CN105409767A - Transitional culture medium improving somatic embryogenesis and and plant regeneration efficiency of larix olgensis and culture method - Google Patents
Transitional culture medium improving somatic embryogenesis and and plant regeneration efficiency of larix olgensis and culture method Download PDFInfo
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Abstract
The invention provides a transitional culture medium improving somatic embryogenesis and and plant regeneration efficiency of larix olgensis and a culture method, and relates to a culture medium improving somatic embryogenesis and and plant regeneration efficiency of larch and a culture method. The culture medium and the culture method aim to solve the problems that high-frequency somatic embryogenesis in an existing somatic embryogenesis system for larix olgensis needs to use expensive equipment for liquid synchronized culture, the cotyledon embryogenesis frequency is commonly not high and needs a long period, and the somatic embryo germination rate and the plant regeneration rate are low. The culture medium comprises the following components: NH4NO3, KH2PO4, MgSO4.7H2O, CaCl2.4H2O, ZnSO4.7H2O, FeSO4.7H2O, H3BO3, inositol, Gln, casein hydrolysate, GuSO4.5H2O, CoCl2.6H2O, saccharose and the like. According to the culture medium and the culture method provided by the invention, the amount of high-quality somatic embryogenesis of larix olgensis is imporved, and the somatic embryogenesis time is shortened, so that the somatic embryogenesis efficiency of larix olgensis is improved. The culture medium and the culture method are used in the field of plant tissue culture.
Description
Technical field
The present invention relates to a kind of medium and the cultural method that improve the generation of larch somatic embryo and plant regeneration efficiency.
Background technology
Larix olgensis belongs to the larch subfamily Larch in Pinaceae, has that distribution is wide, an advantage such as strong stress resistance and early stage fast-growing, is fast-growing commerical tree species important under mountain area, temperate zone, the Northern Hemisphere and climate of frigid zone condition.Larix olgensis has the very high ecological value and economic worth, but larix olgensis growth cycle is long, there is the problems such as rooting rate is lower in the vegetative propagations such as cuttage, and larix olgensis has the high feature of heterozygosity, the filial generation variation that sexual reproduction produces is larger, a lot of merit is difficult to maintain, the demand of many genetic improvements is not well positioned to meet by traditional breeding method, so utilize as modern biotechnology means such as cell engineerings, foundation is stablized and larix olgensis somatic embryo generation system has important and far-reaching meaning for the genetic breeding of larix olgensis efficiently.
For setting up for plant somatocyte embryo generation system, the link of most critical is the somatic embryo obtaining a large amount of high-quality how fast.Present stage, about body embryo stage of development, there is many problems in the somatic embryo of larix olgensis, mainly the body embryo of of poor quality, the high frequency of embryo callus occur to use expensive equipment to carry out liquid synchronizing culture, embryo callus subculture body embryo generating ability are poor, long, early stage proembryos of required cycle occurs body embryo cotyledon and root restriction growth abnormal.The generating capacity of usual larch embryo callus somatic embryo does not surpass 30/g, and needs more than 80 days occur.Even if system preferably, 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%.
Summary of the invention
It is general not high and the required cycle is long, body embryo germination rate and the low problem of shoot regeneration frequency that the present invention is that the body embryo that will solve existing larix olgensis somatic embryo generation system medium-high frequency occurs to need to use expensive equipment to carry out liquid synchronizing culture, cotyledonary embryos occurrence frequency, provides a kind of larix olgensis somatic embryo that improves to occur and the transitional culture medium of plant regeneration efficiency and cultural method.
The present invention improves the transitional culture medium of the generation of larix olgensis somatic embryo and plant regeneration efficiency, comprises following component:
Wherein the preferred concentration of sucrose is 60g/L, and the preferred concentration of inositol is the preferred concentration of 10g/L, Gln is 0.5g/L, and the preferred concentration of caseinhydrolysate is 0.25g/L.
For ensureing the normal growth metabolism of callus, in medium, also 2mg/LCly, 1mg/LVB1,0.5mg/LVB5 and 0.5mg/LVB6 should be contained.
The method utilizing above-mentioned medium to carry out cultivating is: be inoculated in by larix olgensis embryo callus in above-mentioned medium, in 22-25 DEG C, cultivate 7-21d under light culture condition, wherein preferably incubation time is 14 days.
Beneficial effect of the present invention:
The present invention by cultivating the transition that embryo callus carries out before body embryonal induction, improve larix olgensis high quality bulk embryo generating capacity, shorten body embryo time of origin, thus improve larix olgensis body embryo luminous efficiency, significantly improve body embryo germination rate and rooting rate on this basis.Cultivated by transition, only within 15 days, can obtain the body embryo of cotyledon period, quantity occurs for it can reach 330/g at most, and the germination rate of somatic embryo is about 100%, and rooting rate can reach 50%.
Accompanying drawing explanation
Fig. 1 is larix olgensis embryo callus;
Fig. 2 is that larix olgensis somatic embryo occurs;
Fig. 3 is the sprouting of larix olgensis somatic embryo;
Fig. 4 is taking root of 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 following cited embodiment, also comprises any combination between each embodiment.
Embodiment one: present embodiment improves the transitional culture medium of the generation of larix olgensis somatic embryo and plant regeneration efficiency, comprises following component:
Embodiment two: present embodiment and embodiment one unlike: the concentration of described sucrose is 30-60g/L.Other is identical with embodiment one.
Embodiment three: present embodiment and embodiment one or two unlike: the concentration of described inositol is 1-10g/L.Other is identical with embodiment one or two.
Embodiment four: one of present embodiment and embodiment one to three unlike: the concentration of described Gln is 0.5-1g/L.Other is identical with one of embodiment one to three.
Embodiment five: one of present embodiment and embodiment one to four unlike: the concentration of described caseinhydrolysate is 0.25-0.5g/L.Other is identical with one of embodiment one to four.
Embodiment six: one of present embodiment and embodiment one to five unlike: also containing 2mg/LCly, 1mg/LVB1,0.5mg/LVB5 and 0.5mg/LVB6 in described medium.Other is identical with one of embodiment one to five.
Embodiment seven: the method that present embodiment utilizes the transitional culture medium improving the generation of larix olgensis somatic embryo and plant regeneration efficiency to carry out cultivating is:
Larix olgensis embryo callus is inoculated in improve larix olgensis somatic embryo occur and plant regeneration efficiency transitional culture medium in, in 22-25 DEG C, cultivate 1-28d under light culture condition.
Embodiment eight: present embodiment and embodiment seven unlike: under light culture condition, incubation time is 7-21d.Other is identical with embodiment seven.
Embodiment nine: present embodiment and embodiment seven unlike: under light culture condition, incubation time is 14d.Other is identical with embodiment seven.
For verifying that beneficial effect of the present invention carries out following test:
Embodiment 1:
The formula that the present embodiment improves the transitional culture medium of the generation of larix olgensis somatic embryo and plant regeneration efficiency is as follows:
2 will contained by immature zygotic embryos, the larix olgensis embryo callus (Fig. 1) that the BM medium of 4-D, BA etc. induces, to be inoculated in the medium of the present embodiment containing 30-90g/L sucrose after transition cultivates 14d, again through body embryonal induction, germination rate, the shoot regeneration frequency of somatic embryo (Fig. 2) quantity that every gram of embryo callus can be formed and organizator embryo are as shown in the table:
Table 1. sucrose concentration is on the impact of body embryo generation quantity, body embryo germination rate and shoot regeneration frequency
| Sucrose concentration | Body embryo generation quantity | 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％ |
As can be seen from upper table we, when adding 60g/L sucrose in this medium, the somatic embryo number that every gram of callus can induce is maximum, and the germination rate of body embryo and shoot regeneration frequency are also maximum can reach 100% and 45.00% respectively.
* note: above-mentioned body embryo germination rate refers to an access without in hormone WPM medium, body embryo (Fig. 3) the i.e. cotyledon of sprouting turns green and the ratio of the body embryo extended shared by the body embryo of all inoculations; Shoot regeneration frequency refers to an access in WPM medium, and body embryo (Fig. 4) the i.e. radiculodium of taking root grows the ratio of body embryo shared by the body embryo of all inoculations of red radicle.
Embodiment 2:
Embryo callus described in embodiment 1 is inoculated in containing 1-15g/L inositol, in the medium of other conditions with embodiment 1, embryo callus is after induction, and germination rate, the shoot regeneration frequency of the somatic embryo number that every gram of embryo callus can be formed and organizator embryo are as shown in the table:
Table 2. inositol concentration is on the impact of body embryo generation quantity, body embryo germination rate and shoot regeneration frequency
| Inositol concentration | Body embryo generation number | 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％ |
As can be seen from upper table we, in particular range, the quantity of somatic embryo that embryo callus is formed increases with the interpolation of this medium mysoinositol, and when adding 15g/L inositol, the somatic embryo that every gram of callus can induce is maximum; But the inositol amount of adding in the germination rate of body embryo and shoot regeneration frequency and medium is inverse ratio, and namely body embryo germination rate and shoot regeneration frequency increase with the inositol content in incubation and reduce.
Embodiment 3:
Embryo callus described in embodiment 1 is inoculated in containing 0-1g/L glutamine, 0-0.5g/L caseinhydrolysate, 60g/L sucrose, 10g/L inositol, cultivate in the medium of other conditions with embodiment 1, the combination of its medium GLN and caseinhydrolysate is as shown in the table: unit g/L
The glutamine of table 3. variable concentrations 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 medium transition cultivate the embryo callus of 14d after body embryonal induction, germination rate, the shoot regeneration frequency of the somatic embryo quantity that every gram of embryo callus that statistics is cultivated through the Gln of 0-1g/L can be formed and organizator embryo, its result is as shown in the table:
Table 4. glutamine concentration is on the impact of body embryo generation quantity, body embryo germination rate and shoot regeneration frequency
| Gln concentration | Body embryo generation number | 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, body embryo generation number reduces with the concentration increase of the Gln in medium, namely maximum body embryos can be obtained when not adding Gln, but body embryo germination rate is with the increase of Gln concentration, but the trend first raising and reduce afterwards is shown, the germination rate of the body embryo that embryo callus subculture induces through body embryo after adding the medium culture of 0.5g/L is the highest, can reach 94.44%.And the addition of Gln is proportionate in shoot regeneration frequency and medium, namely shoot regeneration frequency raises with the rising of Gln concentration in medium, and when Gln addition is 1g/L, shoot regeneration frequency is the highest, can reach 19.00%.
Above-mentioned 9 kinds of medium transition cultivate the embryo callus of 14d after body embryonal induction, add up germination rate, the shoot regeneration frequency of somatic embryo quantity that every gram of embryo callus cultivating through the caseinhydrolysate (CH) of 0-0.5g/L can be formed and organizator embryo, its result is as shown in the table:
Table 5. caseinhydrolysate concentration is on the impact of body embryo generation quantity, body embryo germination rate and shoot regeneration frequency
| CH concentration | Body embryo generation number | 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 generation number increases with CH concentration in this medium and increases, when CH concentration is 0.5g/L, obtainable body embryo amount is maximum, be 69.56 ± 30.66/g, and the germination rate of body embryo and shoot regeneration frequency is all raise with the CH concentration processed and first raise rear reduction, namely the sprouting of body embryo when this culture medium C H concentration is 0.25g/L, shoot regeneration frequency reach the highest, and it is respectively 92.75% and 21.75%.
Embodiment 4:
Embryo callus described in embodiment 1 be inoculated in containing or do not contain 1mg/LABA, 0.15mg/L2,4-D, 0.05mg/LBA and 0.05mg/LKT, 60g/L sucrose, 10g/L inositol, other conditions cultivate the embryo callus of 14d after induction with transition in the medium of embodiment 1, the somatic embryo number that every gram of embryo callus can be formed in the medium of the combination of its different growth regulator is as shown in the table:
Table 6. adds the impact of growth regulator on body embryo generation quantity
| ABA | 2，4-D | BA | KT | Body embryo generation number |
| 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 the medium adding 1mg/LABA, obviously can reduce its inducing amount at body embryo stage of development body embryo; Add 2,4-D, BA and KT in medium on the generating capacity of embryo callus subculture body embryo in body embryonal induction almost without impact, but interpolation 2,4-D, BA and KT certain limit can be alleviated inhibitory action when ABA occurs embryo callus subculture body embryo again.
Embodiment 5:
Embryo callus described in embodiment 1 is inoculated in containing 60g/L sucrose, 10g/L inositol, other conditions cultivate 0,1,7,14,21 and 28d with transition in the medium of embodiment 1, induce subsequently, finally add up the germination rate of somatic embryo number that its every gram embryo callus can be formed and organizator embryo, shoot regeneration frequency result as shown in the table:
Table 7. incubation time is on the impact of body embryo generation quantity, body embryo germination rate and shoot regeneration frequency
| Incubation time | Body embryo generation number | 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 have significant change with the prolongation of embryo callus incubation time in the medium described in this invention, and the rooting rate of its body embryo generation number of callus and body embryo that the callus that 1d is cultivated in transition relatively directly carries out the generation of body embryo obviously increases; When transition incubation time extends to 14d from 1d, body embryo generation number and shoot regeneration frequency continue to increase, and when transition incubation time continues overtime from 14d, the body embryo generating ability of callus then obviously declines, transition cultivate its body embryo generation quantity of callus after 21d and shoot regeneration frequency almost identical with the callus without this medium culture.Body embryo germination rate is relatively stable, obvious change does not occur with the prolongation of incubation time.
Find in the incubation of follow-up body embryo stage of development, without cycle of the callus organizator embryo of medium culture of the present invention all more than 45d, and shorten with the prolongation of incubation time in this medium through the time of the embryo callus subculture organizator embryo of medium culture of the present invention, the cycle time of callus organizator blast namely cultivated through 7d is to about 20d; Then 15d is about through the cycle of the callus organizator embryo of 14d cultivation; And when callus is through the cultivation of 21d, within formation cycle of body embryo even can foreshorten to 10d, thus method provided by the present invention can shorten somatic embryo occur required for cycle.
Embodiment 6:
Be inoculated in containing 60g/L sucrose by the embryo callus described in embodiment 1,10g/L inositol, 0.5g/L inositol, 0.25g/LCH, other conditions cultivate 14d with transition in the medium of embodiment 1, and the generating capacity of its body embryo reaches as high as 330/g.Although this individual embryo occurrence frequency and existing Optimal system maintain an equal level, the time shorten 45%-65% needed for body embryo occurs, therefore the more existing Optimal system of body embryo luminous efficiency of this method improves nearly 40% in same time.And the body embryo germination rate to obtain through this method also comparatively existing system improve nearly 30%, shoot regeneration frequency also improves about 20 %.
In sum, adopt medium of the present invention and cultural method thereof can improve the quality of embryo callus before body embryonal induction, shorten body embryo generating period, improve high quality bulk embryo generating capacity and shoot regeneration frequency.In addition, method of the present invention does not need, through liquid synchronizing culture, to eliminate the equipment cost needed for liquid culture.
Claims (9)
1. improve a transitional culture medium for the generation of larix olgensis somatic embryo and plant regeneration efficiency, it is characterized in that this medium comprises following component:
2. a kind of transitional culture medium improving the generation of larix olgensis somatic embryo and plant regeneration efficiency according to claim 1, is characterized in that the concentration of described sucrose is 30-60g/L.
3. a kind of transitional culture medium improving the generation of larix olgensis somatic embryo and plant regeneration efficiency according to claim 1, is characterized in that the concentration of described inositol is 1-10g/L.
4. a kind of transitional culture medium improving the generation of larix olgensis somatic embryo and plant regeneration efficiency according to claim 1, is characterized in that the concentration of described Gln is 0.5-1g/L.
5. a kind of transitional culture medium improving the generation of larix olgensis somatic embryo and plant regeneration efficiency according to claim 1, is characterized in that the concentration of described caseinhydrolysate is 0.25-0.5g/L.
6. a kind of transitional culture medium improving the generation of larix olgensis somatic embryo and plant regeneration efficiency according to claim 1, is characterized in that in described medium also containing 2mg/LCly, 1mg/LVB1,0.5mg/LVB5 and 0.5mg/LVB6.
7. the method utilizing the medium described in claim 1 to carry out cultivating is:
Larix olgensis embryo callus is inoculated in improve larix olgensis somatic embryo occur and plant regeneration efficiency transitional culture medium in, in 22-25 DEG C, cultivate 1-28d under light culture condition.
8. cultural method according to claim 7, is characterized in that incubation time is 7-21d under light culture condition.
9. cultural method according to claim 7, is characterized in that incubation time is 14d under light culture condition.
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| CN110004176A (en) * | 2019-04-12 | 2019-07-12 | 东北林业大学 | The construction method of hybrid larch genetic conversion system |
| CN111280065A (en) * | 2020-04-07 | 2020-06-16 | 北京林业大学 | Method for regenerating larch somatic embryos |
| CN113115706A (en) * | 2020-01-15 | 2021-07-16 | 东北林业大学 | Method for restoring and maintaining embryogenic property of embryogenic callus of larch |
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| CN113115706A (en) * | 2020-01-15 | 2021-07-16 | 东北林业大学 | Method for restoring and maintaining embryogenic property of embryogenic callus of larch |
| 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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