CN102428873A - Method for inducing adventitious buds of hybrid eucalyptus isolated organs through callus tissue differentiation - Google Patents
Method for inducing adventitious buds of hybrid eucalyptus isolated organs through callus tissue differentiation Download PDFInfo
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Abstract
The invention relates to a method for inducing adventitious buds of hybrid eucalyptus isolated organs through callus tissue differentiation. The method comprises: an axillary bud acquisition step: clipping a bud strip from hybrid eucalyptus, disinfecting the bud strip, and inoculating the disinfected bud strip into a culture medium so as to carry out germination culture to grow axillary buds; a rooting induction culture step: cutting the axillary buds, transferring the axillary buds to an MS (Murashige and Skoog) culture medium so as to carry out successive transfer culture to grow sprouts which meet requirements, cutting the sprouts and transferring the sprouts to a 1/2 MS culture medium so as to carry out rooting induction culture to obtain a rooted seedling; a sprout culture step: cutting off the terminal buds of the rooted seedling, reserving 1-4 axillary buds of root and base parts, inoculating the axillary buds onto the MS culture medium so as to carry out sprout culture; a callus tissue induction culture step: inoculating a cut sprout section utilized as an explant onto a callus induction culture medium so as to culture under a dark condition and then under a weak illumination condition, thus obtaining callus tissues; and an adventitious bud differentiation step: transferring the callus tissues onto a differential medium so as to carry out successive transfer culture, thus obtaining the adventitious buds. The method is based on the bud strip of clonal adult eucalyptus, thus the regeneration efficiency reaches about 60%.
Description
Technical field
The present invention relates to biological tissue and cultivate the seedling growing process field, relate in particular to the method for a kind of inducement crossbreeding kind eucalyptus isolated organ through callus differentiation indefinite bud.
Background technology
Eucalyptus be Myrtaceae (Myrtaceae) eucalyptus belong to (
Eucalyptus)The general designation of plant.Be one of three big quick growing species of treess in the world.Be distributed widely in Australia, South America, Asia, areas such as Europe.Eucalyptus is widely used in cellulose industry, pulpwood and fibrous plate, can also be as the raw material that extracts tannin and essential oil, and eucalyptus extracts is applied to chemical industry, and a plurality of fields such as medical treatment are extensively planted by a plurality of countries and regions at present.Therefore eucalyptus has value of exploiting and utilizing widely.
The crossbreed eucalyptus occupies very part and parcel in production field is used, but owing to use the crossbreed eucalyptus to be mostly traditional conventional breeding gained at present, genetic heterozygosity is high; Be difficult to directed the selection and cultivate new varieties, emerging technique for gene engineering has solved the insoluble problem of traditional breeding method, and the genes of interest that it can utilize Protocols in Molecular Biology need to select passes through agriculture bacillus mediated; Modes such as particle gun import recipient cell inside; And make plant cell regeneration through the certain culture method, and develop into whole plant, have high efficiency; Characteristics with strong points are developing direction of good breeding.
The experiment material that the most of success of eucalyptus gene breeding technology is regenerated is to be the basis with the seed, and seed variation is big, is difficult to be applied to clonal hybridization eucalyptus.Therefore the tissue culture, the renovation process that need stability and high efficiency.At present at the hybridization seeds DH32-29 of Chinese extensive use, wide woods waits for No. nine seeds seldom to have report to have the gene breeding that regenerating system efficiently and this system are supported.
Summary of the invention
Embodiment of the invention technical problem to be solved is, the method for a kind of inducement crossbreeding kind eucalyptus isolated organ through callus differentiation indefinite bud is provided, fast, stably to breed and the seed selection improved seeds.
For solving the problems of the technologies described above, the present invention provides following technical scheme: a kind of inducement crossbreeding kind eucalyptus isolated organ comprises the steps: through the method for callus differentiation indefinite bud
Obtain the axillalry bud step: clip rudiment bar from the crossbreed eucalyptus of growth 3-5, normal the no damage by disease and insect of growth, insert after gained rudiment bar is sterile-processed again and sprout cultivation in the medium and grow axillalry bud;
Root induction incubation step: cut axillalry bud and transfer to and under illumination condition, carry out successive transfer culture on the MS medium that is added with 0.1-1.0mg/L 6-benzyl purine and 0.01-0.5mg/L methyl; And every transferring at a distance from 21-28 days continues on the identical medium to cultivate; Until growing satisfactory sprouting, cut sprouting again and transfer to and under illumination condition, carry out root induction on the 1/2MS medium that is added with 0.1-1.0mg/L 3-indolebutyric acid and be cultured to and obtain to take root seedling;
The incubation step of newly sprouting: the seedling of will taking root cuts away terminal bud, keeps 1-4 axillalry bud of root and base portion, is seeded in to carry out newborn bud cultivation on the MS improved culture medium that contains mass percent 3-10% sucrose;
The inducing culture step of callus: with the segment of newly sprouting is explant; Be seeded in and contain 0.1-2.0mg/L N-phenyl-N-1; 2, on the callus of induce medium of 3-thiadiazoles-5-urea, 0.01-0.15mg/L methyl, 20.0-200.0mg/L putrescine and 3.0-30.0mg/L spermidine, secretly cultivated 5-10 days; Transfer to the low light level again according to cultivating 12-14 days, obtain callus;
Differentiation indefinite bud step: with the callus that obtains; Transfer on the differential medium that contains 0.2-2.0mg/L 6-benzyl purine, 0.05-0.5mg/L methyl, 10.0-40.0mg/L putrescine and 0.5-3.0mg/L spermidine successive transfer culture under illumination condition; And every transferring at a distance from 21 days continues on the identical differential medium to cultivate, and cultivates and promptly obtains indefinite bud in 14-100 days.
Further; Obtain in the axillalry bud step, gained rudiment bar is disinfected as follows: the rudiment bar is cut off blade place under the running water flushing 30 minutes, and then the rudiment bar is truncated into every section segment that comprises 1-2 axillalry bud; With 75% alcohol immersion 20-30 second of percent by volume; Soaked 5 minutes with aseptic water washing 2 times and with mass percent 0.1% mercuric chloride again, pour out mercuric chloride, sprout tooth bar segment 4-6 time with aseptic water washing; Be trimmed to the stem section of the long band axillalry bud of 0.5-1.0cm, mass percent 0.1% mercuric chloride soaked 2 minutes; Pour out mercuric chloride, with aseptic water washing 4-6 time.
Further, obtain in the axillalry bud step, the rudiment bar that inserts medium is dark the cultivation 21-28 days under 23-28 ℃ the condition in temperature; Wait for axillary bud sprouting; The medium that is adopted is the MS minimal medium, and it contains mass percent 3% sucrose and mass percent 0.7% agar, pH5.8.
Further, root induction incubation step, the long 0.5 ~ 1.0cm of the axillalry bud that is cut, and the long 1.5 ~ 2.0cm of the sprouting that cuts.
Further, in the root induction incubation step, the condition of culture of the successive transfer culture of axillalry bud is: illumination 16h/ days, and intensity of illumination 1500-10000lx, and temperature is 23-28 ℃.
Further, in the root induction incubation step, the condition of culture of culture of rootage is: 14-30 days, and illumination 16h/ days, intensity of illumination 1500-10000lx, and temperature is 23-28 ℃.
Further, the condition of culture of cultivating of newly sprouting is: secretly cultivated 14-35 days and temperature is 23-28 ℃.
Further, in the inducing culture step of callus, secretly cultivate and the low light level is 23-28 ℃ according to the temperature range of cultivating, the low light level is when cultivating, and illumination 16h/ days and intensity of illumination are 20-100lx.
Further, in the inducing culture step of callus, it is the explant of 3-10mm that the segment of newly sprouting becomes length.
Further, in the differentiation indefinite bud step, condition of culture is: illumination 16h/ days, intensity of illumination 500-3000lx and temperature were 23-28 ℃.
Through adopting technique scheme; The present invention has following beneficial effect at least: the present invention is basis with the grow up rudiment bar of eucalyptus of clone, and the regeneration efficiency of regenerative system reaches about 60%, can be applied to DH32-29, wide excellent strain such as woods No. nine; For utilizing modern biotechnology the genetic improvement of eucalyptus is had laid a good foundation; Have crucial economic worth, one aspect of the present invention can be bred and the seed selection improved seeds fast, for genetic transformation good system is provided on the other hand.
Embodiment
Below in conjunction with instance description embodiment of the present invention are detailed.Need to prove that following instance is illustrative, is not determinate, can not limit protection scope of the present invention with following instance.
The present invention provides the method for a kind of inducement crossbreeding kind eucalyptus isolated organ through callus differentiation indefinite bud, comprises the steps:
S1, obtain the axillalry bud step: clip rudiment bar from the crossbreed eucalyptus of growth 3-5, normal the no damage by disease and insect of growth, insert after gained rudiment bar is sterile-processed again and sprout cultivation in the medium and grow axillalry bud;
S2, root induction incubation step: cut axillalry bud and transfer to and under illumination condition, carry out successive transfer culture on the MS medium that is added with 0.1-1.0mg/L 6-benzyl purine and 0.01-0.5mg/L methyl; And every transferring at a distance from 21-28 days continues on the identical medium to cultivate; Until growing satisfactory sprouting, cut sprouting again and transfer to and under illumination condition, carry out root induction on the 1/2MS medium that is added with 0.1-1.0mg/L 3-indolebutyric acid and be cultured to and obtain to take root seedling;
S3, the new incubation step of sprouting: the seedling of will taking root cuts away terminal bud, keeps 1-4 axillalry bud of root and base portion, is seeded in to carry out newborn bud cultivation on the MS improved culture medium that contains mass percent 3-10% sucrose;
The inducing culture step of S4, callus: with the segment of newly sprouting is explant; Be seeded in and contain 0.1-2.0mg/L N-phenyl-N-1; 2, on the callus of induce medium of 3-thiadiazoles-5-urea, 0.01-0.15mg/L methyl, 20.0-200.0mg/L putrescine and 3.0-30mg/L spermidine, secretly cultivated 5-10 days; Transfer to the low light level again according to cultivating 12-14 days, obtain callus;
S5, differentiation indefinite bud step: with the callus that obtains; Transfer on the differential medium that contains 0.2-2.0mg/L 6-benzyl purine, 0.05-0.5mg/L methyl, 10.0-40.0mg/L putrescine and 0.5-3.0mg/L spermidine successive transfer culture under illumination condition; And every transferring at a distance from 21 days continues on the identical differential medium to cultivate, and cultivates and promptly obtains indefinite bud in 14-100 days.
Wherein, In obtaining the axillalry bud step, specific as follows: as the rudiment bar to be cut off blade placed under the running water flushing 30 minutes, and then the rudiment bar is truncated into every section segment that comprises 1-2 axillalry bud to disinfecting of carrying out of gained rudiment bar; With 75% alcohol immersion 20-30 second of percent by volume; Soaked 5 minutes with aseptic water washing 2 times and with mass percent 0.1% mercuric chloride again, pour out mercuric chloride, sprout tooth bar segment 4-6 time with aseptic water washing; Be trimmed to the stem section of the long band axillalry bud of 0.5-1.0cm, mass percent 0.1% mercuric chloride soaked 2 minutes; Pour out mercuric chloride, with aseptic water washing 4-6 time.
In addition; Obtain in the axillalry bud step, the rudiment bar that inserts medium is dark the cultivation 21-28 days under 23-28 ℃ the condition in temperature, waits for axillary bud sprouting; The medium that is adopted is MS minimal medium+mass percent 3% sucrose+mass percent 0.7% agar, pH5.8.
And at the root induction incubation step, the long 0.5 ~ 1.0cm of the axillalry bud that is cut, and the long 1.5 ~ 2.0cm of the sprouting that cuts; The condition of culture of the successive transfer culture of axillalry bud is: illumination 16h/ days, and intensity of illumination 1500-10000lx, and temperature is 23-28 ℃; And the condition of culture of culture of rootage is: 14-30 days, and illumination 16h/ days, intensity of illumination 1500-10000lx, and temperature is 23-28 ℃.
In the incubation step of newly sprouting, its condition of culture is: secretly cultivated 14-35 days and temperature is 23-28 ℃.
In the inducing culture step of callus, it is the explant of 3-10mm that the segment of newly sprouting becomes length, secretly cultivates and the low light level is 23-28 ℃ according to the temperature range of cultivating, and the low light level is when cultivating, and illumination 16h/ days and intensity of illumination are 20-100lx.
In differentiation indefinite bud step, condition of culture is: illumination 16h/ days, intensity of illumination 500-3000lx and temperature were 23-28 ℃.
Below further specify the specific operation process of the inventive method through several instances.
Instance 1
Choose 3 years living plant of eucalyptus DH32-29 of gathering voluntarily, cut down tree crown, keep the high stub of 0.5-1.5 rice.Gather newbornly after 20-30 days, be cut into the stem section of the long band axillalry bud of 2.0cm, soak 20s in percent by volume 70% ethanol not with the rudiment bar of axillalry bud; Aseptic water washing 2 times; The 5min that in mass percent 0.1% mercuric chloride, sterilizes then, aseptic water washing 4 times is trimmed to the stem section of the long band axillalry bud of 1.0cm; The 2min that in mass percent 0.1% mercuric chloride, sterilizes again, aseptic water washing 6 times.Be seeded in after drying in the MS medium, every bottle graft 1-2, secretly cultivated the axillalry bud that obtains sprouting 28 days for 25 ℃.
Cut about 1.0cm long axillalry bud, transfer on the medium of MS medium+0.1mg/L 6-benzyl purine+0.01mg/L methyl, every at a distance from 3-4 week transferring to successive transfer culture on the identical medium, illumination 16h/ days, intensity of illumination 3000lx, 25 ℃.Long bud about the selected 1.5cm that newly grows out is transferred on the medium of 1/2MS+0.5mg/L3-indolebutyric acid and is carried out 3 weeks of root induction, illumination 16h/d, intensity of illumination 3000lx, 25 ℃.
The seedling of will taking root keeps 2 axillalry buds of root and base portion, cuts away remainder, is seeded in to carry out newborn bud cultivation on the MS medium that contains sucrose 7% (mass percent), secretly cultivates 21d, 24 ℃.
The segment that is cut to the 3-5 millimeter with newly sprouting is transferred to and is contained 0.1mg/L N-phenyl-N-1, and 2, in the callus of induce medium of 3-thiadiazoles-5-urea+0.01mg/L methyl, secretly cultivated 25 ℃ 7 days; Transfer to the low light level according to cultivation 14 days, illumination 16h/ days, intensity of illumination 50lx, 25 ℃.
With the callus that obtains, transfer in the differential medium that contains 0.5mg/L 6-benzyl purine+0.1mg/L methyl, whenever transferred to successive transfer culture on the identical differential medium at a distance from 21 days, illumination 16h/ days, intensity of illumination 1500lx, 25 ℃.Successive transfer culture 20-100 days generation indefinite buds on differential medium.
Instance 2
Choose No. nine 3 years living plant of the wide woods of eucalyptus of gathering voluntarily; Except at the inducing culture medium that step is used of callus for containing 1.0mg/L N-phenyl-N-1; 2; Outside the callus of induce medium of 3-thiadiazoles-5-urea+0.1mg/L methyl, all the other steps are identical with instance 1 corresponding step.
Instance 3
Choose 3 years living plant of eucalyptus DH32-29 of purchase; Except at the inducing culture medium that step is used of callus for containing 2.0mg/L N-phenyl-N-1; 2, outside the callus of induce medium of 3-thiadiazoles-5-urea+0.15mg/L methyl, all the other steps are identical with instance 1 corresponding step.
The differentiation callus quantity that above-mentioned three instances are obtained respectively is as shown in table 1 below:
Table 1: the differentiation effect of experiment material in the instance 1 ~ 3
| The instance sequence number | The explant sum | The callus sum | Differentiation callus number | Regeneration efficiency (%) |
| Instance 1 | 598 | 586 | 358 | 61.09 |
| Instance 2 | 570 | 563 | 336 | 60.98 |
| Instance 3 | 501 | 501 | 306 | 61.08 |
Can find by last table; Regeneration efficiency of the present invention reaches about 60%; Can be applicable to excellent strains such as No. nine, DH32-29, wide woods, breed fast and the seed selection improved seeds, the genetic improvement of eucalyptus has been established good basis for utilizing modern biotechnology; For genetic transformation provides good system, has crucial economic worth.
Claims (10)
1. an inducement crossbreeding kind eucalyptus isolated organ is characterized in that through the method that callus breaks up indefinite bud, comprises the steps:
Obtain the axillalry bud step: clip rudiment bar from the crossbreed eucalyptus of growth 3-5, normal the no damage by disease and insect of growth, insert after gained rudiment bar is sterile-processed again and sprout cultivation in the medium and grow axillalry bud;
Root induction incubation step: cut axillalry bud and transfer to and under illumination condition, carry out successive transfer culture on the MS medium that is added with 0.1-1.0mg/L 6-benzyl purine and 0.01-0.5mg/L methyl; And every transferring at a distance from 21-28 days continues on the identical medium to cultivate; Until growing satisfactory sprouting, cut sprouting again and transfer to and under illumination condition, carry out root induction on the 1/2MS medium that is added with 0.1-1.0mg/L 3-indolebutyric acid and be cultured to and obtain to take root seedling;
The incubation step of newly sprouting: the seedling of will taking root cuts away terminal bud, keeps 1-4 axillalry bud of root and base portion, is seeded in to carry out newborn bud cultivation on the MS improved culture medium that contains mass percent 3-10% sucrose;
The inducing culture step of callus: with the segment of newly sprouting is explant; Be seeded in and contain 0.1-2.0mg/L N-phenyl-N-1; 2, on the callus of induce medium of 3-thiadiazoles-5-urea, 0.01-0.15mg/L methyl, 20.0-200.0mg/L putrescine and 3.0-30.0mg/L spermidine, secretly cultivated 5-10 days; Transfer to the low light level again according to cultivating 12-14 days, obtain callus;
Differentiation indefinite bud step: with the callus that obtains; Transfer on the differential medium that contains 0.2-2.0mg/L 6-benzyl purine, 0.05-0.5mg/L methyl, 10.0-40.0mg/L putrescine and 0.5-3.0mg/L spermidine successive transfer culture under illumination condition; And every transferring at a distance from 21 days continues on the identical differential medium to cultivate, and cultivates and promptly obtains indefinite bud in 14-100 days.
2. inducement crossbreeding kind eucalyptus isolated organ according to claim 1 is through the method for callus differentiation indefinite bud; It is characterized in that: obtain in the axillalry bud step; Gained rudiment bar is disinfected as follows: the rudiment bar is cut off blade place under the running water flushing 30 minutes; And then the rudiment bar is truncated into every section segment that comprises 1-2 axillalry bud, and using percent by volume was 75% alcohol immersion 20-30 second, using aseptic water washing again 2 times and using mass percent is that 0.1% mercuric chloride soaked 5 minutes; Pour out mercuric chloride, sprout tooth bar segment 4-6 time with aseptic water washing; Be trimmed to the stem section of the long band axillalry bud of 0.5-1.0cm, mass percent is that 0.1% mercuric chloride soaked 2 minutes; Pour out mercuric chloride, with aseptic water washing 4-6 time.
3. inducement crossbreeding kind eucalyptus isolated organ according to claim 1 and 2 is through the method for callus differentiation indefinite bud; It is characterized in that: obtain in the axillalry bud step; The rudiment bar that inserts medium is dark the cultivation 21-28 days under 23-28 ℃ the condition in temperature; Wait for axillary bud sprouting, the medium that is adopted is MS minimal medium+mass percent 3% sucrose+mass percent 0.7% agar, pH5.8.
4. inducement crossbreeding kind eucalyptus isolated organ according to claim 1 is characterized in that through the method for callus differentiation indefinite bud: root induction incubation step, the long 0.5 ~ 1.0cm of the axillalry bud that is cut, and the long 1.5 ~ 2.0cm of the sprouting that cuts.
5. pass through the method that callus breaks up indefinite bud according to claim 1 or 4 described inducement crossbreeding kind eucalyptus isolated organs; It is characterized in that: in the root induction incubation step; The condition of culture of the successive transfer culture of axillalry bud is: illumination 16h/ days; Intensity of illumination 1500-10000lx, and temperature is 23-28 ℃.
6. pass through the method that callus breaks up indefinite bud according to claim 1 or 4 described inducement crossbreeding kind eucalyptus isolated organs; It is characterized in that: in the root induction incubation step; The condition of culture of culture of rootage is: 14-30 days; Illumination 16h/ days, intensity of illumination 1500-10000lx, and temperature is 23-28 ℃.
7. inducement crossbreeding kind eucalyptus isolated organ according to claim 1 is characterized in that through the method for callus differentiation indefinite bud: the condition of culture of cultivating of newly sprouting is: secretly cultivated 14-35 days and temperature is 23-28 ℃.
8. inducement crossbreeding kind eucalyptus isolated organ according to claim 1 is through the method for callus differentiation indefinite bud; It is characterized in that: in the inducing culture step of callus; The dark cultivation and the low light level are 23-28 ℃ according to the temperature range of cultivating; The low light level is when cultivating, and illumination 16h/ days and intensity of illumination are 20-100lx.
9. inducement crossbreeding kind eucalyptus isolated organ according to claim 1 is through the method for callus differentiation indefinite bud, and it is characterized in that: in the inducing culture step of callus, it is the explant of 3-10mm that the segment of newly sprouting becomes length.
10. inducement crossbreeding kind eucalyptus isolated organ according to claim 1 is through the method for callus differentiation indefinite bud; It is characterized in that: in the differentiation indefinite bud step; Condition of culture is: illumination 16h/ days, intensity of illumination 500-3000lx and temperature were 23-28 ℃.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102696482A (en) * | 2012-06-18 | 2012-10-03 | 湖南省林业科学院 | Method for detoxifying clonal tissue culture explant of Camellia oleifera Abel |
| CN105900829A (en) * | 2016-04-15 | 2016-08-31 | 中国林业科学研究院热带林业研究所 | Rapid breeding method of Eucapyptus urophylla + E. tereticornis artificial hydrids based on ion implantation |
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| CN101897298A (en) * | 2010-08-10 | 2010-12-01 | 普罗米绿色能源(深圳)有限公司 | Method for inducing eucalyptus to generate calli and differentiating calli into buds |
| CN102165918A (en) * | 2011-01-20 | 2011-08-31 | 湛江师范学院 | Tissue culturing and regenerating method of eucalyptus pellita |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101897298A (en) * | 2010-08-10 | 2010-12-01 | 普罗米绿色能源(深圳)有限公司 | Method for inducing eucalyptus to generate calli and differentiating calli into buds |
| CN102165918A (en) * | 2011-01-20 | 2011-08-31 | 湛江师范学院 | Tissue culturing and regenerating method of eucalyptus pellita |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102696482A (en) * | 2012-06-18 | 2012-10-03 | 湖南省林业科学院 | Method for detoxifying clonal tissue culture explant of Camellia oleifera Abel |
| CN105900829A (en) * | 2016-04-15 | 2016-08-31 | 中国林业科学研究院热带林业研究所 | Rapid breeding method of Eucapyptus urophylla + E. tereticornis artificial hydrids based on ion implantation |
| CN105900829B (en) * | 2016-04-15 | 2018-05-11 | 中国林业科学研究院热带林业研究所 | A kind of rapid breeding method based on the thin eucalyptus artificial hybridization kind of ion implanting tail |
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