CN113951144B - Method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds - Google Patents
Method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds Download PDFInfo
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Abstract
Provides a method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds. The method comprises the steps of collecting and disinfecting fruit pods, germinating seeds, differentiating protocorms, rooting and the like. According to the invention, different culture media are designed at different development stages by optimizing the culture media, so that a tissue culture and rapid propagation system of paphiopedilum sanctum is established, the germination rate of paphiopedilum sanctum seeds can reach 80%, the seedling rate can reach 94%, and the survival rate after half a year of transplantation can reach 90.5%. By adopting the method, the large-scale reproduction of the paphiopedilum sanfranch can be realized, and the method has important significance for protecting and utilizing the paphiopedilum sanfranch germplasm resources.
Description
Technical Field
The invention belongs to the technical field of plant biology, particularly relates to rapid paphiopedilum propagation, and more particularly relates to a method for promoting aseptic germination and seedling formation of paphiopedilum sansevieri seeds.
Background
Paphiopedilum (Paphiopedilum) is a famous ornamental orchid in the world, and is also called mullen because the labial lobe of the flower is specialized in a pocket shape. At present, the wild population number of paphiopedilum is reduced sharply due to excessive collection and habitat loss, and all species of the paphiopedilum are listed in appendix I of International convention on the trade of endangered species of wild animals and plants (CITES), and are protected to the highest degree. The solution of the seedling breeding technology has important significance for the protection and utilization of germplasm resources. Due to the lack of endosperm, seeds of orchids are difficult to germinate under natural conditions, and symbiotic fungi are usually required to provide the seeds with the nutrition required by seed germination, and the natural germination rate is less than 5%. The seed sterile germination technology and the asexual cloning technology are main seedling artificial breeding modes of orchids, but the asexual cloning of paphiopedilum is very difficult, and although some progress is made in the technology at present, the seed sterile germination technology and the asexual cloning technology cannot be put into commercial application. The seed sterile germination technology has been successful on various paphiopedilum, but the seedling rate of the technology is greatly different among species, and the sterile germination of seeds of some species is very difficult, such as paphiopedilum sanctum (P.tigrinum). Zeng Songjun and the like (2011) perform sterile sowing on a plurality of paphiopedilum, test tube seedlings are obtained except paphiopedilum sansevieri and paphiopedilum elegans (P.venustum), and thus the difficulty of seedling breeding of paphiopedilum sansevieri is seen. The paphiopedilum frutescens is a rare species with high commercial value, and the lack of the seedling breeding technology greatly limits the large-scale production and cultivation and the return to the original land. The aseptic germination and seedling technology of the seeds of the paphiopedilum hirsutissimum will have important significance for the protection and utilization of special rare germplasm resources.
Disclosure of Invention
The invention develops the optimal culture medium formula of the paphiopedilum schrenckii in each stage of sterile germination of seeds, protocorm differentiation, seedling subculture, strong seedling rooting and the like so as to improve the germination rate and the seedling rate of the paphiopedilum schrenckii seeds and provide theoretical support and technical support for protection regression and large-scale propagation of the paphiopedilum schrenckii.
Aiming at the problems of low germination rate and difficult seedling and rooting of paphiopedilum sansevieri in the prior art, the invention aims to provide a method for sterile germination and seedling rapid propagation of paphiopedilum sansevieri seeds, and the germination rate and the seedling rate of the seeds are greatly improved.
The above purpose of the invention is realized by the following technical scheme:
a method for promoting aseptic germination and seedling formation of paphiopedilum sansevieri seeds comprises the following steps:
(1) Collecting and disinfecting fruit pods: selecting ripe and uncracked fruit pods of paphiopedilum micranthum, washing, soaking in 0.1% mercuric chloride solution for 10-15min, sterilizing the surface of 75% ethanol for 30s, slightly burning the fruit pods by an alcohol lamp, and placing on sterile paper for later use;
(2) And (3) sterile germination of seeds: cutting the sterilized fruit pods on sterile paper, uniformly scattering the cut sterilized fruit pods on the surface of a seed germination culture medium, starting germination of seeds in 13-19 days, taking the period of 3-6 weeks as the peak period of seed germination, finishing germination in 7 weeks, and performing dark culture for 2-3 months to obtain white and healthy protocorms;
(3) Protocorm differentiation: transferring the protocorm obtained in the step (2) into a protocorm differentiation culture medium, gradually turning green after light culture, and culturing for 3-4 months to obtain a seedling with 1-2 leaves;
(4) Seedling subculturing: transferring the plantlets with 1-2 leaves obtained in the step (3) into a plantlet subculture medium, and culturing for 3-4 months to obtain plantlets with 2-3 leaves;
(5) Rooting and strengthening seedlings: transferring the plantlets with 2-3 leaves obtained in the step (4) into a rooting and seedling strengthening culture medium, and culturing for 4-6 months to obtain plantlets with 4-5 leaves, 5-7 roots and the height of about 4 cm;
(6) Transplanting seedlings: and (4) hardening the seedlings obtained in the step (5) for 2 weeks under the greenhouse condition, taking the plants out of the bottle, washing the culture medium attached to the roots of the seedlings, and transferring the seedlings into a crushed bark substrate for culture.
According to the method for promoting sterile germination and seedling formation of paphiopedilum sanfranch seeds, the paphiopedilum sanfranch fruit pods in the step (1) are washed by soapy water and tap water.
According to the method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds, the seed germination culture medium in the step (2) is as follows: modified Harvais +1mg/L KT +0.1g/L active carbon +100mL/L coconut milk +20g/L sucrose +6g/L agar.
According to a method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds, the seed germination in the step (2) is cultured under the complete dark condition.
According to the method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds, the protocorm differentiation medium in the step (3) is as follows: 1/4MS +2mg/L KT +0.3g/L active carbon + 50-100 mL/L coconut milk +20g/L sucrose +6g/L agar.
According to the method for promoting sterile germination and seedling formation of paphiopedilum sanctum seeds, the seedling subculture medium in the step (4) is as follows: 1/2MS + 0.5-1.5 g/L of activated carbon +100mL/L of coconut milk +20g/L of sucrose +6g/L of agar.
According to the method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds, the rooting and seedling strengthening culture medium in the step (5) is as follows: 1/2MS +15g/L potato +30g/L banana +1.0g/L active carbon +0.5g/L dolomite powder +20g/L sucrose +6g/L agar.
According to the method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds, the steps (3), (4) and (5) are cultured under the conditions of 25 +/-2 ℃,12 h/d of illumination and 1600-2000 Lx of illumination, and the step (6) is carried out under the conditions of the environmental temperature of 18-24 ℃, the relative air humidity of 50-70% and the shading of 75-80%.
According to the method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds, the PH values of the seed germination culture medium, the protocorm differentiation culture medium, the seedling subculture culture medium and the rooting and seedling strengthening culture medium in the steps (2), (3), (4) and (5) are all 5.6-5.8.
A method for promoting aseptic germination and seedling formation of paphiopedilum sansevieri seeds comprises the following specific operation steps:
(1) Collecting and disinfecting fruit pods: selecting ripe and uncracked fruit pods of paphiopedilum hirsutissimum, scrubbing the fruit pods with soapy water, washing the fruit pods with tap water, soaking the fruit pods on a super-clean workbench for 10-15min with 0.1% mercuric chloride solution, sterilizing the surfaces of 75% ethanol for 30s, slightly burning the fruit pods with an alcohol lamp, and placing the fruit pods on sterile paper for later use.
(2) And (3) sterile germination stage of seeds: the sterilized fruit pods were cut open with a scalpel on sterile paper and evenly spread on the surface of the seed germination medium. The seeds start to germinate in 13 to 19 days, the peak period of the seed germination is 3 to 6 weeks, and the germination is finished about 7 weeks. After dark culture for 2-3 months, white and healthy protocorms (figure 1) are obtained, and the effective germination rate can reach 82%. The seed germination culture medium comprises: modified Harvais +1mg/LKT +0.1g/L active carbon +100mL/L coconut milk +20g/L sucrose +6g/L agar.
(3) Protocorm differentiation stage: transferring the protocorm into a protocorm differentiation culture medium, gradually turning green after light culture, and culturing for 3-4 months to obtain a seedling with 1-2 leaves (figure 2), wherein the differentiation rate is as high as 94%. The protocorm differentiation culture medium comprises: 1/4MS +2mg/L KT +0.3g/L active carbon + 50-100 mL/L coconut milk +20g/L sucrose +6g/L agar.
(4) Seedling subculture stage: transferring the plantlets with 1-2 leaves into a plantlet subculture medium, and culturing for 3-4 months to obtain plantlets with 2-3 leaves (figure 3). The seedling subculture medium comprises: 1/2MS + 0.5-1.5 g/L of active carbon +100mL/L of coconut milk +20g/L of sucrose +6g/L of agar.
(5) And (3) rooting and seedling strengthening stage: transferring the plantlets with 2-3 leaves into a rooting and seedling strengthening culture medium, and culturing for 4-6 months to obtain plantlets with 4-5 leaves, 5-7 roots and the height of about 4 cm. The rooting and seedling strengthening culture medium comprises: 1/2MS +15g/L potato +30g/L banana +1.0g/L active carbon +0.5g/L dolomite powder +20g/L sucrose +6g/L agar.
(6) Transplanting seedlings: the seedlings are placed in a greenhouse condition (the ambient temperature is 18-24 ℃, the relative air humidity is 50-70%, and the shading is 75-80%) to be trained for 2 weeks, then the plants are taken out of the bottle, the culture medium attached to the roots of the seedlings is cleaned, and the seedlings are transplanted into a crushed bark substrate to be cultured. The survival rate can reach 90.5% after half a year of transplantation.
And (3) culturing the seed germination in complete darkness, culturing the seeds in the steps (3), (4) and (5) under the conditions of 25 +/-2 ℃,12 h/d of illumination and 1600-2000 Lx of illumination, and culturing the seeds in the step (6) under the conditions of the ambient temperature of 18-24 ℃, the relative air humidity of 50-70% and the shading of 75-80%. The PH values of the seed germination culture medium, the protocorm differentiation culture medium, the seedling subculture medium and the rooting and seedling strengthening culture medium are all between 5.6 and 5.8.
Compared with the prior art, the invention has the following advantages:
according to the invention, different culture media are designed at different development stages, a tissue culture and rapid propagation system of paphiopedilum sanforbesii is established, the germination rate of seeds of paphiopedilum sanforbesii can reach 80%, the seedling rate can reach 94%, and the survival rate after half a year of transplantation can reach 90.5%. By adopting the method, the large-scale reproduction of the paphiopedilum sanfranchetii can be realized, and the method has important significance for protecting and utilizing the paphiopedilum sanfranchetii germplasm resources.
Drawings
FIG. 1 is a schematic diagram of the germination and cultivation of seeds according to the present invention;
FIG. 2 is a schematic representation of the differentiation of protocorms according to the present invention;
FIG. 3 is a schematic diagram of the seedling subculture of the present invention;
FIG. 4 is a schematic diagram of the strong seedling rooting culture of the present invention;
fig. 5 is a schematic diagram of the survival status of the transplanting.
Detailed Description
The material of the present invention will be further described with reference to the following examples, but the present invention is not limited thereto.
Example 1
1. And (3) sterilizing the surfaces of the fruit pods: scrubbing paphiopedilum micranthum (P.tigrinum) pods with soapy water for three times, washing the paphiopedilum micranthum (P.tigrinum) pods with tap water, soaking the paphiopedilum micranthum (P.tigrinum) pods in 0.1% mercuric chloride solution for 10-15min on a super clean workbench, sterilizing the surfaces of 75% ethanol for 30s, slightly burning the paphiopedilum micranthum by an alcohol lamp, and placing the paphiopedilum micranthum on sterile paper for later use.
2. Seed germination: cutting the disinfected fruit pod of the step (1) by a scalpel, and sowing seeds in a pre-sterilized 50mL triangular flask containing 20-30 mL of culture medium per flask (sterilized in an autoclave at 121 ℃ for 15 min). The components of the culture medium are as follows: the basic culture medium is cHa, and 1.0mg/L KT and 100mL/L coconut juice are added. The concentrations of activated carbon were set to 0.1, 0.5, 1.0, and 2.0g/L, respectively, as compared with a treatment without adding activated carbon.
Each treatment inoculated 3 to 5 flasks, with approximately 100 seeds per flask. The culture was carried out in dark. The germination rate of seeds in each medium was counted at 90d after sowing, while the browning rate at the completion of germination was counted. In this study, embryo enlargement is regarded as seed germination, and the individual after germination and before leaf/root formation is called protocorm.
Total germination (%) = number of all germinated seeds/number of seeds sown in each bottle
Browning rate (%) = number of vitrified and browned protocorms/number of germinated protocorms
Effective germination rate (%) = number of healthy protocorms/number of seeds sown in each bottle
The results show (table 1) that the total germination rate of paphiopedilum sanfranch increases and then decreases with the increase of the concentration of the activated carbon, and the browning rate gradually increases. The highest effective germination rate of the culture medium added with 0.1g/L of activated carbon reaches 88.10 +/-2.38%, and protocorm grows healthily.
TABLE 1 influence of activated carbon on the germination of paphiopedilum sansevieri seeds
Note: the basic culture medium is cHa +1.0mg/L KT +100mL/L coconut juice. Data are mean ± sem (n = 3-5). Different letters in the same column indicate significant differences (P < 0.05).
3. Protocorm differentiation: dark culturing the protocorm obtained in the step (2) for 90d, and transferring the protocorm to the following culture medium: KT (0.5, 1.0, 2.0, 4.0 mg/L) and TDZ (0.1, 0.5, 1.0, 2.0, 4.0 mg/L) with different concentrations are added by taking 1/4MS as a basic culture medium, and 50mL/L of coconut juice and 0.3g/L of activated carbon are added in the culture medium. After 90d, the differentiation rate was counted. All media were supplemented with 20g/L sucrose and 6g/L agar and pH was adjusted to 5.8. The culture temperature is 25 +/-2 ℃. Except special illumination requirements, the illumination time is 12h/d, and the illumination intensity is 1600-2000 Lx. A50 mL Erlenmeyer flask was used. The data were collated in Excel and the significance of differences was analyzed in SPSS 20. The result shows (Table 2), in the culture medium of 1/4MS +2.0mg/L KT +50mL/L coconut juice +0.3g/L active carbon, the differentiation rate of paphiopedilum is the highest and reaches 94%, the rooting rate reaches 66%, each seedling can be differentiated into 2 leaves on average, and the growth and development of the seedling are very healthy.
TABLE 2 influence of TDZ and KT on the differentiation of paphiopedilum sanctum protocorms
Note: the components of the culture medium are 1/4MS +0.3g/L active carbon +50mL/L coconut juice. Each treated 4 flasks, 30 protocorms per flask. Data are mean ± sem (n = 4). Different letters in the same column indicate significant differences (P < 0.05).
4. Seedling subculturing: transferring the paphiopedilum heulans seedlings with 1-2 leaves obtained in the step (3) into the following culture medium: taking 1/2MS as a basic culture medium, setting four gradients of activated carbon concentrations of 0, 0.5, 1.0 and 1.5g/L, and adding 1.0mg/L KT and 100mL/L coconut juice into the above culture media.
Each treatment consisted of 10 flasks of each, 10 seedlings of paphiopedilum regolium. And after 120d, counting the survival rate, the rooting rate and the average leaf number.
Survival (%) = number of surviving seedlings per bottle/total number of seedlings inoculated per bottle
Rooting rate (%) = number of plants rooted per bottle/total number of seedlings inoculated per bottle
Average leaf number = leaf number per bottle/number of surviving seedlings per bottle.
The results show (Table 3) that the survival rate, rooting rate and average leaf number of seedlings are obviously improved in the culture medium added with the activated carbon. In conclusion, the culture medium added with 0.5g/L of activated carbon has the best effect, the survival rate reaches 75%, the rooting rate reaches 66.25%, the average leaf number is the most, and the seedling growth performance is better.
TABLE 3 influence of activated carbon in the subculture of paphiopedilum sanfranchetii seedlings
5. Rooting and strengthening seedlings: transferring the paphiopedilum schroederi seedling with 2-3 leaves obtained in the step (4) into a rooting culture medium. 1/2MS is used as a basic culture medium, hormones and dolomite powder with different concentrations are added, and 1.0g/L of active carbon, 15g/L of potatoes and 30g/L of bananas are added into the culture medium. And counting the number of the survived seedlings and the leaf number, the longest leaf length, the number of roots, the longest root length, the longest root width and the plant height of each seedling after 120 days after each 40 seedlings are treated.
The results show (table 4) that the overground part treated with dolomite powder had a significantly better growth vigor, and the number of leaves, the length of leaves and the plant height were significantly higher than those treated without dolomite powder. In conclusion, in the culture medium added with 0.5g/L dolomite powder, the number of the largest seedlings of the paphiopedilum schroederi is 5.20 +/-0.29, the longest leaf length is 2.77 +/-0.08, and indexes such as the average number of the seedlings (6.15 +/-0.42), the longest root length (1.87 +/-0.12), the plant height (3.91 +/-0.10) and the like are on the same level.
6. Transplanting seedlings: and (3) putting the rooted seedlings obtained in the step (5) into a greenhouse condition (the environmental temperature is 18-24 ℃, the relative air humidity is 50-70%, and the shading is 75-80%) for hardening for 2 weeks, then taking the plants out of the bottle, cleaning a culture medium attached to the roots of the seedlings, and transferring the culture medium into a crushed bark substrate for culturing. The total number of 200 seedlings are transplanted, 181 surviving seedlings are obtained by statistics after half a year of transplantation, and the survival rate reaches 90.5%.
According to the invention, a technical scheme suitable for germination, protocorm differentiation, seedling subculture and rooting of paphiopedilum sanfranchetii seeds is summarized by optimizing a culture medium formula, so that the germination rate of the paphiopedilum sanfranch seeds and the seedling survival rate of seedlings are remarkably improved, the germination rate of the seeds can reach 80% (the natural germination rate is only 5%), the seedling survival rate can reach 94%, and the survival rate after half a year of transplanting can reach 90.5%. The invention is beneficial to realizing the large-scale propagation of paphiopedilum sanfranch and has important significance for protecting and utilizing the germplasm resources of paphiopedilum sanfranch.
Claims (6)
1. A method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds is characterized by comprising the following steps:
(1) Collecting and disinfecting fruit pods: selecting ripe and uncracked fruit pods of paphiopedilum sanctum, washing, soaking in 0.1% mercuric chloride solution for 10 to 15min, sterilizing the surfaces of the fruits by using 75% ethanol by using 30s, slightly burning the fruit pods by using an alcohol lamp, and placing the fruits on sterile paper for later use;
(2) Sterile germination of seeds: cutting the sterilized fruit pods on sterile paper, uniformly scattering the cut sterilized fruit pods on the surface of a seed germination culture medium, starting seed germination in 13-19 days, wherein 3~6 weeks is the peak period of seed germination, after finishing germination in 7 weeks, carrying out dark culture for 2~3 months to obtain protocorms, wherein the seed germination culture medium is as follows: cHa +1mg/L KT +0.1g/L activated carbon +100mL/L coconut milk +20g/L sucrose +6g/L agar;
(3) Protocorm differentiation: transferring the protocorm obtained in the step (2) into a protocorm differentiation culture medium, gradually turning green under light transfer culture, and culturing for 3~4 months to obtain a plantlet with 1~2 leaves, wherein the protocorm differentiation culture medium is as follows: 1/4MS +2mg/L KT +0.3g/L activated carbon +50 to 100mL/L coconut milk +20g/L sucrose +6g/L agar;
(4) Seedling subculturing: transferring the plantlets with 1~2 leaves obtained in the step (3) into a plantlet subculture medium, and culturing 3~4 months to obtain the plantlets with 2~3 leaves, wherein the plantlet subculture medium is as follows: 1/2MS +0.5 to 1.5g/L of activated carbon +100mL/L of coconut milk +20g/L of cane sugar +6g/L of agar;
(5) Rooting and strengthening seedlings: transferring the plantlets with 2~3 leaves obtained in the step (4) into a rooting and seedling strengthening culture medium, and culturing 4~6 months to obtain seedlings with 4~5 leaves, 5~7 roots and 4-6 cm high; the rooting and seedling strengthening culture medium comprises: 1/2MS +15g/L potato +30g/L banana +1.0g/L activated carbon +0.5g/L dolomite powder +20g/L sucrose +6g/L agar;
(6) Transplanting seedlings: and (4) hardening the seedlings obtained in the step (5) for 2 weeks under the greenhouse condition, taking the plants out of the bottle, washing the culture medium attached to the roots of the seedlings, and transferring the seedlings into a crushed bark substrate for culture.
2. The method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds as claimed in claim 1, wherein the paphiopedilum sansevieri fruit pod is washed by soap water and tap water in the step (1).
3. The method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds according to claim 1, wherein the seed germination in step (2) is cultured in complete darkness.
4. The method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds as claimed in claim 1, wherein the steps (3), (4) and (5) are all cultured under the conditions of 25 +/-2 ℃,12 h/d of illumination and 1600-2000 Lx of illumination, and the step (6) is carried out under the conditions of the environmental temperature of 18-24 ℃, the relative air humidity of 50-70% and the shading of 75-80%.
5. The method for promoting sterile germination and seedling establishment of paphiopedilum sansevieri seeds as claimed in claim 1, wherein the pH values of the seed germination culture medium, the protocorm differentiation culture medium, the seedling subculture culture medium and the rooting and seedling strengthening culture medium in steps (2), (3), (4) and (5) are 5.6-5.8.
6. A method for promoting aseptic germination and seedling formation of paphiopedilum sanctum seeds is characterized by comprising the following steps:
(1) Collecting and disinfecting fruit pods: selecting ripe and uncracked fruit pods of paphiopedilum micranthum, washing the fruit pods with soapy water, washing the fruit pods with tap water, soaking the fruit pods in 0.1% mercury bichloride solution for 10 to 15min on an ultra-clean workbench, sterilizing the surface of 75% ethanol for 30s, slightly burning the fruit pods with an alcohol lamp, and placing the fruit pods on sterile paper for later use;
(2) And (3) sterile germination stage of seeds: cutting the sterilized fruit pods on sterile paper by using a scalpel, uniformly scattering the cut fruit pods on the surface of a seed germination culture medium, starting germination of seeds in 13-19 days, wherein 3~6 weeks is the peak period of seed germination, and finishing germination in 7 weeks; the seed germination is cultured under the complete dark condition, white and healthy protocorms are obtained after the dark culture for 2~3 months, and the effective germination rate reaches 82 percent; the seed germination culture medium comprises: cHa +1mg/L KT +0.1g/L activated carbon +100mL/L coconut milk +20g/L sucrose +6g/L agar, and the PH is between 5.6 and 5.8;
(3) Protocorm differentiation stage: transferring the protocorm into a protocorm differentiation culture medium, gradually turning the protocorm green after light transferring culture, and culturing for 3~4 months to obtain a plantlet with 1~2 leaves, wherein the differentiation rate reaches 94%; the protocorm differentiation culture medium comprises: 1/4MS +2mg/L KT +0.3g/L activated carbon +50 to 100mL/L coconut milk +20g/L sucrose +6g/L agar; culturing at 25 + -2 deg.C under illumination of 12h/d and 1600-2000Lx, with a pH of 5.6-5.8;
(4) Seedling subculture stage: transferring the plantlets with 1~2 leaves into a plantlet subculture medium, and culturing 3~4 months to obtain plantlets with 2~3 leaves, wherein the plantlet subculture medium is as follows: 1/2MS +0.5 to 1.5g/L of activated carbon +100mL/L of coconut milk +20g/L of cane sugar +6g/L of agar; culturing at 25 + -2 deg.C under illumination of 12h/d and 1600-2000Lx, with a pH of 5.6-5.8;
(5) And (3) rooting and seedling strengthening stage: transferring the plantlets with 2~3 leaves into a rooting and strong seedling culture medium, and culturing 4~6 months to obtain plantlets with 4~5 leaves, 5~7 roots and a height of about 4cm, wherein the rooting and strong seedling culture medium is as follows: 1/2MS +15g/L potato +30g/L banana +1.0g/L activated carbon +0.5g/L dolomite powder +20g/L sucrose +6g/L agar; culturing at 25 + -2 deg.C under illumination of 12h/d and 1600-2000Lx, with a pH of 5.6-5.8;
(6) Transplanting seedlings: and (3) hardening the seedlings under the greenhouse condition for 2 weeks, keeping the environmental temperature at 18-24 ℃, keeping the relative air humidity at 50-70%, shading the light at 75-80%, taking the plants out of the bottle, cleaning a culture medium attached to the roots of the seedlings, transferring the culture medium into a crushed bark substrate for culture, and culturing the culture medium until the survival rate reaches 90.5% after transplanting for half a year.
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