CN105409767B - A kind of transitional culture medium and cultural method for improving larix olgensis somatic embryo occur and plant regeneration efficiency - Google Patents
A kind of transitional culture medium and cultural method for improving larix olgensis somatic embryo occur and plant regeneration efficiency Download PDFInfo
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- A—HUMAN NECESSITIES
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Abstract
A kind of transitional culture medium and cultural method for improving larix olgensis somatic embryo occur and plant regeneration efficiency, is related to a kind of culture medium and cultural method for improving larch somatic embryo occur and plant regeneration efficiency.It is that the body embryo that solve existing larix olgensis body embryo generation system medium-high frequency needs to use the problem of equipment of costliness carries out the universal not high and required cycle length of liquid synchronizing culture, cotyledonary embryos occurrence frequency, body embryo germination rate and low shoot regeneration frequency.The culture medium includes following components：NH4NO3、KH2PO4、MgSO4·7H2O、CaCl2·4H2O、ZnSO4·7H2O、FeSO4·7H2O、H3BO3, inositol, Gln, caseinhydrolysate, CuSO4·5H2O、CoCl2·6H2O, sucrose etc..The present invention improves the high-quality body embryo generating capacity of larix olgensis, shortens body embryo time of origin, so as to improve larix olgensis body embryo luminous efficiency.For field of plant tissue culture.
Description
Technical field
The present invention relates to a kind of culture medium and cultural method for improving larch somatic embryo occur and plant regeneration efficiency.
Background technology
The fallen leaves abieteae Larch that larix olgensis belongs in Pinaceae, with distribution is wide, strong stress resistance and early stage speed
Raw the advantages of, be Northern Hemisphere temperate zone mountain area and fast-growing commerical tree species important under the conditions of climate of frigid zone.Larix olgensis has very high
The ecological value and economic value, but larix olgensis growth cycle is long, and it is more low that the vegetative propagation such as cuttage has rooting rate
Problem, and larix olgensis has the characteristics of heterozygosity is high, the filial generation variation that zoogamy is produced is larger, and many merits are difficult
To maintain, the demand of many genetic improvements is not well positioned to meet by traditional breeding method, so using such as cell work
The modern biotechnology means such as journey, set up stable and efficient larix olgensis somatic embryo and occur system for long white fallen leaves
The genetic breeding of pine has important and far-reaching meaning.
For setting up plant somatocyte embryo generation system, the link of most critical is how quickly to obtain a large amount of excellent
The somatic embryo of matter.At this stage, on body embryo stage of development, there is many and ask in the somatic embryo of larix olgensis
Topic, mainly embryo callus are of poor quality, high frequency body embryo generation need to use expensive equipment progress liquid synchronizing culture,
Cycle length, the cotyledon of early stage proembryos and root restriction needed for embryo callus subculture body embryo generating ability is poor, body embryo occurs develop abnormal.It is logical
The generating capacity of normal larch embryo callus somatic embryo does not surpass 30/g, and occurs to need more than 80 days.It is even preferable
System, the generation of cotyledonary embryos also needs 40-60 days；The germination rate of body embryo is no more than 70%；Shoot regeneration frequency is also below 30%.
The content of the invention
Needs, which occur, for the body embryo for occurring system medium-high frequency the present invention is to solve existing larix olgensis somatic embryo makes
The universal not high and required cycle length of liquid synchronizing culture, cotyledonary embryos occurrence frequency, body embryo germination rate are carried out with expensive equipment
And shoot regeneration frequency it is low the problem of there is provided a kind of transition training for improving larix olgensis somatic embryo occur and plant regeneration efficiency
Support base and cultural method.
The present invention improves the transitional culture medium of larix olgensis somatic embryo occur and plant regeneration efficiency, including with the following group
Point：
Wherein the preferred concentration of sucrose is 60g/L, and the preferred concentration of inositol is 10g/L, and Gln preferred concentration is 0.5g/
L, the preferred concentration of caseinhydrolysate is 0.25g/L.
To ensure also contain 2mg/L Cly, 1mg/L VB1 in the normal growth metabolism of callus, culture medium,
0.5mg/L VB5 and 0.5mg/L VB6.
The method cultivated using above-mentioned culture medium is：Larix olgensis embryo callus is inoculated in above-mentioned culture
In base, 7-21d is cultivated under the conditions of 22-25 DEG C, light culture, wherein it is preferred that incubation time is 14 days.
Beneficial effects of the present invention：
The present invention improves larix olgensis high by the transition culture carried out before body embryo is induced to embryo callus
Quality body embryo generating capacity, shortens body embryo time of origin, so as to improve larix olgensis body embryo luminous efficiency, on this basis
Significantly improve body embryo germination rate and rooting rate.By transition culture, the body embryo of cotyledon period can be obtained within only 15 days, it occurs number
Amount at most can be up to 330/g, and the germination rate of somatic embryo is about 100%, and rooting rate is up to 50%.
Brief description of the drawings
Fig. 1 is larix olgensis embryo callus；
Fig. 2 occurs for larix olgensis somatic embryo；
Fig. 3 is the sprouting of larix olgensis somatic embryo；
Fig. 4 is taken root for larix olgensis somatic embryo；
Fig. 5 is the plant regeneration of larix olgensis somatic embryo.
Embodiment
Technical solution of the present invention is not limited to act embodiment set forth below, in addition between each embodiment
Any combination.
Embodiment one：Present embodiment improves the mistake of larix olgensis somatic embryo occur and plant regeneration efficiency
Cross culture medium, including following components：
Embodiment two：Present embodiment from unlike embodiment one：The concentration of the sucrose is 30-
60g/L.It is other identical with embodiment one.
Embodiment three：Present embodiment from unlike embodiment one or two：The concentration of the inositol
For 1-10g/L.It is other identical with embodiment one or two.
Embodiment four：Unlike one of present embodiment and embodiment one to three：The Gln's is dense
Spend for 0.5-1g/L.It is other identical with one of embodiment one to three.
Embodiment five：Unlike one of present embodiment and embodiment one to four：The hydrolysis junket
The concentration of albumen is 0.25-0.5g/L.It is other identical with one of embodiment one to four.
Embodiment six：Unlike one of present embodiment and embodiment one to five：The culture medium
In also contain 2mg/L Cly, 1mg/L VB1,0.5mg/L VB5 and 0.5mg/L VB6.Other and embodiment one to five
One of it is identical.
Embodiment seven：Present embodiment utilizes and improves larix olgensis somatic embryo occur and plant regeneration efficiency
The method cultivated of transitional culture medium be：
Larix olgensis embryo callus is inoculated in raising larix olgensis somatic embryo occur and plant regeneration effect
In the transitional culture medium of rate, 1-28d is cultivated under the conditions of 22-25 DEG C, light culture.
Embodiment eight：Present embodiment from unlike embodiment seven：When being cultivated under the conditions of light culture
Between be 7-21d.It is other identical with embodiment seven.
Embodiment nine：Present embodiment from unlike embodiment seven：When being cultivated under the conditions of light culture
Between be 14d.It is other identical with embodiment seven.
Tests below is carried out for checking beneficial effects of the present invention：
Embodiment 1：
The present embodiment improves the formula of the transitional culture medium of larix olgensis somatic embryo occur and plant regeneration efficiency such as
Under：
The larix olgensis embryo callus subculture that will be induced by immature zygotic embryos in the BM culture mediums containing 2,4-D, BA etc.
Organize (Fig. 1), be inoculated in the culture medium of the present embodiment containing 30-90g/L sucrose after transition culture 14d, then lured through body embryo
Lead, somatic embryo (Fig. 2) quantity and form the germination rate of body embryo, shoot regeneration frequency such as that every gram of embryo callus can be formed
Shown in following table：
The sucrose concentration of table 1. body embryo occurs the influence of quantity, body embryo germination rate and shoot regeneration frequency
| Sucrose concentration | Quantity occurs for body embryo | Body embryo germination rate | Shoot regeneration frequency |
| 30g/L | 77.75 ± 4.83/g | 95.88% | 20.75% |
| 60g/L | 80.40 ± 5.07/g | 100% | 45.00% |
| 90g/L | 74.41 ± 5.32/g | 72.33% | 25.00% |
From upper table it will be seen that in the culture medium add 60g/L sucrose when, what every gram of callus can be induced
Somatic embryo number is most, and also maximum can respectively reach 100% and 45.00% for the germination rate and shoot regeneration frequency of body embryo.
* note：Above-mentioned body embryo germination rate is referred to the accession in no hormone WPM culture mediums, the body embryo (Fig. 3) of sprouting be cotyledon greening and
The body embryo of elongation ratio shared in all body embryos of inoculation；Shoot regeneration frequency is referred to the accession in WPM culture mediums, the body embryo taken root
(Fig. 4) is that radiculodium grows ratio of the body embryo of red radicle shared by all body embryos of inoculation.
Embodiment 2：
Embryo callus described in embodiment 1 is inoculated in containing 1-15g/L inositols, other conditions be the same as Example 1
Culture medium in, embryo callus is after induction, somatic embryo number and formation that every gram of embryo callus can be formed
Germination rate, the shoot regeneration frequency of body embryo are as shown in the table：
The inositol concentration of table 2. body embryo occurs the influence of quantity, body embryo germination rate and shoot regeneration frequency
| Inositol concentration | Number occurs for body embryo | Body embryo germination rate | Shoot regeneration frequency |
| 1g/L | 69.15 ± 4.83/g | 90.57% | 34.57% |
| 10g/L | 74.67 ± 5.07/g | 90.78% | 31.44% |
| 15g/L | 88.77 ± 5.32/g | 85.22% | 25.11% |
From upper table it will be seen that in particular range, the quantity of the somatic embryo of embryo callus formation with
The addition of the culture medium mysoinositol and increase, add 15g/L inositols when, the somatic embryo that every gram of callus can be induced
Tire is most；But the inositol amount added in the germination rate of body embryo and shoot regeneration frequency and culture medium is in inverse ratio, i.e. body embryo germination rate
And shoot regeneration frequency increases and reduced with the inositol content in incubation.
Embodiment 3：
Embryo callus described in embodiment 1 is inoculated in containing 0-1g/L glutamine, 0-0.5g/L hydrolysis junket
In albumen, 60g/L sucrose, 10g/L inositols, the culture medium of other conditions be the same as Example 1 cultivate, its culture medium GLN with
The combination of caseinhydrolysate is as shown in the table：Unit g/L
The glutamine of the various concentrations of table 3. and the assembled scheme of caseinhydrolysate
| Combination 1 | Combination 2 | Combination 3 | Combination 4 | Combination 5 | Combination 6 | Combination 7 | Combination 8 | Combination 9 | |
| Gln | 0 | 0 | 0 | 0.5 | 0.5 | 0.5 | 1 | 1 | 1 |
| CH | 0 | 0.25 | 0.5 | 0 | 0.25 | 0.5 | 0 | 0.25 | 0.5 |
Above-mentioned 9 kinds of culture mediums transition culture 14d embryo callus is counted through 0-1g/L's after body embryo is induced
Somatic embryo quantity and germination rate, the shoot regeneration frequency of formation body embryo that every gram of embryo callus of Gln cultures can be formed,
Its result is as shown in the table：
The glutamine concentration of table 4. body embryo occurs the influence of quantity, body embryo germination rate and shoot regeneration frequency
| Gln concentration | Number occurs for body embryo | Body embryo germination rate | Shoot regeneration frequency |
| 0g/L | 72.16 ± 30.22/g | 74.11% | 11.01% |
| 0.5g/L | 67.53 ± 29.31/g | 94.44% | 14.78% |
| 1g/L | 52.20 ± 9.99/g | 75.00% | 19.00% |
As seen from the above table, number, which occurs, for body embryo increases and reduces with the concentration of the Gln in culture medium, i.e., when without Gln
Most body embryos can be obtained, but body embryo germination rate but shows first to raise the trend reduced afterwards with the increase of Gln concentration,
The germination rate highest of the body embryo induced after the added 0.5g/L of embryo callus subculture medium culture through body embryo, it is reachable
94.44%.And the addition of shoot regeneration frequency and Gln in culture medium is proportionate, i.e., shoot regeneration frequency is dense with Gln in culture medium
The rise of degree and raise, when Gln additions are 1g/L, shoot regeneration frequency highest, up to 19.00%.
Above-mentioned 9 kinds of culture mediums transition culture 14d embryo callus is counted through 0-0.5g/L's after body embryo is induced
The somatic embryo quantity that can be formed of every gram of embryo callus of caseinhydrolysate (CH) culture and formed body embryo germination rate,
Shoot regeneration frequency, its result is as shown in the table：
The caseinhydrolysate concentration of table 5. body embryo occurs the influence of quantity, body embryo germination rate and shoot regeneration frequency
| CH concentration | Number occurs for body embryo | Body embryo germination rate | Shoot regeneration frequency |
| 0g/L | 60.29 ± 30.85/g | 68.75% | 4.13% |
| 0.25g/L | 62.04 ± 13.08/g | 92.75% | 21.75% |
| 0.5g/L | 69.56 ± 30.66/g | 83.33% | 17.56% |
As seen from the above table, in certain concentration range, larix olgensis body embryo occurs number and increased with CH concentration in the culture medium
Plus and increase, when CH concentration is 0.5g/L, obtainable body embryo amount is 69.56 ± 30.66/g at most, and body embryo is sprouted
Hair rate and shoot regeneration frequency are the CH concentration rises with processing and first raise reduces afterwards, i.e., be in culture medium C H concentration
The sprouting of body embryo, shoot regeneration frequency reach highest during 0.25g/L, and it is respectively 92.75% and 21.75%.
Embodiment 4：
Embryo callus described in embodiment 1 is inoculated in or without 1mg/LABA, 0.15mg/L 2,4-D,
Transition in 0.05mg/L BA and 0.05mg/L KT, 60g/L sucrose, 10g/L inositols, the culture medium of other conditions be the same as Example 1
14d embryo callus is cultivated after induction, every gram of embryo is cured in the culture medium of the combination of its different growth regulator
The somatic embryo number that injured tissue can be formed is as shown in the table：
Table 6. adds growth regulator and body embryo occurs the influence of quantity
| ABA | 2,4-D | BA | KT | Number occurs for body embryo |
| 1mg/L | 0.15mg/L | 0.05mg/L | 0.05mg/L | 25.63 ± 4.26/g |
| - | 0.15mg/L | 0.05mg/L | 0.05mg/L | 37.27 ± 11.6/g |
| 1mg/L | - | - | - | 11.03 ± 2.91/g |
| - | - | - | - | 37.39 ± 3.84/g |
As seen from the above table, embryo callus is cultivated in addition 1mg/L ABA culture medium, can substantially reduce it in body
The inducing amount of embryo stage of development body embryo；2,4-D, BA and KT body embryo in body embryo induction to embryo callus subculture are added in culture medium
Generating capacity is almost without influence, but addition 2, and 4-D, BA and KT can alleviate ABA in certain limit again, and embryo callus subculture body embryo occurs
When inhibitory action.
Embodiment 5：
Embryo callus described in embodiment 1 is inoculated in containing 60g/L sucrose, 10g/L inositols, other conditions are same
Transition culture 0,1,7,14,21 and 28d, are then induced in the culture medium of embodiment 1, finally count its every gram embryo callus subculture
Organize the somatic embryo number that can be formed and formed the germination rate of body embryo, shoot regeneration frequency result it is as shown in the table：
The incubation time of table 7. body embryo occurs the influence of quantity, body embryo germination rate and shoot regeneration frequency
| Incubation time | Number occurs for body embryo | Body embryo germination rate | Shoot regeneration frequency |
| 0d | 32.33 ± 8.39/g | 62.67% | 11.00% |
| 1d | 51.0 ± 17.78/g | 83.33% | 33.33% |
| 7d | 68.0 ± 18.68/g | 100% | 33.33% |
| 14d | 88.00 ± 1.73/g | 100% | 50.00% |
| 21d | 35.67 ± 1.53/g | 100% | 16.67% |
| 28d | 34.5 ± 23.17/g | 100% | 16.67% |
As seen from the above table, the generation quantity of body embryo and shoot regeneration frequency with embryo callus in the culture described in the invention
The extension of incubation time has a significant change in base, the transition culture 1d relatively direct callus for carrying out body embryo generation of callus its
Number and the rooting rate of body embryo, which occur, for body embryo substantially increases；When transition incubation time extends to 14d from 1d, number occurs for body embryo with planting
Strain regeneration rate continues to increase, and when transition incubation time continues to extend from 14d, the body embryo generating ability of callus is then obvious
Decline, callus its body embryo after transition culture 21d occur quantity and shoot regeneration frequency almost with without the medium culture
Callus is identical.Body embryo germination rate is stablized relatively, and significantly change does not occur with the extension of incubation time.
Found in the incubation of follow-up body embryo stage of development, the callus without medium culture of the present invention
The cycle of body embryo is formed more than 45d, and the time of the embryo callus subculture formation body embryo Jing Guo medium culture of the present invention
Shorten with the extension of incubation time in the culture medium, i.e., form the cycle time of somatic embryo extremely by the 7d callus cultivated
20d or so；The cycle for the callus formation body embryo cultivated by 14d is about then 15d；And when callus passes through 21d culture, body embryo
The formation cycle can even foreshorten within 10d, therefore required for method provided by the present invention can shorten somatic embryo occur
Cycle.
Embodiment 6：
Embryo callus described in embodiment 1 is inoculated in containing 60g/L sucrose, 10g/L inositols, 0.5g/L fleshes
Transition culture 14d in alcohol, 0.25g/L CH, the culture medium of other conditions be the same as Example 1, the generating capacity of its body embryo is reached as high as
330/g.Although this body embryo occurrence frequency maintains an equal level with existing Optimal system, the time needed for occurring due to body embryo shortens
45%-65%, therefore in same time the more existing Optimal system of body embryo luminous efficiency of this method improves nearly 40%.And
The body embryo germination rate obtained through this method also relatively has system and improves nearly 30%, and shoot regeneration frequency also improves 20 or so %.
In summary, can be before body embryo be induced to embryo callus using culture medium of the present invention and its cultural method
Quality improved, shorten body embryo generating period, improve high-quality body embryo generating capacity and shoot regeneration frequency.In addition, this hair
Bright described method needs not move through liquid synchronizing culture, eliminates the equipment cost needed for Liquid Culture.
Claims (2)
1. a kind of transitional culture medium for improving larix olgensis somatic embryo occur and plant regeneration efficiency, it is characterised in that the training
Support base component be：
2. the method cultivated using the culture medium described in claim 1, it is characterised in that this method is：
Larix olgensis embryo callus is inoculated in raising larix olgensis somatic embryo occur as claimed in claim 1
In the transitional culture medium of plant regeneration efficiency, 14d is cultivated under the conditions of 22-25 DEG C, light culture.
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| CN110004176B (en) * | 2019-04-12 | 2023-03-10 | 东北林业大学 | Construction method of hybrid larch genetic transformation system |
| CN113115706B (en) * | 2020-01-15 | 2022-03-15 | 东北林业大学 | Method for restoring and maintaining embryogenic property of embryogenic callus of larch |
| CN111280065A (en) * | 2020-04-07 | 2020-06-16 | 北京林业大学 | Method for regenerating larch somatic embryos |
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| CN1922985A (en) * | 2006-09-04 | 2007-03-07 | 中国科学院植物研究所 | Larix plants embryo callus subculture method and dedicated culture medium |
| CN101218895A (en) * | 2008-01-30 | 2008-07-16 | 东北林业大学 | Method for regenerating isolated culture adventive bud evoked plant strain of larix olgensis |
| CN104839028A (en) * | 2015-06-02 | 2015-08-19 | 东北林业大学 | Method for inducing hybrid larch plant regeneration through in vitro culture of adventitious buds |
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| KR100720338B1 (en) * | 2005-11-09 | 2007-05-22 | 대한민국 | Propagation Method of Japanese larchLarix leptolepis through Somatic Embryogenesis Technique |
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| CN1922985A (en) * | 2006-09-04 | 2007-03-07 | 中国科学院植物研究所 | Larix plants embryo callus subculture method and dedicated culture medium |
| CN101218895A (en) * | 2008-01-30 | 2008-07-16 | 东北林业大学 | Method for regenerating isolated culture adventive bud evoked plant strain of larix olgensis |
| CN104839028A (en) * | 2015-06-02 | 2015-08-19 | 东北林业大学 | Method for inducing hybrid larch plant regeneration through in vitro culture of adventitious buds |
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