CN109526748B - Tissue culture method for anthurium andraeanum inflorescence - Google Patents
Tissue culture method for anthurium andraeanum inflorescence Download PDFInfo
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- CN109526748B CN109526748B CN201811633203.9A CN201811633203A CN109526748B CN 109526748 B CN109526748 B CN 109526748B CN 201811633203 A CN201811633203 A CN 201811633203A CN 109526748 B CN109526748 B CN 109526748B
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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
- A01H4/001—Culture apparatus for tissue culture
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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 discloses a method for culturing anthodium taro inflorescence tissues, which comprises the following steps: the rhinacanthus nasutus is obtained by disinfection and cleaning, induction and proliferation (culture and subculture) of cluster buds of inflorescence, rooting induction of the cluster buds, and transplantation and hardening of rooted seedlings. The method can obtain the rhinacanthus nasutus tissue culture seedlings with stable cultivation properties and consistent growth, and select the test-tube seedlings with vigorous growth from turfy soil or turfy soil: the seedling domestication is carried out in a ratio of vermiculite =4:1, the survival rate of the test-tube seedlings is 98%, the growth vigor is good, and technical support is provided for industrial production and cost saving.
Description
Technical Field
The invention relates to a tissue culture method of a taro inflorescence, belonging to the technical field of taro inflorescence tissue culture.
Background
The plant of the genus Rhinacanthus in the family Araceae, the plant is an ornamental plant cultivated indoors in recent years; the varieties are various, and the ornamental leaves and the inflorescences are the main ones. Because the pollen of the hairyvein agrimony is degraded and is not easy to seed, the artificial pollination is needed; the conventional breeding of the taro is mainly based on the plant division breeding, the slow breeding speed and the long breeding period are not suitable for large-scale production and seedling culture. So far, the tissue culture of the taro has a great breakthrough, and the prior art mainly obtains certain achievements through the propagation of leaves, stem tips and the like, the research of embryoids of inflorescences and the research of germination of panicles. However, the technique for culturing the anthus nasutus inflorescence is not described.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a tissue culture method of the anthodium taro inflorescence, which solves the problems in the prior art.
The technical scheme adopted by the invention is as follows: a method for culturing the tissue of the anthodium of the hairyvein agrimonia, which comprises the following steps:
(1) collecting inflorescences, collecting parent plant inflorescences which grow vigorously, have no diseases and insect pests and are young and tender from 8 o 'clock to 10 o' clock in early summer, wherein the length of the parent plant inflorescences is not more than 2cm, and cleaning and disinfecting the inflorescences;
(2) cutting each inflorescence into 4 parts by using a blade on average, wherein an induction culture medium is MS, the MS induction culture medium is added with 6-BA, KT, NAA, agar and sucrose, the culture condition is dark culture, the temperature is 25 +/-2 ℃, subculture is induced in an original culture medium, the culture condition is carried out under the condition of light, the illumination intensity is 2000lux, 16h/d, and the temperature is 25 +/-2 ℃;
(3) cutting off and rooting after the cluster buds of the inflorescence grow well and the differentiated buds grow to be more than 2cm, taking 1/2MS as a basic rooting culture medium, adding auxin IBA with the concentration of 0.5mg/L, and adding sucrose with the concentration of 20 g/L;
(4) transplanting domesticated seedlings selects rooting seedlings with good emerald leaves, transplanting substrates are subjected to high-temperature sterilization treatment, and the substrates are 100% turfy soil or turfy soil: 1: 4, transferring a culture bottle from an incubator to an indoor culture frame for a week at the seedling domesticating initial stage, opening a sealing film to domesticate the seedlings for more than one week, transplanting and domesticating the test-tube seedlings before a large amount of fungi are bred in the culture bottle, and spraying water to the test-tube seedling leaves in the morning and at the evening every day.
Preferably, the cleaning and disinfecting in the step (1): adding 4 drops of liquid detergent, washing with tap water for half an hour, transferring to an ultra-clean bench, treating with 75% ethanol for 30s, and treating with 0.1% HgCl2The treatment is carried out for 6 min.
Preferably, the amounts of 6-BA, KT, NAA, agar and sucrose in the above step (2) are 3mg/L, 0.5mg/L, 0.1mg/L, 6g/L and 30g/L, respectively.
The invention has the beneficial effects that: compared with the prior art, the anthurium andraeanum tissue culture seedling with stable cultivation property and consistent growth can be obtained by taking the inflorescence of the anthurium andraeanum as an explant, sterilizing, cleaning, inducing the cluster buds of the inflorescence and multiplying (culturing and subculture), inducing the rooting of the cluster buds, transplanting and hardening the seedling of the rooted seedling, adding 6-BA3mg/L, KT0.5mg/L, NAA0.1mg/L, agar 6g/L and sucrose 30g/L into an MS culture medium for culturing and subculture, wherein the germination rate of the anthurium andraeanum reaches 89%, carrying out subculture differentiation on the original culture medium, and the number of the differentiated cluster buds can reach more than 20, and in the rooting stage, taking 1/2MS as a basic culture medium, adding 0.5mg/LIBA, and taking 20g/L as a proper cluster bud rooting culture medium; selecting test-tube seedlings which grow vigorously and transplanting the test-tube seedlings to 100% turfy soil or turfy soil: the vermiculite is domesticated in a ratio of 4:1, the survival rate of the test-tube plantlets is over 98 percent, the test-tube plantlets grow well, and technical support is provided for industrial production and cost saving.
Detailed Description
The invention is further described below with reference to specific examples.
Example 1: a method for culturing the tissue of the anthodium of the hairyvein agrimonia, which comprises the following steps:
(1) collecting inflorescences, collecting parent plant inflorescences which grow vigorously, have no diseases and insect pests and are young and tender from 8 o 'clock to 10 o' clock in early summer, wherein the length of the parent plant inflorescences is not more than 2cm, and cleaning and disinfecting the inflorescences;
(2) cutting each inflorescence into 4 parts by using a blade on average, wherein an induction culture medium is MS, the MS induction culture medium is added with 6-BA, KT, NAA, agar and sucrose, the culture condition is dark culture, the temperature is 25 +/-2 ℃, subculture is induced in an original culture medium, the culture condition is carried out under the condition of light, the illumination intensity is 2000lux, 16h/d, and the temperature is 25 +/-2 ℃;
(3) cutting off and rooting after the cluster buds of the inflorescence grow well and the differentiated buds grow to be more than 2cm, taking 1/2MS as a basic rooting culture medium, adding auxin IBA with the concentration of 0.5mg/L, and adding sucrose with the concentration of 20 g/L;
(4) transplanting domesticated seedlings selects rooting seedlings with good emerald leaves, transplanting substrates are subjected to high-temperature sterilization treatment, and the substrates are turfy soil or turfy soil and vermiculite, the turfy soil: 1: 4, transferring a culture bottle from an incubator to an indoor culture frame for a week at the seedling domesticating initial stage, opening a sealing film to domesticate the seedlings for more than one week, transplanting and domesticating the test-tube seedlings before a large number of fungi are bred in the culture bottle, and spraying water to the test-tube seedling leaves in the morning and at the evening every day.
Preferably, the cleaning and disinfecting in the step (1): adding 4 drops of liquid detergent, washing with tap water for half an hour, transferring to an ultra-clean bench, treating with 75% ethanol for 30s, and treating with 0.1% HgCl2The treatment is carried out for 6 min.
Preferably, the amounts of 6-BA, KT, NAA, agar and sucrose in the above step (2) are 3mg/L, 0.5mg/L, 0.1mg/L, 6g/L and 30g/L, respectively.
To further illustrate the effects of the present invention, the following experiments were performed:
test materials and methods
1.1 test materials and treatments
The test material is taken from a No. 12 Erythrophorum chinense cultivation greenhouse in Tianxin flower breeding base in Nengyun Chifeng city, the best season of the inflorescence is collected as early summer, and the collection time is 8-10 points earlier. The mother plant of the collected inflorescence grows vigorously without diseases and insect pests, and the length of the young inflorescence is preferably not more than 2 cm. The sample is taken back to a laboratory for cleaning, then 4 drops of liquid detergent is added for cleaning, the sample is washed under tap water for half an hour and transferred to an ultra-clean workbench for 30s of treatment by 75 percent alcohol and 0.1 percent HgCl2The treatment is carried out for 6 min.
1.2 Induction of Cluster buds of inflorescence and proliferation thereof
Cutting each inflorescence into 4 parts by a blade on average, wherein an induction culture medium is MS; wherein 6-BA (1.0mg/L, 2.0mg/L, 3.0mg/L), KT (0.5mg/L) and NAA (0.1mg/L, 0.5mg/L) are added into the culture medium, the experiment is repeated for 3 times, each group is inoculated with 10 bottles, and each bottle is inoculated with 2 explants. The culture condition is dark culture, subculture is induced in the original culture medium, and the culture condition is carried out under light conditions.
1.3 rooting Induction of Cluster shoots with different sucrose concentrations
Cutting off and rooting after the cluster buds of the inflorescence grow well and the differentiated buds grow to more than 2 cm. 1/2MS is used as a basic rooting culture medium, 0.5mg/L of auxin IBA is added, and different sucrose concentrations of 20g/L, 30g/L, 40g/L and 50g/L are added; and observing the rooting condition of the differentiated bud and the growth condition of the seedling in 10-20 days.
1.4 transplanting and hardening of the rooting seedlings under different matrixes
Transplanting domesticated seedlings selects rooting seedlings with good emerald leaves, transplanting substrates are subjected to high-temperature sterilization treatment, and the substrates are 100% turfy soil or turfy soil: and (4) 1: 4 for vermiculite, and 300 seedlings are domesticated in each group. In the initial stage of domesticating the seedlings, the culture bottle is firstly moved from the incubator to an indoor culture frame for transition for one week, then the sealing film is opened to domesticate the seedlings for more than one week, and the test-tube seedlings are transplanted and domesticated before a large amount of fungi are bred in the culture bottle. The leaves of the test tube seedlings are sprayed with water in the morning and at night every day to prevent the leaves from drying up.
1.5 culture conditions
Dark culture conditions: the temperature is 25 +/-2 ℃; the light culture comprises the following steps: the illumination intensity is 2000lux, the illumination time is 16h/d, and the temperature is 25 +/-2 ℃. Sucrose (30 g/L) and agar (6 g/L) are added into the culture medium.
2 results and analysis
2.1 Effect of different hormone combinations on inflorescence Cluster bud Induction
Inoculating the inflorescence into a culture medium, and culturing on the 10 th day, wherein the surface begins to swell and has bulges; when the surface of the inflorescence is obviously enlarged and has differentiation signs on the day 21 of culture, the culture bottle is transferred to a light incubator for light culture; the white inflorescences became green on the day 7 of the light culture and a small part of seedlings differentiated from the inflorescences; when the seedlings are cultured to the 40 th day, the differentiated seedlings have good leaf opening and growth, and the culture medium consumes more nutrients and needs subculture; culturing for 49 days, and successively differentiating the plantlets from the base of the inflorescence cluster; when the culture time exceeds 60 days, the cluster buds occupy the whole culture flask, and the number of the cluster buds can be up to 20. However, it can be derived from table 1: with the increase of the concentration of 6-BA, the induction rate of the cluster buds is increased, and the proliferation number of the cluster buds is also increased (shown in a table 1); when the concentration of 6-BA is 3.0mg/L and the concentration of NAA is 0.1mg/L, the cluster bud inductivity can reach 89% and the cluster bud inductivity can be used as a culture medium for inducing cluster buds by inflorescences.
TABLE 1 Effect of different hormone combinations on inflorescence cluster shoot Induction
Note that different lower case letters in the same column represent significance levels (P <0.05), respectively.
2.2 Effect of different sucrose concentrations on Cluster bud rooting
Cutting off the well-grown cluster buds, inoculating the cluster buds to rooting culture media with different sucrose concentrations, culturing the cluster buds in the rooting culture media for about 10 days, and differentiating bud primordia from the base parts. As can be seen from table 2, the rooting rates of the cluster buds are all 100% as the sucrose concentration increases, the sucrose concentration has no significant difference (P <0.05) to the rooting rates of the cluster buds, and also has no significant difference (P <0.05) to the root number; however, the growth conditions of the rooted seedlings are relatively consistent, and no obvious difference exists. Therefore, 20g/L was selected as the sucrose concentration in the rooting medium from the economical point of view.
TABLE 2 Effect of different sucrose concentrations on rooting of clumpy buds
2.3 Effect of different substrates on growth of test-tube plantlets
The culture bottle is removed from the incubator at the seedling training initial stage, the size of the opening of the bottle is continuously increased along with the increase of time in the seedling training process (the first day when the sealing film is opened), the propagation speed of fungi breeding is extremely high when the cultivation starts in the culture bottle about 5 days after the seedlings are trained, and the test-tube seedlings are damaged and die if the cultivation is not carried out in time. Transplanting the grass peat soil into the grass peat soil by statistics: the survival rate of 294 plants which survive in 300 test-tube plantlets in 4:1 is as high as 98 percent, and the growth is good; while the survival rate of 295 plants survived from 300 test-tube seedlings transplanted in the turfy soil is 98.3 percent (35 days of transplanting in the turfy soil). The test-tube plantlets grown in the two matrixes grow well without obvious difference and can be used as transplanting matrixes of the test-tube plantlets.
3 conclusion and discussion
Adding hormones of 3mg/L6-BA, 0.5mg/LKT and 0.1mg/LNAA into an MS culture medium to ensure that the germination rate of inflorescences reaches 89 percent, and carrying out subculture differentiation on the original culture medium to ensure that the highest number of differentiated cluster buds reaches more than 20; in the rooting stage, 1/2MS is used as a basic culture medium, 0.5mg/LIBA is added, and 20g/L is the concentration of sucrose suitable for rooting cluster buds; selecting test-tube plantlets which grow vigorously from turfy soil and turfy soil: domesticating the test-tube plantlets in a ratio of 4:1, wherein the survival rate of the test-tube plantlets is 98 percent and the growth vigor is good.
6-BA, KT and NAA are added in the initiation of the cluster buds of the inflorescence in the experiment, and the experiment shows that the high-concentration cytokinin is more beneficial to the initiation, proliferation and differentiation of the inflorescence, and the induction rate is up to 89%. Rooting induction is carried out on the cluster buds under the condition of 0.5mg/LIBA by using different sucrose concentrations, however, the rooting induction rate is 100 percent, no obvious difference exists between the growth vigor and the number of rooting roots of the test-tube plantlets, and more economic sucrose concentration of 20g/L is selected. Transplanting the gradually domesticated test-tube plantlets to turfy soil or turfy soil: in the case of vermiculite 4:1, the survival rate is more than 98%.
The above description is only an embodiment 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 changes or substitutions within the technical scope of the present invention, and therefore, the scope of the present invention should be determined by the scope of the claims.
Claims (1)
1. A method for culturing the tissue of the anthodium of the white crane is characterized in that: the method comprises the following steps:
(1) collecting inflorescences, collecting parent plant inflorescences which grow vigorously, have no diseases and insect pests and are young and tender from 8 o 'clock to 10 o' clock in early summer, wherein the length of the parent plant inflorescences is not more than 2cm, and cleaning and disinfecting the inflorescences; cleaning and disinfecting: adding 4 drops of liquid detergent, washing with tap water for half an hour, transferring to an ultra-clean bench, treating with 75% ethanol for 30s, and treating with 0.1% HgCl2Treating for 6 min;
(2) averagely cutting each inflorescence into 4 parts by using a blade, wherein an induction culture medium comprises MS, 3mg/L6-BA, 0.5mg/L KT, 0.1mg/L NAA, 6g/L agar and 30g/L sucrose, the culture condition is dark culture, the temperature is 25 +/-2 ℃, subculture is induced in an original culture medium, the culture condition is illumination culture, the illumination intensity is 2000lux, 16h/d, and the temperature is 25 +/-2 ℃;
(3) cutting off and rooting when the cluster buds of the inflorescence grow well and the differentiated buds grow to be more than 2cm, wherein the rooting culture medium is 1/2MS, 0.5mg/L auxin IBA, 20g/L sucrose and 6g/L agar;
(4) transplanting domesticated seedlings selects rooting seedlings with good emerald leaves, transplanting substrates are subjected to high-temperature sterilization treatment, and the substrates are 100% turfy soil or turfy soil: vermiculite =4:1, the culture bottle is moved from the incubator to an indoor culture frame for a week at the seedling domesticating initial stage, the sealing film is opened to domesticate the seedlings for more than one week, and the test tube seedling leaves are sprayed with water in the morning and evening every day.
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| CN101238795A (en) * | 2008-03-07 | 2008-08-13 | 阳光国际集团科技发展有限公司 | Artificial breeding method for spathiphyllum floribundum |
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