CN108849511B - Tissue culture method of young populus tomentosa seedlings - Google Patents
Tissue culture method of young populus tomentosa seedlings Download PDFInfo
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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
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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
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Abstract
The invention provides a tissue culture method of young populus tomentosa seedlings, which comprises the following steps: (1) taking a Populus tomentosa explant, and sterilizing to obtain a detoxified explant; (2) inoculating the detoxified explant into an induction culture medium for induction culture to obtain an adventitious bud; (3) inoculating the adventitious bud into a subculture medium for subculture to obtain a subculture seedling; (4) and inoculating the subculture seedling into a rooting culture medium for rooting culture to obtain a rooted seedling. The method solves the problems that the propagation efficiency of the populus tomentosa is low and the industrial production cannot be realized, efficiently induces the adventitious buds of the populus tomentosa, screens out the culture medium formula with the propagation coefficient reaching 3.8, and shortens the propagation period of the populus tomentosa.
Description
Technical Field
The invention relates to the technical field of poplar breeding, in particular to a tissue culture method of young populus tomentosa seedlings.
Background
The branches of the Populus makinoi are from campus of university of agriculture in China, are varieties obtained by hybridizing Populus makinoi and Populus Sinkiangensis cultivated by professor xu weyayingying and the like in the fifty years, and have the excellent characteristics of the Populus makinoi. Compared with Chinese white poplar, the Chinese white poplar does not fly, and two canopy width of the Chinese white poplar grows in a rhombus shape. Therefore, the cultivation of the populus tomentosa is worth popularizing.
At present, the poplar is bred by using a tissue culture method in China. Namely, a stem section or a stem tip is used as an explant, and the explant is inoculated on a culture medium after being sterilized to obtain a test-tube seedling with high rooting rate. However, there is currently no corresponding tissue culture method for populus tomentosa.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a tissue culture method of young populus tomentosa seedlings.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention relates to a tissue culture method of young populus tomentosa seedlings, which comprises the following steps:
(1) taking a Populus tomentosa explant, and sterilizing to obtain a detoxified explant;
(2) inoculating the detoxified explant into an induction culture medium for induction culture to obtain an adventitious bud;
(3) inoculating the adventitious bud into a subculture medium for subculture to obtain a subculture seedling;
(4) and inoculating the subculture seedling into a rooting culture medium for rooting culture to obtain a rooted seedling.
Preferably, in the step (1), the explant is semi-lignified twig of populus tomentosa or 2-3 leaves below the top bud of populus tomentosa.
Preferably, in the step (1), the sterilization treatment is to wash the explant with sterile water after soaking the explant with an alcohol solution and a mercuric chloride solution in sequence.
Preferably, in the step (1), the volume fraction of the alcohol solution is 75%, and the mass concentration of the mercuric chloride solution is 0.2%.
Preferably, in the step (2), the induction medium is based on an MS medium and is obtained by adding 6-BA, IBA, sucrose and agar, wherein the concentration of the 6-BA in the induction medium is 0.3mg/L, the concentration of the IBA is 0.2mg/L, the concentration of the sucrose is 30g/L, the concentration of the agar is 6g/L, and the pH value of the induction medium is 5.8-6.0.
Preferably, in the step (2), the induction culture is to inoculate the detoxified explant into an induction culture medium, and culture is carried out for 15-25 days under the conditions that the temperature is 25 +/-1 ℃, the illumination intensity is 1800-2200 Lx and the illumination time is 12-16 h/day until adventitious buds are generated.
Preferably, in the step (3), the subculture medium is based on an MS culture medium and is obtained by adding 6-BA, IBA, sucrose and agar, wherein the concentration of the 6-BA in the subculture medium is 0.2mg/L, the concentration of the IBA is 0.1mg/L, the concentration of the sucrose is 30g/L, the concentration of the agar is 6g/L, and the pH value of the subculture medium is 5.8-6.0.
Preferably, in the step (3), the subculture is to inoculate the adventitious bud into a subculture medium, culture is carried out under the conditions that the temperature is 25 +/-1 ℃, the illumination intensity is 1800-2200 Lx and the illumination time is 12-16 h/day, subculture is carried out once every 20-25 days, the culture medium is changed for each subculture, and the adventitious bud is differentiated until a subculture seedling is obtained.
Preferably, in the step (4), the rooting medium is obtained by taking 1/2MS medium as a basis and adding NAA, IBA, sucrose and agar, wherein the concentration of NAA in the rooting medium is 0.1mg/L, the concentration of IBA is 0.1mg/L, the concentration of sucrose is 30g/L, the concentration of agar is 6g/L, and the pH value of the rooting medium is 5.8-6.0.
Preferably, in the step (4), the rooting culture is to inoculate the subculture seedling into a rooting culture medium, and culture for 20-30 days under the conditions that the temperature is 25 +/-1 ℃, the illumination intensity is 1800-2200 Lx and the illumination time is 12-16 h/day, so as to obtain the rooting seedling.
Preferably, after the step (4) is finished, the method further comprises the step (5): and placing the rooted seedlings and the rooting culture medium on a seedling bed of a greenhouse, culturing for 7-10 days under natural light, cleaning the rooted seedlings, and culturing in a matrix for 30-40 days to obtain transplanted seedlings.
Preferably, the volume ratio of the grass carbon to the vermiculite to the perlite in the matrix is 3: 2: 1.
the invention has the beneficial effects that:
the invention provides a method for culturing seedlings by aiming at standardized tissues of populus tomentosa, which comprises the following specific operation modes: sterilizing young and tender stem segments of the current year of the populus tomentosa, cutting off two ends, inoculating the cut off two ends into an induction culture medium, and performing induction culture until adventitious buds are generated; then, cutting the adventitious buds into blocks, and placing the blocks in a subculture medium for subculture to obtain subcultured seedlings; cutting the subculture seedling into single plants, and placing the single plants in a rooting culture medium for rooting culture to obtain rooted seedlings; finally, domesticating the rooted seedlings and planting the domesticated rooted seedlings to obtain transplanted seedlings, wherein the survival rate reaches more than 90%. The method solves the problems that the propagation efficiency of the populus tomentosa is low and the industrial production cannot be realized, efficiently induces the adventitious buds of the populus tomentosa, screens out the culture medium formula with the propagation coefficient reaching 3.8, and shortens the propagation period of the populus tomentosa.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
The embodiment of the invention relates to a tissue culture method of young populus tomentosa seedlings, which comprises the following steps:
(1) and (3) explant sterilization treatment:
and (3) taking the populus tomentosa explant and carrying out sterilization treatment to obtain the detoxified explant.
Wherein, the explant is a segment of plant organ or tissue as an isolated culture material, and has the characteristics of vigorous metabolism and strong regeneration capacity. In one embodiment of the invention, the explant is a semi-lignified shoot of Populus deltoides, or 2-3 leaves under the apical bud of Populus deltoides.
In one embodiment of the invention, the sterilization treatment is to soak the explant with an alcohol solution and a mercuric chloride solution in sequence, and then wash the explant with sterile water. Mercuric chloride is known as mercuric chloride, and is in the form of white crystals, granules or powder, and can be used as disinfectant and antiseptic. The volume fraction of the alcohol solution can be 75%, and the mass concentration of the mercuric chloride solution can be 0.2%.
In one embodiment of the present invention, step (1) comprises: sterilizing annual young stems of populus tomentosa in 75% alcohol for 40-60 seconds, transferring the young stems into 0.2% mercuric chloride solution for sterilization for 10-15 minutes, and then washing with sterile water for 3-5 times to obtain the detoxified explants.
(2) Culturing adventitious buds:
inoculating the detoxified explant into an induction culture medium for induction culture to obtain an adventitious bud.
In one embodiment of the invention, the induction culture medium is obtained by taking an MS culture medium as a base and adding 6-BA, IBA, sucrose and agar, wherein the concentration of the 6-BA in the induction culture medium is 0.3mg/L, the concentration of the IBA is 0.2mg/L, the concentration of the sucrose is 30g/L, the concentration of the agar is 6g/L, and the pH value of the induction culture medium is 5.8-6.0.
Wherein IBA is indolebutyric acid, which has promoting effect on the top shoot apex formation of plant twigs or buds, seedlings and the like, and can be replaced by indoleacetic acid. The 6-BA is benzylamino adenine, is an artificially synthesized cytokinin, and has the characteristics of high efficiency, stability, low price, easy use and the like. The main function of 6-BA is to promote the formation of buds and also to induce callus formation.
In one embodiment of the present invention, the specific conditions of the induction culture are: culturing for 15-25 days under the conditions that the temperature is 25 +/-1 ℃, the illumination intensity is 1800-2200 Lx and the illumination time is 12-16 h/day until adventitious buds are generated.
In one embodiment of the present invention, step (2) comprises: in an aseptic working environment, cutting off two ends of a disinfected stem segment which are contacted with a disinfectant, then cutting the stem segment into segments with the length of 0.5 cm-1 cm and 1-2 growing points, and inoculating the segments into an induction culture medium: the induction culture medium is obtained by taking an MS culture medium as a base and adding 6-BA, IBA, sucrose and agar, wherein the concentration of the 6-BA in the induction culture medium is 0.3mg/L, the concentration of the IBA is 0.2mg/L, the concentration of the sucrose is 30g/L, the concentration of the agar is 6g/L, and the pH value is 5.8-6.0. Culturing for 15-25 days at the temperature of 25 +/-1 ℃, the illumination intensity of 1800-2200 Lx and the illumination time of 12-16 h/day until adventitious buds are generated.
(3) Subculturing:
inoculating the adventitious bud into a subculture medium for subculture to obtain a subculture seedling.
In one embodiment of the invention, the subculture medium is based on an MS medium and is obtained by adding 6-BA, IBA, sucrose and agar, wherein the concentration of the 6-BA in the subculture medium is 0.2mg/L, the concentration of the IBA is 0.1mg/L, the concentration of the sucrose is 30g/L, the concentration of the agar is 6g/L, and the pH value of the subculture medium is 5.8-6.0.
In one embodiment of the present invention, the specific conditions of the subculture are: culturing at the temperature of 25 +/-1 ℃, the illumination intensity of 1800-2200 Lx and the illumination time of 12-16 h/day, carrying out subculture every 20-25 days, replacing a culture medium for each subculture, and carrying out segmentation and differentiation on adventitious buds until obtaining subculture seedlings.
In one embodiment of the present invention, step (3) comprises: in a sterile working environment, the adventitious buds are cut into pieces and placed in a subculture medium: the subculture medium is obtained by taking an MS culture medium as a base and adding 6-BA, IBA, sucrose and agar, wherein the concentration of the 6-BA in the subculture medium is 0.2mg/L, the concentration of the IBA is 0.1mg/L, the concentration of the sucrose is 30g/L, the concentration of the agar is 6g/L, and the pH value of the subculture medium is 5.8-6.0. And carrying out subculture under the conditions that the temperature is 25 +/-1 ℃, the illumination intensity is 1800-2200 Lx and the illumination time is 12-16 h/day, carrying out subculture once every 20-25 days, replacing the culture medium for each subculture, and differentiating the adventitious buds until obtaining the subcultured seedlings. The inventors found that if NAA (naphthylacetic acid, a broad-spectrum plant growth regulator) is added to the secondary culture medium, the plant height and the multiplication factor of populus tomentosa are reduced, and therefore it is preferable that NAA is not added to the secondary culture medium.
(4) Rooting culture:
and inoculating the subculture seedling into a rooting culture medium for rooting culture to obtain a rooted seedling.
In one embodiment of the invention, the rooting medium is obtained by taking 1/2MS medium as a basis and adding NAA, IBA, sucrose and agar, wherein the concentration of NAA in the rooting medium is 0.1mg/L, the concentration of IBA is 0.1mg/L, the concentration of sucrose is 30g/L, the concentration of agar is 6g/L, and the pH value of the rooting medium is 5.8-6.0.
In one embodiment of the present invention, the specific conditions for rooting culture are: culturing for 20-30 days under the conditions that the temperature is 25 +/-1 ℃, the illumination intensity is 1800-2200 Lx and the illumination time is 12-16 h/day to obtain the rooting seedlings.
In one embodiment of the present invention, the step (4) comprises: the subculture seedling is cut into individual plants and placed in a rooting medium. The rooting medium is obtained by taking 1/2MS medium as a base and adding NAA, IBA, sucrose and agar, wherein the concentration of NAA in the rooting medium is 0.1mg/L, the concentration of IBA is 0.1mg/L, the concentration of sucrose is 30g/L, the concentration of agar is 6g/L, and the pH value of the rooting medium is 5.8-6.0. And culturing for 20-30 days under the conditions that the temperature is 25 +/-1 ℃, the illumination intensity is 1800-2200 Lx and the illumination time is 12-16 h/day to obtain the rooting seedlings.
In an embodiment of the invention, after the step (4) is finished, the method further comprises a step (5) of hardening and transplanting the seedlings. The "hardening off" refers to a process of gradually adapting the rooted tissue culture seedling to the atmospheric environment under the conditions of properly controlling temperature, humidity, gas exchange, etc. The specific process is as follows:
placing the rooted seedlings and a rooting culture medium on a seedbed of a greenhouse together, culturing for 7-10 days under natural light, cleaning the rooted seedlings, soaking in carbendazim with the mass concentration of 0.1-0.2% for 2-5 min, transplanting in a matrix, and obtaining transplanted seedlings after 30-40 days, wherein the survival rate is more than 90%.
In one embodiment of the invention, the volume ratio of the turf, the vermiculite and the perlite in the matrix is 3: 2: 1. as the main component of the matrix, the vermiculite is a natural, inorganic and nontoxic mineral substance, has good water permeability, and can effectively promote the growth of plant roots and the stable development of seedlings. Provides water and nutrition necessary for plant growth for a long time, and can keep the temperature of the sunlight of the roots stable. The vermiculite can make crops obtain sufficient water and mineral substances from the early growth stage, promote the plants to grow faster and increase the yield. As the transplanted rooted seedlings grow at a high speed, the matrix needs to contain more nutrients, and turf is added into the matrix.
Example 1
Culturing populus tomentosa by adopting an in-bottle rooting method:
(1) and (3) explant sterilization treatment:
sterilizing annual young stems of populus tomentosa in 75% alcohol for 40-60 seconds, transferring the young stems into 0.2% mercuric chloride solution for sterilization for 10-15 minutes, and then washing with sterile water for 3-5 times to obtain the detoxified explants.
(2) Culturing adventitious buds:
in an aseptic working environment, cutting off two ends of a disinfected stem segment which are contacted with a disinfectant, then cutting the stem segment into segments with the length of 0.5 cm-1 cm and 1-2 growing points, and inoculating the segments into an induction culture medium: the induction culture medium is obtained by taking an MS culture medium as a base and adding 6-BA, IBA, sucrose and agar, wherein the concentration of the 6-BA in the induction culture medium is 0.3mg/L, the concentration of the IBA is 0.2mg/L, the concentration of the sucrose is 30g/L, the concentration of the agar is 6g/L, and the pH value is 5.8-6.0. Culturing for 15-25 days at the temperature of 25 +/-1 ℃, the illumination intensity of 1800-2200 Lx and the illumination time of 12-16 h/day until adventitious buds are generated.
(3) Subculturing:
in a sterile working environment, the adventitious buds are cut into pieces and placed in a subculture medium: the subculture medium is obtained by taking an MS culture medium as a base and adding 6-BA, IBA, sucrose and agar, wherein the concentration of the 6-BA in the subculture medium is 0.1-0.3 mg/L, the concentration of the IBA is 0.1-0.3 mg/L, the concentration of the sucrose is 30g/L, the concentration of the agar is 6g/L, and the pH value of the subculture medium is 5.8-6.0. And carrying out subculture under the conditions that the temperature is 25 +/-1 ℃, the illumination intensity is 1800-2200 Lx and the illumination time is 12-16 h/day, carrying out subculture once every 20-25 days, replacing the culture medium for each subculture, and differentiating the adventitious buds until obtaining the subcultured seedlings.
(4) Rooting culture:
the subculture seedling is cut into individual plants and placed in a rooting medium. The rooting medium is obtained by taking 1/2MS medium as a base and adding NAA, IBA, sucrose and agar, wherein the concentration of NAA in the rooting medium is 0.1mg/L, the concentration of IBA is 0.1mg/L, the concentration of sucrose is 30g/L, the concentration of agar is 6g/L, and the pH value of the rooting medium is 5.8-6.0. And culturing for 20-30 days under the conditions that the temperature is 25 +/-1 ℃, the illumination intensity is 1800-2200 Lx and the illumination time is 12-16 h/day to obtain the rooting seedlings.
(5) Hardening and transplanting seedlings:
placing the rooted seedlings and a rooting culture medium on a seedbed of a greenhouse together, culturing for 7-10 days under natural light, cleaning the rooted seedlings, soaking in carbendazim with the mass concentration of 0.1-0.2% for 2-5 min, transplanting in a matrix, and obtaining transplanted seedlings after 30-40 days. The volume ratio of the grass carbon, the vermiculite and the perlite in the matrix is 3: 2: 1.
examples 1-1 to 1-5
And (3) changing the types and the use amounts of the reagents in the subculture medium in the step (3), and observing the influence of different hormone combinations on the propagation coefficient of the adventitious buds of the populus tomentosa, wherein the results are shown in a table 1. Wherein the proliferation multiple is the total proliferation number of the growth points of the explants/the total number of the differentiated explants, and is calculated by adopting an averaging method.
TABLE 1
As can be seen from Table 1, in comparative examples 1-1 to 1-3, the plant height and the multiplication factor of Populus tomentosa were the best when the concentration of 6-BA was 0.2 mg/L. However, it is understood from examples 1-3 to 1-5 that the plant height and the growth effect are most significant when the concentration of IBA is 0.1mg/L and the concentration of 6-BA is 0.2mg/L, and particularly when NAA is not used, the multiplication factor is 3.8 or more. Thus for the subculture step, the most suitable subculture medium formulation is MS +0.2mg/L of 6-BA +0.1mg/L of IBA.
Example 2
In the subculture of the step (3), the concentration of 6-BA in the subculture medium is 0.2mg/L, the concentration of IBA is 0.1mg/L, the concentration of sucrose is 30g/L, the concentration of agar is 6g/L, NAA is not used, and the pH value of the subculture medium is 5.8-6.0.
In the rooting culture of the step (4), the concentration of NAA in the rooting culture medium is 0.1-0.3 mg/L, the concentration of IBA is 0.1-0.5 mg/L, the concentration of sucrose is 30g/L, the concentration of agar is 6g/L, and the pH value of the rooting culture medium is 5.8-6.0. The other steps and operation are the same as in example 1.
Example 2-1 to example 2-5
And (3) changing the types and the use amounts of the reagents in the rooting culture medium in the step (4), and observing the influence of different hormone combinations on the rooting rate and the number of roots of the populus tomentosa, wherein the results are shown in a table 2.
TABLE 2
As can be seen from Table 2, the populus tomentosa can root on the rooting medium of examples 2-1 to 2-5, but the rooting rate is low when the concentration of NAA and IBA is not satisfactory, indicating that the populus tomentosa is a plant which is not easy to root. Comparing the above examples, it can be seen that the most suitable rooting medium formulation is 1/2MS +0.1mg/L NAA +0.1mg/L IBA.
Comparative example 1
The procedure of example 1 was followed to grow populus tomentosa, and the procedure of example 1 was repeated except that the species of the populus tomentosa was changed, and the results are shown in Table 3.
TABLE 3
The average plant height and the multiplication factor in table 3 are greatly reduced compared with those in table 1, which shows that the method of the invention is only suitable for cultivating populus tomentosa and is not suitable for other populus varieties.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (7)
1. A tissue culture method of young populus tomentosa seedlings is characterized by comprising the following steps:
(1) taking a Populus tomentosa explant, and sterilizing to obtain a detoxified explant; the explant is a semi-lignified twig of the populus tomentosa or 2-3 leaves below the top bud of the populus tomentosa;
(2) inoculating the detoxified explant into an induction culture medium for induction culture to obtain an adventitious bud; the induction culture medium is obtained by taking an MS culture medium as a basis and adding 6-BA, IBA, sucrose and agar, wherein the concentration of the 6-BA in the induction culture medium is 0.3mg/L, the concentration of the IBA is 0.2mg/L, the concentration of the sucrose is 30g/L, the concentration of the agar is 6g/L, and the pH value of the induction culture medium is 5.8-6.0;
(3) inoculating the adventitious bud into a subculture medium for subculture to obtain a subculture seedling; the subculture medium is obtained by taking an MS culture medium as a basis and adding 6-BA, IBA, sucrose and agar, wherein the concentration of the 6-BA in the subculture medium is 0.2mg/L, the concentration of the IBA is 0.1mg/L, the concentration of the sucrose is 30g/L, the concentration of the agar is 6g/L, and the pH value of the subculture medium is 5.8-6.0;
(4) inoculating the subculture seedling into a rooting culture medium for rooting culture to obtain a rooted seedling; the rooting medium is obtained by taking 1/2MS medium as a basis and adding NAA, IBA, sucrose and agar, wherein the concentration of NAA in the rooting medium is 0.1mg/L, the concentration of IBA is 0.1mg/L, the concentration of sucrose is 30g/L, the concentration of agar is 6g/L, and the pH value of the rooting medium is 5.8-6.0.
2. The method according to claim 1, wherein in the step (1), the sterilization treatment is that the explant is soaked by using an alcohol solution and a mercuric chloride solution in sequence and then washed by using sterile water; the volume fraction of the alcohol solution is 75%, and the mass concentration of the mercuric chloride solution is 0.2%.
3. The method according to claim 1, wherein in the step (2), the induction culture is carried out by inoculating the detoxified explant into an induction medium, and culturing for 15-25 days under the conditions of temperature of 25 +/-1 ℃, illumination intensity of 1800-2200 Lx and illumination time of 12-16 h/day until adventitious buds are generated.
4. The method according to claim 1, wherein in the step (3), the adventitious bud is inoculated into a subculture medium, the subculture is carried out at a temperature of 25 ± 1 ℃, a light intensity of 1800-2200 Lx and a light time of 12-16 h/day, the subculture is carried out every 20-25 days, the medium is changed for each subculture, and the adventitious bud is differentiated until a subculture seedling is obtained.
5. The method according to claim 1, wherein in the step (4), the rooting culture is to inoculate the subculture seedling into a rooting culture medium, and culture for 20-30 days under the conditions of temperature of 25 +/-1 ℃, illumination intensity of 1800-2200 Lx and illumination time of 12-16 h/day to obtain the rooting seedling.
6. The method of claim 1, wherein after the step (4) is finished, the method further comprises the step (5): and placing the rooted seedlings and the rooting culture medium on a seedling bed of a greenhouse, culturing for 7-10 days under natural light, cleaning the rooted seedlings, and culturing in a matrix for 30-40 days to obtain transplanted seedlings.
7. The method according to claim 6, wherein the volume ratio of the turf, the vermiculite and the perlite in the matrix is 3: 2: 1.
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| CN106561458A (en) * | 2016-11-09 | 2017-04-19 | 金陵科技学院 | Production method for regenerated plants of good single plant leaves of hybrid F1 generation of populus deltoids 69 and populus euramericana 72 |
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