CN109430056A - A method of induction alum root adventitious shoot regeneration - Google Patents
A method of induction alum root adventitious shoot regeneration Download PDFInfo
- Publication number
- CN109430056A CN109430056A CN201811373025.0A CN201811373025A CN109430056A CN 109430056 A CN109430056 A CN 109430056A CN 201811373025 A CN201811373025 A CN 201811373025A CN 109430056 A CN109430056 A CN 109430056A
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- Prior art keywords
- alum root
- explant
- alum
- shoot regeneration
- petiole
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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
-
- 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
Abstract
A method of induction alum root adventitious shoot regeneration is inoculated in induced medium using the petiole of alum root aseptic seedling or blade as explant, carries out Fiber differentiation, obtains alum root adventitious bud；Wherein, the induced medium adds sucrose 2-4wt.%, agar powder 0.5-0.7wt.%, methyl α-naphthyl acetate 0.05-0.15mg/L, Thidiazuron 0.1-0.2mg/L or 6-benzyl aminopurine 0.2-1.0mg/L, pH5.5-6.0 using MS as minimal medium；The step of being forwarded to adventitious bud differentiation and culture base this invention simplifies self-induction callus tissue culture base, and efficiently solve the Vitrification Occurred of adventitious bud, it is easy to operate, economical and effective, it can be quick breeding, the cultivation of new varieties of alum root seedling, and provide technical support using the improvement that genetic engineering means carry out alum root character.
Description
Technical field
The invention belongs to the technical field of tissue culture of plant, and in particular to a kind of side for inducing alum root adventitious shoot regeneration
Method.
Background technique
Alum root (Heuchera spp.) is Saxifragaceae heuchera, is a kind of perennial evergreen herbage flower, is originated in
In the middle part of America.Because of its floral leaf and beauty, leaf color is beautiful and various colors, is rare new excellent color leaf ornamental plant, can make
For edging, group planting, the materials application such as quilt, potting, constantly heat up on China flowers market.
Most alum root kinds carry out seeling industry by asexual reproduction method, and division propagation and cutting propagation are because of the period
Long, the deficiencies of breeding coefficient is low, big by seasonal effect, there is certain limitation.
Currently, tissue culture technique is the main method of propagation in scale alum root seedling.Existing alum root tissue-culturing rapid propagation skill
Art expands numerous material using bud as starting, there are material materials difficulty, negligible amounts, it is big to maternal plant destructiveness the problems such as.
In the prior art, expand numerous material using alum root ' Paris ' petiole as starting, by Fiber differentiation and differentiation culture two
A stage carries out Fiber differentiation on the induction medium, and petiole generates callus, is further forwarded to adventitious buds differentiation training
It supports and carries out differentiation culture on base, obtain adventitious bud.In this method, the differentiation rate of adventitious bud is relatively low, and must pass through more
The switching process of injured tissue, complicated for operation, higher cost, and increase the pollution probability during sterile working.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for inducing alum root adventitious shoot regeneration, simplify self-induction callus
Culture medium is forwarded to the step of adventitious bud differentiation and culture base, and adventitious shoot regeneration rate, should 62.5% or more, up to 89.58%
Method simplicity can be quick breeding, the cultivation of new varieties of alum root seedling, and utilize genetic engineering means economically and efficiently
The improvement for carrying out alum root character provides technical support.
In order to achieve the above object, the invention provides the following technical scheme:
A method of induction alum root adventitious shoot regeneration, comprising the following steps:
1) it is inoculated with
Using the petiole of alum root aseptic seedling or blade as explant, it is seeded in induced medium；
2) Fiber differentiation
By the explant elder generation that is seeded in induced medium dark culture 14-28 days, illumination cultivation 25-40 days, were obtained later
Alum root adventitious bud；
Wherein, cultivation temperature is 24 ± 2 DEG C, and the light application time of illumination cultivation is 10-14h/d；
Wherein, the induced medium adds sucrose 2-4wt.%, agar powder 0.5- using MS as minimal medium
0.7wt.%, methyl α-naphthyl acetate 0.05-0.15mg/L, Thidiazuron 0.1-0.2mg/L or 6-benzyl aminopurine 0.2-1.0mg/L,
pH5.5-6.0。
Further, in step 1), when the explant is blade, blade is cut into 0.5~0.8cm × 0.5~0.8cm
Leaf dish is inoculated with.
Also, when the explant is petiole, the length of petiole is cut to 0.8~1cm in step 1), it is inoculated in induction training
It supports on base.
Preferably, in step 2), the dark culture time is 21 days, and the illumination cultivation time is 30 days.
Also, the cultivation temperature is 25 DEG C in step 2), the light application time of illumination cultivation is 12h/d.
Further, when the explant is petiole, in the induced medium, 6-benzyl aminopurine 0.2-0.5mg/L,
pH5.8。
Preferably, when the explant is blade, in the induced medium, 6-benzyl aminopurine 0.5-1.0mg/L,
pH5.8。
Also, the kind of the alum root is ' Paris ' or ' dodging the autumn ' in step 1).
The present invention is merely with a kind of culture medium, the dark culture and illumination cultivation for being 50 days or so by cultivation cycle, energy
It is enough to regenerate good alum root adventitious bud from petiole and blade both explants, simplify self-induction callus tissue culture
Base is forwarded to the step of adventitious bud differentiation and culture base, reduces technical operation difficulty, meanwhile, toxigenic capacity is also greatly reduced,
Efficiently solve the Vitrification Occurred of adventitious bud.
The formation of Illumination on Plant organ have inducing action, the present invention in, first carry out dark culture, then carry out illumination training
It supports, explant carries out dedifferentiation under dark culture, is broken up again under illumination cultivation, and callus group is sequentially formed on explant
It knits and adventitious bud；During the cultivation process, using the plant growth regulator of low concentration in induced medium, promote explant not
Normal bud and calli induction reach higher adventitious shoot regeneration rate, and efficiently solve the Vitrification Occurred of adventitious bud.
In culture medium of the invention, adds Thidiazuron or 6-benzyl aminopurine promotes the bud differentiation of alum root, the naphthalene second of addition
Acid has and promotes cell division and widened effect, and the differentiation for promoting callus can be played in the tissue cultures of alum root
Effect, meanwhile, the additive amount of these basic elements of cell division and auxin is also to adjust explant to form callus or adventitious bud
Important factor in order be also easy to produce Vitrification Occurred if excessive concentration.
Compared with prior art, the invention has the following beneficial effects:
In the present invention, alum root petiole and blade can be used as explant, merely with a kind of culture medium in incubation, pass through
50 days or so cultivation cycles, during which no replacement is required new culture medium is easy to operate and substantially reduce production cost, and adventitious bud is again
Raw rate efficiently solves the Vitrification Occurred of adventitious bud 62.50% or more.
Using the present invention, the relaying step of self-induction callus tissue culture base to adventitious bud differentiation and culture base can be saved, letter
Just, economical and effective, adventitious shoot regeneration rate can be the quick breeding of alum root seedling, newly up to 89.58% 62.50% or more
The cultivation of kind and the improvement offer more preferably technical support that alum root character is carried out using genetic engineering means.
Detailed description of the invention
Fig. 1 is state of the petiole explant in ' Paris ' in the embodiment of the present invention 2 when cultivating 30 days.
Fig. 2 is state of the blade explant in ' Paris ' in the embodiment of the present invention 7 when cultivating 30 days.
Fig. 3 is state of the blade explant in ' Paris ' in the embodiment of the present invention 7 when cultivating 50 days.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1-5
Make explant with the petiole of alum root ' Paris ' aseptic seedling, the length of petiole is cut to 0.8~1cm, induction is inoculated in
On culture medium, Fiber differentiation is carried out, directly induces and breaks up acquisition adventitious bud, incubation: first dark culture 21 days, later illumination
Culture 30 days, cultivation temperature are 24 ± 2 DEG C.
Wherein, the induced medium is added to methyl α-naphthyl acetate (Naphthylacetic using MS as minimal medium
Acid, NAA) 0.1mg/L, sucrose 3wt.% and agar powder 0.6wt.%, and it is added to the Thidiazuron of different content
(Thidiazuron, TDZ) or 6-benzyl aminopurine (6-BA), and it is provided with comparative example 1 and comparative example 2, induced medium
PH5.8, and the autoclave sterilization through 121 DEG C, 0.11MPa.
The additive amount of TDZ and 6-BA and cultivation results are referring to 1 He of table in embodiment 1-5 and comparative example 1-2 induced medium
Fig. 1, wherein adventitious bud number is that 12 explants of every ware amount to effective adventitious bud quantity that differentiation obtains；Adventitious shoot regeneration rate=
Differentiation obtains explant number/inoculation explant number × 100% of effective adventitious bud.
Table 1
Seen from table 1, when the TDZ concentration added in culture medium is 0.1,0.2mg/L, the induction of alum root ' Paris ' petiole is produced
The regeneration rate of raw adventitious bud is 65% or more, and the regeneration rate of adventitious bud is higher, and without glassy state；When TDZ concentration is
The regeneration rate that petiole induction in 0.5mg/L, alum root ' Paris " generates adventitious bud is 2.08%, and the regeneration rate of adventitious bud is very low.
When the 6-BA concentration added in culture medium is 0.2mg/L, 0.5mg/L and 1.0mg/L, alum root ' Paris ' petiole is lured
The regeneration rate of the raw adventitious bud of artificial delivery is 85% or more, and the regeneration rate of adventitious bud is higher, as seen from Figure 1, when cultivating 30 days, in petiole
Both ends have generated the adventitious bud of many greens；When 6-BA concentration is 2.0mg/L, the induction of alum root ' Paris ' petiole generates adventitious bud
Regeneration rate be 62.50%, the regeneration rate of adventitious bud is lower, and part adventitious bud present glassy state.
In addition, ' dodging autumn ' petiole when the 6-BA concentration added in culture medium is 0.2mg/L, 0.5mg/L and 1.0mg/L
The regeneration rate that induction generates adventitious bud is 77.08%, 62.50% and 72.9%, and without glassy state.
Embodiment 6-8
With alum root ' Paris ' and ' dodge autumn ' tests for sterility make explant, by blade be cut into 0.5~0.8cm × 0.5~
0.8cm leaf dish, is inoculated in induced medium, carries out Fiber differentiation.
The induced medium using MS as minimal medium, be added to methyl α-naphthyl acetate NAA0.1mg/L, sucrose 3wt.% and
Agar powder 0.6wt.%, and be added to the 6-BA of different content, is provided with comparative example 3, induced medium pH5.8, and through 121
DEG C, the autoclave sterilization of 0.11MPa.
The additive amount of 6-BA and cultivation results are referring to table 2 and Fig. 2-3 in 3 induced medium of embodiment 6-8 and comparative example.
Table 2
As can be seen from Table 2, when the 6-BA concentration added in culture medium is 0.2mg/L, 0.5mg/L and 1.0mg/L, alum root
The regeneration rate that the induction of ' Paris ' blade generates adventitious bud is 60% or more, and ' dodging autumn ' blade induces the regeneration rate for generating adventitious bud to exist
66% or more, the regeneration rate of adventitious bud is higher, when 6-BA concentration is 2.0mg/L, alum root ' Paris ' and ' sudden strain of a muscle autumn ' petiole induction
The regeneration rate for generating adventitious bud is respectively 58.33% and 58.33%, but glassy state is presented in part adventitious bud.
By Fig. 2-Fig. 3 as it can be seen that at leaf culture 30 days, many bottle-green adventitious buds have been generated in blade surrounding；Training
When supporting 50 days, the adventitious bud generated around blade through induction has formed more apparent blade-carrying regeneration plant.
Claims (8)
1. a kind of method for inducing alum root adventitious shoot regeneration, comprising the following steps:
1) it is inoculated with
Using the petiole of alum root aseptic seedling or blade as explant, it is seeded in induced medium；
2) Fiber differentiation
By the explant elder generation that is seeded in induced medium dark culture 14-28 days, illumination cultivation 25-40 days, obtained alum root later
Adventitious bud；
Wherein, cultivation temperature is 24 ± 2 DEG C, and the light application time of illumination cultivation is 10-14h/d；
Wherein, the induced medium adds sucrose 2-4wt.%, agar powder 0.5-0.7wt.% using MS as minimal medium,
Methyl α-naphthyl acetate 0.05-0.15mg/L, Thidiazuron 0.1-0.2mg/L or 6-benzyl aminopurine 0.2-1.0mg/L, pH5.5-6.0.
2. inducing the method for alum root adventitious shoot regeneration according to claim 1, which is characterized in that in step 1), the explant
When body is blade, blade is cut into 0.5~0.8cm × 0.5~0.8cm leaf dish, is inoculated with.
3. inducing the method for alum root adventitious shoot regeneration according to claim 1, which is characterized in that in step 1), the explant
When body is petiole, the length of petiole is cut to 0.8~1cm, is inoculated in induced medium.
4. inducing the method for alum root adventitious shoot regeneration according to claim 1, which is characterized in that in step 2), the dark training
Supporting the time is 21 days, and the illumination cultivation time is 30 days.
5. inducing the method for alum root adventitious shoot regeneration according to claim 1, which is characterized in that in step 2), the culture
Temperature is 25 DEG C, and the light application time of illumination cultivation is 12h/d.
6. inducing the method for alum root adventitious shoot regeneration according to claim 1, which is characterized in that the explant is petiole
When, in the induced medium, 6-benzyl aminopurine 0.2-0.5mg/L, pH5.8.
7. inducing the method for alum root adventitious shoot regeneration according to claim 1, which is characterized in that the explant is blade
When, in the induced medium, 6-benzyl aminopurine 0.5-1.0mg/L, pH5.8.
8. the method for any one of -7 induction alum root adventitious shoot regenerations according to claim 1, which is characterized in that in step 1),
The kind of the alum root is ' Paris ' or ' dodging the autumn '.
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Cited By (3)
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| CN110199885A (en) * | 2019-07-16 | 2019-09-06 | 上海市农业科学院 | A method of separation alum root secondary color leaf obtains pure color aseptic seedling |
| CN111034615A (en) * | 2019-12-21 | 2020-04-21 | 北京花乡花卉科技研究所有限公司 | Tissue culture method for rapid propagation of alum root "tiramisu |
| CN111513061A (en) * | 2020-05-22 | 2020-08-11 | 上海市农业生物基因中心 | Ultralow-temperature preservation and recovery culture method for alum root clump buds |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110199885A (en) * | 2019-07-16 | 2019-09-06 | 上海市农业科学院 | A method of separation alum root secondary color leaf obtains pure color aseptic seedling |
| CN111034615A (en) * | 2019-12-21 | 2020-04-21 | 北京花乡花卉科技研究所有限公司 | Tissue culture method for rapid propagation of alum root "tiramisu |
| CN111513061A (en) * | 2020-05-22 | 2020-08-11 | 上海市农业生物基因中心 | Ultralow-temperature preservation and recovery culture method for alum root clump buds |
| CN111513061B (en) * | 2020-05-22 | 2022-05-03 | 上海市农业生物基因中心 | Ultralow-temperature preservation and recovery culture method for alum root clump buds |
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