CN1778168A - Cultivation of peony germinal dislocation - Google Patents
Cultivation of peony germinal dislocation Download PDFInfo
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- CN1778168A CN1778168A CN 200510086592 CN200510086592A CN1778168A CN 1778168 A CN1778168 A CN 1778168A CN 200510086592 CN200510086592 CN 200510086592 CN 200510086592 A CN200510086592 A CN 200510086592A CN 1778168 A CN1778168 A CN 1778168A
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Abstract
An in-vitro culture method for peony embryo bud includes such steps as disinfecting the explant, culturing in starting culture medium for 30-40 days, culturing in secondary culture medium A for 35-45 days, culturing in the secondary culture media B and C, and growing in green house.
Description
Technical field
The present invention relates to method for plant tissue culture, relate in particular to a kind of tree peony embryo cultured in vitro method.
Background technology
Tree peony is the traditional famous flower that originates in China, is unique candidate's's " national flower " prestige at present, not only occupies critical role in the flowers market at home, also is China important export flowers simultaneously.But difficulty (Buchheim JAT et al is all compared in its breeding and breeding, 1992), wherein breeding is still carried out in traditional hybridization mode, but the Paeonia suffruticosa seed plumular axis all has the dormancy phenomenon, only seed germination will 1～2 year under the nature, and germination rate is lower, variation is big, is difficult to obtain seedling (Zilis MR et al, 1976 of neat and consistent; Barton LV et al, 1957).Can break hypocotylar dormancy on the part Paeonia suffruticosa seed though GA3 handles, make sprout time that shortening (Jing Xinming etc., 1995) be arranged slightly, handle most some anamorphosis (BartonLV et al, 1957) of seedling, each kind also need be sought suitable working concentration.Tree peony obtains seed from hybridization, forms seedling to seed germination, and blooming at last selects kind, needs the time (becoming imitative cloud etc., 2005) more than 10 years at least.Therefore, the shortening breeding cycle has become the tree peony breeding and has pressed for one of important topic of solution, and stripped embryo culture technique provides a very valuable approach.Simultaneously, the tree peony kind of China nearly all is in the product population or the selection cross between the product population goes out, and the tree peony distant hybridization breeding has the history in more than 100 year abroad, developed into the main direction of tree peony breed improvement from now on, China will try hard to catch up, just must manage to capture the difficult problem of distant hybridization abortion, on this problem, embryo cultured in vitro technology demonstrates great application prospect.
At present, the rataria culture technique in the plant distant hybridization, overcome and be able to extensive use (Raghavan V.et al2003) aspect stupid stubborn seed dormancy and the researchs such as Developmental Biology and genetics, this technology to the germination rate that improves plant seed, shorten sprout time and in time carry out the rescue of hybrid embryo, quicken breeding process with expand fast numerous all significant.The successful utilization of this technology has also indicated the possibility of tree peony distant hybridization embryo rescue in all kinds of crops, fruit tree, vegetables and multiple flower breeding, and particularly immature rataria is cultivated, and cultivation period is early more favourable more to the hybrid embryo rescue in the hybridization abortion.Cultured in vitro to zygotic embryo, epicotyl and mature seed etc. in tree peony has some reports (Demoise CF et al, 1969, Xie Jingxuan, 1987, Wang Het al, 2001), but planting percent is still all lower, and technology path is still very unripe.The rataria cultured in vitro is not reported so far, more lack the research that this technology is used in breeding.
Summary of the invention
(1), the technical problem that will solve
The object of the present invention is to provide a kind of tree peony rataria cultured in vitro method.
(2), technical scheme
The present invention is a kind of tree peony embryo cultured in vitro method, may further comprise the steps: select explant, be inoculated into after cleaning, the sterilization and start medium culture 30-40 days, move in the subculture I medium and cultivated 35-45 days, subculture is to subculture III medium again, continuous 2-3 time successive transfer culture, the greenhouse hardening.
Tree peony embryo cultured in vitro method of the present invention comprises subculture II put under 15 ± 1 ℃ of left and right sides dark conditions and carried out root induction 15 days that move to subculture I medium culture after 30-40 days, subculture is to subculture III medium.
Tree peony embryo cultured in vitro method of the present invention comprises and the seedling that takes root on the subculture III medium was put into 4 ℃ of refrigerations of refrigerator after 30-90 days, the room temperature hardening.
In the tree peony embryo cultured in vitro method of the present invention, explant is rataria or mature embryo.。
In the tree peony embryo cultured in vitro method of the present invention, starting medium is 1/2MS+BAP 0-0.2mg/L+IAA 0-2.0mg/L+IBA 0-1.0mg/L+GA
30-1.0mg/L+Vc 0 or 100mg/L.
In the tree peony embryo cultured in vitro method of the present invention, subculture I medium is 1/2MS+BAP 0-0.05mg/L+IAA 0-0.1mg/L+IBA 0-0.4mg/L.
In the tree peony embryo cultured in vitro method of the present invention, subculture III medium is 1/2MS+BAP 0-0.2mg/L+IAA 0-0.2mg/L+IBA 0-0.2mg/L+Ac 0.05%.
In the tree peony embryo cultured in vitro method of the present invention, the 1/2MS medium is that macroelement reduces by half, Ca
2+Double.
In the tree peony embryo cultured in vitro method of the present invention, condition of culture: 25 ± 1 ℃ of cultivation temperature, illumination 16～20h/d, light intensity 1600～2000Lx.
The acquisition of explant: gather carpel from healthy and strong plant and bring back the laboratory, flowing water flushing 24h strips out ovule after dish detergent (white cat board) is thoroughly scrubbed cleaning, takes on the super-clean bench and sterilizes.Ovule is earlier with 70% Ethanol Treatment 3min, and aseptic distillation is handled 15-20min with 0.2%NaClo after washing 3 times again, after aseptic distillation is washed 3 times, peels off kind of a skin, breaks endosperm, chooses the embryo inoculation with dissecting needle.
Condition of culture: 25 ± 1 ℃ of cultivation temperature, illumination 16-20h/d, light intensity 1600-2000Lx.
(3), beneficial effect
The present invention provides a kind of effective tree peony rataria cultured in vitro method.It has broken the dormancy of tree peony embryo plumular axis, makes it very fast sprouting, cultivates into seedling in 3 months rapidly, and the rate of development of not only planting percent height, and seedling is higher than seedling far away, very likely blooms in advance in field cultivation.This has just solved in the conventional seed planting seed selection process, and the Paeonia suffruticosa seed sprout time is long, germination rate is low, variation is big, be difficult to obtain the neat and consistent seedling, and a series of problems such as growth of seedling is slow.Therefore, the present invention is applied in the tree peony crossbreeding, can shorten breeding cycle greatly, in distant hybridization, can be used for simultaneously the rescue of hybrid embryo, overcome the hybridization abortion, thereby can improve breeding efficiency, the acceleration breeding process, the input of manpower and materials in the reduction breeding is simultaneously also for studying the tree peony Developmental Biology and carrying out genetics operation, control etc. and created condition.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Medium sees Table 1.
Growth regulator combination in table 1 medium
| Conditioning agent kind mg/l | The medium combination | ||||||||||||
| Start and cultivate (Q) | Subculture I cultivates (J) | Subculture III (J ") | |||||||||||
| Q-1 | Q-2 | Q-3 | Q-4 | Q-5 | Q-6 | J-1 | J-2 | J-3 | J-4 | J-5 | J”-1 | J”-2 | |
| BAP | - | 0.1 | 0.1 | 0.2 | - | 0.2 | - | 0.05 | - | - | - | - | 0.2 |
| IAA | - | - | - | 0.5 | 1.0 | 2.0 | - | 0.1 | - | - | - | - | 0.5 |
| IBA | - | 0.5 | 1.0 | - | - | - | - | - | 0.1 | 0.2 | 0.4 | - | 0.5 |
| GA 3 | - | - | 0.5 | 0.5 | 1.0 | - | - | - | - | - | - | - | 0.5 |
| - | - | - | - | Vc100 | - | Subculture II | Ac 0.05％ | Ac 0.05％ | |||||
Embodiment 1 explant obtains
Adopt to such an extent that the Paeonia papaveracea carpel is brought back the laboratory from healthy and strong plant, flowing water flushing 24h after dish detergent (white cat board) is thoroughly scrubbed cleaning, strips out ovule, takes on the super-clean bench and sterilizes.Ovule is earlier with 70% Ethanol Treatment 3min, and aseptic distillation is handled 20min with 0.2%NaClo after washing 3 times again, after aseptic distillation is washed 3 times, peels off kind of a skin, breaks endosperm, chooses the embryo inoculation with dissecting needle.
Embodiment 2
(1) explant starts medium Q-1 and goes up and cultivated 30 days, and subculture (I) was cultivated 42 days to J-2 fully then, J-2 is gone up true leaf extraction, root grow up seedling subculture (II) in 1.5cm to J " on-1; J-2 is gone up root is shorter than the seedling subculture (II) of 1.5cm to J " on-2;
(2) with J " 4 ℃ of refrigerations of seedling 60 days on-1, the greenhouse hardening; With J " the seedling that takes root on-2 put into 4 ℃ of refrigerator cold-storages 90 days, the greenhouse hardening; The seedling that do not take root discards.Condition of culture: 25 ± 1 ℃ of cultivation temperature, illumination 20h/d, light intensity 1600Lx.
Embodiment 3
(1) explant is divided into 4 parts of difference subcultures (I) to J-1,3,4,5 after cultivating 40 days on the startup medium Q-2,3,4;
(2) seedling on the J-3,4,5 is put into immediately under the dark condition about 15 ± 1 ℃ and carried out root induction 15 days;
(3) and then with seedling again all subcultures (II) to J-1; After 30 days, whole again subcultures (III) are to J "-1;
(4) J " on-1 growth choose the well-grown seedling hardening of root and leaf after 30 days.Condition of culture: 25 ± 1 ℃ of cultivation temperature, illumination 20h/d, light intensity 2000Lx.
Embodiment 4
(1) explant is divided into 6 parts after cultivating 40 days on the startup medium Q-5, and the difference subculture (I) 1/2,1/6,1/6,1/6 on the J-1,3,4,5;
(2) seedling on the J-3,4,5 is put into immediately under the dark condition 15 ± 1 ℃ and carried out root induction 15 days;
(3) and then with seedling all subculture (II) is to J-1 again, and whole again subcultures (III) are to J after 40 days "-1;
(4) J " growth of seedling was chosen the well-grown seedling of root and leaf in hardening after 35 days on-1.Condition of culture: 25 ± 1 ℃ of cultivation temperature, illumination 16h/d, light intensity 1600Lx.
Embodiment 5
(1) explant start cultivate 30 days on the medium Q-6 after, fully subculture (I) to J-1, after 45 days more all subcultures (II) to J " on-1;
(2) at J " growth after 35 days on-1, the seedling that will take root is in 4 ℃ of refrigerations hardening after 30 days; To not take root seedling again subculture (III) to J " on-1, cultivate and chose the seedling hardening of taking root in 35 days.Condition of culture: 25 ± 1 ℃ of cultivation temperature, illumination 16h/d, light intensity 2000Lx.
Table 2 Paeonia papaveracea rataria is cultivated into the seedling statistics
| Kind | Medium | The embryo number | Total number of seedling | Planting percent (%) |
| Paeonia papaveracea | Q-1 | 30 | 28 | 93.33 |
| Q-2 | 30 | 25 | 83.33 | |
| Q-3 | 30 | 22 | 73.33 | |
| Q-4 | 30 | 22 | 73.33 | |
| Q-5 | 40 | 31 | 77.5 | |
| Q-6 | 25 | 21 | 84.0 |
Table 3 subculture (II) is cultivated the every growth indexes analysis of seedling (Duncan method) in back 30 days
| Medium | The embryo number | Go out the true leaf rate | The true number of blade | Handle is long | The rate of putting forth | Stem is long | Rooting rate | Radical | Root is long | Planting percent |
| 1 | 75 | 0.85± 0.08ab | 2.15± 0.08ab | 2.62± 0.63ab | 0.45± 0.16ab | 0.42± 0.16ab | 0.42± 0.05b | 0.97± 0.20abc | 2.59± 0.46ab | 0.41± 0.05A |
| 2 | 41 | 0.93± 0.03a | 1.49± 0.16b | 0.31± 0.06b | 0.00± 0.00b | 0.00± 0.00b | 0.69± 0.05a | 0.88± 0.14bc | 3.41± 0.20ab | 0.59± 0.04A |
| 3 | 44 | 0.95± 0.03a | 2.38± 0.28a | 3.24± 0.98a | 0.69± 0.20a | 0.50± 0.20a | 0.61± 0.07a | 1.49± 0.45ab | 4.32± 1.47a | 0.59± 0.09A |
| 4 | 35 | 0.98± 0.03a | 2.18± 0.26ab | 2.51± 0.83ab | 0.59± 0.15a | 0.36± 0.12ab | 0.63± 0.07a | 1.77± 0.35a | 3.29± 0.76ab | 0.56± 0.08A |
| 5 | 45 | 0.73± 0.04b | 1.77± 0.34ab | 2.65± 1.17ab | 0.33± 0.15ab | 0.30± 0.15ab | 0.27± 0.04b | 0.57± 0.14c | 1.07± 0.22b | 0.15± 0.06B |
| Amount to | 240 | 0.89± 0.03 | 2.00± 0.11 | 2.29± 0.38 | 0.41± 0.08 | 0.32± 0.07 | 0.52± 0.04 | 1.12± 0.14 | 2.91± 0.37 | 0.46± 0.04 |
Claims (9)
1, a kind of tree peony embryo cultured in vitro method, it is characterized in that may further comprise the steps: select explant, be inoculated into after cleaning, the sterilization and start medium culture 30-40 days, move in the subculture I medium and cultivated 35-45 days, subculture is to subculture III medium again, continuous 2-3 time successive transfer culture, the greenhouse hardening.
2, the method for claim 1 is characterized in that also comprising subculture II put under 15 ± 1 ℃ of dark conditions and carried out root induction 15 days that move to subculture I medium culture after 30-40 days, subculture is to subculture III medium.
3, the method for claim 1 is characterized in that also comprising and the seedling that takes root on the subculture III medium was put into 4 ℃ of refrigerations of refrigerator after 30-90 days, the room temperature hardening.
4,, it is characterized in that described explant is rataria or mature embryo as the arbitrary described method of claim 1-3.
5, the method for claim 1 is characterized in that described startup medium is 1/2MS+BAP 0-0.2mg/L+IAA 0-2.0mg/L+IBA 0-1.0mg/L+GA
30-1.0mg/L+Vc 0 or 100mg/L.
6, the method for claim 1 is characterized in that described subculture I medium is 1/2MS+BAP 0-0.05mg/L+IAA 0-0.1mg/L+IBA 0-0.4mg/L.
7, the method for claim 1 is characterized in that described subculture III medium is 1/2MS+BAP 0-0.2mg/L+IAA 0-0.5mg/L+IBA 0-0.5mg/L+GA
30-0.5mg/L+Ac 0.05%.
8,, it is characterized in that described 1/2MS medium is that MS medium macroelement reduces by half, Ca as the arbitrary described method of claim 5-7
2+Double.
9, the method for claim 1 is characterized in that condition of culture: 25 ± 1 ℃ of cultivation temperature, illumination 16-20h/d, light intensity 1600-2000Lx.
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| CNB2005100865924A CN100403882C (en) | 2005-10-12 | 2005-10-12 | Cultivation of peony germinal dislocation |
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|---|---|---|---|
| CNB2005100865924A CN100403882C (en) | 2005-10-12 | 2005-10-12 | Cultivation of peony germinal dislocation |
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| CN1778168A true CN1778168A (en) | 2006-05-31 |
| CN100403882C CN100403882C (en) | 2008-07-23 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102599051A (en) * | 2011-01-20 | 2012-07-25 | 河南省农业科学院 | Sesame callus induction and differentiation and plant regeneration method |
| CN102144563B (en) * | 2009-05-11 | 2012-09-26 | 中国林业科学研究院林业研究所 | Method for inducing calluses of peony anther |
| CN102934611A (en) * | 2012-10-23 | 2013-02-20 | 河南科技大学 | Method for collecting embryo from paeonia suffruticosa seed and for inoculation |
| CN110476807A (en) * | 2019-07-31 | 2019-11-22 | 河南科技大学 | A method of establishing oil tree peony ' Feng Dan ' Mature Embryos Among sterile culture system |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1600082A (en) * | 2004-09-30 | 2005-03-30 | 北京林业大学 | Tissue culture technique for peony |
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2005
- 2005-10-12 CN CNB2005100865924A patent/CN100403882C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102144563B (en) * | 2009-05-11 | 2012-09-26 | 中国林业科学研究院林业研究所 | Method for inducing calluses of peony anther |
| CN102599051A (en) * | 2011-01-20 | 2012-07-25 | 河南省农业科学院 | Sesame callus induction and differentiation and plant regeneration method |
| CN102599051B (en) * | 2011-01-20 | 2015-04-22 | 河南省农业科学院 | Sesame callus induction and differentiation and plant regeneration method |
| CN102934611A (en) * | 2012-10-23 | 2013-02-20 | 河南科技大学 | Method for collecting embryo from paeonia suffruticosa seed and for inoculation |
| CN102934611B (en) * | 2012-10-23 | 2014-02-05 | 河南科技大学 | Method for collecting embryo from paeonia suffruticosa seed and for inoculation |
| CN110476807A (en) * | 2019-07-31 | 2019-11-22 | 河南科技大学 | A method of establishing oil tree peony ' Feng Dan ' Mature Embryos Among sterile culture system |
| CN110476807B (en) * | 2019-07-31 | 2021-06-08 | 河南科技大学 | Method for establishing sterile culture system of mature seed embryo of peony 'paeonia ostii' for oil |
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| CN100403882C (en) | 2008-07-23 |
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