CN107980637A - Tea-tree tissue culture method - Google Patents
Tea-tree tissue culture method Download PDFInfo
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- CN107980637A CN107980637A CN201711391223.5A CN201711391223A CN107980637A CN 107980637 A CN107980637 A CN 107980637A CN 201711391223 A CN201711391223 A CN 201711391223A CN 107980637 A CN107980637 A CN 107980637A
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- tea
- tissue culture
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- bud
- culture method
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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
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides a kind of tea-tree tissue culture method, comprises the following steps：Step 1, materials and methods, testing of materials material newly sprout the 2nd~3 axillary bud of branch for tea tree spring, pick up from tea place tested by the Chinese Academy of Agricultural Sciences's tealeaves research, kind is Dragon Well tea 43.The branch of 2~3 axillary buds of band of collection is carefully cleaned up, is sterilized, the stem section of the 1cm long of one axillary bud of band is cut into, is sprouted in inoculation medium, transfers 3~4 times in new same medium after sprouting, that is, Multiple Buds group is obtained, as experiment material.Herein for the purpose of the proliferation rate and planting percent that improve tea tree Multiple Buds, optimize the hormone combinations of tea-tree tissue culture fast breeding culture medium, the proliferation rate of every 20 days a cycles is set to can reach more than 2.3 times, the bud more than 20% can grow up to more than height 5cm, be adapted to the seedling of transplanting.
Description
Technical field
The present invention relates to tea tree technology, more particularly to a kind of tea-tree tissue culture method.
Background technology
The Study on tissue culture of tea tree just has begun to the eighties from twentieth century, explant generally use blade
Leaf, stem section, axillary bud etc..But since tea tree is perennial xylophyta, the speed of growth is slow [5], thus there are proliferation rate, into
The problem of seedling rate is relatively low.
The tissue cultures of tea tree have just had scientist to begin one's study from or so eighties in 19th century, and foreign countries had in recent years
Indra Sandal [7], Mondal [8] etc. study the tissue culturing system of tea tree, and domestic relevant research report
Road almost without.But up to now, the problem of its proliferation rate is low, and the speed of growth is slow, and cultivation cycle is long, be still tissue cultures
Technology is used for the bottleneck of tea Culture nursery.
The content of the invention
It is an object of the present invention in view of the above-mentioned problems, propose a kind of tea-tree tissue culture method, grown thickly herein with improving tea tree
For the purpose of the proliferation rate and planting percent of bud, the hormone combinations of tea-tree tissue culture fast breeding culture medium are optimized, make every 20 day week
The proliferation rate of phase can reach more than 2.3 times, and the bud more than 20% can grow up to more than height 5cm, be adapted to the seedling of transplanting.
To achieve the above object, the technical solution adopted by the present invention is：A kind of tea-tree tissue culture method, comprises the following steps：
Step 1, materials and methods
Testing of materials material newly sprouts the 2nd~3 axillary bud of branch for tea tree spring, picks up from tealeaves research institute of the Chinese Academy of Agricultural Sciences
Tea place is tested, kind is Dragon Well tea 43.The branch of 2~3 axillary buds of band of collection is carefully cleaned up, is sterilized, is cut into one, band
The stem section of the 1cm long of axillary bud, is sprouted in inoculation medium, is transferred 3~4 times in new same medium after sprouting,
Multiple Buds group is obtained, as experiment material；
Step 2, experiment condition subculture method are the new sorites that the sorite that grows thickly is cut into each 1~3 bud of band, are inoculated into
On new different culture media 3；
Step 3, cytokinin concentration are to the influence auxin breeding and grow, totally 6 concentration gradients.Each processing is set
Put 5 repetitions, each 10 materials of repeated inoculation.After inoculation the 30th day, respectively the bud head number to each sorite and
Highly up to more than 5cm, it is adapted to the seedling number of transplanting to carry out counting statistics, calculates proliferation rate (bud during bud number/inoculation during observation
Number) and planting percent (higher than 5cm can transplanted seedling number/observation when total bud number)；
Step 4, data analysis carry out the one-factor analysis of variance and Multiple range test using statistic software SPSS.
Further, step 1 sterilizes：With 0.1% HgCl2 (adding 2~3 drop Tween-20s) surface sterilization 10min.
Further, step 2 cultivation temperature is 25 ± 2 DEG C, when light application time is 12 small, 2000~3000lx of light intensity.It is real
Test and use MS minimal medium formulas.Hormone concentration unit is mg/L.
Further, step 3 auxin uses NAA (heteroauxin) 0.5mg/L, and the basic element of cell division uses BA (6- benzyl ammonia
Base adenine).
Further, 6 concentration gradients 0.5,1.0,2.0,4.0,6.0,8.0mg/L.
Tea-tree tissue culture method of the present invention, has the following advantages compared with prior art：
Hormone is bred by bud in tea tree tissue cultures and the influence of growth, finds the propagation for tea tissue-cultured seedling, BA concentration
It is proper in 2.0mg/L or so；During less than 1.0, due to basic element of cell division dosage deficiency, it is impossible to promote propagation as early as possible；It is higher than
When 2.0, inhibit on the contrary propagation in addition cause death, it may be possible to due to concentration too it is high cause to produce around tufted seedling base portion it is poisonous
Harmful material (such as phenols secondary metabolic product etc.).And NAA concentration is preferable below 0.1, promote the effect of propagation more apparent,
Seedling base portion can produce callus when more than 0.2, so as to hinder to grow.GA3 has preferable promotion to breeding and growing
Effect, particularly preferably can promote bud head to be grown to serve as seedling, and effect is best when its concentration is 3.0 or so, and tea shoot not only increases
Reproductive growth is fast, and more healthy and strong.Analyze the dynamic data of tissue culture seedling proliferation, it can be seen that the 0th~10 day after subinoculation,
There is a resting stage, growth rate of the tea tissue-cultured seedling within this period is relatively low, and is within the 20th~30 day the fast fast-growing of its propagation
For a long time, thereafter, the growth rate of tea shoot can progressively slow down again.Therefore, efficiency is maximumlly bred to try to achieve, it may be considered that with 20
It or so is a subculture cycle, when not only can shorten the culture in each cycle on the basis of a certain amount of proliferation rate is ensured
Between, and the material in fast growing period was used for followed by generation, it can further shorten the resting stage of next cultivation cycle
Length.
Herein for the purpose of the proliferation rate and planting percent that improve tea tree Multiple Buds, tea-tree tissue culture fast breeding culture medium is optimized
Hormone combinations, the proliferation rate of every 20 days a cycles is can reach more than 2.3 times, the bud more than 20% can grow up to height 5cm
Above, it is adapted to the seedling of transplanting.Research deeper into a step is it is contemplated that continue to improve the fast numerous squamous subculture skill of tea-tree tissue culture
Art and transplantation rooting technology, promote tissue culture technique to be applied to the quick numerous of tea tree breed Upgrading or new varieties early
Educate.
Embodiment
The present invention is further described with reference to embodiments：
Embodiment 1
Present embodiment discloses a kind of tea-tree tissue culture method, comprise the following steps：
Step 1, materials and methods
Testing of materials material newly sprouts the 2nd~3 axillary bud of branch for tea tree spring, picks up from tealeaves research institute of the Chinese Academy of Agricultural Sciences
Tea place is tested, kind is Dragon Well tea 43.The branch of 2~3 axillary buds of band of collection is carefully cleaned up, is sterilized, is cut into one, band
The stem section of the 1cm long of axillary bud, is sprouted in inoculation medium, is transferred 3~4 times in new same medium after sprouting,
Multiple Buds group is obtained, as experiment material；Disinfection：With 0.1% HgCl2 (adding 2~3 drop Tween-20s) surface sterilization
10min。
Step 2, experiment condition subculture method are the new sorites that the sorite that grows thickly is cut into each 1~3 bud of band, are inoculated into
On new different culture media 3；Cultivation temperature is 25 ± 2 DEG C, when light application time is 12 small, 2000~3000lx of light intensity.Experiment is adopted
With MS minimal medium formulas.Hormone concentration unit is mg/L.
Step 3, cytokinin concentration are to the influence auxin breeding and grow, totally 6 concentration gradients.Each processing is set
Put 5 repetitions, each 10 materials of repeated inoculation.After inoculation the 30th day, respectively the bud head number to each sorite and
Highly up to more than 5cm, it is adapted to the seedling number of transplanting to carry out counting statistics, calculates proliferation rate (bud during bud number/inoculation during observation
Number) and planting percent (higher than 5cm can transplanted seedling number/observation when total bud number)；Auxin uses NAA (heteroauxin)
0.5mg/L, the basic element of cell division use BA (6- benzyls aminoadenine).6 concentration gradients 0.5,1.0,2.0,4.0,6.0,
8.0mg/L。
Step 4, data analysis carry out the one-factor analysis of variance and Multiple range test using statistic software SPSS.
Hormone is bred by bud in tea tree tissue cultures and the influence of growth, finds the propagation for tea tissue-cultured seedling, BA concentration
It is proper in 2.0mg/L or so；During less than 1.0, due to basic element of cell division dosage deficiency, it is impossible to promote propagation as early as possible；It is higher than
When 2.0, inhibit on the contrary propagation in addition cause death, it may be possible to due to concentration too it is high cause to produce around tufted seedling base portion it is poisonous
Harmful material (such as phenols secondary metabolic product etc.).And NAA concentration is preferable below 0.1, promote the effect of propagation more apparent,
Seedling base portion can produce callus when more than 0.2, so as to hinder to grow.GA3 has preferable promotion to breeding and growing
Effect, particularly preferably can promote bud head to be grown to serve as seedling, and effect is best when its concentration is 3.0 or so, and tea shoot not only increases
Reproductive growth is fast, and more healthy and strong.Analyze the dynamic data of tissue culture seedling proliferation, it can be seen that the 0th~10 day after subinoculation,
There is a resting stage, growth rate of the tea tissue-cultured seedling within this period is relatively low, and is within the 20th~30 day the fast fast-growing of its propagation
For a long time, thereafter, the growth rate of tea shoot can progressively slow down again.Therefore, efficiency is maximumlly bred to try to achieve, it may be considered that with 20
It or so is a subculture cycle, when not only can shorten the culture in each cycle on the basis of a certain amount of proliferation rate is ensured
Between, and the material in fast growing period was used for followed by generation, it can further shorten the resting stage of next cultivation cycle
Length.
Herein for the purpose of the proliferation rate and planting percent that improve tea tree Multiple Buds, tea-tree tissue culture fast breeding culture medium is optimized
Hormone combinations, the proliferation rate of every 20 days a cycles is can reach more than 2.3 times, the bud more than 20% can grow up to height 5cm
Above, it is adapted to the seedling of transplanting.Research deeper into a step is it is contemplated that continue to improve the fast numerous squamous subculture skill of tea-tree tissue culture
Art and transplantation rooting technology, promote tissue culture technique to be applied to the quick numerous of tea tree breed Upgrading or new varieties early
Educate.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe is described in detail the present invention with reference to foregoing embodiments, it will be understood by those of ordinary skill in the art that：Its according to
Can so modify to the technical solution described in foregoing embodiments, either to which part or all technical characteristic into
Row equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention technology
The scope of scheme.
Claims (5)
- A kind of 1. tea-tree tissue culture method, it is characterised in that comprise the following steps：Step 1, materials and methods, testing of materials material newly sprout the 2nd~3 axillary bud of branch for tea tree spring, pick up from Chinese agriculture Tea place tested by the tealeaves research of institute of section, kind is Dragon Well tea 43.The branch of 2~3 axillary buds of band of collection is carefully cleaned up, is disappeared Poison, is cut into the stem section of the 1cm long of one axillary bud of band, is sprouted in inoculation medium, in new same medium after sprouting It is upper to transfer 3~4 times, that is, Multiple Buds group is obtained, as experiment material；Step 2, experiment condition subculture method are the new sorites that the sorite that grows thickly is cut into each 1~3 bud of band, are inoculated into new On different culture media 3；Step 3, cytokinin concentration are to the influence auxin breeding and grow, totally 6 concentration gradients.Each processing sets 5 A repetition, each 10 materials of repeated inoculation.After inoculation the 30th day, respectively to the bud head number and height of each sorite Up to more than 5cm, it is adapted to the seedling number of transplanting to carry out counting statistics, calculates proliferation rate and planting percent；Step 4, data analysis carry out the one-factor analysis of variance and Multiple range test using statistic software SPSS.
- 2. tea-tree tissue culture method according to claim 1, it is characterised in that step 1 sterilizes：With 0.1% HgCl2Surface disappears Malicious 10min.
- 3. tea-tree tissue culture method according to claim 1, it is characterised in that step 2 cultivation temperature is 25 ± 2 DEG C, during illumination Between for 12 it is small when, 2000~3000lx of light intensity.
- 4. tea-tree tissue culture method according to claim 1, it is characterised in that step 3 auxin uses NAA0.5mg/L, cell Mitogen uses BA.
- 5. tea-tree tissue culture method according to claim 1, it is characterised in that 6 concentration gradients 0.5,1.0,2.0,4.0, 6.0、8.0mg/L。
Priority Applications (1)
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| CN201711391223.5A CN107980637A (en) | 2017-12-21 | 2017-12-21 | Tea-tree tissue culture method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711391223.5A CN107980637A (en) | 2017-12-21 | 2017-12-21 | Tea-tree tissue culture method |
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| CN107980637A true CN107980637A (en) | 2018-05-04 |
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| CN201711391223.5A Withdrawn CN107980637A (en) | 2017-12-21 | 2017-12-21 | Tea-tree tissue culture method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114190251A (en) * | 2021-12-03 | 2022-03-18 | 云南农业大学 | Induction method for promoting proliferation of babysbreath lateral buds |
-
2017
- 2017-12-21 CN CN201711391223.5A patent/CN107980637A/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114190251A (en) * | 2021-12-03 | 2022-03-18 | 云南农业大学 | Induction method for promoting proliferation of babysbreath lateral buds |
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Application publication date: 20180504 |