) | Trichoderma harzianum composite bacteria culture and application of trichoderma harzianum composite bacteria culture in aspect of plant protection | |
| CN104928201B (en) | A kind of bacillus amyloliquefaciens HN-11 and its microbial inoculum | |
| CN101525587B (en) | Streptomyces strain and application thereof | |
| CN1325635C (en) | Endogenetic polymexa bacillus of plant for prophyiaxis and promoting growth and application thereof | |
| CN101250495A (en) | Plant pathogenic fungi antagonistic bacteria strain and its use in control of plant diseases | |
| CN104789509B (en) | Raw bacillus pumilus and its application in one plant of bark of eucommia | |
| CN108148794A (en) | A kind of the bacillus subtilis DYr3.3 and preparation method and application of broad-spectrum antibacterial activity | |
| CN103756930A (en) | Peanut rhizosphere biocontrol bacterium, and preparation method and application thereof | |
| CN113789274A (en) | Grape rhizosphere antagonistic growth-promoting streptomyces F2 and application thereof | |
| CN101519644B (en) | Sinorhizobium sp. and application thereof | |
| CN113564065A (en) | Streptomyces fradiae G-1 and application thereof | |
| CN110295129A (en) | A kind of biocontrol microorganisms and application for preventing and treating gray mold of cucumber and powdery mildew | |
| CN102787084A (en) | Bacillus methylotrophicus 4-L-16 for prevention and control of banana vascular wilt and its application | |
| Chen et al. | Biocontrol of Fusarium wilt disease in strawberries using bioorganic fertilizer fortified with Bacillus licheniformis X-1 and Bacillus methylotrophicus Z-1 | |
| CN110317747A (en) | A kind of bacillus amyloliquefaciens JT68 and its application in prevention and treatment tea anthracnose | |
| CN104152372B (en) | The biocontrol bacterial strain G68 of controlling plant diseases and its bacterial preparation process and application | |
| CN101942403A (en) | Bacillus pumilus as well as culture method and application thereof | |
| CN106754529B (en) | The bacillus amyloliquefaciens of one plant of antagonism fructus lycii root rot and its application | |
| CN116676234A (en) | Salt-tolerant bacillus JK-25, microbial inoculum, preparation method and application thereof | |
| CN104593284B (en) | One plant of Endophyte of walnut and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110824 Termination date: 20180519 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |