CN1675992A - Chinese herbeceous peony invitro tissue culture method - Google Patents
Chinese herbeceous peony invitro tissue culture method Download PDFInfo
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- CN1675992A CN1675992A CN 200510039010 CN200510039010A CN1675992A CN 1675992 A CN1675992 A CN 1675992A CN 200510039010 CN200510039010 CN 200510039010 CN 200510039010 A CN200510039010 A CN 200510039010A CN 1675992 A CN1675992 A CN 1675992A
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Abstract
The isolated tissue culture method of Chinese herbaceous peony includes the following steps: shearing and using Chinese herbaceous peony shoot tip with bud scale, cleaning and removing its water content, disinfecting in ethyl alcohol, removing bud scale and cutting shoot tip, disinfecting by using NaHClO solution, washing with sterile water, inoculating in 1/2 MS culture medium containing Vc50mg.L to the power-1 with pH5.6, its culture condition is as follows: temperature is 25 deg.C minus or plus 1 deg.C, light duration is 12-14 h/d and intensity of illumination is 2000Lx minus or plus 100Lx.
Description
Technical field
The present invention relates to the tissue culture technique field of Chinese herbaceous peony.
Background technology
Chinese herbaceous peony (Paeonia lactiflora Pall.) is the perennial perennial root herbaceous plant of Paeoniaceae (Paeoniaceae) Paeonia (Paeonia), also be China famous view and admire, medicinal plant, cultivation history is long, large flower and brilliant color, give off a strong fragrance, regarded as " flower phase ".Through artificial cultivation seed selection in several thousand, the breeding of Chinese herbaceous peony improved seeds was mainly based on asexual offshoot, plant division in 3～5 years once, the breeding cycle is long, reproduction coefficient is low, brings difficulty for the industrialized development of Chinese herbaceous peony.
Summary of the invention
The object of the invention is intended to set up Chinese herbaceous peony tissue culture regenerative system, for the fast asexual propagation of Chinese herbaceous peony improved seeds provides a kind of efficient feasible technological approaches.
Clip band chlamydia-chlamydia stem apex of the present invention is cleaned the back and is inhaled the branch that anhydrates, and places alcohol to sterilize, and removes perula, cuts stem apex, uses the NaHClO solution disinfection, after aseptic water washing is clean, is seeded to and adds V
C50mgL
-1, pH5.6 the 1/2MS medium in.Culturing room's condition is: temperature 25 ℃ ± 1, light application time 12～14h/d, intensity of illumination 2000lx ± 100lx.
The present invention provides a kind of efficient feasible technological approaches for the fast asexual propagation of Chinese herbaceous peony improved seeds.
It is 70% alcohol that smart concentration is spilt in above-mentioned sterilization, and disinfecting time is 10～15s.
The concentration of above-mentioned NaHClO solution is 0.1%, and disinfecting time is 10～15min.
Towards the Xian Shui sterile water, be seeded in the medium after washing 3～5 times.
Bud proliferated culture medium of the present invention is with 1/2MS+0.5BAmgL
-1+ 1.0GAmgL
-1For preferably.
Wherein, mineral salt macroelement weight is 50% of MS medium normal contents in the 1/2MS medium.
The time of drawing materials of Shoot Tip Culture is in 10～November, or in late September the turn up stem eye put refrigerate under 4 ℃ of conditions 5～6 week the back inoculated and cultured.
Adopt the Chinese herbaceous peony stem apex to carry out in vitro tissue and cultivate, young shoot propagation was promptly arranged after 35～40 days, can realize the quick breeding of improved seeds, the industrialized development of cultivating to Chinese herbaceous peony brings deciding the issue of the battle property foundation.
Instantiation:
1 materials and methods
The Shoot Tip Culture material is selected from polyphyll type kind " big rich and honour (P.lactiflora ' Da Fu Gui ') " sleeping bud.
In planting the Chinese herbaceous peony sleeping bud 10-11 month for for the examination material with getting, or in late September the turn up stem eye put 4 ℃ of refrigerations and get the bud cultured in vitro after 5 weeks of preliminary treatment.Clip band perula stem apex cleans up in liquid detergent solution with hairbrush, place flowing water flushing 30min after, blotting paper is inhaled the branch that anhydrates, and changes the 15s that sterilizes in 70% alcohol over to; Remove perula behind the aseptic water washing, cut stem apex, with 0.1%NaHClO solution disinfection 15min, aseptic water washing 3～5 times is seeded to 1/2MS+0.5BAmgL
-1+ 1.0GAmgL
-1In the medium; Wherein, in the 1/2MS medium mineral salt macroelement weight be MS medium normal contents 50%, sucrose 3%, agar 0.8%, V
C50mgL
-1, pH5.6.Culturing room's condition is: temperature 25 ℃ ± 1, light application time 14h/d, intensity of illumination 2000lx.
2 results and analysis
2.1 different sample times are to the influence of Shoot Tip Culture
Table 1 influence of period of drawing materials to Shoot Tip Culture
| Draw materials the time | The inoculation number | Pollute number | Survival rate % | Differentiation rate % | Draw materials the time | The inoculation number | Pollute number | Survival rate % | Differentiation rate % |
| Outdoor cropping | 4 ℃ of refrigerations | ||||||||
| September | ??30 | ??13 | ??56.7 | ??64.7 | 4 weeks | ??30 | ??13 | ??56.7 | ??64.7 |
| October | ??30 | ??8 | ??73.3 | ??86.3 | 5 weeks | ??30 | ??8 | ??73.3 | ??86.3 |
| November | ??30 | ??10 | ??66.7 | ??85.0 | 6 weeks | ??30 | ??10 | ??66.7 | ??85.0 |
| December | ??30 | ??16 | ??46.7 | ??42.9 | 7 weeks | ??30 | ??16 | ??46.7 | ??42.9 |
| January | ??30 | ??21 | ??30.0 | ??33.3 | 8 weeks | ??30 | ??21 | ??30.0 | ??33.3 |
| February | ??30 | ??30 | ??0 | ??0 | 9 weeks | ??30 | ??30 | ??0 | ??0 |
*Medium: 1/2MS+0.5BAmgL
-1+ 1.0GAmgL
-1
As can be seen from Table 1, outdoor cropping kind suitable draw materials the time 10～November or in late September the turn up stem eye put 4 ℃ of refrigerations and handled for 5～6 weeks, the survival rate and the differentiation rate of cultured in vitro are the highest.Inoculation this moment, sleeping bud is sprouted and is grown rapidly; Or in late September the turn up stem eye put 4 ℃ of refrigerations and handle 5～6 weeks, the test-tube plantlet stalwartness of differentiation.The time of drawing materials handled in 8～9 weeks of February or 4 ℃ refrigeration, and the perula of most of material is split, bud extends, caused trying material sterilization difficulty, pollution rate is high.Thoroughly do not remove as yet because of the dormancy of bud At All Other Times, there is inhibiting substances in inside, poor growth, and survival rate and differentiation rate are all lower.
2.2 different medium are to the influence of bud propagation
The different medium of table 2 are to the influence of Shoot Tip Culture
| Medium 1/2MS | Successive transfer culture number of times (bud of growing thickly breaks up required fate) | The bud quantity of growing thickly | Leaf length | Blade quantity |
| ????0.5BAmg·L -1+1.0GAmg·L -1 | ????1(35) | ????3.2 | ????3.1 | ????5.2 |
| ????2(32) | ????2.8 | ????2.9 | ????4.7 | |
| ????3(36) | ????2.1 | ????2.4 | ????4.1 | |
| ????1.5BAmg·L -1+0.5KTmg·L -1 | ????1(40) | ????2.0 | ????4.5 | ????4.0 |
| ????2(38) | ????1.8 | ????4.2 | ????3.8 | |
| ????3(39) | ????1.9 | ????4.1 | ????3.6 | |
| ????1.0BAmg·L -1+0.5GAmg·L -1 | ????1(36) | ????2.7 | ????2.8 | ????4.7 |
| ????2(38) | ????2.6 | ????2.6 | ????4.2 | |
| ????3(34) | ????1.8 | ????2.4 | ????3.7 |
*The time of drawing materials is January, and every winding kind is several 30, assembly average.
Result of the test (table 2) shows that the medium bud differentiation effect of BA and GA combination is better than BA and KT combination.1.5BAmgL
-1+ 0.5KTmgL
-1The bud of growing thickly in the medium, blade quantity are less, and the seedling growth is higher.In adding the medium of BA, GA, the bud of growing thickly, blade quantity are more, and seedling is strong.In the successive transfer culture, cut the simple bud inoculation, base portion forms sprouting behind 35～40d; But along with algebraically increases, the bud of growing thickly, number of sheets amount reduce gradually, and a little less than the seedling growth was changeed, part bud point, leaf were withered, the albefaction seedling occurs; Substantially, transfer glass seedling, albefaction seedling to the 4th generation, and brown stain is serious.The bud enrichment culture is with 1/2MS+0.5BAmgL
-1+ 1.0GAmgL
-1Be optimal medium.
3 sum up
The draw materials culture effect of time of Chinese herbaceous peony stem apex cultured in vitro, difference is different, with 10-11 month or in late September the turn up stem eye put 4 ℃ of refrigerations to handle for 5～6 weeks be the best.The too early dormancy of drawing materials is not removed as yet, and it is slower to grow, a little less than the seedling; After late then bud grow sterilization difficulty.Clump bud enrichment culture cycle 35～40d, optimum multiplication medium: 1/2MS+0.5BAmgL
-1+ 1.0GAmgL
-1
Full name of above-mentioned MS medium is: Murashige and Skoon (1962) medium
The chinesization formal name used at school of BA is: the 6-benzylaminopurine
The chinesization formal name used at school of GA is: gibberellin
The chinesization formal name used at school of KT is: 6-furfuryl group aminopurine.
Claims (6)
1, Chinese herbeceous peony invitro tissue culture method is characterized in that clip band chlamydia-chlamydia stem apex, cleans the back and inhales the branch that anhydrates, and places alcohol to sterilize, and removes perula, cuts stem apex, uses the NaHClO solution disinfection, after aseptic water washing is clean, is seeded to and adds Vc50mgL
-1, pH5.6 the 1/2MS medium in, culturing room's condition is: 25 ℃ ± 1 ℃ of temperature, light application time 12～14h/d, intensity of illumination 2000lx ± 100lx.
2, according to the described Chinese herbeceous peony invitro tissue culture method of claim 1, it is characterized in that above-mentioned rubbing alcohol concentration is 70% alcohol, disinfecting time is 10～15s.
3, according to the described Chinese herbeceous peony invitro tissue culture method of claim 1, the concentration that it is characterized in that above-mentioned NaHClO solution is 0.1%, and disinfecting time is 10～15min.
4,, it is characterized in that being seeded in the medium with behind the aseptic water washing 3～5 times according to the described Chinese herbeceous peony invitro tissue culture method of claim 1.
5,, it is characterized in that also containing in the medium 0.5BAmgL according to the described Chinese herbeceous peony invitro tissue culture method of claim 1
-1And 1.0GAmgL
-1
6, according to claim 1 or 2 or 3 or 4 or 5 described Chinese herbeceous peony invitro tissue culture methods, the time of drawing materials that it is characterized in that Shoot Tip Culture is in 10～November, or in late September the turn up stem eye put refrigerate under 4 ℃ of conditions 5～6 week the back inoculated and cultured.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102440192A (en) * | 2011-10-14 | 2012-05-09 | 扬州大学 | Culture media for peony high frequency embryogenic callus differentiation, and culture method thereof |
| CN102124946B (en) * | 2010-01-27 | 2012-11-21 | 北京林业大学 | Method for tissue culture of paeonia lactiflora |
| CN103651123A (en) * | 2013-11-20 | 2014-03-26 | 宁波立华制药有限公司 | Method for producing paeonia lactiflora double glycosides |
| CN103733995A (en) * | 2013-12-20 | 2014-04-23 | 北京林业大学 | Peony callus induction method |
| CN104304029A (en) * | 2014-11-02 | 2015-01-28 | 杨业容 | Peony tissue culture and rapid propagation method |
| CN108184598A (en) * | 2018-01-10 | 2018-06-22 | 中国农业科学院蔬菜花卉研究所 | A kind of Chinese herbaceous peony propagation method |
| CN109496766A (en) * | 2019-01-02 | 2019-03-22 | 山东省林业科学研究院 | A set of method for improving Chinese herbaceous peony reproductive efficiency |
| CN113133410A (en) * | 2021-06-04 | 2021-07-20 | 河南省农业科学院园艺研究所 | Method for breeding Chinese herbaceous peony by tissue culture technology |
| CN116918701A (en) * | 2020-09-30 | 2023-10-24 | 伽蓝（集团）股份有限公司 | Callus culture medium and extract of rosa tenuifolia, preparation method and application |
-
2005
- 2005-04-19 CN CN 200510039010 patent/CN1675992A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102124946B (en) * | 2010-01-27 | 2012-11-21 | 北京林业大学 | Method for tissue culture of paeonia lactiflora |
| CN102440192A (en) * | 2011-10-14 | 2012-05-09 | 扬州大学 | Culture media for peony high frequency embryogenic callus differentiation, and culture method thereof |
| CN103651123A (en) * | 2013-11-20 | 2014-03-26 | 宁波立华制药有限公司 | Method for producing paeonia lactiflora double glycosides |
| CN103651123B (en) * | 2013-11-20 | 2015-08-12 | 宁波立华制药有限公司 | A kind of method of producing the two glycosides of Chinese herbaceous peony |
| CN103733995A (en) * | 2013-12-20 | 2014-04-23 | 北京林业大学 | Peony callus induction method |
| CN103733995B (en) * | 2013-12-20 | 2015-07-22 | 北京林业大学 | Peony callus induction method |
| CN104304029A (en) * | 2014-11-02 | 2015-01-28 | 杨业容 | Peony tissue culture and rapid propagation method |
| CN108184598A (en) * | 2018-01-10 | 2018-06-22 | 中国农业科学院蔬菜花卉研究所 | A kind of Chinese herbaceous peony propagation method |
| CN109496766A (en) * | 2019-01-02 | 2019-03-22 | 山东省林业科学研究院 | A set of method for improving Chinese herbaceous peony reproductive efficiency |
| CN109496766B (en) * | 2019-01-02 | 2021-07-09 | 山东省林业科学研究院 | Method for improving peony propagation efficiency |
| CN116918701A (en) * | 2020-09-30 | 2023-10-24 | 伽蓝（集团）股份有限公司 | Callus culture medium and extract of rosa tenuifolia, preparation method and application |
| CN113133410A (en) * | 2021-06-04 | 2021-07-20 | 河南省农业科学院园艺研究所 | Method for breeding Chinese herbaceous peony by tissue culture technology |
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