Rhododendrons are rhododendrons. The azaleas are evergreen or deciduous shrubs with many varieties, early flowering, rich flowers, and long flowering period. In recent years, due to the development of cutting propagation technology, the production of azaleas in China has increased. However, rare breeds are still unable to meet the needs of the community due to low maternity and low breeding coefficient. Tissue culture is an effective measure to achieve azalea plant nursery. The following is a brief description of it:
A. Rhododendron tissue culture of the basic technical links Rhododendron tissue culture, the commonly used explants are shoot tips, stem sections and buds. Due to the difficulty of bud differentiation, the cycle is long, and the extraction of stem tips is limited. Therefore, stem segments are often used as primary explants in the country.
When sterilizing, mercury-enhanced mercury (mercuric chloride), which has a strong bactericidal capacity, can easily cause browning and extermination of explant materials, and should not be used. Tests have shown that the use of saturated calcium hypochlorite supernatant or 5% sodium hypochlorite solution as a sterilizer, the effect is good, the processing time is 15 to 20 minutes. To enhance the sterilization effect, it is also possible to add 1% capsaicin or 0.1% Tween-20 (sorbitol). The pre-sterilization pre-treatment of the material is usually done with 70% alcohol soaking for no more than 30 seconds. In addition, the pretreatment of materials with neutral detergents can also effectively reduce the contamination rate after sterilization. Sterilization process of explant material: washing powder soaking for 20 minutes? Rinse with water until it is clear, 70% alcohol soak for 30 seconds, sterile water for 1 minute, 5% sodium hypochlorite for 15 minutes, sterile water, rinse 3 times 1 minute. In this way, the explant contamination rate can be reduced to less than 15%.
The culture medium for azalea tissue culture was Andeson modified MS medium: the amount of ammonium nitrate and potassium nitrate in the MS medium was reduced to 1/4, and the potassium iodide component was eliminated, and the amount of iron salt was doubled. Since the rhododendron family is native to high acid soils, the medium should have a pH of 5.0 to 5.4. The incubation room temperature was 25°C, the light intensity was 3000 lux, and the illumination time was 12 to 14 hours.
The hormones used in the budding culture of azaleas are different from those of ordinary plants and require more active cytokinins to achieve satisfactory results. The experiment showed that the bud induction effect was most significant when using ZT; the treatment with KT had almost no buds or lateral buds, and only the elongation of the original shoot tips; and the use of 6-BA treatment resulted in increased browning of the culture materials. It's better than control. In addition, in the subsequent proliferation culture, the requirement for the species of cytokinin in azaleas is still to add ZT.
In foreign countries, 2ip is used as an exogenous cytokinin in tissue culture of azaleas, and the effect is better than that of ZT. However, from an economic point of view, ZT is still recommended.
Second, the first generation and secondary propagation of rhododendron culture buds culture of the first generation of azaleas, NAA concentration can not exceed 0.1mg/L, ZT dosage has a significant role in promoting the budding effect. However, when the concentration of ZT is increased, the concentration of NAA should not increase synchronously, and the ZT/NAA value should be large. Based on the significance of economic cultivation, the ratio of hormones cultured in the first generation is preferably ZT5+NAA0.01 (mg/L). When cultured for one month or so, the induction rate is 100% and the budding coefficient is 6.5.
Rhododendron subcultured and cultured had lower cytokinin requirements than primary cultures. Although the proliferation effect still increased with the increase of the amount of ZT, it was not as obvious as in the initial culture. In addition, the materials used for proliferation and culture have different properties and their proliferation effects are also different. There are also significant differences among varieties. Among the test cultivars, 'Peach Blossom' had a higher proliferation coefficient (7.6) when the clustered shoots were used as a propagating culture, while 'Flower Spring Rain' was the opposite, and the reaction when taking a bud shoot was preferred (7.5).
Rhododendron tissue culture, from the inoculation to the bud, the time required is significantly longer than the rose and chrysanthemum, etc., so the cycle of subculture proliferation is also longer. In the test, 30 days and 45 days were taken as the culture period. The total number of proliferating shoots in each hormone leveling treatment was higher by 50% to 80% than that of the 30th day in culture. Therefore, in the rhododendron tissue culture process, cutting and transformation are often conducted too often, and the purpose of rapid propagation is not achieved on the contrary.
Third, the rooting of test tube seedlings and transplanting Rhododendron root culture is not very difficult, but the requirements of the medium is still different from the general plant. First, the amount of inorganic elements in basic culture cannot be halved; second, the amount of sucrose is still 30 g/L, and the amount of auxin NAA added is 1.5 to 2.0 mg/L, and the rooting rate is 45 days. About 60%.
The test tube seedlings for rooting need to be more than 15 cm tall, with more than 7 true leaves and robust development. Rooting was observed after 2 weeks of rooting, and the highest rooting rate was reached at 6 weeks. In the experiment, it was found that when KT or 6-BA was used to replace ZT in the proliferation and culture process, the buds and proliferation effects were poor, but the buds developed more stoutly. Therefore, after proliferating culture reaches a certain number of groups, subculture with KT or 6-BA can significantly increase the rooting rate and transplanting survival rate. In addition, the addition of 2g/L of activated carbon to the rooting medium can significantly increase the individual development of the test tube seedlings, and the rooting rate can reach more than 90% after one month.
According to foreign research data, some plants that are difficult to root in conventional propagation (cutting and cutting) can be easily rooted by tissue culture. Some can be transplanted directly into rooting medium without rooting and the rooting rate can reach 100%. After 3 to 4 weeks, they can be transplanted into the greenhouse for potting. This rooting medium is a mixture of peat, vermiculite, and perlite, with a pH of 4 to 4.5 being preferred.
The rooting test tube seedlings were transplanted into the soil and the cultivation substrate was a 1:1 mixture of peat moss + vermiculite. The surface layer is required to be sieved to facilitate the attachment of the root system; the lower layer is covered with coarse particles to facilitate the permeability of the substrate. When transplanting, use tweezers to remove the root attached to the medium (not to damage the roots). Use a tip to set a hole in the substrate, place the root system, cover the soil, lightly compact and pour enough water. The key to survival of transplanting test-tube seedlings is to ensure certain temperature and humidity conditions. The conditions for transplanting and test-tube culture vary widely, often resulting in discomfort or death of transplanted seedlings.
Although the transplanting of tube rooting plants can be carried out throughout the year, it is safer and more convenient for the spring and fall seasons without the rooting room.
Fourth, the application of tissue culture technology in the breeding of new varieties of rhododendron at the beginning of the test, given that the azalea flower explants are difficult to sterilize, the inoculation groups available for testing are too small to meet the normal test. In order to screen out the appropriate medium formulation and the corresponding culture conditions as soon as possible, the author combined the methods of cross breeding, using hybrid seed tubes to germinate, and then used the test tube seedlings to study the epicotyl for explants, achieving a multiplier effect. The authors found that the hybrid seedlings of Rhododendron need 3 to 5 years for flowering. However, the hybrid seedlings cultured in the epicotyl can be flowered in the second year after transplanting, greatly shortening the childhood of hybrid seedlings. It is of great significance to speed up the process of breeding flowers and fruit plants.
In general, seed sterilization is much easier than other organs or tissues. The sterilized treatment of azaleas hybrid seeds was as follows: 70% alcohol was soaked on the surface for 1 minute, rinsed with sterile water, then transferred to 0.1% mercury-mercuring solution for 10 minutes, and sterilized water was rinsed 3 times for more than 1 minute each time. After sterilization, seeds were taken and seedlings were inoculated and cultivated. Seeding was initiated within two weeks after inoculation. After emergence, the embryonic shoots were propagated using the epicotyl for rapid propagation and the propagation coefficient was high. After emergence, the shoot tip or stem section may be re-cultivated. The culture conditions and methods are the same as above. Domestic tissue culture involves a small number of new breeds, especially in ornamental horticultural plants. From the development point of view, in order to maintain strong vitality, tissue culture techniques should not leave the breeding of new varieties. Therefore, organically combining tissue culture technology with the selection and breeding of new varieties of azaleas will be very beneficial to improving the level of greenery plant landscape construction and promoting the industrialization of azaleas.
A. Rhododendron tissue culture of the basic technical links Rhododendron tissue culture, the commonly used explants are shoot tips, stem sections and buds. Due to the difficulty of bud differentiation, the cycle is long, and the extraction of stem tips is limited. Therefore, stem segments are often used as primary explants in the country.
When sterilizing, mercury-enhanced mercury (mercuric chloride), which has a strong bactericidal capacity, can easily cause browning and extermination of explant materials, and should not be used. Tests have shown that the use of saturated calcium hypochlorite supernatant or 5% sodium hypochlorite solution as a sterilizer, the effect is good, the processing time is 15 to 20 minutes. To enhance the sterilization effect, it is also possible to add 1% capsaicin or 0.1% Tween-20 (sorbitol). The pre-sterilization pre-treatment of the material is usually done with 70% alcohol soaking for no more than 30 seconds. In addition, the pretreatment of materials with neutral detergents can also effectively reduce the contamination rate after sterilization. Sterilization process of explant material: washing powder soaking for 20 minutes? Rinse with water until it is clear, 70% alcohol soak for 30 seconds, sterile water for 1 minute, 5% sodium hypochlorite for 15 minutes, sterile water, rinse 3 times 1 minute. In this way, the explant contamination rate can be reduced to less than 15%.
The culture medium for azalea tissue culture was Andeson modified MS medium: the amount of ammonium nitrate and potassium nitrate in the MS medium was reduced to 1/4, and the potassium iodide component was eliminated, and the amount of iron salt was doubled. Since the rhododendron family is native to high acid soils, the medium should have a pH of 5.0 to 5.4. The incubation room temperature was 25°C, the light intensity was 3000 lux, and the illumination time was 12 to 14 hours.
The hormones used in the budding culture of azaleas are different from those of ordinary plants and require more active cytokinins to achieve satisfactory results. The experiment showed that the bud induction effect was most significant when using ZT; the treatment with KT had almost no buds or lateral buds, and only the elongation of the original shoot tips; and the use of 6-BA treatment resulted in increased browning of the culture materials. It's better than control. In addition, in the subsequent proliferation culture, the requirement for the species of cytokinin in azaleas is still to add ZT.
In foreign countries, 2ip is used as an exogenous cytokinin in tissue culture of azaleas, and the effect is better than that of ZT. However, from an economic point of view, ZT is still recommended.
Second, the first generation and secondary propagation of rhododendron culture buds culture of the first generation of azaleas, NAA concentration can not exceed 0.1mg/L, ZT dosage has a significant role in promoting the budding effect. However, when the concentration of ZT is increased, the concentration of NAA should not increase synchronously, and the ZT/NAA value should be large. Based on the significance of economic cultivation, the ratio of hormones cultured in the first generation is preferably ZT5+NAA0.01 (mg/L). When cultured for one month or so, the induction rate is 100% and the budding coefficient is 6.5.
Rhododendron subcultured and cultured had lower cytokinin requirements than primary cultures. Although the proliferation effect still increased with the increase of the amount of ZT, it was not as obvious as in the initial culture. In addition, the materials used for proliferation and culture have different properties and their proliferation effects are also different. There are also significant differences among varieties. Among the test cultivars, 'Peach Blossom' had a higher proliferation coefficient (7.6) when the clustered shoots were used as a propagating culture, while 'Flower Spring Rain' was the opposite, and the reaction when taking a bud shoot was preferred (7.5).
Rhododendron tissue culture, from the inoculation to the bud, the time required is significantly longer than the rose and chrysanthemum, etc., so the cycle of subculture proliferation is also longer. In the test, 30 days and 45 days were taken as the culture period. The total number of proliferating shoots in each hormone leveling treatment was higher by 50% to 80% than that of the 30th day in culture. Therefore, in the rhododendron tissue culture process, cutting and transformation are often conducted too often, and the purpose of rapid propagation is not achieved on the contrary.
Third, the rooting of test tube seedlings and transplanting Rhododendron root culture is not very difficult, but the requirements of the medium is still different from the general plant. First, the amount of inorganic elements in basic culture cannot be halved; second, the amount of sucrose is still 30 g/L, and the amount of auxin NAA added is 1.5 to 2.0 mg/L, and the rooting rate is 45 days. About 60%.
The test tube seedlings for rooting need to be more than 15 cm tall, with more than 7 true leaves and robust development. Rooting was observed after 2 weeks of rooting, and the highest rooting rate was reached at 6 weeks. In the experiment, it was found that when KT or 6-BA was used to replace ZT in the proliferation and culture process, the buds and proliferation effects were poor, but the buds developed more stoutly. Therefore, after proliferating culture reaches a certain number of groups, subculture with KT or 6-BA can significantly increase the rooting rate and transplanting survival rate. In addition, the addition of 2g/L of activated carbon to the rooting medium can significantly increase the individual development of the test tube seedlings, and the rooting rate can reach more than 90% after one month.
According to foreign research data, some plants that are difficult to root in conventional propagation (cutting and cutting) can be easily rooted by tissue culture. Some can be transplanted directly into rooting medium without rooting and the rooting rate can reach 100%. After 3 to 4 weeks, they can be transplanted into the greenhouse for potting. This rooting medium is a mixture of peat, vermiculite, and perlite, with a pH of 4 to 4.5 being preferred.
The rooting test tube seedlings were transplanted into the soil and the cultivation substrate was a 1:1 mixture of peat moss + vermiculite. The surface layer is required to be sieved to facilitate the attachment of the root system; the lower layer is covered with coarse particles to facilitate the permeability of the substrate. When transplanting, use tweezers to remove the root attached to the medium (not to damage the roots). Use a tip to set a hole in the substrate, place the root system, cover the soil, lightly compact and pour enough water. The key to survival of transplanting test-tube seedlings is to ensure certain temperature and humidity conditions. The conditions for transplanting and test-tube culture vary widely, often resulting in discomfort or death of transplanted seedlings.
Although the transplanting of tube rooting plants can be carried out throughout the year, it is safer and more convenient for the spring and fall seasons without the rooting room.
Fourth, the application of tissue culture technology in the breeding of new varieties of rhododendron at the beginning of the test, given that the azalea flower explants are difficult to sterilize, the inoculation groups available for testing are too small to meet the normal test. In order to screen out the appropriate medium formulation and the corresponding culture conditions as soon as possible, the author combined the methods of cross breeding, using hybrid seed tubes to germinate, and then used the test tube seedlings to study the epicotyl for explants, achieving a multiplier effect. The authors found that the hybrid seedlings of Rhododendron need 3 to 5 years for flowering. However, the hybrid seedlings cultured in the epicotyl can be flowered in the second year after transplanting, greatly shortening the childhood of hybrid seedlings. It is of great significance to speed up the process of breeding flowers and fruit plants.
In general, seed sterilization is much easier than other organs or tissues. The sterilized treatment of azaleas hybrid seeds was as follows: 70% alcohol was soaked on the surface for 1 minute, rinsed with sterile water, then transferred to 0.1% mercury-mercuring solution for 10 minutes, and sterilized water was rinsed 3 times for more than 1 minute each time. After sterilization, seeds were taken and seedlings were inoculated and cultivated. Seeding was initiated within two weeks after inoculation. After emergence, the embryonic shoots were propagated using the epicotyl for rapid propagation and the propagation coefficient was high. After emergence, the shoot tip or stem section may be re-cultivated. The culture conditions and methods are the same as above. Domestic tissue culture involves a small number of new breeds, especially in ornamental horticultural plants. From the development point of view, in order to maintain strong vitality, tissue culture techniques should not leave the breeding of new varieties. Therefore, organically combining tissue culture technology with the selection and breeding of new varieties of azaleas will be very beneficial to improving the level of greenery plant landscape construction and promoting the industrialization of azaleas.
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