CN107006376A - A kind of paulownia method for plant tissue culture - Google Patents
A kind of paulownia method for plant tissue culture Download PDFInfo
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- CN107006376A CN107006376A CN201710467843.6A CN201710467843A CN107006376A CN 107006376 A CN107006376 A CN 107006376A CN 201710467843 A CN201710467843 A CN 201710467843A CN 107006376 A CN107006376 A CN 107006376A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
A kind of paulownia method for plant tissue culture, comprises the following steps：The explant of " green paulownia No. 1 " plant is taken to carry out disinfection；It is then seeded into specific primary inducing culture and induces Multiple Buds；The Multiple Buds of induction are subjected to Multiplying culture in specific subculture multiplication medium, mass efficient Multiple Buds are obtained；Effective bud is transferred on specific root media and carries out culture of rootage, complete aseptic seedling is obtained.The specific primary inducing culture of present invention selection, subculture medium and root media, using tissue culture technique, the aseptic seedling of " green paulownia No. 1 " plant is obtained by a kind of new way, establishes stabilization, efficient tissue culture quick breeding system.The method of the present invention is easy to operate, and production cost is low, is adapted to industrial seedling rearing, and yield is high, and quality better is free from environmental pollution, it is possible to achieve batch production.
Description
Technical field
The present invention relates to field of plant tissue culture, specifically a kind of paulownia method for plant tissue culture.
Background technology
Paulownia are Scrophulariaceae Paulownia deciduous tree seeds, are originated in the range of 20 ° -40 ° of China's north latitude, 98 ° -125 ° of east longitude
23 provinces (area), be an excellent fast-growing commerical tree species." green paulownia No. 1 " is the paulownia new varieties of natural hybridization, in 2016
The Nian Huo State Administration of Forestry protects new variety of plant mandate (new varieties warrant number：20160153)." green paulownia No. 1 " botanical character
The characteristics of with high trunk under rapid, dry-shaped straight, branch, strong adaptability is grown.Height of tree during 5 year cutting cycle reaches 16-18m, chest
Footpath 28-30cm, individual volume reaches 0.7m3-0.8m3." green paulownia No. 1 " strong adaptability, in China north to Xinjiang, Heilungkiang, Hebei,
South establishes experiment and demonstration woods to Guangxi, Guangdong, Hainan and other places, excellent growth traits is all shown in various regions, with special
Property, uniformity and stability.The wood-fibred content of timber is high, and resistance to compression, bending resistance, wind loading rating are strong, widely used, be particieboard,
The good raw material material of fiberboard, papermaking etc., wide market.Method most effective and rapid and low seedling cost at present is to build
Vertical " green paulownia No. 1 " Paulownia Clones group culturation rapid propagating technology system, to improve " green paulownia No. 1 " productivity rapidly.
At present, the existing more report of the tissue culture technique of Paulownia trees, such as fortune paulownia, " excellent No. 1 of paulownia ", " bubble
The clonal tissue culture technology of the paulownia kind such as paulownia 9501 ", " miscellaneous No. one of Henan ", but the tissue culture technology of paulownia new varieties " green paulownia No. 1 "
Not yet there is open report.Using modern organization culture biotechnology, the nursery production base of modernization is set up, can not only greatly
Fast reproduction speed, makes the quality balance of nursery stock consistent, is more suitable for standardized management, moves towards nursery industry.Popularization and application new product
Plant " green paulownia No. 1 ", realize artificial forest cultivation seeds variation and national timber strategic core deposit construction of base target and ecology
Environmental construction target, its is significant.Therefore it provides a kind of have from the induction of bud, Multiplying culture, rooting induction all stage
The method for tissue culture of preferably fast numerous effect, produces Quality and economy nursery stock, is that " green paulownia No. 1 " fast growing is built in popularization and application
Woods, the only way increased economic efficiency.
The content of the invention
, can be in training it is an object of the invention to provide a kind of paulownia new varieties " green paulownia No. 1 " method for plant tissue culture
There is higher Differentiation ration of adventitious buds, value-added coefficient and rooting rate during supporting.
To achieve the above object, the technical scheme is that：A kind of paulownia method for plant tissue culture, it includes as follows
Step：
(1), explant selection and aseptic process
Paulownia new varieties " green paulownia No. 1 " plant shoots stem section is chosen, as the explant of tissue cultures, to be rinsed with running water
2-3 hours, blade is cut off, in distilled water flushing, is put in superclean bench, it is alcohol-pickled 30-60 seconds with 75%, alcohol is removed,
Sterile water wash 3-5 times, is then handled 5-7 minutes with 0.1% mercuric chloride solution, removes chlorization mercury liquid, use sterile water wash 3-5
It is secondary, explant is placed in suck dry moisture on aseptic filter paper, aseptically by the explant disinfected from terminal bud from top to bottom
Clip band terminal bud section, the second stem section, 1 stipes of every section of 1.0-1.5 centimetres of band；
(2), inducing clumping bud culture
Explant Jing Guo aseptic process is inoculated into inducing culture and carries out tissue induction Multiple Buds, cultivation cycle：
15-30 days, cultivation temperature：20-28 DEG C, illumination cultivation time：10-14 hours/day, intensity of illumination：2000-3000lux；It is described
Inducing culture be：MS culture mediums+sucrose 10-30g/L+ agar 6-8g/L+NAA 0.1-0.5mg/L+6-BA 1.0-
5.0mg/L, pH value is 5.0-7.0；
(3), Multiplying culture
The Multiple Buds induced are inoculated into proliferated culture medium and carry out Multiplying culture, enough effective seedlings, culture is obtained
Cycle：15-30 days, cultivation temperature：20-28 DEG C, illumination cultivation time：10-14 hours/day, intensity of illumination：2000-
3000lux；Described subculture medium is：MS culture mediums+sucrose 10-30g/L+ agar 6-8g/L+NAA 0.1-0.5mg/L+
6-BA 1.0-5.0mg/L, pH value is 5.0-7.0；
(4), rooting induction：
Effective seedling that Multiplying culture is obtained, which is transferred on root media, carries out culture of rootage, obtains complete aseptic seedling,
Cultivation cycle：20-30 days, cultivation temperature：20-28 DEG C, illumination cultivation time：10-14 hours/day, intensity of illumination：2000lux；
Described root media is：MS culture mediums+sucrose 10-30g/L+ agar 6-8g/L+NAA 0.1-0.5mg/L+IBA 0.1-
0.5mg/L, pH value is 5.0-7.0.
The segment that the explant disinfected is aseptically cut into 1 stipes of 1.0-1.5 centimetres of band refer to by
The explant disinfected from terminal bud from top to bottom clip band terminal bud section, the second stem section, 1 stipes of every section of 1.0-1.5 centimetres of band.
Preferably, inducing clumping bud cultivation cycle 15-20 days, 15-20 days Multiplying culture cycle, culture of rootage culture week
10-20 days phase.
Unless otherwise indicated, percentage of the present invention is mass percent, and each component content percentage sum is
100%.
The prominent advantage of the present invention is：
By Plant Tissue Breeding, the merit of plant is kept.
This method is easy to operate, and production cost is low, free from environmental pollution.
Stabilization, efficient tissue culture rapid propagation system are established using the method for the invention, industrial seedling rearing can be achieved.
Embodiment
Technical scheme is described further below by way of specific embodiment.
Embodiment 1
The explant selection of paulownia new varieties " green paulownia No. 1 " plant and aseptic process
Paulownia new varieties " green paulownia No. 1 " plant shoots stem section is chosen, as the explant of tissue cultures, to be rinsed with running water
2-3 hours, blade is cut off, in distilled water flushing, is put in superclean bench.
Set with 75% alcohol-pickled A1 (30 seconds), A2 (60 seconds), remove alcohol, sterile water wash 3-5 times.Then set
0.1% mercuric chloride solution processing B1 (5 minutes), B2 (7 minutes), remove chlorization mercury liquid, with sterile water wash 3-5 times, by explant
Body is placed in suck dry moisture on aseptic filter paper, aseptically by the explant disinfected from terminal bud clip C1 (bands from top to down
Terminal bud section), C2 (the second stem section), 1 stipes of every section of 1.0-1.5 centimetres of band.If A1B1C1, A1B1C2, A1B2C1, A1B2C2,
A2B1C1, A2B1C2, A2B2C1, A2B2C2 totally 8 kinds of processing, are shown in Table 1, every kind of processing is inoculated with 10 bottles, and every bottle connects 1 explant,
Every kind of processing is repeated 3 times.
Above-mentioned process aseptic process explant is seeded in inducing culture and is：MS culture mediums+sucrose 20g/L+ agar 6g/L+
NAA 0.1mg/L+6-BA 3.0mg/L, pH value is 6.5.Cultivation temperature：25 DEG C, illumination cultivation time：12 hours/day, illumination
Intensity：3000lux.Culture counts the growth potential of pollution rate, germination rate and bud after 7 days.
Table 1：Different explant selections and aseptic process result
The explant number of pollution rate=pollution/explant inoculation number × 100%,
The explant number of germination rate=rudiment/explant inoculation number × 100%,
Growth potential=+ shows the long potential difference of adventitious bud, and color chlorosis is light brown, ++ show adventitious bud growing way preferably, color is light green ,+
++ show adventitious bud growing way very well, color is dark green.
Experimental result finds that disinfecting time length (75% concentration alcohol 60 seconds, 0.1% mercuric chloride 7 minutes) can significantly reduce dirt
Dye rate.Under identical disinfecting time, C1 stem sections are higher than the pollution rate of C2 stem section.In terms of the result of germination rate, disinfecting time is long
Although pollution rate is reduced, rudiment is substantially inhibited, germination rate, the long potential difference of bud is reduced.(used with handling 2 i.e. A1B1C2
75% alcohol 30 seconds, the C2 stem sections of 0.1% mercury chloride sterilization in 5 minutes) although also there is 24.5% pollution rate, germination rate reaches
72.8%, and adventitious bud growing way is very well, can meet the adventitious bud needed for next step continuation is cultivated.
Embodiment 2
The inducing clumping bud culture of paulownia new varieties " green paulownia No. 1 "
2 sterile " the green paulownia No. 1 " explants obtained, which are handled, with embodiment 1 proceeds inducing clumping bud.Inducing clumping bud
Culture uses MS culture mediums, NAA 0.1mg/L, add respectively 6-BA 1.0mg/L, 2.0mg/L, 3.0mg/L, 4.0mg/L,
5 kinds of processing of 5.0mg/L various concentrations, sucrose 20g/L, agar 6.5g/L, pH value is 6.5.Every kind of 20 bottles, every bottle of processing inoculation
1 explant is connect, every kind of processing is repeated 3 times.25 DEG C of cultivation temperature, the illumination cultivation time 12 hour/day, intensity of illumination
3000lux.Culture counts differentiation number, differentiation rate, growth potential after 25 days.
Table 2：Influence of the different 6-BA concentration to differentiation
Note：Growth coefficient=adventitious bud sum/inoculation number.Same column English alphabet difference represents significant difference.+ show adventitious bud
Long potential difference, color is slightly yellow, ++ show adventitious bud growing way preferably, color is light green, +++ show adventitious bud growing way very well, color is dark green.
Result of the test shows that the present invention is using the inducing culture of 5 kinds of concentration of 6-BA to sterile " green paulownia No. 1 " explant
Evoking adventive bud is carried out, there is significant difference.Induction 10 days i.e. it is visible have adventitious bud, have within 15 days Multiple Buds appearance, 20 days buds
Number reach that at most basic afterwards there is no adventitious bud generation.Add 6-BA3.0mg/L preferably, growth coefficient highest reaches
2.85.Addition 6-BA 4.0mg/L take second place, and growth coefficient is 2.45.The growing way of two kinds of processing buds is all fine.
Embodiment 3
The Multiplying culture of paulownia new varieties " green paulownia No. 1 "
The good bud of growing way obtained with embodiment 2 proceeds Multiplying culture.Proliferated culture medium is using two kinds of culture mediums
(MS culture mediums and MS improved culture mediums), two kinds of concentration NAA (0.1mg/L, 0.2mg/L), two kinds of concentration 6-BA (3.0mg/L,
4.0mg/L) carry out contrast test.Sucrose 20g/L, agar 6.5g/L, pH value is 6.5.Every kind of 20 bottles of processing inoculation, every bottle connects 1
Strain, every kind of processing is repeated 3 times.25 DEG C of cultivation temperature, the illumination cultivation time 12 hour/day, intensity of illumination 3000lux.Culture 20
Proliferation rate, growth potential are counted after it.
Table 3：" green paulownia No. 1 " Multiplying culture result
Note：MS improvement is 1/2MS a great number of elements, the same MS of remaining composition.
Adventitious bud sum before adventitious bud sum/Multiplying culture after growth coefficient=Multiplying culture.
Result of the test shows that the present invention is using two kinds of different culture medias and adds the induction training that the hormone of various concentrations is carried out
Base is supported to the Multiplying culture of " green paulownia No. 1 ", there were significant differences for its proliferation rate.The propagation bud of MS culture mediums induction is in green, growing way
Good, the proliferation rate of bud is higher than MS improved culture medium.To handle 2 (MS culture mediums, 6-BA 3.0mg/L, NAA0.2mg/L) increasing
Rate highest is grown, up to 6.7；Processing 3 (MS culture mediums, 6-BA 4.0mg/L, NAA0.2mg/L) take second place, proliferation rate is 5.8, and this two
The bud for planting processing is sturdy, and in green, growing way is good, and this seed bud is conducive to rooting induction culture.And the induction of MS improved culture mediums is not
Growth coefficient with HORMONE TREATMENT slightly has difference, but it is elongated all to show propagation bud, green decline, and long potential difference is unfavorable for taking root
Induction.
Embodiment 4
The culture of rootage of paulownia new varieties " green paulownia No. 1 "
The propagation bud that growing way is good, green is sturdy obtained with the processing 2 and processing 3 of embodiment 3 carries out culture of rootage.Take root
Culture medium uses two kinds of IBA concentration (0.1mg/L, 0.2mg/L) and two kinds of NAA concentration (0.1mg/L, 0.2mg/L), individually with two
Hormone combinations are planted to use.Sucrose 20g/L, agar 6.5g/L, pH value is 6.5.Every kind of 20 bottles of processing inoculation, every bottle connects 3 plants, every kind of
Processing is repeated 3 times.25 DEG C of cultivation temperature, the illumination cultivation time 12 hour/day, intensity of illumination 3000lux.Culture is counted after 20 days
Rooting rate.
Table 4：" green paulownia No. 1 " culture of rootage result
The present invention induces " green paulownia No. 1 " culture of rootage using the culture medium of two kinds of hormones, three kinds of concentration combinations.Result of the test
Prove, IBA, NAA are used alone or two kinds of hormone combinations can induce " green paulownia No. 1 " to take root, cultivate 7 days it is seen that having
Root growth point is grown.A kind of independent hormone or two kinds of hormone combinations are in 0.1-0.2mg/L, and the root system lateral root of induction is more,
Be conducive to surviving for transplanted seedling.When hormonal readiness is more than 0.2mg/L, the root system of induction is although sturdy, but lateral root is few, transplanted seedling
Survival rate is low, therefore, and this kind of rooted seedling is not suitable for transplanting.With average most, the side per the effective root of young plant of the processing 1 and processing 7
Root is flourishing, is adapted to transplant, and only uses 0.1mg/L IBA or NAA, and cost is low.
Claims (5)
1. a kind of paulownia method for plant tissue culture, it is characterised in that comprise the following steps：
(1), explant selection and aseptic process
Paulownia new varieties " green paulownia No. 1 " plant shoots stem section is chosen as the explant of tissue cultures, 2-3 is rinsed with running water small
When, blade is cut off, in distilled water flushing, is put in superclean bench, it is alcohol-pickled 30-60 seconds with 75%, remove alcohol, sterilized water
Cleaning 3-5 time, then with 0.1% mercuric chloride solution processing 5-7 minutes, removes chlorization mercury liquid, with sterile water wash 3-5 times, general
Explant is placed in suck dry moisture on aseptic filter paper, aseptically by the explant disinfected from terminal bud clip band from top to bottom
Terminal bud section, the second stem section, 1 stipes of every section of 1.0-1.5 centimetres of band；
(2), inducing clumping bud culture
Explant Jing Guo aseptic process is inoculated into inducing culture and carries out tissue induction Multiple Buds, cultivation cycle：15-30
My god, cultivation temperature：20-28 DEG C, illumination cultivation time：10-14 hours/day, intensity of illumination：2000-3000lux；Described lures
Leading culture medium is：MS culture mediums+sucrose 10-30g/L+ agar 6-8g/L+NAA 0.1-0.5mg/L+6-BA 1.0-5.0mg/L,
PH value is 5.0-7.0；
(3), Multiplying culture
The Multiple Buds induced are inoculated into proliferated culture medium and carry out Multiplying culture, enough effective seedlings, cultivation cycle is obtained：
15-30 days, cultivation temperature：20-28 DEG C, illumination cultivation time：10-14 hours/day, intensity of illumination：2000-3000lux；It is described
Subculture medium be：MS culture mediums+sucrose 10-30g/L+ agar 6-8g/L+NAA 0.1-0.5mg/L+6-BA 1.0-
5.0mg/L, pH value is 5.0-7.0；
(4), rooting induction：
Effective seedling that Multiplying culture is obtained, which is transferred on root media, carries out culture of rootage, obtains complete aseptic seedling, culture
Cycle：20-30 days, cultivation temperature：20-28 DEG C, illumination cultivation time：10-14 hours/day, intensity of illumination：2000lux；It is described
Root media be：MS culture mediums+sucrose 10-30g/L+ agar 6-8g/L+NAA 0.1-0.5mg/L+IBA 0.1-
0.5mg/L, pH value is 5.0-7.0.
2. paulownia method for plant tissue culture according to claim 1, it is characterised in that described aseptically to disappear
The segment that the good explant of poison is cut into 1 stipes of 1.0-1.5 centimetres of band refers to the explant that will be disinfected from terminal bud from top to bottom
Clip band terminal bud section, the second stem section, 1 stipes of every section of 1.0-1.5 centimetres of band.
3. paulownia method for plant tissue culture according to claim 1, it is characterised in that preferably, inducing clumping bud
Cultivation cycle 15-20 days, 15-20 days Multiplying culture cycle, culture of rootage cultivation cycle 10-20 days.
4. paulownia method for plant tissue culture according to claim 1, it is characterised in that preferably, the sterile place
Reason is, with 75% alcohol-pickled 30 seconds, to remove alcohol, and then sterile water wash 3-5 times handles 5 points with 0.1% mercuric chloride solution
Clock, removes mercuric chloride solution, with sterile water wash 3-5 times.
5. paulownia method for plant tissue culture according to claim 1, it is characterised in that preferably, inducing clumping bud
Cultivation cycle is addition 6-BA 3.0mg/L in 20 days, described inducing culture.
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Cited By (8)
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| CN111202002A (en) * | 2020-01-17 | 2020-05-29 | 广东省林业科学研究院 | Tissue culture and rapid propagation method of clerodendrum japonicum |
| CN111374056A (en) * | 2020-04-17 | 2020-07-07 | 山东省林业科学研究院 | Proliferation medium formula for culturing stout tetraploid paulownia seedlings and application |
| CN111406648A (en) * | 2020-04-17 | 2020-07-14 | 山东省林业科学研究院 | Efficient starting culture medium for directly inducing tetraploid paulownia field stem section to regenerate adventitious bud and application |
| CN111406647A (en) * | 2020-04-17 | 2020-07-14 | 山东省林业科学研究院 | Efficient starting culture medium for directly inducing tetraploid paulownia petioles to regenerate adventitious buds and application |
| CN112690211A (en) * | 2021-01-04 | 2021-04-23 | 海南品优种苗科技有限公司 | Method for virus-free tissue culture and domestication cultivation of paulownia saplings |
| CN112841032A (en) * | 2021-02-01 | 2021-05-28 | 中国科学院华南植物园 | Infinite bud multiplication medium for mallotus maritima and method for in-vitro rapid propagation of mallotus maritima |
| CN113349055A (en) * | 2021-07-07 | 2021-09-07 | 广东粤恬生物科技有限公司 | Tissue culture method of paulownia |
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| CN111202002A (en) * | 2020-01-17 | 2020-05-29 | 广东省林业科学研究院 | Tissue culture and rapid propagation method of clerodendrum japonicum |
| CN111202002B (en) * | 2020-01-17 | 2022-04-19 | 广东省林业科学研究院 | Tissue culture and rapid propagation method of clerodendrum japonicum |
| CN111374056B (en) * | 2020-04-17 | 2021-05-18 | 山东省林业科学研究院 | Proliferation medium formula for culturing stout tetraploid paulownia seedlings and application |
| CN111406647A (en) * | 2020-04-17 | 2020-07-14 | 山东省林业科学研究院 | Efficient starting culture medium for directly inducing tetraploid paulownia petioles to regenerate adventitious buds and application |
| CN111406648B (en) * | 2020-04-17 | 2021-05-18 | 山东省林业科学研究院 | Efficient starting culture medium for directly inducing tetraploid paulownia field stem section to regenerate adventitious bud and application |
| CN111406648A (en) * | 2020-04-17 | 2020-07-14 | 山东省林业科学研究院 | Efficient starting culture medium for directly inducing tetraploid paulownia field stem section to regenerate adventitious bud and application |
| CN111374056A (en) * | 2020-04-17 | 2020-07-07 | 山东省林业科学研究院 | Proliferation medium formula for culturing stout tetraploid paulownia seedlings and application |
| CN112690211A (en) * | 2021-01-04 | 2021-04-23 | 海南品优种苗科技有限公司 | Method for virus-free tissue culture and domestication cultivation of paulownia saplings |
| CN112841032A (en) * | 2021-02-01 | 2021-05-28 | 中国科学院华南植物园 | Infinite bud multiplication medium for mallotus maritima and method for in-vitro rapid propagation of mallotus maritima |
| CN112841032B (en) * | 2021-02-01 | 2022-03-22 | 中国科学院华南植物园 | Infinite bud multiplication medium for mallotus maritima and method for in-vitro rapid propagation of mallotus maritima |
| CN113349055A (en) * | 2021-07-07 | 2021-09-07 | 广东粤恬生物科技有限公司 | Tissue culture method of paulownia |
| CN116135010A (en) * | 2023-04-10 | 2023-05-19 | 江苏省农业科学院宿迁农科所(宿迁市农业科学研究院) | Tissue culture and rapid propagation method for paulownia virus-free seedlings |
| CN116135010B (en) * | 2023-04-10 | 2024-05-28 | 江苏省农业科学院宿迁农科所(宿迁市农业科学研究院) | Tissue culture and rapid propagation method for paulownia virus-free seedlings |
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