CN1292645C - Method of inducing small liwan moss to produce callus and differentiate to form gametocyte - Google Patents
Method of inducing small liwan moss to produce callus and differentiate to form gametocyte Download PDFInfo
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- CN1292645C CN1292645C CN 200510026616 CN200510026616A CN1292645C CN 1292645 C CN1292645 C CN 1292645C CN 200510026616 CN200510026616 CN 200510026616 CN 200510026616 A CN200510026616 A CN 200510026616A CN 1292645 C CN1292645 C CN 1292645C
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Abstract
The present invention relates to a method for inducting physcomitrella patens calluses and generating gametophyte by differentiation. The method comprises the following steps: step 1, physcomitrella patens is sterilized, stem apexes of the physcomitrella patens are cut out and are inoculated into a Knop culture medium for induction culture, the induction culture lasts for 28 days, wherein the Knop culture medium contains one or two kinds of hormones of 0 to 0.5 mg/L of 6-BA and 0 to 0.5 mg/L of KT, and the sugar concentration is from 0 to 2%; step 2, the physcomitrella patens calluses generated after 28 days of culture are inoculated into a Knop differential medium for differential culture, or into a Knop differential medium which contains 0.5 mg/L of 6-BA for differential culture, or a Knop differential medium which contains 0.5 mg/L2 and 4-D for differential culture, or Knop differential medium which contains 0.5 mg/L of KT for differential culture, and the culture lasts for 21 days. In the present invention, a tissue culture technique is adopted, phytohormones of different types and in different concentration proportions are added to the improved Knop culture medium for inducting the physcomitrella patens calluses, normal plants are generated by differentiation, and therefore, physcomitrella patens is propagated in large scale, and heavy demand for the physcomitrella patens is met.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of method of inducing small liwan moss to produce callus and differentiate to form gametocyte.
Background technology
Small liwan moss is subordinate to Herba Funariae Hygrometricae section, and small liwan moss belongs to, and is the highest terrestrial plant of homologous recombination frequency up to now, and its genome is 460mb, is that 4 times of arabidopsis and rice genome are more or less the same, and chromosome is 27.In recent years, research institutes such as Schaefer DG are similar through experimental results show that its homology class frequency and yeast, are 1000 times of arabidopsis.Small liwan moss has become behind arabidopsis another model plant in the vegetative kingdom.Because small liwan moss has higher homologous recombination rate, more and more scholars with it as research object, carry out extensive studies at aspects such as functional genomics, plant physiology, Developmental Biology, phyletic evolution, this just needs a large amount of small liwan moss, can not satisfy the demand far away by self-sow like this.But at present, also lack culture technique both at home and abroad about the tissue and the cell of small liwan moss, new through looking into, also there not be appearance about inducing the small liwan moss callus and generating the report of gametophytic method.
Summary of the invention
The purpose of this invention is to provide a kind of method of inducing small liwan moss to produce callus and differentiate to form gametocyte, by evoked callus and differentiate to form gametocyte, thereby obtain prolific small liwan moss, satisfy needs at aspects such as functional genomics, plant physiology, Developmental Biology, phyletic evolution.
Purpose of the present invention can be achieved through the following technical solutions:
The invention provides a kind of method of inducing small liwan moss to produce callus and differentiate to form gametocyte, this method has following steps:
1, with after the small liwan moss sterilization, it is 0-2% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, and contains on the Knop medium of one or both hormones of KT of the 6-BA of 0-0.5mg/L and 0-0.5mg/L and carry out inducing culture, and the inducing culture fate is 28 days;
2, will cultivate after 28 days the small liwan moss callus that produces is transferred to and carries out differentiation culture on the Knop differential medium; Or be transferred on the Knop differential medium of the 6-BA that contains 0.5mg/L and carry out differentiation culture; Or be transferred to and contain 2 of 0.5mg/L, carry out differentiation culture on the Knop differential medium of 4-D; Or be transferred on the Knop differential medium of the KT that contains 0.5mg/L and carry out differentiation culture, cultivating fate is 21 days.
The condition of above-mentioned inducing culture and differentiation culture is: cultivation temperature is 24-26 ℃, and periodicity of illumination is 12h/d, and illuminance is 3000-4000lx.
Above-mentioned steps 1) in the small liwan moss callus being carried out the employed sugared concentration of inducing culture is in 0.5% the Knop medium, add the KT hormone of 0.1mg/L or sugared concentration and be in 2% the Knop medium, add the KT hormone of 0.05mg/L or sugared concentration and be in 2% the Knop medium, the 6-BA hormone that adds 0.05mg/L is best, induces the effect of small liwan moss callus ideal.The callus structure is comparatively loose, and color is light green, and brownization rate is low.
Above-mentioned steps 2) it is best in the small liwan moss callus that induces being transferred in the Knop medium that does not add any hormone, and the gametophyte that differentiates is maximum.
A kind of method of inducing small liwan moss callus and differentiate to form gametocyte provided by the invention, it is the utilization tissue culture technique, the plant hormone that adds variety classes and concentration proportioning in the Knop medium of improvement induces the callus of small liwan moss to make it differentiate normal plant, thereby breed small liwan moss in a large number, as the material of various tests, to satisfy wilderness demand to small liwan moss.
Description of drawings
Fig. 1 is the small liwan moss callus induction culture effect figure of the embodiment of the invention 1;
Fig. 2 is the small liwan moss callus induction culture effect figure of the embodiment of the invention 2;
Fig. 3 is the small liwan moss callus induction culture effect figure of the embodiment of the invention 3;
Fig. 4 is the small liwan moss callus induction culture effect figure of the embodiment of the invention 4;
Fig. 5 is the small liwan moss callus induction culture effect figure of the embodiment of the invention 5;
Fig. 6 is the small liwan moss callus induction culture effect figure of the embodiment of the invention 6;
Fig. 7 is the small liwan moss callus induction culture effect figure of the embodiment of the invention 7;
Fig. 8 is the small liwan moss callus induction culture effect figure of the embodiment of the invention 8;
Fig. 9 is the small liwan moss callus induction culture effect figure of the embodiment of the invention 9;
Figure 10 is the small liwan moss callus induction culture effect figure of the embodiment of the invention 10;
Figure 11 is the small liwan moss callus induction culture effect figure of the embodiment of the invention 11;
Figure 12 is the small liwan moss callus induction culture effect figure of the embodiment of the invention 12;
Figure 13 is the small liwan moss callus induction culture effect figure of the embodiment of the invention 13;
Figure 14 is the small liwan moss callus induction culture effect figure of the embodiment of the invention 14;
Figure 15 is the small liwan moss callus induction culture effect figure of the embodiment of the invention 15;
Figure 16 is the small liwan moss callus induction culture effect figure of the embodiment of the invention 16;
Figure 17 is the design sketch behind the small liwan moss callus differentiation culture of the embodiment of the invention 17;
Figure 18 is another design sketch behind the small liwan moss callus differentiation culture of the embodiment of the invention 17;
Figure 19 is the design sketch behind the small liwan moss callus differentiation culture of the embodiment of the invention 19;
Figure 20 is the design sketch behind the small liwan moss callus differentiation culture of the embodiment of the invention 20;
Embodiment
A kind of method of inducing small liwan moss to produce callus and differentiate to form gametocyte, this method has following steps:
1, with after the small liwan moss sterilization, it is 0-2% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, and contains on the Knop medium of one or both hormones of KT of the 6-BA of 0-0.5mg/L and 0-0.5mg/L and carry out inducing culture, and the inducing culture fate is 28 days;
2, will cultivate after 28 days the small liwan moss callus that produces is transferred to and carries out differentiation culture on the Knop differential medium; Or be transferred on the Knop differential medium of the 6-BA that contains 0.5mg/L and carry out differentiation culture; Or be transferred to and contain 2 of 0.5mg/L, carry out differentiation culture on the Knop differential medium of 4-D; Or be transferred on the Knop differential medium of the KT that contains 0.5mg/L and carry out differentiation culture.
The condition of inducing culture of the present invention and differentiation culture is: cultivation temperature is 24-26 ℃, and periodicity of illumination is 12h/d, and illuminance is 3000-4000lx.
Knop medium of the present invention contains KCL0.07604g, MgSO for plant culture commonly used among promptly every 1L Knop
47H
2O 0.8282g, KH
2PO
40.2504g, Ca (NO
3)
24H
2O 1.0013g, CuSO
45H
2O 0.027mg, KI 0.014mg, ZnSO
47H
2O 0.027mg, H
3BO
30.309mg, Na
2MoO
42H
2O 0.012mg, MnCl
24H
2O 0.198mg, CoCl
26H
2O 0.027mg, FeSO
47H
2O 12.5088mg, tartaric acid ammonia 0.4604mg, and to add percentage by weight be 0.8% agar, and adopt 1N NaOH that its pH value is adjusted to 6.5.
Contain KCL 0.07604g, MgSO among the every 1L Knop of Knop differential medium of the present invention
47H
2O0.8282g, KH
2PO
40.2504g, Ca (NO
3)
24H
2O 1.0013g, CuSO
45H
2O 0.027mg, KI0.014mg, ZnSO
47H
2O 0.027mg, H
3BO
30.309mg, Na
2MoO
42H
2O 0.012mg, MnCl
24H
2O 0.198mg, CoCl
26H
2O 0.027mg, FeSO
47H
2O 12.5088mg, tartaric acid ammonia 0.4604mg, and to add percentage by weight be that 0.5% glucose and percentage by weight are 0.8% agar, and adopt 1N NaOH that its pH value is adjusted to 6.5.
Further specify the present invention by the following examples, but should be understood that these embodiment are exemplary, the present invention does not limit to this.Following examples are the sugared concentration difference of employed Knop medium, and the 6-BA hormone concentration that adds is different with the KT hormone concentration, or employed Knop differential medium does not add any growth substance, adds different 6-BA hormones, 2,4-D hormone, KT hormone.
The embodiment that following embodiment 1 to embodiment 16 cultivates for small liwan moss callus induction of the present invention.
Embodiment 1:
With after the small liwan moss sterilization, it is 0% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, does not add on the Knop medium of 6-BA hormone and KT hormone and carries out inducing culture, after the inducing culture fate is 28 days; Small liwan moss is not induced the formation callus.Can not carry out differentiation culture.
Embodiment 2:
With after the small liwan moss sterilization, it is 0% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, adds the 6-BA hormone of 0.05mg/L and the KT hormone of 0.05mg/L, after the inducing culture fate is 28 days; Small liwan moss is induced the callus of formation little, and brownization is serious, and brownization rate is 100%.
Embodiment 3:
With after the small liwan moss sterilization, it is 0% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, adds the 6-BA hormone of 0.1mg/L and the KT hormone of 0.1mg/L, after the inducing culture fate is 28 days; Small liwan moss is induced the callus of formation little, and brownization is serious, and brownization rate is 100%.
Embodiment 4:
With after the small liwan moss sterilization, it is 0% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, adds the 6-BA hormone of 0.5mg/L and the KT hormone of 0.5mg/L, after the inducing culture fate is 28 days; Small liwan moss is induced the callus of formation little, and brownization is serious, and brownization rate is 100%.
Embodiment 5:
With after the small liwan moss sterilization, it is 0.5% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, adds the 6-BA hormone of 0.1mg/L, does not add the KT hormone, after the inducing culture fate is 28 days; Small liwan moss is induced the callus of formation bigger, and structure is tight, but brownization is serious, and brownization rate is 95%.
Embodiment 6:
With after the small liwan moss sterilization, it is 0.5% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, adds the 6-BA hormone of 0.5mg/L and the KT hormone of 0.05mg/L, after the inducing culture fate is 28 days; Small liwan moss is induced the callus of formation bigger, and structure is tight, but brownization is serious, and brownization rate is 95%.
Embodiment 7:
With after the small liwan moss sterilization, it is 1% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, adds the 6-BA hormone of 0.5mg/L, does not add the KT hormone, after the inducing culture fate is 28 days; Small liwan moss is induced the callus of formation bigger, and structure is tight, but brownization is serious, and brownization rate is 95%.
Embodiment 8:
With after the small liwan moss sterilization, it is 1% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, does not add the 6-BA hormone, adds the KT hormone of 0.5mg/L, after the inducing culture fate is 28 days; Small liwan moss is induced the callus of formation bigger, and structure is comparatively loose, and brownization rate is about 85%.
Embodiment 9:
With after the small liwan moss sterilization, it is 1% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, adds the 6-BA hormone of 0.1mg/L and the KT hormone of 0.05mg/L, after the inducing culture fate is 28 days; Small liwan moss is induced the callus of formation bigger, and structure is comparatively loose, and brownization rate is about 85%.
Embodiment 10:
With after the small liwan moss sterilization, it is 2% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, adds the 6-BA hormone of 0.1mg/L and the KT hormone of 0.5mg/L, after the inducing culture fate is 28 days; Small liwan moss is induced the callus of formation bigger, and structure is comparatively loose, and brownization rate is about 85%.
Embodiment 11:
With after the small liwan moss sterilization, it is 2% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, adds the 6-BA hormone of 0.5mg/L and the KT hormone of 0.1mg/L, after the inducing culture fate is 28 days; Small liwan moss is induced the callus of formation bigger, and structure is comparatively loose, and brownization rate is about 85%.
Embodiment 12:
With after the small liwan moss sterilization, it is 0.5% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, adds the 6-BA hormone of 0.05mg/L and the KT hormone of 0.5mg/L, after the inducing culture fate is 28 days; Small liwan moss is induced the callus volume of formation big, and structure is tight, and brownization rate is about 75%.
Embodiment 13:
With after the small liwan moss sterilization, it is 1% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, adds the 6-BA hormone of 0.05mg/L and the KT hormone of 0.1mg/L, after the inducing culture fate is 28 days; Small liwan moss is induced the callus volume of formation big, and structure is tight, and brownization rate is about 75%.
Embodiment 14:
With after the small liwan moss sterilization, it is 0.5% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, does not add the 6-BA hormone, adds the KT hormone of 0.1mg/L, after the inducing culture fate is 28 days; Small liwan moss is induced the callus of formation big, loosely organized, and brownization rate is lower, and brownization rate is about 50%.
Embodiment 15:
With after the small liwan moss sterilization, it is 2% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, adds the 6-BA hormone of 0.05mg/L, does not add the KT hormone, after the inducing culture fate is 28 days; Small liwan moss is induced the callus of formation big, loosely organized, and brownization rate is lower, and brownization rate is about 50%.
Embodiment 16:
With after the small liwan moss sterilization, it is 2% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, does not add the 6-BA hormone, adds the KT hormone of 0.05mg/L, after the inducing culture fate is 28 days; Small liwan moss is induced the callus of formation big, and structure is tight, and brownization rate is minimum, only is 15%.
As can be seen, it is bigger to add the callus that induces in the medium of glucose from above embodiment, and that is because glucose provides required carbon source for callus growth, but the size of sugared concentration there is no obvious influence to callus Growth.
And as can be seen, can successfully induce callus as long as add the medium that 6-BA or any hormone of KT or two kinds of hormones all add.Find that by embodiment that adds single hormone induces effect comparatively desirable, especially the effect of embodiment 5, embodiment 15 and embodiment 16 is ideal.
Embodiment 17 to embodiment 20 is after small liwan moss callus induction of the present invention is cultivated, and carries out the embodiment of differentiation culture.
Embodiment 17:
The foregoing description 2 to embodiment 16 is cultivated the small liwan moss callus that produces after 28 days to be transferred on the Knop differential medium that does not add any hormone and to carry out differentiation culture; Cultivating just to can be observed from the small liwan moss callus after 21 days has normal gametophyte to grow, and the gametophyte that this embodiment generates is maximum.
Embodiment 18:
The foregoing description 2 to embodiment 16 is cultivated the small liwan moss callus that produces after 28 days to be transferred on the Knop differential medium that adds the 0.5mg/L6-BA hormone and to carry out differentiation culture; Cultivating just to can be observed from the small liwan moss callus after 21 days has normal gametophyte to grow.
Embodiment 19:
The foregoing description 2 to embodiment 16 is cultivated the small liwan moss callus that produces after 28 days to be transferred on the Knop differential medium that adds the 0.5mg/LKT hormone and to carry out differentiation culture; Cultivating just to can be observed from the small liwan moss callus after 21 days has the minority gametophyte to grow.
Embodiment 20:
The small liwan moss callus that the foregoing description 2 to embodiment 16 cultivations were produced after 28 days is transferred to interpolation 0.5mg/L2, carries out differentiation culture on the Knop differential medium of 4-D hormone; Cultivate and just can be observed from the small liwan moss callus after 21 days, the protonema that grows is maximum, but gametophyte is less.
In step 1) of the present invention, adopt embodiment 5 or embodiment 15 or embodiment 16 to induce the effect of little upright bowl callus ideal, in step 2) in to adopt embodiment 17 to break up the gametophyte that obtains maximum.
Claims (9)
1, a kind of method of inducing small liwan moss to produce callus and differentiate to form gametocyte, it is characterized in that: this method has following steps:
1), with after the small liwan moss sterilization, it is 0-2% that the stem apex that cuts small liwan moss is inoculated into sugared concentration, and contains on the Knop medium of one or both hormones of KT of the 6-BA of 0-0.5mg/L and 0-0.5mg/L and carry out inducing culture, the inducing culture fate is 28 days;
2), will cultivate after 28 days the small liwan moss callus that produces is transferred to and carries out differentiation culture on the Knop differential medium; Or be transferred on the Knop differential medium that contains 0.5mg/L6-BA and carry out differentiation culture; Or be transferred to and contain 2 of 0.5mg/L, carry out differentiation culture on the Knop differential medium of 4-D; Or be transferred on the Knop differential medium that contains 0.5mg/LKT and carry out differentiation culture, cultivating fate is 21 days;
The condition of described inducing culture and differentiation culture is: cultivation temperature is 24-26 ℃, and periodicity of illumination is 12h/d, and illuminance is 3000-4000lx.
2, a kind of method of inducing small liwan moss to produce callus and differentiate to form gametocyte according to claim 1, it is characterized in that: above-mentioned steps 1) the small liwan moss callus being carried out the employed sugared concentration of inducing culture is in 0.5% the Knop medium, add the KT hormone of 0.1mg/L, above-mentioned steps 2) in the small liwan moss callus that induces is transferred in the Knop differential medium that does not add any hormone.
3, a kind of method of inducing small liwan moss to produce callus and differentiate to form gametocyte according to claim 1, it is characterized in that: above-mentioned steps 1) the small liwan moss callus being carried out the employed sugared concentration of inducing culture is in 2% the Knop medium, add the KT hormone of 0.05mg/L, above-mentioned steps 2) in the small liwan moss callus that induces is transferred in the Knop differential medium that does not add any hormone.
4, a kind of method of inducing small liwan moss to produce callus and differentiate to form gametocyte according to claim 1, it is characterized in that: above-mentioned steps 1) the small liwan moss callus being carried out the employed sugared concentration of inducing culture is in 2% the Knop medium, add the 6-BA hormone of 0.05mg/L, above-mentioned steps 2) in the small liwan moss callus that induces is transferred in the Knop differential medium that does not add any hormone.
5, a kind of method of inducing small liwan moss to produce callus and differentiate to form gametocyte according to claim 1, it is characterized in that: described Knop medium adding percentage by weight is 0.8% agar, and above-mentioned Knop medium pH value is adjusted to 6.5; Contain KCL0.07604g, MgSO among the every 1L Knop of described Knop differential medium
47H
2O 0.8282g, KH
2PO
40.2504g, Ca (NO
3)
24H
2O 1.0013g, CuSO
45H
2O 0.027mg, KI 0.014mg, ZnSO
47H
2O 0.027mg, H
3BO
30.309mg, Na
2MoO
42H
2O 0.012mg, MnCl
24H
2O 0.198mg, CoCl
26H
2O 0.027mg, FeSO
47H
2O12.5088mg, tartaric acid ammonia 0.4604mg, and to add percentage by weight be that 0.5% glucose and percentage by weight are 0.8% agar, and above-mentioned Knop differential medium pH value is adjusted to 6.5.
6, a kind of method of inducing small liwan moss to produce callus and differentiate to form gametocyte according to claim 2, it is characterized in that: described Knop medium adding percentage by weight is 0.8% agar, and above-mentioned Knop medium pH value is adjusted to 6.5; Contain KCL 0.07604g, MgSO among the every 1L Knop of described Knop differential medium
47H
2O 0.8282g, KH
2PO
40.2504g, Ca (NO
3)
24H
2O 1.0013g, CuSO
45H
2O 0.027mg, KI 0.014mg, ZnSO
47H
2O 0.027mg, H
3BO
30.309mg, Na
2MoO
42H
2O 0.012mg, MnCl
24H
2O 0.198mg, CoCl
26H
2O 0.027mg, FeSO
47H
2O12.5088mg, tartaric acid ammonia 0.4604mg, and to add percentage by weight be that 0.5% glucose and percentage by weight are 0.8% agar, and above-mentioned Knop differential medium pH value is adjusted to 6.5.
7, a kind of method of inducing small liwan moss to produce callus and differentiate to form gametocyte according to claim 3, it is characterized in that: described Knop medium adding percentage by weight is 0.8% agar, and above-mentioned Knop medium pH value is adjusted to 6.5; Contain KCL 0.07604g, MgSO among the every 1L Knop of described Knop differential medium
47H
2O 0.8282g, KH
2PO
40.2504g, Ca (NO
3)
24H
2O 1.0013g, CuSO
45H
2O 0.027mg, KI 0.014mg, ZnSO
47H
2O 0.027mg, H
3BO
30.309mg, Na
2MoO
42H
2O 0.012mg, MnCl
24H
2O 0.198mg, CoCl
26H
2O 0.027mg, FeSO
47H
2O12.5088mg, tartaric acid ammonia 0.4604mg, and to add percentage by weight be that 0.5% glucose and percentage by weight are 0.8% agar, and above-mentioned Knop differential medium pH value is adjusted to 6.5.
8, a kind of method of inducing small liwan moss to produce callus and differentiate to form gametocyte according to claim 4, it is characterized in that: described Knop medium adding percentage by weight is 0.8% agar, and above-mentioned Knop medium pH value is adjusted to 6.5; Contain KCL 0.07604g, MgSO among the every 1L Knop of described Knop differential medium
47H
2O 0.8282g, KH
2PO
40.2504g, Ca (NO
3)
24H
2O 1.0013g, CuSO
45H
2O 0.027mg, KI 0.014mg, ZnSO
47H
2O 0.027mg, H
3BO
30.309mg, Na
2MoO
42H
2O 0.012mg, MnCl
24H
2O 0.198mg, CoCl
26H
2O 0.027mg, FeSO
47H
2O12.5088mg, tartaric acid ammonia 0.4604mg, and to add percentage by weight be that 0.5% glucose and percentage by weight are 0.8% agar, and above-mentioned Knop differential medium pH value is adjusted to 6.5.
9, according to the described a kind of method of inducing small liwan moss to produce callus and differentiate to form gametocyte of the arbitrary claim of claim 5 to 8, it is characterized in that: adopt 1N NaOH that the pH value of above-mentioned Knop medium and Knop differential medium is adjusted to 6.5.
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|---|---|---|---|
| CN 200510026616 CN1292645C (en) | 2005-06-09 | 2005-06-09 | Method of inducing small liwan moss to produce callus and differentiate to form gametocyte |
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| CN1292645C true CN1292645C (en) | 2007-01-03 |
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| CN106359092B (en) * | 2016-08-30 | 2018-12-14 | 中国科学院昆明植物研究所 | Small liwan moss protonema rapid propagation method |
| CN106119185A (en) * | 2016-08-30 | 2016-11-16 | 中国科学院昆明植物研究所 | A kind of preparation method of small liwan moss protoplast |
| CN112852864A (en) * | 2021-02-13 | 2021-05-28 | 中国科学院新疆生态与地理研究所 | Stable genetic transformation method of agrobacterium-mediated moss |
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