CN104285787A - Method for inducing peony receptacle callus tissues and differentiating adventitious buds - Google Patents
Method for inducing peony receptacle callus tissues and differentiating adventitious buds Download PDFInfo
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- CN104285787A CN104285787A CN201410478283.0A CN201410478283A CN104285787A CN 104285787 A CN104285787 A CN 104285787A CN 201410478283 A CN201410478283 A CN 201410478283A CN 104285787 A CN104285787 A CN 104285787A
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Abstract
The invention relates to a method for inducing peony receptacle callus tissues and differentiating adventitious buds. The method comprises the following steps: selecting receptacle explants, pre-treating and normally disinfecting; putting sterile receptacles on a start culture medium for carrying out induction culture, firstly culturing for one week in a dark environment, then transferring, culturing in a weak light environment for 30-50 days, and generating an initial callus tissue; transferring the initial callus tissue to a multiplication culture medium for carrying out multiplication culture; and transferring the initial or proliferated callus tissue to an adventitious bud culture medium for carrying out differentiation culture of the adventitious buds, culturing in the weak light environment for 4 days firstly, then transferring the cultured buds on the adventitious bud culture medium in the normal environment for culturing, and differentiating so as to obtain the adventitious buds. By virtue of the method, a large quantity of receptacle callus tissues and adventitious buds can be quickly prepared; the induction rates of the receptacle callus tissues and the adventitious buds respectively reaches 100% and 91%, so that the technical problems that the peony receptacle callus tissues are low in induction rate and poor in induction quality and the adventitious buds are difficult to differentiate can be overcome; a good foundation is laid for variety improvement and biotechnological breeding of peony.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to a kind of tree peony holder callus induction and differentiation indefinite bud method.
Background technology
Tree peony (Paeonia suffruticosa) is world-renowned flowers, have the laudatory title of " aromatic ", " king in spending ", it spent greatly, shape is beautiful, look gorgeous, aromatic, praise alleged by successive dynasties people, there is very high viewing and admiring and medical value, in Landscape Application, occupy critical positions.Some important ornamental plantss as on orchid, tissue culture technique has started application, and large-scale commercial production, utilize transgenic technology to import genes of interest simultaneously in the ornamental plantss such as petunia and cultivated transgenosis new lines, this is utilize the special tree peony new lines of transgenic technology acquisition strong stress resistance, pattern to provide possibility.The research mushroom development at home and abroad that peony tissue is cultivated since the eighties in last century, but fail so far to obtain the breakthrough of essence.Research shows that tree peony plantlet in vitro forming feature is mainly based on callus dedifferentiation, and the quality of callus is the basis of dedifferentiation.Owing to there is higher aldehydes matter in tree peony body, there is the more difficult induction of callus, inductivity is low, the problems such as the callus quality difference of formation, cause tree peony callus to be induced with the progress of regeneration slow.So far, with the embryo of tree peony, cotyledon, blade, petiole, petal, for explant carries out the induction of callus, there are reports, but in in-vitro inducing tree peony holder callus and differentiation indefinite bud, there is not yet progress, therefore, take holder as explant, improve tree peony callus inductivity, induction quality and Differentiation ration of adventitious buds, the development of tree peony industry will be promoted to a great extent, bring huge economic benefit.
Summary of the invention
The present invention is to overcome the deficiencies in the prior art, set up a kind of tree peony holder callus induction and differentiation indefinite bud method, improving the inductivity of tree peony callus, induction quality and Differentiation ration of adventitious buds, providing a kind of new technology platform for carrying out the research of tree peony biotechnology breeding in a deep going way.
The technical solution adopted for the present invention to solve the technical problems is: first, chooses applicable tree peony holder explant, carries out pretreatment and routine disinfection; Then, explant is carried out to the induction of initial callus, obtain initial callus; Again, Multiplying culture is carried out to initial callus, make callus fast breeding; Finally, differentiation adventitious buds induction is carried out to the callus of propagation, has reached the object of regeneration.
Tree peony holder callus induction and differentiation indefinite bud method of the present invention comprises the steps:
1) the choosing and sterilization of holder explant: choose the bud that diameter is 0.5 ~ 0.6cm, after 4 DEG C of refrigerator pretreatment 5 ~ 6d, routine disinfection, strips aseptic holder, and holder diameter is 0.3 ~ 0.5cm;
2) Primary culture: by aseptic holder light culture one week on Primary culture base, cultivates 30 ~ 50d under then going to the low light level, produces initial callus; Condition of culture under the low light level is: cultivation temperature 25 ± 1 DEG C, illumination 14h/d, low light level intensity of illumination 800 ~ 1000Lx; Described Primary culture base is MS+2,4-D 0.5 ~ 1.0mg/L+KT 2.0 ~ 2.5mg/L+ glutamine 0.5g/L+ serine 0.1g/L;
3) Multiplying culture: callus initial incubation obtained, is transferred in proliferated culture medium and carries out Multiplying culture; Condition of culture is: cultivation temperature 25 ± 1 DEG C, illumination 14h/d, intensity of illumination 1500Lx; Described proliferated culture medium is MS+2,4-D 0.5 ~ 1.0mg/L+KT 1.5 ~ 2.0mg/L+NAA 1.5 ~ 2.0mg/L+ glutamine 0.5g/L+ serine 0.1g/L;
4) differentiation adventitious buds is cultivated: callus initial incubation or Multiplying culture obtained, and is transferred to low light environment on differentiation adventitious buds medium and cultivates 4d, cultivate under being then placed in home; Described differentiation adventitious buds medium is MS+ZT2.0 ~ 3.0mg/L+6-BA 10.0 ~ 15.0mg/L+ lactoalbumin hydrolysate 10.0mg/L.
As a kind of preferred version, described holder diameter is 0.3 ~ 0.5cm, described step 2) in, described Primary culture base is MS+2,4-D 0.8mg/L+KT 2.3mg/L+ glutamine 0.5g/L+ serine 0.1g/L; Described initial callus culture environment is the low light level: intensity of illumination 800 ~ 1000Lx.
As a kind of preferred version, described step 3) in, described proliferated culture medium is MS+2,4-D 0.8mg/L+KT 1.5mg/L+NAA 2.0mg/L+ glutamine 0.5g/L+ serine 0.1g/L; Described step 4) in, described differentiation adventitious buds medium is MS+ZT 2.5mg/L+6-BA 12.0mg/L+ lactoalbumin hydrolysate 10.0mg/L.
As a kind of preferred version, described step 4) in, described low light environment condition of culture is: cultivation temperature 15 ± 1 DEG C, illumination 14h/d, intensity of illumination 500 ~ 600Lx; Described step 4) in, described home condition of culture is: cultivation temperature 20 ± 1 DEG C, illumination 14h/d, intensity of illumination 2000Lx.
Above-mentioned various medium is all minimal medium with MS, and containing agar 5.4g/L, sucrose 30g/L, before medium sterilization, pH is 5.8 ~ 6.0.
Beneficial effect of the present invention is: adopt the method for the invention, a large amount of holder callus can be obtained fast, inductivity is up to 100%, the callus of induction adopts the differentiation-inducing indefinite bud of the method for the invention, differentiation rate reaches 91%, thus captured tree peony callus inductivity low, induce of poor quality and Differentiation ration of adventitious buds this technical barrier low, for Varieties of Peony improvement and biotechnology breeding have laid a good foundation.
Accompanying drawing explanation
Fig. 1 is the initial callus that tree peony holder obtains on Primary culture base; Fig. 2 is the indefinite bud that callus obtains on differentiation adventitious buds medium.
Embodiment
Following examples are used for the present invention is described, but are not used for limiting the scope of the invention.
In following embodiment, experiment material is tree peony " oolong holds in both hands victory " and " pearl and jade necklace mound " ', and the experimental result of two kinds of Varieties of Peonies is consistent.Minimal medium is MS, and containing agar 5.4g/L, sucrose 30g/L, before medium sterilization, pH is 5.8 ~ 6.0.Condition of culture is (except being otherwise noted): cultivation temperature 25 ± 1 DEG C, illumination 14h/d, intensity of illumination 2000Lx.
Choosing and sterilization of embodiment 1 holder explant
In 3 ~ April, choose the tree peony bud that field diameter is 0.5 ~ 0.6cm, a bud just ready to burst, 4 DEG C of refrigerator pretreatment 6d, running water 1 ~ 2h, bud is put into 70% ethanol 30s by superclean bench, then proceeds to 8min in 0.1%HgCl2 solution, aseptic water washing 3 ~ 4 times, peel off calyx, petal, androecium and gynoecium, be cut into 4 pieces for subsequent use.
Embodiment 2: variable concentrations 2,4-D and the impact of KT combination on callus induction
The aseptic holder stripping and slicing of tree peony is inoculated into containing variable concentrations 2,4-D (0.5,0.8,1.0mg/L) with KT (2,2.3,2.5mg/L) first light culture one week on the MS medium that combines, under then going to the low light level, (800 ~ 1000Lx) cultivates.The impact of exogenous hormone on callus induction is compared after 30d.Its result is as shown in table 1: there were significant differences for the inducing effect of each Primary culture base, medium MS+2, the inductivity of 4-D 0.8mg/L+KT 2.3mg/L is the highest, induce best in quality, callus is yellow green, discrete particles shape (as shown in Figure 1), and increase gradually with the prolongation of incubation time, can 50d be cultured to.Therefore, the suitableeest Primary culture base of tree peony holder callus induction is MS+2,4-D 0.8mg/L+KT 2.3mg/L, and applicable induction time is cultivate 30 ~ 50d under the low light level.
Table 1 variable concentrations 2,4-D, KT are on the impact of callus induction
Embodiment 3 different explants is on the impact of callus induction
In Plant Tissue Breeding, the explant that evoked callus is conventional is blade, holder, petiole, stem section.The present embodiment explant used is: blade, takes from the 3rd, plant top leaf; Petiole, takes from the 2nd, plant top leaf petiole; Holder, takes from the bud of little finger size; Petal, takes from the plant top full-bloom stage flower petal that a bud just ready to burst.Above-mentioned explant is inoculated in the suitableeest Primary culture base (MS+2 respectively, 4-D 0.8mg/L+KT 2.3mg/L+ glutamine 0.5g/L+ serine 0.1g/L) in carry out Fiber differentiation, first light culture one week, then under going to the low light level, (800 ~ 1000Lx) cultivates 30d, the callus induction result of different explants is as shown in table 2: when explant is petiole, petiole place expands and occurs callus, and inductivity is 75%, and callus is little; When explant is blade, petal, callus induction rate is respectively 70%, 80%, and callus is less; When explant is holder, callus induction rate is 100%, and callus is large and density is suitable for.Therefore, holder is the best explant of tree peony ' oolong holds in both hands victory ' and ' pearl and jade necklace mound ' callus induction.
The different tree peony explant of table 2 is on the impact of callus induction
| Explant | Petiole | Blade | Holder | Petal |
| Callus induction rate, size | 75%, little | 70%, less | 100%, greatly | 80%, less |
The pretreatment of embodiment 4 low light level is on the impact of Calli Differentiation indefinite bud
In Plant Tissue Breeding, Calli Differentiation indefinite bud is the key that plant regenerates indirectly.Be transferred in the yellow green of the suitableeest Primary culture base induction gained, discrete particles shape initial callus on differentiation adventitious buds medium (MS+ZT 2.5mg/L+6-BA 13mg/L+ lactoalbumin hydrolysate 10mg/L), adopt 2 kinds of culture scheme: one directly cultivates 30d in home; One is that first low light environment cultivates 4d, then in home, cultivates 26d; Described low light environment condition of culture is: cultivation temperature 15 ± 1 DEG C, illumination 14h/d, intensity of illumination 500 ~ 600Lx; Described home condition of culture is: cultivation temperature 20 ± 1 DEG C, illumination 14h/d, intensity of illumination 2000Lx.Its result is as shown in table 3: 2 kinds of culture scheme adventitious bud induction frequencies are widely different, and after the first low light level, the normal inductivity cultivated is the highest, induces best in quality.
The process of table 3 varying environment is on the impact of callus induction
| Culture scheme | Home | Normal illumination environment after the first low light level |
| Callus induction rate, size | 63%, quality is general | 91%, quality is good |
Claims (4)
1. a tree peony holder callus induction and differentiation indefinite bud method, is characterized in that, comprise the steps:
1) the choosing and sterilization of holder explant: choose the bud that diameter is 0.5 ~ 0.6cm, after 4 DEG C of refrigerator pretreatment 5 ~ 6d, routine disinfection, strips aseptic holder, and holder diameter is 0.3 ~ 0.5cm;
2) Primary culture: by aseptic holder light culture one week on Primary culture base, cultivates 30 ~ 50d under then going to the low light level, produces initial callus; Condition of culture under the low light level is: cultivation temperature 25 ± 1 DEG C, illumination 14h/d, low light level intensity of illumination 800 ~ 1000Lx; Described Primary culture base is MS+2,4-D 0.5 ~ 1.0mg/L+KT 2.0 ~ 2.5mg/L+ glutamine 0.5g/L+ serine 0.1g/L;
3) Multiplying culture: callus initial incubation obtained, is transferred in proliferated culture medium and carries out Multiplying culture; Condition of culture is: cultivation temperature 25 ± 1 DEG C, illumination 14h/d, intensity of illumination 1500Lx; Described proliferated culture medium is MS+2,4-D 0.5 ~ 1.0mg/L+KT 1.5 ~ 2.0mg/L+NAA 1.5 ~ 2.0mg/L+ glutamine 0.5g/L+ serine 0.1g/L;
4) differentiation adventitious buds is cultivated: callus initial incubation or Multiplying culture obtained, and is transferred to low light environment on differentiation adventitious buds medium and cultivates 4d, cultivate under being then placed in home; Described differentiation adventitious buds medium is MS+ZT2.0 ~ 3.0mg/L+6-BA 10.0 ~ 15.0mg/L+ lactoalbumin hydrolysate 10.0mg/L.
2. the method for claim 1, is characterized in that, described holder diameter is 0.3 ~ 0.5cm, described step 2) in, described Primary culture base is MS+2,4-D 0.8mg/L+KT 2.3mg/L+ glutamine 0.5g/L+ serine 0.1g/L; Described initial callus culture environment is the low light level: intensity of illumination 800 ~ 1000Lx.
3. the method for claim 1, is characterized in that, described step 3) in, described proliferated culture medium is MS+2,4-D0.8mg/L+KT 1.5mg/L+NAA 2.0mg/L+ glutamine 0.5g/L+ serine 0.1g/L; Described step 4) in, described differentiation adventitious buds medium is MS+ZT 2.5mg/L+6-BA 12.0mg/L+ lactoalbumin hydrolysate 10.0mg/L.
4. the method for claim 1, is characterized in that, described step 4) in, described low light environment condition of culture is: cultivation temperature 15 ± 1 DEG C, illumination 14h/d, intensity of illumination 500 ~ 600Lx; Described step 4) in, described home condition of culture is: cultivation temperature 20 ± 1 DEG C, illumination 14h/d, intensity of illumination 2000Lx.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104604690A (en) * | 2015-02-10 | 2015-05-13 | 重庆文理学院 | Oil peony tissue culture method and improved basic culture medium |
| CN107372125A (en) * | 2017-09-14 | 2017-11-24 | 河南科技学院 | A kind of indefinite bud method of rhizome of Chinese monkshood holder callus induction and differentiation |
| CN116267612A (en) * | 2023-03-16 | 2023-06-23 | 中国热带农业科学院热带作物品种资源研究所 | Tissue culture propagation method of hippeastrum |
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| CN102577968A (en) * | 2012-03-06 | 2012-07-18 | 河南科技大学 | Adventitious bud induction method for tree peony |
| CN103355165A (en) * | 2012-04-09 | 2013-10-23 | 上海植物园 | Culture method of peony embryonic callus as well as culture medium |
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| CN102577968A (en) * | 2012-03-06 | 2012-07-18 | 河南科技大学 | Adventitious bud induction method for tree peony |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104604690A (en) * | 2015-02-10 | 2015-05-13 | 重庆文理学院 | Oil peony tissue culture method and improved basic culture medium |
| CN107372125A (en) * | 2017-09-14 | 2017-11-24 | 河南科技学院 | A kind of indefinite bud method of rhizome of Chinese monkshood holder callus induction and differentiation |
| CN107372125B (en) * | 2017-09-14 | 2019-11-05 | 河南科技学院 | A kind of indefinite bud method of rhizome of Chinese monkshood holder callus induction and differentiation |
| CN116267612A (en) * | 2023-03-16 | 2023-06-23 | 中国热带农业科学院热带作物品种资源研究所 | Tissue culture propagation method of hippeastrum |
| CN116267612B (en) * | 2023-03-16 | 2024-01-09 | 中国热带农业科学院热带作物品种资源研究所 | Tissue culture propagation method of hippeastrum |
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